WO2015132333A1 - Methods for predicting the response to treatment and for treating cancer - Google Patents

Abstract

The present invention relates to methods for predicting the response to treatment and for treating cancer, in particular non-small cell lung cancer.

Description

Methods for predicting the response to treatment and for treating cancer FIELD OF THE INVENTION

The present invention relates to methods for predicting the response to treatment and for treating cancer, in particular non-small cell lung cancer.

BACKGROUND OF THE INVENTION

Cancer is a class of diseases in which a group of cells display the traits of uncontrolled growth (growth and division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). Cancer represents one of the leading causes of death in the world.

Cancers can be classified according to the organ, tissue and cell-type from which the cancerous cells originate: lung, colon, liver, skin etc.

Lung cancer is the leading cause of cancer-related deaths throughout the world. Among those, non-small cell lung cancer (NSCLC) represents 80% of the cases. The size of the primary tumor, the invasion of loco-regional nodes and, the presence of distant metastases, determine the survival rate. These parameters are used to define the stage of the disease and to decide the optimal patient management.

In spite of the progress in medical and surgical treatments, long term survival remains poor, with overall values ranging from 20 to 30% at 5 years after surgery. Patients generally relapse within 3 years, with the development of metastases.

Currently available therapies for lung cancer are neo-adjuvant chemotherapy followed by surgery (the chemotherapy being decided if the patient is not operable or presents an invasion of the mediastinal ganglions), or surgery alone. In both cases, the surgical intervention can be followed by chemotherapy in order to eradicate any residual tumor cells.

In a previous study (Cherfils-Vicini et al, 2010), the inventors have shown that TLR7, a receptor for ssRNA, is expressed by lung cancer cell lines and primary NSCLC tumor cells and that in vitro stimulation of tumor cells with different TLR7 agonists increases the expression of anti-apoptotic molecules, tumor cell survival and resistance to chemotherapeutic drugs including cisplatin, carboplatin, doxorubicin and vironelbine (Cherfils-Vicini et al, 2010). However, there remains a need in the art for a method for predicting the response to treatment with an anti-cancer agent in a patient suffering from cancer, in particular NSCLC.

In addition, methods for predicting the response to an anti-cancer agent in a patient, by analyzing a sample other than a primary tumor sample are lacking.

SUMMARY OF THE INVENTION

The inventors have discovered that a high expression of TLR7 confers poor overall survival and impacts response to treatment in patients treated with neo-adjuvant chemotherapy, making TLR7 a strong prognostic marker.

In addition, the inventors have shown that TLR7 could also be a predictive marker for response to chemotherapy. Indeed, they have shown that high TLR7 expression is associated with resistance to chemotherapy in patients.

Also, the inventors have observed a good correlation of TLR7 expression on tumor cells present in the metastatic lymph nodes before and after chemotherapy and in primary tumors after chemotherapy. This indicates that mediastinal lymph nodes can be used as a representative sample for TLR7 expression in the primary tumor.

Therefore, the present invention relates a method of prognosis of cancer in a patient, comprising the step of detecting the expression of TLR7 in tumor cells of said patient.

In another aspect, the invention relates to method for predicting the response to treatment with an anti-cancer agent in a patient suffering from cancer, comprising the step of detecting the expression of TLR7 in tumor cells of said patient.

In another aspect, the invention also relates to a method for treating a patient suffering from cancer with an anti-cancer agent, comprising the steps of:

- selecting patients who do not express TLR7 in tumor cells in the primary tumor and/or who do not express TLR7 in tumor cells in metastatic lymph nodes,

- administering a therapeutically effective amount of anti-cancer agent to said patient.

The invention also relates to the use of TLR7 in lymph nodes as a bio marker. DETAILED DESCRIPTION OF THE INVENTION

Definitions As used herein, the term "cancer" refers to the pathological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to lung cancer, breast cancer, colorectal cancer, renal carcinoma, prostate cancer, melanoma, and lymphoma.

In a preferred embodiment, said cancer is lung cancer. Even more preferably, said cancer is a non-small cell lung cancer (NSCLC).

In a preferred embodiment, said cancer is diffuse large B-cell lymphoma.

The expression "cancer cells" refers to the population of cells which display uncontrolled growth. It falls within the ability of the skilled artisan to identify the cancer cells which are characteristic of each type of cancer. Many morphological markers and biomarkers are available that allow the identification of cancer cells. For example, in the case of NSCLC, the cancer cells express Epithelial Membrane Antigen (EMA), BrEp4 and AE1-AE3.

Cancer cells can be present in the primary tumor, i.e. in the tissue from which the cancer originates. In case of metastatic cancers, cancer cells can also be found in secondary tumors, metastatis, or lymph nodes.

As used herein, the term "patient" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human. In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies. As used herein, the expression "anti-cancer agent" or "chemotherapeutic agent" refers to compounds which are used in the treatment of cancer.

Anti-cancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin.

In one embodiment, the anti-cancer agent is selected for the group consisting of taxol; taxotere; platinum complexes such as cisplatin, carboplatin and oxaliplatin; doxorubicin; taxanes such as docetaxel and paclitaxel; vinca alkaloids such as vinblastine, vincristine and vinorelbine; genistein; erbstatin; and lavendustin.

In a preferred embodiment, said anti-cancer agent is cisplatin, used either alone or in combination with gemcitabine or vironelbine.

By a "therapeutically effective amount" of anti-cancer agent is meant a sufficient amount to treat cancer, preferably non-small cell lung cancer, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of anti- cancer agent will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject in need thereof will depend upon a variety of factors including the stage of non-small cell lung cancer being treated and the activity of the specific anti-cancer agent employed, the age, body weight, general health, sex and diet of the subject, the time of administration, route of administration, the duration of the treatment; drugs used in combination or coincidental with the and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

Method for predicting response to treatment

According to the invention, a biological sample is a sample comprising tumor cells obtained from the patient according to methods known in the art. In the case of solid tumors, the biological sample can be a biopsy. Typically, the biological sample according to the invention is a tumor sample obtained after medical surgery. Typically, the biological sample is a purified cancer cell sample obtained from a tissue sample.

For example, in the case of lung cancer, the biological sample can be a human primary lung tumor sample or purified primary lung tumor cells. In the case of blood cancers, the biological sample can be a blood sample or purified blood cells. In the case of skin cancer, the biological sample can be a skin biopsy or purified melanocytes.

In one embodiment, the biological sample is a primary tumor sample.

In another embodiment, the biological sample is a metastatic lymph node sample. Advantageously, in this embodiment, it is not necessary to perform invasive surgery in order to obtain the sample. Lymph node samples (also called lymph biopsy samples) can be obtained in different ways, such as, but not limited to:

Fine-needle aspiration biopsy: a thin needle is inserted into a lymph node in order to remove a sample of cells.

Core needle biopsy: a needle with a special tip is inserted in order to remove a small sample of tissue.

Open (surgical) biopsy, a small cut is made in the skin in order to remove a lymph node. If more than one lymph node is taken, the biopsy is called a lymph node dissection.

Detecting the expression of TLR7 in cancer cells can be performed by a variety of techniques.

More preferably, the detection comprises contacting the cancer cells of the biological sample with selective reagents such as probes, primers, ligands or antibodies, and thereby detecting the presence of nucleic acids or proteins of interest originally in the sample.

In a preferred embodiment, the expression may be detected by detecting the presence of mR A.

Methods for detecting the presence of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., isolated cancer cells prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). In a preferred embodiment, the expression of the TLR7 gene is detected by RT-PCR, preferably quantitative or semi-quantitative RT-PCR, even more preferably real-time quantitative or semi-quantitative RT-PCR.

Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

Probes typically comprise single- stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise contacting the cancer cells of the biological sample with a binding partner capable of selectively interacting with the TLR7 protein present in the biological sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal. In another embodiment, the binding partner may be an aptamer.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985).

Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti-TLR7 single chain antibodies. Antibodies useful in practicing the present invention also include anti-TRL7 fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to TLR7. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

Antibodies against TLR7 are available from Alexis (Grunberg, Germany) (rabbit polyclonal anti-TLR7). In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. 1997. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

The binding partners of the invention, such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88.

The aforementioned assays generally involve the binding of the binding partner (ie. Antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. The expression of the TLR7 protein in cancer cells may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. In such embodiments, cancers cells are purified from the isolated biological sample Such assays include, but are not limited to, agglutination tests; enzyme- labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against TLR7. The cancer cells of the biological sample that are suspected of containing TLR7 are then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Other standard method for isolating cancer cells expression TLR7 consists in collecting cancer cells of the biological sample and using differential antibody binding, wherein cancer cells expressing TLR7 are bound by antibodies directed to TLR7. Fluorescence activated cell sorting (FACS) may be therefore used to isolate and quantify the cancer cells expressing TLR7 In another embodiment, magnetic beads may be used to isolate cancer cells expressing TLR7 (MACS). For instance, magnetic beads labelled with monoclonal cell type specific antibodies may be used for the positive selection of cancer cells expressing TLR 7.

In a preferred embodiment, detecting the expression of TLR7 in cancer cells of the patient is carried out by immunohistochemistry performed on a biopsy or after medical surgery.

In another embodiment, detecting the expression of TLR7 in the cancer cells of a patient can be performed by subjecting said patient to imaging after administration of a quantity sufficient for imaging of a labelled agent which binds to TLR7. It falls within the ability of the skilled artisan to carry out such an imaging method. Typically, the label may be a fluorophore, a radioactive isotope or a paramagnetic agent.

The term "agents which bind to TLR7" includes agonists of TLR7 and antagonists of TLR7. On the one hand, agonists of TLR7 have been described above. On the other hand, 2'-0- methyl-modified RNAs act as TLR7 antagonists (Robbins et al, Molecular Therapy). In a particular embodiment, the cancer is selected from the group consisting of breast cancer, colorectal cancer, renal carcinoma, prostate cancer, melanoma, and lymphoma, such as lung cancer, for example non-small cell lung cancer.

In some embodiments, the anti-cancer agent is selected from taxol; taxotere; platinum complexes such as cisplatin, carboplatin and oxaliplatin; doxorubicin; taxanes such as docetaxel and paclitaxel; vinca alkaloids such as vinblastine, vincristine and vinorelbine; genistein; erbstatin; and lavendustin.

Therapeutic method

The invention also relates to a method for treating a patient suffering from cancer with an anti-cancer agent, comprising the steps of:

- selecting patients who do not express TLR7 in tumor cells in the primary tumor and/or who do not express TLR7 in tumor cells in metastatic lymph nodes,

- administering a therapeutically effective amount of anti-cancer agent to said patient.

In a preferred embodiment, the anti-cancer agent is selected from taxol; taxotere; platinum complexes such as cisplatin, carboplatin and oxaliplatin; doxorubicin; taxanes such as docetaxel and paclitaxel; vinca alkaloids such as vinblastine, vincristine and vinorelbine; genistein; erbstatin; and lavendustin.

The present invention relates to a method for treating a cancer patient comprising administering a therapeutically effective amount of an antagonist of TLR7, wherein said patient expresses TLR7 respectively in cancer cells.

Methods for determining that whether the patient expresses TLR7 in cancer cells or not can be performed as described above.

Typically medicaments according to the invention comprise an anti-cancer agent together with a pharmaceutically-acceptable carrier. A person skilled in the art will be aware of suitable carriers. Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known text such as Remington; The Science and Practice of Pharmacy.

A method of treatment according to the invention may be used in combination with any other therapeutic strategy for treating cancer, in particular non-small cell lung cancer, e.g. surgery, external radiotherapy, chemotherapy or hormone therapy or cytokine therapy.

In a particular embodiment, the cancer is selected from the group consisting of breast cancer, colorectal cancer, renal carcinoma, prostate cancer, melanoma, and lymphoma, and lung cancer, for example non-small cell lung cancer.

FIGURE LEGENDS

Figure 1: Poor prognostic value conferred by high tumor TLR7 expression in NSCLC patients treated by primary surgery. (A) Representative images of immunohistochemical staining of TLR7 on tumor cells among NSCLC patients (0%, 20%, 50% and 100% TLR7). Red perinuclear staining indicates TLR7 expression. Images were taken at 20X magnification. (B) Percentages of TLR7 expressing tumor cells among the cohort of NSCLC stage I to III patients who did not receive any treatment before the surgery (n = 352). (C) Kaplan-Meier survival curve for overall survival (OS) for the 352 patients according to the stratification of TLR7 expression, using the optimal cutoff of 82%. P value was determined by log-rank test. The table shows the number of patients at risk, events and censored between the TLR7 >82% and < 82% groups.

Figure 2: Determining the optimal cutoff for the cohort of patients not treated with neoadjuvant chemotherapy. The optimal cutoff of 82% expression of TLR7 for the cohort of 352 patients not treated with neo-adjuvant chemotherapy was chosen based on the most significant P value.

Figure 3: Poor prognostic value conferred by high tumor TLR7 expression in NSCLC patients treated by neo-adjuvant chemotherapy and surgery. Correlation of TLR7 expression among tumor cells before and after treatment. (A) Distribution of percentage of TLR7 expression by tumor cells among the cohort of stage III patients treated with chemotherapy before the surgery (n = 210). (B) Kaplan-Meier survival curves for overall survival (OS) for the 210 patients according to the stratification of TLR7 expression, using the optimal cutoff of 81%. P value was determined by log-rank test. The table shows the number of patients at risk, events and censored, between the TLR7 >81% and < 81% groups. (C) Distribution of percentage of TLR7 positive tumor cells for 55 patients with global downstaging and 1 1 1 patients without global downstaging. Table represents the mean and median TLR7 expression on tumor cells for the responder and the non-responders to chemotherapy. P values were calculated by t-test for the means and Mann- Whitney test for the medians.

Figure 4: Determining the optimal cutoff for the cohort of patients treated with neoadjuvant chemotherapy. The optimal cutoff of 81% expression of TLR7 for the cohort of 210 patients treated with neo-adjuvant chemotherapy was chosen based on the most significant P value.

Figure 5: Non-responders patients to neo-adjuvant chemotherapy have a poor prognosis. Kaplan-Meier survival curve for overall survival (OS) for the 166 patients for whom we have the information regarding the response to treatment, determined as responders and non-responders to chemotherapy with global downstaging. P value was determined by log-rank test.

Figure 6: Correlation graph for TLR7 expression by tumor cells in metastatic lymph nodes and in primary lung tumor, after treatment with neo-adjuvant chemotherapy.

Wilcoxon matched pairs signed-rank test was used to compare the percentage of TLR7 positive tumor cells in the lymph node before and after chemotherapy and in lungs from the same patients after chemotherapy. Correlations were obtained by the Spearman test.

Figure 7: High TLR7 expression on tumor cells is associated with low response to neoadjuvant chemotherapy. (A) Distribution of percentage of TLR7 positive tumor cells for 41 patients with tumor downstaging and 125 patients without tumor downstaging. (B) Distribution of percentage of TLR7 positive tumor cells for 64 patients with lymph node downstaging and 102 patients without lymph node downstaging. (C) Table represents the mean and median of TLR7 expression on tumor cells for the responders and the non- responders to chemotherapy. P values were calculated by two-tailed unpaired t test for the means and two-tailed Mann- Whitney test for the medians.

Figure 8: High TLR7 expression on tumor cells is associated with low response to different types of neo-adjuvant chemotherapy. (A) Distribution of percentage of TLR7 positive tumor cells for 25 patients with global downstaging and 59 patients without downstaging treated with cisplatin and gemcitabine combination. (B) Distribution of percentage of TLR7 positive tumor cells for 14 patients with global downstaging and 70 patients without downstaging treated with cisplatin and vironelbine combination. Tables represent the mean and median of TLR7 expression on tumor cells for the responders and the non-responders to chemotherapy. P values were calculated by two-tailed unpaired t test for the means and two-tailed Mann- Whitney test for the medians. EXAMPLES

Material and methods Patients

A retrospective series of 352 NSCLC stage I-III patients who underwent primary surgery (without neo-adjuvant chemotherapy) and who were operated between 2001-2005 was obtained from Hotel-Dieu hospital (Paris). A retrospective cohort of 210 stage III NSCLC patients who received neo-adjuvant chemotherapy and who were operated between 2000- 2007 was obtained from Hotel-Dieu hospital. Pathological staging of lung cancer was reviewed and classified according to the new TNM classification 2009 (Detterbeck et al. 2009), and histological types were determined according to the classification of the WHO (Brambilla et al, 2001).

Lung tumor samples were analyzed with the agreement of the French ethic committee (agreement 2008-133 and 2012 06-12) in accordance with article L.l 121-1 of French law. Immunohistochemistry and quantification of TLR7 positive tumor cells

Tumor samples were fixed in neutral buffered 10% formalin solution and paraffin-embedded. TLR7 staining was performed as previously described (Cherfils-Vicini et al, 2010) using TLR7-specific polyclonal antibody (ENZO Lifesciences) at 10 μg/ml. Positive staining was identified as a clear red perinuclear staining of the tumor cells. The expression level of TLR7 for each patient was determined as the average percentage of TLR7 expression by tumor cells for 10 fields at 20X magnification under a light microscope (Nikon eclipse, 80i), by three independent readers (SC, DD and IC), one of them being a pathologist (DD).

Statistical analysis

Overall survival (OS) curves were estimated by the Kaplan-Meier method and compared by the log-rank test. Survival comparison was adjusted for either imbalanced or prognostic baseline covariates using a Cox model. Groups of patients were obtained using the cutoff at minimal p value. Because the p values obtained might be overestimated, OS log-rank p values were corrected using the formula proposed by Altman et al (Altman et al., 1994). The overall survival was defined from the date of the surgery until the date of death or the last day of the patient's visit to the hospital. Univariate and multivariate analysis with Cox-proportional hazards regression model was carried out to identify possible factors that could influence the survival of the patients. Parameters identified at univariate analysis as possibly influencing outcome ( <0.05) were introduced in a multivariate Cox-proportional hazards regression model.

Comparisons of the mean and the median TLR7 expression in tumor cells in responders and non-responders were performed using t-test and Mann- Whitney test, respectively.

Wilcoxon matched pairs signed-rank test was used to compare TLR7 expression in lymph nodes and in primary tumors for the same patients after neo-adjuvant chemotherapy. Correlations were obtained by Spearman test. All P values were two-tailed.

P value of < 0.05 was considered statistically significant for all experiments.

Results

High TLR7 expression on tumor cells is associated with poor clinical outcome

To determine the relevance of TLR7 expression in NSCLC patients, we quantified TLR7 expressing tumor cells in a cohort of 352 untreated patients with stages I to III disease (Table 1). We found an heterogeneity of TLR7 positive tumor cells among different patients, ranging from 0 to 100% (Figure 1A). Twenty seven percent of the patients had no expression of TLR7 on their tumor cells, and 33% patients had more than 80% of tumor cells expressing TLR7 (Figure IB).

We determined whether such expression of TLR7 on tumor cells had an impact on the clinical outcome of the patients. We divided the patients in two groups based on the TLR7 distribution. The optimal cutoff, found at 82%, was determined taking into consideration the least significant P value (Figure 2), and was validated by the AUC (Area Under Curve) method. We found a significant worse outcome ( =0.0021) among patients who had more than 82%o tumor cells expressing TLR7 compared to the patients expressing less than 82% (Figure 1C). The mean overall survival was 36 months for the TLR7 >82% group and increased to 72 months for the TLR7 <82% group. Univariate and multivariate proportional hazard Cox analyses (Table 2) revealed that among parameters tested (gender, age, smoking history, histological type, pathological stage), the pathological stage and TLR7 are strong and independent predictors of survival for resected NSCLC patients. Table 1

Summary of the cohort of 352 stages I-III NSCLC patients not treated with neo-adjuvant chemotherapy based on different clinical parameters indicated as numbers and percentages. All parameters were evaluated among the 352 NSCLC patients enrolled in the retrospective study. Age was calculated at the date of the surgery. Chi-square P values were determined using the Fisher's and the Bonferroni-Dunn exact tests. Abbreviations: ADC, adenocarcinoma; SCC, squamous cell carcinoma; NA, not available.

Table 2

Univariate and Multivariate analysis for 352 NSCLC patients not treated with neo-adjuvant chemotherapy (treated by primary surgery). (A) Univariate Cox- proportional hazards analysis for overall survival according to clinical parameters. (B) Multivariate Cox proportional hazards analyses for overall survival according to clinical parameters. Parameters identified in univariate analysis as possibly influencing outcome were introduced in multivariate Cox- proportional hazards regression model. Table 3

Summary of a series of 210 stage III NSCLC patients treated with neo-adjuvant chemotherapy based on different clinical parameters indicated as numbers and percentages. All parameters were evaluated among the 210 NSCLC patients enrolled in the retrospective study. Age was calculated at the date of the surgery. Chi-square P values were determined using the Fisher's and the Bonferroni-Dunn exact tests. Abbreviations: ADC, adenocarcinoma; SCC, squamous cell carcinoma; NA, not available. * Pathological stage was obtained after chemotherapy. High TLR7 expression on tumor cells is a marker of chemoresistance

We quantified the percentages of TLR7 expressing tumor cells in a cohort of 210 stage III patients treated with chemotherapy before the surgery (Table 3). We observed a heterogeneous distribution of TLR7 expression among different patients, ranging from 0 to 100%. Twenty eight percent of the patients had no expression of TLR7 on their tumor cells and 30%) patients had more than 80% of tumor cells expressing TLR7 (Figure 3A). We also observed that high TLR7 expression (more than 81% of tumor cells expressing TLR7 determined as the optimal cutoff for this cohort, using the minimal P value approach, (Figure 4) confers poor clinical outcome (i^O.0032) (Figure 3B). The mean survival was 17 months for the TLR7>81% group and increased up to 34 months for the TLR7 <81% group.

Having observed that TLR7 triggering conferred chemoresistance both in vitro and in mice, we searched whether the percentage of TLR7 expressing tumor cells was associated with response to neo-adjuvant chemotherapy. At the end of the treatment, the global response, called "downstaging", was estimated by pathologists to determine if the tumor had regressed of not in response to chemotherapy. Among the 166 patients for whom this information was available, tumor regression was observed only in 55 patients. These patients, considered as responders, were stage III before the treatment and stage I or II after chemotherapy. As expected, the patients for whom no downstaging was observed have a poor clinical outcome as compared to the others (Figure 5). We observed strong differences in the mean and the median percentages of TLR7 expressing tumor cells when we compared patients who responded or not to the chemotherapy. The mean of TLR7 expressing cells was 27% and 50% for the responders and the non-responders, respectively (P=0.0004) and the median TLR7 was 3% and 50%, respectively P=0.0003) (Figure 5C). In addition, pathological examination of the resected lung and nodal dissection after treatment also allowed the evaluation of pathological lymph node and tumor response and assessment of possible downstaging. For few patients (n=27), we compared the percentages of TLR7 expressing tumor cells in the metastatic lymph node and in the primary lung tumor. We observed a strong positive correlation between TLR7 expression on tumor cells in lymph node and lung of the same patient (r²=0.5084, =0.0009) (Figure 6). A gain, a significant difference in the percentage of TLR7 expressing cells was observed in the responders and the non-responder patients both in tumor and lymph node samples (Figure 7). We also showed significant differences in global, tumor and lymph node downstaging in patients having more than 81% of tumor cells expressing TLR7 compared to patients having less than 81% of TLR7 expression. We compared TLR7 expression in responders and non-responder patients treated with gemcitabine or with vironelbine, in combination with cisp latin, and observed similar levels of mean and median TLR7 expression among the responders whatever the type of chemotherapy (Figure 8).

We performed univariate and multivariate proportional hazard Cox analyses on the 166 patients (Table 2). We observed in univariate analyses that among all clinical parameters tested, the number of cycles of chemotherapy, the global downstaging and the TLR7 expression were strong predictors of survival for NSCLC patients. However, in multivariate analyses, tumor TLR7 expression is not an independent prognostic marker.

Altogether, these results underline a major role of TLR7 in conferring resistance to chemotherapy consisting of platinum salt combined with gemcitabine or vironelbine.

Conclusion

The inventors have demonstrated that a high expression of TLR7 confers poor overall survival and impacts response to treatment in patients treated with neo-adjuvant chemotherapy.

In the cohort of resected patients not treated with neo-adjuvant chemotherapy, the level of TLR7 expression and the pathological stage were found to have a significant impact on the outcome of the patients. Multivariate analysis revealed TLR7 as a strong prognostic marker. In addition, the inventors have shown that TLR7 could also be a predictive marker for response to chemotherapy. The analysis of pathologic specimens indicates that most of the patients who did not respond to treatment expressed high levels of TLR7 compared to the patients who display good response. This demonstrates that high TLR7 expression confers resistance to chemotherapy in patients. There was no major difference in the expression of TLR7 among the patients based on the type of chemotherapy administered, cisplatin plus vironelbine or gemcitabine, suggesting a broad effect of TLR7 on different chemotherapeutic treatments.

In addition, the inventors have observed a good correlation of TLR7 expression on tumor cells present in the metastatic lymph nodes before and after chemotherapy and in primary tumors after chemotherapy. This indicates that mediastinal lymph nodes can be used as a representative sample for TLR7 expression in the primary tumor. REFERENCES

Throughout this application, various references describe the state of the art to which the invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

1. A method for predicting the response to treatment with an anti-cancer agent in a patient suffering from cancer, comprising the step of detecting the expression of TLR7 in tumor cells of said patient.

2. A method according to claim 1 wherein the expression of TLR7 is detected in tumors cells obtained from the primary tumor of said patient.

3. A method according to claim 1 wherein the expression of TLR7 is detected in tumors cells obtained from a metastatic lymph node of said patient.

4. The method according to any one of claims 1 to 3, wherein said cancer is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, renal carcinoma, prostate cancer, melanoma, and lymphoma.

5. The method according to claim 4, wherein said cancer is lung cancer.

6. The method or compound according to claim 5, wherein said cancer is non-small cell lung cancer ( SCLC).

7. The method according to any of the above claims, wherein said anti-cancer agent is selected from taxol; taxotere; platinum complexes such as cisplatin, carboplatin and oxaliplatin; doxorubicin; taxanes such as docetaxel and paclitaxel; vinca alkaloids such as vinblastine, vincristine and vinorelbine; genistein; erbstatin; and lavendustin.

8. The method according to any of the above claims, wherein the step of detecting the expression of TLR7 is carried out by detecting the amount of mR A encoding TLR7.

9. The method according to any of the above claims, wherein the step of detecting the expression of TLR7 is carried out by detecting the amount of TLR7 protein.

10. A method for treating a patient suffering from cancer with an anti-cancer agent, comprising the steps of: - selecting patients who do not express TLR7 in tumor cells in the primary tumor and/or who do not express TLR7 in tumor cells in metastatic lymph nodes,

- administering a therapeutically effective amount of anti-cancer agent to said patient.

11. Use of TLR7 in lymph nodes as a biomarker.