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Despite a growing incidence in developed countries and a recent improved understanding of its pathogenesis, anal cancer management has not evolved over the past decades and drug combination used as first-line regimen still largely depends on clinician preferences. Aiming at paving the way for precision medicine, a large cohort of 372 HIV-negative patients diagnosed over a 20-year time period with locally advanced anal carcinoma was collected and carefully characterized at the clinical, demographic, histopathologic, immunologic, and virologic levels. Both the prognostic relevance of each clinicopathological parameter and the efficacy of different concurrent chemoradiation strategies were determined. Overall, the incidence of anal cancer peaked during the sixth decade (mean: 63.4) and females outnumbered males (ratio: 2.51). After completion of treatment, 95 (25.5%) patients experienced progression of persistent disease or local/distant recurrence and 102 (27.4%) died during the follow-up period (median: 53.8 months). Importantly, uni-multivariate analyses indicated that both negative HPV/p16ink4a status and aberrant p53 expression were far better predictors for reduced progression-free survival than traditional risk factors such as tumor size and nodal status. As for overall survival, the significant influences of age at diagnosis, p16ink4a status, cTNM classification as well as both CD3+ and CD4+ T-cell infiltrations within tumor microenvironment were highlighted. Cisplatin-based chemoradiotherapy was superior to both radiotherapy alone and other concurrent chemoradiation therapies in the treatment of HPV-positive tumors. Regarding their HPV-uninfected counterparts, frequent relapses were observed, whatever the treatment regimen administered. Taken together, our findings reveal that current anal cancer management and treatment have reached their limits. A dualistic classification according to HPV/p53 status should be considered with implications for therapy personalization and optimization.

Background In the past decade, immunotherapy using immune checkpoint inhibitors (especially targeting the PD-1/PD-L1 axis) has been demonstrated as a promising strategy for the treatment of cancers that do not respond to classical chemoradiotherapy. Given that cancer cells have the potential to express many immunosuppressive molecules other than PD-L1, the combination of immune checkpoint inhibitors with other drugs thwarting tumor immunosuppressive microenvironment could represent a promising strategy. Among these immunosuppressive molecules, RANKL, a member of the TNF superfamily, which mainly affects the immune system and bone remodeling, has been shown to be a key factor promoting the progression of breast cancer. In addition, RANKL induces the formation of tolerogenic dendritic cells and Treg cells, which promotes immunotolerance to the tumor. The aim of this research project is to study the impact of several RANKL inhibitors on triple negative breast cancer and to analyze the efficiency of their association with anti-PD-1/PD-L1 agents. Materials and Methods We studied RANKL and PD-L1 expression in several murine and human breast cancer cell lines by immunohistochemistry. The secretion of RANKL was analyzed by ELISA. Inhibitors of RANKL were then tested in vitro. We selected several RANKL inhibitors: anti-RANKL antibody, RANK-Fc, Isoliquiritigenin and Gallocatechin gallate. The efficacy of these inhibitors was indirectly evaluated with the murine macrophage RAW264.7 cell line which undergoes, in the presence of RANKL, an osteoclast differentiation (TRAP and Cathepsin K expression). The efficacy of RANKL inhibitors was then evaluated, in this model, by RT-qPCR. Apoptosis and proliferation of the cancer cell lines in the presence of the inhibitors were also analyzed. Results RANKL/PD-L1 expression profile on specimens from each breast cancer subtypes showed that both immunosuppressive molecules are expressed by all breast cancers with a significantly more intense immunoreactivity for triple negative breast cancers. Most of the cell lines expressed both proteins. We found that RANKL is secreted in their extracellular environment. RANKL inhibitors are efficient and will be tested in vivo. Conclusions Several murine triple negative breast cancer cell lines will be sub-cutaneously injected in mice and the efficacy of both RANKL and PD-L1 inhibitors will be evaluated (separately or in combination). The infiltration of tumor microenvironment by different immune cell populations, the presence of metastasis and the tumor growth will be analyzed. Disclosure Information C. Pilard: None. P. Roncarati: None. E. Hendrick: None. A. Lebeau: None. D. Bruyere: None. T. Lerho: None. M. Ancion: None. C. Reynders: None. P. Delvenne: None. M. Herfs: None. P. Hubert: None.

Background Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels. Methods In this multi-institutional study, we first examined the histological features displayed by each collected tumour (n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets (CD4, CD8, Foxp3), the expression of immune checkpoints (PD-1, PD-L1), the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed. Results Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa. Conclusions Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits.

Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV-related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region-specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease-free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cervical carcinoma is a major cause of morbidity and mortality in the United States and throughout the world. Associated to the infection with one or several high-risk human papillomavirus (HPV) genotypes, the incidence of cervical cancer increases progressively from age 30 and peaks between 45 and 50. Due to the implementation of screening programs, the number of diagnosed invasive cervical neoplasms has significantly decreased in the last decades in developed countries. The present chapter summarizes the current knowledge on HPV-related carcinogenesis, discusses the histology, diagnosis and outcome of HPV-positive cervical (pre)neoplastic lesions and, finally, reviews the different methods of cervical cancer screening/prevention.

BackgroundHigh-mobility group box 1 (HMGB1) is a multifunctional redox-sensitive protein involved in various intracellular (eg, chromatin remodeling, transcription, autophagy) and extracellular (inflammation, autoimmunity) processes. Regarding its role in cancer development/progression, paradoxical results exist in the literature and it is still unclear whether HMGB1 mainly acts as an oncogene or a tumor suppressor.MethodsHMGB1 expression was first assessed in tissue specimens (n=359) of invasive breast, lung and cervical cancer and the two distinct staining patterns detected (nuclear vs cytoplasmic) were correlated to the secretion profile of malignant cells, patient outcomes and the presence of infiltrating immune cells within tumor microenvironment. Using several orthotopic, syngeneic mouse models of basal-like breast (4T1, 67NR and EpRas) or non-small cell lung (TC-1) cancer, the efficacy of several HMGB1 inhibitors alone and in combination with immune checkpoint blockade antibodies (anti-PD-1/PD-L1) was then investigated. Isolated from retrieved tumors, 14 immune cell (sub)populations as well as the activation status of antigen-presenting cells were extensively analyzed in each condition. Finally, the redox state of HMGB1 in tumor-extruded fluids and the influence of different forms (oxidized, reduced or disulfide) on both dendritic cell (DC) and plasmacytoid DC (pDC) activation were determined.ResultsAssociated with an unfavorable prognosis in human patients, we clearly demonstrated that targeting extracellular HMGB1 elicits a profound remodeling of tumor immune microenvironment for efficient cancer therapy. Indeed, without affecting the global number of (CD45+) immune cells, drastic reductions of monocytic/granulocytic myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes, a higher M1/M2 ratio of macrophages as well as an increased activation of both DC and pDC were continually observed following HMGB1 inhibition. Moreover, blocking HMGB1 improved the efficacy of anti-PD-1 cancer monoimmunotherapy. We also reported that a significant fraction of HMGB1 encountered within cancer microenvironment (interstitial fluids) is oxidized and, in opposite to its reduced isoform, oxidized HMGB1 acts as a tolerogenic signal in a receptor for advanced glycation endproducts-dependent manner.ConclusionCollectively, we present evidence that extracellular HMGB1 blockade may complement first-generation cancer immunotherapies by remobilizing antitumor immune response.