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Gynecologic carcinosarcomas (CS) are biphasic neoplasms composed of carcinomatous (C) and sarcomatous (S) malignant components. Because of their rarity and histologic complexity, genetic and functional studies on CS are scarce and the mechanisms of initiation and development remain largely unknown. Whole-genome analysis of the C and S components reveals shared genomic alterations, thus emphasizing the clonal evolution of CS. Reconstructions of the evolutionary history of each tumor further reveal that C and S samples are composed of both ancestral cell populations and component-specific subclones, supporting a common origin followed by distinct evolutionary trajectories. However, while we do not find any recurrent genomic features associated with phenotypic divergence, transcriptomic and methylome analyses identify a common mechanism across the cohort, the epithelial-to-mesenchymal transition (EMT), suggesting a role for nongenetic factors in inflicting changes to cellular fate. Altogether, these data accredit the hypothesis that CS tumors are driven by both clonal evolution and transcriptomic reprogramming, essential for susceptibility to transdifferentiation upon encountering environmental cues, thus linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences. Significance: We have provided a detailed characterization of the genomic landscape of CS and identified EMT as a common mechanism associated with phenotypic divergence, linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences.

Background: MOST-plus is a multicenter, randomized, open-label, adaptive Phase II trial evaluating the clinical benefit of targeted treatments matched to molecular alteration in advanced/metastatic solid tumors. Sorafenib was tested on patients with tumors harboring sorafenib-targeted genes. Methods: The MOST-plus trial used a randomized discontinuation design. After 12 weeks of sorafenib (400 mg, po BID), patients with progressive disease discontinued study, patients with objective response were proposed to continue sorafenib, whereas patients with stable disease (SD) were randomly assigned (1:1) to the maintenance or interruption of treatment. The primary endpoint was RECIST version 1.1 progression-free rate at 16 weeks after randomization (PFR-16w). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. Statistical analyses used a sequential Bayesian approach with interim efficacy analyses. The enrolment could be stopped in the case of a 95% probability for the estimated PFR-16w to be higher in the maintenance than in the interruption arm (NCT02029001). Results: 151 patients were included, of whom 35 had SD at 12 weeks of Sorafenib. For the 35 patients with SD on sorafenib, the PFR-16w was 65% [95% credibility interval 43.4–83.7] in the continuation arm and 25% [7.8–48.1] in the interruption arm. Median PFS and OS were improved in the maintenance versus the interruption arm (mPFS: 5.6 [95%CI 1.97–6.77] months versus 2.0 [95%CI 1.61–3.91] months (p = 0.0231) and mOS: 14.3 [95%CI 8.9–23.8] versus 8.0 months [95%CI 3.5–15.2] (p = 0.0857)). Conclusion: Sorafenib showed activity in progressive patients with solid tumors harboring somatic genomic alterations in sorafenib-targeted genes. Continuing sorafenib when SD is achieved improves PFR compared to interruption.

Background: MOST-plus is a multicenter, randomized, open-label, adaptive Phase II trial evaluating the clinical benefit of targeted treatments matched to molecular alteration in advanced/metastatic solid tumors. Sorafenib was tested on patients with tumors harboring sorafenib-targeted genes Methods: The MOST-plus trial used a randomized discontinuation design. After 12 weeks of sorafenib (400 mg, po BID), patients with progressive disease dis-continued study, patients with objective response were proposed to continue sorafenib, whereas patients with stable disease (SD) were randomly assigned (1:1) to maintenance or interruption of treatment. Primary endpoint was RE-CIST version 1.1 progression-free rate at 16 weeks after randomization (PFR-16w). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. Statistical analyses used a sequential Bayes-ian approach with interim efficacy analyses. The enrolment could be stopped in the case of a 95% probability for the estimated PFR-16w to be higher in the maintenance than in the interruption arm (NCT02029001). Results: 151 patients were included, of whom 35 had SD at 12 weeks of Soraf-enib. For the 35 patients with SD on sorafenib, the PFR-16w was 65% [95% credibility interval 43.4–83.7] in the continuation arm and 25% [7.8–48.1] in the interruption arm. Median PFS and OS were improved in maintenance versus interruption arm (mPFS: 5.6 [95%CI 1.97–6.77] months versus 2.0 [95%CI 1.61–3.91] months (p =0.0231) and mOS : 14.3 [95%CI 8.9–23.8] versus 8.0 months [95%CI 3.5–15.2] (p =0.0857)). Conclusion: Sorafenib showed activity in progressive patients with solid tu-mors harboring somatic genomic alterations in sorafenib-targeted genes. Con-tinuing sorafenib when SD is achieved improves PFR compared to interruption

Heterogeneity of genomic rearrangements in carcinosarcoma.

Curated list of chromatin remodeling genes.

Genomic landscape of CS. A, Tissue origin and histological subtypes for each tumor (from P01 to P15). B, Main component and detailed percentage content for both samples (a and b) derived from the same tumor. C, CNA analysis: tumor purity (%), ploidy, FGA, and CNA breakpoints number. D, SV number, classified as deletion, duplication, inversion, and interchromosomal translocation. E, SNV and small indel number. F, Proportion of each of four mutational signatures deciphered in the whole cohort. G, MSI phenotype: MSI score (representing the percentage of unstable microsatellite loci), MLH1 promoter methylation (beta-value), and mRNA expression level (TPM – transcripts per million). H, Kataegis number illustrating APOBEC error-prone DNA repair process. I, BRCAness phenotype: number of LST, (white stars highlight samples with LST number ≥20), BRCA1 promoter methylation level (beta-value), BRCA1/2 genes alterations and TDP score, collectively summarized by the HRD status track.

List of differentially methylated gene promoters (paired analysis of 5 selected tumors).

Key gene alterations in uterine and ovarian CS. Oncoprint of alterations identified in the 50 most altered genes from TCGA cancer pathways and custom lists of CRG. The type of genomic alteration (deletion, amplification, fusion, broken by SV, missense, truncated) is described in the legend. Number and proportions of alteration types are summarized by gene (horizontal bars on right) and by sample (vertical bars on top). The samples are classified into MSI and CN-high according to TCGA endometrial carcinoma classification.

Evolutionary histories of CS. Clonal lineage histories of P02 MSI phenotype tumor (A), P12 BRCAness-phenotype tumor (B), and P15 ovarian bilateral tumor (C). Left, Clonal lineage inference for the whole tumor: subclonal populations (Ci), main cancer gene alterations, genome doubling events, and representative mutational signature proportions are represented. Right, Subclonal population frequencies projection in each tumor sample. Clones colored in gray are found in both derived samples, pink (or green) clones are specific to sarcoma- (or carcinoma-) derived samples, respectively.

Differential expression and methylation analysis (C vs. S). A, Heatmap and pathway overrepresentation analysis of differentially expressed genes between C and S (or undifferentiated) components of the 10 samples derived from the five selected CS tumors. Gene clustering method: Ward's; distance: Spearman. FDR: false discovery rate. B, Heatmaps of EMT and stemness pathways scores (left) and methylation level of EMT-related miRNAs (right). mRNA level: scaled TPM. Pathway scores: scaled ssGSEA scores. Methylation level: scaled beta-values.

List of differentially expressed genes (paired analysis of 5 selected tumors).

Substitution signature exposures estimated in 640 breast cancer WGS and the ovarian sample (AOCS-166).

Mismatch repair (MMR)–deficient cancers have been discovered to be highly responsive to immune therapies such as PD-1 checkpoint blockade, making their definition in patients, where they may be relatively rare, paramount for treatment decisions. In this study, we utilized patterns of mutagenesis known as mutational signatures, which are imprints of the mutagenic processes associated with MMR deficiency, to identify MMR-deficient breast tumors from a whole-genome sequencing dataset comprising a cohort of 640 patients. We identified 11 of 640 tumors as MMR deficient, but only 2 of 11 exhibited germline mutations in MMR genes or Lynch Syndrome. Two additional tumors had a substantially reduced proportion of mutations attributed to MMR deficiency, where the predominant mutational signatures were related to APOBEC enzymatic activity. Overall, 6 of 11 of the MMR-deficient cases in this cohort were confirmed genetically or epigenetically as having abrogation of MMR genes. However, IHC analysis of MMR-related proteins revealed all but one of 10 samples available for testing as MMR deficient. Thus, the mutational signatures more faithfully reported MMR deficiency than sequencing of MMR genes, because they represent a direct pathophysiologic readout of repair pathway abnormalities. As whole-genome sequencing continues to become more affordable, it could be used to expose individually abnormal tumors in tissue types where MMR deficiency has been rarely detected, but also rarely sought. Cancer Res; 77(18); 4755–62. ©2017 AACR.

Mutations in MMR genes

Detection of MMR deficient breast cancers in whole exome sequencing data (WES)

Details of 11 MMR deficient breast cancers.

This file contains the following Supplementary figures and Supplementary Material and Methods: Supplementary Figure 1: Contribution of base substitution signatures and indels in 640 breast cancer WGS. Supplementary Figure 2: Whole genome plots for 11 MMR deficient breast cancers Supplementary Figure 3: Genome plots for the two samples with germline PMS2 mutations. Supplementary Figure 4: Scatter plot demonstrating the relationship between total number of substitutions associated with the three MMR signatures 6, 20, and 26 and number of indels. Supplementary Materials and Methods 1. Mutational signatures background 2. Detection of MMR deficient cancers in other data sets 2.1 Detection of a genomic profile characteristic of PMS2 deficient tumours in Ovarian cancer. 2.2 Detection of MMR deficient breast cancers in WES

Background: High body mass index (BMI) is an established risk factor for developing breast cancer (BC), especially estrogen receptor (ER)-positive, and also has been associated with adverse survival. Still, patients with BC are currently treated independently of their BMI given limited understandings of the association between BC biology and patient adiposity. In this study, using retrospective data retrieved from two large BC studies, we aimed to identify genomic alterations of primary BC that are associated with BMI in the most common histological BC subtype - invasive carcinoma of no special type (NST). Patients, Data and Methods: Clinicopathological and genomic alteration data were retrieved from two study cohorts: METABRIC (Pereira et al. 2016) and ICGC (Nik-Zainal et al. 2016), with BMI recorded at the time of diagnosis and represented as either a continuous variable or a categorical variable of three categories - lean, overweight and obese. Stratification according to ER and HER2 status resulted in two focused subgroups: NST ER+/HER2- (n=392) and NST ER-/HER2- (n=152). Mutations classified as oncogenic using a set of predefined criteria were used to determine gene-level mutation status. Copy number alteration (CNA) calls were distinguished into three event types: amplification, hemizygous deletion and homozygous deletion. We used multivariable Firth’s logistic regression models with the presence of a genomic alteration as the response variable, BMI as the predicting variable of interest, and data cohort (METABRIC vs ICGC), age group (≤50 vs >50) and tumor grade (I & II vs III) as covariates, to assess the associations between BMI and recurrent gene-level genomic alterations, including gene mutations and CNAs. In a similar manner, we performed multivariable linear regression analysis, adjusting for age and tumor grade, to evaluate the associations of BMI with mutational signatures (MS) and tumor mutational burden in the ICGC NST subsets where these data are available. Results: Considering BMI as a categorical variable, we observed in the NST ER+/HER2- subgroup that PIK3CA was significantly less frequently mutated in obese compared to lean patients (33% vs 46%, odds ratio (OR) = 0.57 (95% confidence interval = (0.33, 0.97)), p = .039), while PTEN and TBX3 showed an increased frequency in overweight (6% vs 1%, OR = 4.14 (1.1, 22.34), p = .034) and obese (8% vs 1%, OR = 7.41 (1.82, 70.65), p = .008) patients, respectively. Regression analyses with BMI as a continuous variable revealed an increased prevalence of mutations in CDH1 and TBX3 genes as BMI increases by 1kg/m2 (OR = 1.14 (1.05, 1.24), p = .002, and OR = 1.13 (1.04, 1.22), p = .005, respectively) in patients with NST ER+/HER2- BC. No associations between BMI and oncogenic mutations was observed in the NST ER-/HER2- subgroup. Interrogation of gene-level CNAs in both subgroups demonstrated differences according to BMI in the prevalence of CNAs affecting a number of genes, many of which are known or have been presented with evidence to be involved in regulation of or regulated by hallmark pathways of BC, such as the MAPK/ERK, JAK/STAT and Wnt/β-catenin signaling pathways. We report a strong positive association between the single-base substitution signature 1 (SBS1), an age-correlated MS, and both continuous (coefficient (coef) = 18.3 (7.7, 28.9), p < .001) and categorical BMI (obese vs lean, coef = 336.3 (187.9, 484.8), p < .001) in the ICGC NST ER+/HER2- subgroup. Conclusion: This exploratory retrospective study suggests that the genomic profiles of primary BC may differ according to BMI. Clinical implications of these differences, especially the decreased prevalence of PIK3CA mutations in obese patients in the context of alpelisib, warrant further investigation. These results however indicate that patient adiposity should be taken into account in the era of personalized medicine. Citation Format: Ha-Linh Nguyen, Tatjana Geukens, Marion Maetens, Karen Van Baelen, Maxim De Schepper, Sophia Leduc, Edoardo Isnaldi, Sam Aparicio, Ake Borg, Jane Brock, Annegien Broeks, Carlos Caldas, Andrew Green, Hazem Khout, Eyfjörð Jórunn, Stian Knappskog, Savitri Krishnamurthy, Sunil Lakhani, Anita Langerod, John WM Martens, Leigh Murphy, Serena Nik-Zainal, Colin Purdie, Emad Rakha, Andrea Richardson, Anne Salomon, Peter Simpson, Christos Sotiriou, Paul Span, Benita Kiat-Tee Tan, Alastair Thompson, Stefania Tommasi, Marc Van de Vijver, Steven Van Laere, Alain Viari, Giuseppe Floris, Elia Biganzoli, François Richard, Christine Desmedt. The association between genomic alterations and body mass index in patients with early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-18.

Worldwide, there is a growing proportion of women who are overweight or obese. While obesity has been associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet the impact of adiposity (abnormal or excess body fat) on BC biology remains understudied in humans. This retrospective study aimed to investigate how the biology of primary BC would differ according to patients’ body mass index (BMI). We examined clinicopathological data (including BMI at the time of diagnosis) and molecular data (including genomic, bulk and single-cell transcriptomic data) of treatment-naïve (early stage) BC patients from five cohorts (N = 2071). We identified several genomic alterations considered actionable or of potential clinical relevance which had a different prevalence in overweight or obese patients compared to lean patients, for instances, less PIK3CA gene mutations, and more CCND1, CCNE1 and IGFR1 amplifications. Moreover, we found evidence supporting an ageing accelerating effect of obesity at the genetic level, through its association with an age-associated mutational signature. We showed that BMI-associated differences in transcriptomic profile were subtle at the bulk resolution while single cell profiling allowed detection of more pronounced changes in different cell compartments. Investigation at the single cell resolution revealed an elevated and unresolved inflammation of the BC tumor microenvironment (TME) associated with obesity, which had distinct characteristics contingent on the estrogen receptor status. Collectively, analyses at both genomic and transcriptomic levels implied that obesity is associated with an inflammaging-like phenotype of the TME. Our results indicate that patient adiposity might play a significant role in the heterogeneity of BC and should be considered in the context of precision medicine.

Background and ObjectivesParaneoplastic cerebellar degeneration (PCD) with anti-Yo antibodies is a cancer-related autoimmune disease directed against neural antigens expressed by tumor cells. A putative trigger of the immune tolerance breakdown is genetic alteration of Yo antigens. We aimed to identify the tumors' genetic and immune specificities involved in Yo-PCD pathogenesis.MethodsUsing clinicopathologic data, immunofluorescence (IF) imaging, and whole-transcriptome analysis, 22 breast cancers (BCs) associated with Yo-PCD were characterized in terms of oncologic characteristics, genetic alteration of Yo antigens, differential gene expression profiles, and morphofunctional specificities of their in situ antitumor immunity by comparing them with matched control BCs.ResultsYo-PCD BCs were invasive carcinoma of no special type, which early metastasized to lymph nodes. They overexpressed human epidermal growth factor receptor 2 (HER2) but were hormone receptor negative. All Yo-PCD BCs carried at least 1 genetic alteration (variation or gain in copy number) on CDR2L, encoding the main Yo antigen that was found aberrantly overexpressed in Yo-PCD BCs. Analysis of the differentially expressed genes found 615 upregulated and 54 downregulated genes in Yo-PCD BCs compared with HER2-driven control BCs without PCD. Ontology enrichment analysis found significantly upregulated adaptive immune response pathways in Yo-PCD BCs. IF imaging confirmed an intense immune infiltration with an overwhelming predominance of immunoglobulin G–plasma cells.DiscussionThese data confirm the role of genetic alterations of Yo antigens in triggering the immune tolerance breakdown but also outline a specific biomolecular profile in Yo-PCD BCs, suggesting a cancer-specific pathogenesis.

Background & aimsThe unexplained interpatient variation in hepatocellular carcinoma (HCC) remains a major challenge. We aimed at addressing the under-explored association between the disease and the hepatic autonomic nervous system (ANS).Methods & ResultsWe in-depth characterized the innervation of French biobanks HCC samples by conventional biochemistry methods. We also applied bioinformatics approaches to the TCGA dataset in order to stratify samples according to neural features and molecular correlates. We highlighted the predominant parasympathetic polarity of HCC nerves, and demonstrated that a cirrhotic rat model of aggressive HCC hosts liver neurogenesis with cholinergic features. Using the TCGA dataset, we then defined an HCC neural signature, derived from adrenergic and cholinergic receptor levels, that allowed patient stratification into two classes. Cholinergic tumors correlated with TP53 mutations (p ≤ 0.05), shorter progression-free interval (PFI) and overall survival (OS), displayed more pathogenic molecular traits (e.g., AFP-rich, proliferative tumors, mitotic functions including DNA repair, EMT, Ras, and Akt/mTOR pathways), aggressive HCC signatures and B cell accumulation. Instead, adrenergic tumors, predominant in patients aged >60 and with mutated CTNNB1, were correlated with better OS and PFI (p < 0.05), and numerous immune pathways.ConclusionsOur results depict neural features of HCC and how the existing tumor classification may also be shaped by neural inputs. Altogether, we show that the parasympathetic branch of the ANS is implicated in the pathobiology of HCC, and advocate for the use of ANS-targeting drugs in HCC research, many of which are clinically safe and well characterized.

International audience; Importance: Breast cancer (BC) diagnosis and treatment expose patients to a 5-fold higher risk of depression compared with the general population, with an estimated prevalence of 10% to 25%. A depressive episode in patients with BC has implications for the tolerance of and adherence to treatment, impairing quality of life and reducing life expectancy.Objective: To identify and characterize distinct longitudinal patterns of depressive symptoms in patients with BC from diagnosis to 3 years after treatment.Design, settings, and participants: The CANTO-DEePRESS (Deeper in the Understanding and Prevention of Depression in Breast Cancer Patients) cohort study included women in the French multicenter CANTO (CANcer TOxicities) cohort study (conducted between March 20, 2012 and December 11, 2018), who were 18 years or older with invasive stage I to III BC and no previous BC treatment. The study aimed to characterize toxicities over a 5-year period following stage I to III primary BC treatment. Assessments of depressive symptoms were performed on a subset of patients with available data at diagnosis and at least 2 other time points. All data were extracted from the CANTO database on October 1, 2020.Main outcomes and measures: The primary outcome was the level of depressive symptoms at each assessment time point measured with the Hospital Anxiety and Depression Scale and depression subscale at BC diagnosis and at 3 to 6, 12, and 36 months after the end of treatment. The group-based trajectory modeling was used to identify trajectory groups, and multinomial logistic regression models were used to characterize the following factors associated with trajectory group affiliation: demographic, socioeconomic, clinical, lifestyle, and quality-of-life data.Results: A total of 4803 women (mean [SD] age, 56.2 [11.2] years; 2441 patients [50.8%] with stage I BC) were included in the study. Six trajectory groups that described the heterogeneity in the expression of depressive symptoms were identified: noncases with no expression of symptoms (n = 2634 [54.8%]), intermediate worsening (1076 [22.4%]), intermediate improvement (480 [10.0%]), remission (261 [5.4%]), delayed occurrence (200 [4.2%]), and stable depression (152 [3.2%]). HADS-D scores at diagnosis were consistently associated with the 5 depressive trajectory group affiliations, with an estimated higher probability per point increase of experiencing subthreshold or clinically significant depressive symptoms between diagnosis and the 3 years after the end of BC treatment. The higher probabilities ranged from 1.49 (95% CI, 1.43-1.54) for the intermediate worsening group to 10.53 (95% CI, 8.84-12.55) for the stable depression group. Trajectory groups with depressive symptoms differed from the noncases group without symptoms by demographic and clinical factors, such as having dependent children, lower household income, cancer stage, family history of BC, previous psychiatric hospitalizations, obesity, smoking status, higher levels of fatigue, and depression at diagnosis.Conclusions and relevance: In this cohort study, nearly a third of patients with BC experienced temporary or lasting significant depressive symptoms during and after treatment. Improving early identification of women at risk of developing long-term or delayed depression is therefore critical to increase quality of life and overall survival. Subjected to validation, this study is an important first step toward personalized care of patients with BC at risk of depression.

ImportanceWhile comprehensive tumor molecular profile by whole exome and RNA sequencing (WES/RNA-Seq) is now feasible in routine practice, it remains unclear whether this increases therapeutic options as compared to a more limited targeted gene panel (TGP) plus array-based comparative genomic hybridization (aCGH) in advanced cancer patients.ObjectiveTo determine the added value of WES/RNA-Seq in advanced and refractory cancer patients who had no molecular-based treatment recommendation (MBTR) based on a TGP/aCGH in the course of a clinical trial.DesignRetrospective analysis.SettingSingle center.ParticipantsWe selected 50 patients previously included in the PROFILER trial (NCT01774409) for which no molecular-based therapy could be recommended in the course of the clinical trial based on a targeted 90-gene panel and aCGH. For each patient, the frozen tumor sample mirroring the FFPE sample used for TGP/aCGH analysis were processed for WES and RNA-Seq. Data from TGP/aCGH were reanalyzed and together with WES/RNA-Seq, findings were simultaneously discussed at a new molecular tumor board (MTB).Main outcomes and MeasuresMBTR based on TGP/aCGH versus WES/RNA-Seq were compared.ResultsAfter exclusion of variants of unknown significance, a total of 167 somatic molecular alterations were identified in 50 patients (median: 3; range: 1-10). Out of these 167 relevant molecular alterations reported by the biologist, 51 (31%) were common to both TGP/aCGH and WES/RNA-Seq, 19 (11%) were identified by the TGP/aCGH only and 97 (58%) were identified by WES/RNA-Seq only, including 2 fusion transcripts in two patients. A MBRT was provided in 4/50 (8%) patients using the information from TGP/aCGH vs. 9/50 (18%) patients using WES/RNA-Seq findings. Three patients had similar recommendations based on TGP/aCGH and WES/RNA-Seq.Conclusion and RelevanceIn advanced and refractory cancer patients in whom no MBRT was recommended from TGP/aCGH, WES/RNA-Seq allowed to identify more alterations which may in turn, in a limited fraction of patients, lead to new MBRT.Key PointsQuestionDoes WES/RNA-Seq provide additional targeted treatment guidance for advanced cancer patients with no molecular-based treatment recommendation (MBTR) from a 90-tumor gene panel (TGP) sequencing and array-based comparative genomic hybridization (aCGH)?FindingsFor fifty advanced cancer patients included in the PROFILER trial with no treatment recommendation based on a TGP/aCGH, frozen tumor sample was processed for WES and RNA-Seq. MBTR was given in 4/50 (8%) patients using the reanalyzed TGP/aCGH vs. 9/50 (18%) patients using WES/RNA-Seq findings.MeaningsWES/RNA-Seq increased the number of patients with MBTR as compared to a TGP/aCGH screening to yet only a minority of patients.

International audience; Beyond their critical role in hemostasis, platelets physically interact with neutrophils to form neutrophil-platelet aggregates (NPAs), enhancing neutrophil effector functions during inflammation. NPAs may also promote disease worsening in various inflammatory diseases. However, characterization of NPAs in cancer remains totally unexplored. Using ImageStreamX (ISX) imaging flow cytometer, we were not only allowed able to detect CD15+ CD14- CD36+ ITGA2B+ NPAs in both healthy donors' (HDs) and cancer patients' bloods, but we also showed that NPAs result from the binding of platelets preferentially to low-density neutrophils (LDNs) as opposed to normal-density neutrophils (NDNs). By reanalyzing two independent public scRNAseq data of whole blood leukocytes from cancer patients and HDs, we could identify a subset of neutrophils with high platelet gene expression that may correspond to NPAs. Moreover, we showed that cancer patients' derived NPAs possessed a distinct molecular signature compared to the other neutrophil subsets, independently of platelet genes. Gene ontology (GO) term enrichment analysis of this NPAs-associated neutrophil transcriptomic signature revealed a significant enrichment of neutrophil degranulation, chemotaxis and trans-endothelial migration GO terms. Lastly, using The Cancer Genome Atlas (TCGA), we could show by multivariate Cox analysis that the NPAs-associated neutrophil transcriptomic signature was associated with a worse patient prognosis in several cancer types. These results suggest that neutrophils from NPAs are systemically primed by platelets empowering them with cancer progression capacities once at tumor site. NPAs may therefore hold clinical utility as novel noninvasive blood prognostic biomarker in cancer patients with solid tumors.

Human endogenous retroviruses (HERVs) represent 8% of the human genome. HERV products may represent tumor antigens relevant for cancer immunotherapy. We developed a bioinformatic approach to identify shared CD8 + T cell epitopes derived from cancer-associated HERVs in solid tumors. Six candidates among the most commonly shared HLA-A2 epitopes with evidence of translation were selected for immunological evaluation. In vitro priming assays confirmed the immunogenicity of these epitopes, which induced high-avidity CD8 + T cell clones. These T cells specifically recognize and kill HLA-A2 + tumor cells presenting HERV epitopes on HLA molecules, as demonstrated by mass spectrometry. Furthermore, epitope-specific CD8 + T cells were identified by dextramer staining among tumor-infiltrating lymphocytes from HLA-A2 + patients with breast cancer. Last, we showed that HERV-specific T cells lyse patient-derived organoids. These shared virus-like epitopes are of major interest for the development of cancer vaccines or T cell–based immunotherapies, especially in tumors with low/intermediate mutational burden.

International audience; Human endogenous retroviruses (HERVs) represent 8% of the human genome. The expression of HERVs and their immune impact have not been extensively studied in Acute Myeloid Leukemia (AML). In this study, we used a reference of 14 968 HERV functional units to provide a thorough analysis of HERV expression in normal and AML bone marrow cells. We show that the HERV retrotranscriptome accurately characterizes normal and leukemic cell subpopulations, including leukemia stem cells, in line with different epigenetic profiles. We then show that HERV expression delineates AML subtypes with different prognoses. We finally propose a method to select and prioritize CD8 + T cell epitopes derived from AML-specific HERVs and we show that lymphocytes infiltrating patient bone marrow at diagnosis contain naturally

Beyond their critical role in hemostasis, platelets physically interact with neutrophils to form neutrophil-platelet aggregates (NPAs), enhancing neutrophil effector functions during inflammation. NPAs may also promote disease worsening in various inflammatory diseases. However, characterization of NPAs in cancer remains totally unexplored. Using ImageStream®X (ISX) imaging flow cytometer, we were not only allowed able to detect CD15+ CD14− CD36+ ITGA2B+ NPAs in both healthy donors’ (HDs) and cancer patients’ bloods, but we also showed that NPAs result from the binding of platelets preferentially to low-density neutrophils (LDNs) as opposed to normal-density neutrophils (NDNs). By re-analyzing two independent public scRNAseq data of whole blood leukocytes from cancer patients and HDs, we could identify a subset of neutrophils with high platelet gene expression that may correspond to NPAs. Moreover, we showed that cancer patients’ derived NPAs possessed a distinct molecular signature compared with the other neutrophil subsets, independently of platelet genes. Gene ontology (GO) term enrichment analysis of this NPAs-associated neutrophil transcriptomic signature revealed a significant enrichment of neutrophil degranulation, chemotaxis and trans-endothelial migration GO terms. Lastly, using The Cancer Genome Atlas (TCGA), we could show by multivariate Cox analysis that the NPAs-associated neutrophil transcriptomic signature was associated with a worse patient prognosis in several cancer types. These results suggest that neutrophils from NPAs are systemically primed by platelets empowering them with cancer progression capacities once at tumor site. NPAs may therefore hold clinical utility as novel non-invasive blood prognostic biomarker in cancer patients with solid tumors.Novelty and ImpactPlatelets physically interact with peripheral blood neutrophils to form neutrophil-platelet aggregates (NPAs), known to promote disease worsening in various inflammatory diseases. However, characterization of NPAs in cancer remains totally unexplored. We showed that NPAs-associated neutrophils were a yet-unreported unique subset of circulating neutrophils associated with a worse patient prognosis in several cancer types. NPAs may hold clinical utility as novel non-invasive blood prognostic biomarker in cancer patients with solid tumors.

Human endogenous retroviruses (HERVs) represent 8% of the human genome. HERVs are silenced by epigenetic mechanisms in normal cells but are aberrantly expressed by tumor cells. Given their viral origin, HERV products may represent tumor antigens relevant for cancer immunotherapy. We developed a systematic bioinformatics-based approach to identify shared CD8+ T cell epitopes derived from cancer-associated HERVs in solid tumors. Six HLA-A2 epitopes among the most shared epitope candidates with evidence of translation were selected for further immunological evaluation. In vitro priming assays showed the induction of specific CD8+ T cells leading to polyfunctional T cell responses. The functionality of the sorted T cell clones was confirmed by Elispot (GrzB+ IFN-γ+) before TCR sequencing. Interestingly, these TCRs were predicted to interact with a high affinity with their respective MHC-peptide complexes in 3D models. This was confirmed by measurement of the functional avidity, which was in the same order as CMV-specific T cell clones. HERV-specific CD8+ T cells induced specific cell death of HLA-A2+ cancer cell lines presenting HERV epitopes on HLA molecules, as demonstrated by mass spectrometry. Furthermore, HERV-specific CD8+ T cells were identified by dextramer-staining among tumor infiltrating lymphocytes (TILs) from HLA-A2+ breast and ovarian cancer patients. Finally, we showed that HERV-specific T cells can lyse patient-derived organoids (Bonaventura et al. Sci Adv 2022). Synthetic long peptides containing these HERV epitopes have been validated for the development of a cancer vaccine. TCR engineered T cells specific to these HERV epitopes have been generated and their functionality and specificity have been confirmed. In parallel, we also evaluated HERV expression in Acute Myeloid Leukemia (AML). We used a complete database of 14,968 HERVs functional units to provide a thorough analysis of HERVs in normal and AML bone marrow cells. We found that HERV retrotranscriptome characterizes normal and leukemic cell subpopulations and HERV expression separates distinct AML subtypes of different prognosis. We showed that patients’ bone marrow infiltrating lymphocytes at diagnosis also contain naturally occurring CD8+ T cells against AML-specific HERV epitopes. Furthermore, we demonstrated that HERV-specific CD8+ T cells specifically recognize AML cells (Alcazer et al. Am J Hematol 2022). Our bioinformatic approach allowed us to identify shared HERV-derived CD8+ T cell epitopes specifically expressed by tumor cells and inducing high-avidity T cell clones able to kill tumor cells in a class I-restricted manner. The detection of TILs recognizing HERV peptides suggests natural presentation of these epitopes in the tumors. These HERV-derived epitopes may thus represent relevant targets for the development of new immunotherapeutic approaches, especially in tumors with a low or moderate mutational burden. We are currently developing a therapeutic vaccine as well as TCR engineered T cells specific to these HERV epitopes. Citation Format: Paola Bonaventura, Vincent Alcazer, Virginie Mutez, Laurie Tonon, Juliette Martin, Nicolas Chuvin, Emilie Michel, Rasha Boulos, Yann Estornes, Jenny Valladeau-Guilemond, Alain Viari, Quing Wang, Christophe Caux, Stephane Depil. Human Endogenous Retroviruses represent a source of shared tumor epitopes inducing high-avidity cytotoxic T cells for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B49.

International audience; Immunohistochemistry and recent molecular technologies progressively guided access to personalized anti-tumoral therapies. We explored the feasibility, efficacy, and the impact of molecular profiling in patients with advanced brain tumors. This multicentric prospective trial ProfiLER enrolled patients with primary brain tumors, who have been pre-treated with at least one line of anti-cancer treatment, and for whom molecular profiles had been achieved using next-generation sequencing and/or comparative genomic hybridization on fresh or archived samples from tumor, relapse, or biopsies. A molecular tumor board weekly analyzed results and proposed molecular-based recommended therapy (MBRT). From February 2013 to December 2015, we enrolled 141 patients with primary brain tumor and analyzed 105 patients for whom tumor genomic profiles had been achieved. Histology mainly identified glioblastoma (N = 46, 44%), low-grade glioma (N = 26, 25%), highgrade glioma (N = 12, 11%), and atypical and anaplastic meningioma (N = 8, 8%). Forty-three (41%) patients presented at least one actionable molecular alteration. Out of 61 alterations identified, the most frequent alterations occurred in CDKN2A (N = 18), EGFR (N = 12), PDGFRa (N = 8), PTEN (N = 8), CDK4 (N = 7), KIT (N = 6), PIK3CA (N = 5), and MDM2 (N = 3). Sixteen (15%) patients could not be proposed for a MBRT due to early death (N = 5), lack of available clinical trials (N = 9), or inappropriate results (N = 2). Only six (6%) of the 27 (26%) patients for whom a MBRT had been proposed finally initiated MBRT (everolimus (N = 3), erlotinib (N = 1), ruxolitinib (N = 1), and sorafenib (N = 1)), but discontinued treatment for toxicity (N = 4) or clinical progression (N = 2). High-throughput sequencing in patients with brain tumors may be routinely performed, especially when macroscopic surgery samples are available; nevertheless delays should be reduced. Criteria for clinical trial enrollment should be reconsidered in patients with brain tumors, and a panel of genes specifically dedicated to neurologic tumors should be developed to help decision-making in clinical practice.