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Objective: Tumors harboring a POLE pathogenic variant, associated with high tumor mutational burden, are good candidates for immunotherapy. However, POLE pathogenic variants are not currently screened in routine clinical practice. Can these tumors be identified by means of an already available test? Methods: We describe seven tumors harboring a POLE pathogenic variant, among eight patients with tumors harboring multiple BRCA1/2 variants (from 4 to 20). All patients were managed at Institut Curie, Paris. Five patients were selected because of unexpected tumor BRCA testing results with multiple variants and another three patients were selected because of a POLE pathogenic variant detected by large tumor testing. We looked for other tumor variants by Next-Generation Sequencing in tumors harboring multiple BRCA1/2 variants, and for multiple BRCA1/2 variants in tumors harboring a POLE pathogenic variant. Results: Four of the five tumors selected because of multiple BRCA1/2 variants exhibited a POLE pathogenic variant, and all three tumors selected for POLE pathogenic variants exhibited multiple BRCA1/2 variants. Conclusions: Tumor BRCA testing could be a way to detect tumors harboring a highly mutagenic POLE pathogenic variant.

Breast cancers (BC) are rare in men and are often caused by constitutional predisposing factors. In women, mosaic BRCA1 promoter methylations (MBPM) are frequent events, detected in 4-8% of healthy subjects. This constitutional epimutation increases risk of early-onset and triple-negative BC. However, the role of MBPM in male BC predisposition has never been assessed. We screened 40 blood samples from men affected by BC, and performed extensive tumour analysis on MBPM-positive patients. We detected two patients carrying MBPM. Surprisingly, tumour analysis revealed that neither of these two male BCs were caused by the constitutional BRCA1 epimutations carried by the patients., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Background: The involvement of thrombin receptor PAR1 in blood vessel development has been largely demonstrated in knockout mice; however, its implication in adult mouse angiogenesis seems very moderate., Objectives: We aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony-forming cells (ECFCs)., Methods and Results: PAR1 activation on ECFCs using the selective PAR1-activating peptide induced a significant decrease in CD133 expression (RTQ-PCR analysis). In line, silencing of PAR1 gene expression with siRNA increased CD133 mRNA as well as intracellular CD133 protein expression. To confirm the link between CD133 and PAR1, we explored the association between PAR1 and CD133 levels in fast and slow fibroblasts prone to reprogramming. An imbalance between PAR1 and CD133 levels was evidenced, with a decreased expression of PAR1 in fast reprogramming fibroblasts expressing a high CD133 level. Regarding in vitro ECFC angiogenic properties, PAR1 silencing with specific siRNA induced cell proliferation evidenced by the overexpression of Ki67. However, it did not impact migration properties nor ECFC adhesion on smooth muscle cells or human arterial endothelial cells. In a mouse model of hind-limb ischemia, PAR1 silencing in ECFCs significantly increased postischemic revascularization compared to siCtrl-ECFCs along with a significant increase in cutaneous blood flows (P < .0001), microvessel density (P = .02), myofiber regeneration (P < .0001), and human endothelial cell incorporation in muscle (P < .0001)., Conclusion: In conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Oral cavity is the most prevalent site of head and neck squamous cell carcinomas (HNSCCs). Most often diagnosed at a locally advanced stage, treatment is multimodal with surgery as the cornerstone. The aim of this study was to explore the molecular landscape of a homogenous cohort of oral cavity squamous cell carcinomas (OCSCCs), and to assess the prognostic value of tumor mutational burden (TMB), along with classical molecular and clinical parameters., Patients and Methods: One hundred and fifty-one consecutive patients with OCSCC treated with upfront surgery at the Institut Curie were analyzed. Sequencing of tumor DNA from frozen specimens was carried out using an in-house targeted next-generation sequencing panel (571 genes). The impact of molecular alterations and TMB on disease-free survival (DFS) and overall survival (OS) was evaluated in univariate and multivariate analyses., Results: Pathological tumor stage, extranodal spread, vascular emboli, and perineural invasion were associated with both DFS and OS. TP53 was the most mutated gene (71%). Other frequent molecular alterations included the TERT promoter (50%), CDKN2A (25%), FAT1 (17%), PIK3CA (14%), and NOTCH1 (15%) genes. Transforming growth factor-β pathway alterations (4%) were associated with poor OS (P = 0.01) and DFS (P = 0.02) in univariate and multivariate analyses. High TMB was associated with prolonged OS (P = 0.01 and P = 0.02, in the highest 10% and 20% TMB values, respectively), but not with DFS. Correlation of TMB with OS remained significant in multivariate analysis (P = 0.01 and P = 0.005 in the highest 10% and 20% TMB values, respectively). Pathological tumor stage combined with high TMB was associated with good prognosis., Conclusion: Our results suggest that a high TMB is associated with a favorable prognosis in patients with OCSCC treated with upfront surgery., Competing Interests: Disclosure CLT participated in advisory boards from MSD, BMS, Merck Serono, AstraZeneca, Nanobiotix, Celgene, GSK, Roche, Rakuten, and Seattle Genetics. All other authors have declared no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Better knowledge of the tumor genomic landscapes has helped to develop more effective targeted drugs. However, there is no tool to interpret targetability of genomic alterations assessed by next-generation sequencing in the context of clinical practice. Our aim is to rank the level of evidence of individual recurrent genomic alterations observed in breast cancer based on the ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) in order to help the clinicians to prioritize treatment. Analyses of databases suggested that there are around 40 recurrent driver alterations in breast cancer. ERBB2 amplification, germline BRCA1/2 mutations, PIK3CA mutations were classified tier of evidence IA based on large randomized trials showing antitumor activity of targeted therapies in patients presenting the alterations. NTRK fusions and microsatellite instability (MSI) were ranked IC. ESR1 mutations and PTEN loss were ranked tier IIA, and ERBB2 mutations and AKT1 mutations tier IIB. Somatic BRCA 1/2 mutations, MDM2 amplifications and ERBB 3 mutations were ranked tier III. Seventeen genes were ranked tier IV based on preclinical evidence. Finally, FGFR1 and CCND1 were ranked tier X alterations because previous studies have shown lack of actionability., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The cyclin-dependent kinase 4 (CDK4)/6 inhibitor Palbociclib is a new standard treatment in hormone-receptor positive breast cancer patients. No predictive biomarkers have been identified and no pharmacodynamics has properly been described so far., Patients and Methods: Patients with early-breast cancer were randomized 3 : 1 to oral palbociclib 125 mg daily for 14 days until the day before the surgery versus no treatment. Primary objective was antiproliferative response defined as a natural logarithm of Ki67 expression at day 15 below 1. Secondary end points were subgroups analyses and safety. Exploratory analyses included search for predictive biomarkers. Immunostainings (Ki67, RB, pRB, p16, pAKT, pER, pCDK2, CyclinD1), FISH (CCND1) and gene expression (GE) arrays were carried out at baseline and at surgery. In addition, activating PIK3CA and AKT1 mutations were assessed at baseline., Results: 74 patients were allocated to palbociclib and 26 to control. Most patients (93%) were hormone-receptor (HR)-positive, whereas 8% were HER2-positive. Palbociclib led to significantly more antiproliferative responses when compared with control (58% versus 12%, P < 0.001), and to a significantly higher Ki67 decrease (P < 0.001). In the HR-positive/HER2-negative subgroup, this antiproliferative effect was even more marked in the palbociclib arm when compared with control (70% versus 9%, P < 0.001). Palbociclib treatment led also to a significantly higher decrease from baseline in phospho-Rb when compared with control (P < 0.001). Among treated patients, changes in Ki67 correlated with changes in phospho-Rb (Spearman rank r = 0.41, P < 0.0001). GE analyses confirmed a major effect on proliferation and cell cycle genes. Among treated patients, CCNE2 expression was significantly more decreased in antiproliferative responders versus nonresponders (P = 0.006)., Conclusion: Short-term preoperative palbociclib decreases Ki67 in early-breast cancer patients. Early decrease of Rb phosphorylation correlates with drug's effect on cell proliferation and could potentially identify patients with primary resistance., Clinical Trial Registration: NCT02008734.

Background: High throughput molecular screening techniques allow the identification of multiple molecular alterations, some of which are actionable and can be targeted by molecularly targeted agents (MTA). We aimed at evaluating the relevance of using this approach in the frame of Institut Curie Molecular Tumor Board (MTB) to guide patients with cancer to clinical trials with MTAs., Patients and Methods: We included all patients presented at Institut Curie MTB from 4 October 2014 to 31 October 2017. The following information was extracted from the chart: decision to perform tumour profiling, types of molecular analyses, samples used, molecular alterations identified and those which are actionable, and inclusion in a clinical trial with matched MTA., Results: 736 patients were presented at the MTB. Molecular analyses were performed in 442 patients (60%). Techniques used included next-generation sequencing, comparative genomic hybridisation array and/or other techniques including immunohistochemistry in 78%, 51% and 58% of patients, respectively. Analyses were performed on a fresh frozen biopsy in 91 patients (21%), on archival tissue (fixed or frozen) in 326 patients (74%) and on both archival and fresh frozen biopsy in 25 patients (6%). At least one molecular alteration was identified in 280 analysed patients (63%). An actionable molecular alteration was identified in 207 analysed patients (47%). Forty-five analysed patients (10%) were enrolled in a clinical trial with matched MTA and 29 additional patients were oriented and included in a clinical trial based on a molecular alteration identified prior to the MTB analysis. Median time between date of specimen reception and molecular results was 28 days (range: 5-168)., Conclusions: The implementation of an MTB at Institut Curie enabled the inclusion of 10% of patients into a clinical trial with matched therapy., Competing Interests: Competing interests: None declared.

Background: Recent clinical results support the use of new immune checkpoint blockers (ICB), such as anti-PD-1 (e.g. nivolumab and pembrolizumab) and anti-PD-L1 antibodies. Radiological evaluation of ICB efficacy during therapy is challenging due to tumor immune infiltration. Changes of circulating tumor DNA (ctDNA) levels during therapy could be a promising tool for very accurate monitoring of treatment efficacy, but data are lacking with ICB., Patients and Methods: This prospective pilot study was conducted in patients with nonsmall cell lung cancer, uveal melanoma, or microsatellite-instable colorectal cancer treated by nivolumab or pembrolizumab monotherapy at Institut Curie. ctDNA levels were assessed at baseline and after 8 weeks (w8) by bidirectional pyrophosphorolysis-activated polymerization, droplet digital PCR or next-generation sequencing depending on the mutation type. Radiological evaluation of efficacy of treatment was carried out by using immune-related response criteria., Results: ctDNA was detected at baseline in 10 out of 15 patients. At w8, a significant correlation (r = 0.86; P = 0.002) was observed between synchronous changes in ctDNA levels and tumor size. Patients in whom ctDNA levels became undetectable at w8 presented a marked and lasting response to therapy. ctDNA detection at w8 was also a significant prognostic factor in terms of progression-free survival (hazard ratio = 10.2; 95% confidence interval 2.5-41, P < 0.001) and overall survival (hazard ratio = 15; 95% confidence interval 2.5-94.9, P = 0.004)., Conclusion: This proof-of-principle study is the first to demonstrate that quantitative ctDNA monitoring is a valuable tool to assess tumor response in patients treated with anti-PD-1 drugs., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

As a result of recent progress in detection techniques, circulating tumor DNA (ctDNA) and circulating tumor cells (CTC) can now be accurately detected in the blood of most cancer patients. While these new biomarkers can provide a better understanding of key biological mechanisms underlying cancer growth and dissemination, they also open up a wide range of possible clinical applications in medical oncology, radiation oncology and surgical oncology. In this review, we summarize the results obtained with ctDNA and CTC together with their potential future clinical applications in the field of surgical oncology, with particular focus on the perioperative setting of various types of cancer. These applications include, but are not limited to, cancer screening, early diagnosis, prognostic assessment, evaluation and management of preoperative systemic or local therapies, post-surgical detection of minimal residual disease and early detection of cancer relapse., (Copyright © 2017 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Objectives: Immunotherapy for bladder cancer seems to have promising results. Here, we evaluated the association between messenger RNA (mRNA) and protein levels and possible prognostic value of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) immune checkpoint pathways during bladder carcinogenesis., Methods and Materials: Tumor samples were obtained from 155 patients (84 with muscle-invasive bladder cancer [MIBC], and 71 non-muscle-invasive bladder cancer [NMIBC]) and normal bladder tissue from 15 patients. We evaluated the mRNA expression of 3 genes in the PD-1 pathway (PD-1, PD-L1, and PD-L2) and 4 in the CTLA4 pathway (CTLA4, CD28, CD80, and CD86) in normal and tumoral human bladder samples by quantitative real-time reverse transcription polymerase chain reaction, with immunohistochemistry used to evaluate the protein expression of PD-1 and PD-L1 in tumor and immune cells. Results of molecular analyses were compared with survival analyses., Results: As compared with normal bladder tissue, MIBC tissue showed PD-1, PD-L1, CTLA4, and CD80 overexpression (59.5%, 60.7%, 84.5%, and 92.9%, respectively), whereas overexpression was lower in NMIBC tissue (22.5%, 4.2%, 35.2%, and 46.5%, respectively). The results of reverse transcription polymerase chain reaction analysis were confirmed by immunohistochemistry, with a high correlation between mRNA and protein expression. On multivariate analyses, overexpression of the studied genes was not associated with prognosis in relapse or progression of NMIBC or in recurrence-free and overall survival of MIBC., Conclusions: The CTLA4 pathway appears to be deregulated along with the PD-1/PD-L1 pathway in bladder carcinogenesis, with good correlation between mRNA and protein expression endorsing the useful role of immune checkpoints, especially for a large subgroup of MIBC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The mechanisms regulating megakaryopoiesis and platelet production (thrombopoiesis) are still incompletely understood. Identification of a progenitor with enhanced thrombopoietic capacity would be useful to decipher these mechanisms and to improve our capacity to produce platelets in vitro. Differentiation of peripheral blood CD34(+) cells in the presence of bone marrow-human mesenchymal stromal cells (MSCs) enhanced the production of proplatelet-bearing megakaryocytes (MKs) and platelet-like elements. This was accompanied by enrichment in a MK precursor population exhibiting an intermediate level of CD41 positivity while maintaining its expression of CD34. Following sorting and subculture with MSCs, this CD34(+)CD41(low) population was able to efficiently generate proplatelet-bearing MKs and platelet-like particles. Similarly, StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AhR) transcription factor known to maintain CD34 expression of progenitor cells, led to an enriched CD34(+)CD41(low) fraction and to an increased capacity to generate proplatelet-producing MKs and platelet-like elements ultrastructurally and functionally similar to circulating platelets. The effect of MSCs, like that of SR1, appeared to be mediated by an AhR-dependent mechanism because both culture conditions resulted in repression of its downstream effector CYP1B1. This newly described isolation of a precursor exhibiting strong MK potential could be exploited to study normal and abnormal thrombopoiesis and for in vitro platelet production., (© 2016 by The American Society of Hematology.)

Bladder cancer is the sixth cause of cancer mortality in France and prognosis of muscle-invasive tumors remains poor due to lack of effective treatments. Recent advances in molecular biology applied to tumors and results of recent genome-wide studies have brought a important impact on the understanding of bladder carcinogenesis. Main molecular alterations concern FGFR3, TP53 and HER2, and it is now possible to distinguish three subgroups of tumors according to molecular profile. This paper proposes a review of different genetic and epigenetic alterations in bladder cancer, their potential role as theranostic markers in clinical oncology and new targeted therapies according to the concept of personalized medicine., (Copyright © 2015 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Background: Transforming growth factor-β1 (TGF-β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF-β1 modulation of EPC angiogenic properties., Methods: TGF-β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF-β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony-forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF-β1 in ECFCs by using short interfering RNA., Results: Total TGF-β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF-β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF-β1 receptors., Conclusions: TGF-β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF-β1 may play a role during vascular remodeling in fibrotic disease states via EPCs., (© 2012 International Society on Thrombosis and Haemostasis.)

Background & Aims: Based on the success of sorafenib, several anti-angiogenic therapies are currently evaluated in advanced hepatocellular carcinomas. Few biological data are currently available from patients that may help understanding mechanisms of acquired resistance to these drugs. Herein, we report translational data from a post-treatment surgical specimen in a patient who experienced acquired resistance to sunitinib., Methods: Clinical, radiological, and pathological data were collected before treatment, under treatment, and at the time of tumor progression. In addition, a biomolecular analysis was performed at the time of progression., Results: In this patient with non-alcoholic steatohepatitis, initial response to sunitinib was followed by tumor progression within 6 months of treatment, requesting salvage surgical resection. Surprisingly, pathological examination on post-treatment specimens revealed the presence of two juxtaposed tissue components containing either sarcomatoid-like mesenchymal cells or well- to moderately-differentiated hepatocellular carcinoma cells. Cancer cells retain a high α-fetoprotein expression in both components. However, while cells from carcinoma expressed E-cadherin but no vimentin, cancer cells from the mesenchymal component highly expressed vimentin and lost E-cadherin protein expression as measured by immunostaining. HMGA2 and Ki67 mRNA were also expressed at higher levels in mesenchymal than in carcinoma cells., Conclusion: This case report suggests the occurrence of an epithelial-to-mesenchymal transition in discrete areas of hepatocellular carcinomas developing resistance to sunitinib., (Copyright © 2011. Published by Elsevier B.V.)

Vascular endothelial growth factor (VEGF) is essential for breast cancer progression and is a relevant target in anti-angiogenesis. Although VEGF121 and VEGF165, the fully or partially secreted isoforms, respectively, have been the focus of intense studies, the role of the cell-associated VEGF189 isoform is not understood. To clarify the contribution of VEGF189 to human mammary carcinogenesis, we established several clones of MDA-MB-231 cells stably overexpressing VEGF189 (V189) and VEGF165 (V165). V189 and V165 clones increased tumor growth and angiogenesis in vivo. Remarkably, V165 induced the most rapid tumor uptake, whereas V189 increased vasodilation. In vitro overexpression of VEGF165 and VEGF189 increases the proliferation and chemokinesis of these cancer cells. Interestingly, overexpression of VEGF189 increased cell adhesion on fibronectin (1.9-fold) and vitronectin (1.6-fold), as compared to VEGF165, through alpha5beta1 and alphavbeta5 integrins. Using the BIACore system we demonstrated for the first time that VEGF189 binds directly to neuropilin-1, which is strongly expressed in MDA-MB-231 cells. In contrast, VEGF-R2 was not significantly expressed and VEGF-R1 was expressed at low level. Our in vitro results suggest an autocrine effect of VEGF189 on breast cancer cells, probably through neuropilin-1. In conclusion, our data indicate that VEGF189 participates in mammary tumor growth through both angiogenesis and nonangiogenic functions. Whether VEGF189 overexpression is correlated to prognosis in human breast tumors remains to be established.