METHOD FOR THE PROGNOSIS OF SURVIVAL TIME OF A PATIENT SUFFERING FROM A SOLID CANCER

Abstract

The present invention relates to an in vitro method for the prognosis of the survival time of a patient suffering from a solid cancer, comprising the quantification of the cell density of follicular B cells present in tumor-induced lymphoid structures from said patient, wherein a high density of follicular B cells indicates that the patient has a favorable prognosis and a low density of follicular B cells indicates that the patient has a poor prognosis.

Claims

1. A method of treating a patient with a solid tumor containing tumor-induced lymphoid structures, comprising the following steps: a) obtaining a sample of the solid tumor that contains tumor-induced lymphoid structures from the patient; b) quantifying within the tumor-induced lymphoid structures of the sample, a cell density of follicular B cells; and within the whole tumor sample, a cell density of mature dendritic cells; c) comparing the cell density of follicular B cells and the cell density of mature dendritic cells with predetermined reference values; and d) providing the patient with chemotherapy and/or radiotherapy when the cell density of follicular B cells and the cell density of mature dendritic cells are lower than the predetermined reference values.

2. The method of claim 1, wherein the solid tumor is a lung cancer tumor, a colorectal cancer tumor or a breast cancer tumor.

3. The method of claim 2 wherein the lung cancer tumor is a non-small cell lung cancer tumor.

4. The method of claim 1 wherein the patient is a patient with an early stage of cancer.

5. The method of claim 4 wherein the patient did not receive neo-adjuvant or adjuvant therapy.

6. The method of claim 1 wherein the patient is a patient with an advanced stage of cancer.

7. The method of claim 6 wherein, the patient has received adjuvant therapy.

8. The method of claim 1 wherein said step of quantifying is performed by immunohistochemistry with an anti-CD20 and anti-CD208 antibodies.

Description

FIGURE LEGENDS

[0037] FIG. 1. Characterization of B cell subsets into and outside Ti-BALT

[0038] Single (A-B, E-F, M-N) and double (C-D, G-L) immunostainings on paraffin-embedded lymph node (left column) and lung tumor (right column) sections. Ti-BALT B-cell areas present features of secondary follicles of reactive lymph nodes, as they are composed by: a mantle of IgD+ nave B cells (dark grey, B) surrounding a germinal centre. As in lymph nodes, CD20+ germinal centre B cells (dark grey, D, H, J, L) express the CD23 (black, D), AID (dark grey, F), Ki67 (black, H), and Bc16 (dark, J) but not Bc12 (dark, L). CD138+ plasma cells (dark grey) are exclusively detected in the stroma reaction (N) but never in Ti-BALT (data not shown). Original magnification: (A-D, K-L), 100; (E-J, M-N), 200. Abbreviation: T, Tumor nest.

[0039] FIG. 2A-C. Comparison of the subsets of B cells infiltrating lung tumors, conventional secondary lymphoid organs and peripheral blood

[0040] Flow cytometry analysis of CD19+ CD14 B cell subsets on lung tumors (n=7), lymph nodes (n=3) and peripheral blood (n=5). Representative dot plots (A) and means (B) of B cell subsets based on the expression of IgD and CD38 on the 3 localizations. Each bar represents a mean+/SD of different samples. Statistical significance of B cell subsets between sites was calculated by Mann-Whitney test. *, P<0.05. (C) Comparison of the ratio of the different differentiation stages of nave B cells (CD23CD27CD38Bm1 and CD23+ CD27CD38Bm2, left panel), germinal centre B cells (CD23CD27CD77+ CD38+Bm3 and CD23CD27CD77CD38+Bm4, centre panel) and memory B cells (CD23CD27+ CD38+ early Bm5 and CD23CD27+ CD38 late Bm5, right panel). Abbreviations: pre-GC, pre-germinal centre; GC, germinal centre.

[0041] FIG. 3A-D. Prognostic value of Ti-BALT B cells in NSCLC patients

[0042] (A) Correlation between the density of DC-Lamp+ mature DC and the density of follicular CD20+ B cells, both populations located in Ti-BALT. Kaplan-Meier curves of disease-specific survival for 74 patients with early-stage NSCLC according to the density of CD20+ follicular B cells (B), the density of DC-Lamp+ mature DC (C), and the density of both cell populations (D). P value was determined using the log-rank test. Abbreviation: DSS, Disease-Specific Survival.

[0043] FIG. 4. Prognostic value of mature DC in late-stage NSCLC patients who received neo-adjuvant chemotherapy.

[0044] Kaplan-Meier curves of disease-specific survival of 56 patients according to the density of tumor-infiltrating DC-Lamp+ mature DC. Significant differences between the two groups of patients were evaluated using the log-rank test. The median DSS was 17 months for the patients with DC-Lamp low tumors, whereas it was 36 months for the patients characterized as having DC-Lamp High tumors. P value significant when <0.05.

[0045] FIG. 5: Prognostic value of tumor-infiltrating follicular B cells and/or mature DC in late-stage NSCLC patients who received neo-adjuvant chemotherapy.

[0046] Kaplan-Meier curves of overall survival of 122 patients with advanced-stage of NSCLC and treated by neo-adjuvant chemotherapy according to the presence of a high or low density of tumor-infiltrating (A) CD20+ follicular B cells, (B) DC-Lamp+ mature DC, or (C) both CD20+ follicular B cells and DC-Lamp+ mature DC. Significant differences between the groups of patients were evaluated using the Log-rank test. P value significant when <0.05. Abbreviation: OS, Overall Survival.

TABLES

[0047]

TABLE-US-00001 TABLE 1 Clinical, histological and immunological parameters in patients with Foll- B cells low versus Foll-B cells High tumors Foll-B cells low Foll-B cells High Significance No. % No. % (P value) Density of follicular CD20+ B <0.0001 cells mean SEM 0.011 0.002 0.097 0.014 range 0.000-0.029 0.031-0.442 Gender 0.0553 male/female 34/4 89/11 26/10 72/28 Age 0.9665 mean (years) SEM 67 1 67 2 range 49-83 43-81 Smoking history 0.8374 current/never smokers 35/2 95/5 31/5 86/14 pack-years (years) SEM 45 4 45 5 range 0-100 0-100 Histological type 0.8560 ADC 24 63 22 61 SCC 14 37 14 39 Tumor differentiation 0.0819 well 19 50 9 25 intermediate 10 26 12 33 poorly 9 24 15 42 pTNM stage 0.3623 pT1N0M0 27 71 21 58 pT2N0M0 7 18 7 20 pT1N1M0 4 11 8 22 % fibrosis 0.1877 mean SEM 22 3 28 3 range 0-75 0-85 % necrosis 0.6888 mean SEM 13 3 11 3 range 1-75 1-50 % Ki67+ tumor cells 0.8895 mean SEM 36 4 36 4 range 1-80 2-80 Density of DC-Lamp+ mature 0.0002 DC mean SEM 2.652 0.444 7.049 1.034 range 0-12.935 0.500-31.667 Density of CD3+ T cells (centre 0.2067 of tumor) mean SEM 1.579 0.184 1.931 0.206 range 0.000-5.000 0.000-5.000 Density of CD3+ T cells (invasive <0.0001 margin) mean SEM 1.487 0.112 2.542 0.172 <0.0001 range 0.000-3.000 0.500-5.000 Pathologic staging and histologic types of lung cancer were determined according to the TNM staging system (Piemonte. NY, Wiley-Liss, 2002) and to the histologic classification of the WHO (Brambilla et al., Eur Respir. J., 2001), respectively. Abbreviations: NSCLC, Non-Small Cell Lung Cancer; ADC, Adenocarcinoma; SCC, Squamous Cell Carcinoma; pTNM, pathologic TNM. All parameters were evaluated among 74 early-stage NSCLC. P-values were obtained using the Fisher's and the Bonferroni-Dunn exact tests. Abbreviation: SEM, Standard Error of Measurements.

TABLE-US-00002 TABLE 2 Clinical and pathological features of NSCLC patients included in the retrospective study Total No. % gender male/female 60/14 81/19 age mean (years) SEM 64 1 range 41-79 smoking history current/never smokers 67/7 91/9 pack-years (years) SEM 45 3 range 0-100 vital status of patients alive 54 73 disease-free 51 in relapse 3 dead 20 27 from metastasis of NSCLC 9 from other causes 11 histological type ADC 46 62 SCC 28 38 pTNM stage pT1N0M0 48 65 pT2N0M0 14 19 pT1N1M0 12 16 tumor differentiation well 28 38 intermediate 22 30 poorly 24 32 % fibrosis mean SEM 25 2 range 0-85 % necrosis mean SEM 12 2 range 1-75 % Ki67.sup.+ tumor cells* mean SEM 36 3 range 1-80 Pathologic staging and histologic types of lung cancer were determined according to the TNM staging system (Piemonte. NY, Wiley-Liss, 2002) and to the histologic classification of the WHO (Brambilla et al., Eur Respir. J., 2001), respectively. Abbreviations: NSCLC, Non-Small Cell Lung Cancer; ADC, Adenocarcinoma; SCC, Squamous Cell Carcinoma; pTNM, pathologic TNM.

TABLE-US-00003 TABLE 3 Prognostic parameters for survival in univariate analysis for patients with advanced disease and treated by neo-adjuvant chemotherapy. Hazard Variable ratio 95% CI P value Clinical Sex 0.75 0.39 to 1.43 0.383 parameters Age 1.45 0.74 to 2.84 0.283 Smoker history 0.83 0.43 to 1.58 0.565 COPD 0.42 0.75 to 1.97 0.418 Side 1.17 0.69 to 1.96 0.562 Intervention 1.38 1.07 to 1.78 0.012 Resection quality 2.65 1.33 to 5.29 0.00585 Histological subtype 0.90 0.79 to 1.02 0.109 Chemotherapy drugs 0.92 0.71 to 1.20 0.548 Number of cycles 0.50 0.31 to 0.81 0.0044 Time between 0.96 0.64 to 1.42 0.826 chemotherapy and surgery % viable tumor cells 1.66 1.05 to 2.63 0.0313 TNM stage after 1.53 1.12 to 2.08 0.00678 chemotherapy Immune DC-LAMP+ cell density 0.50 0.30 to 0.83 0.00721 parameters CD20 + cell density 0.41 0.24 to 0.70 0.00095 Immune score 0.50 0.36 to 0.72 0.000097

EXAMPLES

[0048] In the following description, all molecular biology experiments for which no detailed protocol is given are performed according to standard protocol.

Abbreviations

[0049] ADC, adenocarcinoma; BALT, Bronchus-Associated Lymphoid Tissue; DC, Dendritic Cell; NSCLC, Non-Small-Cell Lung Cancer; SCC, Squamous-Cell Carcinoma; TIL, Tumor-Infiltrating Lymphocyte.

Example 1

Summary

[0050] The aim of the present study was to determine whether a protective humoral immune response takes place within Ti-BALT.

[0051] Here, we studied B-cell differentiation and migration to Ti-BALT by using complementary approaches (immunohistochemistry, flow cytometry, laser-capture micro-dissection, PCR low density array) on a series of 104 NSCLC patients. We have shown that B cell follicles of Ti-BALT present the same cellular composition and organization as in canonical secondary lymphoid organs. All stages of differentiation could be detected among tumor-infiltrating B cells. Interestingly, the major B cell compartments were compartments comprising effector cells (memory B cells and plasma cells). We have also shown that the somatic mutation and isotype switching machineries are activated in B cell follicles of Ti-BALT in accordance with the presence of Bm3 and Bm4 cells. We have also studied B cell recruitment to Ti-BALT in order to identify chemoattractants orchestrating this migration. Interestingly, intra-tumoral PNAd+ high endothelial venules were exclusively associated with Ti-BALT. The PNAd+ ligand, CD62L, was selectively detected on most Ti-BALT lymphocytes including all B cells but not on germinal center B cells, as reported for conventional secondary lymphoid organs. The analysis of the chemokine receptor profiles indicated that CXCR5 is expressed by nave B cells, which parallels the expression of its unique ligand, CXCL13 in the B-cell areas of Ti-BALT. Finally, CXCR5 expression decreased on fully differentiated B cells, a known key regulatory process that allows effector cells to leave the germinal centre, as previously described in lymph nodes.

[0052] Finally, we demonstrated that the density of Ti-BALT B cells is correlated with a long-term survival for lung cancer patients.

[0053] All together, these data indicate that Ti-BALT present strong similarity with canonical lymphoid organ, a site specialized in the induction and memory establishment of adaptive immune responses. Thus, Ti-BALT represents an active site for the initiation of a protective humoral immunity. The quantification of follicular B-cells would allow the identification of high-risk patients.

Materials and Methods

Patients

[0054] Fresh and paraffin-embedded lung tumor samples were obtained from NSCLC patients undergoing surgery at Institut Mutualiste Montsouris, Hotel Dieu, Tenon and European Georges Pompidou Hospitals (Paris, France). Pre-operative evaluation of patients included lung, brain, and adrenal CT scan and liver ultrasound echography. They all underwent complete surgical resection of their tumors, including multilevel lymph node sampling or lymphadenectomy, but none received pre-operative chemotherapy or radiotherapy. Patients with an Eastern Cooperative Oncology Group performance status (Finkelstein et al., JCO, 1988)1 were eligible. For the retrospective study, paraffin-embedded tumor biopsies with representative areas of tumor and adjacent lung parenchyma were retrieved from 74 successive patients diagnosed between 1998 and 2002 with early-stage NSCLC (Mountain, Cancer Chest, 1997). The main clinical and pathological features of the patients for the retrospective study are presented in Table 2. Patients with mixed histologic features, a T3 tumor, or pleural invasion were ineligible. At the completion of the study, the minimal clinical follow-up was 48 months for the last patient included in the cohort. Non-tumoral lymph nodes were obtained after surgery from patients suffering from cardiac diseases. Peripheral blood was obtained from healthy volunteers at the Centre National de la Transfusion Sanguine (Paris, France). The protocol was approved by local ethic and human investigations committee (n 2008-133), and by the Assistance Publique-Hopitaux de Paris (AP-HP), in application with the article L.1121-1 of French law. A written informed consent was obtained from the patients prior to inclusion in the study.

[0055] Immunohistochemistry

[0056] Serial 5 m tissue sections of paraffin-embedded lung tumors were deparaffinized, rehydrated, and pretreated in appropriate buffer for antigen retrieval. Then, the sections were incubated with 5% human serum for 30 min before adding the appropriate antibodies or isotype controls. Enzymatic activity was revealed as described previously (MCD, JCO, 2008). When necessary, sections were counterstained with hematoxylin. Images were acquired using a Nikon Eclipse 80i microscope (Nikon, Champigny-sur-Marne, France) operated with Nikon NIS Elements BR software.

[0057] Method for Cell Quantification

[0058] The cell quantification was measured quantitatively in the tumoral areas of the entire tissue section (original magnification: 100) using the Nikon Eclipse 80i microscope and operated with Nikon NIS Elements BR software. The density of follicular B cells of Ti-BALT was expressed as a surface of follicular CD20+ B cells per tumor intermediate power field (IPF) with SEM calculated. The number of DC-Lamp.sup.+ mature DC was lower than the number of cells described above allowing us to realize a quantitative counting (mean DC per tumor IPF with SEM calculated). According to the standard evaluation by pathologists, the necrosis and fibrosis were counted as the percentage of the positive areas among the whole tumor mass section.

[0059] Enrichment of Tumor-Infiltrating Lymphocytes

[0060] Fresh lung tumor specimens were mechanically dissociated and incubated in a non-enzymatic solution (Cell Recovery solution, BD Biosciences, Le Pont-de-Claix, France) for 1 h at 4 C. The cell suspension was then filtrated through a 70 m filter (BD Biosciences), and the mononuclear cells were isolated by centrifugation over Ficoll Hypaque.

[0061] Flow Cytometry

[0062] Multiple stainings were performed using antibodies against B cell markers. Briefly, after saturation with 2% human serum, mononuclear cells were incubated with the primary antibodies or appropriate isotype controls for 30 min at +4 C. in the dark. Then, cells were washed and fixed in 1% formaldehyde before the analysis on a LSRII cytometer (BD Biosciences). Flow cytometry data were analyzed with the Diva software (BD Biosciences).

[0063] Ex Vivo Culture of Tumor-Infiltrating B Cells

[0064] Total tumor-infiltrating B cells were isolated from mononuclear cells through a positive selection. Briefly, cells were incubated in presence of anti-CD19 microbeads (Miltenyi Biotec), and then loaded onto a Macs columns (Miltenyi Biotec). Freshly isolated B cells were cultured in presence of Pansorbin (Staphylococcus aureus cells extract, Calbiochem) or murine CD40 ligand transfected fibroblastic cell line (CD4O-L L cells were kindly provided by Schering-Plough, Laboratory for Immunological Research, Dardilly, France). The supernatant was recovered every 3 days until day 9, and cryopreserved.

[0065] Statistical Analysis

[0066] Variables taken into account for statistical analysis included clinical (age, sex, smoking, tumor relapse, and vital status), histopathological (histology, pTNM, tumor differentiation, localization of the primary tumor, necrosis, fibrosis, proliferation of the tumor) and immunological parameters (see markers described above). To perform univariate analysis, groups of patients were defined according to the bimodal distribution of the density of positive cells which appointed the following cut-offs. (follicular CD20+ B cells: 0.029 mm.sup.2/tumor IPF, DC-Lamp: 1.65 mean cells/tumor IPF). Chi-square test with Yates correction and ANOVA test (post-hoc tests with Fisher and Bonferonni methods) were used for univariate analysis. Disease-specific survival (DSS) curve were estimated by Kaplan-Meier method and the significance of differences between groups of patients was evaluated by the Logrank test. An event affecting the OS was defined as death from any cause, DSS as death from NSCLC and DFS as relapse of the primary tumor. Statistical analysis was performed using StatView software. A P value <0.05 was considered statistically significant.

[0067] Results

[0068] 1Ti-BALT Contain the Same Contingent of Immune Cells as that Observed in Secondary Lymphoid Organs

[0069] In order to study the role of the B-cell compartment of Ti-BALT, we first characterized by immunohistochemistry B cells and other immune cells in which they will be in contact in these tertiary lymphoid structures. As described in conventional lymphoid organs, we observed that CD3+ T cells and DC-Lamp+ mature DC cluster to form the T-cell rich areas whereas most CD20+ B cells segregate into B follicles. Within the B-cell areas, different non-B cells were present. We detected few CD3+ T cells which probably represent follicular helper T cells, follicular dendritic cells which are organized into a network and a specialized population of CD68+ macrophages also called tangible-body macrophages.

[0070] In conclusion, the segregation of immune cells in Ti-BALT is the same as that observed in secondary lymphoid organs, suggesting that immune responses may take place within ectopic lymphoid structures in human lung cancer.

[0071] 2the B-Cell Areas of Ti-BALT have Features of an Ongoing Humoral Immune Response

[0072] The segregation of T and B-cell areas is a mandatory for the development of both high-affinity class-switched antibodies and memory humoral immune response. Thus, we assessed the stage of B-cell differentiation within the lung tumors, and compared it to secondary lymphoid organs. By immunohistochemistry, we first characterized B-cell subsets according to the Bm classification proposed by Dr. D. Capra (Pascual et al., J. Exp. Med., 1994). As observed in the secondary follicles of lymph nodes (FIG. 1A), we shown an accumulation of IgD+nave B cells in a restricted areas called the mantle (FIG. 1B). This mantle surrounded a germinal centre (GC) as defined by the presence of CD23+ cells which comprised a network of follicular dendritic cells and Bm2 nave B cells (FIG. 1C-D). GC-B cells were also characterized by the expression of AID (FIG. 1E-F), the enzyme critical for the somatic hypermutation, class switch recombination and gene conversion of immunoglobulin (Ig) genes. In a similar manner, GC-B cells were positive for the proliferation marker Ki67 (FIGS. 1G-H) and Bc16 (FIGS. 1I-J), but did not express the anti-apoptotic protein Bc12 (FIGS. 1K-L) in Ti-BALT and lymph nodes. Based on the expression of CD138, no plasma cell (PC) was detected in Ti-BALT B follicles (data not shown), in accordance with the situation seen in lymph nodes (FIG. 1M). However, CD138+PC were observed in the stroma and the fibrosis of the tumor (FIG. 1N).

[0073] We next further compared the proportion of intra-tumoral B cell subsets to lymph nodes as well as peripheral blood by flow cytometry. According to the expression of IgD and CD38 among total CD19+ cells, the distribution of the five B cell subsets was completely distinct in NSCLC compared to lymph nodes and blood (FIG. 2A). Blood and LN-B cells were mainly of nave and memory phenotypes (IgD+ CD38low nave B cells, IgD CD38low memory B cells, respectively). In contrast to blood and lymph nodes, every stage of B cell differentiation was detected in NSCLC. We shown that the percentage of memory B cells and PC was statistically higher, and nave B cells lower in NSCLC compared to the two other sites (FIG. 2B; 18%, 48%, and 62% of memory cells; 1%, 0.5% and 28% of PC; 75%, 42% and 4% of nave B cells in blood, lymph nodes and NSCLC). The differential expression of additional B cell markers (CD23, CD27, CD77) allowed us to study the distribution of subsets of nave (Bm1 and Bm2), GC (Bm3 and Bm4) and memory (early Bm5 and late Bm5) B cells in NSCLC and lymph nodes. As shown in FIG. 2C, the main difference between the 2 sites was the ration of Bm1/Bm2 which was in favor of Bm1 in lung tumor. The other ratios analyzed (Bm3/Bm4 and early Bm5/late Bm5) were not statistically different in the 2 anatomic sites. All together, these data demonstrate that the differentiation stage of tumor-infiltrating B cells is in accordance with their in situ localization, i.e. in or out of ectopic lymphoid structures. Early differentiated B cells are organized into B follicles of Ti-BALT where the somatic mutation and isotype switching machineries are activated, suggesting that Ti-BALT may be an active site for the generation of memory B cells and antibody-secreting cells.

[0074] 3Density of Follicular B Cells is Correlated with Long-Term Survival, and its Prognostic Value is Enhanced when Associated with the Density of Mature DC

[0075] The presence of reactive B follicles in some lung tumors prompted us to investigate their immunological function. As we have shown that the density of mature DC was associated with a favourable clinical outcome (Dieu-Nosjean et al., J. Clin. Oncol., 2008), we investigated whether the densities of follicular B cells and mature DC were correlated to each other as well as their prognostic value. FIG. 3A shows that, even if the global increase of the density of follicular B cells was associated with a global increase of the density of mature DC, these two parameters were not statistically related to each other (R.sup.2=0.1224) suggesting their reciprocal independence. By univariate analysis, we next investigated the prognostic value of follicular B cells alone or in combination with mature DC. The four-year disease-specific survival rates were 97% among patients with high density of follicular B cells (Foll-CD20 High), and 65% among patients with low density of follicular B cells (Foll-CD20 low) (FIG. 3B). Thus, the patients with Foll-CD20 High have a longer survival then patients with Foll-CD20 low (P=0.0099) demonstrating that the number of follicular B cells was associated with a favorable prognosis. There were no distinguishable clinical (sex, age, smoking history), tumor (tumor differentiation, pTNM staging, fibrosis, necrosis, and proliferating tumor cells), or histologic characteristics between the patients with Foll-CD20 High versus Foll-CD20 low tumors (Table 1). A similar result was obtained for the density of DC-Lamp+ mature DC (FIG. 3C) indicating that both professional antigen-presenting cells were predictive for survival. We next tested whether the patients with DC-Lamp High tumors and patients with Foll-CD20 High tumors were the same, and vise versa for patients with DC-Lamp low tumors and Foll-CD20 low tumors. Among the 74 patients, 31 patients (42% of patients) belonged to both groups of DC-Lamp High tumors and Foll-CD20 High tumors, 17 (23% of patients) belonged to both groups of DC-Lamp low tumors and Foll-CD20 low tumors, and 26 (35% of patients) were mixed. Among this mix group called Foll-CD20/DC-Lamp mix, 21 patients were characterized as having DC-Lamp High tumors and Foll-CD20 low tumors, and 5 patients with DC-Lamp low tumors and Foll-CD20 High tumors. The presence of this non-overlapping group is in favour of an independence between these 2 parameters. Due to the limited number of patients within each sub-group of the mix group, we decided to keep the 26 patients within a unique group. The Kaplan-Meier curves indicated that 100% of patients with Foll-CD20/DC-Lamp high were alive (none event among the 31 patients) after a follow-up of 48 months (FIG. 3D). We found that patients with low density of both intra-tumoral mature DC and follicular B cells (Foll-CD20/DC-Lamp low group) had a very poor prognosis with 38% of surviving patients after 48 months (6 events out of 17 patients). The survival curves of patients with Foll-CD20/DC-Lamp mix tumors were between these two curves with 86% of alive patients (3 events out of 26 patients). The median disease-specific survival was only reached for the patients with Foll-CD20/DC-Lamp low tumors (42 months, P<0.0099).

[0076] In conclusion, we demonstrated that the density of tumor-infiltrating follicular B cells is highly predictive of disease-specific survival in early-stage NSCLC. Compared to the density of each cell type, the combination of both mature DC and follicular B cells allows the identification of a group of patients without any event and a group with most events.

Example 2

[0077] 1Prognostic Value of Follicular B Cells and of Mature Dendritic Cells in Patients Treated by Neo-Adjuvant Chemotherapy

[0078] The five-year survival of patients with early-stage NSCLC is 70%, and drops to 15% for late-stage metastastic NSCLC. Studies have shown that two-drug combinations are more efficacious than single-agent treatment (Schiller et al., 2000). Presently, patients with advanced NSCLC receive a neo-adjuvant polychemotherapy (cisplatin plus gemcitabine or carboplatin plus paclitaxel) in many North American and European hospitals (Bunn et al., 2002; Rosell et al., 2002). Now, more and more patients with early-stage lung cancer also receive neo-adjuvant chemotherapy. The response rate of two-drug combinations is between 20 to 30% in advanced NSCLC. Recent reports indicate that cytotoxic drugs are not only targeting tumor cells, but can indirectly promote tumor control by facilitating the development of an immune response within the tumor microenvironment (reviewed in Zitvogel et al., 2008).

[0079] Lung cancer contains tumor cells as well as stroma components: the vasculature, connective tissue and immune infiltrating cells. Among immune cell infiltrate, some of them are professionals for the presentation of processed antigens, like DC, B cells and macrophages. Recently, we have shown that the density of different DC subsets (epithelial Langerhans cells, stromal interstitial DC and mature DC) was associated with a favorable outcome in NSCLC patients (Dieu-Nosjean et al., 2008; Fridman et al., 2011). Now, we show that the density of mature DC is still correlated with a longer-term survival for advanced NSCLC patients treated by neo-adjuvant chemotherapy (n=56 patients, P=0.0373, see FIG. 4).

[0080] As Ti-BALT are present in patients treated by chemotherapy, the density of B cells, another antigen-presenting cell population, can be associated with the outcome of patients treated by chemotherapy. The density of B cells may also be impacted by the type of the two-drug combinations. Thus, the B-cell marker will be correlated to clinical response to treatment. This biomarker can be used to discriminate between patients who will respond and patients who will not respond to the treatment, and thus allowing a more rational and targeted therapy design.

Example 3

[0081] Here, we evaluated the prognostic value of either follicular B cells, mature DC, or the combination of both types of immune cells in a retrospective study of 122 patients with advanced-stage of NSCLC and treated by neo-adjuvant chemotherapy. We demonstrated that the density of each immune parameter was correlated with a favorable outcome. The Kaplan-Meier curves indicated that the density of CD20+ follicular B cells was associated with longer overall survival (OS, P=0.007) (FIG. 5A). The median OS was 55 months for the patients characterized as having Foll-CD20 High tumors whereas the median OS was 18 months for patients with Foll-CD20 low tumors. The density of DC-Lamp+ mature DC was also correlated with a better clinical outcome (FIG. 5B, P=0.04). The median OS was 55 months for the patients with DC-Lamp High tumors whereas the median OS was 24 months for patients with DC-Lamp low tumors.

[0082] As Foll-CD20 and DC-Lamp positively influence the patient survival, we stratified the patients into 3 groups according to the high/low densities of each marker (Foll-CD20/DC-Lamp High, Foll-CD20/DC-Lamp mix, and Foll-CD20/DC-Lamp low). Patients with high density of both types of immune populations were at low risk of death (median OS was not reached) (FIG. 5C, P=0.003). Patients with low densities of both follicular B cells and mature DC were at very high risk of death (median OS was 18 months), demonstrating that the combination of both immune markers allows the identification of a subgroup of patients with a very poor outcome despite chemotherapy. Patients with Foll-CD20/DC-Lamp mix tumors were at intermediate risk of death (median OS was 34 months).

[0083] These data were in accordance with univariate analyses (Table 3) showing that the density of each immune cell type is highly associated with the survival of patients treated by chemotherapy (Foll-CD20 with a Hazard Ratio (HR)=0.41 and P=0.00095; DC-Lamp with a HR=0.50 and P=0.00721). More interestingly, the combination of both biomarkers was the better predictor for survival (HR=0.50, P=0.000097) compared to standard clinical parameters.

[0084] All together, we have demonstrated that the combination of CD20 and DC-Lamp markers is able to discriminate patients with very high risk of death among advanced-stage NSCLC patients treated by neo-adjuvant chemotherapy.

REFERENCES

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