Immune checkpoints are predominantly co-expressed by clonally expanded CD4+FoxP3+ intratumoral T-cells in primary human cancers.

BACKGROUND: In addition to anti-PD(L)1, anti-CTLA-4 and anti-LAG-3, novel immune checkpoint proteins (ICP)-targeted antibodies have recently failed to demonstrate significant efficacy in clinical trials. In these trials, patients were enrolled without screening for drug target expression. Although these novel ICP-targeted antibodies were expected to stimulate anti-tumor CD8 + T-cells, the rationale for their target expression in human tumors relied on pre-clinical IHC stainings and transcriptomic data, which are poorly sensitive and specific techniques for assessing membrane protein expression on immune cell subsets. Our aim was to describe ICP expression on intratumoral T-cells from primary solid tumors to better design upcoming neoadjuvant cancer immunotherapy trials. METHODS: We prospectively performed multiparameter flow cytometry and single-cell RNA sequencing (scRNA-Seq) paired with TCR sequencing on freshly resected human primary tumors of various histological types to precisely determine ICP expression levels within T-cell subsets. RESULTS: Within a given tumor type, we found high inter-individual variability for tumor infiltrating CD45 + cells and for T-cells subsets. The proportions of CD8+ T-cells (~ 40%), CD4+ FoxP3- T-cells (~ 40%) and CD4+ FoxP3+ T-cells (~ 10%) were consistent across patients and indications. Intriguingly, both stimulatory (CD25, CD28, 4-1BB, ICOS, OX40) and inhibitory (PD-1, CTLA-4, PD-L1, CD39 and TIGIT) checkpoint proteins were predominantly co-expressed by intratumoral CD4+FoxP3+ T-cells. ScRNA-Seq paired with TCR sequencing revealed that T-cells with high clonality and high ICP expressions comprised over 80% of FoxP3+ cells among CD4+ T-cells. Unsupervised clustering of flow cytometry and scRNAseq data identified subsets of CD8+ T-cells and of CD4+ FoxP3- T-cells expressing certain checkpoints, though these expressions were generally lower than in CD4+ FoxP3+ T-cell subsets, both in terms of proportions among total T-cells and ICP expression levels. CONCLUSIONS: Tumor histology alone does not reveal the complete picture of the tumor immune contexture. In clinical trials, assumptions regarding target expression should rely on more sensitive and specific techniques than conventional IHC or transcriptomics. Flow cytometry and scRNAseq accurately characterize ICP expression within immune cell subsets. Much like in hematology, flow cytometry can better describe the immune contexture of solid tumors, offering the opportunity to guide patient treatment according to drug target expression rather than tumor histological type.

Multiomic analysis of malignant pleural mesothelioma identifies molecular axes and specialized tumor profiles driving intertumor heterogeneity.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with rising incidence and challenging clinical management. Through a large series of whole-genome sequencing data, integrated with transcriptomic and epigenomic data using multiomics factor analysis, we demonstrate that the current World Health Organization classification only accounts for up to 10% of interpatient molecular differences. Instead, the MESOMICS project paves the way for a morphomolecular classification of MPM based on four dimensions: ploidy, tumor cell morphology, adaptive immune response and CpG island methylator profile. We show that these four dimensions are complementary, capture major interpatient molecular differences and are delimited by extreme phenotypes that-in the case of the interdependent tumor cell morphology and adapted immune response-reflect tumor specialization. These findings unearth the interplay between MPM functional biology and its genomic history, and provide insights into the variations observed in the clinical behavior of patients with MPM.

Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. METHODS: Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). RESULTS: Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. CONCLUSIONS: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next-generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods.

Involvement of SUR2/Kir6.1 channel in the physiopathology of pulmonary arterial hypertension.

Aims: We hypothesized that the ATP-sensitive K+ channels (KATP) regulatory subunit (ABCC9) contributes to PAH pathogenesis. ABCC9 gene encodes for two regulatory subunits of KATP channels: the SUR2A and SUR2B proteins. In the KATP channel, the SUR2 subunits are associated with the K+ channel Kir6.1. We investigated how the SUR2/Kir6.1 channel contributes to PAH pathogenesis and its potential as a therapeutic target in PAH. Methods and results: Using in vitro, ex vivo, and in vivo approaches, we analyzed the localization and expression of SUR2A, SUR2B, and Kir6.1 in the pulmonary vasculature of controls and patients with PAH as in experimental pulmonary hypertension (PH) rat models and its contribution to PAH physiopathology. Finally, we deciphered the consequences of in vivo activation of SUR2/Kir6.1 in the monocrotaline (MCT)-induced PH model. We found that SUR2A, SUR2B, and Kir6.1 were expressed in the lungs of controls and patients with PAH and MCT-induced PH rat models. Organ bath studies showed that SUR2 activation by pinacidil induced relaxation of pulmonary arterial in rats and humans. In vitro experiments on human pulmonary arterial smooth muscle cells and endothelial cells (hPASMCs and hPAECs) in controls and PAH patients showed decreased cell proliferation and migration after SUR2 activation. We demonstrated that SUR2 activation in rat right ventricular (RV) cardiomyocytes reduced RV action potential duration by patch-clamp. Chronic pinacidil administration in control rats increased heart rate without changes in hemodynamic parameters. Finally, in vivo pharmacological activation of SUR2 on MCT and Chronic-hypoxia (CH)-induced-PH rats showed improved PH. Conclusion: We showed that SUR2A, SUR2B, and Kir6.1 are presented in hPASMCs and hPAECs of controls and PAH patients. In vivo SUR2 activation reduced the MCT-induced and CH-induced PH phenotype, suggesting that SUR2 activation should be considered for treating PAH.

[Thymoma and squamous thymic carcinoma diagnosis; experience from the RYTHMIC network]. / Diagnostic des thymomes et carcinomes thymiques malpighiens ; expérience du réseau RYTHMIC.

The RYTHMIC network, supported by the French National Cancer Institute is dedicated to the management of patients with thymic epithelial tumors through regional and national multidisciplinary tumor boards. Tumor board decisions are based on the initial pathology diagnoses. However, following clinical inclusion in the network, a central pathology review is organized, implicating a panel of pathologists, for histotype and stage classification, which is different from a classical second opinion from pathologist to pathologist for a difficult case. Thanks to the participation of all French pathologists, more than 1000 cases have been reviewed by the panel. The aim of this review is to share with the French pathology community, the experience of the group. It underlines the importance of macroscopy and surgeon-pathologist involvement to allow a good central review, the main histopathological and immunophenotypical patterns of the most frequent thymomas and thymic carcinoma types, the differential diagnoses, as well as the difficulties for the panel to reproducibly assess on slides, stage, for some cases.

Natural History of Localized and Locally Advanced Atypical Lung Carcinoids after Complete Resection: A Joined French-Italian Retrospective Multicenter Study.

BACKGROUND: The natural history and the best modality of follow-up of atypical lung carcinoids (AC) remain ill defined. The aim of this study was to analyze recurrence-free survival (RFS) after complete resection (R0) of stage I-III pulmonary AC. Secondary objectives were prognostic parameters, the location of recurrences, and the modality of follow-up. METHODS: A retrospective review of 540 charts of AC patients treated between 1998 and 2008 at 10 French and Italian centers with experience in lung neuroendocrine tumor management was undertaken. The exclusion criteria were MEN1-related tumor, history of another cancer, referral after tumor relapse, and being lost to follow-up. A central pathological review was performed in each country. RESULTS: Sixty-two patients were included. After a median follow-up time of 91 months (mean 85, range 6-165), 35% of the patients experienced recurrence: 16% were regional recurrences and 19% were distant metastases. Median RFS was not reached. The 1-, 3-, and 5-year RFS rate was 90, 79, and 68%, respectively. In univariate analysis, lymph node involvement (p = 0.0001), stage (p = 0.0001), mitotic count (p = 0.004), and type of surgery (p = 0.043) were significantly associated with RFS. In multivariate analysis, lymph node involvement was significantly associated with RFS (HR 95% CI: 0.000-0.151; p = 0.004). During follow-up, somatostatin receptor scintigraphy, fibroscopy, and abdominal examination results were available for 22, 12, and 25 patients, respectively. The median time interval for imaging follow-up was 10 months. CONCLUSIONS: After complete resection of AC, recurrences were observed mostly within the first 5 years of follow-up, within bronchi, mediastinal nodes, the liver, and bones. In R0 patients, lymph node involvement could help to stratify follow-up intervals. Suboptimal imaging is evidenced.

Osteopontin and thrombospondin-1 play opposite roles in promoting tumor aggressiveness of primary resected non-small cell lung cancer.

BACKGROUND: Osteopontin (OPN) and thrombospondin-1 (TSP-1) are extracellular matrix proteins secreted by stromal and tumor cells. These proteins appear to have a key role in the tumor microenvironment for cancer development and metastasis. There is little information regarding the prognostic value of the combination of these two proteins in human cancers. Our aim was to clarify clinical significance and prognostic value of each circulating protein and their combination in primary resected non-small cell lung cancer (NSCLC) patients. METHODS: We retrospectively reviewed 171 patients with NSCLC following curative intent surgery from January to December of 2012. Preoperative serums, demographics, clinical and pathological data and molecular profiling were analyzed. Pre-treatment OPN and TSP-1 serum levels were measured by ELISA. Tissue protein expression in primary tumor samples was determined by immunohistochemical analysis. RESULTS: OPN and TSP-1 serum levels were inversely correlated with survival rates. For each 50 units increment of serum OPN, an increased risk of metastasis by 69 % (unadjusted HR 1.69, 95 % CI 1.12-2.56, p = 0.01) and an increased risk of death by 95 % (unadjusted HR 1.95, 95 % CI 1.15-3.32, p = 0.01) were observed. Conversely, for each 10 units increment in TSP-1, the risk of death was decreased by 85 % (unadjusted HR 0.15, 95 % CI 0.03-0.89; p = 0.04). No statistically significant correlation was found between TSP-1 serum level and distant metastasis-free survival (p = 0.2). On multivariate analysis, OPN and TSP-1 serum levels were independent prognostic factors of overall survival (HR 1.71, 95 % CI 1.04-2.82, p = 0.04 for an increase of 50 ng/mL in OPN; HR 0.18, 95 % CI 0.04-0.87, p = 0.03 for an increase of 10 ng/mL in TSP-1). In addition, the combination of OPN and TSP-1 serum levels remained an independent prognostic factor for overall survival (HR 1.31, 95 % CI 1.03-1.67, p = 0.03 for an increase of 6 ng/mL in OPN/TSP-1 ratio). CONCLUSIONS: Our results show that pre-treatment OPN and TSP-1 serum levels may reflect the aggressiveness of the tumor and might serve as prognostic markers in patients with primary resected NSCLC.

SMARCA4 inactivation defines a group of undifferentiated thoracic malignancies transcriptionally related to BAF-deficient sarcomas.

While investigating cohorts of unclassified sarcomas by RNA sequencing, we identified 19 cases with inactivation of SMARCA4, which encodes an ATPase subunit of BAF chromatin-remodeling complexes. Clinically, the cases were all strikingly similar, presenting as compressive mediastino-pulmonary masses in 30- to 35-year-old adults with a median survival time of 7 months. To help define the nosological relationships of these tumors, we compared their transcriptomic profiles with those of SMARCA4-mutated small-cell carcinomas of the ovary, hypercalcemic type (SCCOHTs), SMARCB1-inactivated malignant rhabdoid tumors (MRTs) and lung carcinomas (of which 10% display SMARCA4 mutations). Gene profiling analyses demonstrated that these tumors were distinct from lung carcinomas but related to MRTs and SCCOHTs. Transcriptome analyses, further validated by immunohistochemistry, highlighted strong expression of SOX2, a marker that supports the differential diagnosis of these tumors from SMARCA4-deficient lung carcinomas. The prospective recruitment of cases confirmed this new category of 'SMARCA4-deficient thoracic sarcomas' as readily recognizable in clinical practice, providing opportunities to tailor their therapeutic management.

Thoracic endometriosis: clinicopathologic updates and issues about 18 cases from a tertiary referring center.

Thoracic endometriosis is defined as the ectopic presence of endometrial glands and stroma in lung or pleura and constitutes an uncommon cause of spontaneous pneumothorax in nonsmoker women in childbearing age. From 2000 to 2014, 18 (7.3%) women of 246 had a histologically proven endometriosis-related pneumothorax. The examination of thoracic samples was performed on formalin-fixed, paraffin-embedded hematoxylin and eosin-stained slides. Immunohistochemistry was carried out on all samples. The pathologic findings included endometriosis foci appearing as a triad of endometrial glands, stroma, and hemosiderin-laden macrophages in 8 patients. In the remaining 10 patients, minute nests of endometrial stroma were recognized at histologic examination and immunohistochemistry. One patient displayed diaphragmatic endometriosis and pulmonary lymphangioleiomyomatosis. Thoracic endometriosis is an uncommon cause of recurrent pneumothorax necessitating histologic confirmation. The histologic diagnosis of endometriosis may be challenging on small pleural or lung biopsies because endometriosis mostly appears as minute nests of endometrial stromal cells. Here, we present our experience on thoracic endometriosis: considering frequent interpretation difficulties of biopsy samples, we propose a multidisciplinary diagnostic algorithm.

Sub-aortic obstruction of left ventricular outflow tract secondary to benfluorex-induced endocardial fibrosis.

Patients exposed to benfluorex have an increased risk of restrictive organic valvular heart disease. Aortic and mitral regurgitations caused by fibrotic valve disease are the most common features observed in exposure to fenfluramine derivatives in general and benfluorex in particular. We report here, for the first time to our knowledge, a well-documented case in which obstructive sub-aortic endocardium fibrosis within the left ventricular outflow tract is related with exposure to a drug that modifies the metabolism of serotonin. It now remains to be established whether extensive fibrosis of the myocardium in addition to well-documented valvular fibrosis may develop in patients exposed to amphetamine-derived drugs affecting the serotonin system.

Intimal sarcoma is the most frequent primary cardiac sarcoma: clinicopathologic and molecular retrospective analysis of 100 primary cardiac sarcomas.

We report novel molecular and pathologic features of sarcomas involving the heart. Intimal sarcoma appears as the most frequent primary cardiac sarcoma within the largest described series of 100 primary cardiac sarcomas. Immunohistochemical analysis, fluorescence in situ hybridization, real-time polymerase chain reaction, and array-comparative genomic hybridization were performed on materials from 65 women and 35 men, aged 18 to 82 years (mean 50 y), retrieved from the French Departments of Pathology, between 1977 and early 2013. Right and left heart was involved in 44 and 56 cases, respectively. There were 42 intimal sarcomas, 26 angiosarcomas, 22 undifferentiated sarcomas, 7 synovial sarcomas, 2 leiomyosarcomas, and 1 peripheral neuroectodermal tumor. All but 1 angiosarcomas originated from the right heart, whereas 83% of the intimal sarcomas and 72% of the undifferentiated sarcomas were from the left heart. MDM2 overexpression was immunohistochemically observed in all intimal sarcomas, as well as in 10 of the 22 undifferentiated sarcomas and in 5 of the 26 angiosarcomas. MDM2 amplification was only demonstrated in intimal sarcomas. Genomic analysis showed a complex profile, with recurrent 12q13-14 amplicon involving MDM2, 4q12 amplicon involving KIT and PDGFRA, 7p12 gain involving EGFR, and 9p21 deletion targeting CDKN2A. Immunohistochemical detection of MDM2 overexpression can easily detect intimal sarcoma, provided that molecular aberration is proved. As resections are limited to the left atrium, this histologic subtype could benefit from therapies targeting PDGFRA or MDM2.

Effect of chemotherapy in patients with resected small-cell or large-cell neuroendocrine carcinoma.

INTRODUCTION: Surgical resection of an undiagnosed lung lesion may lead to unintentional removal of small-cell lung cancer (SCLC). The benefit of perioperative chemotherapy in resected SCLC or large-cell neuroendocrine carcinomas (LCNEC) is not clear. METHODS: This retrospective analysis included limited disease SCLC and LCNEC that had been surgically removed between 1979 and 2007 at a single institution. Perioperative treatments were analyzed, and survival followed up. Log rank tests were used to compare overall survival. RESULTS: Among 74 patients who had a tumor resection, 45 received chemotherapy, four had preoperative radiotherapy, and 21 had postoperative radiotherapy. Eleven patients were women. The median age was 64 in the surgery group and 58 in the surgery plus chemotherapy group, and four and 11 patients in these groups, respectively, had LCNEC. There were 10 node positive tumors and only two incomplete resections in the surgery group versus 27 node positive tumors and three incomplete resections in the surgery plus chemotherapy group. The median follow-up was shorter in the group with surgery alone: 4.5 years (1.4-7) versus 5.8 years (0.6-19.6). Among the patients with a survival or a follow-up of at least 6 months, the median survival was 2.3 and 6.1 years in the surgery (n = 20) and surgery plus chemotherapy (n = 39) groups, respectively, such that the hazard ratio for death was 0.48 (95% confidence interval, 0.24-0.99, p = 0.04). CONCLUSION: These results suggest that perioperative chemotherapy may be beneficial in patients with resected SCLC or LCNEC.

Paraganglioma after maternal transmission of a succinate dehydrogenase gene mutation.

CONTEXT: Inactivating mutations of SDHD, which is mapped to 11q23 and encodes the cybS subunit of succinate dehydrogenase, predispose to hereditary paraganglioma (PGL) and/or pheochromocytoma. So far no disease was shown to occur in case of maternal transmission of a SDHD mutation, suggesting the existence of genomic imprinting. A hypothetic model, involving the loss of the maternal copy of a tumor suppressor gene mapped to 11p15 in the tumoral tissue, has been proposed to explain this mode of inheritance. OBJECTIVE: Our objective was to investigate the possibility of maternal transmission of SDHD-linked PGL. DESIGN: A three-generation family carrying the SDHD W43X mutation was studied at the clinical, pathological, and genetical levels. RESULTS: The germline's mutation was probably inherited from the grandfather. In the second generation, three carriers (two females and one male), who had the same at risk 11q13-q23 haplotype, developed multiple cervical PGLs. In the third generation, one boy received the mutation from his mother and developed a glomus tympanicum PGL at 11 yr. He shared only the 11q23 haplotype with the other affected members of the family. Methylation analysis of the differentially methylated region upstream of the maternally expressed H19 gene, mapped to 11p15, showed that the seventh CTCF binding site is hypermethylated in the germline of the affected boy suggesting a gain of imprinting. CONCLUSION: Our data show that maternal transmission of a SDHD-linked PGL, even if a rare event, can occur. Therefore, we propose that children who inherited a pathogenic mutation from their mother should be considered as at risk of PGL.

Paraganglioma with ganglioneuromatous component located in the posterior mediastinum.

A 4-cm paravertebral mediastinal tumor was resected in a 70-year-old male patient treated for hypertension. The tumor displayed both paraganglioma and ganglioneuroma areas that were in equal proportion and often merged one into the other. Paraganglioma areas contained synaptophysin and chromogranin-positive chief cells and PS100-positive sustentacular cells. Ganglioneuroma areas contained neurofilament-positive mature ganglion cells and PS100-positive Schwann cells. Such pheochromocytoma-ganglioneuroma has not been previously reported in the mediastinum and appears as the adrenal and aorticosympathetic counterpart of gangliocytic paraganglioma described in other anatomic sites.

Endocardial location of familial myofibromatosis revealed by cerebral embolization: cardiac counterpart of the frequent intravascular growth of the disease?

Myofibromatosis is a rare infantile benign neoplasia, which may involve the heart in the rare and usually fatal generalized form of the disease. Diagnosis of endocardial myofibromas was made on two surgically excised lesions of the mitral valve that were reveled by a cerebral embolization in a 12-month-old female infant. Surprisingly, the patient had no other obvious lesion of myofibromatosis. However, her father had a histologically proven neonatal history of myofibromatosis. This case confirms the likely autosomal dominant mode of inheritance of myofibromatosis. It highlights the embolization risk of the previously unreported endocardial location. We suggest that these clinically isolated non-invasive endocardial myofibromas did not represent a true visceral form of myofibromatosis. They were, rather, similar to the frequent intravascular growth of the disease.