BIOMARKERS FOR RADIATION TREATMENT
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.
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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. § 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 FILEThe 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. §§ 1.821 to 1.825, is hereby incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTIONNo 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-β is a pleiotropic cytokine that is important in normal tissue homeostasis, regulates inflammation and immune responses, and controls proliferation and differentiation. TGF-β 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-β 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-β plays a key role in the response to ionizing radiation. TGF-β is activated in irradiated tissues and plays a pivotal role in development of radiation induced fibrosis.
BRIEF SUMMARY OF THE INVENTIONThe 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.
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.
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.
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.
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.
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.
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.
In one embodiment, the method comprises:
-
- (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
- (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,
- thereby treating the tumor in the subject.
In one embodiment, the method comprises:
-
- (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
- (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,
- thereby treating the tumor in the subject.
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:
-
- (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
- (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,
- thereby treating the tumor in the subject.
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.
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.
In another aspect, the disclosure provides a method for treating a tumor in a subject in need thereof, the method 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
- 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.
In another aspect, a method of identifying a subject as a candidate for treatment with ionizing radiation is disclosed, the method 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, hyaluronan, 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.
In another aspect, a method of treating a subject having a tumor is disclosed, the method comprising:
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- 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;
- wherein the two or more biomarkers are selected from the group consisting of CD44, MMP9, ALDH1A1, Vimentin, hyaluronan, beta-catenin, MFG-E8, and CD68;
- thereby treating the tumor in the subject.
In another aspect, a method for selecting a treatment for a subject having a tumor is disclosed, the method 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.
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.
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.
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.
In some embodiments, the normal tissue sample comprises non-tumor cells from the same tissue type as the tumor.
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.
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.
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.
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.
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.
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.
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
-
- (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
- (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.
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:
-
- (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
- (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.
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.
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.
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.
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.
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.).
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.
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-β inhibitors with ionizing radiation to treat cancer patients.
I. MethodsThe 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-β signalling pathway. Thus, in some embodiments, the therapeutic agent is an inhibitor of TGF-β or an inhibitor of a component of the TGF-β signalling pathway.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In another aspect, the expression level of at least one, two, three, four or more of the biomarkers described herein is determined.
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.
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.
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.
The biomarkers used in the method will now be described.
A. Biomarkers
The biomarkers described herein correlate with TGF-β 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-β inhibitors in patients. The biomarker signature is associated with but not limited to the correlation with TGF-β 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, β-catenin MFG-E8, and CD68.
MMP9: A clear correlation can be shown between MMP9, EMT and TGF-β. MMP9 regulates TGF-β and TGF-β 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.).
Vimentin (VIM): Vimentin is upregulated when TGF-β 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.
Hyaluronan (HA): Hyaluronan is an abundant glycosaminoglycan component of the extracellular matrix. It is induced by TGF-β, 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.
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.
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.
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).
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.
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.
Nuclear β-catenin: β-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.
The GenBank Accession Nos. for the biomarkers described herein are provided in the Table below.
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).
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.
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.
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-β.
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.
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).
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.
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.
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.
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.
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.
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).
B. Treatments
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.
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.
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.
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.
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.
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.
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.
In some embodiments, the treatment plan is developed and/or modified based on the expression levels of the biomarkers described herein.
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.
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.
C. Therapeutic Radiation Doses
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.
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.
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.
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™ 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.
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.
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.
D. Chemotherapeutic Agents
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.
1. Radiosensitizers
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.
2. Anti-Tumor Agents
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.
3. TGF-β Inhibitors
There is substantial evidence that TGF-β plays a crucial role in the response to ionizing radiation. TGF-β is a pleiotropic cytokine that is important in normal tissue homeostatis, regulates inflammation and immune responses, and suppresses epithelial proliferation. TGF-β 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-β 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-β 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.
4. Immune Modulators
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 1This example describes the association between the biomarkers described herein and clinical outcomes (survival and local tumor control) for lung cancer patients treated with radiation.
Statistical Methods
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.
Results
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
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 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.
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.
Predictive Ability of Biomarkers for Local Tumor Control
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
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
Subgroup Analyses for Stage II and III Patients
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).
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.
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.
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.
Type: Application
Filed: Oct 14, 2020
Publication Date: Mar 4, 2021
Applicant: Varian Medical Systems, Inc. (Palo Alto, CA)
Inventor: Renate Parry (Oakland, CA)
Application Number: 17/070,701
