Clonal expansion of intra-epithelial T cells in breast cancer revealed by spatial transcriptomics.

The spatial distribution of tumor-infiltrating lymphocytes (TIL) predicts breast cancer outcome and response to systemic therapy, highlighting the importance of an intact tissue structure for characterizing tumors. Here, we present ST-FFPE, a spatial transcriptomics method for the analysis of formalin-fixed paraffin-embedded samples, which opens the possibility of interrogating archival tissue. The method involves extraction, exome capture and sequencing of RNA from different tumor compartments microdissected by laser-capture, and can be used to study the cellular composition of tumor microenvironment. Focusing on triple-negative breast cancer (TNBC), we characterized T cells, B cells, dendritic cells, fibroblasts and endothelial cells in both stromal and intra-epithelial compartments. We found a highly variable spatial distribution of immune cell subsets among tumors. This analysis revealed that the immune repertoires of intra-epithelial T and B cells were consistently less diverse and more clonal than those of stromal T and B cells. T-cell receptor (TCR) sequencing confirmed a reduced diversity and higher clonality of intra-epithelial T cells relative to the corresponding stromal T cells. Analysis of the top 10 dominant clonotypes in the two compartments showed a majority of shared but also some unique clonotypes both in stromal and intra-epithelial T cells. Hyperexpanded clonotypes were more abundant among intra-epithelial than stromal T cells. These findings validate the ST-FFPE method and suggest an accumulation of antigen-specific T cells within tumor core. Because ST-FFPE is applicable for analysis of previously collected tissue samples, it could be useful for rapid assessment of intratumoral cellular heterogeneity in multiple disease and treatment settings.

Primordial germ cells as a potential shared cell of origin for mucinous cystic neoplasms of the pancreas and mucinous ovarian tumors.

Mucinous ovarian tumors (MOTs) morphologically and epidemiologically resemble mucinous cystic neoplasms (MCNs) of the pancreas, sharing a similar stroma and both occurring disproportionately among young females. Additionally, MOTs and MCNs share similar clinical characteristics and immunohistochemical phenotypes. Exome sequencing has revealed frequent recurrent mutations in KRAS and RNF43 in both MOTs and MCNs. The cell of origin for these tumors remains unclear, but MOTs sometimes arise in the context of mature cystic teratomas and other primordial germ cell (PGC) tumors. We undertook the present study to investigate whether non-teratoma-associated MOTs and MCNs share a common cell of origin. Comparisons of the gene expression profiles of MOTs [including both the mucinous borderline ovarian tumors (MBOTs) and invasive mucinous ovarian carcinomas (MOCs)], high-grade serous ovarian carcinomas, ovarian surface epithelium, Fallopian tube epithelium, normal pancreatic tissue, pancreatic duct adenocarcinomas, MCNs, and single-cell RNA-sequencing of PGCs revealed that both MOTs and MCNs are more closely related to PGCs than to either eutopic epithelial tumors or normal epithelia. We hypothesize that MCNs may arise from PGCs that stopped in the dorsal pancreas during their descent to the gonads during early human embryogenesis, while MOTs arise from PGCs in the ovary. Together, these data suggest a common pathway for the development of MCNs and MOTs, and suggest that these tumors may be more properly classified as germ cell tumor variants. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Report on the first SLFN11 monothematic workshop: from function to role as a biomarker in cancer.

SLFN11 is a recently discovered protein with a putative DNA/RNA helicase function. First identified in association with the maturation of thymocytes, SLFN11 was later causally associated, by two independent groups, with the resistance to DNA damaging agents such as topoisomerase I and II inhibitors, platinum compounds, and other alkylators, making it an attractive molecule for biomarker development. Later, SLFN11 was linked to antiviral response in human cells and interferon production, establishing a potential bond between immunity and chemotherapy. Recently, we demonstrated the potential role of SLN11 as a biomarker to predict sensitivity to the carboplatin/taxol combination in ovarian cancer. The present manuscript reports on the first international monothematic workshop on SLFN11. Several researchers from around the world, directly and actively involved in the discovery, functional characterization, and study of SLFN11 for its biomarker and medicinal properties gathered to share their views on the current knowledge advances concerning SLFN11. The aim of the manuscript is to summarize the authors' interventions and the main take-home messages resulting from the workshop.

Assessment of chemical stability of monoclonal antibody and antibody drug conjugate administered by pressurized intraperitoneal aerosol chemotherapy.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a new therapeutic approach for patients with peritoneal cancer. So far, most published studies investigated the administration of established cytostatic agents through PIPAC. This study aimed to evaluate the effect of PIPAC on two breakthrough anti-cancer agents, specifically anti-PD1 pembrolizumab, and anti-HER2 antibody-drug conjugate (ADC) - trastuzumab-deruxtecan. We conducted systematic analyses on samples of pembrolizumab and trastuzumab-deruxtecan at clinically relevant concentrations before and after PIPAC administration using an experimental setup of a hermetic container system, mimicking the abdominal cavity and using identical features as in clinical use. We utilized a range of chromatographic and spectroscopic techniques to explore potential alterations in the primary, secondary, and tertiary structures of the drugs, focusing on post-translational modifications resulting from the aerosolization. Our findings indicate that PIPAC did not compromise the integrity of tested biopharmaceuticals. The size variants of both drugs, assessed by size exclusion chromatography (SEC), remained unchanged. Reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC) revealed no significant differences in hydrophobicity variants, the average drug-to-antibody ratio (DAR), or DAR distribution before and after PIPAC treatment. Circular dichroism (CD) spectroscopy confirmed that the secondary and tertiary structures were preserved. While pembrolizumab showed no change in charge variants post-PIPAC, trastuzumab-deruxtecan exhibited a non-negligible change in the quantity of charge variants on the monoclonal antibody itself, while the payload remained unchanged. This shift could possibly be related to the metallic composition of the CapnoPen® device (made of nickel and chromium) used in PIPAC and for these experiments. Together, our results suggest that PIPAC does not alter the structure of pembrolizumab and trastuzumab-deruxtecan, paving the way for future clinical trials.

Breast cancer-derived transforming growth factor-ß and tumor necrosis factor-α compromise interferon-α production by tumor-associated plasmacytoid dendritic cells.

We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon-α (IFN-α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor-associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN-α, the production by Toll-like receptor (TLR)-activated healthy pDC of IFN-ß and TNF-α but not IP-10/CXCL10 nor MIP1-α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF-ß and TNF-α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF-ß1 and TNF-α synergistically blocked IFN-α production of TLR-activated pDC, and neutralization of TGF-ß and TNF-α in tumor-derived supernatants restored pDCs' IFN-α production. The involvment of tumor-derived TGF-ß was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF-7 expression and nuclear translocation in pDC after their exposure to tumor-derived supernatants or recombinant TGF-ß1 and TNF-α. Our findings indicate that targeting TApDC to restore their IFN-α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9-based immunotherapy with TGF-ß and TNF-α antagonists.

Characteristics of long-survivor metastatic melanoma after polychemotherapy and interferon: a retrospective study.

BACKGROUND: The development of immunotherapy and tyrosine kinase inhibitors dramatically improved the prognosis of metastatic melanoma. Consequently, chemotherapy is now rarely used. Here, we describe the characteristics of long-surviving patients with metastatic melanoma treated with immunochemotherapy. MATERIAL AND METHODS: We retrieved retrospective clinical and pathological data for patients diagnosed with metastatic melanoma between January 1993 and December 2015 who received the CVD-INF (cisplatin, vinblastine, dacarbazine, and interferon α-2b) regimen at the Hôpitaux Universitaires de Genève. We estimated their progression-free survival and overall survival. This ad hoc study's primary aim was to describe the clinical and biological characteristics of long-term survivors, defined as patients surviving more than two years after immunochemotherapy initiation. The spatial distribution pattern of CD8+ T cells (inflamed, excluded, or desert) was immunohistochemically determined. RESULTS: Ninety patients received CVD-INF. Their median age at metastatic melanoma diagnosis was 55 years (20-75). Their median progression-free survival was 2.8 months, and median overall survival was 7.2 months. Eleven (12%) patients were long-term survivors. In multivariate analysis, central nervous system metastases (hazard ratio [HR]: 2.66; 95% confidence interval [CI]: 1.43-4.95; p = 0.001), multiple metastases (HR: 1.82; 95% CI: 1.01-3.29; p = 0.047), and elevated lactate dehydrogenase (LDH) (HR: 1.92; 95% CI: 1.12-3.30; p = 0.016) were independently associated with shorter survival. Most long-survivors (6/8; 75%) had a tumour-inflamed pattern compared to 25% of non-long survivors (5/20; Fisher's test p = 0.030). CONCLUSIONS: A subset of patients with metastatic melanoma and a tumour-inflamed phenotype treated with CVD-INF survived over two years. Factors associated with prolonged survival are consistent with those previously reported in metastatic melanoma.

Chemotherapy for metastatic breast cancer. Comparison of clinical practice and cost of drugs in two cohorts of patients: 1994-1998 and 2003-2006.

Although new chemotherapeutic drugs for metastatic breast cancer (MBC) have been approved over the past decade, it is unclear whether this has changed the overall outcome of patients. This study assessed the clinical and economic impacts of these drugs. We retrospectively studied MBC patients receiving chemotherapy in our institution over two time periods, 1994-1998 and 2003-2006. Patient characteristics and outcomes, and treatment characteristics and costs (€, 2008) were compared. Three hundred and one patients were identified, 149 patients in the first cohort and 152 in second one. The median number of lines of chemotherapy was similar in the two cohorts (three lines). The median costs of chemotherapy per patient nearly doubled over time, from 6,272 € in the 1994-1998 cohort to 13,035 € in the 2003-2006 cohort (P < 0.001). No survival difference was observed between the two groups, with a 3-year survival rate estimated to 41% in the 1994-1998 cohort and 44% in the 2003-2006 cohort (P = 0.52). In multivariate analysis, prognostic factors associated with longer overall survival were single metastatic site (HR 0.48; P < 10⁻³), bone metastases (HR = 0.67; P = 0.007) and positive hormone receptors (HR 0.56; P = 0.0002). New chemotherapeutic agents induced a significant cost increase over time. The limited size and heterogeneity of our cohort do not allow any conclusion concerning their impact on survival.

PARP-inhibitors in epithelial ovarian cancer: Actual positioning and future expectations.

Poly-(ADP)-ribose polymerase inhibitors (PARPi) are a class of oral anticancer drugs first developed as "synthetically lethal" in cancers harboring BRCA1/BRCA2 inactivating mutations. In high-grade serous or endometrioid ovarian cancers (HGOC), PARPi demonstrated benefit as maintenance therapy in relapsing BRCA-mutated and non-mutated tumors. Recently, they extended their indications to frontline maintenance therapy. This review summarizes the current place of PARPi (i) as maintenance or single agent in recurrent disease and (ii) frontline maintenance with different settings. We reviewed the course of biomarker identification, the challenge of overcoming resistance to PARPi and future combinations with targeted therapies, including anti-angiogenic, immune checkpoint inhibitors and DNA damage response inhibitors.

Plasmacytoid dendritic cells infiltrating ovarian cancer are associated with poor prognosis.

Using two different and complementary approaches (flow cytometry and immunohistochemistry) on two independent cohorts of ovarian cancer patients, we found that accumulation of plasmacytoid dendritic cells (pDC) in tumors is associated with early relapse. This deleterious effect of tumor-associated pDC was evident when they are present in cancer epithelium but not in lymphoid aggregates.

Impaired IFN-α production by plasmacytoid dendritic cells favors regulatory T-cell expansion that may contribute to breast cancer progression.

Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3(+) Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer.

First-line endocrine therapy alone could be a reasonable treatment option for hormone-positive, HER2-positive metastatic breast cancer.

PURPOSE: The treatment strategy for hormone receptor-positive (ER+) HER2-positive (HER2+) metastatic breast cancer has been modified since several randomized trials have proven the effectiveness of anti-HER2 targeted therapy. Previously validated clinical practice guidelines recommending the use of endocrine therapy alone in first line might be changed. METHODS: This study focused on the outcomes of women with ER+ HER2+ metastatic breast cancer receiving first-line endocrine therapy alone at the Léon-Bérard Centre, Lyon, France. RESULTS: Of 290 patients with ER+ HER2+ tumors, 32 (11%) met the criteria for inclusion. The median age was 54 years (29-79 years). Eighteen patients (56%) had only bone and/or soft tissue metastases. Most patients (n = 21; 65%) had only one metastatic site. Fifteen (47%) had histological grade III disease. The median progression free survival (PFS) was 8.2 months (95% CI: 0.1-16.3) and the median overall survival (OS) was 48 months (95% CI: 22.9-72.9). The overall response rate was 25% (95% CI: 11-49%), including one patient with complete response and seven with partial responses. Ten patients (31%) had stable disease. After failure of endocrine therapy, all patients received trastuzumab. The median PFS after first-line chemotherapy was 8.4 months (95% CI: 5.1-11.8). We identified a group of 10 patients with good prognostic factor (tumor grade < 3 tumors and no visceral metastases), for whom median PFS was 15.5 months (95% CI: 7-23). CONCLUSIONS: Our result suggests that first-line endocrine therapy is a viable therapeutic option for a selected population of metastatic breast cancer patients with HER2-positive tumors. Genomic and transcriptomic signature could help to identify tumors that remain dependant of estrogen-signaling pathway.

Therapeutic pipeline for soft-tissue sarcoma.

INTRODUCTION: Soft-tissue sarcomas (STS) represent a heterogeneous group of malignant tumors originating from connective tissues. Over recent years, this heterogeneity has led to a molecular breakdown of STS and subsequent use of targeted agents in several molecularly defined subgroups. After the initial success of imatinib in gastrointestinal stromal tumors, several other compounds have shown promising activity in some but not all subgroups of sarcoma. AREAS COVERED: This review discusses the rational and clinical results, when available, that support this subtype-directed approach. In the vast majority of cases, these agents have been tested only in patients with advanced disease; as chemotherapeutic agents are developed as non-histotype-specific therapies, they are not discussed here. The PubMed literature was searched using the terms 'sarcoma', 'angiogenesis', 'mTOR' and 'targeted agents'. Proceedings of the annual meeting of the American Society of Clinical Oncology as well as those of the Connective Tissue Oncology Society were also searched for relevant information. EXPERT OPINION: Many agents are currently developed in a subtype-specific manner in STS and this represents a significant leap forward. However, much remains to be done to improve our understanding of the molecular biology of this heterogeneous group of diseases.

Quantitative and functional alterations of plasmacytoid dendritic cells contribute to immune tolerance in ovarian cancer.

In ovarian cancer, the immune system fails to eradicate established tumors partly due to the induction of immune tolerance within tumor microenvironment. In this study, we investigated the contribution of plasmacytoid dendritic cells (pDC) in the establishment of immune tolerance in a cohort of 44 ovarian cancer patients. In the tumor and malignant ascites, CD4(+)CD123(+)BDCA2(+) pDC were the most abundant dendritic cell subset; however, they were profoundly depleted in peripheral blood. The presence of pDC in primary ovarian cancer, but not ascites, was an independent prognostic factor associated with early relapse. Following chemotherapy, we observed a partial restoration of blood pDC levels in patients in complete remission. These findings show preferential recruitment of pDC into tumors where they express a partially mature phenotype that may reflect an in situ activation. Importantly, compared with pDC found in ascites or blood, tumor-associated pDC (TApDC) produced less IFN-α, TNF-α, IL-6, macrophage inflammatory protein-1ß, and RANTES in response to toll-like receptor stimulation, and alterations in pDC functions were mainly mediated through tumor-derived TNF-α and TGF-ß. Unlike ascites-derived pDC, TApDC induced IL-10 production from allogeneic naive CD4(+) T lymphocytes, suggesting the existence of a paracrine immunosuppressive loop. Taken together, our findings indicate that both local and systemic dysfunction of pDC play a critical role in the progression of ovarian cancer via induction of immune tolerance.