JP7062125B1 - A method for predicting the prognosis of patients with lung adenocarcinoma based on the expression of basic fibroblast growth factor in the stroma. - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for predicting the prognosis of a lung adenocarcinoma patient and a method for treating the lung adenocarcinoma patient.
The present invention comprises detecting the expression of basic fibroblast growth factor (bFGF) in lung adenocarcinoma tissue obtained from a patient with lung adenocarcinoma, wherein the prognosis is positive if bFGF is positive. A method that indicates that the prognosis is good or possible, and if it is negative, the prognosis is poor or possible. It is also a pharmaceutical composition for use in treating patients with lung adenocarcinoma and contains a therapeutically effective amount of an anti-vascular endothelial growth factor (VEGF) drug.
[Selection diagram] None

Description

The present disclosure relates to a method of predicting the prognosis of a lung adenocarcinoma patient based on the expression of basic fibroblast growth factor in the present, and a method of treating lung adenocarcinoma in a subject determined to have a poor prognosis.

In 2018, the number of deaths from cancer in Japan was 1.36 million, of which 370,000 (27%) died from cancer. In addition, 980,000 new people will be diagnosed with cancer in 2018, and accurate diagnosis of cancer is a social issue.

Non-Patent Document 1 investigates the relationship between stage II and III non-small cell lung cancer and FGF2 expression, and discloses that FGF2 negative and improvement in overall survival (OS) are correlated. Non-Patent Document 2 examines the relationship between lung squamous cell carcinoma and FGF2 expression, and discloses that there is no clear correlation between FGF2 expression and OS. Non-Patent Document 3 shows that overexpression of bFGF in lung cancer is associated with a poor prognosis in resectable non-small cell lung cancer.

Int. J. Radiation Oncology Biol. Phys. , 82 (1): 442-447, 2012 Int J Clin Exp Pathol. , 8 (9): 9760-9771, 2015 PLoS One, 11 (1): e0147374. 2016

The present disclosure is a method of predicting the prognosis of a lung adenocarcinoma patient based on the expression of basic fibroblast growth factor (bFGF) in, and a method of treating lung adenocarcinoma in a subject determined to have a poor prognosis. I will provide a.

According to the present inventors, it has been found that the expression of bFGF in stromal cells during lung adenocarcinoma biopsy obtained from a lung adenocarcinoma patient affects the prognosis after cancer resection in the patient. ..

According to the present invention, the following inventions are provided.
[1] A method for predicting the prognosis of patients with lung adenocarcinoma.
It involves detecting the expression of basic fibroblast growth factor (bFGF) in lung adenocarcinoma tissue obtained from patients with lung adenocarcinoma, where the prognosis is good if bFGF is positive. A method in which the possibility is indicated and, if negative, the prognosis is poor or the possibility is indicated.
[2] The method according to the above [1], wherein the cancer patient is a patient after cancer resection.
[3] The method according to [1] or [2] above, wherein the level of expression is determined based on the ratio of positive cells to cancer cells.
[4] The method according to any one of [1] to [3] above, wherein the expression is expression of a protein.
[5] The method according to any one of [1] to [4] above, wherein the prognosis is at least one selected from the group consisting of recurrence rate and survival rate.
[6] The method according to any one of [1] to [5] above, wherein the lung adenocarcinoma is an early stage cancer selected from the group consisting of stages I and II.
[7] A pharmaceutical composition for use in treating patients with lung adenocarcinoma.
Contains therapeutically effective amounts of anti-vascular endothelial growth factor (VEGF) drug,
The pharmaceutical composition, wherein the lung adenocarcinoma patient is a patient whose prognosis is evaluated to be poor by the method according to any one of the above [1] to [6].
[8] The pharmaceutical composition according to the above [7], wherein the anti-VEGF drug is bevacizumab.
[9] A method of treating cancer in a subject having cancer.
Predicting the prognosis of the subject by the method according to any one of the above [1] to [6], and
A pharmaceutical composition comprising an anti-angiogenic agent for use in a method comprising administering a therapeutically effective amount of an anti-angiogenic agent to a subject with a predicted poor prognosis.
[10] The method according to any one of the above, wherein when the expression level of bFGF is equal to or higher than a predetermined value, it is evaluated as positive, and when it is lower than a predetermined value, it is evaluated as negative.
[11] The method according to any one of the above, wherein when the expression level of bFGF exceeds a predetermined value, it is evaluated as positive, and when it is equal to or less than a predetermined value, it is evaluated as negative.

FIG. 1 is an immunohistochemical staining image for bFGF of a cancer tissue biopsy obtained from a lung adenocarcinoma patient. FIG. 2 shows the recurrence-free survival time of the stage I patient group and the stage II-IV patient group. FIG. 3 shows the recurrence-free survival time of the stage I and II patient groups and the stage III and IV patient groups. FIG. 4 shows the survival time of the stage I patient group and the stage II-IV patient group. FIG. 5 shows the survival time of the stage I and II patient groups and the stage III and IV patient groups. FIG. 6 shows the recurrence-free survival time of bFGF-positive and bFGF-positive cases in all cases. FIG. 7 shows the recurrence-free survival time of bFGF-positive and bFGF-positive cases in the stage I and II patient groups. FIG. 8 shows the recurrence-free survival time of bFGF-positive and bFGF-positive cases in the stage I patient group. FIG. 9 shows the survival time of bFGF positive cases and negative cases in all cases. FIG. 10 shows the survival time of bFGF positive and negative cases in the stage I and II patient groups. FIG. 11 shows the survival time of bFGF positive and negative cases in the stage I patient group.

Detailed description of the invention

<Definition>
As used herein, the "subject" can be a mammal, preferably a human. The subject can be a subject who has or is at risk of having a tumor or cancer. Subjects may include subjects over the age of 70 and under the age of 70. Subjects may include smokers (eg, BI index of 700 and above) and nonsmokers.

As used herein, "cancer" is a disease characterized by uncontrolled proliferation of cells. Cancer cells may spread locally or throughout the body through the bloodstream or lymphatic system. Non-limiting examples of cancer include, for example, solid tumors. Non-limiting examples of cancer include, for example, hematopoietic tumors. Solid tumors also include, for example, lung cancer. As used herein, the term "cancerous tissue" is a tissue containing cancer. Cancer tissue can be obtained by biopsy and cancer resection. As used herein, the term "prognosis" can mean the recurrence rate (or time to recurrence) and survival rate of cancer. Recurrence includes recurrence in the primary lesion and recurrence after metastasis. The recurrence rate can be the recurrence rate after a period of treatment. The fixed period is, for example, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, or 15 years. It can be a year, or a period greater than or equal to any of these periods.

As used herein, "lung adenocarcinoma" is a type of lung cancer. Lung cancer is roughly classified into small cell lung cancer and non-small cell lung cancer, and non-small cell lung cancer is roughly classified into three cancers including lung adenocarcinoma. Non-small cell lung cancer accounts for about 80% of lung cancer, and lung adenocarcinoma accounts for about 60% of non-small cell lung cancer.

As used herein, "gene expression" or similar expressions means both messenger RNA (mRNA) expression and protein expression from genes present on the cell's genome. For mRNA, total RNA is extracted from cells by a method well known to those skilled in the art, and for example, synthesis of cDNA first strand using the poly A sequence unique to mRNA is followed by amplification (for example, polymerase chain reaction). ) Can be detected. A protein can be detected using an antibody that specifically binds to the protein (eg, polyclonal antibody or monoclonal antibody).

As used herein, "positive" in the context of gene expression in a cell means that mRNA or protein is detected at detectable levels, unless otherwise noted. Also, "negative" means that no mRNA or protein is detected at detectable levels unless otherwise noted. In the present specification, in order to further clarify the classification of "positive" and "negative" in the context of gene expression in a tissue, a threshold value is set, and a threshold value indicating an expression level equal to or higher is defined as "positive". Those showing an expression level below the threshold value are referred to as "negative". Thus, in the present specification, the criteria for determining gene expression "positive" at the cellular level and "positive" for gene expression at the tissue level are different.

As used herein, expression level means the amount of mRNA or protein expressed in a tissue or a given cell population. The expression level of mRNA can be quantified by various methods well known to those skilled in the art for quantifying mRNA such as quantitative PCR. The protein can be quantified by a method well known to those skilled in the art such as immunohistochemical staining (IHC). In IHC, an antibody binds to a protein in a tissue section, and the antibody can be colored by a dye. The intensity of color development corresponds to the expression level of the protein. Therefore, the expression level of the protein can be quantified by the intensity of color development. Further, in IHC, the expression level of the protein can be quantified based on the number of expressed cells or the cell ratio, not on the intensity of color development. Whether the protein expression level is quantified based on the intensity of color development or based on the number of expressed cells or the cell ratio is selected according to the situation and purpose.

As used herein, "basic fibroblast growth factor" (bFGF) is a growth factor belonging to the FGF family. bFGF is also called FGF2. In addition to angiogenesis, bFGF has been suggested to be involved in the development of the nervous system, lungs, muscles, bones, skin and the like. Human bFGF can have, for example, an amino acid sequence registered under NCBI Reference Sequence: NP_0019977.5. Human bFGF may have an amino acid sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more with the amino acid sequence registered under NCBI Reference Sequence: NP_0019977.5. ..

As used herein, "antibody" means immunoglobulin. The antibody can be an antibody of various isotypes, for example IgG. The antibody may preferably be a monoclonal antibody. The antibody can be a human chimeric antibody, a humanized antibody, or a human antibody. Human chimeric antibodies can be made by replacing the constant regions of non-human antibodies with the constant regions of human antibodies. Humanized antibodies can be made by replacing the 6 CDRs of a human antibody with the 6 corresponding CDRs of a non-human antibody. Human antibodies can be made using animals (eg, mice) in which at least the heavy chain variable region of immunoglobulin is replaced with the corresponding region of the human locus. When the constant region is non-human, a human antibody can be obtained by replacing the constant region with the amino acid sequence of the human antibody. As used herein, the antibody can preferably be a humanized antibody. As used herein, the antibody can preferably be a human antibody. The antibody has a signal peptide when it is produced intracellularly, but the signal peptide is excised when it is secreted extracellularly. Therefore, when administered as a drug, the antibody does not require a signal peptide.

As used herein, "CDR" is a complementarity determining region present in the heavy and light chain variable regions of an antibody. There are three in each of the heavy and light chain variable regions, and they are called CDR1, CDR2, and CDR3 from the N-terminus. The CDRs are, for example, numbered by Kabat et al. (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. Can be determined based on.

As used herein, "competing" means competing with other binding antibodies for binding to an antigen. Competition can occur when the binding sites of two antibodies overlap for an antigen. Such antibodies can be obtained by immunization with the epitope as described above, and / or by competing assays to determine if the binding of one antibody to the antigen is reduced by the other antibody. Obtainable. Competing antibodies include antibodies that compete with each other.

As used herein, the term "antibody-drug conjugate" (hereinafter, also referred to as "ADC") means a substance in which an antibody and a cytotoxic agent are linked. In the ADC, the antibody and the cytotoxic agent can be linked via a suitable linker. Chemotherapeutic agents, radioisotopes, and toxins can be used as cytotoxic agents. The ADC also includes a conjugate of the antigen-binding fragment of the antibody with the drug.

As used herein, the term "antigen-binding fragment of an antibody" means a fragment of an antibody that maintains its binding property to an antigen. Examples of the antigen-binding fragment include Fab, Fab', F (ab') ₂ , Fv, scFv (single chain Fv), diabodies, sc (Fv) ₂ (single chain (Fv) ₂ ). For example, digesting an antibody with papain can give Fab. Alternatively, digestion of the antibody with pepsin can give F (ab') ₂ , and further reduction can give Fab'. Antigen-binding fragments of other antibodies can also be prepared by methods well known to those of skill in the art. In the present invention, such an antigen-binding fragment of an antibody can be used.

<Inspection method and prediction method of the present disclosure>
According to this disclosure
(A) A method for predicting the prognosis of cancer patients.
Including the step of detecting the expression of FGF (particularly bFGF) in cancer tissue obtained from a cancer patient.
The method is provided.

In the above method (A), it can be predicted that the prognosis is good or may be good if the expression of bFGF in the cancer tissue is positive. Alternatively, in the above method, negative expression of bFGF in cancer tissue can be predicted to have poor or possible prognosis. Further, in the above method, if the expression of bFGF in the cancer tissue is positive, the prognosis can be predicted to be good or possible, and if the expression of bFGF in the cancer tissue is negative, the prognosis can be predicted. Can be predicted to be bad or possible.

In some embodiments, whether bFGF is positive or negative can be determined based on the level of bFGF protein or mRNA in the tissue (or section thereof or cell disruption solution).

In some embodiments, bFGF levels can be determined using an antibody that binds to bFGF. Under the conditions under which a complex of bFGF protein and antibody is formed, the tissue (or section thereof or cell disruption solution) is brought into contact with the antibody that binds to bFGF, and when the complex is formed, the bFGF protein is formed. Detected. Detection can be performed by methods well known to those skilled in the art, such as ELISA method, Western blotting method and protein array method. Based on this detection amount, it can be determined that the cancer tissue is bFGF positive or negative. In certain preferred embodiments, positive or negative bFGF in cancer tissue can be confirmed by immunohistochemical staining. Protein levels of bFGF can be measured using techniques well known to those of skill in the art.

In some embodiments, bFGF mRNA levels can be measured using techniques well known to those of skill in the art. For example, the mRNA level of bFGF can be determined by a quantitative measurement method such as quantitative PCR, digital PCR, microarray, or next-generation sequencer.

According to the present disclosure, in the above method (A), when the expression level of the bFGF protein is equal to or higher than the first predetermined value, it is determined to be bFGF positive, and when the expression level is less than the first predetermined value. In addition, it can be determined to be bFGF negative. Further, in the above method (A), when the expression level of bFGF mRNA is equal to or higher than the second predetermined value, it is determined to be bFGF positive, and when it is less than the second predetermined value, bFGF is determined. It can be determined to be negative.

The first predetermined value can be less than or equal to the maximum value of the expression level of bFGF protein in a cancer patient having a good prognosis, less than or equal to the third quartile, less than or equal to the average value, or less than or equal to the median value. The predetermined value can be equal to or greater than the minimum value of the expression level of bFGF protein in a group of cancer patients having a poor prognosis, greater than or equal to the first quartile, greater than or equal to the average value, or greater than or equal to the median value. The first predetermined value is less than or equal to the maximum expression level of bFGF protein in a group of cancer patients with a good prognosis, less than or equal to the third quartile, less than or equal to the mean value, or less than or equal to the median value, and has a poor prognosis. The expression level of bFGF protein in the cancer patient group can be equal to or greater than the minimum value, greater than or equal to the first quartile, greater than or equal to the mean value, or greater than or equal to the median value.

The second predetermined value can be less than or equal to the maximum value of bFGF protein expression in a prognostic cancer patient, less than or equal to the third quartile, less than or equal to the average value, or less than or equal to the median value. Predetermined values can be greater than or equal to the minimum, greater than or equal to the first quartile, greater than or equal to the mean, or greater than or equal to the median expression level of bFGF mRNA in a group of cancer patients with poor prognosis. The first predetermined value is less than or equal to the maximum value of bFGF mRNA expression in a group of cancer patients with a good prognosis, less than or equal to the third quartile, less than or equal to the mean value, or less than or equal to the median value, and has a prognosis. It can be greater than or equal to the minimum, greater than or equal to the first quartile, greater than or equal to the mean, or greater than or equal to the median expression level of bFGF protein mRNA in a group of poor cancer patients.

In the above, whether the prognosis is good or bad can be decided by the doctor. Whether the prognosis is good or bad can be determined, for example, by the length of recurrence-free survival or the length of survival. For example, the recurrence-free survival time is 1,000 hours or more, 1,100 hours or more, 1,200 hours or more, 1,300 hours or more, 1,400 hours or more, 1,500 hours or more after cancer resection. 1,600 hours or more, 1,700 hours or more, 1,800 hours or more, 1,900 hours or more, 2,000 hours or more, 2,100 hours or more, 2,200 hours or more, 2,300 hours or more, 2, Patients with 400 hours or more, 2,500 hours or more, 2,600 hours or more, 2,700 hours or more, 2,800 hours or more, 2,900 hours or more, or 3,000 hours or more are determined to have a good prognosis. And less than that (patients who were not determined to have a good prognosis) can be determined to have a poor prognosis. Or, for example, the survival period is 1,000 hours or more, 1,100 hours or more, 1,200 hours or more, 1,300 hours or more, 1,400 hours or more, 1,500 hours or more after cancer resection. 1,600 hours or more, 1,700 hours or more, 1,800 hours or more, 1,900 hours or more, 2,000 hours or more, 2,100 hours or more, 2,200 hours or more, 2,300 hours or more, 2, Patients with 400 hours or more, 2,500 hours or more, 2,600 hours or more, 2,700 hours or more, 2,800 hours or more, 2,900 hours or more, or 3,000 hours or more are determined to have a good prognosis. And less than that (patients who were not determined to have a good prognosis) can be determined to have a poor prognosis.

In certain preferred embodiments, whether bFGF is positive or negative can be determined by the proportion of bFGF-positive cells in the stromal cells (eg, cancer-related fibroblasts (CAF)) in the cancer tissue. For example, when the proportion of bFGF-positive cells is equal to or higher than a third predetermined value, it can be determined that the subject from which the cancer tissue is derived is bFGF-positive. Further, when the proportion of bFGF-positive cells is less than the third predetermined value, it can be determined that the target from which the cancer tissue is derived is bFGF-negative. Here, whether or not an individual cell is bFGF positive can be determined by immunohistochemical staining using an antibody that binds to bFGF, or by a technique of histochemical staining such as in situ hybridization to bFGF mRNA. .. Usually, these histochemical stainings can make cells with significant staining positive and cells without significant staining negative.

The third predetermined value is a threshold (or cutoff value) in the ratio of bFGF-positive cells to cancer cells. In some embodiments, the third predetermined value is 5% or higher, 6% or higher, 7% or higher, 8% or higher, 9% or higher, 10% or higher, 11% or higher, 12% or higher, 13% or higher, 14%. 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, It can be 27% or higher, 28% or higher, 29% or higher, or 30% or higher. The third predetermined value can be, for example, a value of 5-30%, 5-25%, 5-20%, or 5-15%.

In one preferred embodiment, the cancer patient is a patient after cancer resection.

In one preferred embodiment, the cancer is lung cancer, more preferably lung adenocarcinoma.

In one preferred embodiment, the cancer patient is a patient after cancer resection and the cancer is lung adenocarcinoma.

<Therapeutic method of the present disclosure>
According to this disclosure
(B) A method of treating cancer in a subject having cancer.
Methods are provided that include administering a therapeutically effective amount of an anti-angiogenic agent to a subject with bFGF-negative cancer.

Whether or not bFGF is negative can be determined by the above method (A).

The method (B) may include selecting a subject having a bFGF-negative cancer prior to said administration. The method (B) may further comprise performing the method (A) prior to the administration, in which case the subject with bFGF-negative cancer has a prognosis by the method (A). Can be the subject determined to be bad.

Examples of the anti-angiogenic agent include an anti-vascular endothelial cell growth factor therapeutic agent (anti-VEGF therapeutic agent). Examples of anti-VEGF therapeutic agents include Lucentis ™ (ranibizumab), Eylea ™ (aflibercept or VEGF trap), Avastin ™ (bevacizumab), and McGen ™ (pegaptanib). Will be. In addition, anti-VEGF therapeutic agents include, for example, lapatinib (Tykerb ™), sunitinib (Sutent ™), sorafenib (Nexavar ™), and axitinib. The anti-VEGF therapeutic agent can be, for example, an antibody that binds to VEGF (particularly a monoclonal antibody) or an antigen-binding fragment thereof, and examples thereof include an antibody that can inhibit the activation of VEGF receptor in the presence of VEGF. In some embodiments, the antibody that binds to VEGF can bind to a position on VEGF that can inhibit the binding of VEGF to the VEGF receptor. Examples of the antibody that binds to VEGF include an antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region containing heavy chain CDRs 1 to 3 of the antibody and a light chain variable region containing light chain CDRs 1 to 3 of the antibody. Can be mentioned. Heavy chain CDRs 1-3 and light chain CDRs 1-3 can be identified, for example, using a numbering system by Kabat (Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD). Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883)). In some embodiments, a region that aligns with the heavy and light chain variable regions of the clinical stage antibody group disclosed in PNAS, 114 (5) 944-949, 2017 and is distinct from the framework region (particularly the amino acid sequence). Areas with large fluctuations) can be identified as CDRs. In addition, an antibody that competes for binding to VEGF from any of the above antibodies can be used as an anti-angiogenic agent in the same manner as the above antibody. Competition can be confirmed using a competitive binding assay. For example, when VEGF is immobilized, the first antibody is bound to the immobilized VEGF to form a complex, and then the second antibody is contacted with the complex, the first antibody is used. If the antibody does not inhibit the binding of the second antibody, the first antibody and the second antibody do not compete for binding to VEGF. On the other hand, when the first antibody inhibits the binding of the second antibody, the first antibody and the second antibody compete for binding to VEGF. Competing with each other means that competition is observed even if the first antibody and the second antibody are exchanged. In some embodiments, an antibody that competes with each other for binding of any of the above antibodies to VEGF can be used as the anti-angiogenic agent as well as the above antibodies.

Antiangiogenic agents are, but are not limited to, oral, parenteral (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, intratumoral, mucosal (eg, intranasal) administration. And oral routes) and can be administered by pulmonary administration (eg, aerosolized compounds administered using an inhaler or nebulizer).

A therapeutically effective amount is the dose of the active ingredient that has therapeutic significance. Therapeutic significance can be brought about by slowing the progression of the disease, stopping the progression, reducing the symptoms of the disease, or curing the disease as compared to no administration. The therapeutically effective amount may be determined by the physician in consideration of the degree of disease progression, gender, age, and / or body weight, and may be, for example, 1 to 100 mg / kg body weight. Administration may be performed according to the dosing regimen. The dosing regimen can be tailored to provide the optimal desired response (eg, therapeutic or prophylactic response). The treatment regimen may be a single dose or may include a dosing cycle.

Anti-angiogenic agents are aqueous formulations, lyophilized formulations, or lyophilized and rehydrated formulations (particularly anticancer agents or pharmaceutical compositions for use in treating cancer). be. Anti-angiogenic agents can include pharmaceutically acceptable excipients. In some embodiments, the hydration solution is dextrose and / or saline (eg, dextrose at a concentration of about 5 w / v% and / or saline at a concentration of about 0.9 w / v%).

In some embodiments, the cancer can be lung adenocarcinoma and can be the subject after cancer resection.

In some embodiments, the anti-angiogenic agent may be administered in combination with lung cancer therapy. Lung cancer therapy may include chemotherapy, surgery, and / or radiation therapy. In chemotherapy, subjects include gefitinib (especially if the subject has an EGFR gene mutation), platinum preparations (eg, cisplatin, carboplatin, nedaplatin), metabolic antagonists (eg, pyrimidine metabolic antagonists, eg, pemetrexed, gemcitabine, etc.). TS-1), topoisomerase inhibitors (eg, irinotecan, nogitecan, etoposide), anticancer antibiotics (eg, amrubicin), microtube agonists (eg, docetaxel, pacritaxel, binorelbin), molecular target drugs (eg, eg Bebasismab, Ramsylmab, Nesitumumab, Gefitinib, Erlotinib, Afatinib, Osimertinib, Dakomitinib, Crizotinib, Alektinib, Seritinib, Lorratinib, Dubrafenib, Tramethinib, Entrektibu Can be administered with one or more selected from the following anti-cancer agents.

According to this disclosure
(C) A method of treating cancer in a subject having cancer.
Includes administration of therapeutically effective doses of chemotherapeutic agents other than anti-VEGF therapeutic agents to subjects with bFGF-positive cancer.
The method is provided.

According to this disclosure
(D) A method of treating cancer in a subject having cancer.
Determining if the subject has bFGF-positive cancer and
A therapeutically effective amount of anti-angiogenic agent is administered to a subject having bFGF-negative cancer, and a therapeutically effective amount of an anti-angiogenic agent is administered to a subject having bFGF-positive cancer other than an anti-angiogenic agent (particularly an anti-VEGF therapeutic agent). Including the administration of therapeutically effective doses of chemotherapeutic agents,
The method is provided.

According to this disclosure
(E) A method of treating cancer in a subject having cancer.
Predicting the prognosis of the subject by the method described in (A) above,
Including the administration of therapeutically effective doses of anti-angiogenic agents to subjects with a predicted poor prognosis,
The method is provided.

According to this disclosure
(F) A method of treating cancer in a subject having cancer.
Predicting the prognosis of the subject by the method described in (A) above,
Includes administration of therapeutically effective doses of chemotherapeutic agents other than anti-angiogenic agents (particularly anti-VEGF therapeutic agents) to subjects with a predicted prognosis.
The method is provided.

According to this disclosure
(G) A method of treating cancer in a subject having cancer.
Predicting the prognosis of the subject by the method described in (A) above,
A therapeutically effective amount of anti-angiogenic agent is administered to subjects with a predicted poor prognosis, and subjects other than anti-angiogenic agents (particularly anti-VEGF therapeutic agents) are administered to subjects predicted to have a good prognosis. Including the administration of therapeutically effective doses of chemotherapeutic agents,
The method is provided.

In some embodiments, the cancer can be lung adenocarcinoma and can be the subject after cancer resection.

According to the present disclosure, an anti-angiogenic agent (particularly an anti-VEGF therapeutic agent) is provided for use in the method according to any one of (B), (D), (E), and (G) above. .. According to the present disclosure, a pharmaceutical composition comprising an anti-angiogenic agent (particularly an anti-VEGF therapeutic agent) for use in the method according to any one of (B), (D), (E), and (G) above. Things are provided. According to the present disclosure, anti-angiogenic agents (particularly anti-VEGF therapeutic agents) in the manufacture of pharmaceuticals for use in any of the methods (B), (D), (E), and (G) above. Use is provided. According to the present disclosure, the use of an anti-angiogenic agent (particularly an anti-VEGF therapeutic agent) in the method according to any one of (B), (D), (E), and (G) above is provided.

According to the present disclosure, chemotherapeutic agents other than anti-angiogenic agents are provided for use in the method according to any one of (C), (D), (F), and (G) above. According to the present disclosure, a pharmaceutical composition comprising a chemotherapeutic agent other than an anti-angiogenic agent for use in the method according to any one of (C), (D), (F), and (G) above. Provided. According to the present disclosure, the use of chemotherapeutic agents other than anti-angiogenic agents in the manufacture of a pharmaceutical for use in any of the methods (C), (D), (F), and (G) above. Provided. The present disclosure provides the use of chemotherapeutic agents other than anti-angiogenic agents in any of the methods (C), (D), (F), and (G) above.

INDUSTRIAL APPLICABILITY According to the present invention, a kit for use in immunohistochemical staining of tissue specimens of lung adenocarcinoma can be provided. The kit of the present invention includes, for example, a means for detecting the expression of bFGF in a tissue sample. As a means for detecting the expression of bFGF, for example, an antibody that binds to bFGF can be mentioned. Antibodies that bind to bFGF may or may not be labeled. If the antibody that binds to bFGF is not labeled, a secondary antibody that binds to the antibody can be used. The secondary antibody can be labeled, for example. Accordingly, the kits of the invention are labeled antibodies that contain an antibody that binds to bFGF, or that are labeled or unlabeled and that bind to bFGF and are labeled to bind such antibody. Can include secondary antibodies. In addition, the kit of the present invention may further contain a color-developing substrate, a fluorescent substrate, or a luminescent substrate suitable for detection. As the label, substrates (coloring substrate, fluorescent substrate, and luminescent substrate) and enzymes used in the enzyme antibody method (for example, peroxidase, glucose oxidase, and alkaline phosphatase) can be used. Examples of the substrate include a fluorescent substrate. As the peroxidase, for example, horseradish peroxidase can be used. Horseradish peroxidase produces color, fluorescence or chemiluminescence by adding a color-developing substrate, a fluorescent substrate or a chemiluminescent substrate. Therefore, the presence of a captive molecule labeled with color development, fluorescence or chemiluminescence as an index can be detected. Coloring substrates for horseradish peroxidase include, for example, tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2-azinobis [3-ethylbenzo-thiazolin-6-sulfonic acid (ABTS), and Amplex ( The trademark) Red can be mentioned and used for the detection of labeled molecules in the presence of hydrogen peroxide. Luminescent substrates for alkaline phosphatase include p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP), and AttoPhos ™, which can be used to detect trapping molecules. Glucose oxidase oxidizes glucose to generate gluconic acid and hydrogen peroxide. Hydrogen peroxide can be easily detected by using, for example, a colorimetric probe for detecting hydrogen peroxide (for example, peroxidase). Hydrogen peroxide can develop color, for example, in the presence of peroxidase and its color-developing substrate.

Example 1: Analysis of bFGF expression using a lung adenocarcinoma biopsy In this example, expression of basic fibroblast growth factor (bFGF) in a biopsy or excised tissue obtained from a lung adenocarcinoma patient. I checked.

Of the 182 cases resected from October 2010 to September 2017 at the city general hospital, 102 cases of lung adenocarcinoma that could be immunostained and clinically pathologically examined were used. The average age of the patients was 70.5 years, including patients aged 43 to 89 years. The details of the patients were as shown in Table 1 below.

In Table 1, DM represents diabetes, CEA represents the expression of CEA markers, and EGFR represents the epidermal growth factor receptor. The EGFR mutation is a driver mutation of any of G719X, deletion in exon 19 (del19), S768I, insertion in exon 20, T790M, L858R, and L861Q.

Tissue sections were prepared from biopsies or tissue donations according to conventional methods and subjected to immunohistochemical staining. As the primary antibody, an anti-FGF2 antibody (Abcam; 1/500) was used. As the secondary antibody, the rabbit anti-mouse IgG antibody included in the BOND Polymer Refine Detection (Leica Biosystems) kit was used according to the manufacturer's instructions. Then, staining was performed using a horseradish peroxidase (HRP) -labeled anti-rabbit IgG antibody. Staining was performed by an automatic immunostainer BOND-MAX (Leica Biosystems). The stained image was evaluated by a virtual slide (device used: Nano Zoomer-XR (Hamamatsu Photonics Co., Ltd.), condition: 20x mode, Single Layer).

The results of immunohistochemical staining showed no clear staining of the tumor. However, bFGF-positive cells were found in tumor stromal cells (cancer-related fibroblasts; CAF). FIG. 1 shows the results of immunohistochemical staining of typical lung adenocarcinoma tissues.

Therefore, when more than 10% of the stromal cells in the tumor evaluated the bFGF-positive tissue as bFGF-positive, 13 out of 102 cases were bFGF-positive, and bFGF-positive in 79 stage I-II cases. There were 9 cases. In addition, 9 of the 71 stage I cases were bFGF positive.

As shown in Table 2, there was no clear correlation between bFGF positive frequency with age, gender, smoking status, CEA, EGF receptor mutation, and stage.

In 102 cases of this case, stage II-IV recurred significantly compared to stage I without lymph node metastasis (p = 0.0005) (Fig. 2), and stage III-IV compared to stage I-II, which is a local lung cancer. Recurred significantly (p = 0.0009) (Fig. 3).

In 102 cases of this case, stage II-IV had a significantly poorer prognosis than stage I without lymph node metastasis (p <0.0001) (Fig. 4), and stage III compared to stage I-II, which is a local lung cancer. -IV had a significantly poor prognosis (p <0.0001) (FIG. 5).

In all cases (n = 102), FGF2-positive patients are less likely to relapse (FIG. 6), and even in stage I-II (n = 79), which is a local lung cancer, FGF2-positive patients are less likely to relapse (DFI: Disease Free intervals). (FIG. 7), even in stage I (n = 71) without lymph node metastasis, FGF2-positive patients were less likely to relapse (FIG. 8).

In all cases (n = 102), FGF2-positive patients tend to have a good prognosis (OS: overall survivals) (FIG. 9), and even in stages I to II (n = 79), which are local lung cancers, FGF2-positive patients have a good prognosis. (FIG. 10), and even in stage I (n = 71) without lymph node metastasis, FGF2-positive patients had a good prognosis (FIG. 11).

Thus, in a biopsy of lung adenocarcinoma, a positive bFGF expression in stromal cells indicates a good prognosis, and a negative biopsy indicates a poor prognosis.

Claims (11)

A method for predicting the prognosis of patients with lung adenocarcinoma,
It involves detecting the expression of basic fibroblast growth factor (bFGF) in lung adenocarcinoma tissue obtained from patients with lung adenocarcinoma, where the prognosis is good if bFGF is positive. A method in which the possibility is indicated and, if negative, the prognosis is poor or the possibility is indicated. The method according to claim 1, wherein the cancer patient is a patient after cancer resection. The method according to claim 1 or 2, wherein the level of expression is determined based on the ratio of positive cells to cancer cells. The method according to any one of claims 1 to 3, wherein the expression is expression of a protein. The method according to any one of claims 1 to 4, wherein the prognosis is at least one selected from the group consisting of recurrence rate and survival rate. The method according to any one of claims 1 to 5, wherein the lung adenocarcinoma is an early stage cancer selected from the group consisting of stages I and II. A pharmaceutical composition for use in treating patients with lung adenocarcinoma.
Contains therapeutically effective amounts of anti-vascular endothelial growth factor (VEGF) drug,
The pharmaceutical composition, wherein the lung adenocarcinoma patient is a patient whose prognosis is evaluated to be poor by the method according to any one of claims 1 to 6. The pharmaceutical composition according to claim 7, wherein the anti-VEGF drug is bevacizumab. A method of treating lung adenocarcinoma in subjects with lung adenocarcinoma.
Predicting the prognosis of the subject by the method according to any one of claims 1 to 6.
A pharmaceutical composition comprising an anti-angiogenic agent for use in a method comprising administering a therapeutically effective amount of an anti-angiogenic agent to a subject with a predicted poor prognosis. A pharmaceutical composition for use in treating patients with lung adenocarcinoma.
Contains chemotherapeutic agents for cancer other than anti-vascular endothelial growth factor (VEGF) drugs,
The pharmaceutical composition, wherein the lung adenocarcinoma patient is a patient whose prognosis is evaluated to be good by the method according to any one of claims 1 to 6. A method of treating lung adenocarcinoma in subjects with lung adenocarcinoma.
Predicting the prognosis of the subject by the method according to any one of claims 1 to 6.
The cancer for use in a method comprising administering a therapeutically effective amount of a chemotherapeutic agent for cancer other than an anti-vascular endothelial growth factor (VEGF) drug to a subject with a predicted prognosis. A pharmaceutical composition comprising a chemotherapeutic agent.

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