Patent application title: BIOMARKERS FOR RADIATION TREATMENT
Inventors:
IPC8 Class: AC12Q16886FI
USPC Class:
1 1
Class name:
Publication date: 2021-03-04
Patent application number: 20210062277
Abstract:
The methods described herein allow for the classification of patients
into groups for receiving optimized radiation treatment based on patient
specific biomarker signature. The biomarker signature includes markers
that have been shown to correlate with TGF-B expression and to be
associated with tumor aggressiveness, radioresistance and poor prognosis.
The markers play a key role in the epithelial-mesenchymal transition. The
methods described herein provide the dual benefits of anti-tumor efficacy
plus normal tissue protection when combining TGF-B inhibitors with
ionizing radiation to treat cancer patients.Claims:
1. A method of identifying a subject as a candidate for treatment with
ionizing radiation, comprising: (a) determining an expression level of
two or more biomarkers in a tumor sample from the subject, wherein the
one or more biomarkers are selected from the group consisting of CD44,
MMP9, ALDH1A1, Vimentin, hyaluman, beta-catenin, MFG-E8 and CD68; and (b)
comparing the expression level of the two or more biomarkers to an
expression level in a normal tissue sample; wherein an expression level
of the two or more biomarkers in the tumor sample that is modified
compared to the expression level in the normal tissue sample identifies
the subject as a candidate for treatment with ionizing radiation.
2. The method of claim 1, wherein the expression level of the two of more biomarkers is ranked or weighted.
3. The method of claim 1, wherein the expression level of biomarkers CD44 and CD68 is increased in the tumor environment.
4. The method of claim 1, wherein the tumor sample is a biopsy comprising tumor cells.
5. The method of claim 1, wherein the normal tissue sample comprises non-tumor cells from the same tissue type as the tumor.
6. The method of claim 1, wherein the biomarker is a gene, an RNA, or a protein and the expression level is determined by detecting the expression of an RNA and/or a protein.
7. The method of claim 6, wherein the detecting is selected from the group consisting of immunohistochemistry, ELISA, Western analysis, HPLC, proteomics, PCR, RT-PCR, Northern analysis, and nucleic acid or polypeptide microarrays.
8. The method of claim 1, further comprising treating the subject with ionizing radiation if the expression level of the two or more biomarkers in the tumor sample is modified compared to the expression level in the normal tissue sample.
9. The method of claim 8, wherein the treatment further comprises contacting the tumor with a radiosensitizer.
10. The method of claim 8, wherein the treatment further comprises administering a compound that inhibits TGF-beta signaling to the subject.
11. The method of claim 10, wherein the TGF-beta inhibitor is an antibody or a small molecule that neutralizes or inhibits TGF-beta function, or the TGF-beta inhibitor inhibits the production of TGF-beta.
12. A method for selecting a treatment for a subject having a tumor, comprising: (a) determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8 and CD68; (b) comparing the expression level of the two or more biomarkers to an expression level in a normal tissue sample; and selecting a treatment if the expression level of the two or more biomarkers is modified compared to the expression level in the normal tissue sample.
13. The method of claim 12, wherein the expression level of biomarkers CD44 and CD68 is increased in the tumor environment.
14. The method of claim 12, wherein the tumor sample is a biopsy comprising tumor cells.
15. The method of claim 12, wherein the normal tissue sample comprises non-tumor cells from the same tissue type as the tumor.
16. The method of claim 12, wherein the treatment comprises administering ionizing radiation to the tumor.
17. The method of claim 16, wherein the treatment further comprises contacting the tumor with a radiosensitizer.
18. The method of claim 16, wherein the treatment further comprises administering a compound that inhibits TGF-beta signaling to the subject.
19. The method of claim 18, wherein the TGF-beta inhibitor is an antibody or a small molecule that neutralizes or inhibits TGF-beta function, or the TGF-beta inhibitor inhibits the production of TGF-beta.
20. A kit comprising reagents capable of detecting expression of a biomarker selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8 and CD68.
Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present application is a divisional of, and claims the benefit and priority to U.S. Continuation application Ser. No. 15/913,633, filed Mar. 6, 2018 (now allowed), entitled, "BIOMARKERS FOR RADIATION TREATMENT," which claims the benefit and priority to U.S. application Ser. No. 14/777,209, filed Sep. 15, 2015, now U.S. Pat. No. 9,938,583, entitled "BIOMARKERS FOR RADIATION TREATMENT," which claims the benefit and priority of International Application No. PCT/US2014/029365, filed Mar. 14, 2014, entitled "BIOMARKERS FOR RADIATION TREATMENT," and which claims the benefit and priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Application No. 61/800,011, filed Mar. 15, 2013, the entire contents of which are herein incorporated by reference for all purposes.
REFERENCE TO A "SEQUENCE LISTING" SUBMITTED AS ASCII TEXT FILE
[0002] The Sequence Listing written in file 088389-002920US-079276_SequenceListing.txt created on May 16, 2018, 126,732 bytes, machine format IBM-PC, MS-Windows operating system, in accordance with 37 C.F.R. .sctn..sctn. 1.821 to 1.825, is hereby incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] No validated protein signature is available that has been proved to be sufficiently useful in the clinic to stratify patients into groups that may be treated differently with radiotherapy. Many factors determine the biology of tumors and as such impact prognosis and survival outcome of cancer patients. TGF-.beta. is a pleiotropic cytokine that is important in normal tissue homeostasis, regulates inflammation and immune responses, and controls proliferation and differentiation. TGF-.beta. appears to be key in promoting epithelial-mesenchymal-transition (EMT), a process that leads to increased motility and invasion. Due to these oncogenic properties of TGF-0, several TGF-.beta. signalling inhibitors are in preclinical and clinical trials to treat cancer. Radiotherapy is a corner stone of cancer therapy. There is substantial evidence that TGF-.beta. plays a key role in the response to ionizing radiation. TGF-.beta. is activated in irradiated tissues and plays a pivotal role in development of radiation induced fibrosis.
BRIEF SUMMARY OF THE INVENTION
[0004] The discourse provides biomarkers that are useful for diagnosing and treating tumors or cancer in a subject. The disclosure further provides methods of treating tumors in a subject having modified (i.e., increased or decreased) levels of one or more biomarkers described herein. In some embodiments, methods for treating tumors where the level of one or more biomarkers is increased and the level of another biomarker is decreased are described. The disclosure also provides methods of diagnosing or identifying subjects in need of treatment based on the expression levels of the biomarkers described herein. In some embodiments, the treatment comprises administering ionizing radiation to the subject.
[0005] In one embodiment, the treatment comprises administering an increased dose of ionizing radiation to the subject if the level of one or more biomarkers described herein is modified in the tumor environment, where the dose of ionizing radiation is increased as compared to the standard of care for a subject that does not have modified levels of the biomarker(s) in the tumor environment. Alternatively, the treatment can comprise administering the same or a similar dose of ionizing radiation as the standard of care in combination with a pharmaceutically effective amount of an anti-cancer agent. For example, in some embodiments, if the subject is already undergoing treatment with ionizing radiation, the amount of ionizing radiation administered to the tumor or subject is maintained at the current treatment dose and/or interval, and an anti-cancer agent is administered to the subject if the level of one or more biomarkers described herein is modified in the tumor environment.
[0006] In one aspect, the method comprises modifying the standard radiation treatment protocol if the level of a biomarker described herein is modified in the tumor environment. In some embodiments, the standard radiation treatment protocol is modified by increasing the dose of ionizing radiation administered to the tumor. In some embodiments, the standard radiation treatment protocol is modified by hypofractionation or hyperfractionation of the dose of ionizing radiation. In some embodiments, the standard radiation treatment protocol is modified by further administering an anti-cancer agent or TGF-beta inhibitor to the subject.
[0007] In some embodiments, the method comprises modifying the standard radiation treatment protocol if the level of a biomarker selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8 and CD68 is modified in the tumor environment. The level of a biomarker is modified if the level is increased or decreased compared to the level of the biomarker in a normal (i.e., non-diseased) or control tissue.
[0008] In some embodiments, the method comprises modifying the standard radiation treatment protocol if the level of CD68 is increased in the tumor environment. In some embodiments, the method comprises modifying the standard radiation treatment protocol if the level of CD44 is increased in the tumor environment. In some embodiments, the method comprises modifying the standard radiation treatment protocol if the level of CD44 is increased and the level of MFG-E8 is decreased in the tumor environment.
[0009] In some embodiments, the standard radiation treatment protocol is modified by increasing the dose of ionizing radiation administered to the tumor. In some embodiments, the standard radiation treatment protocol is modified by hypofractionation. In some embodiments, standard radiation treatment protocol is modified by hyperfractionation.
[0010] In some embodiments, the treatment further comprises administering an anti-cancer agent to the subject. In some embodiments, the anti-cancer agent is a chemotherapeutic agent, radiosensitizer, or immune modulator. In some embodiments, the treatment further comprises administering a TGF-beta inhibitor to the subject. In some embodiments, the TGF-beta inhibitor is an antibody or a small molecule that neutralizes or inhibits TGF-beta function. In some embodiments, the TGF-beta inhibitor inhibits the production of TGF-beta.
[0011] In one embodiment, the method comprises:
[0012] (i) administering an increased dose of radiation to the subject, where the dose of radiation is increased compared to the dose administered to a subject that does not have elevated levels of CD68 in the tumor environment; or
[0013] (ii) administering a dose of radiation to the subject that is similar to the dose administered to a subject that does not have elevated levels of CD68 in the tumor environment in combination with a pharmaceutically effective amount of an anti-cancer agent,
[0014] thereby treating the tumor in the subject.
[0015] In one embodiment, the method comprises:
[0016] (i) administering an increased dose of radiation to the subject, where the dose of radiation is increased compared to the dose administered to a subject that does not have elevated levels of CD44 in the tumor environment; or
[0017] (ii) administering a dose of radiation to the subject that is similar to the dose administered to a subject that does not have elevated levels of CD44 in the tumor environment in combination with a pharmaceutically effective amount of an anti-cancer agent,
[0018] thereby treating the tumor in the subject.
[0019] In some embodiments, the disclosure provides a method for treating a tumor in a subject having increased levels of one or more biomarkers and decreased levels of another biomarker described herein. For example, in one embodiment, a method for treating a tumor in a subject having increased levels of CD44 and decreased levels of MFG-E8 in the tumor environment is described, the method comprising:
[0020] (i) administering an increased dose of radiation to the subject, where the dose of radiation is increased compared to the dose administered to a subject that does not have elevated levels of CD44 and decreased levels of MFG-E8 in the tumor environment; or
[0021] (ii) administering a dose of radiation to the subject that is similar to the dose administered to a subject that does not have elevated levels of CD44 and decreased levels of MFG-E8 in the tumor environment in combination with a pharmaceutically effective amount of an anti-cancer agent,
[0022] thereby treating the tumor in the subject.
[0023] In some embodiments, the increased dose of radiation is administered in a hyperfractionated mode. In some embodiments, the increased dose of radiation is administered in a hypofractionated mode.
[0024] In some embodiments, the anti-cancer agent is a chemotherapeutic agent, radiosensitizer, or immune modulator. In some embodiments, the anti-cancer agent is an antibody that neutralizes or inhibits TGF-beta function. In one embodiment, the anti-cancer agent is a small molecule that neutralizes or inhibits TGF-beta function. In some embodiments, the anti-cancer agent inhibits the production of TGF-beta.
[0025] In another aspect, the disclosure provides a method for treating a tumor in a subject in need thereof, the method comprising:
[0026] (a) determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68;
[0027] (b) comparing the expression level of the two or more biomarkers to an expression level in a normal tissue sample; and
[0028] treating the tumor if the expression level of the two or more biomarkers is modified compared to the expression level in the normal tissue sample.
[0029] In another aspect, a method of identifying a subject as a candidate for treatment with ionizing radiation is disclosed, the method comprising:
[0030] (a) determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the one or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68; and
[0031] (b) comparing the expression level of the two or more biomarkers to an expression level in a normal tissue sample;
[0032] wherein an expression level of the two or more biomarkers in the tumor sample that is modified compared to the expression level in the normal tissue sample identifies the subject as a candidate for treatment with ionizing radiation.
[0033] In another aspect, a method of treating a subject having a tumor is disclosed, the method comprising:
[0034] administering ionizing radiation to a subject that has been selected as having an expression level of two or more biomarkers in a tumor sample that is modified relative to an expression level in a normal tissue sample;
[0035] wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68;
[0036] thereby treating the tumor in the subject.
[0037] In another aspect, a method for selecting a treatment for a subject having a tumor is disclosed, the method comprising:
[0038] (a) determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68;
[0039] (b) comparing the expression level of the two or more biomarkers to an expression level in a normal tissue sample; and
[0040] selecting a treatment if the expression level of the two or more biomarkers is modified compared to the expression level in the normal tissue sample.
[0041] In the methods, the expression level of the two or more biomarkers is modified if the expression level of at least one of the biomarkers is increased, or if the expression level of at least one of the biomarkers is decreased, or if the expression level of at least one of the biomarkers is increased and the expression level of at least one of the biomarkers is decreased compared to the expression level in a normal tissue sample.
[0042] In the above aspects, the treatment comprises administering ionizing radiation to the tumor. In some embodiments, the treatment further comprises contacting the tumor with a radiosensitizer. In one embodiment, the treatment further comprises administering a compound that inhibits TGF-beta signaling to the subject.
[0043] In some embodiments, the tumor sample is a biopsy comprising tumor cells. In one embodiment, the tumor is a lung cancer tumor and the tumor sample comprises lung cancer cells. In some embodiments, the biomarker is a gene, an RNA, an extracellular matrix component, or a protein. In some embodiments, the expression level of the biomarker is determined by detecting the expression of an RNA and/or a protein. For example, the expression level can be detected by immunohistochemistry, ELISA, Western analysis, HPLC, proteomics, PCR, RT-PCR, Northern analysis, and/or nucleic acid or polypeptide microarrays.
[0044] In some embodiments, the normal tissue sample comprises non-tumor cells from the same tissue type as the tumor.
[0045] In some embodiments, the expression level of the two of more biomarkers is ranked or weighted. The expression level of each of CD44, MMP9, ALDH1A1, Vimentin, hyalurnan, beta-catenin, MFG-E8 and CD68 can be determined. In one embodiment, the expression level of at least one additional biomarker from the tumor sample is determined.
[0046] In some embodiments, an existing treatment and/or treatment plan is modified if the expression level of the two or more biomarkers is increased or decreased compared to the expression level of the same biomarker in the normal tissue sample. For example, the existing treatment and/or treatment plan can be modified to increase or decrease the effective dose of ionizing radiation administered to the tumor. The effective dose can be increased by increasing the amount of ionizing radiation administered to the tumor and/or contacting the tumor with a radiosensitizer.
[0047] In another aspect, a kit is provided, the kit comprising reagents capable of detecting the expression of a biomarker selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68.
[0048] In some embodiments, one or more of the steps of the methods described herein are carried out in vitro. For example, the expression level of the biomarkers described herein can be determined in vitro using immunohistochemistry techniques on tissue samples isolated from a subject. Thus, the step of determining the expression level of the biomarkers described herein does not require that the determining step be performed in vivo (i.e., in the subject). In certain embodiments, the expression level of the biomarkers described herein is ranked or weighted using software providing instructions to a computer.
[0049] In some aspects, the disclose provides a biomarker composition for use in a method for treating or diagnosing cancer or tumors. In some embodiments, a composition comprising a biomarker selected from CD44, MMP9, ALDH1A1, Vimentin, hyaluman, beta-catenin, MFG-E8 and/or CD68 for use in a method for treating tumors is provided. In some embodiments, the disclosure provides a biomarker in combination with ionizing radiation for use in a method for treating a tumor. For example, a composition comprising a biomarker selected from CD44, MMP9, ALDH1A1, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 in combination with ionizing radiation for use in a method for treating tumors in provided.
[0050] In some embodiments, the disclosure describes a composition comprising a biomarker selected from CD44, MMP9, ALDH1A1, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 for use in a method for treating tumors, the method comprising modifying the standard radiation treatment protocol if the level of a biomarker described herein is increased in the tumor environment. In some embodiments, the standard radiation treatment protocol is modified by increasing the dose of ionizing radiation administered to the tumor. In some embodiments, the standard radiation treatment protocol is modified by hypofractionation or hyperfractionation of the dose of ionizing radiation. In some embodiments, the standard radiation treatment protocol is modified by further administering an anti-cancer agent to the subject.
[0051] In some embodiments, the disclosure describes the use of a composition comprising a biomarker selected from CD44, MMP9, ALDH1A, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 in a method for treating a tumor, the method comprising
[0052] (i) administering an increased dose of radiation to the subject, where the dose of radiation is increased compared to the dose administered to a subject that does not have elevated levels of a biomarker selected from CD44, MMP9, ALDH1A, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 in the tumor environment; or
[0053] (ii) administering a dose of radiation to the subject that is similar to the dose administered to a subject that does not have elevated levels of a biomarker selected from CD44, MMP9, ALDH1A1, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 in the tumor environment in combination with a pharmaceutically effective amount of an anti-cancer agent.
[0054] In some embodiments, a composition comprising a biomarker selected from CD44, MMP9, ALDH1A1, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 for use in a diagnostic method practiced on the human or animal body is provided. In one embodiment, a composition comprising a biomarker selected from CD44, MMP9, ALDH1A1, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 for use in diagnosing or prognosing cancer or tumors is provided. For example, a composition comprising a biomarker selected from CD44, MMP9, ALDH1A, Vimentin, hyalurnan, beta-catenin, MFG-E8 and/or CD68 for use in diagnosing tumors is provided, the use comprising:
[0055] (a) determining an expression level of a biomarker in a biological or tissue sample from the subject, wherein the biomarker is selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68; and
[0056] (b) comparing the expression level of the biomarker(s) to an expression level in a normal biological or tissue sample; wherein an expression level of the biomarker(s) in the biological or tissue sample that is increased or decreased compared to the expression level in the normal biological or tissue sample provides a diagnosis that the subject suffers from a tumor. The use can also provide a prognosis regarding the course of disease, or can be used to identify a subject as a candidate for treatment with ionizing radiation.
Definitions
[0057] The term "treating" refers to administering a treatment to a tumor or the subject diagnosed with a tumor. Examples of treatments include ionizing radiation, a chemotherapeutic treatment, or a combination of both. The treatment can also include a radiosensitizer. The term also includes selecting a treatment or treatment plan, and providing treatment options to a healthcare provider or the subject.
[0058] The term "ionizing radiation" refers to radiation comprising particles having enough kinetic energy to discharge an electron from an atom or molecule, thereby producing an ion. The term includes both directly ionizing radiation, such as that caused by atomic particles such as alpha particles (helium nuclei), beta particles (electrons), and protons, and indirectly ionizing radiation, such as photons, including gamma rays and x-rays. Examples of ionizing radiation used in radiation therapy include high energy x-rays, electron beams, and proton beams.
[0059] The term "tumor environment" or "tumor micro-environment" refers to the immediate small-scale environment of an organism or part of an organism, especially as a distinct part of a larger environment, for example, the immediate small-scale environment of the tumor. The term includes not only the tumor cells themselves, but associated blood-vessels (including endothelial cells and smooth muscle cells), immune system cells and secreted cytokines, epithelial cells, fibroblasts, connective tissue, and/or extracellular matrix that is associated with or surrounds the tumor. The term also refers to the cellular and extracellular environment in which the tumor is located.
[0060] The term "standard of care" or "standard radiation treatment protocol" in radiation therapy generally refers to the ionizing radiation dose and administration interval that is generally accepted in the medical field as appropriate treatment for a given tumor, based on the tumor type, size, tissue location, and various other biological parameters. The standard of care or standard treatment protocol varies and is dependent on several factors. For example, for radiation therapy of lung cancer, the standard of care includes multiple fractions (e.g., approximately 30 fractions of low dose radiation, or approximately 60 Gy over 6 weeks) or a smaller number of fractions (e.g., 1-5 fractions) of biologically active doses (e.g., 54 GY in 3 fractions for peripheral tumors, or 48-60 Gy in 4-8 fractions for central tumors) administered to the tumor.
[0061] The term "similar dose of ionizing radiation" refers to a dose of ionizing radiation that is identical to, nearly the same, or substantially the same as the effective dose administered to a tumor in another subject, or administered to a tumor in the same subject undergoing an existing course of treatment. The term encompasses the normal and expected variation in ionizing radiation doses delivered by a medical technician skilled in the art of administering ionizing radiation to a tumor in a subject. For example, the term encompasses variation in the effective dose administered to a tumor of less than 10%, less than 5%, or less than 1%. The subject can be a human or non-human animal, such as a companion animal (e.g., cat, dog) or farm animal (e.g., cow, horse, etc.).
[0062] The term "small molecule" refers to an organic compound having a molecular weight of less than about 900 daltons, or less than about 500 daltons. The term includes drugs having desired pharmacological properties, and includes compounds that can be taken orally or by injection. The term includes organic compounds that modulate the activity of TGF-beta and/or other molecules associated with enhancing or inhibiting an immune response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1 shows the distribution of ALDH1A1 in biopsy samples from lung cancer patients.
[0064] FIG. 2 shows the distribution of Beta-Cat in biopsy samples from lung cancer patients.
[0065] FIG. 3 shows the distribution of CD44 in biopsy samples from lung cancer patients.
[0066] FIG. 4 shows the distribution of CD68 in biopsy samples from lung cancer patients.
[0067] FIG. 5 shows the distribution of HA in biopsy samples from lung cancer patients.
[0068] FIG. 6 shows the distribution of MFG-E8 in biopsy samples from lung cancer patients.
[0069] FIG. 7 shows the distribution of MMP9 in biopsy samples from lung cancer patients.
[0070] FIG. 8 shows the distribution of VIM in biopsy samples from lung cancer patients.
[0071] FIG. 9 shows the ROC curve for the variables CD44 (Tot), MFG_E8 (Prop) and tumor type from a multivariate model to predict local tumor control.
[0072] FIG. 10 shows the values of CD44 (Tot) and MFG_E8 (Prop) that correspond to a positive and negative test result for predicting local tumor control failure for squamous tumors, as described in the Examples.
[0073] FIG. 11 shows the values of CD44 (Tot) and MFG_E8 (Prop) that correspond to a positive and negative test result for predicting local tumor control failure for adenocarcinoma tumors, as described in the Examples.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The methods described herein allow for the classification of patients into groups for receiving optimized radiation treatment based on patient specific biomarker signature. The biomarker signature includes markers that have been shown to correlate with TGF-0 expression and to be associated with tumor aggressiveness, radioresistance and poor prognosis. The markers play a key role in the epithelial-mesenchymal transition. The methods described herein provide the dual benefits of anti-tumor efficacy+normal tissue protection when combining TGF-.beta. inhibitors with ionizing radiation to treat cancer patients.
I. Methods
[0075] The present disclosure describes methods for treating a tumor in a subject by determining the expression levels of signature biomarkers in a tumor sample, comparing the expression levels in the tumor sample to the expression levels in a normal tissue sample, and treating the tumor if the expression levels in the tumor sample are different from those in the normal tissue sample. In some embodiments, the treatment is ionizing radiation. Thus, the biomarkers provide so called "companion diagnostics" for radiation therapy to treat tumors. The signature biomarkers can also be used to select the appropriate treatment when ionizing radiation is combined with therapeutic tumor treatments such as chemotherapy. Many of the signature biomarkers disclosed herein are associated with the TGF-.beta. signalling pathway. Thus, in some embodiments, the therapeutic agent is an inhibitor of TGF-.beta. or an inhibitor of a component of the TGF-.beta. signalling pathway.
[0076] In one aspect, the method is for treating a tumor. The method comprises determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68. The expression levels of the two or more biomarkers in the tumor sample are compared to the expression levels of the two or more biomarkers in a normal tissue sample. If the expression levels of the two or more biomarkers in the tumor sample are different from the expression levels in the normal tissue sample, for example, increased or decreased relative to the normal tissue level, the tumor is treated.
[0077] Thus, in some embodiments, the method comprises (a) determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68; (b) comparing the expression level of the two or more biomarkers to an expression level in a normal tissue sample; and treating the tumor if the expression level of the two or more biomarkers is increased compared to the expression level in the normal tissue sample.
[0078] In some embodiments, the method comprises (a) determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, and MFG-E8, and CD68; (b) comparing the expression level of the two or more biomarkers to an expression level in a normal tissue sample; and treating the tumor if the expression level of the two or more biomarkers is decreased compared to the expression level in the normal tissue sample.
[0079] In some embodiments, the method comprises determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68; and treating the tumor if the expression level of the two or more biomarkers is increased compared to the expression level in a normal tissue sample. In some embodiments, the method comprises determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44. MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin. MFG-E8, and CD68; and treating the tumor if the expression level of the two or more biomarkers is decreased compared to the expression level in a normal tissue sample.
[0080] In some embodiments, the treatment comprises administering ionizing radiation to the tumor. Thus, in some embodiments, the treatment comprises increasing the effective dose of ionizing radiation if the expression level of the two or more biomarkers is increased compared to the expression level in a normal tissue sample. In some embodiments, the treatment comprises decreasing the effective dose of ionizing radiation if the expression level of the two or more biomarkers is decreased compared to the expression level in a normal tissue sample.
[0081] In a second aspect, the disclosure describes a method for identifying a subject as a candidate for treatment with ionizing radiation. The method comprises determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluman, beta-catenin, MFG-E8, and CD68. As above, the expression levels of the two or more biomarkers in the tumor sample are compared to the expression levels of the two or more biomarkers in a normal tissue sample. If the expression levels of the two or more biomarkers in the tumor sample are different from the expression levels in the normal tissue sample, for example, increased or decreased relative to the normal tissue level, the subject is identified as a candidate for treatment with ionizing radiation.
[0082] In some embodiments, the expression level of the two or more biomarkers is increased compared to the expression level in the normal tissue sample, and the subject is identified as a candidate for a first treatment with ionizing radiation. In other embodiments, the expression level of the two or more biomarkers is decreased compared to the expression level in the normal tissue sample, and the subject is identified as a candidate for a second treatment with ionizing radiation. The first and second treatments can be the same or different. In some embodiments, the first treatment comprises increasing the effective dose of ionizing radiation. In some embodiments, the second treatment comprises decreasing the effective dose of ionizing radiation.
[0083] In a third aspect, a method is provided for treating a subject having a tumor. The method comprises administering ionizing radiation to a subject that has been selected as having an expression level of two or more biomarkers in a tumor sample that is increased or decreased relative to the expression level of the two or more biomarkers in a normal tissue sample. In some embodiments, the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluman, beta-catenin, MFG-E8, and CD68.
[0084] In some embodiments, the method comprises administering ionizing radiation to a subject that has been selected as having an expression level of two or more biomarkers in a tumor sample that is increased relative to the expression level of the two or more biomarkers in a normal tissue sample. In some embodiments, the method comprises administering ionizing radiation to a subject that has been selected as having an expression level of two or more biomarkers in a tumor sample that is decreased relative to the expression level of the two or more biomarkers in a normal tissue sample. In some embodiments, the dose of ionizing radiation administered to the subject is increased if the expression level of two or more biomarkers in a tumor sample is increased relative to the expression level of the two or more biomarkers in a normal tissue sample. In some embodiments, the dose of ionizing radiation administered to the subject is decreased if the expression level of two or more biomarkers in a tumor sample is decreased relative to the expression level of the two or more biomarkers in a normal tissue sample.
[0085] In a fourth aspect, a method is described for selecting a treatment for a subject having a tumor. The method comprises determining an expression level of two or more biomarkers in a tumor sample from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68. As above, the expression levels of the two or more biomarkers in the tumor sample are compared to the expression levels of the two or more biomarkers in a normal tissue sample. If the expression levels of the two or more biomarkers in the tumor sample are different from the expression levels in the normal tissue sample, for example, increased or decreased relative to the normal tissue level, a treatment is selected for the subject having the tumor.
[0086] In another aspect, the biomarkers described herein can also or further be used to determine the prognosis of disease during or after treatment. For example, the expression levels of the biomarkers before and after ionizing radiation therapy can be compared. In some embodiments, if the expression levels of the biomarkers after radiation therapy decrease, then the prognosis is favorable. In some embodiments, if the expression levels of the biomarkers after radiation therapy increase, then the prognosis is unfavorable.
[0087] In another aspect, the biomarkers described herein can also or further be used to assess the responsiveness of a patient to a cancer treatment. For example, the expression levels of the biomarkers before and after ionizing radiation therapy can be compared. The method comprises determining an expression level of two or more biomarkers in a tumor sample obtained from the subject, wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluman, beta-catenin, MFG-E8, and CD68. In some embodiments, if the expression levels of the biomarkers after radiation therapy decrease, then the patient has responded favorably. In some embodiments, if the expression levels of the biomarkers after radiation therapy increase, then the patient response was unfavorable. This information can be used to guide further therapy. Favorable treatments may be repeated or further increased. Unfavorable treatments can be modified or dropped.
[0088] In another aspect, a kit is provided. The kit comprises reagents capable of detecting expression of the biomarkers described herein. In some embodiments, the kit comprises reagents capable of detecting nucleic acid (e.g., RNA) expression of the biomarkers. For example, the kit can comprise oligonucleotide primers that are capable amplifying a nucleic acid expressed by the biomarker genes described herein. In some embodiments, the kit further comprises an oligonucleotide probe that hybridizes to a biomarker nucleic acid or an amplified biomarker nucleic acid, or a complement thereof. Methods of amplifying and detecting nucleic acids are well known in the art, and can comprise PCR, RT-PCR real-time PCR, and quantitative real-time PCR, Northern analysis, sequencing of expressed nucleic acids, and hybridization of expressed and/or amplified nucleic acids to microarrays. In some embodiments, the kit comprises reagents that are capable of detecting proteins expression by the biomarkers described herein. In some embodiments, the reagents are antibodies that specifically bind to biomarker proteins. Methods of detecting protein expression are well known in the art, and include immunoassays, ELISA, Western analysis, and proteomic techniques.
[0089] In some embodiments of any of the above aspects and embodiments, the differences in the expression levels of each of the biomarkers in the tumor sample are increased or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the expression level in normal tissue. In some embodiments, the expression levels of each of the biomarkers in the tumor sample are increased or decreased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10 fold or more relative to the expression level in normal tissue.
[0090] In some embodiments, the average and/or ranked expression level of all the biomarkers in the tumor sample is increased or decreased relative to the expression level in normal tissue. Thus, in some embodiments, the average and/or ranked expression level of all the biomarkers in the tumor sample is increased or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the expression level in normal tissue. In some embodiments, the expression levels in normal tissue are normalized to a control or baseline level. It will be understood that the expression level can also be compared to the expression level in the tumor sample before, after or during a treatment, course of treatment, or treatment plan. Thus, in some embodiments, the expression levels of each of the biomarkers in the tumor sample are increased or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the expression level in the tumor sample before, during or after treatment.
[0091] Further, with regard to any of the above aspects and embodiments, the two or more biomarkers can comprise both CD44 and MMP9; both ALDH1A1 and Vimentin; both hyalurnan and beta-catenin; both CD44 and ALDH1A1; both Vimentin and beta-catenin; both CD44 and hyalurnan; both CD44 and beta-catenin; both CD44 and MFG-E8 or both CD44 and CD68; both MMP9 and hyalurnan; both MMP9 and beta-catenin; both MMP9 and MFG-E8, or both MMP9 and CD68; both ALDH1A1 and hyaluman; both ALDH1A1 and beta-catenin; both ALDH1A1 and MFG-E8, or both ALDH1A1 and CD68; both Vimentin and MFG-E8; both hyalurnan and MFG-E8; both beta-catenin and MFG-E8, or both CD68 and MFG-E8.
[0092] Further, with regard to any of the above aspects and embodiments, the two or more biomarkers can comprise or consist of any combination of the biomarkers, for example any combination of three or more biomarkers, any combination of four or more biomarkers, any combination of five or more biomarkers, any combination of six or more biomarkers, and any combination of seven or more biomarkers. In one embodiment, the combination of biomarkers comprises or consists of CD44, MFG-E8, and CD68.
[0093] In another aspect, the expression level of at least one, two, three, four or more of the biomarkers described herein is determined.
[0094] In some embodiments, the treatment or selected treatment comprises administering ionizing radiation to the tumor. Thus, in some embodiments, the selected treatment comprises increasing the effective dose of ionizing radiation if the expression level of the two or more biomarkers is increased compared to the expression level in a normal tissue sample. In some embodiments, the selected treatment comprises decreasing the effective dose of ionizing radiation if the expression level of the two or more biomarkers is decreased compared to the expression level in a normal tissue sample. Exemplary radiotherapy treatments are further described herein. In all of the methods described herein, the treatment can further comprise contacting the tumor with a radiosensitizer. A radiosensitizer is any substance that makes tumor cells easier to kill with radiation therapy. Exemplary radiosensitizers include hypoxia radiosensitizers such as misonidazole, metronidazole, and trans-sodium crocetinate. Exemplary radiosensitizers also include DNA damage response inhibitors such as Poly (ADP) ribose polymerase (PARP) inhibitors. In all of the methods described herein, the treatment can further comprise contacting the tumor and/or the tumor environment with an immune modulator. Exemplary immune modulators include agents (antibodies or small molecules) involved in priming and activation of the immune systems, and include agents targeting CTLA4, B7 (B7-1 or B7-2), PD-L1/PD-L2, or PD-1, or agents targeting the binding interactions between CTLA4 and B7-1/B7-2, or PD-1 and PD-L1/PD-L2. Agents targeting CTLA4, B7 (B7-1 or B7-2), PD-L1/PD-L2, and PD-1 include antibodies that specifically bind these molecules, such as monoclonal antibodies. In some embodiments, the agent is an antibody that specifically binds to LAG 3, TIM1, TIM3, MFG-E8, IL-10, or Phosphatidylserine.
[0095] Small molecule immune modulators include drugs that enhance or inhibit an immune response, for example, an immune response against a tumor cell. Exemplary small molecule immune modulators include inhibitors of the enzyme Indolamine 2,3-dioxygenase, and inhibitors of alpha-v-beta-3 integrin and alpha-v-beta-5 integrin.
[0096] In some embodiments, the treatment further comprises administering a compound that inhibits TGF-beta signaling to the subject. Suitable compounds are described in more detail below.
[0097] The biomarkers used in the method will now be described.
[0098] A. Biomarkers
[0099] The biomarkers described herein correlate with TGF-.beta. expression, and can be used to stratify patients to receive individualized, tailored radiotherapy. The biomarker signature can also be used to monitor the efficacy of TGF-.beta. inhibitors in patients. The biomarker signature is associated with but not limited to the correlation with TGF-.beta. expression. The expression of the biomarkers is associated with radioresistance, aggressiveness and poor prognosis. The marker set includes, but is not limited to, CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, .beta.-catenin MFG-E8, and CD68.
[0100] MMP9: A clear correlation can be shown between MMP9, EMT and TGF-.beta.. MMP9 regulates TGF-.beta. and TGF-.beta. regulates MMP9 in multiple settings. MMP9 is localized in the extracellular matrix and tumor stroma, within infiltrated immune cells and in tumor cells. The different cellular locations of MMP9 appear to be correlated with different biological outcomes (more/less aggressive tumor, survival etc.).
[0101] Vimentin (VIM): Vimentin is upregulated when TGF-.beta. induces EMT in a variety of cell types, including lung. Vimentin is an intermediate filament protein that characterizes mesenchymal cells as opposed to epithelial cells.
[0102] Hyaluronan (HA): Hyaluronan is an abundant glycosaminoglycan component of the extracellular matrix. It is induced by TGF-.beta., increases MMP9 secretion (likely via CD44), promotes EMT/migration/metastasis, and contributes to chemoresistance and poor prognosis. These findings have been substantiated in a variety of tumor types, including NSCLC. An important receptor for HA is CD44 along with others. The HA-CD44 interaction promotes HER2 signalling and increases Src kinase activity. HA is detected by staining the tissues with a commercially available antibody against Hyaluronic acid, for example, an antibody available from Abcam.
[0103] ALDH1A1: Aldehyde dehydrogenase is a detoxifying enzyme known for its role in the oxidation of intracellular aldehydes, which play a role in stem cell differentiation. It is highly expressed in tumorigenic cell populations of various cancers and elevated protein expression has been shown in putative lung stem cell niches during malignant transformation. Expression of ALDH1A is positively correlated with stage and grade of lung tumors and related to poor prognosis in patients with early stage lung cancer.
[0104] MFG-E8: MFG-E8 is a macrophage-produced protein that promotes engulfment and clearance of apoptotic cells in tumors. Antibodies neutralizing MFG-E8 function have been shown in experimental models to enhance radiation and chemotherapy. It is likely then, that the levels of MFG-E8 in tumor specimens may have predictive value for efficacy of radiotherapy.
[0105] CD68: CD68 is a 110-kD transmembrane glycoprotein that is highly expressed by human monocytes and tissue macrophages. It is a member of the lysosomal/endosomal-associated membrane glycoprotein (LAMP) family. The protein primarily localizes to lysosomes and endosomes with a smaller fraction circulating to the cell surface. It is a type I integral membrane protein with a heavily glycosylated extracellular domain and binds to tissue- and organ-specific lectins or selectins. The protein is also a member of the scavenger receptor family. Scavenger receptors typically function to clear cellular debris, promote phagocytosis, and mediate the recruitment and activation of macrophages (See Entrez listing NCBI).
[0106] CD68 is expressed broadly on macrophages including both M1 and M2 subsets. Numerous studies have suggested that macrophages present in the tumor micro-environment can impact growth of tumor cells and some clinical studies have suggested that the macrophage content and location in the tumor and its micro-environment is predictive of clinical outcome in certain cancer patients.
[0107] M1 macrophages are referred to as pro-inflammatory macrophages and have the ability to activate type 1 T helper cells (Th1) and to promote an anti-tumor response. In contrast, M2 macrophages activate type 2 T helper cells (Th2) and promote an anti-inflammatory, tissue remodeling response and do not lead to an anti-tumor action. As CD68 is expressed on both M1 and M2 macrophages, its presence cannot, a priori, be used to predict anti-tumor responses or clinical outcome. Thus, the present application describes that CD68 is useful as a biomarker determined in a clinical setting.
[0108] Nuclear .beta.-catenin: .beta.-catenin is found associated with E-cadherin at the cell membrane and also in the nucleus, where it accumulates in tumor cells, stem cells or cells undergoing EMT.
[0109] The GenBank Accession Nos. for the biomarkers described herein are provided in the Table below.
TABLE-US-00001 TABLE 1 GenBank GenBank Biomarker Accession # Accession Name Abbreviation (protein) # (nucleotide) Hyaluronate CD44 NP_000601 NM_000610 receptor (SEQ ID (SEQ ID NO: 1) NO: 2) Matrix MMP9 CAC07541 AX011001 metalloproteinase (SEQ ID (SEQ ID NO: 3) NO: 4) Aldehyde ALDH1A1 AAP88039 AY338497 dehydrogenase (SEQ ID (SEQ ID 1A1 NO: 5) NO: 6) Vimentin VIM NP_003371 NM_003380 (SEQ ID (SEQ ID NO: 7) NO: 8) hyaluronan HA Not applicable .beta.-catenin Beta_Cat NP_001091680 NM_001098210 (SEQ ID (SEQ ID NO: 9) NO: 10) Milk fat globule- MFG-E8 NP_005919 NM_005928 EGF factor 8 (SEQ ID (SEQ ID protein NO: 11) NO: 12 CD68 CD68 NP_001242 NM_001251 (SEQ ID (SEQ ID NO: 13) NO: 14)
[0110] When the biomarkers described herein are referred to byname, it is understood that this includes molecules with similar functions and similar amino acid sequences. Thus, the protein biomarkers described herein include the prototype human protein, as well as homologs and polymorphic variations thereof. For example, the name "CD44 protein" includes the prototype protein (e.g., SEQ ID NO: 1), as well as homologs from other species and polymorphic variations thereof. Proteins such as CD44 and CD68 are defined as having similar functions if they have substantially the same biological activity or functional capacity as the wild type protein (e.g., at least 80% of either). Proteins such as CD44 and CD68 are defined as having similar amino acid sequences if they have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the prototype protein. The sequence identity of a protein is determined using the BLASTP program with the defaults wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci USA 89:10915-10919, 1992).
[0111] A conventional test to determine if a protein homolog or polymorphic variant is inclusive of a protein biomarker described herein is by specific binding to polyclonal antibodies generated against the prototype protein. For example, a CD44 protein includes proteins that bind to polyclonal antibodies generated against the protein of SEQ ID NO:1, and an CD68 protein includes proteins that bind to polyclonal antibodies generated against the prototype protein of SEQ ID NO:13.
[0112] Regarding polyclonal antibodies that specifically bind to a protein biomarker described herein, the test protein will bind under designated immunoassay conditions to the specified antibodies at least two times the background, and the specified antibodies do not substantially bind in a significant amount to other proteins present in the sample. For example, polyclonal antibodies raised to CD44, encoded in SEQ ID NO:1, splice variants, or portions thereof, can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with CD44 and not with other proteins, except for polymorphic variants of CD44. This selection may be achieved by subtracting out antibodies that cross-react with other members of the protein family, as appropriate. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see. e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
[0113] In some embodiments, the method comprises determining the expression level of two or more biomarkers in a tumor sample from the subject. In some embodiments, the biomarker is selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and/or CD68. In some embodiments, the expression level of two, three, four, five, six, seven, or eight of the biomarkers is determined. In some embodiments, the expression level of each of the biomarkers is determined. In some embodiments, the expression level of at least one additional biomarker is determined, wherein the additional biomarker is not in the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin. MFG-E8, and CD68. In some embodiments, the additional biomarker is TGF-.beta..
[0114] In some embodiments, the biomarker signature group consists of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68. In some embodiments, the biomarker signature group consists essentially of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68. In some embodiments, the biomarker signature group comprises CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68.
[0115] It will be understood that the expression levels of each of the biomarkers in the tumor sample can increase or decrease relative to the expression level of the biomarker in a normal or control tissue sample. For example, the expression level of one biomarker can increase in the tumor sample compared to the expression level in a normal tissue, whereas the expression level of a second biomarker can decrease in the tumor sample compared to the expression level in a normal tissue. The expression level can also be based on the average, combination or sum of the all the biomarker expression levels in the tumor sample. For example, the expression level of each biomarker in the tumor sample can be ranked or weighted to produce a ranked value that is higher or lower than the normal tissue value (which can be a normalized value, for example, set to 1).
[0116] In some embodiments, biomarker expression is determined in a biological sample from the subject having a tumor. In some embodiments, the biological sample is a tumor sample. The tumor sample can be a biopsy comprising tumor cells from the tumor. In some embodiments, the biological sample comprises a bodily fluid, such as but not limited to blood, serum, plasma, or urine, and/or cells or tissues from the subject. In some embodiments, the biological sample is a formalin-fixed and paraffin embedded tissue or tumor sample. In some embodiments, the biological sample is a frozen tissue or tumor sample. Thus, in some embodiments, one or more steps of the methods described herein are carried out in vitro. For example, in some embodiments, biomarker expression is determined in vitro.
[0117] In some embodiments, the normal tissue sample comprises non-tumor cells from the same tissue type as the tumor. In some embodiments, the normal tissue sample is obtained from the same subject diagnosed with the tumor. A normal tissue sample can also be a control sample of the same tissue-type from a different subject. The expression level of the normal tissue sample can also be an average or mean value obtained from a population of normal tissue samples.
[0118] The level of expression of the biomarkers described herein can be determined using any method known in the art. For example, the level of expression can be determined by detecting the expression of a nucleic acid (e.g., RNA or mRNA) or protein encoded by a biomarker gene.
[0119] Exemplary methods for detecting expression levels of nucleic acids include without limitation Northern analysis, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), real-time PCR, quantitative real-time PCR, and DNA microarrays.
[0120] Exemplary methods for detecting expression levels of proteins (e.g., polypeptides) include without limitation immunohistochemistry, ELISA, Western analysis, HPLC, and proteomics assays. In some embodiments, the protein expression level is determined by immunohistochemistry using the Allred method to assign a score (see, e.g., Allred, D. C., Connection 9:4-5, 2005, which is incorporated by reference herein). For example, formalin-fixed, paraffin embedded tissues are contacted with an antibody that specifically binds a biomarker described herein. The bound antibody is detected with a detectable label or secondary antibody coupled with a detectable label, such as a colorimetric label (e.g., an enzymatic substrate produce by HRP or AP). The antibody positive signal is scored by estimating the proportion of positive tumor cells and their average staining intensity. Both the proportion and intensity scores are combined into a total score that weighs both factors.
[0121] In some embodiments, the protein expression level is determined by digital pathology. Digital pathology methods include scanning images of tissues on a solid support, such as a glass slide. The glass slides are scanned into whole slide images using a scanning device. The scanned images are typically stored in an information management system for archival and retrieval. Image analysis tools can be used to obtain objective quantitative measurements from the digital slides. For example, the area and intensity of immunohistochemical staining can be analyzed using the appropriate image analysis tools. Digital pathology systems can include scanners, analytics (visualization software, information management systems and image analysis platforms), storage and communication (sharing services, software). Digital pathology systems are available from numerous commercial suppliers, for example. Aperio Technologies, Inc. (a subsidiary of Leica Microsystems GmbH), and Ventana Medical Systems, Inc. (now part of Roche). Expression levels can be quantified by commercial service providers, including Flagship Biosciences (CO), Pathology, Inc. (CA), Quest Diagnostics (NJ), and Premier Laboratory LLC(CO).
[0122] B. Treatments
[0123] The expression levels of the biomarkers can be used to determine or select a course of treatment in a subject diagnosed with a tumor. For example, in some embodiments, the treatment comprises administering ionizing radiation to the tumor in the subject. The ionizing radiation can also be administered to the entire subject or a portion thereof, especially if the tumor is dispersed or mobile. In some embodiments, the treatment further comprises contacting the tumor with a radiosensitizer. In some embodiments, the treatment further comprises administering a compound or biologic drug, such as an antibody, that inhibits TGF-beta signaling to the subject. Thus, in some embodiments, the treatment comprises administering a standard radiation treatment protocol in combination with a TGF-beta inhibitor.
[0124] The course of treatment can be selected based on the expression levels of the biomarkers. For example, the expression levels can be used to determine if radiation therapy is appropriate for the subject (i.e., for making a go/no go decision on radiotherapy). Further, if the expression levels of the biomarkers are increased relative to a normal or control value, then the effective radiation dose to the tumor can be increased, and/or the fractionation schedule modified accordingly. The radiation dose to the blood vessels feeding the tumor can also be increased.
[0125] In some embodiments, if the expression levels of the biomarkers are increased relative to a normal or control value, then the treatment can comprise administering ionizing radiation to the tumor. In some embodiments, if the expression levels of the biomarkers are decreased relative to a normal or control value, then the treatment can comprise decreasing the amount of ionizing radiation administered to the tumor.
[0126] The treatment can also comprise modifying an existing course of treatment. For example, in some embodiments, the existing course of treatment is modified to increase the effective dose of the ionizing radiation administered to the tumor. In some embodiments, the effective dose of ionizing radiation is increased by increasing the amount of ionizing radiation administered to the tumor and/or contacting the tumor with a radiosensitizer. In some embodiments, the existing course of treatment is modified to decrease the effective dose of the ionizing radiation administered to the tumor. In some embodiments, the treatment comprises modifying a standard radiation treatment protocol in combination with administering a TGF-beta inhibitor.
[0127] In some embodiments, the effective dose of ionizing radiation administered to the tumor is increased if the level of one or more biomarkers described herein is elevated in the tumor environment. For example, the effective dose of ionizing radiation is increased as compared to the standard of care for a subject that does not have elevated levels of the biomarker(s) in the tumor environment. This applies to subjects who are currently not undergoing radiation therapy as well as modifying an existing course of treatment for subjects undergoing radiation therapy. Tus, the effective dose of ionizing radiation can be increased from the current effective dose if the subject is already undergoing radiation therapy for a tumor. The radiation therapy can be modified to reduce the constraints on neighboring healthy tissue. For example, if the biomarker level in the tumor environment indicates more aggressive radiation therapy is required, the treatment plan can be modified so that the constraints on the border between healthy tissue and tumor tissue are decreased. his would result in a trade-off between damaging some healthy tissue in order to kill more of the tumor tissue.
[0128] In some embodiments, the treatment comprises a combination of radiation therapy and an anti-cancer agent (including a radiosensitizer). In some embodiments, the effective dose of ionizing radiation administered to the tumor is not changed (e.g., relative to the standard of care or relative to an existing course of treatment) when an anti-cancer agent is administered to the subject. For example, in some embodiments, the subject is administered an effective dose of ionizing radiation that is the same or similar to that administered to a subject that does not have elevated levels of one or more biomarkers described herein in the tumor environment, and the subject is further administered an anti-cancer agent. In some embodiments, the effective dose of ionizing radiation administered to the tumor is based on the standard of care for a subject that does not have elevated levels of the biomarker(s) in the tumor environment, and the subject is further administered an anti-cancer agent. In some embodiments involving an existing course of treatment, the effective dose of ionizing radiation is maintained at the current effective dose, and an anti-cancer agent is administered to the subject in combination with the ionizing radiation if the level of one or more biomarkers described herein is elevated in the tumor environment.
[0129] In some embodiments, the subject is administered an increased effective dose of ionizing radiation if the expression of CD44 or CD68 is elevated in the tumor environment. In some embodiments, the subject is administered an effective dose of ionizing radiation that is the same or similar to the effective dose administered to a subject that does not have elevated levels of CD44 or CD68 in the tumor environment (e.g., according to the current standard of care), in combination with a pharmaceutically effective amount of an anti-cancer agent, if the expression of CD44 or CD68 is elevated in the tumor environment. In some embodiments, the subject is administered an increased effective dose of ionizing radiation if the level of CD44 is increased and the level of MFG-E8 is decreased in the tumor environment. In some embodiments, the subject is administered an effective dose of ionizing radiation that is the same or similar to the effective dose administered to a subject that does not have increased levels of CD44 and decreased levels of MFG-E8 in the tumor environment (e.g., according to the current standard of care), in combination with a pharmaceutically effective amount of an anti-cancer agent, if the level of CD44 is increased and the level of MFG-E8 is decreased in the tumor environment. The above embodiments apply to subjects who are currently not undergoing radiation therapy as well as modifying an existing course of treatment for subjects undergoing radiation therapy.
[0130] In some embodiments, the treatment plan is developed and/or modified based on the expression levels of the biomarkers described herein.
[0131] The course of treatment can also be selected by using an algorithm that determines the expression level of the biomarkers in the tumor sample relative to the level in the normal sample. The algorithm can be a linear regression algorithm that includes the biomarker expression levels and coefficients (i.e., weights) for combining the expression levels. In some embodiments, the algorithm comprises a least squares fit to calculate the coefficients. If the algorithm determines that the expression level of the biomarkers in the tumor sample is increased or decreased relative to the normal sample, then the appropriate course of treatment can be assigned. In some embodiments, the algorithm is a nonparametric regression tree. In some embodiments, standard statistical methods were used to analyze the data to determine which biomarkers were most predictive of clinical survival or local tumor control failure.
[0132] In some embodiments, the method described herein is a computer implemented method. In some embodiments, the computer implemented method comprises a linear regression model that assigns a ranked or weighted value to the expression levels of the biomarkers described herein. In some embodiments, the disclosure provides a computer-readable medium, the medium providing instructions to cause a computer to perform a method described herein. For example, the medium can provide instructions to cause a computer to assign a ranked or weighted value to the expression levels of the biomarkers described herein.
[0133] C. Therapeutic Radiation Doses
[0134] The expression levels of the tumor biomarkers described herein can be used to optimize treatment of patients with radiotherapy. For example, the therapeutic dose of the radiation administered to the tumor or subject can be adjusted based on the expression levels of the biomarkers. As is well known in the art, the effective dose of ionizing radiation varies with the type of tumor and stage of cancer that needs to be treated. The effective dose can also vary based on other treatment modalities being administered to the patient, for example chemotherapeutic treatments and surgical treatments, and whether the radiation is administered pre- or post-surgery. In general, a curative therapeutic dose for a solid epithelial tumor ranges from about 60 to 80 gray (Gy), whereas a curative dose for a lymphoma is about 20 to 40 Gy. In general, preventative doses can be 45-60 Gy.
[0135] As is well known in the art, the therapeutic dose can be delivered in fractions. Fractionation refers to spreading out the total dose of radiation over time, for example, over days, weeks or months. The dose delivered in each fraction can be about 1.5-2 Gy per day. The treatment plan can include a fraction treatment one or more times per day, every other day, weekly, etc. depending on the treatment needs of each patient. For example, a hypofractionation schedule comprises dividing the total dose into several relatively large doses, and administering the doses at least one day apart. Exemplary hypofraction doses are 3 Gy to 20 Gy per fraction. An exemplary fractionation schedule that can be used to treat lung cancer is Continuous Hyperfractionated Accelerated Radiation therapy (CHART), which consists of three small fractions per day.
[0136] The biomarkers described herein are useful in developing and modifying treatment plans for patients diagnosed with a tumor or cancer. The treatment plan can include visualizing or measuring the tumor volume that needs to be irradiated, the optimal or effective dose of radiation administered to the tumor, and the maximum dose to prevent damage to nearby healthy tissue or organs at risk. Algorithms can used in treatment planning, and include dose calculation algorithms based on the particular radiotherapy technique parameters employed, e.g., gantry angle, MLC leaf positions, etc., and search algorithms which use various techniques to adjust system parameters between dose calculations to optimize the effectiveness of the treatment. Exemplary dose calculation algorithms include various Monte Carlo ("MC") techniques and pencil beam convolution ("PBC"). Exemplary search algorithms include various simulated annealing ("SA") techniques, algebraic inverse treatment planning ("AITP"), and simultaneous iterative inverse treatment planning ("SIITP"). Such techniques, and others, are well known in the art, and are included within the scope of this disclosure.
[0137] Treatment planning algorithms may be implemented as part of an integrated treatment planning software package which provides additional features and capabilities. For example, a dose calculation algorithm and search algorithm may be used to optimize a set of fluence maps at each gantry angle, with a separate leaf sequencer used to calculate the leaf movements needed to deliver them. Alternatively, a dose calculation algorithm and search algorithm may be used to directly optimize leaf movements and other machine parameters. The Eclipse.TM. Treatment Planning System offered by the assignee of the present invention includes such an integrated software program. Methods for optimizing treatment plans are described in U.S. Pat. No. 7,801,270, which is incorporated by reference herein.
[0138] In some embodiments, the biomarkers described herein can be used to monitor the progress of tumor control after radiation therapy. For example, the expression levels of the biomarkers before and after ionizing radiation therapy can be compared. In some embodiments, if the expression levels of biomarkers increase after radiotherapy, this suggests that the tumor is continuing to grow in size. Thus, the radiation treatment can be modified based on monitoring tumor growth using the biomarkers described herein.
[0139] The biomarkers described herein can be used with any radiation therapy technique known in the art. Radiation therapy techniques include external-beam radiotherapy ("EBRT") and Intensity Modulated Radiotherapy ("IMRT"), which can be administered by a radiotherapy system, such as a linear accelerator, equipped with a multileaf collimator ("MLC"). The use of multileaf collimators and IMRT allows the patient to be treated from multiple angles while varying the shape and dose of the radiation beam, thereby avoiding excess irradiation of nearby healthy tissue. Other exemplary radiation therapy techniques include stereotactic body radiotherapy (SBRT), volumetric modulated arc therapy, three-dimensional conformal radiotherapy ("3D conformal" or "3DCRT"), image-guided radiotherapy (IGRT). The radiation therapy techniques can also include Adaptive radiotherapy (ART), a form of IGRT that can revise the treatment during the course of radiotherapy in order to optimize the dose distribution depending on patient anatomy changes, and organ and tumour shape. Another radiation therapy technique is brachytherapy. In brachytherapy, a radioactive source is implanted within the body of the subject, such that the radioactive source is near the tumor. As used herein, the term radiotherapy should be broadly construed and is intended to include various techniques used to irradiate a patient, including use of photons (such as high energy x-rays and gamma rays), particles (such as electron and proton beams), and radiosurgical techniques. Further, any method of providing conformal radiation to a target volume is intended to be within the scope of the present disclosure.
[0140] D. Chemotherapeutic Agents
[0141] In some embodiments, the radiation therapy is administered in combination with one or more chemotherapeutic agents (i.e., anti-cancer agents). The chemotherapeutic agents include radiosensitizers, anti-tumor or anti-cancer agents, and/or inhibitors of TGF-beta signaling. In some embodiments, the radiation therapy is administered in combination with an immune system modulator.
[0142] 1. Radiosensitizers
[0143] In some embodiments, the chemotherapeutic agent is a radiosensitizer. Exemplary radiosensitizers include hypoxia radiosensitizers such as misonidazole, metronidazole, and trans-sodium crocetinate, a compound that helps to increase the diffusion of oxygen into hypoxic tumor tissue. The radiosensitizer can also be a DNA damage response inhibitor interfering with base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), recombinational repair comprising homologous recombination (HR) and non-homologous end-joining (NHEJ), and direct repair mechanisms. SSB repair mechanisms include BER, NER, or MMR pathways whilst DSB repair mechanisms consist of HR and NHEJ pathways. Radiation causes DNA breaks that if not repaired are lethal. Single strand breaks are repaired through a combination of BER, NER and MMR mechanisms using the intact DNA strand as a template. The predominant pathway of SSB repair is the BER utilizing a family of related enzymes termed poly-(ADP-ribose) polymerases (PARP). Thus, the radiosensitizer can include DNA damage response inhibitors such as Poly (ADP) ribose polymerase (PARP) inhibitors.
[0144] 2. Anti-Tumor Agents
[0145] In some embodiments, the chemotherapeutic agent is an anti-cancer agent. Examples of anti-cancer agents include hypoxic cytotoxins, such as tirapazamine. In some embodiments, the anti-cancer agent is a drug that is currently approved for treating cancer or tumors. In some embodiments, the anti-cancer agent is approved for treating lung cancer, for example, Cisplatin, Taxol, Paclitaxal, Abitrexate, Bevacizumab, Folex, Gemcitabine, or Iressa. In some embodiments, the anti-cancer agent targets a fusion protein, and includes agents such as Crizotinib.
[0146] 3. TGF-.beta. Inhibitors
[0147] There is substantial evidence that TGF-.beta. plays a crucial role in the response to ionizing radiation. TGF-.beta. is a pleiotropic cytokine that is important in normal tissue homeostatis, regulates inflammation and immune responses, and suppresses epithelial proliferation. TGF-.beta. is activated in irradiated tissues, presumably because the latent TGF-0 complex has a specific-redox-sensitive conformation activated by reactive oxygen species, which are generated by radiation. There is significant evidence for activated TGF-.beta. to contribute to metastasis, to drive function-compromising fibrosis, to promote tumor cell proliferation, and to suppress immune surveillance. Thus, in some embodiments, the chemotherapeutic agent is a TGF-.beta. inhibitor. There are four major classes of TGF-0 inhibitors, including ligand traps (e.g. 1D11 or Fresolimumab), antisense oligonucleotides (e.g., Trabedersen), small molecule receptor kinase inhibitors (e.g., LY2109761 or LY2157299), and peptide aptamers (e.g. Trx-SARA). Any suitable TGF-B inhibitor known in the art can be used in the methods, and is considered within the scope of the methods described herein. TGF-beta inhibitors also include agents that inhibit the production of activated TGF-beta.
[0148] 4. Immune Modulators
[0149] Examples of immune modulators include antibodies that bind molecules expressed on the surface of immune system cells, such as antigen presenting cells and T-cells. Immune modulators also include small molecules that inhibit or stimulate the immune system. One non-limiting example of a small molecule immune modulator is an inhibitor of the enzyme Indolamine 2,3-dioxygenase.
EXAMPLES
Example 1
[0150] This example describes the association between the biomarkers described herein and clinical outcomes (survival and local tumor control) for lung cancer patients treated with radiation.
[0151] Statistical Methods
[0152] In order to understand the characteristics of the population under investigation, descriptives of both demographics and biomarker levels (intensity, proportion, and total; abbreviated "Int," "Prop," and "Tot" throughout) were first examined. Biomarker levels were examined using Allred scoring system. The Allred scoring system allows for measurement of biomarker expression as monitored by immunohistochemistry. It takes into account the percentage/proportion of cells that stain by immunohistochemistry (on a scale of 0-5) and the intensity of that staining (on a scale of 0-3), leading to a possible total score of 8. Survival time was then modeled using cox proportional hazards models, defined as date of biopsy to date of death or last follow-up. Univariate models were examined first, followed by multivariate models to determine factors most predictive of survival. Multivariate models were built using stepwise regression, and were also further examined for possible effect modification. We also dichotomized each biomarker using two methods: 1) a cut point suggested by a nonparametric regression tree, where a cut point is found that "best" separates subjects by survival time, and 2) by a visual examination of where clear separation in the distributions exist. Lastly, we examined how predictive biomarkers and clinical characteristics were of local tumor control failure using logistic regression models. Statistical significance was set to level 0.05 for all analyses.
[0153] Results
[0154] A total of 133 deceased lung cancer patients were included in the analysis. The median survival time among all patients was 1.5 years. The majority of patients were white males; most underwent curative radiation therapy, were diagnosed at stage III, and were current smokers (Table 2). The expression pattern of biomarkers varied greatly: ALDH1A1, CD68, HA, and VIM tended to have low values, while Beta_Cat, CD44, MFG_E8 and MMP_9 tended to have high values (See FIGS. 1-8).
TABLE-US-00002 TABLE 2 Descriptive Patient Characteristics (N = 133) N (%) or N, Mean (SD), Variable Level [Min, Max] Gender Female 56 (42%) Male 77 (58%) Race Black 52 (40%) Non-Black 80 (60%) Radiation Therapy Curative 114 (86%) Curative/SBRT 5 (4%) SBRT 14 (10%) Treatment Group RT Alone 42 (33%) Chemo RT 86 (67%) Local Tumor Control Yes 100 (75%) No 33 (25%) Stage at Diagnosis I 25 (20%) II 18 (14%) III 77 (61%) IV 6 (5%) Smoking current smoker 73 (57%) not smoker 2 (2%) past smoker 51 (39%) unknown 3 (2%) Tumor Type Adenocarcinoma 23 (20%) Squamous 95 (80%) Age 133, 78 (11), [52, 98] Median Household Income <$15,000 6 (5%) .gtoreq.$15,000-<$30,000 33 (27%) .gtoreq.$30,000-<$50,000 42 (34%) .gtoreq.$50,000-<$75,000 28 (23%) .gtoreq.$75,000 13 (11%)
[0155] Univariate survival models indicated that the only patient characteristics exhibiting significant differences in risk were race, where blacks had nearly a 1.5 times greater risk of death than non-blacks (p-value=0.038, Table 3). Additionally, crude estimates of differences in survival by biomarker levels indicated that higher levels of CD68 were associated with a statistically significant higher risk of death. Namely, a one unit increase in CD68 Prop increased the risk of death by 49%, while a one unit increase in CD68 Tot increased the risk of death by 25% (p-value=0.008, p-value=0.02 for Prop and Tot, respectively). Further, a marginally significant protective effect was observed for MMP_9 (p-value=0.05, p-value=0.054 for Prop and Tot, respectively). Using the optimal cut point method for each biomarker based on regression trees, CD68 and VIM groups displayed association with survival, and there was some marginal significance of MFG_E8. Using cut points determined by visual examination, MFG_E8 groups were associated with survival. In these plots, CD68 (Prop and Tot) and VIM (Prop and Tot) both increased risk, while MFG_E8 decreased risk.
TABLE-US-00003 TABLE 3 Univariate Survival Estimates Hazard Ratio Comparison Overall Parameter Level (HR) (95% CI) p-value p-value Patient Characteristics Gender Male vs. Female 1.03 (0.72, 1.46) 0.88 Race Black vs. 1.47 (1.02, 2.1) 0.038 0.038 Non-Black Smoking not smoker vs. 2 (0.48, 8.25) 0.34 0.37 current smoker past smoker vs. 1.12 (0.78, 1.61) 0.54 current smoker unknown vs. 2.46 (0.76, 7.92) 0.13 current smoker Stage at Diagnosis II vs. I 1.07 (0.58, 1.96) 0.84 0.98 III vs. I 0.98 (0.62, 1.55) 0.93 IV vs. I 0.9 (0.37, 2.2) 0.82 Stage II or III Yes vs. No 1.16 (0.78, 1.74) 0.46 0.46 at Diagnosis Tumor Type Squamous vs. 1.15 (0.73, 1.82) 0.54 0.54 Adenocarcinoma Treatment Group chemo RT vs. RT 0.72 (0.49, 1.05) 0.088 0.088 Alone Local Tumor Control Yes vs. No 1.06 (0.72, 1.58) 0.76 0.76 Radiation Therapy Curative/SBRT vs. 0.55 (0.22, 1.36) 0.19 0.20 Curative SBRT vs. Curative 1.39 (0.8, 2.44) 0.25 Age 1 (0.99, 1.02) 0.56 Biomarkers ALDH1A1_Int 1 (0.78, 1.29) 1.00 ALDH1A1_Prop 0.98 (0.85, 1.14) 0.82 ALDH1A1_Tot 0.99 (0.9, 1.09) 0.90 Beta_Cat_Int 0.84 (0.66, 1.09) 0.19 Beta_Cat_Prop 0.95 (0.8, 1.13) 0.57 Beta_Cat_Tot 0.95 (0.85, 1.06) 0.32 CD44_Int 1.11 (0.9, 1.36) 0.33 CD44_Prop 1.06 (0.93, 1.2) 0.38 CD44_Tot 1.04 (0.96, 1.14) 0.32 CD68_Int 1.33 (0.89, 1.99) 0.16 CD68_Prop 1.49 (1.11, 1.99) 0.008 CD68_Tot 1.25 (1.04, 1.51) 0.02 HA_Int 0.94 (0.76, 1.17) 0.59 HA_Prop 0.93 (0.83, 1.04) 0.20 HA_Tot 0.96 (0.88, 1.04) 0.27 MFG_E8_Int 0.92 (0.74, 1.14) 0.44 MFG_E8_Prop 0.92 (0.83, 1.02) 0.10 MFG_E8_Tot 0.95 (0.88, 1.02) 0.15 MMP_9_Int 0.83 (0.62, 1.11) 0.21 MMP_9_Prop 0.8 (0.64, 1) 0.05 MMP_9_Tot 0.87 (0.76, 1) 0.054 VIM_Int 1.02 (0.83, 1.25) 0.87 VIM_Prop 1.02 (0.87, 1.19) 0.78 VIM_Tot 1.01 (0.92, 1.11) 0.81
[0156] Table 4 displays the results of a final multivariate model built using stepwise regression. All patient characteristics and 24 biomarker measurements were eligible for model inclusion. The significance level required to both enter the model and be retained in the model was 0.05. This model selection procedure only retained CD68 Prop and race in the model, indicating that these two covariates are most predictive of survival outcomes, among all covariates considered.
TABLE-US-00004 TABLE 4 Final Survival Model Parameter HR (95% CI) p-value CD68 1.61 (1.19, 2.18) 0.002 Prop black vs. 1.59 (1.05, 2.41) 0.028 non-black
[0157] It should be noted that since correlation within a biomarker is high (comparing the three different measures of Int, Prop and Tot), itis unlikely that multiple measurement types of the same biomarker would be retained in the model, due to information redundancy.
[0158] Predictive Ability of Biomarkers for Local Tumor Control
[0159] We also determined if biomarkers, along with clinical characteristics, were predictive of local tumor control failure. We use the Area Under the ROC (Receiver Operating Characteristics) Curve (AUC) as a measure of predictive ability, where the ROC curve is built from various probability cut points from a logistic regression model. An AUC of 0.5 indicates "no better than random chance" and an AUC of 1 indicates "perfect prediction". As seen in Table 5, Tumor Type. CD44, and MFG_E8 all had a significantly better than random chance prediction ability. CD44 and squamous tumors were risk factors for local tumor control failure, while MFG_E8 had a protective effect. To find amore predictive model of local tumor control failure, we then determined which combination of these three factors resulted in the highest AUC. The variables CD44 (Tot), MFG-E8 (Prop) and tumor type combined resulted in an AUC of 0.74 (p-value<0.001, 95% CI=(0.63, 0.85)). The ROC curve from this multivariate model can be seen in FIG. 9.
TABLE-US-00005 TABLE 5 Predictive Ability Of Biomarkers For Local Tumor Control Failure (Univariate) Odds Ratio Comparison Overall Area Under the Curve Parameter Level OR (95% CI) p-value p- value AUG (95% CI) p-value Patient Characteristics Gender Male vs. 1.16 (0.52, 2.59) 0.72 0.52 (0.42, 0.62) 0.72 Female Race Black vs. 1.18 (0.53, 2.63) 0.68 0.52 (0.42, 0.62) 0.69 Non-Black Age 0.98 (0.94, 1.01) 0.18 0.59 (0.47, 0.7) 0.14 Radiation Curative/ 0.7 (0.08, 6.51) 0.75 0.61 0.54 (0.47, 0.6) 0.26 Therapy SBRT vs. SBRT vs. 0.47 (0.1, 2.21) 0.34 Curative Treatment chemo RT 1.64 (0.67, 4.06) 0.28 0.55 (0.46, 0.64) 0.26 Group vs. RT Alone Stage II or Yes vs. No 1.07 (0.43, 2.68) 0.89 0.51 (0.42, 0.59) 0.89 III at Diagnosis Smoking* . . . . . Tumor Type Squamous 4.39 (0.96, 19.98) 0.056 0.59 (0.52, 0.65) 0.008 vs. Median . . . . . Household Income* Biomarkers ALDH1A1 1.48 (0.88, 2.49) 0.14 0.57 (0.46, 0.67) 0.21 Int ALDH1A1 1.27 (0.94, 1.73) 0.12 0.57 (0.47, 0.67) 0.18 Prop ALDH1A1 1.19 (0.97, 1.45) 0.09 0.57 (0.47, 0.68) 0.18 Tot Beta_Cat Int 0.65 (0.36, 1.2) 0.17 0.57 (0.46, 0.69) 0.20 Beta_Cat 0.85 (0.58, 1.26) 0.42 0.56 (0.45, 0.67) 0.30 Prop Beta_Cat Tot 0.85 (0.66, 1.11) 0.24 0.59 (0.47, 0.71) 0.13 CD44 Int 1.94 (1.08, 3.51) 0.027 0.62 (0.52, 0.72) 0.019 CD44 Prop 1.7 (1.11, 2.6) 0.014 0.65 (0.55, 0.76) 0.003 CD44 Tot 1.44 (1.08, 1.92) 0.012 0.66 (0.55, 0.76) 0.004 CD68 Int 1.93 (0.83, 4.49) 0.13 0.56 (0.47, 0.64) 0.18 CD68 Prop 1.16 (0.67, 2) 0.61 0.55 (0.47, 0.63) 0.22 CD68 Tot 1.16 (0.82, 1.66) 0.40 0.55 (0.47, 0.63) 0.23 HA Int 1.03 (0.65, 1.62) 0.91 0.51 (0.4, 0.63) 0.80 HA Prop 0.96 (0.74, 1.26) 0.79 0.5 (0.39, 0.61) 0.97 HA Tot 0.99 (0.83, 1.18) 0.89 0.5 (0.39, 0.61) 0.96 MFG_E8 Int 0.73 (0.47, 1.14) 0.17 0.58 (0.47, 0.7) 0.17 MFG_E8 0.77 (0.62, 0.97) 0.029 0.64 (0.52, 0.75) 0.019 Prop MFG_E8 Tot 0.85 (0.73, 1) 0.046 0.63 (0.52, 0.74) 0.023 MMP_9 Int 1.39 (0.73, 2.67) 0.32 0.56 (0.46, 0.66) 0.23 MMP_9 1.26 (0.69, 2.3) 0.46 0.57 (0.5, 0.65) 0.06 Prop MMP_9 Tot 1.2 (0.83, 1.73) 0.33 0.59 (0.49, 0.7) 0.07 VIM Int 1.15 (0.73, 1.81) 0.56 0.53 (0.43, 0.62) 0.58 VIM Prop 1.07 (0.76, 1.49) 0.71 0.52 (0.43, 0.61) 0.65 VIM Tot 1.05 (0.86, 1.28) 0.63 0.52 (0.43, 0.61) 0.65
[0160] We then chose a probability cut point on this ROC curve that exhibited both high sensitivity and specificity, to act as a"test" for local tumor control failure. We required that both sensitivity and specificity be at least 0.5, and gave more importance to high sensitivity by up-weighting it. This resulted in a probability cut point of 21%, which corresponded to a sensitivity of 82% and a specificity of 55%. The values of CD44 Tot and MFG_E8 Prop that correspond to a positive and negative test using this probability cut point, for each tumor type, can be seen in FIGS. 10 and 11.
[0161] Subgroup Analyses for Stage II and III Patients
[0162] As a sensitivity analysis, univariate survival estimates for patient characteristics and biomarker levels were also recalculated among stage II and III patients only (results not shown). In this analysis, the covariate that achieved statistical significance was race (p-value=0.018).
[0163] This Example demonstrates that, among 133 deceased lung cancer patients, CD68 expression was associated with increased risk of death, while MMP_9 expression was associated with decreased risk of death. Patient characteristics most predictive of survival outcomes were CD68 and race. CD44, MFG_E8, and tumor type were predictive of local tumor control failure.
[0164] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes.
Sequence CWU
1
1
141742PRTHomo sapiensCD44 antigen isoform 1 precursor, hematopoeitic
cell E- and L-selectin ligand (HCELL), chondroitin sulfate
proteoglycan 8 (CSPG8), GP90 lymphocyte homing/adhesion receptor
(LHR), extracellular matrix receptor III (ECMR-III), Hermes antigen
1Met Asp Lys Phe Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro1
5 10 15Leu Ser Leu Ala Gln Ile
Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly 20 25
30Val Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser
Arg Thr Glu 35 40 45Ala Ala Asp
Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met Ala 50
55 60Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr
Cys Arg Tyr Gly65 70 75
80Phe Ile Glu Gly His Val Val Ile Pro Arg Ile His Pro Asn Ser Ile
85 90 95Cys Ala Ala Asn Asn Thr
Gly Val Tyr Ile Leu Thr Ser Asn Thr Ser 100
105 110Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala Pro
Pro Glu Glu Asp 115 120 125Cys Thr
Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro Ile Thr 130
135 140Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr
Val Gln Lys Gly Glu145 150 155
160Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp
165 170 175Asp Val Ser Ser
Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly 180
185 190Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro
Ile Pro Asp Glu Asp 195 200 205Ser
Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Thr Leu 210
215 220Met Ser Thr Ser Ala Thr Ala Thr Glu Thr
Ala Thr Lys Arg Gln Glu225 230 235
240Thr Trp Asp Trp Phe Ser Trp Leu Phe Leu Pro Ser Glu Ser Lys
Asn 245 250 255His Leu His
Thr Thr Thr Gln Met Ala Gly Thr Ser Ser Asn Thr Ile 260
265 270Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn
Glu Asp Glu Arg Asp Arg 275 280
285His Leu Ser Phe Ser Gly Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile 290
295 300Ser Ser Thr Ile Ser Thr Thr Pro
Arg Ala Phe Asp His Thr Lys Gln305 310
315 320Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser His Ser
Asn Pro Glu Val 325 330
335Leu Leu Gln Thr Thr Thr Arg Met Thr Asp Val Asp Arg Asn Gly Thr
340 345 350Thr Ala Tyr Glu Gly Asn
Trp Asn Pro Glu Ala His Pro Pro Leu Ile 355 360
365His His Glu His His Glu Glu Glu Glu Thr Pro His Ser Thr
Ser Thr 370 375 380Ile Gln Ala Thr Pro
Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys385 390
395 400Glu Gln Trp Phe Gly Asn Arg Trp His Glu
Gly Tyr Arg Gln Thr Pro 405 410
415Lys Glu Asp Ser His Ser Thr Thr Gly Thr Ala Ala Ala Ser Ala His
420 425 430Thr Ser His Pro Met
Gln Gly Arg Thr Thr Pro Ser Pro Glu Asp Ser 435
440 445Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser His Pro
Met Gly Arg Gly 450 455 460His Gln Ala
Gly Arg Arg Met Asp Met Asp Ser Ser His Ser Ile Thr465
470 475 480Leu Gln Pro Thr Ala Asn Pro
Asn Thr Gly Leu Val Glu Asp Leu Asp 485
490 495Arg Thr Gly Pro Leu Ser Met Thr Thr Gln Gln Ser
Asn Ser Gln Ser 500 505 510Phe
Ser Thr Ser His Glu Gly Leu Glu Glu Asp Lys Asp His Pro Thr 515
520 525Thr Ser Thr Leu Thr Ser Ser Asn Arg
Asn Asp Val Thr Gly Gly Arg 530 535
540Arg Asp Pro Asn His Ser Glu Gly Ser Thr Thr Leu Leu Glu Gly Tyr545
550 555 560Thr Ser His Tyr
Pro His Thr Lys Glu Ser Arg Thr Phe Ile Pro Val 565
570 575Thr Ser Ala Lys Thr Gly Ser Phe Gly Val
Thr Ala Val Thr Val Gly 580 585
590Asp Ser Asn Ser Asn Val Asn Arg Ser Leu Ser Gly Asp Gln Asp Thr
595 600 605Phe His Pro Ser Gly Gly Ser
His Thr Thr His Gly Ser Glu Ser Asp 610 615
620Gly His Ser His Gly Ser Gln Glu Gly Gly Ala Asn Thr Thr Ser
Gly625 630 635 640Pro Ile
Arg Thr Pro Gln Ile Pro Glu Trp Leu Ile Ile Leu Ala Ser
645 650 655Leu Leu Ala Leu Ala Leu Ile
Leu Ala Val Cys Ile Ala Val Asn Ser 660 665
670Arg Arg Arg Cys Gly Gln Lys Lys Lys Leu Val Ile Asn Ser
Gly Asn 675 680 685Gly Ala Val Glu
Asp Arg Lys Pro Ser Gly Leu Asn Gly Glu Ala Ser 690
695 700Lys Ser Gln Glu Met Val His Leu Val Asn Lys Glu
Ser Ser Glu Thr705 710 715
720Pro Asp Gln Phe Met Thr Ala Asp Glu Thr Arg Asn Leu Gln Asn Val
725 730 735Asp Met Lys Ile Gly
Val 74025748DNAHomo sapiensCD44 antigen transcript variant 1,
hematopoeitic cell E- and L-selectin ligand (HCELL), chondroitin
sulfate proteoglycan 8 (CSPG8), GP90 lymphocyte homing/adhesion
receptor (LHR), extracellular matrix receptor III (ECMR-III), Hermes
antigen 2gagaagaaag ccagtgcgtc tctgggcgca ggggccagtg gggctcggag
gcacaggcac 60cccgcgacac tccaggttcc ccgacccacg tccctggcag ccccgattat
ttacagcctc 120agcagagcac ggggcggggg cagaggggcc cgcccgggag ggctgctact
tcttaaaacc 180tctgcgggct gcttagtcac agcccccctt gcttgggtgt gtccttcgct
cgctccctcc 240ctccgtctta ggtcactgtt ttcaacctcg aataaaaact gcagccaact
tccgaggcag 300cctcattgcc cagcggaccc cagcctctgc caggttcggt ccgccatcct
cgtcccgtcc 360tccgccggcc cctgccccgc gcccagggat cctccagctc ctttcgcccg
cgccctccgt 420tcgctccgga caccatggac aagttttggt ggcacgcagc ctggggactc
tgcctcgtgc 480cgctgagcct ggcgcagatc gatttgaata taacctgccg ctttgcaggt
gtattccacg 540tggagaaaaa tggtcgctac agcatctctc ggacggaggc cgctgacctc
tgcaaggctt 600tcaatagcac cttgcccaca atggcccaga tggagaaagc tctgagcatc
ggatttgaga 660cctgcaggta tgggttcata gaagggcacg tggtgattcc ccggatccac
cccaactcca 720tctgtgcagc aaacaacaca ggggtgtaca tcctcacatc caacacctcc
cagtatgaca 780catattgctt caatgcttca gctccacctg aagaagattg tacatcagtc
acagacctgc 840ccaatgcctt tgatggacca attaccataa ctattgttaa ccgtgatggc
acccgctatg 900tccagaaagg agaatacaga acgaatcctg aagacatcta ccccagcaac
cctactgatg 960atgacgtgag cagcggctcc tccagtgaaa ggagcagcac ttcaggaggt
tacatctttt 1020acaccttttc tactgtacac cccatcccag acgaagacag tccctggatc
accgacagca 1080cagacagaat ccctgctacc actttgatga gcactagtgc tacagcaact
gagacagcaa 1140ccaagaggca agaaacctgg gattggtttt catggttgtt tctaccatca
gagtcaaaga 1200atcatcttca cacaacaaca caaatggctg gtacgtcttc aaataccatc
tcagcaggct 1260gggagccaaa tgaagaaaat gaagatgaaa gagacagaca cctcagtttt
tctggatcag 1320gcattgatga tgatgaagat tttatctcca gcaccatttc aaccacacca
cgggcttttg 1380accacacaaa acagaaccag gactggaccc agtggaaccc aagccattca
aatccggaag 1440tgctacttca gacaaccaca aggatgactg atgtagacag aaatggcacc
actgcttatg 1500aaggaaactg gaacccagaa gcacaccctc ccctcattca ccatgagcat
catgaggaag 1560aagagacccc acattctaca agcacaatcc aggcaactcc tagtagtaca
acggaagaaa 1620cagctaccca gaaggaacag tggtttggca acagatggca tgagggatat
cgccaaacac 1680ccaaagaaga ctcccattcg acaacaggga cagctgcagc ctcagctcat
accagccatc 1740caatgcaagg aaggacaaca ccaagcccag aggacagttc ctggactgat
ttcttcaacc 1800caatctcaca ccccatggga cgaggtcatc aagcaggaag aaggatggat
atggactcca 1860gtcatagtat aacgcttcag cctactgcaa atccaaacac aggtttggtg
gaagatttgg 1920acaggacagg acctctttca atgacaacgc agcagagtaa ttctcagagc
ttctctacat 1980cacatgaagg cttggaagaa gataaagacc atccaacaac ttctactctg
acatcaagca 2040ataggaatga tgtcacaggt ggaagaagag acccaaatca ttctgaaggc
tcaactactt 2100tactggaagg ttatacctct cattacccac acacgaagga aagcaggacc
ttcatcccag 2160tgacctcagc taagactggg tcctttggag ttactgcagt tactgttgga
gattccaact 2220ctaatgtcaa tcgttcctta tcaggagacc aagacacatt ccaccccagt
ggggggtccc 2280ataccactca tggatctgaa tcagatggac actcacatgg gagtcaagaa
ggtggagcaa 2340acacaacctc tggtcctata aggacacccc aaattccaga atggctgatc
atcttggcat 2400ccctcttggc cttggctttg attcttgcag tttgcattgc agtcaacagt
cgaagaaggt 2460gtgggcagaa gaaaaagcta gtgatcaaca gtggcaatgg agctgtggag
gacagaaagc 2520caagtggact caacggagag gccagcaagt ctcaggaaat ggtgcatttg
gtgaacaagg 2580agtcgtcaga aactccagac cagtttatga cagctgatga gacaaggaac
ctgcagaatg 2640tggacatgaa gattggggtg taacacctac accattatct tggaaagaaa
caaccgttgg 2700aaacataacc attacaggga gctgggacac ttaacagatg caatgtgcta
ctgattgttt 2760cattgcgaat cttttttagc ataaaatttt ctactctttt tgttttttgt
gttttgttct 2820ttaaagtcag gtccaatttg taaaaacagc attgctttct gaaattaggg
cccaattaat 2880aatcagcaag aatttgatcg ttccagttcc cacttggagg cctttcatcc
ctcgggtgtg 2940ctatggatgg cttctaacaa aaactacaca tatgtattcc tgatcgccaa
cctttccccc 3000accagctaag gacatttccc agggttaata gggcctggtc cctgggagga
aatttgaatg 3060ggtccatttt gcccttccat agcctaatcc ctgggcattg ctttccactg
aggttggggg 3120ttggggtgta ctagttacac atcttcaaca gaccccctct agaaattttt
cagatgcttc 3180tgggagacac ccaaagggtg aagctattta tctgtagtaa actatttatc
tgtgtttttg 3240aaatattaaa ccctggatca gtcctttgat cagtataatt ttttaaagtt
actttgtcag 3300aggcacaaaa gggtttaaac tgattcataa taaatatctg tacttcttcg
atcttcacct 3360tttgtgctgt gattcttcag tttctaaacc agcactgtct gggtccctac
aatgtatcag 3420gaagagctga gaatggtaag gagactcttc taagtcttca tctcagagac
cctgagttcc 3480cactcagacc cactcagcca aatctcatgg aagaccaagg agggcagcac
tgtttttgtt 3540ttttgttttt tgtttttttt ttttgacact gtccaaaggt tttccatcct
gtcctggaat 3600cagagttgga agctgaggag cttcagcctc ttttatggtt taatggccac
ctgttctctc 3660ctgtgaaagg ctttgcaaag tcacattaag tttgcatgac ctgttatccc
tggggcccta 3720tttcatagag gctggcccta ttagtgattt ccaaaaacaa tatggaagtg
ccttttgatg 3780tcttacaata agagaagaag ccaatggaaa tgaaagagat tggcaaaggg
gaaggatgat 3840gccatgtaga tcctgtttga catttttatg gctgtatttg taaacttaaa
cacaccagtg 3900tctgttcttg atgcagttgc tatttaggat gagttaagtg cctggggagt
ccctcaaaag 3960gttaaaggga ttcccatcat tggaatctta tcaccagata ggcaagttta
tgaccaaaca 4020agagagtact ggctttatcc tctaacctca tattttctcc cacttggcaa
gtcctttgtg 4080gcatttattc atcagtcagg gtgtccgatt ggtcctagaa cttccaaagg
ctgcttgtca 4140tagaagccat tgcatctata aagcaacggc tcctgttaaa tggtatctcc
tttctgaggc 4200tcctactaaa agtcatttgt tacctaaact tatgtgctta acaggcaatg
cttctcagac 4260cacaaagcag aaagaagaag aaaagctcct gactaaatca gggctgggct
tagacagagt 4320tgatctgtag aatatcttta aaggagagat gtcaactttc tgcactattc
ccagcctctg 4380ctcctccctg tctaccctct cccctccctc tctccctcca cttcacccca
caatcttgaa 4440aaacttcctt tctcttctgt gaacatcatt ggccagatcc attttcagtg
gtctggattt 4500ctttttattt tcttttcaac ttgaaagaaa ctggacatta ggccactatg
tgttgttact 4560gccactagtg ttcaagtgcc tcttgttttc ccagagattt cctgggtctg
ccagaggccc 4620agacaggctc actcaagctc tttaactgaa aagcaacaag ccactccagg
acaaggttca 4680aaatggttac aacagcctct acctgtcgcc ccagggagaa aggggtagtg
atacaagtct 4740catagccaga gatggttttc cactccttct agatattccc aaaaagaggc
tgagacagga 4800ggttattttc aattttattt tggaattaaa tacttttttc cctttattac
tgttgtagtc 4860cctcacttgg atatacctct gttttcacga tagaaataag ggaggtctag
agcttctatt 4920ccttggccat tgtcaacgga gagctggcca agtcttcaca aacccttgca
acattgcctg 4980aagtttatgg aataagatgt attctcactc ccttgatctc aagggcgtaa
ctctggaagc 5040acagcttgac tacacgtcat ttttaccaat gattttcagg tgacctgggc
taagtcattt 5100aaactgggtc tttataaaag taaaaggcca acatttaatt attttgcaaa
gcaacctaag 5160agctaaagat gtaatttttc ttgcaattgt aaatcttttg tgtctcctga
agacttccct 5220taaaattagc tctgagtgaa aaatcaaaag agacaaaaga catcttcgaa
tccatatttc 5280aagcctggta gaattggctt ttctagcaga acctttccaa aagttttata
ttgagattca 5340taacaacacc aagaattgat tttgtagcca acattcattc aatactgtta
tatcagagga 5400gtaggagaga ggaaacattt gacttatctg gaaaagcaaa atgtacttaa
gaataagaat 5460aacatggtcc attcaccttt atgttataga tatgtctttg tgtaaatcat
ttgttttgag 5520ttttcaaaga atagcccatt gttcattctt gtgctgtaca atgaccactg
ttattgttac 5580tttgactttt cagagcacac ccttcctctg gtttttgtat atttattgat
ggatcaataa 5640taatgaggaa agcatgatat gtatattgct gagttgaaag cacttattgg
aaaatattaa 5700aaggctaaca ttaaaagact aaaggaaaca gaaaaaaaaa aaaaaaaa
57483707PRTHomo sapiensmatrix metalloproteinase 9 (MMP9,
MMP-9) 3Met Ser Leu Trp Gln Pro Leu Val Leu Val Leu Leu Val Leu Gly Cys1
5 10 15Cys Phe Ala Ala
Pro Arg Gln Arg Gln Ser Thr Leu Val Leu Phe Pro 20
25 30Gly Asp Leu Arg Thr Asn Leu Thr Asp Arg Gln
Leu Ala Glu Glu Tyr 35 40 45Leu
Tyr Arg Tyr Gly Tyr Thr Arg Val Ala Glu Met Arg Gly Glu Ser 50
55 60Lys Ser Leu Gly Pro Ala Leu Leu Leu Leu
Gln Lys Gln Leu Ser Leu65 70 75
80Pro Glu Thr Gly Glu Leu Asp Ser Ala Thr Leu Lys Ala Met Arg
Thr 85 90 95Pro Arg Cys
Gly Val Pro Asp Leu Gly Arg Phe Gln Thr Phe Glu Gly 100
105 110Asp Leu Lys Trp His His His Asn Ile Thr
Tyr Trp Ile Gln Asn Tyr 115 120
125Ser Glu Asp Leu Pro Arg Ala Val Ile Asp Asp Ala Phe Ala Arg Ala 130
135 140Phe Ala Leu Trp Ser Ala Val Thr
Pro Leu Thr Phe Thr Arg Val Tyr145 150
155 160Ser Arg Asp Ala Asp Ile Val Ile Gln Phe Gly Val
Ala Glu His Gly 165 170
175Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe
180 185 190Pro Pro Gly Pro Gly Ile
Gln Gly Asp Ala His Phe Asp Asp Asp Glu 195 200
205Leu Trp Ser Leu Gly Lys Gly Val Val Val Pro Thr Arg Phe
Gly Asn 210 215 220Ala Asp Gly Ala Ala
Cys His Phe Pro Phe Ile Phe Glu Gly Arg Ser225 230
235 240Tyr Ser Ala Cys Thr Thr Asp Gly Arg Ser
Asp Gly Leu Pro Trp Cys 245 250
255Ser Thr Thr Ala Asn Tyr Asp Thr Asp Asp Arg Phe Gly Phe Cys Pro
260 265 270Ser Glu Arg Leu Tyr
Thr Arg Asp Gly Asn Ala Asp Gly Lys Pro Cys 275
280 285Gln Phe Pro Phe Ile Phe Gln Gly Gln Ser Tyr Ser
Ala Cys Thr Thr 290 295 300Asp Gly Arg
Ser Asp Gly Tyr Arg Trp Cys Ala Thr Thr Ala Asn Tyr305
310 315 320Asp Arg Asp Lys Leu Phe Gly
Phe Cys Pro Thr Arg Ala Asp Ser Thr 325
330 335Val Met Gly Gly Asn Ser Ala Gly Glu Leu Cys Val
Phe Pro Phe Thr 340 345 350Phe
Leu Gly Lys Glu Tyr Ser Thr Cys Thr Ser Glu Gly Arg Gly Asp 355
360 365Gly Arg Leu Trp Cys Ala Thr Thr Ser
Asn Phe Asp Ser Asp Lys Lys 370 375
380Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala385
390 395 400His Glu Phe Gly
His Ala Leu Gly Leu Asp His Ser Ser Val Pro Glu 405
410 415Ala Leu Met Tyr Pro Met Tyr Arg Phe Thr
Glu Gly Pro Pro Leu His 420 425
430Lys Asp Asp Val Asn Gly Ile Arg His Leu Tyr Gly Pro Arg Pro Glu
435 440 445Pro Glu Pro Arg Pro Pro Thr
Thr Thr Thr Pro Gln Pro Thr Ala Pro 450 455
460Pro Thr Val Cys Pro Thr Gly Pro Pro Thr Val His Pro Ser Glu
Arg465 470 475 480Pro Thr
Ala Gly Pro Thr Gly Pro Pro Ser Ala Gly Pro Thr Gly Pro
485 490 495Pro Thr Ala Gly Pro Ser Thr
Ala Thr Thr Val Pro Leu Ser Pro Val 500 505
510Asp Asp Ala Cys Asn Val Asn Ile Phe Asp Ala Ile Ala Glu
Ile Gly 515 520 525Asn Gln Leu Tyr
Leu Phe Lys Asp Gly Lys Tyr Trp Arg Phe Ser Glu 530
535 540Gly Arg Gly Ser Arg Pro Gln Gly Pro Phe Leu Ile
Ala Asp Lys Trp545 550 555
560Pro Ala Leu Pro Arg Lys Leu Asp Ser Val Phe Glu Glu Pro Leu Ser
565 570 575Lys Lys Leu Phe Phe
Phe Ser Gly Arg Gln Val Trp Val Tyr Thr Gly 580
585 590Ala Ser Val Leu Gly Pro Arg Arg Leu Asp Lys Leu
Gly Leu Gly Ala 595 600 605Asp Val
Ala Gln Val Thr Gly Ala Leu Arg Ser Gly Arg Gly Lys Met 610
615 620Leu Leu Phe Ser Gly Arg Arg Leu Trp Arg Phe
Asp Val Lys Ala Gln625 630 635
640Met Val Asp Pro Arg Ser Ala Ser Glu Val Asp Arg Met Phe Pro Gly
645 650 655Val Pro Leu Asp
Thr His Asp Val Phe Gln Tyr Arg Glu Lys Ala Tyr 660
665 670Phe Cys Gln Asp Arg Phe Tyr Trp Arg Val Ser
Ser Arg Ser Glu Leu 675 680 685Asn
Gln Val Asp Gln Val Gly Tyr Val Thr Tyr Asp Ile Leu Gln Cys 690
695 700Pro Glu Asp70544506DNAHomo sapiensmatrix
metalloproteinase 9 (MMP9, MMP-9) 4aagcttcaga gccaggcagt tctgggcttg
aacactagtt ctgtggatta actcgctctg 60tgatcacagg caaattcctt aactctctga
gccttagttt ccccctctga aaacaggagg 120gatactcatt aaacttacct tacaggtggt
gaggatgaaa cgagaggctt atagagaact 180tattacggtg cttgacacag taaatctcaa
aaaatgcatt attattatta tggttcagag 240gtaaagtgac ttgcccaagg tcacatagct
ggaaaatgca gagccgggat ggaaatccag 300gacttcgtga cgcaaagcag atgttcattg
gttagtgaac tttagaactt caacttttct 360gtaaaggaag ttaattatct ccatctcaca
gtctcattta ttagataagc atataaaatg 420cctggcacat agtaggccct ttaaatacag
cttattgggc cgggcgccat gctcatgccc 480gtaatcctag cactttggga ggccaggtgg
gcagatcact tgagtcagaa gttcgaaacc 540agcctggtca acgtagtgaa accccatctc
tactaaaaat acaaaaaatt tagccaggcg 600tggtggcgca cctataatac cagctactcg
ggaggctgag gcaggagaat tgcttgaacc 660cgggaggcag atgttgcagt gagccgagat
cacgccactg cactccagcc tgggtgacag 720agtgatacta ccccccccaa aaataaaata
aaataaataa atacaacttt ttgagttgtt 780agcaggtttt tcccaaatag ggctttgaag
aaggtgaata tagaccctgc ccgatgccgg 840ctggctagga agaaaggagt gagggaggct
gctggtgtgg gaggcttggg agggaggctt 900ggcataagtg tgataattgg gcctggagat
ttggctgcat ggaggcaggg ctggaggaac 960taagggctcc tatagattat ttccccatat
cctgccgcaa tttgcagttg aagaatccta 1020agctgagaaa ggggaggcat ttactccagg
ttacactgca gcttagagcc caataacctg 1080gtttggtgat tccaagttag aatcatggtc
ttttggcagg gtctcgctct gttgcccagg 1140ctggagtgca gtgacataat catggctcac
tgtatccttg accttctttc tgggctcaag 1200caatcctccc acctcggcct cccaaagtgc
taagattaca ggaatgagcc accatacctg 1260gccctgaatc ttgggtcttg gccttagtaa
ttaaaaccaa tcaccaccat ccgttgcgga 1320cttacaacct acagtgttct aaacatttta
tatgtttgat ctcatttaat cctcacatca 1380atttagggac aaagagcccc ccaccccccg
tttttttttt tacagctgag gaaacacttc 1440aaagtggtaa gacatttgcc cgaggtcctg
aaggaagaga gtaaagccat gtctgctgtt 1500ttctagaggc tgctactgtc ccctttactg
ccctgaagat tcagcctgcg gaagacaggg 1560ggttgcccca gtggaattcc ccagccttgc
ctagcagagc ccattccttc cgcccccaga 1620tgaagcaggg agaggaagct gagtcaaaga
aggctgtcag ggagggaaaa agaggacaga 1680gcctggagtg tggggagggg tttggggagg
atatctgacc tgggaggggg tgttgcaaaa 1740ggccaaggat gggccagggg gatcattagt
ttcagaaaga agtctcaggg agtcttccat 1800cactttccct tggctgacca ctggaggctt
tcagaccaag ggatggggga tccctccagc 1860ttcatccccc tccctccctt tcatacagtt
cccacaagct ctgcagtttg caaaacccta 1920cccctcccct gagggcctgc ggtttcctgc
gggtctgggg tcttgcctga cttggcagtg 1980gagactgcgg gcagtggaga gaggaggagg
tggtgtaagc cctttctcat gctggtgctg 2040ccacacacac acacacacac acacacacac
acacacacac acaccctgac ccctgagtca 2100gcacttgcct gtcaaggagg ggtggggtca
caggagcgcc tccttaaagc ccccacaaca 2160gcagctgcag tcagacacct ctgccctcac
catgagcctc tggcagcccc tggtcctggt 2220gctcctggtg ctgggctgct gctttgctgc
ccccagacag cgccagtcca cccttgtgct 2280cttccctgga gacctgagaa ccaatctcac
cgacaggcag ctggcagagg aatacctgta 2340ccgctatggt tacactcggg tggcagagat
gcgtggagag tcgaaatctc tggggcctgc 2400gctgctgctt ctccagaagc aactgtccct
gcccgagacc ggtgagctgg atagcgccac 2460gctgaaggcc atgcgaaccc cacggtgcgg
ggtcccagac ctgggcagat tccaaacctt 2520tgagggcgac ctcaagtggc accaccacaa
catcacctat tggatccaaa actactcgga 2580agacttgccg cgggcggtga ttgacgacgc
ctttgcccgc gccttcgcac tgtggagcgc 2640ggtgacgccg ctcaccttca ctcgcgtgta
cagccgggac gcagacatcg tcatccagtt 2700tggtgtcgcg gagcacggag acgggtatcc
cttcgacggg aaggacgggc tcctggcaca 2760cgcctttcct cctggccccg gcattcaggg
agacgcccat ttcgacgatg acgagttgtg 2820gtccctgggc aagggcgtcg tggttccaac
tcggtttgga aacgcagatg gcgcggcctg 2880ccacttcccc ttcatcttcg agggccgctc
ctactctgcc tgcaccaccg acggtcgctc 2940cgacggcttg ccctggtgca gtaccacggc
caactacgac accgacgacc ggtttggctt 3000ctgccccagc gagagactct acacccggga
cggcaatgct gatgggaaac cctgccagtt 3060tccattcatc ttccaaggcc aatcctactc
cgcctgcacc acggacggtc gctccgacgg 3120ctaccgctgg tgcgccacca ccgccaacta
cgaccgggac aagctcttcg gcttctgccc 3180gacccgagct gactcgacgg tgatgggggg
caactcggcg ggggagctgt gcgtcttccc 3240cttcactttc ctgggtaagg agtactcgac
ctgtaccagc gagggccgcg gagatgggcg 3300cctctggtgc gctaccacct cgaactttga
cagcgacaag aagtggggct tctgcccgga 3360ccaaggatac agtttgttcc tcgtggcggc
gcatgagttc ggccacgcgc tgggcttaga 3420tcattcctca gtgccggagg cgctcatgta
ccctatgtac cgcttcactg aggggccccc 3480cttgcataag gacgacgtga atggcatccg
gcacctctat ggtcctcgcc ctgaacctga 3540gccacggcct ccaaccacca ccacaccgca
gcccacggct cccccgacgg tctgccccac 3600cggacccccc actgtccacc cctcagagcg
ccccacagct ggccccacag gtcccccctc 3660agctggcccc acaggtcccc ccactgctgg
cccttctacg gccactactg tgcctttgag 3720tccggtggac gatgcctgca acgtgaacat
cttcgacgcc atcgcggaga ttgggaacca 3780gctgtatttg ttcaaggatg ggaagtactg
gcgattctct gagggcaggg ggagccggcc 3840gcagggcccc ttccttatcg ccgacaagtg
gcccgcgctg ccccgcaagc tggactcggt 3900ctttgaggag ccgctctcca agaagctttt
cttcttctct gggcgccagg tgtgggtgta 3960cacaggcgcg tcggtgctgg gcccgaggcg
tctggacaag ctgggcctgg gagccgacgt 4020ggcccaggtg accggggccc tccggagtgg
cagggggaag atgctgctgt tcagcgggcg 4080gcgcctctgg aggttcgacg tgaaggcgca
gatggtggat ccccggagcg ccagcgaggt 4140ggaccggatg ttccccgggg tgcctttgga
cacgcacgac gtcttccagt accgagagaa 4200agcctatttc tgccaggacc gcttctactg
gcgcgtgagt tcccggagtg agttgaacca 4260ggtggaccaa gtgggctacg tgacctatga
catcctgcag tgccctgagg actagggctc 4320ccgtcctgct ttgcagtgcc atgtaaatcc
ccactgggac caaccctggg gaaggagcca 4380gtttgccgga tacaaactgg tattctgttc
tggaggaaag ggaggagtgg aggtgggctg 4440ggccctctct tctcaccttt gttttttgtt
ggagtgtttc taataaactt ggattctcta 4500accttt
45065501PRTHomo sapiensaldehyde
dehydrogenase 1 family, member A1 (ALDH1A1) 5Met Ser Ser Ser Gly Thr
Pro Asp Leu Pro Val Leu Leu Thr Asp Leu1 5
10 15Lys Ile Gln Tyr Thr Lys Ile Phe Ile Asn Asn Glu
Trp His Asp Ser 20 25 30Val
Ser Gly Lys Lys Phe Pro Val Phe Asn Pro Ala Thr Glu Glu Glu 35
40 45Leu Cys Gln Val Glu Glu Gly Asp Lys
Glu Asp Val Asp Lys Ala Val 50 55
60Lys Ala Ala Arg Gln Ala Phe Gln Ile Gly Ser Pro Trp Arg Thr Met65
70 75 80Asp Ala Ser Glu Arg
Gly Arg Leu Leu Tyr Lys Leu Ala Asp Leu Ile 85
90 95Glu Arg Asp Arg Leu Leu Leu Ala Thr Met Glu
Ser Met Asn Gly Gly 100 105
110Lys Leu Tyr Ser Asn Ala Tyr Leu Asn Asp Leu Ala Gly Cys Ile Lys
115 120 125Thr Leu Arg Tyr Cys Ala Gly
Trp Ala Asp Lys Ile Gln Gly Arg Thr 130 135
140Ile Pro Ile Asp Gly Asn Phe Phe Thr Tyr Thr Arg His Glu Pro
Ile145 150 155 160Gly Val
Cys Gly Gln Ile Ile Pro Trp Asn Phe Pro Leu Val Met Leu
165 170 175Ile Trp Lys Ile Gly Pro Ala
Leu Ser Cys Gly Asn Thr Val Val Val 180 185
190Lys Pro Ala Glu Gln Thr Pro Leu Thr Ala Leu His Val Ala
Ser Leu 195 200 205Ile Lys Glu Ala
Gly Phe Pro Pro Gly Val Val Asn Ile Val Pro Gly 210
215 220Tyr Gly Pro Thr Ala Gly Ala Ala Ile Ser Ser His
Met Asp Ile Asp225 230 235
240Lys Val Ala Phe Thr Gly Ser Thr Glu Val Gly Lys Leu Ile Lys Glu
245 250 255Ala Ala Gly Lys Ser
Asn Leu Lys Arg Val Thr Leu Glu Leu Gly Gly 260
265 270Lys Ser Pro Cys Ile Val Leu Ala Asp Ala Asp Leu
Asp Asn Ala Val 275 280 285Glu Phe
Ala His His Gly Val Phe Tyr His Gln Gly Gln Cys Cys Ile 290
295 300Ala Ala Ser Arg Ile Phe Val Glu Glu Ser Ile
Tyr Asp Glu Phe Val305 310 315
320Arg Arg Ser Val Glu Arg Ala Lys Lys Tyr Ile Leu Gly Asn Pro Leu
325 330 335Thr Pro Gly Val
Thr Gln Gly Pro Gln Ile Asp Lys Glu Gln Tyr Asp 340
345 350Lys Ile Leu Asp Leu Ile Glu Ser Gly Lys Lys
Glu Gly Ala Lys Leu 355 360 365Glu
Cys Gly Gly Gly Pro Trp Gly Asn Lys Gly Tyr Phe Val Gln Pro 370
375 380Thr Val Phe Ser Asn Val Thr Asp Glu Met
Arg Ile Ala Lys Glu Glu385 390 395
400Ile Phe Gly Pro Val Gln Gln Ile Met Lys Phe Lys Ser Leu Asp
Asp 405 410 415Val Ile Lys
Arg Ala Asn Asn Thr Phe Tyr Gly Leu Ser Ala Gly Val 420
425 430Phe Thr Lys Asp Ile Asp Lys Ala Ile Thr
Ile Ser Ser Ala Leu Gln 435 440
445Ala Gly Thr Val Trp Val Asn Cys Tyr Gly Val Val Ser Ala Gln Cys 450
455 460Pro Phe Gly Gly Phe Lys Met Ser
Gly Asn Gly Arg Glu Leu Gly Glu465 470
475 480Tyr Gly Phe His Glu Tyr Thr Glu Val Lys Thr Val
Thr Val Lys Ile 485 490
495Ser Gln Lys Asn Ser 500655461DNAHomo sapiensaldehyde
dehydrogenase 1 family, member A1 (ALDH1A1) genomic DNA 6taagaagtga
gatgacaagc caagtatgtt atgaagcctt gagctttcat cgcctggaca 60tcaaataaac
cagtatttga atccacaaac aactgtgact ctgggagaag aaagcagcaa 120aacacagctg
tttgggcatg gccagagcgc cactctcaag ttatgtaaca gctgttgtgt 180aattgtgcat
ttaactcaaa acacattttt gagagagaaa acaattagac tttccaaaaa 240gaaagaaaca
gagaaatgtt gaaagaacac agaagtttct atctggtcat caaacataag 300acacggggga
gtcaaaggca ttgggaatga tattgcctta gaatctaggg agttggcccg 360tgcaccaaat
ctggcctgcc ttctgtcttt ttaaataaag ttctatagga aaacaactac 420acccattaat
ttacctatca tctacagttt cttttaaata gagtcaagta aattatatca 480gaaaccatcc
agcccagagg ctgaaaatat ttacagaaaa agttttgcaa attcttgtct 540tagatgaata
aaaagttatg tttaaatgcc tgtaagggtg actattcact gagaaaacca 600aagcagttga
tgcttgaacc catgtaggag ttctcttgtg gagaataggg tagaaatagt 660aaacagaaat
aagagtccag gtttgggagt cagactggcc tgaaatgaaa ttctggtcct 720gcactaatag
atctgtgacc ataagtaagc tactcaccct ctgtatgtct ctaaaatggg 780gatatgaaat
gagggtagaa agggaaacaa gtgctgcacc tgatttgggc aaacaattca 840gaaattttta
gtttccctat gtaagtccca tattcagggg aattggcaag tttcactaga 900aaaaaaaaaa
ttggttgatt ctccacaatc agagcatcca gagtatttta tcttgttcct 960attgtaacgt
ttgctagagc tacaatacaa taaagtattg tacttaaaat gggagtaaca 1020ctgtttaaaa
aattgtcctt cagctaaaca ttaatttaag aacttgaatt gtttggaagc 1080cctgcttaaa
tttagtcttt gtacactgcc tatgttgata aatacagaca acttccaaaa 1140ccaggattac
tttcatttta aatgagtatt aatagatgat attgccatat ttctaattgt 1200ggtgattgtg
tgtgacagtg tgttccgaat tccctaaaag tcctgctggc ttttctgttc 1260acatatagaa
aataaagata atttagggct tctgagatca cagtaggtct acttacccag 1320cactgaaaat
acacaagact gatacgatat tttaaaacta acttagggta gggtgtagat 1380aaagggcctt
tcttccccaa acagcacctt gattttctgg gagatggact gatttcctga 1440aagccttgtc
ctgaagacac ctggccaggg ttctctcctc accagcttct actgagaaca 1500agtgcccttt
tagactcttt tcaatcctca aattctctga ttccaagtct gtcagagaac 1560agaaagttac
atagtagcat taaaaagcat gagaagtcaa aaaaataata actggcctta 1620gtggccagag
cagctgctgc atacacttat cacaggtttc ggctttgtaa attaattcat 1680ctgcaaatag
tgcactgtct ccaggtacaa attcgatgct ggagcactgg tttcttaagg 1740atttaagttt
aaagtcaaag gcttcctgcc ctaggtgtta caaataagta gtgtcgtttt 1800ctttttttgc
tctgagtttg ttcatccaat cgtatccgag tatgcaaata aactttagcc 1860cgtgcagata
aaaaaggaac aaataaagcc aagtgctcta tcagaaccaa attgctgagc 1920cagtcacctg
tgttccagga gccgaatcag aaatgtcatc ctcaggcacg ccagacttac 1980ctgtcctact
caccgatttg aagattcaat atactaaggt gagtaaaact tctattttct 2040gctttgactc
gggtttgcaa aaactgcatt tatgtaaagc attaaaggtc aatttaagta 2100aacttgtgta
aagagccctt tgcaaatata aaaataaaag gcatgcaaaa tgcaggtctc 2160agtttagtgt
cctgatcaaa tatgatctaa atcatatcag acaatctttc ataaatttat 2220ttcttaaaat
atttccaagc gaaaggaatt ctcttctgtg tgaaaggatt cttataccag 2280gattcagagg
actttactaa gcacttctat taacttcact atagccggca tgtatgataa 2340gagagcaaaa
tactcactag agaagcttac tggaaagaac caaacgagta ggaacaatag 2400aaagcaaaca
aggagaaaga gagcatactg aaataacata actttccaat atgtttaata 2460aaatgagtgt
taaaataggg ggacttaagt ctgataattg gattcgaaaa tcttcagatg 2520gacttgcatt
attttgcata ttttggtggg aggaagaaaa gcattatttc ttcagccaag 2580tttgttgcca
ttggagcaga tgtgtaattg ttaactgata aatatttaga gaaacaagag 2640caggagctag
atccagactt taaaatccgc agctgggggg aactaggaaa tggatcctgc 2700ttgagtcgtt
ctgagagtaa ctctgaactt tgcctgtttc acactgcttt cctgtcagat 2760cctagaactc
caaaacaaac aaatgaataa aacacaatct aaccaaaatt acttgaagtt 2820atctttatgg
tgtgatgcag aaagtatggt aagaaattaa ctcttgcaaa tcaagagaga 2880gccattttgc
tgtctgaatt agccaacagg aaaaacaaag ggcattataa tttaatgata 2940aagtaaaagt
ccccactgtt atatatttat ttcctcactt gtaaccatgg atggctttga 3000aatcccaagg
tctgttgcat tttcattttc ataaagcaca acaaatgagg aagttgctca 3060gctgattggc
tcttttcctc aggaaagtag cagcagagat cagattgtgc ctttaacatg 3120gttgaccagc
agctatgaag tgtattttaa ggtattgtta tcacgaggtt ttgttaaaga 3180ggaaagattt
tttccattta gttttactac aaatggtagc atgaaaaatg cagtaaatcc 3240ataccgtcaa
acaatttaca tttggcaaag gtatgtccaa aaggttgcaa gagtccagtc 3300tccctcattt
tcttattcca agtttgtcat tcacaagaaa tttcaattga aagtgggtac 3360atttaaaaat
acatttcatg aagctcaaaa ttgtcgacaa tgctcagatt attgtattac 3420tattattaat
gggatttttt ttttaataat actacctagg aagatatttt ggtagtctgg 3480atgcccctgt
ctccccactc ctttggcttt gcagaagttt gcaaagtctt ccttgtgtgt 3540gtatttcaag
gaagctctaa tgtaaataac cttcctgcta aattaagctt cacttagtag 3600tcattaacta
agcatgtaag aagcggtact agacaaatga gatggtccta aaaatctaac 3660atacaagcac
agacctaatg aacccttaac acagagagat gattttgaga ttgaaaccta 3720ttcacttcga
agtcactgtg caacaactcc cctctaggga aagtgactga cctaagtgtc 3780caaaagaagg
caagactcaa agagcaggcg ttttgctctg tgtttaagaa cttttctggg 3840taaaaatact
ccaaacaagt cagtgaattt tgatttctcg tgcatcttct atataaatct 3900gttctgctca
gatagcattt tattgtttaa tttattggtt acagcgtttt cacacttggg 3960aggacgccag
ttatgtatgt aattaaatct aaatgggtga actttttcaa atgccttcgc 4020attttttgtt
tactatgtcc cagtgaagaa ttggattgaa tttgctcttg caagaggagt 4080taaaaaaaaa
atgatggtta ttaactataa aaagttccct ggttacaaac caagtaagca 4140aggagtttat
agtgtctgag gaattatctt ctattaccag cccctaaagt cttgtgttat 4200ttttctgtta
ctattctaat gtcctttttt attattatta tagctagaag tatattggtt 4260gttaggacaa
gttggagaga acagacaggg ttgagtaaaa aaaaaaatat tacaactacg 4320atagaaaagc
tacatactct caagcttggg actgcttttc acccaaacaa aagaatttat 4380tgatgcatat
gtttttatga cttattttct agtacagtct gattatttct ttgttggggt 4440agattaaaga
gagatcagag tttaagaaat gctttctaaa attatacaaa agcttttgga 4500atttaaaacc
tcaaaaacat cgctataaat tcactaatat gaactcatat cccatcctta 4560tcctcattac
tcctctttcc aactctaaat gaaccccaca ttttccattt tattgagcct 4620ttaaatgaag
agattctttg aaagttatgt gatcattggg caagctacat tacttctctc 4680tgcctcggtt
ttcttatgtt taaaatgggg atgataaagg cacgtatttt ctaaggtcga 4740tgaaagtatt
aaacgaactt aatgcgtaca aagcccttga aataatgcct ggacatggga 4800aatgatttaa
cgttagtgat cctagtccta tttttctgaa tcctttccag tttttccaga 4860attttccctt
ttacccaacc tttcctagac ctgtttccta caagtatgcc tcacgtcttc 4920tcctagcttt
tctgtcactc ttttttgtac agtcatgtcc catctgacaa agattgcatc 4980agagtgaagt
tccaagggtc ttccaagatt tcagggatca aaaattttct cctacaggat 5040tcggtaactt
tgtttgccat tctctcttca caatactgtt gttttacctt ttcttttttc 5100tttttttttt
tttataaaag ggaaatagga aattcaagaa gacaagcatt ctcagacaga 5160attcaaacgc
caaaatgttt aacttaatta gtaagcttca cttattaaac catggtttgt 5220tcatgctaca
aaagaccttc atcagctaat gacattttac tcatacatac acacacacac 5280acacacacac
gcacacacac acacaataaa taattatagg taaaatttat tgtgcttact 5340ctgccatgac
tgtttaagtt cttgacaatg attttattta ctcctcataa taaccctatg 5400aggaggttac
tattcctgtt tcacagttca gggaactgag gaacagagcg gctatgtaac 5460ttactaaagt
tacacagcaa gtaagtgaca aagctacact gccaatgctg gcagtttgag 5520ctaacacact
agcaagagac ttgtctaagg tcaattccac tactctggga cactgacttg 5580ccagaatcct
cttgtatcct tcttcatcct gactttcatt tgcccattgc tgattaccat 5640ggtctttcca
gctgctgctc agctgttccc aggccaataa gagaaagttt gcagccaact 5700tagaaaaaga
gaacaatagg gaaattgaaa ggaaataggt tttctgcttt gactgaaaat 5760gtgacgtttg
ggctaggaga ggctatgagg cagaggactt tgacatggat gtgaaatgtc 5820tgtgtgaact
acatttcttc aactcctatg gaatcgattt tggttagagt aggagattgc 5880tttcctagat
ttctggtttt gtttaaccct tctgggttaa agagatattt tcaatggaga 5940agagttatga
atcattgaat aatacgtccc aggcacgatt tgaagtgctt cttttccccc 6000atgattatct
ctttaaattc ttacaacaaa caaattcagc tacatgaact tccaaatacc 6060tcctttgtcc
cctttgcttg tttctccctt ttgcttttcg tttctcccat atttcccttc 6120ctcttattta
acaaggcctt gaatgagtgc cctatgctag ttctcatgat ataagagggc 6180taatatgatt
cacgtctcat gcagcctctg ttcctggaag gtgtgtggaa tagtgtgggg 6240cagaaaataa
gagaatgctt taatcagaaa ttaggataaa tgtttaaagc aaaataactg 6300aaagctgaga
gaatatgagt agaagagctt ttgatgagga tttttgggaa ggtgctttaa 6360aggtaagcct
gagggatgta gagaataagg tggccaagag tagggaagaa caaagatcca 6420gaggtcttac
cacatgcaaa gaccccgagg acaagagcat gttatgctca aagaactatc 6480caaaggctaa
tctggctgga gtaaagtgaa taagggagag aatggatgag atgaaactga 6540agtacaggct
ggggccagat cggctggcgg attttgagga agatcactga gaggtgctta 6600aaacaggaaa
ggacttatgt tcaacgcaaa gctgattgtg ctgtgatgct gctttcactt 6660gggaaatgtt
tttaacttcc ttccaactga agcaatattt tgtttgcttt gggcttggaa 6720gcaggtgaaa
aaaatagctg actggctgca aggtgcagtg actgaacaag ctaaacctgg 6780cttcaatttc
cagctattct atgtatttaa ttatatgacc ttggccaagc ttctcatccc 6840ctggagcttc
agttcctctg tctgtgaatt gggaatgaga atgactacct tggaaagtgg 6900ctgtgagaat
taagtgaggt aatttgtgga atgtggctgg cacttattaa ctacttagtc 6960ctttctctcc
ttgattctct ttcccaagat gactcctaga attatatgat cattctgttc 7020tcaatgtaag
ttatatttat attgcttatc acagctcaaa actgatttca tatatttctc 7080tcatttgagg
tgcatcatta accactgaga ggagctagaa aagatgttag cagagtccca 7140tttcaaagct
gaggagcccg agctcagaga tcatgactga cttaaggtca catagctctt 7200aaaatgcaaa
agtagaataa actggattct aaatcttttt atcttggatc tgatgctttc 7260ccacatgata
tccttttttc cttaaatatc aaaaccagta gtactagaat ctcactcaag 7320aaaacctaat
caagaaaaaa atctgaagta aataacacat atcctaagag aagagggtat 7380tatccccctt
tcccaattag ctctaacaac tgttttcaca cctattactg catttttatc 7440attcattcct
tactgtggtt gaaagaatag atctaggctg aagtctgaga ttctgtgttc 7500ttaacagtct
ttcagctttt gctgatgcta ccaaaatgtc tgttgaccag cagcaagggt 7560ctaaaagttt
taggtgagtg agccttataa cttcaatgca gcaaccacct tccagttact 7620tcacaataga
gcttccagtt tcttccagaa actagcactc agccacatca gaaactttcc 7680tttcagaaag
tgccagagct aagcagaaac atgatgatgg ctacaaacca gtgacagaac 7740caatcattga
ttttaatagt gggagataat catttactaa tatttagaat gctcatctct 7800gaggactgct
gtcttggcaa gttgttttgt tttctggcac ctgcctgact gtaagaaaat 7860ggaattaaca
attctaaagt aaagaacaca tgtcctaaga gagggtattg tccccatttc 7920ataagggctc
tcagtacaac tctttatata actttgtaac agggacttta acctattgaa 7980tttgtcacta
catacttgcc agaatgaggg aaacactctt agagaatatg aaaaataact 8040catttgaaga
ccaacccaaa gtaaaggtaa tgactctgaa atggcatgga taatctatag 8100gatgatatgt
tgcaaatcag cataaaataa atctcacaga aatacagaca tcaaacagag 8160atatctgaaa
atagcaatta tctggcttgg gatggtggtg tgggggggtg cactttggtt 8220tgatttatca
tgatgctatt ataactatag ccaggggata tattatatgt ttacaaagtg 8280taagtgacaa
catagaacca tgttcaaaag aatttgagca aggattgctt cacaaaccct 8340tacttagaat
ccaataaatc agaaaagtaa atctgtgtaa tactggtttt cctgacgctg 8400acatgttttg
ggtgtataga tgtatatagc aaaagtatcc acaaacttag aagcattgct 8460tgagaaggat
gtgaagtgtt gctaatattg ggttttctga tattatctct acctgattca 8520ttgtaacgtt
tgacttacta atggaaattt gttcactggg gaatttgcac aaatctcaca 8580tctctaaaaa
ctttgcctga accgttccct cccccaaccc tggtcccatt tccactgact 8640atttccactg
ctctatcgat acttttttag atgtcaaagg aatacaaata actactgaat 8700tatgctttat
atttggcatt cttttagaag gaagagggct tacatgcgag caattctatt 8760actaggtctt
gatactgcta aaaatgggca gaactttcta tcgcttctac atcttaactc 8820tgatacagaa
aatatatgat gtacagaagt tgctgacaag ttttgttatc atctctagga 8880cacagtgatg
gagtggctca aaacaaatgc tgtggcatct tttctctttc cctgacacaa 8940ttgaaaaggc
tgatggggtt ttgcaaacaa caagaaaaaa gagtatatac tcacgttact 9000aaagtcagac
tatcatttaa agcaaatgaa aggaaaattt tatttctcct attttttgtt 9060ttcgtcattc
tgtcattttg ttttcattca tttgatgaca gttaaaaatt ttctttccct 9120ttgttatttc
ctccttaatt tccttttaca ttttctaact ttgagtctca acattttgct 9180ttcctgcctc
agggttaggg tgcaaagcag ttgccagaga tccagctttg attcttttaa 9240atgtttaaca
ccccacctga cttttgttca gtctctcctc ctcattattc atctccgatc 9300tcatactccc
tgactcccag cggcctttag tactttggta ggttcctaag ctgagaaaag 9360gttgacacat
ttacagcaca caccagtctc cacaggttca caccaccacc catgcagcct 9420atgctgtgac
tcagttctca agtttgaaaa ttcacatgga tgacattaaa tggggactct 9480catcaccctc
cctctcgttt ggaaaatctt aaaagaaatt ctttgtgcat tgaaaatgtg 9540atctcttaaa
atctctctcc caccaagaaa gtattgcctt cctttacaga ctctagggct 9600gctggcccag
ttgctgccct gccgagataa tattaattta aggcactctg agtatctttt 9660gcaaggagtc
aggggctccc cactttagcc cacagactcc aaaatctcat atcagcagat 9720aaggcaaata
gggctttatt ttgccactag tcaaaactag ctttatccaa gccagaaata 9780ttttgagaaa
attatgctta tttatttaga actgtagatt tatacacaat acattaagac 9840ataataactt
ttactctgga aaggcaattt ttttcacaat tatgactaat tcttggacac 9900cagttgaatc
caggaaatgg ctacttgttt atcaaaggta aaatgatgca tttcttaaaa 9960tgtggcatat
ctaaaatctg aagtgtaaaa atccatttct aagttgcctc aacatttcta 10020ttgattgctt
ccccatatag aaagtacttg aagtccaaga gtatgcatag gcatttataa 10080tttccaatca
atatttcaaa cagaaggttg atgtctactt tacatatata ttattcaaaa 10140gtcacctaag
tccaaaattc ataattagaa ttgagttaat atataatggt tacatagaac 10200ccaagaatat
tttactttta tagtatcatc tcttttcctc taagctggct aaaggcaaaa 10260aacaaatgga
aaataatatt gtcaccatta tagaacatta aacaagccta atgtaaattt 10320tataattatt
ataacaaaca ttcattagcc tagacttctc catataacca ttaatataac 10380tataggaaca
gtgattatac aaaaacgttt ggtcacacag ttagctggaa ggcaaatgcc 10440caatgtaaag
ctcctataat taaaacagaa aaaaaatgta tttgaggtag gccctatgaa 10500taaattatct
gtagctaagg ggaagaaaaa gactgggcaa tacctggcct tctaaccatc 10560taggtcatca
cattacctat gccaaattcc cttttcatgc tgaaacgcct ctgtttgtgg 10620gagaatggca
taattcataa tttgtagact gcactgttac atcactctcc ttcaaaagcc 10680tgtgacctta
tgataggatt atgaaattag tctcaaggct aatttcttgg actctggttg 10740gtctacttca
aatctacttg ggatacttta aaatcagtat tcctaaacat ccatttgatc 10800tggtctctct
ccctttttat atccagaata gagcctaaac ttctttctct ggggatcagg 10860atctgccaca
aattgacttt attttcaatg tcaggcctta tctgtgactt atgtccactg 10920caactccatt
ctgttgggag ctgctctgtt tactgtgtta caaacctcca aagatttgtc 10980cctcccttta
ctcttttttt ttccttcttt ccaccaatcc agttccatcc ttcaaagaag 11040cattctttca
gcctatgccc ctctacacca tttattgaca acaccgctgc tttggccatt 11100ttgctccaca
gtctttaatt ctgtactggc ggctcttgat atgaatacat tctcctctta 11160gattataagc
ccctgagagc agaggccatg gctaactcat ctctcatttt tccacaataa 11220ctgccagact
atgtctcaag tatttactca ttattggaca aacacttttc tgataaagca 11280aaagttacta
atcatgatta tataaatgtc aacacaagtt caaacacaag ttgggctcac 11340agtgtcctcc
tgtcttaaag gttaaaactg aataatgcag aacagtgctg gtaatttggg 11400gtcctctaca
gctgatctct tcagttgcct gcaggctgcc agataaacat gtgttattac 11460ttcaggttaa
tgctcattta caaaacattc actggcttta atctaaagtg gcagttttat 11520gtacagctta
gatgcaaatt ctgatcatgg aactagatgg tacatacata ctgtaataat 11580tcttttacaa
aaaagtgtgt aaagagagta aaataagcta taatatattg agagcatatt 11640attgagaact
tgaaaccaca gattatcaca gacctaagga taagaggact cgggcgagaa 11700ctagaaagtc
cgaccacagg cctagcaaaa tgtaattgat tctgtatgtt gttttatttt 11760atttttaatg
aaattacatt gtagaagggt gaaattctgg atctctgcca aaatgaaatc 11820tgtgttccct
tagtttacag gtacaaaatc taaggtggta atacaatctt taggcagaga 11880gaaggcagaa
ttcccaagtg aataaatctg atgcaggttt tgaaagactg atttaaaagt 11940ctacggcagg
tatcgcaata tctttagttt tttgtttgga attttatggg ctgatatgta 12000gatatctaac
attaacacaa agcagaacat aagtaattta tcagtaggat actaacatgt 12060tatgttggaa
agaagaaaaa agagattaaa ttcaattgga gcaactagtc aaatcttcat 12120gaggagatga
cgcagtaagc tagaatttga agtaaaaata gaagaaaata gtggaactct 12180agtctaaaga
acagtgtgaa taaaagcatg ctgcaaaaaa cagcatggtg tttgaggcag 12240tagaatgagg
ctggaacaaa gggtaaactg agggaatggt agagcaaaag actggaaaag 12300catgttggag
caagatcgtg aaccacctcc agtgccatgt taagcagtct gacaatagag 12360gatcactaaa
gtattctgag gagggctgta gacagaatct gaggaagatt atgatgacaa 12420ggtgaaaatt
aaattttaat taggcagagg tagggggcaa agagaacagg gaaagctttt 12480ttatacttgt
atagaacaaa gtgatacaaa agggtaccta gggattattt gtgtcattaa 12540cagaagaagg
aaagtgtgtg agagataggg ctgggggaca gtgtgcatga gcaagcttca 12600gaactttggg
gtgagagcag cataaagcta gagatgacag tgagctgagt tcgggacaag 12660ttgattaagt
tctttcccag ccatcccttg agaagaaaaa tactaagctg taagggcata 12720aatggaaaca
ctctaagaat aattatggga ttctgtaaaa tgtatttctg atagctagag 12780tgttgattca
ctctaagaga cattgggcaa atcagcactt tcgtctgaat ttatttttcc 12840tcaagttgat
ctaagagcct aatgtttttt cccaaaagat aatacaatga attttatggt 12900cttcatctgt
attcagttaa tcttgtttct gtaatctgat agggtttctt gatacttctt 12960ccatctttct
catttcccca tatatgtatt tttgcacatc tagtctcttc atctgaacag 13020ctttgggccc
cttttccctt tatttaggca tgcccatgtg ttttaccatc aatttcaggc 13080tagtactaca
attcctttga atgcttctaa ctatacagga taattttctt gtatggaaat 13140aatgctgttt
tcttgtatgg aaaattatac tggcatgaat gggcaaacaa gacggcttat 13200cctcttcccc
attttgtagg aaatcacaag tgtcatagag tattcatttg cagaacgtta 13260tagatcttat
ccatgagtag ctgatggatt atagatcttt gattcaatta agttagttat 13320tcaagaattt
tttactgagg ctaaaattgc ttgagggctg ttggacactg aagatagact 13380ggaaaaccat
acacagccct tgatggaagg cagacactta aaaatgacta tttaaaaagt 13440gacaaattgt
ccaatggaag tatagaggag accagggaag tatattttgt ctaaataata 13500aatcacagaa
tatagaatga attatattca ggcatttagg gatatatagt aatatacatt 13560ttattactct
gatagaattt taagctatgc tagtgatcac cggggaattt ttaataaata 13620agtaatttgc
atttagcata gtagctaatt taaaccttgg aggagtgttt cattacattc 13680tccctcgggg
ttgaactatt aataacttat ctttagatat ccagtaagaa gggatgtctg 13740aattaggata
tatgttataa tccagcacat atgtcacctt cctttactcc ctgtgttagc 13800tacctctgat
tcaaaataga ataaactagt ccatcttgtc ataagtctaa cagaaagtta 13860tatactatcc
aagactgtat ttatgattgt tttcatattt caaataatta gaaatgtgaa 13920agctggctcc
tcaagaaaag acaaattcag tgacatgaaa gttgaaactg agatgtttta 13980aatgacatac
tgatgctttt aagtatttct attttatagt acacacagac aaaatttcat 14040attttttagt
gcttactgtt tctttagttg catgtctatt tctcatagtt accattaaga 14100aatcttgttc
ctgattttgt ctgaagacaa cgaaattacc aggtaatctt ttgaaaagga 14160gaataaaacc
atatgctttt aaaaagccaa tgaaacaatg tgactagcta attttctcta 14220agtgaactcc
ttttagaatc ctttagcaaa attctttttt taactttaaa acattaaagg 14280attagaaaat
agtctcttat gattccaaat caccagtagc cgaaaatgag gatgttcaga 14340atttaaagtg
gaaaatagaa gcaatcatta ataaagacca agcccacaaa agcctttgct 14400ttgcctcaaa
ttcatggcca tttcaggtaa aagcattgtt ttctagattg ttttctccag 14460tatgtatttt
ctagtgttga actgattgca taccttaaac ttaaaagcat tatgtacttc 14520attttagtac
atgtctatgc atgtgaacac aaaatgtctc aaacagccag aaagttttga 14580gaggtgaaga
gatgtgcatc atttaggcat ggtgaaatgc tttagtttaa gtttatgaaa 14640acttccaaaa
ttttctgcat atttaagcgc cttgtatgta ttttctcttc atctctacag 14700atcttcataa
acaatgaatg gcatgattca gtgagtggca agaaatttcc tgtctttaat 14760cctgcaactg
aggaggagct ctgccaggta gaagaaggag ataaggtgag tttctgaaca 14820ctagtttcat
tttatgccag gtttcttggt tttttgccat tctgagctcc tgaaccccat 14880tgcaagctcc
aaaagacatg ccatgaaaat atggtttctg gggcagctta ggaaaattgt 14940ctaagttgtc
cttgttacca aaaaaaaaaa aaatgctctg tagttatgta ataatgataa 15000acctgtgctt
ctgggtgtca tggtgatttt ttttcacatc attttccttt attttcaatt 15060gaaatagtat
atagatttat tttacagact atataagaag ggattgatca taggttataa 15120agtagagaaa
tcttcagctg ggcgctgtgg ctcacacctg taatcccagc actttgtgag 15180accaaggcag
ttggatcact tgaggtcacg agttcgagac cagcctggcc aacatgggga 15240aacaccgtct
ctactaaaaa tacaaaaatt agccaggcat ggtggcacat gcctctaatc 15300ccagctactc
aggaggctga ggcaggagaa ttgctttagc ccgtgaggca gaggttgcag 15360tgagccgaga
tcataccact gcactccagc caaaaaaaaa aagaaaagga aaaaggagag 15420aaatgcttta
ttcatatgaa attgttcttt ttatgttgct agataaaact aaaagttatt 15480atagaacttt
tagaacaata tattactaag ttattttatt aaaaatcact gcattaatat 15540tattattcaa
cacctgtaaa aattatatta tagaataact taaattgact aacaaattac 15600aagcgttgtt
attttccagc taataataga aacaacaaaa accagttttt atttagtgaa 15660aattgttctc
aagttctaaa tgctatcttc atttaaaact taaagcaacc ctatagggca 15720ggtactattt
tgagtcattt ttacaaataa tgatgagatg gcagagtaga aaggctgagt 15780aatttactca
aggtgacatt gctaataagt aataacctca acattaaaat gctgttcctc 15840tgacactata
acctgtgcct agtagggcta ctgcttcact ttgtgattat tatgaaagta 15900agatagggtt
ggccgggcgc agtggctctc acctgtaatc ccagcacttt gggaggccga 15960ggcgggcaga
tgatctgagg tcaggagttt gagaccaacc tgaccaacat ggtgaaaccc 16020cacctctact
aaaaatacaa aaattagctg ggcatggtgg tgggttcctg taatcccagc 16080tactggggag
gctgacgcag gataattgct tgaacccggg ggtcagaggt tgcagtgagc 16140caagatagtg
ccattgcact ccagcctggg caacaagagt gaagctccat taaaaaaaaa 16200aaattaaata
gtctttctta ttatgaaagt aagatatccc agcactttgg gaggctgagg 16260cgggcagatc
acaaggtcaa gagattgaga ccattctggc caacatggtg aaacctcgtc 16320tctactgaaa
atacaaaaat tagctgggca tggtggcgca tgcctgtaat ttcagctact 16380cgggaggctg
aggtaggaga atcacttgaa cccgggaggc agagcttgca gtgagccgag 16440atcgcatcat
tgcagtccag ctgggtgaca agagcgaaac tccgtctcaa aaaataaaaa 16500ataaaaaatt
agatagcata atattaaata aactttttat tctcctcctg ggtgaggaga 16560gtaaaaaact
tcagaatatt tttctgaaca cgtaagtgtt tctaagtcta atttgaaatg 16620atttttctgc
tctttagttg cccaggggtt gccaagtttt aaatttctgt aaagaagtaa 16680tgttttattt
tattttatta ttattattat tgctgcagga aaagactact gggaaattaa 16740agtcagcaac
acccaaggat aatattattc atgacaatgt gttttagtta attatagcga 16800tcacatgttc
ctgccagtct cctgcccagt cttttccatt actttagccc cagtaaatga 16860gatcacaaat
ctcaacgtac tgggccatgc agatacttat tccaagatag tgacacacct 16920gagggggccc
ttttagattt aggagaggcc cttggttcag atttcagtac gatgaaaaca 16980agtaagaccg
aagtctcaag ccacaggagt acggagttca accatagggg ttaacaattt 17040gagaagtaga
aacaaaataa aatgatgaca aatgtcactg gctttcagat tcagatttac 17100tctttatact
gaagagttca ccaaagcata aattctccat tctgcttctc actgtctgtg 17160ggaaaaagtg
cacaaacaaa ggtcctttat aatattactc aacctttgtt tatggcctct 17220ttcccaggcc
accgcattaa cttaagagtt tcaacttcgt catgcccagt gtccatcccc 17280aacaattgct
tgctagtcac tgcctctgta catgtccaca gtgtagtcct caggacctag 17340ttcaagtcac
catctatagt aagagcgtag attttcaatg tttgtactta accttctttg 17400agattctgtt
ctttttcatg taaaatgagg agatagaaaa tgcttgagga gttaaatata 17460tagagctctt
caacggtgcc aggcatatcc taaagcttca gtcatgcccg ggttttatac 17520ttatttaaag
ccttctctaa ccttctcaag cagacttact ctcttctgtg tggttatagg 17580acattgtatt
tgcctgtatt ataacctttg tattatacag ctataggtta atttgtctga 17640gacaatatgc
ttcttgcaga gatcaacact gcatctctca tttatatcct tggtttctgg 17700atatagtata
tcctataagt atgctatact atattcgata gatatactat actatagata 17760tagtatattc
tataaatcta tgaagtagtc ttctttttca tcaagtgttg aatttccata 17820cattccacat
tcctcacctt aggaatttag tactaacctg actattggct atggcagaca 17880ttctcaaaat
ttagcctgtc tcagaatgtc caggaaggct tgttaaaaca cagatagctg 17940agccccacct
tcccctagat ttcactgcaa taatcttaga atgggacctg aaaattttca 18000ttttttatca
attctttggt aatgctgatg ttgttggttg ggaaggaggt gagggaatgc 18060atcttgaacc
accgaactaa agtgttaagc tgttaagttg tatctcctct caagcgttaa 18120catttaacag
gggtcaaaga gttttgacac ctaagaaagt tagtgattga aaactgtact 18180gtggtgatca
ctctgcacct ctctgaaatg ctaccaaacc aaaattgggc atagaaatac 18240tgatcatccc
tggcaaggtc tataggagcc ccagaagcct gtgccctata agccattccc 18300agaaataaga
gagaaattta ttcttcacct ttcaaagaag aatgaacgcc ttttatcaga 18360gacttcagga
gatccacaca ccaccacctc aaatgttcct ctagttgctt gagtggctct 18420agcaggcctg
ggaaggtgag aaaagcaata aagggcaagg acagagtgag cccttgagag 18480ctacatactt
tctctctcca ttcaagagga aggccagatc tgtttgcagt ttgcaactaa 18540caaacagaaa
aatattattg atgttttaat tctggagcct tgaacatcga cctttgccac 18600tgggattaat
tttattagga aaacaaatct ctatgcccaa agttccagtt gaaaccttgt 18660caggcagtct
gaggactttt cttagtgaag tttgtgggat ttacctcctt cctaaagtat 18720agtaagggaa
agctaatgat catactggaa gacttagaaa tgtttgccac tgatttgctc 18780tgtgcttctt
gacaattact attgcttacc caagagatca aggatgctca ttaacatcta 18840cccaagcttc
aaatcaggtc acaatgccaa ctgagttggc tggtcaactg gttactaaaa 18900ctaaaatgtt
gatggtataa actgttcatg gtaaaccagt ttgaacatct agcactctta 18960tcatcacatt
cttttttgaa ttattttaaa acatattttt aaataattat tgctcttccc 19020acaattttgc
cgacaaatgt atagtaaact ccgacattgc ttagtgaaag gtacaaatct 19080agtaaattac
atagtcacag taaacctaat atgctgtatc tgactcttta gataatgagc 19140caagaggttt
tagggattta atttgtaagt agaatggggc taacaatggg ctatataccg 19200cctttcaacg
gtattataaa gaaaagagga agggcaagaa caaatggagg ttacaaagca 19260aagtacagat
gtgggtggga actgttttct tttcacttca gtggttcttt taggctatat 19320tgactaatcc
tgaaaactgc ttttaatact aaggcatgac ctaacaaata attggtgggg 19380ataaaagtaa
atgtccgtta aagtaagtgc ctttgacatg tgtaaaagta aaactggtct 19440gataactatt
ttctccatct ctgaatgaat ttaatatgga aatgtaccat ctgggagtga 19500tggaagagtt
cagtccttcc ttaactgtgc tttaggaggc atttgatatc tcatcatcat 19560ttgaactgtc
tttttacatt cagcttgtgg cccttacttc aaaaaaaagg agcattttca 19620gtgctataag
caaaattgtt attgatggtg gcaaatctga gttaatgtac caaattgttt 19680tctgtttaat
taaaaaatta tatccctgaa aattccagtt ctgaaataaa aatttaaaaa 19740atgctagtcc
tggaattaag aaaacaataa ctgttatgtc taacagtttt tgaataatgt 19800ataagttttg
ttgtatcatg gaatctatta aaatatgatt tattatgtaa atgccaatgt 19860agttaaatta
attatgtaag gcacccctta tataaaatca gtacaacatg atgagtttat 19920aaaacaggta
accaaattat tcaaaattta tttattgccc attgtagacc cagaattgta 19980agaaatatta
cagtttacaa aacaaatagc agtatatatt tgttgcaaag gaggtcatta 20040ttagggcagc
cttacagaga tgtaagaact agattttgta gtcagatgtg ggatcaaagg 20100ttgattctac
tgtctgctag atttcatatc catgagccat agtctattca tttataaacc 20160aaggctcatg
atacttaata gtaatagtaa ctatggggct ttaaagaact ttccttagtg 20220cctggagctt
agtaggctct tagctagtag ccatcatcaa gctggactag tctggcaagg 20280ttttaggtag
attgatattt gagctgggtc ttaaagtata tataggattt agaaagtaag 20340ggagtatact
ggtataatat tctaggcaag gagaactaca gacacaaaag taagaaggag 20400ttgggtcaga
tagagtagaa aaaatgtaaa cactaagttt ggataggtga gatgtggtct 20460gaaaatcctg
ggccttgaaa accagggagg actttgacct tgagattgta gatgaaaggg 20520aacaggtgta
aacatttgaa agacaaagtg ataagataaa attatttgca gaagattagc 20580ctggtgatag
tagccaagat agtttgagga gagaagagaa tgaacttgag acagcattga 20640caatatgaaa
taatagggcc taaaattgtt cagagcagtg aaaactgaaa ggaaggatta 20700aaatacatta
gaacttgcta ggtgcagcaa tacacaccta taatccctgt gtgcctgtaa 20760tcccaccacg
ttggaaggca aaggcagcca ggttgcttga acccaggaat tttagaccag 20820ctttgacaac
atggcaaaac cccatctcta caaaaaaatt aggcagacgg tggctcatgc 20880ctgcaatcat
agcacttcgg aaggtcaagg tgagaggatc acttgagccc aggggtttga 20940gaccagcctg
gacaacatag caagacctca tctttactaa aaataaacaa ttagatgggc 21000atggtgatgc
actcctgtag tcccagctac tccagaggct gaggtgggag gatcacctga 21060ggctcgtcag
gaggtctagg ctttggtgag ccatgtttgt gccactgcac accagcctag 21120gcaacagagt
gaatctccct ctattgagag agggagaaat tgctgagagt tatattaaaa 21180tttaaagttt
taactgctag gacaacagta acaattgcca ccattgaata atacataatg 21240ttctaacata
gaaaatgttt aaatgttatc ttattaatct tcattacaac cctgtgaggt 21300aacatacccc
aatttctttt ttcttttgtc ttgctttttc tttttttttt tttttttttt 21360tgttttgaga
tgagggtctt actctatcac ccaggctgga gtgcagtggt gcaatctcag 21420ctcactgcaa
cctgcacctc tcaggttcaa acgatcctcc cacttcagcc tcctgggtag 21480ctgggactac
aggcacacgc caccacacca agcaacatac cttgatttta aagaaaagaa 21540agctgaacct
tagagaagtc atgccacaac atatttcatg gtttataata atgttttggg 21600atagtgattt
gattgtatgt ctctcccatt tgattgaata ttccaaaagg acaatcttct 21660tacttcactc
tgctgtcttt agcggaggga tgatgagaat gctgcagagt cgaatattta 21720tcaaaccttt
agctagaggg atgaatggtt agatgcctca aggtttacac agctaatgaa 21780gaggaatcag
aattcaaatc tgggttgttt tcatatcaaa ctatatatta actaacatta 21840ctgtatttct
gcatctgcaa gattacatat aagatgtgag aaaagggggc gcatgatcca 21900aaggacataa
tcataagaac atctttattt atagcagcat tctttaggga tgtgctttcc 21960atatgggaga
gttacttatc ttgaagccag ttctccgtat ccctttggga ggccaagatc 22020cctatgatag
cccatatttg gctaagacca atagcaggac actgaacaga aagatccaaa 22080tgaaagctgt
gtcatacaga tctgtttaaa cacagaccag catgtatttt ataagctctg 22140gtatccacag
atcatgctga caatctcaga tttttgtggt ttgctttttc tttttttcaa 22200acagtgctct
gtaaatttag gaaaaagtta gaacgtgtgg catgaaacaa tgtctaaata 22260taagcaatca
atgtgattaa ctgaatgccg caaatatgca ctgtatatgt atttctcagg 22320aaaggcaggc
aataaataca tatattttgt atatatcatc attctctgaa aagttagaca 22380gtgagatata
tcagtgtgtg atgtattaca ctgaaagaat ctttgcttgt agtcagtaaa 22440tattattact
taatcagacc agattgtgct tctaaaatat ctctcttgcc ccagatcata 22500ttattttggt
gataaatact agaagaatag tttttttgta ttttaattca cagacttgta 22560agtgtgggtc
ttcttgattg aatatagcct aaggaggagg atgttaattt gaggtccatt 22620tgaggggaag
atatctcctt agatgggatt tagcttaatt gtgtacaatt ttttaaaaaa 22680atctagggtc
attacttttt ctctccccca attcttaaag ggaattgtgt ctcctgcaaa 22740atattgcaac
ctactggcct gtgatatagc cactatttgt ggagctatac aattccatgt 22800agtggccagt
tctatgtaca aaagaacact ggaatgatat attcctttat ggtgtttgca 22860gtagtagtag
gtatgttctg taagagctca gaagaggtct gaacagggat ctgcagggga 22920gtggagtggt
cagggcaata tttgtaactg aggtgaattg caaaagaacc agagaatata 22980tctggtgtag
aaaaacacaa cagagaaaga aattgagtga aggcactatg agagtgaagg 23040acaggaggct
acactgaatg tggagtcact ttactctttg acttttactt gaattaatgt 23100agttttaagc
catgcatctt taataagaaa ctgacttctt tatcggatta gggatttaaa 23160aggatctctt
tgtacccagt tgggaaatga gaggttgtca catgcacatc ctactctatt 23220agttccacta
tttccatgcc ctgttccctc tgcacaagag gaatgagcac atgtttctaa 23280ggtttattct
tcaacagcaa agcaaagttc ttattcaccc ttcaataggc tcttcatgaa 23340agtggacagg
ctgttgagtc accagagacc aaggaagata ttcattattt tggatcttga 23400gcttgacctg
gaattcctgt aacccagtca ctaagaaagt ataatgtgtg gtaaacagca 23460aagattctac
aatcagatgg gctcccttta aaactcagtt cagccacttt tagttattaa 23520ccttgagcaa
agctcccgtc tctctttgct ttagtttctt catctgtaaa acagggaaca 23580tatttgtgcc
ataaaattaa tgagaattaa atgagctaat aggtataaag tcatcagaac 23640attacttgaa
acagggcaag cactcagatg atagcaacta ttaaaatcat aaaacactta 23700tattctagga
aatccagcat gtgctaggta cgtggagctc atcatgtaaa gattattcag 23760caggagtaat
tccaacatac agtagttgaa tgtgcgcaat gtgttagact ttgggataat 23820ggagacacag
tgcctgctta tgaggcacta cagtctattt tacagggtta ttggcattaa 23880atttgcttgc
ctaatatgtt tagcttactg aattgaattc ctgttgactt acaggaggat 23940gttgacaagg
cagtgaaggc cgcaagacag gcttttcaga ttggatcccc gtggcgtact 24000atggatgctt
ccgagagggg gcgactatta tacaagttgg ctgatttaat cgaaagagat 24060cgtctgctgc
tggcggtgag tattatccaa gctggatggg tagctagagc tctcaaaagc 24120attcagcgtt
tgaaatggca agttgttttg attttaggga tcactatatg ctctctacaa 24180acaaaatgaa
aacatttttt gtcccaatga ataggcccct taacattgaa ctattctcaa 24240atagtaatct
gcatttcaat tctggatgat gttaatttct gctcccataa tagattctac 24300ggtctaaact
ttagcaaacc caagttaaac caaattttag aaatttcttt accttaaaac 24360ttctcagagg
tttcaataag atcacatgaa gtttgagtct cctagaatga tatattaggt 24420ttattcaagc
atttgaccac tgagctcttt tgatggaaaa actcaagttt gttaagggtg 24480cccaaaatat
ttctctaaaa taaatcttat ttctcattag tctagctcgc taagatattt 24540aataaatggc
tacttttttt cttcctgaaa tgtgtctgtt cacaagggtc ataattaaat 24600gatgttcttt
ttacagatga aagaggcaaa aaataaaaaa aaatccaatt attaatgtgg 24660tttatcaaaa
tcatgttttt atgaatacta tttttgtttg tttatgtttt agcagttaga 24720tgagtcagag
cataatatag ttgggggagg gtatttcctt gtttctgttg tctcaattgg 24780gcattatgat
gaagccaatt taacataaac caataccaag atcaggtttc aagcaaattt 24840catctttaga
atctgaaagt ggcagtaaca aagaagtcta catttttaaa aaatcaacat 24900tagcatgtat
ggttaatagc aagtatggtt aatcaaagga ccatttatta ctcaaatatt 24960caacataatt
tgaaatacac aaaaattcag aacgagcagc tatgtgcaat aaaactatag 25020taataaaaat
gacctgtagg aagaaagcag aaaatgctaa aacttggctt ttctcaatta 25080tctgatttgt
tgactgcctg tcagcataag atcctataga gagaaaagta caggcataca 25140aaagtcacat
ttggttaaat tttgacatga tagagagtgt agtacagaaa caaatagctt 25200taacagttca
tcacctgtgc atttctgcca ggtaaccacc ccagcagaat attagatctc 25260aaagagctta
aggtcctgct taagaagaga agccaaaggg gagataggtc atcttataat 25320ggttagggca
catgactaga aaatgttgaa ctttacctga ccattaaaac ggcaattatg 25380acaataatgg
caacactggt agtttcctaa ttaaaattct gctgaaggaa attcatggat 25440gaaaaatcca
ggcattactt aagtttgtgt gagtagactg tgtatatcca gatggaatgt 25500aaaaattaaa
aatgattact gagccatcat taatagtcac tgacactaag ttgtcaccca 25560cagttgatta
caaaataaga gaaaccttgt tggaattctg ataggactta gaagactagg 25620tttcattctc
agatgttatg tgtatatcaa attgtgaccg attcctagaa agctggttag 25680caggagaaca
tcaaaatgag atagaaagag cactggggtt tagaaatcag aatctcacta 25740ccctttgtgc
ccagatcttg ctatttgacc taaaggaagt cattttaact cttaaaaagt 25800ggaggttaga
caagatgaca agatttcttc tatttcaaaa attccctagc acatgattgc 25860tgaaaatatt
tactacatat ttttaatcta aaacttttat ggaattttag atttggtgtt 25920tgacatgttt
ttcagacaat ggagtcaatg aatggtggaa aactctattc caatgcatat 25980ctgaatgatt
tagcaggctg catcaaaaca ttgcgctact gtgcaggttg ggctgacaag 26040atccagggcc
gtacaatacc aattggtaag tatctttgag aaaccactaa tggtgaggat 26100aggagcgagg
agtttactat agagctgaat aatttcaaac tctccctttt aaagatgtca 26160accaaataag
gcaaaattat tttcctcttg actttgagac aacacagttt tcaacttaga 26220agttctatta
aaattcataa aaggtctttt aaagttgttt cagcataatc atgaagataa 26280cattatagat
attttagaaa atgtcaaagt aagaacattc ctttggcagt aattactgat 26340ctgaggcaat
tgcctctcta aggtcccaaa ctttttaaag ctgacctctg aaattattta 26400tttgggtttt
attccaatac tgacatttat atcctttctg taatgtaatt atttaagcct 26460ctttacatgt
tccagtataa gtcagttatg gtcatctgtg cctggtcaaa ctcagtgtag 26520ttagaccaga
ctaatcaaac aagccagttc acacggactc gtttcaaata tcttcaaagc 26580aagatgggac
tgtgtttgca gcctaagtta agggtttgtg tgtgtgaatc tgtacgtatg 26640tgttatttta
gtggggtatg cacataatgg agtagaaata ctaaagatat ttcatgtgaa 26700acatgaaaac
acacacattt aggaaacagt atgggtggaa tttaattcag gcaaacctat 26760gaactttagg
aatacaatac cctccaggac tggccctgat gtgcacttcc aatccaattg 26820gagcatgcag
gaggtcagaa gtgatagcag aaagttgaga ggagagtaag acctcggtgg 26880tactaagaaa
tgtggctact ttagaactgt cctactttac tccgggacaa aatgggagaa 26940gtcacttaaa
acacaaatag atcttgatga agagagtcct tgctgaaacc aggaagcttc 27000tgtgggaact
agaacagaat tgaatgtaaa gcataatgta tctctctgga tgaccacatt 27060aaacttcggg
gcacataaat tggtcaaatt tagattgaaa ataaatgtct tatactcctg 27120tcattttctt
catgtattta gagaatttgc tattattgtt tggtaggcaa ataaaataca 27180ttttagaaca
agtcagtagg aaaggcttat tactcatagg gagattttct ttttaattct 27240actgttttta
attttactat ttctaaaaat tatattataa cttttaaaaa gaattctctt 27300attggtagaa
ctttgagcta ccagaaacaa attggaatgt ggttcttctc ttcgccagag 27360acctcctact
tcctttctcg tccctttttt ataacactta gagaataaag ataattttca 27420aatcaggcct
cctagaagat gaaaggtgga attaattatc tttcagattt ttgccaattt 27480tgcctctttg
atttcaccta actcaatttt atggttccta tcatttcgaa ttactatttt 27540agggcttcac
ttaccatttt gggaatagtt ggagatatag atggtctcga tctcttgacc 27600tcgtgatctg
cctgcctcgg cctcccaaag tgctgggatt acaggcgtga gccaccacac 27660ctggccagat
atatgttttt aaaattattg tctacacctt tctctcacat caccaaccac 27720cattttgtga
atgtcaaata catttgttta tatatgattg tgtgtgtttt atttttcaga 27780tggaaatttt
tttacatata caagacatga acctattggt gtatgtggcc aaatcattcc 27840tgtaagcttt
tctcctatat aattctcaat tttaaaaaga agagttctta ttctatctaa 27900tgatgaagct
ttctctaaaa cagatggatg cttatgtatt tgttaaatgt ggagtaaatg 27960taagatttgt
tgaacttggt ctccctgtga ctaaacttct tgcatatgaa ttaggtagtc 28020cgaaaaccta
tctatattct ggcatatctc ttgagaatgt tttaatacta catcatccat 28080aaatgttaac
cctttactct atgggttgtt ttattaaaag ttgtattatg tactctttat 28140aagagccaag
ctttacatgt atatggcaag gtgctctcca ctgcacatct gcaggatagc 28200attttggtgg
aatgtagtgg ggcctactat tctccaacgc actttctcat tgagacatct 28260catcatgcag
aacagtagct cccagagtgt tcaactcatg acacacattt tttaatcaaa 28320atggaaaaaa
gaataccaga ggcatttgaa gcactgcatg gaatatattt tacctttaga 28380taagaggctc
agatcctcca tattactctt agaggaaagt agccttttta agaggattgt 28440gaagattttt
taagcccaac aaacaggaca ctatctaaat gattttattt taactgacat 28500gctttaaaag
gccaaacaaa aaaatggtag cgaagtcctt atgtgaagaa atggtcaaga 28560cttatctttg
gctccaatga gattgaaata tattcatatc aatattttag tgaaattgct 28620tattaattca
aaagtcccaa taaatcgata aaaactatat aattatgaac actgataata 28680gctgatatgt
actacttgtc tatgctagac cagtcattgt gttgagcccc tggccctctt 28740tcttggttaa
tgctcaagac agccttctga ggcagagagg actattatca gcactcacag 28800attatgaaat
cagatacaga gagcacgtaa cctgtgtgag ttcacatggc tggtcagtga 28860ttgagctgag
atttgaaccc aagtacttgg cctctggacc ctggacatct ttgtctttag 28920caaggtattt
ttcctggaaa taaaagctac taaataatat tattgggtaa ataattacaa 28980taaatgactt
atggagaagg agagtaatca tcctttttaa acatttttag ataacttaca 29040gtgtgctcca
taataagcca aaccatgcag tgattttttt tttcctggta gaaaaagaac 29100tcaacagtac
ttgattaaat tttgaggttc tctcctattt ccttctcata tctcatatct 29160ggtttattca
gagcgtttta gcatttacta gttgctttta gcaatgaagt ataatatgcc 29220agatgatttc
acaaaactac gcaattatta cataacatct acagggggtg tataatatgg 29280aagttggatt
agcaaatatg ccttttgcaa tacaaagaat ctattgttac atattgcttt 29340ctagtggaat
ttcccgttgg ttatgctcat ttggaagata gggcctgcac tgagctgtgg 29400aaacacagtg
gttgtcaaac cagcagagca aactcctctc actgctctcc acgtggcatc 29460tttaataaaa
gaggtaagtc tcccgaaatc aaaatatgct caagaactca agaatcctaa 29520attacaatag
gaagacctca tttgttgcta ctataaagta cattatttac agatggctcc 29580tgtccagtgg
ggggaataca tttagcatga cggctggctg caatttctgg cagtcacccc 29640aaattcatct
ctgcccaaat gcagacagga agccaaacac aaaggtttgg tgtcaaacag 29700tcaacttggg
atcacatttt tgcttctttg tccaactctc atgaacataa attcatgttg 29760aaattaatgt
agcattcttt caaatgttga aacattaagt tggtttgtac ctgccactga 29820tggcctagtg
ttttctgcaa aattgtgtaa actaatctat gtaaggttga aagggcctct 29880atgccaatat
gcttgtttgt aatattgggc cacattattt ccaaacactt ttcaatctac 29940tcatgagtgg
atatgtttat attcagtttt ctattatgag ttcctctgca tttatcttcc 30000tgttcaaaaa
cagtaaaaga acatgtaaaa cattttcatc agctatctag attgtgttaa 30060tatacttgca
aacaattcat tgtccttttt tcttcatctt accaaatctt aaaaaataaa 30120tgttaatcac
tagaaactta aagttacaaa actgaactta tcttttaaag atattattta 30180ttgaaattaa
aaacagggaa aagagatgtc aaaatgaaac atttcgatgc aaataacatg 30240aaattgtaat
gtgcccaact cagttcccat gccttcacct gaattcttta atgaagattg 30300aaatcaacct
gtgatgttga atcatgaatg ggatgacagt atttacttaa caatttacta 30360agtaatgcca
atgggaattt gcaactcagc agttatgccc tcaacaatca acaacagcta 30420ggaactaaaa
aatgttagat cccttcagct tcttattttt gctgatggga ataataaaca 30480tgttacttac
tttctacata agtaacttac caaacacttt tttacataca atctttttga 30540gttagcatta
tgaaatttga aagagccatc actggagcaa tgattcatag agtatggact 30600taccctgaca
agaaattgtc ctttaataat gcaacccttg aagttattct cttatgtgtg 30660cttagaaatg
acataataag actaaagaat agagccagtt gggtgaatta tttttttcta 30720gagcatctca
taaggttggc attaagagac aatctcaatc ccctactctc ctcctttttg 30780aattaaattc
ttatactgta acttttaaac tttttatctt ttaggcaggg tttcctcctg 30840gagtagtgaa
tattgttcct ggttatgggc ctacagcagg ggcagccatt tcttctcaca 30900tggatataga
caaagtagcc ttcacaggat caacagaggt aatattattt actcagggca 30960aaagttaaga
atgtctgcat tgccagctat gaagtatgtt ttatgtaact atttttgagc 31020caacaatttt
aaaacaaaaa ctctttttaa tgatttgtta cttattttag ccttcaaaca 31080tatggataga
atataagcag atgtagtttg aatgacatga tgcacctatt ttaaatcaga 31140atgagagtaa
actattttta gtatgttcat tactttctct gacgagaaat atggctgctg 31200agaccttgtc
atattattag gaagatttat aatagaaaat taggccatag tgatttgtat 31260gaatgaatat
gattttatcc tataggttat gaaaattaaa tagtagagtc tcaccctttg 31320caccttattt
ggggttgtct ttctcctgga cactgtgaaa gagagccaga aactcagctt 31380ccctcagtgg
tacctagact aacttttctg cctctctggc ttgaacttga ccatcctaaa 31440ggttaaagaa
aaagacccca accacaaagg agaagtggcg ggcaggaggg agagcagact 31500agaaggtgaa
gtttgggaaa atagatcccc atttattgat gatgttctat cctaagcaaa 31560agaaagtggt
ggaagcatac tgtaccatgc aaaaaaaatt attgagtcat tctattaaat 31620tacgtgactt
tgttagatgc tcagtgcaaa aatgttattc attggtacac agtatagaca 31680gtgaagagtg
agcttcactc catcttagaa agctgctgac aacaagagac caggcagggt 31740gtgataaggt
actaaggcat gtacaaaatg ctgtaaaaaa ggttcacaag aaggaatgat 31800gatgtccaaa
tgggtactag aactagctct gggttttgag gaattgttac ttttttaact 31860tcctggaagt
tgagaagcaa aaaagacatt ccagatagtg tggtgagcat aatttgatta 31920ttttataatc
ttaattattt actccagaat aaaggcaaat ttacatagca aaaagaaatg 31980gtggaaaatc
aaactttatt tttcttggtc tttttaaaat gaagtaaata aatatatata 32040tgtcttcttt
gtttttcaca gacgtagagt aaaaaaagtt tgcattctgc aactccctaa 32100ttgagatagg
gaaaagcaaa ccccacagtc aagatttatt gtagacagaa caagtgggaa 32160tgttcaagat
atggcaatac atttaaaaat ggtgacacca caggttgaaa tcataccaga 32220ggaataaagg
tgaaaattcc tcagcctaga attctagacc ctccccaata gggaatccat 32280gtacctgttg
aggtctgtcc aacccatggc cagggatggc tttgaatgta gcccgacaca 32340tttttttttc
attctcatca gctatcttta ctgttagtgt attttttgtg tggcccaaga 32400caattcttct
tcccatgtag cccagggaag ccaaaagatt gaacaaccct ggtctagatc 32460ctatgcctac
tccttctcca ggtcatctgg accaggagct actgtttgct gatctcatct 32520tcagcatcag
tttgtcttcg ctcatccatg atcccctata acattggggg agcactttac 32580ctacttaaaa
aaatccctgg agattaaaag tctagaaaat atcttggttt aaattctgac 32640tctacccctc
ctagcagtgt gtctttaaaa agttaagtag ccttactaaa tttgagtttc 32700ttcatttgta
aaatggagat gagatcaaat ttactgagtt gtacagaata caattaaatg 32760agctaatgag
tagtaaaaca tttatcaccc tactgagcgc tcagtacatg ccatatttcc 32820tctttcctcc
catacttttt aatttcgcac actagtttac acaatgtgag aaaaaagctt 32880cctgtcccaa
tatttctttt tctccatctc tgccactctg ccccatctct tcctgattat 32940tccatgtatc
ttttcattcc cccaggcttt tagttcatcc ttggccataa cctactctaa 33000acttgctccc
acacgttccc tctctggctt tctccttgcc tctacagtcc caggagcaga 33060gcagcctaaa
ttttaaaaca ccccatctga gttcccattg tccaccacat ataataacag 33120tgcatgccta
ttgccttatg tgtctctctc tcttgctgat tatcctcaaa gacagtctta 33180ttcattgtta
tctccagaaa ccatcacagt aagaaattct aaataagtat taaagaatta 33240aatgtgatat
cacactctgg ggactgtggt ggggtcgggg gaggggggag ggatagcatt 33300gggagatata
cctaatgcta gatgacacat tagtgggtgc agcgcaccag catggcacat 33360gtatacatat
gtaactaacc tgcacaatgt gcacatgtac cctaaaactt agagtataat 33420taaaaaaaaa
aaaatgaaaa aaaaaaaaaa aaagaattaa atgtgaataa cacgatttta 33480ggagccacaa
aacccatcct actttcaact cctgaaactt agttccatac tccctggaac 33540ttctttctgg
attttctttt agcttcacat ttgttgcttg gcagatatta aggaacttga 33600taaatgtcat
gaaagaggca aacaatagac tgtatccttt gtgttcattg tagcaaagac 33660cctccattct
aacaacactt taggcaaggt aagaattttt tttttttttt ttttttgaga 33720ggagtcgtgc
actgtcaccc aggctggagt gcagtggcgt gatcttggct caagctccac 33780ctccgtggtt
catgccattc tcctgcctca gcctcccgag tagctgggac tacaggcgcc 33840caccaccacg
cccagctaat tttttgtact tttagtagag ttggggtttc accgtgttag 33900ccaggatggt
ctcgatctct tgacctcatg atctgcccgc ctcagcctcc cagagtgctg 33960ggattacagg
cgtgagccac cgcaatatcc cattagattc atataaaata gaactctacc 34020cacattatgc
aagacataat aagctagacc attttcatgg gcatttgcta cttctgctcc 34080acatggcaat
gccacagtat catggaatac actgtttagc catagtctta atagttctta 34140tcttttcttc
attgcatact cctttgagaa aaatgataaa cgtttacttt tctccaaata 34200agggaatgaa
atcatgagac aaaaattaat gtctctttgt tttgacttta gctagacttt 34260tgggaaaact
caagcctttt atttgccatc taaacttgtg ctaagcaaaa gtgtagactt 34320tctttctcat
tcatacttct aaccattctt catcctagtt cttttgacgt ttgtagcttt 34380tacaaaaata
tatttgattt tattaccttc tccaaggcaa aagttatcta ctttaatgga 34440gttacttccg
taaacctaaa ttaaacaaat aaaaaaaatt cagaaagaat ttagagaaat 34500caaagggtga
caggtaagta aattttttat ctacaaaatt aaaaactcct taacatctta 34560aatgaaaatt
cagtttctga aagtggtcat aagaagtgtg ttatagaatg gcatataaga 34620gaaatttcag
acaggatttc cctagtgcaa atctcctttt aatttatttt ttattaatct 34680tttaggggtt
tgatttagaa acataaaata tttaaatagg aggaaaacat tagcatatta 34740atgccctatt
ctgtaattca cagtagcagt tagttcagta accacataaa atctaaacat 34800acactcacga
aacatctatt tctctcttgt tcagttggga tagaaacttt ttattaaaat 34860ctgacatgca
gagtaaaaaa aagaaaccta gaaaataaaa gaagcaaaat agtgaattgc 34920ctttaaaaaa
gaaaggcaaa gaacatgttg gtggattaag gctgccatac aaataataaa 34980aaagaaataa
ttgtagaacc agccaagttt ttagagatag aaaaaccaga aacaactata 35040gtaaataatc
ttgatgggaa gtgatccaca aaggaaaaaa taattgaaaa caaagtcttc 35100ctctgaaaag
ggcaaactag cattaaagaa gaaaccagaa ctgtaggatt ccagctaact 35160gaagtagctg
cacagatccc atttctttca tccagttcca ctgttcaaag gtgtgtgtgt 35220tgggtgggaa
gacagagaaa gaaagagata gagagacgac tgtcaatccg caattaaatg 35280agccttctgt
catagatact aatgaggaga ggtatgcagt gatacgttat tgttactgct 35340accacagagt
taacaatcta tttgggaaat gtatttctat acaaataata caatctggat 35400cagactctga
aggagacttc atgaggtttt ttgggagact agaaaaagaa atggtcctgt 35460catttcttct
agcatctgac ctagtgtcat cagagaagga tttaaggaga agctaagata 35520tgagctggac
ccctaagaaa aatagaactt gaaggcagtc caggttgggg tggacaaata 35580atgagaagaa
ggatgagatt caagaggtag tgaatatgtc agtttgactg gatagagtta 35640tctggatgcc
agaagaggga taaaaaatga aatagctaag ttgcgactgg gtcatgataa 35700gtctgatcaa
tctgtagaaa gatgatattc tgattaaaat taaaaataac ctccgtagcc 35760atcaatatca
cttatccgtg ttctatatct ctgctttctt gctgatgaaa aatcagcatt 35820tttctgattc
cttttatgtc tgtatgcttg gatgagggag ctttacatat ctaaaagtgc 35880cagatcttta
aaaatgccat ttgccagaga atcacttgaa cccgggaggc ggaggttgca 35940gtgagccgag
attgcgccac tgcactccag cccagtcgac agtgtgagac ttcgtctcaa 36000aaaaaaaagc
catttggtgt ttctcccaaa tgaaatttta ctgtggcaca aacatcacat 36060aaatttagaa
tgctaaagca acctgtgttc atgagagcag ggtaagcctc ctcctttgta 36120atgctccctt
tcaggttggc aagttgatca aagaagctgc cgggaaaagc aatctgaaga 36180gggtgaccct
ggagcttgga ggaaagagcc cttgcattgt gttagctgat gccgactgtg 36240agtagaaacc
actttgttaa cttttcgtcc ttcatcgttt ttggtgtctg ataatgccaa 36300aagtgaactt
gaactttaca aaaaaaaaaa aaaaaaaaaa aaagcatttg cttctaacat 36360caaagtgtaa
agagtccaaa attactcttg aaaatctctt aaatcatcct aaagttataa 36420cacatagata
atagacacaa tttcttcctc gtttagaata gattgctatt tcctcagctg 36480aaccctagat
gatgtaaaca ttttgcattt aaatgccatg gtgtattttt taaattccat 36540atctgtgatt
attagcattt cattgagcta atgagatact ccagaatggg acttggattg 36600agaccaactg
aggagacagc agtttcacat ttgccctctc ccttcactct gggtcataca 36660ctcattaaat
gaaatgatca gaagaaaatt atacctaata tttaaatatt ttttcctagc 36720aagtatagtg
gagtttatgt acattaaacc tacaaatttg atataacccc actatataat 36780attttaggtt
cattctgatt atattaccaa gtagaatttc ccaattattt cctaaataga 36840agttaattag
cagaaatgca tggtcaaaca ttcttttttc tttcacaact gcattatctg 36900ttagaggctg
taaatacaaa tggttcatgg aattagccta tgttgctgtt ttgatcttaa 36960ttgaagtatt
ttagaacaca ctaccttatg aaatattcta taaacatgaa agatttctaa 37020ttttactaag
actgtctata aactgcttct gctacaactt aagcagatgt aattagttca 37080caaaataaat
aataatcata cccagttgct ataacattga attgaatctt cagcatttag 37140aaaattaaaa
tgcaaagaaa attttattct tgtttagagt ctactaagga agcaacctat 37200ttcttccttt
taatgcacat tttatcgcct aaaataaata agagtttata ggcacatatg 37260aaataatacg
gtatacttta gatagatttt gaaaatgtct ttgatggaat aaaatcataa 37320ggtgagtgag
tgattctgag tattagtgtt ttttgttact ttttagttgt ttttgtttta 37380ttttgttttt
ctttcccaca aagatcacat ttcctgttga gtagagtatt taagaatctt 37440ttgaaattat
ttaattcgat agacatgtca ggtttttttt attatctgtt taaaatctgg 37500ggcctgaaag
aaataatcac aatcatctaa ttatataatt ggagatattt atataaatca 37560tttccaaaga
gatttggtct catgatcaaa tctacattct tggtgatgtc catattaaag 37620ttgtaatagt
aaccactatt ttttcagatt ctgtatgttt ctattttttc catggtaaat 37680gcaggatcag
aaagattaat ttgccacctg gctggtagga ggtaaactat actgagccca 37740tgtctgacta
cacaactcat aatttttctt tatctccatg ttgcctctga gaaacatata 37800aaatatataa
tttttataaa atatatcttt aatgtgaatg tcttcttcgc agtggacaat 37860gctgttgaat
ttgcacacca tggggtattc taccaccagg gccagtgttg tatagccgca 37920tccaggattt
ttgtggaaga atcaatttat gatgagtttg ttcgaaggag tgttgagcgg 37980gctaagaagt
atatccttgg aaatcctctg accccaggag tcactcaagg ccctcaggta 38040agtataaaat
agaaaggata gcattttcag ggcacaggaa taaagtatcc tctttagacc 38100tagattttat
tgagtaagat tacttcccat ctgcacacct tcctaggtga caatgctgtg 38160ccagtttggt
gtttaaaagc aatctaactc ccagtgagaa tgaaatcatc ctgtttttgt 38220gttgcccagt
tttcatgtct agaaacagtt taccggccat gctgagaatt aaagacatga 38280ggaaaaataa
caaatgttct acatttttgg ctttcctcaa tctgcacacc tcttgattcc 38340tatatacagt
tctctgtcca ctttgaaatt cttatacatt cctaccattt tttcctcctg 38400aatccttcat
ctccacagcc atcccacagt tataaccagc actcccctaa atcctctgca 38460agtcatcggt
tactctgacc ccctaatcgt agcatggggt cctaaaatgt cctgagtttc 38520ctttgcggca
aacttcgtat ttcagttgtt aaagcccaat tattccccta ggcactcagt 38580tttctttgta
gactttatta tcagtaaagg tctctctctc tcttatccaa agatcatctt 38640tccctcttcc
ttgcatagtt ctctgttcat tgtttgttta agcttctgga tctttggttg 38700tgaatttctt
cctgcccttt gggtcttgtt ctagagcagt gattctaaac tggggcaatt 38760ttgctcccct
caccccgggg acatttggca atgtctaggg atattttaag ttgtcactac 38820cttggtgaga
agcggtggct ctacaggcat ctagtgagta gaggccagag atgctgctaa 38880acatcttgca
gtgaccagga cagccccatg acaaagaatt atctggccca aaatgccaac 38940aggacaactg
ccgagaaatc ctgctctgga gtctagacat taagcttaga ggctctgttc 39000tttcacaata
aaggttttac actgatgcgt gctgtcaatt tgaaaccaaa cgctattctt 39060gactcattca
ctaagccaaa agtaaattca attaactaaa agttagtgaa cgttgctatc 39120agctaaaact
tgagatgggt acctggcaat gccaaaaaga acaaagagga cttgatcccg 39180gacctcatgg
agtttacagt ccaagtaagt tggacagtct tggattttcc tgttcatctc 39240aagacatcca
agtaggaaaa tcttaggact taggcagtgt aacatattga aatctaaagg 39300ccctaaaata
agcggcaagt gaggtaatac tttgttttat cttaatccca ttatattttg 39360tatttgtctt
tgagtttaat gagtacttaa aagtagttgc cctttttttc atatttggta 39420tatggatgcc
aagagatata ttcaaatcag ttttgacttg gtctcccatt tcaaaaaaga 39480atggaattct
atgttttttt gttgtgatta tttatctatc taactaaaag tgaagcactt 39540ttcttatttt
gttgatggat tgatggatga ttgattgata gatctgaaga gaggagagaa 39600tacctaagat
acctaatgcc taattgttgg gcagtacttt cctccattag gacatcttca 39660caattcaacc
aaagactcaa aaattgagct ggaatacctc attgatttta agataaggaa 39720gttaaaatag
acttaaaaat tcctatttgt aagattctaa agtaaataat ccagtgatct 39780agaatttagt
ttctataaat gaatagaatc cagtgatctg attaatataa gaatttagtt 39840tctataagtg
aatagatttt tatcacaagt tccattgagt cagtaggata agtaggtatg 39900ttagaatttg
gagaaacaaa agactaaaaa gccctgggct cctttaccat taaatattca 39960tctattgtta
ggagaaaaaa cttacgcaaa ggggatactc actcactaag ctttagccta 40020ttgcctcatc
caggggtggt tgagtgaact gaggctttta aatacaatga tggaatcttc 40080ctcattaaat
ataacatcaa aaaggttata gaaaaaataa tggtcaaaac gatttgtttt 40140aggggaaaaa
attaaagttt aaaaaaattt gaacattaca aattatttgg gggttagagc 40200aattacaaaa
tatatatata tatatacttt gcatatttgt attatatctg tggattcatg 40260tgtgtatctt
tttcatattt caagtgattt tcctatttga ctttatagaa aaaatatata 40320ttgtcttaaa
cttggaaaaa ttagtatttt catttgaaac agaaattgtg atagccacat 40380aaacgatact
aaaattatca cagagtagca ccttataata gtgtggaaag atggactttg 40440gagttacagt
tatataggat ttaaactgga tttaaaatcc agcctccctg aaactattgt 40500cttatgtgta
gaatgtgcat agaaacctca atcttgaaga ataattctgc agtgcaattg 40560gataatctat
aaaatgtatc catcagagtg cttggtccac atgctcaaat gtctagtaaa 40620taaataaaaa
ttacatgctt ccatttatac ccatggtgac aattatcata cagatcaatt 40680acttctatat
ctattcattg cttttgttga tacatggttt ctgaataatt tgagaacatg 40740taagtgcttt
ttcccaattc atcttcttta acccattaag cctaaggaat caaagaattt 40800acccattcaa
acaccttaaa atgtttcctt tgagactgtt accatatcat gacctaccta 40860atacatttca
tagactagcc cataggccaa tggcccatca catgaaagag ccccagctac 40920atcctagcat
tattggacag tcccaggtgt ccagaaagaa ggtcaagttt atatatggtc 40980agcctgatgc
cttctgaaga aggtctgtat ttgtggtgaa tcaaaattag ccagacaaaa 41040ctgaccagtc
tctaagtaga gcctaatttt ttgtttgtag tcccaagctc caccacagtg 41100ttcctcaatc
tttcccgtgc ccggggattc agtagatctg aggtggggtc aaagaagcac 41160atttggctac
caagtgatgc cacaactgct ggttgtagga ccacattgtc ggtagcaagg 41220cactgaaata
caaaggcctg tagttttata agttagcgac catctaagtt ttccaacaaa 41280taaggctttt
cagccctttt ctccagtatg tatgcaggga tttgattggc actggttatt 41340caacgtggtc
acagacagga aaacatgact tgggaaatac atcttgaaat aaaaaccaga 41400tatcatgatg
ttgagggcat agtcagatta atatgttcca ataagacaga aagctttatg 41460tggagtgaca
tggacccttg actatttatt gagaaactag aataccttac ccaggcagaa 41520tttatctcac
cccacccctt agtctgctac tattcaacct atgtagtaat ttaaatattt 41580cttcttaaca
ctattttttt cttaatctgc tggaaatgaa ggaatatttt ccttctggga 41640tattatatta
aatattgcac taaagactgt tgagcaacat tgtaccaaat attgtgccaa 41700attttagaaa
atagtttcta gtttcaaata gtgcttaatc tagttagata tgggggctga 41760gctgtattca
gttagaaata actttatgga agctttttct tggctttgga agataaagaa 41820gatttgcaga
gtcagagaag agtaagagaa agtgaaacat aaaacttttc aagtagtggg 41880aagttaaacc
tacaagagct tccaccctcc aacccatctt agctcctaga tctcctgttt 41940ttcctttaat
ccttaacccc tgctacacct gagataggca gccctcaccg gttacttgga 42000atccaagagt
cacccagtta ttcaatgtca caaaatagaa attgagacaa aaattagccc 42060cagatttcag
tgcttgaaga ggtacgtgtt cctggtgttc acaaccagaa ctccaaactc 42120atttattttc
cttataaaaa tagtgtttaa ataaagacga tttaagttct gtaatacttg 42180gcacatagtt
agatgtggct catttaatga agcaggatgg tgacattcag gacaggccag 42240aaattggaag
cagatttact gactgcccca gtgggaccag aactgggaag ggtgcgcaga 42300ctatggtcag
aatgggatat ccaagggcat cggacaggtc agagtgggtg attcagaaat 42360ggggagatca
aacagaggct cgggagtctt ctaagaacag tcttaaagca tctgcttccc 42420aacagatcta
attaaactca gtatcaaagt tctgtttttc tcttttccct cctttcattt 42480cagccgggac
aatagtttcc aggtaaatgt atttagatta acaggcattt cttccagagt 42540gaataaatga
tgattgtcat ttatgtgttt tttatgtgtt tgcagtgctg aatgctttcc 42600tatataacta
taaaacaaat gttaaataca aatatctgtc ttcaagagat tttcccctaa 42660aagtcatgag
aagaaaatat atccaaagaa tgaatctgaa cctctgttag ctgtttcgat 42720tcaatatttg
gtttaattgc aatattttag cattttgaag cccttgttaa caggcccagc 42780aatgtgttat
ctttacaagt gactactttt tttcttttca tgtgatattt ttcaaataga 42840ttgacaagga
acaatatgat aaaatacttg acctcattga gagtgggaag aaagaagggg 42900ccaaactgga
atgtggagga ggcccgtggg ggaataaagg ctactttgtc cagcccacag 42960tgttctctaa
tgttacagat gagatgcgca ttgccaaaga ggaggtaaat ggcttcattc 43020tgttctgttc
tttttgttgc catgttttgt ctgtttgtgt gtatacaaag tgtcacttta 43080aaattcccag
ctctttggaa catctttccc tctaaacctt actctttatt ctgttcttga 43140tagaggttta
agttatttgt gatagatact aaaaagtagt aagggatcca tggggccagc 43200cacaaatgtt
cagccaacac agatctggat gcttaacaat tttcaggtgc tgccttcaca 43260gctttaaaac
aatggaaaag aatcctgtca tttgcagcaa caacctggaa aatttatgct 43320ccatgaaata
tgacaggcat aaaaagacaa ataccgcagg atctcacttt tatgtgggat 43380ctcaaagagt
agaactcata gaagcagaga ttggaatggt ggttaccagg ggcttggggg 43440agtggaggat
gagggttggg aaatgttggt caaaggattc aaactttcaa ttaggaggag 43500taagttcaag
agagctatcg taaatatcct gactgtaatt aataacaatg tattgtatac 43560ttgaatattg
ctaagagagt caattttaag tgttctcacc acaaaagaga taaatatata 43620ggttctactt
tctggttcca cctttgccct atagactctt tctatataac agccatccat 43680gtatttgaaa
acagcattca tcccctctat cactccctac cttctatgtg ctcttagtcc 43740tatttgtttc
agctgttaga cttcctgatt ttgtatattg cagttaagtg tttggtgagt 43800gtatatatgt
gtgtctgtat acatatataa tgtgtatata cattatatat atgctcacca 43860agtatttaac
tacaatatac aaaattagaa aaaaaatgag ctgtcattct tgcatatctt 43920gattaatacc
agataagacg tggaaaaaaa tcttcaatta aggaaacatt tattgtgttc 43980ctataatata
ttaatatata gtagaacagc tagcttgcta cttcaaagta ggagcctgga 44040gctatgtgct
gtgttcaatt aaaattatgt aaaatatgaa cacatttatt agagtagctg 44100ctgtgtatat
ggttcctatg ttcaaatagg attagattta taacgttaag attcacactc 44160aacctctatt
gaacagcagc ccctcccttc taacatttaa atcagtggtg aacaccagaa 44220aactctttgt
catttctgat ttttgtcttc cttcattcag atttcccaga aaattctggt 44280aatttcagga
atctttatct gatcttaata aatattttat tgaaatgcag gagcttaatt 44340ttaaagagaa
aatccagttg tctttctttt acctgaggct gagatacagg gtgaattgag 44400tttgggctgc
agtaaccagg aacttagtga aagcaaaaga gtactttacg ttagaataag 44460caaattgtga
tttttgtttt ctgtcctgca attatagcca ttaccaatga actcatgctt 44520tgattagaat
aggatggttt agaatgtata agctcttgca gtaaggaaca attctgtttg 44580atatatttaa
gttgcctaag attttgctac agagtagact aaaagttcgt gatgtttacc 44640taacttggct
aattatgaaa agtaattagt aactattcta ctgagtacag tagaatagtt 44700aatattttac
caatggcata caggtattta aagcattatt attatcaatc attgcctatt 44760catttatcca
gcagattatc attgcctatt catttatcca gtagattgct ttctgggact 44820tgttactatg
tgtagtgact tggaagataa gaaaaactag agagtgaatt taaaatattc 44880ccaagtgaaa
aagaaattct gaagttaatg atttgttgca caatcataag ttcttgaaag 44940cttaattcca
acatctagaa acttaatatt gccttaatta tttgtaccct ttcttcttcc 45000aaaggaaatt
taacacctga ccattatctg ttttcaccta ccatttttgc agttacctat 45060ttggattctc
acacataggt ttgagatgag aagaaaaatc ttaatcaatt taggatagcg 45120tgtaggaaaa
aaaatctacg atatcattta aaatatttca ttttaaccta ttatttttac 45180ttatttattt
cttgtggtat agatttttgg accagtgcag caaatcatga agtttaaatc 45240tttagatgac
gtgatcaaaa gagcaaacaa tactttctat ggcttatcag caggagtgtt 45300taccaaagac
attgataaag ccataacaat ctcctctgct ctgcaggcag gaacagtgtg 45360gtaagtccaa
cctaaggaat gtagcctttt cagtaataac cacattaaca gattactacc 45420ttgaactttt
tcagacttgg atttttcatt tggaattacc tatccttcta gaaaagcagt 45480tgctgccttg
aaaaacaaac aaaaggctgg gtgcggtggc tcatgcctgt aatcccagca 45540ctttgggagg
ctgaggtggg tggatcagct gactgaggtc aggagtttga gaccagcctg 45600gccaacatgg
tgaaacgcca tgtctactaa aaatacaaaa attagatggg tgtgatgcct 45660gtaatcccag
ctacatggag gatgaggcag gagaattgct tgagcctggg aggcggaggt 45720tgcagtgagc
cgagatcatg ccattgcact ctagcctcag caacaagagc aaaactccgt 45780ctcaaaaaaa
aaaaaaaaaa aaaaaagctg tattggaaga actttaggga ggatattttc 45840tttaacttta
tctagcttct tgaaattgct taccaaaaat attgtattga tgtttgatta 45900atacaatata
agaattgcca agtaatttct gagcacgtgg tactatgctg tatacaggga 45960ggtaaaagag
taagaacaat atttacttgg tacccttgtg tatgcagata ttcttatatc 46020ggccttctta
ctctaggatt attagagata attgaagtta tttttgaaag attgaatttt 46080gaagataccc
tccctctccc atttttgacc tagtttatat ctcttatttt tatactttaa 46140tcaagaggat
ataaacatga agtctgtgcc tctcaaactg ttgcattctg tactcagctg 46200tcagtctcta
gactatgtct ttggtcactt tggtcccatt agcctaattt tggcccctca 46260gtcctggaaa
aagcacaaga ttattttcct tcccaacact aagtcacacc tagatcagac 46320ctatgcaata
ttctctttct ttctttcttt ctttctttct ttctttcttt ctttctttct 46380ttctttcttt
ctttctttct ttctttcttc tttcttttct ttctttcttt ctttctttct 46440ttctttcttt
ctttcgtctc tctctctctc tttctctttc tctcttttct tttctttctt 46500cttttttttt
tttttttttt tttttttgag ctggagtctc actctgtcac ccaggctgga 46560gtgcaatggt
gcgatcttgg atcactgcaa cctctgcctc ctgggttcaa gtgattctcc 46620tgcctcagcc
tcccaactag ctgggactac aggcatgtac caccaagccc agctaatctt 46680tgtattttgt
ttttttcttt ttttagtaga gacagggttt caccatgttg gccggactgg 46740tctcgaactc
ctgacctcaa gtgatccatc cgccttggcc tcccaaggtg ctgggattac 46800aggcctgagc
caccatgctg gcctggacct atacaatatt ctaaggctgt ggttctcagc 46860cctggctgtt
cattagaatc atctcggggg ctttaaaaat gtataaactg atttggggca 46920ggggagggta
tacattaaca caaattatat cagtgtatag tttaaatttc tcctccaaag 46980cccatgtggc
aatttaaatg ccattgtaac agcaatagga agtgggccta atgggagact 47040cttaaataaa
attcagcaag ttcttcttga atagtcggta tagctagtga tgaatataga 47100atttgcctat
gataaaagca ttaataaaat tcagcatagt tactgtgtgt atgtatatta 47160tcgccactta
ctgaagatat ttataccaag tactacaaca agaatactat tcacgcaatc 47220ttacgcatac
cttgaaacaa tcctattaaa tattatcatc cccactttac acatgaggaa 47280acgcttgcag
aagacagata acattttgaa ctcaaagttt ttgccaagtg aacatttctc 47340agttccctgt
taattcacag taaaatagtt tcatttctag ttgacagtaa aatgagcaaa 47400tttactctct
gcacttttta acaaagacaa atttcagttt tgtattttcg actagcagaa 47460tgttccatgc
ttgtttgatg aagcttgtta tgacatcacc ccactgaggg tcttgggaat 47520ccctgatcag
gaatttcctc tattatgaaa aacagagggg accatcttgt tactacatat 47580cctagaagat
aataattttc tggcattgtc ccagacctaa tagctgcttg gtgttttcag 47640gaaaattatt
gaactccaga gttgcaggaa gtttactcat aatttcaaag tagtatttga 47700ttttgcaatt
taaagggcaa ttaaacaaag ctgagacaat actgcctttt gtaagtgtaa 47760atttattaaa
ggaattatcc ctccaggatg agagacatag ttaagaactt tatggtccag 47820tgagaatgtg
gaacttcggg aactgggata gtgttttaac aaagaaaaca tctgaatatt 47880atctcaggat
aaaagtaatg accaaacctt caaagtataa tacaatttat ctggaagcca 47940ccctggtttt
ttgtttgttt gtttggttgg ttttctaaat ttgttttgta gaggcaaggg 48000tttgctatgt
tgcccaggct ggtctcaaac tcttgggctc aagtgatcct cctgcctcag 48060tctccagcta
ctaacccagg ctctggaagc cacatttttg tccacttatt aaaatagcat 48120gaagggagag
aaaagtataa acataaatct cctttaaaaa gtgtttttct cttgcgtata 48180atgagaatca
ggtaaataat tagattttgc taacttttgg ccaccagcaa atttaaagct 48240aaacaaattg
tttatggtct gcaaaactgc attctaattt tccccatcta gatcttctat 48300tgtaattttt
tgttggattc taattactac tattcatgta attatgtttg tttatataag 48360catctttaaa
cactttatga aatatgacag ggaataaatt ataattaaac ccaactcctg 48420ccaaggcctc
tgtttaagca atttatttaa ccaattaatg ggaaaaaaaa ttaactgagt 48480aatatgatac
atattacaat tgcaaaagaa aattgattct agcctgttaa gtacagaaac 48540atcaagagaa
ttacaagctg atccatatat aagcagtaat gaatactgat ccaacaaatt 48600ctagcttcta
gcaaaccata tctttccccc tacagcatag tgactattct atcttttata 48660aattcagact
accacttctc agggacacaa tgtacgacca aaatggctat ccatatatat 48720agttgcacta
ttctatttgt ttctaggtag tgtgtatcca tatatgtctg cattctaatt 48780tatagtgcta
ctttttttta tcaagatgag tatgttataa tgaaattaat ggagtttgta 48840aggagctttc
caaatttcta aaactgccaa aaaggatttt tttttcacac cttaagtatt 48900gtctataatg
cacttttctt ttccttcagg gtgaattgct atggcgtggt aagtgcccag 48960tgcccctttg
gtggattcaa gatgtctgga aatggaagag aactgtaaga ttaacgttct 49020attaagataa
atatttattt ttatgaaaat gattttcatt cccagggaat taactcatag 49080ttttcacctt
acataaaacc tgcctctgtt ctttcctgga gattcatagc accaaatagc 49140ttattaaatg
tgggatgtac tccataagtg caaaaggtga ttgcagaaca gcaacataat 49200ttactaaatt
cctactatat tttagggact atattaagca acttacattg gtgattgtaa 49260tactgcttgc
ttttagaatt gcttgctata tatatgaatg taatccattt gggaaacatt 49320tccagaaaag
aggtggaaac tatgcattgg tgattgcaag aaaggtgcaa atgtaaacac 49380tctagttatt
tgaataaacc atttaaataa gtaaattagg tgacttatgt aaaggaaatt 49440tcagtgtaag
aggaatgatt cccatttgtt gagatattat tgatgtagac tttaacattt 49500tttattacca
taagaagtta tttttagtat taaaacttaa acctaaaagg aaatttgagg 49560aaaacatgtt
ttatagtagt gactagatta agaatattta tatataaatg catttatgaa 49620atttcaattg
cttttgaaac tagggtatat agacttttta agtatgcaca tatatatgta 49680aatacagaaa
gtagtggcaa tatgcttagt tgattttgtt taggcaagtt tagtcatttt 49740atttcttaaa
acttttttac atgtattaat gtaaacattt atgaagaaaa aagaaataga 49800aaccttatag
tatcaaacac agatcgtaaa cttgctcatt ttgtaattag gaagtgaaaa 49860tgtgcctttc
tcaatctcta aatgtattaa atggttgagt accgtactct aacaaatcag 49920caaaattaca
aggaaacaaa tatctctgaa gagtcagtga aatgggagaa ttataaaatg 49980accagtctac
tttctcaata tgctttcatt tattaagcac ttgctatgtg taaggtccta 50040ggaaccccag
agacatgaca ccatgttcca tgctctacat agctgtggtg gcaggacatg 50100caaaataacc
tacatacact atgaaataag tagtaataca ctgggattcc atatcctatg 50160gaggtagaga
agcaattaaa gtaccgagca aagtattgaa gaaaacctag acactaccag 50220agtataagag
tcaggatggc atctctctaa ccatcactat tttcatctgt cagacaaggc 50280caaggattgt
gagtgtggat aagagtggaa gaagaaaata gatcttttaa cctctattcc 50340atgcttaaga
ttctatagtt ctgaccccac tggaaatcca gtggttgaaa acttaatggc 50400agtttataga
gaagaaaact tctcctaaca gaagagatga aacttcctct agggcagaaa 50460gcaagtgtag
gatatcagtt agtagagaag agggctttct tgcatggaac attacatgtg 50520gtatgttgag
gggctagtga attttgttga ttggtgtgga tttatgggga tgaaaaatat 50580gatttcagag
taacatatga tacagattaa gaagtcctgc atttaagaaa tttgacttag 50640attttattag
tggagtagtg aaaagccatg gatatattta ggatgttgct gaagtagaac 50700aaaaagttag
cccatcaaaa atgcatggcg tagctgagaa aacattactg gagtctgacc 50760agctgaattt
gattcctaat tctgccactt gctagccatg caccccagtt caagctacct 50820aacttctgag
cctctacttt ctcatatttg gtggagatgt tcacattcct cttaagatgg 50880aatgagagtt
cttgtgggag ttaaatgaca tactgaatat taaatcccta gccacataac 50940tgtcacaaag
tgtaaattca acaaatgggt ttcttccttc cctagtttct tctctttcta 51000ttactggaga
atctcttggc aaaactaatt actttgtaca aaaagtaaga acaaaggtta 51060gattactctg
aagggcagtt ggttcgtttc tggcctggtt atctaacaac acatttcact 51120gagaacatta
aagataaaat aatcaaaagg aatgcaagac taaagtagac caaacatgtc 51180aatttcgttg
aacataattt ggtcagactt taaggtgaag gcataaagaa atcaaataag 51240agaaggactg
aaaacaggaa tcaaccatca gtaatttcct aaaaatctag aacatgagta 51300tagataattt
ctttcaaata caaaggaaaa agttgtgggt tttttttttt ttacttcaaa 51360aattcaacta
gagaatgcta cagttaaagt gtacctgaaa ccaaatttgg tgaatttaat 51420aaccaagtca
tttgctgcaa gatgttccaa gagttacaag ttattactca ggcgataacc 51480tcaaatgact
cccaagagtt aaattataaa ttttcctcaa caatagagag aattgatcag 51540ttgagaacag
agtcctcaaa gagcgaaaaa tggtgttatg cagaccccag tgtgtttgaa 51600ggtgatacag
agtatgcaaa tcttgatttt gcatctctga tcatagtgtt ggctctggga 51660ttagttaaaa
gacagaaatt cctccctttg tgtaactgta ctgtttcact taggttaacg 51720taggtccttt
aagactttgc tagtatgcca ttttaaactt gattggctct tagcccctcc 51780caccatattt
ttttcctcct gtcccataga aaagggagca aggaagtgta aactaatggt 51840cgtatagtga
gaacacaaat gaattttttt tcttgatatt taccaagtgt gttcagcaaa 51900cagttcttct
gtgcctactt aattagtgcc agtcttaatc ctgggtacag agaggaaatc 51960agatgtaatg
tggtactagt gccttttttt tttttacagt gtggaccctg gacaagaagc 52020ataggttgat
atcacttgca agtttattac caatgccaac tcaggcttca ctcgagacct 52080gctaattcag
aatcagtatt tgaataaggt cccaggtgat ttgtacattg aagattttag 52140aagtatacaa
acagtaactc acagacatag taagatgaag agaaatccaa acagatgttt 52200ggggctatgt
cgtagatata gtataggcat aggaatccta aggagcagga catatttcag 52260cctgaatatg
acaaaaatcc cacttattac tctcctgaga gcttcaagtg cctatatgac 52320ccaaaataca
atggaaagcc tattggcgaa agtcatgggt tgattgatct aaccctgaga 52380taaaatactt
attaaaatat tatcttttaa tgggtttcag acaactgagg ctaagccctt 52440taattacttt
aaggaccatg ctcactgaag cttttaaaag gtattttcaa aagcttaatt 52500gcccaagaaa
ataatcagtg taagagtatt aggttaccca gcagaaaatg atgtcttcta 52560catacctgtc
tacatcacaa gaagggaggg gtaaaaaagg atcaagatct tattcttctg 52620taagcctaca
tgtgcatgag tgttatgatt ttgagactac tcttatatac atgtaatttg 52680atcctcttat
caaaacaata tagagaataa ctgagcccaa tctttttagt catctcttca 52740acaaggggta
aatcagtcag tttctaaaac tggtgggagg tctccataaa cctgataaca 52800agatcccaaa
atccaaactg attgactgag ttaattcctg atcatttggg ttgaacttaa 52860gagttataca
agaaaatggt aggggacgag gaggttgtat aaaggggaaa aaacaacaac 52920tgcaaaaagc
ccaagagcct gaatttagac caatctatca tcttcctcct cttaaaaaga 52980aaacaattta
aaagttttaa ataaaaaata aaggtcatgt ttttgttttg ccaagaatca 53040aaagattttg
ctgaaactac tgctgcaaaa tattttgttt caagccatct tagggcacct 53100cagactaaaa
atgaaaccat gatcaatttc tatcccctta ccacttctat gacaatcaca 53160catggtaaat
acaagctctg ctctagtact acaataaaac tgtagaacta gagtagacct 53220tgtagtgatc
atatattact acttctcctc ttgtcctatg tccatcttat ttcaatctaa 53280actagaaatg
acaaaattct tgttgggtcg aattgtcttt tgagtagttt tagagctttt 53340tgttttcatt
tctccaagat gatcttgttg catctgcagt tcgagttttt aaaatcaagt 53400agaataatgt
caaaagggga caatttatgt gagaaatcaa ctgacagtac atgattattt 53460aaaatgaaag
ttttaaagaa aattttcccc acaagggaat ctgtgtaaga ccagaaatct 53520tatgattggc
tagctactat ataaaatgct ctgtacacaa gaaatatttt ctattgtccc 53580tagccagtaa
aacaagaaca aactcgtacc aaacatgaac tacagtatat ttatactgct 53640gtgctaaatg
gtgtttgtgg gtatgttttt cttctctcta ggggagagta cggtttccat 53700gaatatacag
aggtcaaaac agtcacagtg aaaatctctc agaagaactc ataaagaaaa 53760tacaagagtg
gagagaagct cttcaatagc taagcatctc cttacagtca ctaatatagt 53820agattttaaa
gacaaaattt ttcttttctt gattttttta aacataagct aaatcatatt 53880agtattaata
ctacccatag aaaacttgac atgtagcttc ttctgaaaga attatttgcc 53940ttctgaaatg
tgacccccaa gtcctatcct aaataaaaaa agacaaattc ggatgtatga 54000tctctctagc
tttgtcatag ttatgtgatt ttcctttgta gctacttttg caggataata 54060attttataga
aaaggaacag ttgcatttag cttctttccc ttagtgactc ttgaagtact 54120taacatacac
gttaactgca gagtaaattg ctctgttccc agtagttata aagtccttgg 54180actgttttga
aaagtttcct aggatgtcat gtctgcttgt caaaagaaat aatccctgta 54240atatttagct
gtaaactgaa tataaagctt aataaaaaca accttgcatg attcttgtta 54300cttttgaatt
tttttaagta caagttttgg ttacagtgat ttcttcttgt cacttaaaaa 54360cagtgttaaa
ctgagcataa aggtacattt aaaagtaaaa gtctaatcca cctattctca 54420aataggtaaa
gaaacatgct gtattttcca aaagaattct caaaatcagt ggattttatc 54480tgaaatagat
ggcctcagtc cttcagtaag caattattga gttcctacaa agttttgggt 54540atgtgttaag
tgttgtagaa aagaggtgaa taatgaatgg tccattatct gaagatcttt 54600aatttagtgg
ttaataaaga cacaatccct gcaccacaga aaggaggggt cataaaaaaa 54660atgaggattt
tagaaacttg tagtgacttg cagaagtggt catgatgaag gctaactgag 54720ggaatcagga
atggcctcag gtaggaaatt tgactgaata taaaccttaa taatgggcaa 54780atttgcaatg
aataaagggg aaagaggttc tacaaaattt atcaggacat gttccattga 54840aaaaacattc
ggaaaaattc tatacaatat attttcctct cttggaattt cagattgcac 54900atcggcagca
tattataggt ttcgagaagt cctgcaacct tcttagtttt aatagagccc 54960cccctttttt
tttccaaatt agcagagact ttgtctcctc tccctcacct cctcagttac 55020caccaattaa
cattaagaaa cccatgctgg gctgaataca gtggctcact cctgtaatcc 55080tagcactttg
ggaggccaca gcaggtggat ctgttgagcc caggagtttg agaccagcct 55140gggcaatggg
caaaatccca cctttattta aaaaaaaaaa ttagccaggc ataggggcac 55200aaacctgtgg
tcccatctac atgagaggct gaggcaggag gatggcttga acctgggagg 55260tcgaggctgc
agtgagccat gatcatacta ctgcactcta gcctgggtga cagagtggga 55320ccctgtttca
aaaataaaaa taaaaataaa aacccttgct gtcctatgca atgagggaac 55380ccttagagtt
ctaaggagag atctgggaat caaaagagag cagctgaaaa aaatgtcctc 55440cacatgaaac
tacaccaagc t 554617466PRTHomo
sapiensvimentin (VIM), epidiymis luminal protein 113 (HEL113),
CTRCT30 7Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly1
5 10 15Gly Pro Gly Thr
Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr 20
25 30Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala
Leu Arg Pro Ser Thr 35 40 45Ser
Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg 50
55 60Ser Ser Ala Val Arg Leu Arg Ser Ser Val
Pro Gly Val Arg Leu Leu65 70 75
80Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu
Phe 85 90 95Lys Asn Thr
Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100
105 110Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg
Phe Leu Glu Gln Gln Asn 115 120
125Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser 130
135 140Arg Leu Gly Asp Leu Tyr Glu Glu
Glu Met Arg Glu Leu Arg Arg Gln145 150
155 160Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu
Val Glu Arg Asp 165 170
175Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu
180 185 190Met Leu Gln Arg Glu Glu
Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln 195 200
205Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg
Lys Val 210 215 220Glu Ser Leu Gln Glu
Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu225 230
235 240Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln
Glu Gln His Val Gln Ile 245 250
255Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val
260 265 270Arg Gln Gln Tyr Glu
Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu 275
280 285Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu
Ala Ala Asn Arg 290 295 300Asn Asn Asp
Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg305
310 315 320Arg Gln Val Gln Ser Leu Thr
Cys Glu Val Asp Ala Leu Lys Gly Thr 325
330 335Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu
Glu Asn Phe Ala 340 345 350Val
Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355
360 365Ile Gln Asn Met Lys Glu Glu Met Ala
Arg His Leu Arg Glu Tyr Gln 370 375
380Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr385
390 395 400Arg Lys Leu Leu
Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro 405
410 415Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr
Asn Leu Asp Ser Leu Pro 420 425
430Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu
435 440 445Thr Arg Asp Gly Gln Val Ile
Asn Glu Thr Ser Gln His His Asp Asp 450 455
460Leu Glu46582151DNAHomo sapiensvimentin (VIM), epidiymis luminal
protein 113 (HEL113), CTRCT30 8gcctctccaa aggctgcaga agtttcttgc
taacaaaaag tccgcacatt cgagcaaaga 60caggctttag cgagttatta aaaacttagg
ggcgctcttg tcccccacag ggcccgaccg 120cacacagcaa ggcgatggcc cagctgtaag
ttggtagcac tgagaactag cagcgcgcgc 180ggagcccgct gagacttgaa tcaatctggt
ctaacggttt cccctaaacc gctaggagcc 240ctcaatcggc gggacagcag ggcgcgtcct
ctgccactct cgctccgagg tccccgcgcc 300agagacgcag ccgcgctccc accacccaca
cccaccgcgc cctcgttcgc ctcttctccg 360ggagccagtc cgcgccaccg ccgccgccca
ggccatcgcc accctccgca gccatgtcca 420ccaggtccgt gtcctcgtcc tcctaccgca
ggatgttcgg cggcccgggc accgcgagcc 480ggccgagctc cagccggagc tacgtgacta
cgtccacccg cacctacagc ctgggcagcg 540cgctgcgccc cagcaccagc cgcagcctct
acgcctcgtc cccgggcggc gtgtatgcca 600cgcgctcctc tgccgtgcgc ctgcggagca
gcgtgcccgg ggtgcggctc ctgcaggact 660cggtggactt ctcgctggcc gacgccatca
acaccgagtt caagaacacc cgcaccaacg 720agaaggtgga gctgcaggag ctgaatgacc
gcttcgccaa ctacatcgac aaggtgcgct 780tcctggagca gcagaataag atcctgctgg
ccgagctcga gcagctcaag ggccaaggca 840agtcgcgcct gggggacctc tacgaggagg
agatgcggga gctgcgccgg caggtggacc 900agctaaccaa cgacaaagcc cgcgtcgagg
tggagcgcga caacctggcc gaggacatca 960tgcgcctccg ggagaaattg caggaggaga
tgcttcagag agaggaagcc gaaaacaccc 1020tgcaatcttt cagacaggat gttgacaatg
cgtctctggc acgtcttgac cttgaacgca 1080aagtggaatc tttgcaagaa gagattgcct
ttttgaagaa actccacgaa gaggaaatcc 1140aggagctgca ggctcagatt caggaacagc
atgtccaaat cgatgtggat gtttccaagc 1200ctgacctcac ggctgccctg cgtgacgtac
gtcagcaata tgaaagtgtg gctgccaaga 1260acctgcagga ggcagaagaa tggtacaaat
ccaagtttgc tgacctctct gaggctgcca 1320accggaacaa tgacgccctg cgccaggcaa
agcaggagtc cactgagtac cggagacagg 1380tgcagtccct cacctgtgaa gtggatgccc
ttaaaggaac caatgagtcc ctggaacgcc 1440agatgcgtga aatggaagag aactttgccg
ttgaagctgc taactaccaa gacactattg 1500gccgcctgca ggatgagatt cagaatatga
aggaggaaat ggctcgtcac cttcgtgaat 1560accaagacct gctcaatgtt aagatggccc
ttgacattga gattgccacc tacaggaagc 1620tgctggaagg cgaggagagc aggatttctc
tgcctcttcc aaacttttcc tccctgaacc 1680tgagggaaac taatctggat tcactccctc
tggttgatac ccactcaaaa aggacacttc 1740tgattaagac ggttgaaact agagatggac
aggttatcaa cgaaacttct cagcatcacg 1800atgaccttga ataaaaattg cacacactca
gtgcagcaat atattaccag caagaataaa 1860aaagaaatcc atatcttaaa gaaacagctt
tcaagtgcct ttctgcagtt tttcaggagc 1920gcaagataga tttggaatag gaataagctc
tagttcttaa caaccgacac tcctacaaga 1980tttagaaaaa agtttacaac ataatctagt
ttacagaaaa atcttgtgct agaatacttt 2040ttaaaaggta ttttgaatac cattaaaact
gctttttttt ttccagcaag tatccaacca 2100acttggttct gcttcaataa atctttggaa
aaactcaaaa aaaaaaaaaa a 21519781PRTHomo sapiensbeta-catenin
(CTNNB), catenin beta-1 (CTNNB1), cadherin-associated protein,
armadillo homolog, MRD19 9Met Ala Thr Gln Ala Asp Leu Met Glu Leu Asp Met
Ala Met Glu Pro1 5 10
15Asp Arg Lys Ala Ala Val Ser His Trp Gln Gln Gln Ser Tyr Leu Asp
20 25 30Ser Gly Ile His Ser Gly Ala
Thr Thr Thr Ala Pro Ser Leu Ser Gly 35 40
45Lys Gly Asn Pro Glu Glu Glu Asp Val Asp Thr Ser Gln Val Leu
Tyr 50 55 60Glu Trp Glu Gln Gly Phe
Ser Gln Ser Phe Thr Gln Glu Gln Val Ala65 70
75 80Asp Ile Asp Gly Gln Tyr Ala Met Thr Arg Ala
Gln Arg Val Arg Ala 85 90
95Ala Met Phe Pro Glu Thr Leu Asp Glu Gly Met Gln Ile Pro Ser Thr
100 105 110Gln Phe Asp Ala Ala His
Pro Thr Asn Val Gln Arg Leu Ala Glu Pro 115 120
125Ser Gln Met Leu Lys His Ala Val Val Asn Leu Ile Asn Tyr
Gln Asp 130 135 140Asp Ala Glu Leu Ala
Thr Arg Ala Ile Pro Glu Leu Thr Lys Leu Leu145 150
155 160Asn Asp Glu Asp Gln Val Val Val Asn Lys
Ala Ala Val Met Val His 165 170
175Gln Leu Ser Lys Lys Glu Ala Ser Arg His Ala Ile Met Arg Ser Pro
180 185 190Gln Met Val Ser Ala
Ile Val Arg Thr Met Gln Asn Thr Asn Asp Val 195
200 205Glu Thr Ala Arg Cys Thr Ala Gly Thr Leu His Asn
Leu Ser His His 210 215 220Arg Glu Gly
Leu Leu Ala Ile Phe Lys Ser Gly Gly Ile Pro Ala Leu225
230 235 240Val Lys Met Leu Gly Ser Pro
Val Asp Ser Val Leu Phe Tyr Ala Ile 245
250 255Thr Thr Leu His Asn Leu Leu Leu His Gln Glu Gly
Ala Lys Met Ala 260 265 270Val
Arg Leu Ala Gly Gly Leu Gln Lys Met Val Ala Leu Leu Asn Lys 275
280 285Thr Asn Val Lys Phe Leu Ala Ile Thr
Thr Asp Cys Leu Gln Ile Leu 290 295
300Ala Tyr Gly Asn Gln Glu Ser Lys Leu Ile Ile Leu Ala Ser Gly Gly305
310 315 320Pro Gln Ala Leu
Val Asn Ile Met Arg Thr Tyr Thr Tyr Glu Lys Leu 325
330 335Leu Trp Thr Thr Ser Arg Val Leu Lys Val
Leu Ser Val Cys Ser Ser 340 345
350Asn Lys Pro Ala Ile Val Glu Ala Gly Gly Met Gln Ala Leu Gly Leu
355 360 365His Leu Thr Asp Pro Ser Gln
Arg Leu Val Gln Asn Cys Leu Trp Thr 370 375
380Leu Arg Asn Leu Ser Asp Ala Ala Thr Lys Gln Glu Gly Met Glu
Gly385 390 395 400Leu Leu
Gly Thr Leu Val Gln Leu Leu Gly Ser Asp Asp Ile Asn Val
405 410 415Val Thr Cys Ala Ala Gly Ile
Leu Ser Asn Leu Thr Cys Asn Asn Tyr 420 425
430Lys Asn Lys Met Met Val Cys Gln Val Gly Gly Ile Glu Ala
Leu Val 435 440 445Arg Thr Val Leu
Arg Ala Gly Asp Arg Glu Asp Ile Thr Glu Pro Ala 450
455 460Ile Cys Ala Leu Arg His Leu Thr Ser Arg His Gln
Glu Ala Glu Met465 470 475
480Ala Gln Asn Ala Val Arg Leu His Tyr Gly Leu Pro Val Val Val Lys
485 490 495Leu Leu His Pro Pro
Ser His Trp Pro Leu Ile Lys Ala Thr Val Gly 500
505 510Leu Ile Arg Asn Leu Ala Leu Cys Pro Ala Asn His
Ala Pro Leu Arg 515 520 525Glu Gln
Gly Ala Ile Pro Arg Leu Val Gln Leu Leu Val Arg Ala His 530
535 540Gln Asp Thr Gln Arg Arg Thr Ser Met Gly Gly
Thr Gln Gln Gln Phe545 550 555
560Val Glu Gly Val Arg Met Glu Glu Ile Val Glu Gly Cys Thr Gly Ala
565 570 575Leu His Ile Leu
Ala Arg Asp Val His Asn Arg Ile Val Ile Arg Gly 580
585 590Leu Asn Thr Ile Pro Leu Phe Val Gln Leu Leu
Tyr Ser Pro Ile Glu 595 600 605Asn
Ile Gln Arg Val Ala Ala Gly Val Leu Cys Glu Leu Ala Gln Asp 610
615 620Lys Glu Ala Ala Glu Ala Ile Glu Ala Glu
Gly Ala Thr Ala Pro Leu625 630 635
640Thr Glu Leu Leu His Ser Arg Asn Glu Gly Val Ala Thr Tyr Ala
Ala 645 650 655Ala Val Leu
Phe Arg Met Ser Glu Asp Lys Pro Gln Asp Tyr Lys Lys 660
665 670Arg Leu Ser Val Glu Leu Thr Ser Ser Leu
Phe Arg Thr Glu Pro Met 675 680
685Ala Trp Asn Glu Thr Ala Asp Leu Gly Leu Asp Ile Gly Ala Gln Gly 690
695 700Glu Pro Leu Gly Tyr Arg Gln Asp
Asp Pro Ser Tyr Arg Ser Phe His705 710
715 720Ser Gly Gly Tyr Gly Gln Asp Ala Leu Gly Met Asp
Pro Met Met Glu 725 730
735His Glu Met Gly Gly His His Pro Gly Ala Asp Tyr Pro Val Asp Gly
740 745 750Leu Pro Asp Leu Gly His
Ala Gln Asp Leu Met Asp Gly Leu Pro Pro 755 760
765Gly Asp Ser Asn Gln Leu Ala Trp Phe Asp Thr Asp Leu
770 775 780103256DNAHomo
sapiensbeta-catenin (CTNNB), catenin beta-1 (CTNNB1) transcript
variant 3, cadherin-associated protein, armadillo homolog, MRD19
10aggatacagc ggcttctgcg cgacttataa gagctccttg tgcggcgcca ttttaagcct
60ctcggtctgt ggcagcagcg ttggcccggc cccgggagcg gagagcgagg ggaggcggag
120acggaggaag gtctgaggag cagcttcagt ccccgccgag ccgccaccgc aggtcgagga
180cggtcggact cccgcggcgg gaggagcctg ttcccctgag ggtatttgaa gtataccata
240caactgtttt gaaaatccag cgtggacaat ggctactcaa gctgatttga tggagttgga
300catggccatg gaaccagaca gaaaagcggc tgttagtcac tggcagcaac agtcttacct
360ggactctgga atccattctg gtgccactac cacagctcct tctctgagtg gtaaaggcaa
420tcctgaggaa gaggatgtgg atacctccca agtcctgtat gagtgggaac agggattttc
480tcagtccttc actcaagaac aagtagctga tattgatgga cagtatgcaa tgactcgagc
540tcagagggta cgagctgcta tgttccctga gacattagat gagggcatgc agatcccatc
600tacacagttt gatgctgctc atcccactaa tgtccagcgt ttggctgaac catcacagat
660gctgaaacat gcagttgtaa acttgattaa ctatcaagat gatgcagaac ttgccacacg
720tgcaatccct gaactgacaa aactgctaaa tgacgaggac caggtggtgg ttaataaggc
780tgcagttatg gtccatcagc tttctaaaaa ggaagcttcc agacacgcta tcatgcgttc
840tcctcagatg gtgtctgcta ttgtacgtac catgcagaat acaaatgatg tagaaacagc
900tcgttgtacc gctgggacct tgcataacct ttcccatcat cgtgagggct tactggccat
960ctttaagtct ggaggcattc ctgccctggt gaaaatgctt ggttcaccag tggattctgt
1020gttgttttat gccattacaa ctctccacaa ccttttatta catcaagaag gagctaaaat
1080ggcagtgcgt ttagctggtg ggctgcagaa aatggttgcc ttgctcaaca aaacaaatgt
1140taaattcttg gctattacga cagactgcct tcaaatttta gcttatggca accaagaaag
1200caagctcatc atactggcta gtggtggacc ccaagcttta gtaaatataa tgaggaccta
1260tacttacgaa aaactactgt ggaccacaag cagagtgctg aaggtgctat ctgtctgctc
1320tagtaataag ccggctattg tagaagctgg tggaatgcaa gctttaggac ttcacctgac
1380agatccaagt caacgtcttg ttcagaactg tctttggact ctcaggaatc tttcagatgc
1440tgcaactaaa caggaaggga tggaaggtct ccttgggact cttgttcagc ttctgggttc
1500agatgatata aatgtggtca cctgtgcagc tggaattctt tctaacctca cttgcaataa
1560ttataagaac aagatgatgg tctgccaagt gggtggtata gaggctcttg tgcgtactgt
1620ccttcgggct ggtgacaggg aagacatcac tgagcctgcc atctgtgctc ttcgtcatct
1680gaccagccga caccaagaag cagagatggc ccagaatgca gttcgccttc actatggact
1740accagttgtg gttaagctct tacacccacc atcccactgg cctctgataa aggctactgt
1800tggattgatt cgaaatcttg ccctttgtcc cgcaaatcat gcacctttgc gtgagcaggg
1860tgccattcca cgactagttc agttgcttgt tcgtgcacat caggataccc agcgccgtac
1920gtccatgggt gggacacagc agcaatttgt ggagggggtc cgcatggaag aaatagttga
1980aggttgtacc ggagcccttc acatcctagc tcgggatgtt cacaaccgaa ttgttatcag
2040aggactaaat accattccat tgtttgtgca gctgctttat tctcccattg aaaacatcca
2100aagagtagct gcaggggtcc tctgtgaact tgctcaggac aaggaagctg cagaagctat
2160tgaagctgag ggagccacag ctcctctgac agagttactt cactctagga atgaaggtgt
2220ggcgacatat gcagctgctg ttttgttccg aatgtctgag gacaagccac aagattacaa
2280gaaacggctt tcagttgagc tgaccagctc tctcttcaga acagagccaa tggcttggaa
2340tgagactgct gatcttggac ttgatattgg tgcccaggga gaaccccttg gatatcgcca
2400ggatgatcct agctatcgtt cttttcactc tggtggatat ggccaggatg ccttgggtat
2460ggaccccatg atggaacatg agatgggtgg ccaccaccct ggtgctgact atccagttga
2520tgggctgcca gatctggggc atgcccagga cctcatggat gggctgcctc caggtgacag
2580caatcagctg gcctggtttg atactgacct gtaaatcatc ctttaggagt aacaatacaa
2640atggattttg ggagtgactc aagaagtgaa gaatgcacaa gaatggatca caagatggaa
2700tttatcaaac cctagccttg cttgttaaat tttttttttt ttttttttaa gaatatctgt
2760aatggtactg actttgcttg ctttgaagta gctctttttt tttttttttt tttttttttg
2820cagtaactgt tttttaagtc tctcgtagtg ttaagttata gtgaatactg ctacagcaat
2880ttctaatttt taagaattga gtaatggtgt agaacactaa ttcataatca ctctaattaa
2940ttgtaatctg aataaagtgt aacaattgtg tagccttttt gtataaaata gacaaataga
3000aaatggtcca attagtttcc tttttaatat gcttaaaata agcaggtgga tctatttcat
3060gtttttgatc aaaaactatt tgggatatgt atgggtaggg taaatcagta agaggtgtta
3120tttggaacct tgttttggac agtttaccag ttgcctttta tcccaaagtt gttgtaacct
3180gctgtgatac gatgcttcaa gagaaaatgc ggttataaaa aatggttcag aattaaactt
3240ttaattcatt cgattg
325611387PRTHomo sapiensmilk fat globule-EGF factor 8 protein (MFGE8,
MFG-E8), lactadherin isoform a preprotein, sperm associated antigen
10 (SPAG10), O-acetyl disialogangliocide synthase (OAcGD3S), medin,
sperm surface protein hP47, breast epithelial antigen BA46 11Met Pro
Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys1 5
10 15Ala Pro Ser Leu Leu Val Ala Leu
Asp Ile Cys Ser Lys Asn Pro Cys 20 25
30His Asn Gly Gly Leu Cys Glu Glu Ile Ser Gln Glu Val Arg Gly
Asp 35 40 45Val Phe Pro Ser Tyr
Thr Cys Thr Cys Leu Lys Gly Tyr Ala Gly Asn 50 55
60His Cys Glu Thr Lys Cys Val Glu Pro Leu Gly Leu Glu Asn
Gly Asn65 70 75 80Ile
Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu
85 90 95Gly Leu Gln His Trp Val Pro
Glu Leu Ala Arg Leu Asn Arg Ala Gly 100 105
110Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro
Trp Ile 115 120 125Gln Val Asn Leu
Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln 130
135 140Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys
Ala Phe Lys Val145 150 155
160Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn
165 170 175Lys Lys His Lys Glu
Phe Val Gly Asn Trp Asn Lys Asn Ala Val His 180
185 190Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr
Val Arg Leu Tyr 195 200 205Pro Thr
Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly 210
215 220Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly
Leu Lys Asn Asn Ser225 230 235
240Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly
245 250 255Leu His Leu Phe
Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln 260
265 270Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr
Gly Asn Asp Gln Trp 275 280 285Leu
Gln Val Asp Leu Gly Ser Ser Lys Glu Val Thr Gly Ile Ile Thr 290
295 300Gln Gly Ala Arg Asn Phe Gly Ser Val Gln
Phe Val Ala Ser Tyr Lys305 310 315
320Val Ala Tyr Ser Asn Asp Ser Ala Asn Trp Thr Glu Tyr Gln Asp
Pro 325 330 335Arg Thr Gly
Ser Ser Lys Ile Phe Pro Gly Asn Trp Asp Asn His Ser 340
345 350His Lys Lys Asn Leu Phe Glu Thr Pro Ile
Leu Ala Arg Tyr Val Arg 355 360
365Ile Leu Pro Val Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu 370
375 380Leu Gly Cys385122005DNAHomo
sapiensmilk fat globule-EGF factor 8 protein (MFGE8, MFG-E8),
lactadherin transcript variant 1, sperm associated antigen 10
(SPAG10), O-acetyl disialogangliocide synthase (OAcGD3S), medin,
sperm surface protein hP47, breast epithelial antigen BA46
12agtgggaggt gctgagccgc ctgatttatt ccggtcccag aggagaaggc gccagaaccc
60cgcggggtct gagcagccca gcgtgcccat tccagcgccc gcgtccccgc agcatgccgc
120gcccccgcct gctggccgcg ctgtgcggcg cgctgctctg cgcccccagc ctcctcgtcg
180ccctggatat ctgttccaaa aacccctgcc acaacggtgg tttatgcgag gagatttccc
240aagaagtgcg aggagatgtc ttcccctcgt acacctgcac gtgccttaag ggctacgcgg
300gcaaccactg tgagacgaaa tgtgtcgagc cactgggcct ggagaatggg aacattgcca
360actcacagat cgccgcctcg tctgtgcgtg tgaccttctt gggtttgcag cattgggtcc
420cggagctggc ccgcctgaac cgcgcaggca tggtcaatgc ctggacaccc agcagcaatg
480acgataaccc ctggatccag gtgaacctgc tgcggaggat gtgggtaaca ggtgtggtga
540cgcagggtgc cagccgcttg gccagtcatg agtacctgaa ggccttcaag gtggcctaca
600gccttaatgg acacgaattc gatttcatcc atgatgttaa taaaaaacac aaggagtttg
660tgggtaactg gaacaaaaac gcggtgcatg tcaacctgtt tgagacccct gtggaggctc
720agtacgtgag attgtacccc acgagctgcc acacggcctg cactctgcgc tttgagctac
780tgggctgtga gctgaacgga tgcgccaatc ccctgggcct gaagaataac agcatccctg
840acaagcagat cacggcctcc agcagctaca agacctgggg cttgcatctc ttcagctgga
900acccctccta tgcacggctg gacaagcagg gcaacttcaa cgcctgggtt gcggggagct
960acggtaacga tcagtggctg caggtggacc tgggctcctc gaaggaggtg acaggcatca
1020tcacccaggg ggcccgtaac tttggctctg tccagtttgt ggcatcctac aaggttgcct
1080acagtaatga cagtgcgaac tggactgagt accaggaccc caggactggc agcagtaaga
1140tcttccctgg caactgggac aaccactccc acaagaagaa cttgtttgag acgcccatcc
1200tggctcgcta tgtgcgcatc ctgcctgtag cctggcacaa ccgcatcgcc ctgcgcctgg
1260agctgctggg ctgttagtgg ccacctgcca cccccaggtc ttcctgcttt ccatgggccc
1320gctgcctctt ggcttctcag cccctttaaa tcaccatagg gctggggact ggggaagggg
1380agggtgttca gaggcagcac caccacacag tcacccctcc ctccctcttt cccaccctcc
1440acctctcacg ggccctgccc cagcccctaa gccccgtccc ctaaccccca gtcctcactg
1500tcctgttttc ttaggcactg agggatctga gtaggtctgg gatggacagg aaagggcaaa
1560gtagggcgtg tggtttccct gcccctgtcc ggaccgccga tcccaggtgc gtgtgtctct
1620gtctctccta gcccctctct cacacatcac attcccatgg tggcctcaag aaaggcccgg
1680aagcgccagg ctggagataa cagcctcttg cccgtcggcc ctgcgtcggc cctggggtac
1740catgtggcca caactgctgt ggccccctgt ccccaagaca cttccccttg tctccctggt
1800tgcctctctt gccccttgtc ctgaagccca gcgacacaga agggggtggg gcgggtctat
1860ggggagaaag ggagcgaggt cagaggaggg catgggttgg cagggtgggc gtttggggcc
1920ctctatgctg gcttttcacc ccagaggaca caggcagctt ccaaaatata tttatcttct
1980tcacgggaaa aaaaaaaaaa aaaaa
200513354PRTHomo sapiensmacrophage antigen CD68, microsialin isoform A
precursor, lysosomal/endosomal-associated membrane glycoprotein 4
(LAMP4), scavenger receptor class D, member 1 (SCARD1), GP110 13Met Arg
Leu Ala Val Leu Phe Ser Gly Ala Leu Leu Gly Leu Leu Ala1 5
10 15Ala Gln Gly Thr Gly Asn Asp Cys
Pro His Lys Lys Ser Ala Thr Leu 20 25
30Leu Pro Ser Phe Thr Val Thr Pro Thr Val Thr Glu Ser Thr Gly
Thr 35 40 45Thr Ser His Arg Thr
Thr Lys Ser His Lys Thr Thr Thr His Arg Thr 50 55
60Thr Thr Thr Gly Thr Thr Ser His Gly Pro Thr Thr Ala Thr
His Asn65 70 75 80Pro
Thr Thr Thr Ser His Gly Asn Val Thr Val His Pro Thr Ser Asn
85 90 95Ser Thr Ala Thr Ser Gln Gly
Pro Ser Thr Ala Thr His Ser Pro Ala 100 105
110Thr Thr Ser His Gly Asn Ala Thr Val His Pro Thr Ser Asn
Ser Thr 115 120 125Ala Thr Ser Pro
Gly Phe Thr Ser Ser Ala His Pro Glu Pro Pro Pro 130
135 140Pro Ser Pro Ser Pro Ser Pro Thr Ser Lys Glu Thr
Ile Gly Asp Tyr145 150 155
160Thr Trp Thr Asn Gly Ser Gln Pro Cys Val His Leu Gln Ala Gln Ile
165 170 175Gln Ile Arg Val Met
Tyr Thr Thr Gln Gly Gly Gly Glu Ala Trp Gly 180
185 190Ile Ser Val Leu Asn Pro Asn Lys Thr Lys Val Gln
Gly Ser Cys Glu 195 200 205Gly Ala
His Pro His Leu Leu Leu Ser Phe Pro Tyr Gly His Leu Ser 210
215 220Phe Gly Phe Met Gln Asp Leu Gln Gln Lys Val
Val Tyr Leu Ser Tyr225 230 235
240Met Ala Val Glu Tyr Asn Val Ser Phe Pro His Ala Ala Gln Trp Thr
245 250 255Phe Ser Ala Gln
Asn Ala Ser Leu Arg Asp Leu Gln Ala Pro Leu Gly 260
265 270Gln Ser Phe Ser Cys Ser Asn Ser Ser Ile Ile
Leu Ser Pro Ala Val 275 280 285His
Leu Asp Leu Leu Ser Leu Arg Leu Gln Ala Ala Gln Leu Pro His 290
295 300Thr Gly Val Phe Gly Gln Ser Phe Ser Cys
Pro Ser Asp Arg Ser Ile305 310 315
320Leu Leu Pro Leu Ile Ile Gly Leu Ile Leu Leu Gly Leu Leu Ala
Leu 325 330 335Val Leu Ile
Ala Phe Cys Ile Ile Arg Arg Arg Pro Ser Ala Tyr Gln 340
345 350Ala Leu141872DNAHomo sapiensmacrophage
antigen CD68, microsialin transcript variant 1,
lysosomal/endosomal-associated membrane glycoprotein 4 (LAMP4),
scavenger receptor class D, member 1 (SCARD1), GP110 14ttaattacaa
aaactaatga ctaagagaga ggtggctaga gctgaggccc ctgagtcagg 60ctgtgggtgg
gatcatctcc agtacaggaa gtgagacttt catttcctcc tttccaagag 120agggctgagg
gagcagggtt gagcaactgg tgcagacagc ctagctggac tttgggtgag 180gcggttcagc
catgaggctg gctgtgcttt tctcgggggc cctgctgggg ctactggcag 240cccaggggac
agggaatgac tgtcctcaca aaaaatcagc tactttgctg ccatccttca 300cggtgacacc
cacggttaca gagagcactg gaacaaccag ccacaggact accaagagcc 360acaaaaccac
cactcacagg acaaccacca caggcaccac cagccacgga cccacgactg 420ccactcacaa
ccccaccacc accagccatg gaaacgtcac agttcatcca acaagcaata 480gcactgccac
cagccaggga ccctcaactg ccactcacag tcctgccacc actagtcatg 540gaaatgccac
ggttcatcca acaagcaaca gcactgccac cagcccagga ttcaccagtt 600ctgcccaccc
agaaccacct ccaccctctc cgagtcctag cccaacctcc aaggagacca 660ttggagacta
cacgtggacc aatggttccc agccctgtgt ccacctccaa gcccagattc 720agattcgagt
catgtacaca acccagggtg gaggagaggc ctggggcatc tctgtactga 780accccaacaa
aaccaaggtc cagggaagct gtgagggtgc ccatccccac ctgcttctct 840cattccccta
tggacacctc agctttggat tcatgcagga cctccagcag aaggttgtct 900acctgagcta
catggcggtg gagtacaatg tgtccttccc ccacgcagca cagtggacat 960tctcggctca
gaatgcatcc cttcgagatc tccaagcacc cctggggcag agcttcagtt 1020gcagcaactc
gagcatcatt ctttcaccag ctgtccacct cgacctgctc tccctgaggc 1080tccaggctgc
tcagctgccc cacacagggg tctttgggca aagtttctcc tgccccagtg 1140accggtccat
cttgctgcct ctcatcatcg gcctgatcct tcttggcctc ctcgccctgg 1200tgcttattgc
tttctgcatc atccggagac gcccatccgc ctaccaggcc ctctgagcat 1260ttgcttcaaa
ccccagggca ctgagggggt tggggtgtgg tgggggggta cccttatttc 1320ctcgacacgc
aactggctca aagacaatgt tattttcctt ccctttcttg aagaacaaaa 1380agaaagccgg
gcatgacggc tcatgcctgt aatcccagca ctttgggagg ctgaggcagg 1440tggatcactg
gaggtcagga gtttgagacc agcctggcca acatggtgaa accctgtctc 1500tactaaaaat
acaattagcc aggtgtggcg gcgtaatccc agctggcctg taatcccagc 1560tacttgggag
gctgaggcag aactgcttga acccaggagg tggaggttgc agtgagccgt 1620catcgcgcca
ctaagccaag atcgcgccac tgcactccag cctgggcgac agagccagac 1680tgtctcaaat
aaataaatat gagataatgc agtcgggaga agggagggag agaattttat 1740taaatgtgac
gaactgcccc cccccccccc ccagcaggag agcagcaaaa tttatgcaaa 1800tctttgacgg
ggttttcctt gtcctgccag gattaaaagc catgagtttc ttgtcaaaaa 1860aaaaaaaaaa
aa 1872
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