A Vision Transformer-Based Framework for Knowledge Transfer From Multi-Modal to Mono-Modal Lymphoma Subtyping Models.

Determining lymphoma subtypes is a crucial step for better patient treatment targeting to potentially increase their survival chances. In this context, the existing gold standard diagnosis method, which relies on gene expression technology, is highly expensive and time-consuming, making it less accessibility. Although alternative diagnosis methods based on IHC (immunohistochemistry) technologies exist (recommended by the WHO), they still suffer from similar limitations and are less accurate. Whole Slide Image (WSI) analysis using deep learning models has shown promising potential for cancer diagnosis, that could offer cost-effective and faster alternatives to existing methods. In this work, we propose a vision transformer-based framework for distinguishing DLBCL (Diffuse Large B-Cell Lymphoma) cancer subtypes from high-resolution WSIs. To this end, we introduce a multi-modal architecture to train a classifier model from various WSI modalities. We then leverage this model through a knowledge distillation process to efficiently guide the learning of a mono-modal classifier. Our experimental study conducted on a lymphoma dataset of 157 patients shows the promising performance of our mono-modal classification model, outperforming six recent state-of-the-art methods. In addition, the power-law curve, estimated on our experimental data, suggests that with more training data from a reasonable number of additional patients, our model could achieve competitive diagnosis accuracy with IHC technologies. Furthermore, the efficiency of our framework is confirmed through an additional experimental study on an external breast cancer dataset (BCI dataset).

Performance of non-invasive tests for liver fibrosis resolution after bariatric surgery.

BACKGROUND & AIMS: The value of non-invasive tests for monitoring the resolution of significant liver fibrosis after treatment is poorly investigated. We compared the performances of six non-invasive tests to predict the resolution of significant fibrosis after bariatric surgery. METHODS: Participants were individuals with obesity submitted to needle liver biopsy at the time of bariatric surgery, and 12 and/or 60 months after surgery. We calculated the fibrosis-4 index (FIB-4), NAFLD fibrosis score (NFS), AST to platelet ratio index (APRI), Hepatic fibrosis score (HFS), Fibrotic NASH index (FNI), and Liver risk score (LRS) at each time point, and compared their performances for predicting significant fibrosis (F ≥ 2) and its resolution following surgery. RESULTS: At baseline, 2436 patients had liver biopsy, including 261 (10.7 %) with significant fibrosis. Overall, 672 patients had pre- and post-operative biopsies (564 at M12 and 328 at M60). The fibrosis stage decreased at M12 and M60 (p < 0.001 vs M0). Resolution of significant fibrosis occurred in 58/121 (47.9 %) at M12 and 32/50 (64 %) at M60. The mean value of all tests decreased after surgery, except for FIB-4. Performances for predicting fibrosis resolution was higher at M60 than at M12 for all tests, and maximal at M60 for FNI and LRS: area under the curve 0.843 (95%CI 0.71-0.95) and 0.92 (95%CI 0.84-1.00); positive likelihood ratio 3.75 (95 % CI 1.33-10.59) and 4.58 (95 % CI 1.65-12.70), respectively. CONCLUSIONS: Results showed the value and limits of non-invasive tests for monitoring the evolution of liver fibrosis after an intervention. Following bariatric surgery, the best performances to predict the resolution of significant fibrosis were observed at M60 with tests combining liver and metabolic traits, namely FNI and LRS.

Time-of-day-dependent variation of the human liver transcriptome and metabolome is disrupted in MASLD.

Background & Aims: Liver homeostasis is ensured in part by time-of-day-dependent processes, many of them being paced by the molecular circadian clock. Liver functions are compromised in metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), and clock disruption increases susceptibility to MASLD progression in rodent models. We therefore investigated whether the time-of-day-dependent transcriptome and metabolome are significantly altered in human steatotic and MASH livers. Methods: Liver biopsies, collected within an 8 h-window from a carefully phenotyped cohort of 290 patients and histologically diagnosed to be either normal, steatotic or MASH hepatic tissues, were analyzed by RNA sequencing and unbiased metabolomic approaches. Time-of-day-dependent gene expression patterns and metabolomes were identified and compared between histologically normal, steatotic and MASH livers. Results: Herein, we provide a first-of-its-kind report of a daytime-resolved human liver transcriptome-metabolome and associated alterations in MASLD. Transcriptomic analysis showed a robustness of core molecular clock components in steatotic and MASH livers. It also revealed stage-specific, time-of-day-dependent alterations of hundreds of transcripts involved in cell-to-cell communication, intracellular signaling and metabolism. Similarly, rhythmic amino acid and lipid metabolomes were affected in pathological livers. Both TNFα and PPARγ signaling were predicted as important contributors to altered rhythmicity. Conclusion: MASLD progression to MASH perturbs time-of-day-dependent processes in human livers, while the differential expression of core molecular clock components is maintained. Impact and implications: This work characterizes the rhythmic patterns of the transcriptome and metabolome in the human liver. Using a cohort of well-phenotyped patients (n = 290) for whom the time-of-day at biopsy collection was known, we show that time-of-day variations observed in histologically normal livers are gradually perturbed in liver steatosis and metabolic dysfunction-associated steatohepatitis. Importantly, these observations, albeit obtained across a restricted time window, provide further support for preclinical studies demonstrating alterations of rhythmic patterns in diseased livers. On a practical note, this study indicates the importance of considering time-of-day as a critical biological variable which may significantly affect data interpretation in animal and human studies of liver diseases.

A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Background: Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). Materials and methods: CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. Results: IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. Conclusion: We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

CDK4 phosphorylation status and rational use for combining CDK4/6 and BRAF/MEK inhibition in advanced thyroid carcinomas.

Background: CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers. Methods: We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays. Results: CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes. Conclusion: Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.

Combining diabetes, sex, and menopause as meaningful clinical features associated with NASH and liver fibrosis in individuals with class II and III obesity: A retrospective cohort study.

OBJECTIVE: Steatotic liver disease (SLD) is frequent in individuals with obesity. In this study, type 2 diabetes (T2D), sex, and menopausal status were combined to refine the stratification of obesity regarding the risk of advanced SLD and gain further insight into disease physiopathology. METHODS: This study enrolled 1446 participants with obesity from the ABOS cohort (NCT01129297), who underwent extensive phenotyping, including liver histology and transcriptome profiling. Hierarchical clustering was applied to classify participants. The prevalence of metabolic disorders associated with steatohepatitis (NASH) and liver fibrosis (F ≥ 2) was determined within each identified subgroup and aligned to clinical and biological characteristics. RESULTS: The prevalence of NASH and F ≥ 2 was, respectively, 9.5% (N = 138/1446) and 11.7% (N = 159/1365) in the overall population, 20.3% (N = 107/726) and 21.1% (N = 106/502) in T2D patients, and 3.4% (N = 31/920) and 6.1% (N = 53/863) in non-T2D patients. NASH and F ≥ 2 prevalence was 15.4% (33/215) and 15.5% (32/206) among premenopausal women with T2D vs. 29.5% (33/112) and 30.3% (N = 36/119) in postmenopausal women with T2D (p < 0.01); and 21.0% (21/100) / 27.0% (24/89) in men with T2D ≥ age 50 years and 17.9% (17/95) / 18.5% (17/92) in men with T2D < age 50 years (NS). The distinct contribution of menopause was confirmed by the interaction between sex and age with respect to NASH among T2D patients (p = 0.048). Finally, several NASH-associated biological traits (lower platelet count; higher serum uric acid; gamma-glutamyl transferase; aspartate aminotransferase) and liver expressed genes AKR1B10 and CCL20 were significantly associated with menopause in women with T2D but not with age in men with T2D. CONCLUSIONS: This study unveiled a remarkably high prevalence of advanced SLD after menopause in women with T2D, associated with a dysfunctional biological liver profile.

Factors Associated with Survival in Anaplastic Thyroid Carcinoma: A Multicenter Study from the ENDOCAN-TUTHYREF Network.

Background: Anaplastic thyroid carcinoma (ATC) is a rare and frequently fatal type of thyroid cancer. The degree of heterogeneity in survival rates for ATC is incompletely studied. This study evaluated the factors associated with overall survival (OS) of patients with ATC using multicenter real-world data from a national tertiary care center network in France. Methods: In this multicenter, retrospective cohort study, all patients with ATC diagnosed between 2010 and 2020 were identified from the national database of the French ENDOCAN-TUTHYREF network. Factors associated with OS were examined in multivariable analyses using Cox proportional hazards models. Results: The study included 360 patients. Of these, 220 (61%) were female and the median age was 72 years (interquartile range: 62-80). The percentages of patients with pure and mixed (synchronously-transformed) ATC (p-ATC and st-ATC) were 62.5% and 26.7%, respectively. The median OS was 6.8 months [confidence interval, CI: 5.5-8.1]: not reached for stage IVa, 11.4 months [8.2-17.8] for IVb, and 4.6 months [3.5-5.7] for IVc. Surgery, radiation therapy to the neck, chemotherapy, and best supportive care were administered to 69 (19.2%), 214 (59.4%), 254 (70.6%), and 66 (18.3%) patients, respectively. In a multivariable analysis, including stage IVb-IVc patients, significantly higher OS was observed in patients with Eastern Cooperative Oncology Group performance-status of 0-1 (hazard ratio [HR], 0.6; [CI, 0.4-0.9], p < 0.02), stage IVb [HR, 0.5; CI, 0.4-0.8, p < 0.001], and multimodal treatment (surgery and chemoradiotherapy) [HR, 0.07; CI, 0.04-0.1, p < 0.001]. Variables associated with significantly worse OS included: p-ATC (vs. st-ATC) [HR, 1.83; CI, 1.33-2.51, p = 0.001] and a neutrophil-to-lymphocyte ratio (NLR) >5.05 [HR, 2.05, CI, 1.39-3.05, p < 0.001]. Conclusions: Factors independently associated with improved OS in ATC included: European Cooperative Oncology Group performance status, disease stage, multimodality treatment, synchronously transformed ATC, and lower NLR. Long-term OS was observed in selected patients with ATC who underwent multimodal treatment.

Serum Succinate/Fumarate Ratio in Patients With Paraganglioma/Pheochromocytoma Attending an Endocrine Oncogenetic Unit.

CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) with SDHx pathogenic variants (PVs) are characterized by a higher intratissular succinate/fumarate ratio (RS/F) than non-SDHx-mutated ones. Also, an increase in serum succinate levels has been reported in patients with germline SDHB or SDHD PV. OBJECTIVE: To assess whether measurement of serum succinate, fumarate levels, and RS/F might aid identification of an SDHx germline PV/likely pathogenic variant (LPV) in patients with PPGL or in asymptomatic relatives; and to guide identification of a PV/LPV among the variants of unknown significance (VUS) identified in SDHx by next-generation sequencing. METHODS: This prospective monocentric study included 93 patients attending an endocrine oncogenetic unit for genetic testing. Succinate and fumarate were measured in serum by gas chromatography coupled to mass spectrometry. The RS/F was calculated to assess SDH enzymatic function. Diagnostic performance was assessed by receiver operating characteristic analysis. RESULTS: RS/F had a higher discriminant power than succinate alone to identify an SDHx PV/LPV in patients with PPGL. However, SDHD PVs/LPVs are frequently missed. Only RS/F differed between asymptomatic SDHB/SDHD PV/LPV carriers and SDHB/SDHD-linked patients with PPGL. Finally RS/F could be helpful to easily evaluate the functional impact of VUS in SDHx. CONCLUSION: Measurement of serum RS/F in patients with PPGL and in asymptomatic relatives is a valuable initial workup tool to detect those carrying a germline PV/LPV in SDHx. Its discriminative power is equal or superior to those of succinate measured alone. SDHD PVs/LPVs are less frequently identified by these biochemical tools. Use of RS/F for SDHx VUS reclassification needs to be evaluated further.

Complete pathological response following chemotherapy and radiotherapy in two cases of advanced anaplastic thyroid carcinoma.

Introduction: Anaplastic thyroid carcinoma (ATC) is the most aggressive form of thyroid cancer with a bleak prognosis. Favorable outcomes are rare but help decipher molecular pathophysiology, investigate prognosis factors, and discover new therapeutic targets. Case presentation: Two patients were diagnosed with locally advanced nonresectable ATC, one with metastatic extension. Each patient received chemotherapy and radiotherapy, allowing thyroid surgical resection. In both cases, the pathological examination was consistent with complete response with no viable tumor cells. After follow-ups of 48 and 70 months, both patients remain disease-free. Molecular explorations on thyroid biopsies revealed microsatellite instability (MSI) and alterations on mismatch repair-gene complex, also PTEN and ATM variants in both cases. Both also presented with non-classical immune infiltrate composed of equal parts T CD4+ lymphocytes and macrophages. Conclusion: We report two cases of patients cured from advanced ATC and for the first time provide genetic and immunological explorations in this setting. It seems with these two cases that MSI-ATCs may indicate a better prognosis. Our study hypothesizes different responsible mechanisms including increased sensitivity to chemoradiotherapy and/or immune tumor infiltrate modulation.

Impact of Lymph Node Dissection on Postoperative Complications of Total Thyroidectomy in Patients with Thyroid Carcinoma.

Background: Lymph node dissection (LND) in primary treatment of differentiated thyroid carcinoma is controversial. The aim of our retrospective study was to analyse the risk factors of post-thyroidectomy complications and to assess the morbidity of lymph node dissection, especially in the central neck compartment, since prophylactic central lymph node dissection has not been proven to bring an overall survival benefit. Methods: We performed a retrospective analysis of postoperative complications from 1547 consecutive patients with differentiated thyroid carcinoma in an academic department of endocrine surgery over a period of 10 years. Results: A total of 535 patients underwent lymph node dissection, whereas the other 1012 did not. The rate of postoperative hypoparathyroidism was higher in patients with LND (17.6% vs. 11.4%, p = 0.001). No significant difference in the rate of permanent hypoparathyroidism (2.4% vs. 1.3%, p = 0.096) was observed between these two groups. A multivariate analysis was performed. Female gender, ipsilateral and bilateral central LND (CLND), parathyroid autotransplantation, and the presence of the parathyroid gland on the resected thyroid were associated with transient hypoparathyroidism. Bilateral CLND and the presence of the parathyroid gland on specimen were associated with permanent hypoparathyroidism. The rate of transient recurrent laryngeal nerve (RLN) injury (15.3% vs. 5.4%, p < 0.001) and permanent RLN injury (6.5% vs. 0.9%, p < 0.001) were higher in the LND group. In multivariate analysis, ipsilateral and bilateral lateral LND (LLND) were the main predictive factors of transient and permanent RLN injury. Bilateral RLN injury (2.6% vs. 0.4%, p < 0.001), chyle leakage (2.4% vs. 0%, p < 0.001), other nerve injuries (2.2% vs. 0%, p < 0.001), and abscess (2.4% vs. 0.5%, p = 0.001) were higher in the patients with LND. Conclusions: The surgical technique and the extent of lymph node dissection during surgery for thyroid carcinoma increase postoperative morbidity. A wider knowledge of lymph-node-dissection-related complications associated with thyroid surgery could help surgeons to carefully evaluate the surgical and medical therapeutic options.

Functional genomics uncovers the transcription factor BNC2 as required for myofibroblastic activation in fibrosis.

Tissue injury triggers activation of mesenchymal lineage cells into wound-repairing myofibroblasts, whose unrestrained activity leads to fibrosis. Although this process is largely controlled at the transcriptional level, whether the main transcription factors involved have all been identified has remained elusive. Here, we report multi-omics analyses unraveling Basonuclin 2 (BNC2) as a myofibroblast identity transcription factor. Using liver fibrosis as a model for in-depth investigations, we first show that BNC2 expression is induced in both mouse and human fibrotic livers from different etiologies and decreases upon human liver fibrosis regression. Importantly, we found that BNC2 transcriptional induction is a specific feature of myofibroblastic activation in fibrotic tissues. Mechanistically, BNC2 expression and activities allow to integrate pro-fibrotic stimuli, including TGFß and Hippo/YAP1 signaling, towards induction of matrisome genes such as those encoding type I collagen. As a consequence, Bnc2 deficiency blunts collagen deposition in livers of mice fed a fibrogenic diet. Additionally, our work establishes BNC2 as potentially druggable since we identified the thalidomide derivative CC-885 as a BNC2 inhibitor. Altogether, we propose that BNC2 is a transcription factor involved in canonical pathways driving myofibroblastic activation in fibrosis.

Second-line treatment and prognostic factors in neuroendocrine carcinoma: the RBNEC study.

Neuroendocrine carcinomas (NEC) are aggressive malignant diseases. Etoposide-based rechallenge (EBR) and the prognostic role of RB transcriptional corepressor 1 (RB1) status in second-line chemotherapy (2L) have not been studied. The objectives of this study were to report the results of 2L including EBR as well as prognostic factors in a national retrospective multicentre study. NEC patients treated with 2L and further, with tissue samples available, were included. RB1 status and morphological classification were reviewed centrally. Among the 121 NEC patients (40% female, median age 61 years) included, there were 73 small-cell NEC (60%), 34 large-cell NEC (28%) and 14 NEC (not otherwise specified, 12%). Primary sites were lung (39%), gastroenteropancreatic (36%), other (13%) and unknown (12%). Median Ki-67 index was 80%. Median progression-free survival (PFS) and overall survival (OS) under 2L were 2.1 and 6.2 months, respectively. No difference was observed between patients who received an 'adenocarcinoma-like' or a 'neuroendocrine-like' 2L or according to the RB1 status. Thoracic NEC primary was the only adverse prognostic factor for OS. EBR, administered to 31 patients, resulted in a 62% disease control rate with a median PFS and OS of 3.2 and 11.7 months, respectively. In the 94 patients with a relapse-free interval of ≥3 months after first-line platinum-etoposide chemotherapy, the median OS was 12 months in patients who received EBR as compared to 5.9 months in patients who did not (P = 0.043). EBR could be the best 2L option for patient with initial response to first-line platinum-etoposide lasting at least 3 months. RB1 status does not provide prognostic information in this setting.

A new pancreatic adenocarcinoma-derived organoid model of acquired chemoresistance to FOLFIRINOX: First insight of the underlying mechanisms.

BACKGROUND INFORMATION: Although improvements have been made in the management of pancreatic adenocarcinoma (PDAC) during the past 20 years, the prognosis of this deadly disease remains poor with an overall 5-year survival under 10%. Treatment with FOLFIRINOX, a combined regimen of 5-fluorouracil, irinotecan (SN-38) and oxaliplatin, is nonetheless associated with an excellent initial tumour response and its use has allowed numerous patients to go through surgery while their tumour was initially considered unresectable. These discrepancies between initial tumour response and very low long-term survival are the consequences of rapidly acquired chemoresistance and represent a major therapeutic frontier. To our knowledge, a model of resistance to the combined three drugs has never been described due to the difficulty of modelling the FOLFIRINOX protocol both in vitro and in vivo. Patient-derived tumour organoids (PDO) are the missing link that has long been lacking in the wide range of epithelial cancer models between 2D adherent cultures and in vivo xenografts. In this work we sought to set up a model of PDO with resistance to FOLFIRINOX regimen that we could compare to the paired naive PDO. RESULTS: We first extrapolated physiological concentrations of the three drugs using previous pharmacodynamics studies and bi-compartmental elimination models of oxaliplatin and SN-38. We then treated PaTa-1818x naive PDAC organoids with six cycles of 72 h-FOLFIRINOX treatment followed by 96 h interruption. Thereafter, we systematically compared treated organoids to PaTa-1818x naive organoids in terms of growth, proliferation, viability and expression of genes involved in cancer stemness and aggressiveness. CONCLUSIONS: We reproductively obtained resistant organoids FoxR that significantly showed less sensitivity to FOLFORINOX treatment than the PaTa-1818x naive organoids from which they were derived. Our resistant model is representative of the sequential steps of chemoresistance observed in patients in terms of growth arrest (proliferation blockade), residual disease (cell quiescence/dormancy) and relapse. SIGNIFICANCE: To our knowledge, this is the first genuine in vitro model of resistance to the three drugs in combined therapy. This new PDO model will be a great asset for the discovery of acquired chemoresistance mechanisms, knowledge that is mandatory before offering new therapeutic strategies for pancreatic cancer.

Antagonistic Roles of the Tumor Suppressor miR-210-3p and Oncomucin MUC4 Forming a Negative Feedback Loop in Pancreatic Adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is a deadly cancer with an extremely poor prognosis. MUC4 membrane-bound mucin is neoexpressed in early pancreatic neoplastic lesions and is associated with PDAC progression and chemoresistance. In cancers, microRNAs (miRNAs, small noncoding RNAs) are crucial regulators of carcinogenesis, chemotherapy response and even metastatic processes. In this study, we aimed at identifying and characterizing miRNAs activated downstream of MUC4-associated signaling in pancreatic adenocarcinoma. MiRnome analysis comparing MUC4-KD versus Mock cancer cells showed that MUC4 inhibition impaired miR-210-3p expression. Therefore, we aimed to better understand the miR-210-3p biological roles. METHODS: miR-210-3p expression level was analyzed by RT-qPCR in PDAC-derived cell lines (PANC89 Mock and MUC4-KD, PANC-1 and MiaPACA-2), as well as in mice and patients tissues. The MUC4-miR-210-3p regulation was investigated using luciferase reporter construct and chromatin immunoprecipitation experiments. Stable cell lines expressing miR-210-3p or anti-miR-210-3p were established using CRISPR/Cas9 technology or lentiviral transduction. We evaluated the biological activity of miR-210-3p in vitro by measuring cell proliferation and migration and in vivo using a model of subcutaneous xenograft. RESULTS: miR-210-3p expression is correlated with MUC4 expression in PDAC-derived cells and human samples, and in pancreatic PanIN lesions of Pdx1-Cre; LstopL-KrasG12D mice. MUC4 enhances miR-210-3p expression levels via alteration of the NF-κB signaling pathway. Chromatin immunoprecipitation experiments showed p50 NF-κB subunit binding on miR-210-3p promoter regions. We established a reciprocal regulation since miR-210-3p repressed MUC4 expression via its 3'-UTR. MiR-210-3p transient transfection of PANC89, PANC-1 and MiaPACA-2 cells led to a decrease in cell proliferation and migration. These biological effects were validated in cells overexpressing or knocked-down for miR-210-3p. Finally, we showed that miR-210-3p inhibits pancreatic tumor growth and proliferation in vivo. CONCLUSION: We identified a MUC4-miR-210-3p negative feedback loop in early-onset PDAC, but also revealed new functions of miR-210-3p in both in vitro and in vivo proliferation and migration of pancreatic cancer cells, suggesting a complex balance between MUC4 pro-oncogenic roles and miR-210-3p anti-tumoral effects.

Prognostic factors of metastatic neuroendocrine carcinoma under first-line treatment with platinum etoposide with a focus on NEC score and Rb expression: Results from the multicentre RBNEC study of the Groupe d'Etude des Tumeurs Endocrines (GTE) and the ENDOCAN-RENATEN network.

INTRODUCTION AND AIM: Neuroendocrine carcinomas (NECs) are aggressive malignant diseases. Platinum-etoposide (PE) combination is the standard first-line treatment, whatever the primary location. The NEC score and also retinoblastoma protein (Rb) status have been suggested to be predictive/prognostic factors in NEC. The primary objective of our multicentric retrospective study was to evaluate the prognostic relevance of the NEC score and Rb status, assessed by immunohistochemistry in PE-treated patients with metastatic NEC. METHODS: Seven centres participated. The inclusion criteria were NEC, whatever the primary site, metastatic stage, first-line treatment with PE and tissue samples available. Rb status was determined centrally. RESULTS: We report multicentric data from 185 metastatic patients (37% women, median age 63). There were 108 small-cell NECs (SCNECs, 58.4%), 50 large-cell NECs (LCNECs, 27%) and 27 not otherwise specified NECs (nosNECs, 14.6%). The primary sites were the thorax (37%), gastroenteropancreatic sites (38%), unknown (15%) and other (9%). The mean Ki-67 index was 76% (range 20-100). Rb status was interpretable in 122 cases. Rb expression was lost in 74% of the cases: 84% of SCNEC vs. 60% and 63% of LCNEC and nosNEC, respectively (p = 0.016). Objective response was seen in 70% of SCNEC, 45% of LCNEC and 48% of nosNEC (p < 0.001) and in 62% of Rb-negative tumours vs. 46% of Rb-positive tumours (p = 0.3). There was no difference in median progression-free survival or overall survival (OS) as per Rb status. Age, NEC score and response to chemotherapy were the main factors associated with OS in our cohort. CONCLUSION: In our series, Rb status had no prognostic impact in PE-treated metastatic patients with NEC, whereas age, NEC score and response to chemotherapy were the main factors associated with OS.

Quantitative dual isotope 123iodine/99mTc-MIBI scintigraphy: A new approach to rule out malignancy in thyroid nodules.

BACKGROUND: The aim of this study was to evaluate the role of dual isotope 123Iodine/99mTc-MIBI thyroid scintigraphy (IMS) in discriminating between malignant and benign lesions in indeterminate nodules using quantitative analysis methods. METHODS: Thirty-five consecutive patients with thyroid nodules of indeterminate or non-diagnostic cytology and cold on 123Iodine scintigraphy (10 Bethesda I, 24 Bethesda III-IV, 1 in which cytology was impossible) underwent IMS between 2017 and 2019 with uptake quantification at two time points ahead of thyroidectomy: early and late. Images were analyzed by two blinded physicians. RESULTS: Twelve nodules were malignant and 23 benign on histopathology. Mean uptake values were lower in benign than in malignant nodules at both time points: early, 8.7±4.1 versus 12.9±3.5 (P=0.005); and late, 5.3±2.7 versus 7.7±1.1 (P=0.008). Interobserver reproducibility was excellent. The intraclass correlation coefficient was 0.86 in benign and 0.92 in malignant lesions for early uptake result (ER) and 0.94 and 0.85 respectively for late uptake result (LR). The optimal LR cut-off  to exclude a diagnosis of malignancy was set at 5.9 . The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of this cut-off were, respectively, 100%, 65.2%, 60%, 100% and 77.1%. CONCLUSION: Despite some study limitations, quantitative analysis of 99mTc-MIBI thyroid scintigraphy had a good reproducibility, which could help to rule out malignancy in non-diagnostic or indeterminate thyroid nodules and thereby reducing the number of patients undergoing unnecessary surgery when LR is below 5.9.

A tool to predict progression of non-alcoholic fatty liver disease in severely obese patients.

BACKGROUND & AIMS: Severely obese patients are a growing population at risk of non-alcoholic fatty liver disease (NAFLD). Considering the increasing burden, a predictive tool of NAFLD progression would be of interest. Our objective was to provide a tool allowing general practitioners to identify and refer the patients most at risk, and specialists to estimate disease progression and adapt the therapeutic strategy. METHODS: This predictive tool is based on a Markov model simulating steatosis, fibrosis and non-alcoholic steatohepatitis (NASH) evolution. This model was developped from data of 1801 severely obese, bariatric surgery candidates, with histological assessment, integrating duration of exposure to risk factors. It is then able to predict current disease severity in the absence of assessment, and future cirrhosis risk based on current stage. RESULTS: The model quantifies the impact of sex, body-mass index at 20, diabetes, age of overweight onset, on progression. For example, for 40-year-old severely obese patients seen by the general practitioners: (a) non-diabetic woman overweight at 20, and (b) diabetic man overweight at 10, without disease assessment, the model predicts their current risk to have NASH or F3-F4: for (a) 5.7% and 0.6%, for (b) 16.1% and 10.0% respectively. If those patients have been diagnosed F2 by the specialist, the model predicts the 5-year cirrhosis risk: 1.8% in the absence of NASH and 6.0% in its presence for (a), 10.3% and 26.7% respectively, for (b). CONCLUSIONS: This model provides a decision-making tool to predict the risk of liver disease that could help manage severely obese patients.

The Impact of Modern Chemotherapy and Chemotherapy-Associated Liver Injuries (CALI) on Liver Function: Value of 99mTc-Labelled-Mebrofenin SPECT-Hepatobiliary Scintigraphy.

BACKGROUND: Chemotherapy is increasingly used before hepatic resection, with controversial impact regarding liver function. This study aimed to assess the capacity of 99mTc-labelled-mebrofenin SPECT-hepatobiliary scintigraphy (HBS) to predict liver dysfunction due to chemotherapy and/or chemotherapeutic-associated liver injuries (CALI), such as sinusoidal obstruction syndrome (SOS) and nonalcoholic steatohepatitis (NASH) activity score (NAS). METHODS: From 2011 to 2015, all consecutive noncirrhotic patients scheduled for a major hepatectomy (≥ 3 segments) gave informed consent for preoperative SPECT-HBS allowing measurements of segmental liver function. As primary endpoint, HBS results were compared between patients with versus without (1) preoperative chemotherapy (≤ 3 months); and (2) CALI, mainly steatosis, NAS (Kleiner), or SOS (Rubbia-Brandt). Secondary endpoints were (1) other factors impairing function; and (2) impact of chemotherapy, and/or CALI on hepatocyte isolation outcome via liver tissues. RESULTS: Among 115 patients, 55 (47.8%) received chemotherapy. Sixteen developed SOS and 35 NAS, with worse postoperative outcome. Overall, chemotherapy had no impact on liver function, except above 12 cycles. In patients with CALI, a steatosis ≥ 30% significantly compromised function, as well as NAS, especially grades 2-5. Conversely, SOS had no impact, although subjected to very low patients number with severe SOS. Other factors impairing function were diabetes, overweight/obesity, or fibrosis. Similarly, chemotherapy in 73 of 164 patients had no effect on hepatocytes isolation outcome; regarding CALI, steatosis ≥ 30% and NAS impaired the yield and/or viability of hepatocytes, but not SOS. CONCLUSIONS: In this first large, prospective study, HBS appeared to be a valuable tool to select heavily treated patients at risk of liver dysfunction through steatosis or NAS.

Genetic predisposition to neural crest-derived tumors: revisiting the role of KIF1B.

OBJECTIVE: We previously described a family in which predisposition to pheochromocytoma (PCC) segregates with a germline heterozygous KIF1B nucleotide variant (c.4442G>A, p.Ser1481Asn) in three generations. During the clinical follow-up, one proband's brother, negative for the KIF1B nucleotide variant, developed a bilateral PCC at 31 years. This prompted us to reconsider the genetic analysis. DESIGN AND METHODS: Germline DNA was analyzed by next-generation sequencing (NGS) using a multi-gene panel plus MLPA or by whole exome sequencing (WES). Tumor-derived DNA was analyzed by SnapShot, Sanger sequencing or NGS to identify loss-of-heterozygosity (LOH) or additional somatic mutations. RESULTS: A germline heterozygous variant of unknown significance in MAX (c.145T>C, p.Ser49Pro) was identified in the proband's brother. Loss of the wild-type MAX allele occurred in his PCCs thus demonstrating that this variant was responsible for the bilateral PCC in this patient. The proband and her affected grandfather also carried the MAX variant but no second hit could be found at the somatic level. No other pathogenic mutations were detected in 36 genes predisposing to familial PCC/PGL or familial cancers by WES of the proband germline. Germline variants detected in other genes, TFAP2E and TMEM214, may contribute to the multiple tumors of the proband. CONCLUSION: In this family, the heritability of PCC is linked to the MAX germline variant and not to the KIF1B germline variant which, however, may have contributed to the occurrence of neuroblastoma (NB) in the proband.