Autologous T cell responses to primary human colorectal cancer spheroids are enhanced by ectonucleotidase inhibition.

OBJECTIVE: T cells are major effectors of the antitumoural immune response. Their activation by tumour-associated antigens can unleash their proliferation and cytotoxic functions, leading to tumour cell elimination. However, tumour-related immunosuppressive mechanisms including the overexpression of immune checkpoints like programmed cell death protein-1 (PD-1), are also engaged, promoting immune escape. Current immunotherapies targeting these pathways have demonstrated weak efficacy in colorectal cancer (CRC). It is thus crucial to find new targets for immunotherapy in this cancer type. DESIGN: In a prospective cohort of patients with CRC, we investigated the phenotype of tumour-related and non-tumour related intestinal T cells (n=44), particularly the adenosinergic pathway, correlating with clinical phenotype. An autologous coculture model was developed between patient-derived primary tumour spheroids and their autologous tumour-associated lymphocytes. We used this relevant model to assess the effects of CD39 blockade on the antitumour T cell response. RESULTS: We show the increased expression of CD39, and its co-expression with PD-1, on tumour infiltrating T cells compared with mucosal lymphocytes. CD39 expression was higher in the right colon and early-stage tumours, thus defining a subset of patients potentially responsive to CD39 blockade. Finally, we demonstrate in autologous conditions that CD39 blockade triggers T cell infiltration and tumour spheroid destruction in cocultures. CONCLUSION: In CRC, CD39 is strongly expressed on tumour infiltrating lymphocytes and its inhibition represents a promising therapeutic strategy for treating patients.

Comprehensive landscape of immune-checkpoints uncovered in clear cell renal cell carcinoma reveals new and emerging therapeutic targets.

Clear cell renal cell carcinoma (ccRCC) constitutes the most common renal cell carcinoma subtype and has long been recognized as an immunogenic cancer. As such, significant attention has been directed toward optimizing immune-checkpoints (IC)-based therapies. Despite proven benefits, a substantial number of patients remain unresponsive to treatment, suggesting that yet unreported, immunosuppressive mechanisms coexist within tumors and their microenvironment. Here, we comprehensively analyzed and ranked forty-four immune-checkpoints expressed in ccRCC on the basis of in-depth analysis of RNAseq data collected from the TCGA database and advanced statistical methods designed to obtain the group of checkpoints that best discriminates tumor from healthy tissues. Immunohistochemistry and flow cytometry confirmed and enlarged the bioinformatics results. In particular, by using the recursive feature elimination method, we show that HLA-G, B7H3, PDL-1 and ILT2 are the most relevant genes that characterize ccRCC. Notably, ILT2 expression was detected for the first time on tumor cells. The levels of other ligand-receptor pairs such as CD70:CD27; 4-1BB:4-1BBL; CD40:CD40L; CD86:CTLA4; MHC-II:Lag3; CD200:CD200R; CD244:CD48 were also found highly expressed in tumors compared to adjacent non-tumor tissues. Collectively, our approach provides a comprehensible classification of forty-four IC expressed in ccRCC, some of which were never reported before to be co-expressed in ccRCC. In addition, the algorithms used allowed identifying the most relevant group that best discriminates tumor from healthy tissues. The data can potentially assist on the choice of valuable immune-therapy targets which hold potential for the development of more effective anti-tumor treatments.

[Primary epithelioid adrenal angiosarcoma mimicking undifferentiated carcinoma]. / Angiosarcome épithélioïde surrénalien primitif simulant un carcinome peu différencié.

The adrenal primary epithelioid angiosarcoma (ASE) is a rare malignant tumor which can be histologically confused with other neoplasms. We report one case in a 79-year-old man who underwent adrenal tumor surgery for a mass fortuitly discovered by imaging for examination of an inflammatory syndrome associated with anemia. The histological diagnosis was difficult because of the undifferentiated and epithelioid appearance of tumor cells, whose immunohistochemical epithelial markers positivity led to frequent confusion with a metastatic carcinoma. Careful research for vascular differentiation at histopathological study and expression of immunohistochimical endothelial markers, are crucial to confirm the diagnosis.

Computed Tomography Evaluation of Esophagogastric Necrosis After Caustic Ingestion.

BACKGROUND: Endoscopy is the standard of care for emergency patient evaluation after caustic ingestion. However, the inaccuracy of endoscopy in determining the depth of intramural necrosis may lead to inappropriate decision-making with devastating consequences. Our aim was to evaluate the use of computed tomography (CT) for the emergency diagnostic workup of patients with caustic injuries. METHODS: In a prospective study, we used a combined endoscopy-CT decision-making algorithm. The primary outcome was pathology-confirmed digestive necrosis. The respective utility of CT and endoscopy in the decision-making process were compared. Transmural endoscopic necrosis was defined as grade 3b injuries; signs of transmural CT necrosis included absence of postcontrast gastric/ esophageal-wall enhancement, esophageal-wall blurring, and periesophageal-fat blurring. RESULTS: We included 120 patients (59 men, median age 44 years). Emergency surgery was performed in 24 patients (20%) and digestive resection was completed in 16. Three patients (3%) died and 28 patients (23%) experienced complications. Pathology revealed transmural necrosis in 9/11 esophagectomy and 16/16 gastrectomy specimens. Severe oropharyngeal injuries (P = 0.015), increased levels of blood lactate (P = 0.007), alanine aminotransferase (P = 0.027), bilirubin (P = 0.005), and low platelet counts (P > 0.0001) were predictive of digestive necrosis. Decision-making relying on CT alone or on a combined CT-endoscopy algorithm was similar and would have spared 19 unnecessary esophagectomies and 16 explorative laparotomies compared with an endoscopy-alone algorithm. Endoscopy did never rectify a wrong CT decision. CONCLUSIONS: Emergency decision-making after caustic injuries can rely on CT alone.

Molecular apocrine breast cancers are aggressive estrogen receptor negative tumors overexpressing either HER2 or GCDFP15.

INTRODUCTION: Molecular apocrine (MA) tumors are estrogen receptor (ER) negative breast cancers characterized by androgen receptor (AR) expression. We analyzed a group of 58 transcriptionally defined MA tumors and proposed a new tool to identify these tumors. METHODS: We performed quantitative reverse transcription PCR (qRT-PCR) for ESR1, AR, FOXA1 and AR-related genes, and immunohistochemistry (IHC) for ER, PR, Human Epidermal Growth Factor Receptor 2 (HER2), CK5/6, CK17, EGFR, Ki67, AR, FOXA1 and GCDFP15 and we analyzed clinical features. RESULTS: MA tumors were all characterized by ESR1(-) AR(+) FOXA1(+) and AR-related genes positive mRNA profile. IHC staining on these tumors showed 93% ER(-), only 58% AR(+) and 90% FOXA1(+). 67% and 57% MA tumors were HER2(3+) and GCDFP15(+), respectively. Almost all MA tumors (94%) had the IHC signature HER2(3+) or GCDFP15(+) but none of the 13 control basal-like (BL) tumors did. Clinically, MA tumors were rather aggressive, with poor prognostic factors. CONCLUSION: MA tumors could be better defined by their qRT-PCR-AR profile than by AR IHC. In addition, we found that HER2 or GCDFP15 protein overexpression is a sensitive and specific tool to differentiate MA from BL in the context of ER negative tumors. A composite molecular and IHC signature could, therefore, help to identify MA tumors in daily practice.

Assessment of the reliability of MSI status and dMMR proteins deficiency screening on endoscopic biopsy material in esophagus and gastric adenocarcinoma.

BACKGROUND: Microsatellite instability (MSI) is a negative predictive factor for neoadjuvant chemotherapy in resectable oesogastric adenocarcinoma and a crucial determinant for immunotherapy. We aimed to evaluate reliability of dMMR/MSI status screening performed on preoperative endoscopic biopsies. METHODS: Paired pathological samples from biopsies and surgical specimen of oesogastric adenocarcinoma were retrospectively collected between 2009 and 2019. We compared dMMR status obtained by immunohistochemistry (IHC) and MSI status by PCR. dMMR/MSI status on surgical specimen was considered as reference. RESULTS: PCR and IHC were conclusive on biopsies respectively for 53 (96.4%) and 47 (85.5%) of the 55 patients enrolled. IHC was not contributive for 1 surgical specimen. A third reading of IHC was carried out for 3 biopsies. MSI status was observed in 7 (12.5%) surgical specimens. When analyses were contributive, sensitivity and specificity of biopsies for dMMR/MSI were respectively 85% and 98% for PCR vs. 86% and 98% for IHC. Concordance rate between biopsies and surgical specimen was 96.2% for PCR and 97.8% for IHC. CONCLUSIONS: Endoscopic biopsies are a suitable source of tissue for dMMR/MSI status determination in oesogastric adenocarcinoma which should be routinely performed at diagnosis to better adapt neoadjuvant treatment. MINIABSTRACT: By comparison of dMMR phenotype obtained by immunohistochemistry and MSI status by PCR between match-paired samples of oesogastric cancer's endoscopic biopsies and surgical specimen, we observed that biopsies are a suitable source of tissue for dMMR/MSI status determination.

Large-scale analysis of cell-cell communication reveals angiogenin-dependent tumor progression in clear cell renal cell carcinoma.

Cellular crosstalk in the tumor microenvironment (TME) is still largely uncharacterized, while it plays an essential role in shaping immunosuppression or anti-tumor response. Large-scale analyses are needed to better decipher cell-cell communication in cancer. In this work, we used original and publicly available single-cell RNA sequencing (scRNAseq) data to characterize in-depth the communication networks in human clear cell renal cell carcinoma (ccRCC). We identified 50 putative communication channels specifically used by cancer cells to interact with other cells, including two novel angiogenin-mediated interactions. Expression of angiogenin and its receptors was validated at the protein level in primary ccRCC. Mechanistically, angiogenin enhanced ccRCC cell line proliferation and down-regulated secretion of IL-6, IL-8, and MCP-1 proinflammatory molecules. This study provides novel biological insights into molecular mechanisms of ccRCC, and suggests angiogenin and its receptors as potential therapeutic targets in clear cell renal cancer.

Methods for Establishing a Renal Cell Carcinoma Tumor Spheroid Model With Immune Infiltration for Immunotherapeutic Studies.

Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies.

Micromolecular methods for diagnosis and therapeutic strategy: a case study.

An intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffin-embedding of the sample. Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We laser-microdissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2-overexpressing breast cancer cell line BT-474. We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy.

18FDG-PET/CT and molecular markers to predict response to neoadjuvant chemotherapy and outcome in HER2-negative advanced luminal breast cancers patients.

BACKGROUND: The efficacy of neoadjuvant chemotherapy regimens in advanced luminal breast cancer patients is difficult to predict. Intrinsic properties of breast tumors, including altered gene expression profile and dynamic evaluation of metabolic properties of tumor cells using positron emission tomography/computed tomography (PET/CT) of tumor cells, have been identified to guide patient's prognosis. The aim of this study is to determine if both analyses may improve the prediction of response to neoadjuvant chemotherapy in ER-positive / HER2-negative breast cancers (BCs) patients. METHODS: We used metabolic PET parameters, at diagnosis and after two cycles of chemotherapy and proliferation gene expression profile on biopsy at diagnosis, in particular, the genomic grade index (GGI) analyzed by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The pathological response was the surrogate endpoint. RESULTS: The change of FDG uptake between baseline PET and interim PET after 2 cycles of neoadjuvant chemotherapy (ΔSUVmax) was highly associated with pCR (p=0.008). We also observed an ability of P53 mutated status (p=0.042), in addition to histological grade (p=0. 0004), and PR expression (p=0.01) to predict pCR in ER-positive BCs, whereas no proliferation marker predicted pCR (P=0.39 for GGI). Finally, only ΔSUVmax was significantly associated with event free survival (p=0.047). CONCLUSIONS: Our results confirm the predictive and prognostic value of tumor ΔSUVmax in ER-positive /HER2-negative advanced BCs patients. These findings can be helpful to select high-risk patients within trials investigating novel treatment strategies.

Phenotypic analysis of T cells infiltrating colon cancers: Correlations with oncogenetic status.

Colorectal cancers (CRC) develop in the face of an important immune system associated with the intestinal mucosal tissue. The immune response against the tumor has been proposed to affect the prognosis of patients undergoing treatment for CRC. In this study T cells infiltrating the tumor were compared with T cells populating the unaffected neighboring mucosal tissue and cells from the peripheral blood. We observed that T cells from the tumor harbor an activated phenotype, with engagement of the NKG2D pathway in CD8 T cells. We show that mucosal and tumor-infiltrating T cells are enriched in NKG2D CD4 T cells, which exhibit cytotoxic functions. Finally, T cell populations in the tumor were modified according to its oncogenetic status, with higher percentages of CD8 T cells isolated from patients with microsatellite instable tumor status.

The High Expression of the microRNA 17-92 Cluster and its Paralogs, and the Downregulation of the Target Gene PTEN, Is Associated with Primary Cutaneous B-Cell Lymphoma Progression.

The oncogenic microRNA (miR) 17-92 cluster has a causative role in the lymphomagenesis of nodal B-cell lymphomas, by activating proliferation and inhibiting apoptosis. Here we analyzed primary cutaneous B-cell lymphomas for the miR-17-92 cluster and its paralogs miR-106a-363 and miR-106b-25. In 22 primary cutaneous diffuse large B-cell lymphomas, leg type (PCLBCL-LT) compared with 22 primary cutaneous follicle center lymphomas (PCFCLs), we found that miR-20a and miR-106a were overexpressed. Multivariate Cox analysis showed that higher miR-20a and miR-20b expression levels were associated with shorter disease-free and overall survival, independently from histological type. Gene expression profiling also showed a downregulation of 8 candidate target genes of miR-20a, miR-20b, and miR-106a in PCLBCL-LT compared with PCFCL. Among the candidate target genes, PTEN, NCOA3, and CAPRIN2 were confirmed to be underexpressed in PCLBCL-LT using quantitative reverse transcriptase-PCR on CD20-positive laser-microdissected tumor cells. In multivariate Cox analysis, lower PTEN mRNA expression level was associated with shorter disease-free survival (DFS), independently from the histological type. Altogether, this molecular and bioinformatic study of 44 patient skin biopsy samples showed that the oncogenic miR-17-92 cluster and its paralogs were involved in cutaneous B-cell lymphoma progression, and that the downregulation of the target gene PTEN was associated with shorter DFS.

Towards better digital pathology workflows: programming libraries for high-speed sharpness assessment of Whole Slide Images.

BACKGROUND: Since microscopic slides can now be automatically digitized and integrated in the clinical workflow, quality assessment of Whole Slide Images (WSI) has become a crucial issue. We present a no-reference quality assessment method that has been thoroughly tested since 2010 and is under implementation in multiple sites, both public university-hospitals and private entities. It is part of the FlexMIm R&D project which aims to improve the global workflow of digital pathology. For these uses, we have developed two programming libraries, in Java and Python, which can be integrated in various types of WSI acquisition systems, viewers and image analysis tools. METHODS: Development and testing have been carried out on a MacBook Pro i7 and on a bi-Xeon 2.7GHz server. Libraries implementing the blur assessment method have been developed in Java, Python, PHP5 and MySQL5. For web applications, JavaScript, Ajax, JSON and Sockets were also used, as well as the Google Maps API. Aperio SVS files were converted into the Google Maps format using VIPS and Openslide libraries. RESULTS: We designed the Java library as a Service Provider Interface (SPI), extendable by third parties. Analysis is computed in real-time (3 billion pixels per minute). Tests were made on 5000 single images, 200 NDPI WSI, 100 Aperio SVS WSI converted to the Google Maps format. CONCLUSIONS: Applications based on our method and libraries can be used upstream, as calibration and quality control tool for the WSI acquisition systems, or as tools to reacquire tiles while the WSI is being scanned. They can also be used downstream to reacquire the complete slides that are below the quality threshold for surgical pathology analysis. WSI may also be displayed in a smarter way by sending and displaying the regions of highest quality before other regions. Such quality assessment scores could be integrated as WSI's metadata shared in clinical, research or teaching contexts, for a more efficient medical informatics workflow.

Active chronic sarcoidosis is characterized by increased transitional blood B cells, increased IL-10-producing regulatory B cells and high BAFF levels.

BACKGROUND: Sarcoidosis is a multisystemic disease of unknown etiology characterized by a disproportionate Th1 granulomatous immune response in the organs involved. Plasmatic hypergammaglobulinemia and B cell accumulation in granulomatous lesions suggest the possible role of humoral immune responses in the pathogenesis of sarcoidosis. The purpose of this study is to describe B cell peripheral compartment in sarcoidosis. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed blood B cell subsets and BAFF levels in 33 patients with chronic sarcoidosis (active sarcoidosis n = 18; inactive sarcoidosis n = 15) and 18 healthy donors. Active chronic sarcoidosis patients had significantly less circulating memory B cells (p<0.01), more transitional (p<0.01) and increased numbers of IL-10-producing regulatory B cells (p<0.05) compared with healthy donors and patients with inactive sarcoidosis. BAFF serum levels were significantly higher in patients with active sarcoidosis (p<0.01 versus healthy donors and inactive sarcoidosis patients) and strongly correlated with serum hypergammaglobulinemia (r = 0.53, p<0.01) and angiotensin converting enzyme levels (r = 0.61, p = <0.01). CONCLUSIONS/SIGNIFICANCE: These data show that there is an altered B cell homeostasis in active sarcoidosis and suggest BAFF antagonist drugs as potential new treatments of this disease.

Pericapillary hemorrhage as criterion of severe human digestive graft-versus-host disease.

In an experimental model we demonstrated that endothelial cells of all organs are targets of the alloimmune reaction. Here, in 68 digestive biopsies, we found endothelial lesions by immunohistochemistry and ultrastructure in patients with severe acute graft-versus-host disease (GVHD). In contrast, no such endothelial cell alterations were found either in patients without GVHD or in nongrafted controls. In the biopsies with severe GVHD lesions, ultrastructure showed rupture of the capillary basal membrane and extravased red blood cells. These pericapillary hemorrhages were highly correlated with GVHD severity. In a separate cohort of 39 patients who underwent an allogeneic transplantation after a nonmyeloablative conditioning, 8 patients had intestinal biopsies. Three of these latter patients had both severe pathologic lesions of GVHD and similar endothelial lesions, thus, strengthening the concept that endothelial lesions are linked to GVHD severity and not to the intensity of the conditioning regimen. (Blood. 2004;103:4681-4684)