Pierre Validire

Profound Coordinated Alterations of Intratumoral NK Cell Phenotype and Function in Lung Carcinoma

Both the innate and adaptive immune systems contribute to tumor immunosurveillance in mice and humans; however, there is a paucity of direct evidence of a role for natural killer (NK) cells in this important process. In this study, we investigated the intratumoral phenotypic profile and functions of NK cells in primary human tumor specimens of non-small cell lung carcinoma (NSCLC). We used in situ methods to quantify and localize NK cells using the NKp46 marker and we characterized their phenotype in blood, tumoral, and nontumoral samples of NSCLC patients. Intratumoral NK cells displayed a profound and coordinated alteration of their phenotype, with a drastic reduction of NK cell receptor expression specifically detected in the tumoral region. According to their altered phenotype, intratumoral NK cells exhibited profound defects in the ability to activate degranulation and IFN-γ production. We found that the presence of NK cells did not impact the clinical outcome of patients with NSCLC. Finally, we showed that tumor cells heterogeneously express ligands for both activating and inhibitory NK receptors. Taken together, our results suggest that the NSCLC tumor microenvironment locally impairs NK cells, rendering them less tumorcidal and thereby supportive to cancer progression.

Matrix architecture defines the preferential localization and migration of T cells into the stroma of human lung tumors.

Appropriate localization and migration of T cells is a prerequisite for antitumor immune surveillance. Studies using fixed tumor samples from human patients have shown that T cells accumulate more efficiently in the stroma than in tumor islets, but the mechanisms by which this occurs are unknown. By combining immunostaining and real-time imaging in viable slices of human lung tumors, we revealed that the density and the orientation of the stromal extracellular matrix likely play key roles in controlling the migration of T cells. Active T cell motility, dependent on chemokines but not on β1 or β2 integrins, was observed in loose fibronectin and collagen regions, whereas T cells migrated poorly in dense matrix areas. Aligned fibers in perivascular regions and around tumor epithelial cell regions dictated the migratory trajectory of T cells and restricted them from entering tumor islets. Consistently, matrix reduction with collagenase increased the ability of T cells to contact cancer cells. Thus, the stromal extracellular matrix influences antitumor immunity by controlling the positioning and migration of T cells. Understanding the mechanisms by which this collagen network is generated has the potential to aid in the development of new therapeutics.

No significant predictive value of c- erbB-2 or p53 expression regarding sensitivity to primary chemotherapy or radiotherapy in breast cancer

To document whether c‐erbB‐2 over‐expression or p53 accumulation in tumour cells was predictive of response to chemo‐ or radiotherapy, we analyzed a population of patients with breast cancer assigned to neo‐adjuvant therapy (median follow‐up: 54 months). T2/T3‐N0N1b‐M0 tumours (329 cases) were treated either by FAC chemotherapy or by radiotherapy before surgery, and the clinical response was classified as complete or incomplete. Expression of c‐erbB‐2 and p53 was retrospectively evaluated by immunohistochemistry. Proliferation rate was assessed by means of MIB‐1 antibody and by S‐phase fraction. A complete response to chemotherapy was observed in 38/167 patients (23%). Complete response rate was 20% in c‐erbB‐2‐negative tumours, and rose to 31% in tumours with c‐erbB‐2 over‐expression, but this trend was not statistically significant. There was no correlation between p53 staining and response to treatment, whereas chemosensitivity was found correlated with histological grade and S‐phase. A complete response to radiotherapy was observed in 64 of the 156 evaluable patients (41%). Complete response rate was 41% in c‐erbB‐2‐ or p53‐negative tumours, 54% in tumours with c‐erb‐B‐2 over‐expression, and 44% in tumours with p53 accumulation. There was no correlation between response to radiotherapy and histological grade or proliferative rate. No prognostic value was found for c‐erbB‐2 or p53 expression, whereas the 5‐year survival rate was 85% for patients presenting a tumour with a low proliferating index (MIB‐1 < 10%), and 68% for patients presenting a tumour with a high proliferative index. In multivariate analysis, node status (RR = 2), MIB‐1 immunostaining (RR = 2), and tumour size (RR = 1.8) were found to be associated with survival. These results indicate that c‐erbB‐2 or p53 expression is not significantly associated with tumour response to neo‐adjuvant chemo/radiotherapy in our series of breast cancers. Int. J. Cancer (Pred. Oncol.) 79:27–33, 1998.

Dendritic Cells in Tumor-Associated Tertiary Lymphoid Structures Signal a Th1 Cytotoxic Immune Contexture and License the Positive Prognostic Value of Infiltrating CD8+ T Cells

Tumor-infiltrating T cells, particularly CD45RO(+)CD8(+) memory T cells, confer a positive prognostic value in human cancers. However, the mechanisms that promote a protective T-cell response in the tumor microenvironment remain unclear. In chronic inflammatory settings such as the tumor microenvironment, lymphoid neogenesis can occur to create local lymph node-like structures known as tertiary lymphoid structures (TLS). These structures can exacerbate a local immune response, such that TLS formation in tumors may help promote an efficacious immune contexture. However, the role of TLS in tumors has yet to be investigated carefully. In lung tumors, mature dendritic cells (DC) present in tumor-associated TLS can provide a specific marker of these structures. In this study, we evaluated the influence of TLS on the characteristics of the immune infiltrate in cohorts of prospective and retrospective human primary lung tumors (n = 458). We found that a high density of mature DC correlated closely to a strong infiltration of T cells that are predominantly of the effector-memory phenotype. Moreover, mature DC density correlated with expression of genes related to T-cell activation, T-helper 1 (Th1) phenotype, and cytotoxic orientation. Lastly, a high density of TLS-associated DC correlated with long-term survival, which also allowed a distinction of patients with high CD8(+) T-cell infiltration but a high risk of death. Taken together, our results show how tumors infiltrated by TLS-associated mature DC generate a specific immune contexture characterized by a strong Th1 and cytotoxic orientation that confers the lowest risk of death. Furthermore, our findings highlight the pivotal function of TLS in shaping the immune character of the tumor microenvironment, in promoting a protective immune response mediated by T cells against cancer.

Presence of B Cells in Tertiary Lymphoid Structures Is Associated with a Protective Immunity in Patients with Lung Cancer

RATIONALE It is now well established that immune responses can take place outside of primary and secondary lymphoid organs. We previously described the presence of tertiary lymphoid structures (TLS) in patients with non-small cell lung cancer (NSCLC) characterized by clusters of mature dendritic cells (DCs) and T cells surrounded by B-cell follicles. We demonstrated that the density of these mature DCs was associated with favorable clinical outcome. OBJECTIVES To study the role of follicular B cells in TLS and the potential link with a local humoral immune response in patients with NSCLC. METHODS The cellular composition of TLS was investigated by immunohistochemistry. Characterization of B-cell subsets was performed by flow cytometry. A retrospective study was conducted in two independent cohorts of patients. Antibody specificity was analyzed by ELISA. MEASUREMENTS AND MAIN RESULTS Consistent with TLS organization, all stages of B-cell differentiation were detectable in most tumors. Germinal center somatic hypermutation and class switch recombination machineries were activated, associated with the generation of plasma cells. Approximately half of the patients showed antibody reactivity against up to 7 out of the 33 tumor antigens tested. A high density of follicular B cells correlated with long-term survival, both in patients with early-stage NSCLC and with advanced-stage NSCLC treated with chemotherapy. The combination of follicular B cell and mature DC densities allowed the identification of patients with the best clinical outcome. CONCLUSIONS B-cell density represents a new prognostic biomarker for NSCLC patient survival, and makes the link between TLS and a protective B cell-mediated immunity.

Triggering of TLR7 and TLR8 expressed by human lung cancer cells induces cell survival and chemoresistance

Compelling evidence suggests that inflammation, cell survival, and cancer are linked, with a central role played by NF-kappaB. Recent studies implicate some TLRs in tumor development based on their ability to facilitate tumor growth; however, to our knowledge, involvement of neither TLR7 nor TLR78 has yet been demonstrated. Here we have demonstrated expression of TLR7 and TLR8, the natural receptors for single-stranded RNA, by tumor cells in human lung cancer in situ and in human lung tumor cell lines. Stimulation with TLR7 or TLR8 agonists led to activated NF-kappaB, upregulated expression of the antiapoptotic protein Bcl-2, increased tumor cell survival, and chemoresistance. Transcriptional analysis performed on human primary lung tumor cells and TLR7- or TLR8-stimulated human lung tumor cell lines revealed a gene expression signature suggestive of chronic stimulation of tumor cells by TLR ligands in situ. Together, these data emphasize that TLR signaling can directly favor tumor development and further suggest that researchers developing anticancer immunotherapy using TLR7 or TLR8 agonists as adjuvants should take into account the expression of these TLRs in lung tumor cells.

Human papillomavirus genotype as a major determinant of the course of cervical cancer.

PURPOSE To determine whether the prognosis of invasive cancers of the uterine cervix is related to the type of human papillomavirus (HPV) associated with the tumor. PATIENTS AND METHODS Two hundred ninety-seven patients with invasive cervical cancer were prospectively registered from 1986 to 1994. HPV typing was performed on DNA extracted from frozen tumor specimens by means of Southern blot hybridization (SBH) and polymerase chain reaction (PCR) techniques. The median follow-up was 38 months. RESULTS HPV sequences were detected in 246 patients (83%): 150 patients had HPV16, 31 patients had HPV18, and 14 patients had one of the intermediate-oncogenic-risk HPV types (HPV31, 33, 35, 52, 58). In 51 patients, HPV type remained undetermined, and in 51 patients, no viral sequences were found. No significant associations were observed between virologic data and tumor stage or node status. The 5-year disease-free survival (DFS) rate was 100% for patients with intermediate-risk HPV-associated tumors, 58% for patients with HPV16-positive tumors, and 38% for patients with HPV18-positive tumors (P = .02). In multivariate analysis, patients with HPV18-associated tumors had a relative risk (RR) of death 2.4 times greater (95% confidence interval [CI], 1.29-4.59) than that for patients with HPV16, and 4.4 times greater (95% CI, 3.48-5.32) than that for patients with a tumor associated with a viral type different from HPV16/18. CONCLUSION The prognosis for invasive cancers of the uterine cervix is dependent on the oncogenic potential of the associated HPV type. HPV typing may provide a prognostic indicator for individual patients and is of potential use in defining specific therapies against HPV-harboring tumor cells.

Ex-vivo characterization of human colon cancer by Mueller polarimetric imaging

Cancerous and healthy human colon samples have been analyzed ex-vivo using a multispectral imaging Mueller polarimeter operated in the visible (from 500 to 700 nm) in a backscattering configuration with diffuse light illumination. Three samples of Liberkühn colon adenocarcinomas have been studied: common, mucinous and treated by radiochemotherapy. For each sample, several specific zones have been chosen, based on their visual staging and polarimetric responses, which have been correlated to the histology of the corresponding cuts. The most relevant polarimetric images are those quantifying the depolarization for incident linearly polarized light. The measured depolarization depends on several factors, namely the presence or absence of tumor, its exophytic (budding) or endophytic (penetrating) nature, its thickness (its degree of ulceration) and its level of penetration in deeper layers (submucosa, muscularis externa and serosa). The cellular density, the concentration of stroma, the presence or absence of mucus and the light penetration depth, which increases with wavelength, are also relevant parameters. Our data indicate that the tissues with the lowest and highest depolarizing powers are respectively mucus-free tumoral tissue with high cellular density and healthy serosa, while healthy submucosa, muscularis externa as well as mucinous tumor probably feature intermediate values. Moreover, the specimen coming from a patient treated successfully with radiochemotherapy exhibited a uniform polarimetric response typical of healthy tissue even in the initially pathological zone. These results demonstrate that multi-spectral Mueller imaging can provide useful contrasts to quickly stage human colon cancer ex-vivo and to distinguish between different histological variants of tumor.

High-dose Chemotherapy with Carboplatin, Etoposide and Cyclophosphamide Followed by a Haematopoietic Stem Cell Rescue in Patients with High-risk Retinoblastoma: a SFOP and SFGM Study

This study investigates the role of high-dose chemotherapy with haematopoietic stem cell rescue as consolidation treatment in high-risk retinoblastoma (extraocular disease at diagnosis or relapse or invasion of cut end of optic nerve). 25 patients received high-dose chemotherapy including carboplatin (250 mg/m2/day from day 1 to day 5 for the 6 first patients and 350 mg/m2/day from day 1 to day 5 for the other patients), etoposide (350 mg/m2/day from day 1 to day 5) and cyclophosphamide (1.6 g/m2/day from day 2 to day 5) (CARBOPEC) followed by autologous haematopoietic stem cell rescue. 19 patients received this drug combination for chemosensitive extraocular relapse. The other 6 patients with histological high-risk factors were given this treatment as consolidation after enucleation and conventional chemotherapy. The three year disease-free survival was 67.1%. In 7 of the 9 relapsing patients, the first site of relapse was the central nervous system. All patients with central nervous system disease died except one. The main toxicity was haematological and digestive (mucositis and diarrhoea). 2 of the 13 evaluable patients had grade III and IV ototoxicity. One patient experienced an acute grade I reversible cardiotoxicity. The CARBOPEC regimen seems to be a promising therapeutic strategy in patients with high-risk retinoblastoma, especially those with bone and/or bone marrow involvement. This treatment did not improve the outcome of patients with central nervous system disease.

CD8+CD103+ Tumor–Infiltrating Lymphocytes Are Tumor-Specific Tissue-Resident Memory T Cells and a Prognostic Factor for Survival in Lung Cancer Patients

We had previously demonstrated the role of CD103 integrin on lung tumor-infiltrating lymphocyte (TIL) clones in promoting specific TCR-mediated epithelial tumor cell cytotoxicity. However, the contribution of CD103 on intratumoral T cell distribution and functions and the prognosis significance of TIL subpopulations in non–small cell lung carcinoma (NSCLC) have thus far not been systematically addressed. In this study, we show that an enhanced CD103+ TIL subset correlates with improved early stage NSCLC patient survival and increased intraepithelial lymphocyte infiltration. Moreover, our results indicate that CD8+CD103+ TIL, freshly isolated from NSCLC specimens, display transcriptomic and phenotypic signatures characteristic of tissue-resident memory T cells and frequently express PD-1 and Tim-3 checkpoint receptors. This TIL subset also displays increased activation-induced cell death and mediates specific cytolytic activity toward autologous tumor cells upon blockade of the PD-1–PD-L1 interaction. These findings emphasize the role of CD8+CD103+ tissue-resident memory T cells in promoting intratumoral CTL responses and support the rationale for using anti–PD-1 blocking Ab to reverse tumor-induced T cell exhaustion in NSCLC patients.