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Cancer immunotherapy combinations have recently been shown to improve the overall survival of advanced mesotheliomas, especially for patients responding to those treatments. We aimed to characterize the biological correlates of malignant pleural mesotheliomas’ primary resistance to immunotherapy and antiangiogenics by testing the combination of pembrolizumab, an anti–PD-1 antibody, and nintedanib, a pan-antiangiogenic tyrosine kinase inhibitor, in the multicenter PEMBIB trial (NCT02856425). Thirty patients with advanced malignant pleural mesothelioma were treated and explored. Unexpectedly, we found that refractory patients were actively recruiting CD3+CD8+ cytotoxic T cells in their tumors through CXCL9 tumor release upon treatment. However, these patients displayed high levels of somatic copy-number alterations in their tumors that correlated with high blood and tumor levels of IL6 and CXCL8. Those proinflammatory cytokines resulted in higher tumor secretion of VEGF and tumor enrichment in regulatory T cells. Advanced mesothelioma should further benefit from stratified combination therapies adapted to their tumor biology. Significance: Sequential explorations of fresh tumor biopsies demonstrated that mesothelioma resistance to anti–PD-1 + antiangiogenics is not due to a lack of tumor T-cell infiltration but rather due to adaptive immunosuppressive pathways by tumors, involving molecules (e.g., IL6, CXCL8, VEGF, and CTLA4) that are amenable to targeted therapies. This article is highlighted in the In This Issue feature, p. 799

Metastatic relapse after treatment is the leading cause of cancer mortality, and known resistance mechanisms are missing for most treatments administered to patients. To bridge this gap, we analyze a pan-cancer cohort (META-PRISM) of 1,031 refractory metastatic tumors profiled via whole-exome and transcriptome sequencing. META-PRISM tumors, particularly prostate, bladder, and pancreatic types, displayed the most transformed genomes compared with primary untreated tumors. Standard-of-care resistance biomarkers were identified only in lung and colon cancers—9.6% of META-PRISM tumors, indicating that too few resistance mechanisms have received clinical validation. In contrast, we verified the enrichment of multiple investigational and hypothetical resistance mechanisms in treated compared with nontreated patients, thereby confirming their putative role in treatment resistance. Additionally, we demonstrated that molecular markers improve 6-month survival prediction, particularly in patients with advanced breast cancer. Our analysis establishes the utility of the META-PRISM cohort for investigating resistance mechanisms and performing predictive analyses in cancer.Significance:This study highlights the paucity of standard-of-care markers that explain treatment resistance and the promise of investigational and hypothetical markers awaiting further validation. It also demonstrates the utility of molecular profiling in advanced-stage cancers, particularly breast cancer, to improve the survival prediction and assess eligibility to phase I clinical trials.This article is highlighted in the In This Issue feature, p. 1027

These figures provide complementary information for the figures presented in the main text and illustrations of the methods section.

These tables present clinical data, sample data, whole-exome and transcriptome sequencing results, literature references, and external data sources that were used for the analyses presented in the main text.

Vaccination against coronavirus disease 2019 (COVID-19) relies on the in-depth understanding of protective immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We characterized the polarity and specificity of memory T cells directed against SARS-CoV-2 viral lysates and peptides to determine correlates with spontaneous, virus-elicited, or vaccine-induced protection against COVID-19 in disease-free and cancer-bearing individuals. A disbalance between type 1 and 2 cytokine release was associated with high susceptibility to COVID-19. Individuals susceptible to infection exhibited a specific deficit in the T helper 1/T cytotoxic 1 (Th1/Tc1) peptide repertoire affecting the receptor binding domain of the spike protein (S1-RBD), a hotspot of viral mutations. Current vaccines triggered Th1/Tc1 responses in only a fraction of all subject categories, more effectively against the original sequence of S1-RBD than that from viral variants. We speculate that the next generation of vaccines should elicit Th1/Tc1 T-cell responses against the S1-RBD domain of emerging viral variants.Significance:This study prospectively analyzed virus-specific T-cell correlates of protection against COVID-19 in healthy and cancer-bearing individuals. A disbalance between Th1/Th2 recall responses conferred susceptibility to COVID-19 in both populations, coinciding with selective defects in Th1 recognition of the receptor binding domain of spike.See related commentary by McGary and Vardhana, p. 892.This article is highlighted in the In This Issue feature, p. 873

Immuno-Histo-Chemistry scoring of baseline tumor biopsies according to their DCB status.

Supplementary Figure from Cancer Induces a Stress Ileopathy Depending on β-Adrenergic Receptors and Promoting Dysbiosis that Contributes to Carcinogenesis

Supplementary Figure from The Polarity and Specificity of Antiviral T Lymphocyte Responses Determine Susceptibility to SARS-CoV-2 Infection in Patients with Cancer and Healthy Individuals

Gut dysbiosis has been associated with intestinal and extraintestinal malignancies, but whether and how carcinogenesis drives compositional shifts of the microbiome to its own benefit remains an open conundrum. Here, we show that malignant processes can cause ileal mucosa atrophy, with villous microvascular constriction associated with dominance of sympathetic over cholinergic signaling. The rapid onset of tumorigenesis induced a burst of REG3γ release by ileal cells, and transient epithelial barrier permeability that culminated in overt and long-lasting dysbiosis dominated by Gram-positive Clostridium species. Pharmacologic blockade of β-adrenergic receptors or genetic deficiency in Adrb2 gene, vancomycin, or cohousing of tumor bearers with tumor-free littermates prevented cancer-induced ileopathy, eventually slowing tumor growth kinetics. Patients with cancer harbor distinct hallmarks of this stress ileopathy dominated by Clostridium species. Hence, stress ileopathy is a corollary disease of extraintestinal malignancies requiring specific therapies.Significance:Whether gut dysbiosis promotes tumorigenesis and how it controls tumor progression remain open questions. We show that 50% of transplantable extraintestinal malignancies triggered a β-adrenergic receptor–dependent ileal mucosa atrophy, associated with increased gut permeability, sustained Clostridium spp.–related dysbiosis, and cancer growth. Vancomycin or propranolol prevented cancer-associated stress ileopathy.This article is highlighted in the In This Issue feature, p. 873

Supplementary Data from The Polarity and Specificity of Antiviral T Lymphocyte Responses Determine Susceptibility to SARS-CoV-2 Infection in Patients with Cancer and Healthy Individuals

Cytotoxic response in tumor biopsies of patients with No DCB upon combination treatment.

Cancer immunotherapy combinations have recently been shown to improve the overall survival of advanced mesotheliomas, especially for patients responding to those treatments. We aimed to characterize the biological correlates of malignant pleural mesotheliomas’ primary resistance to immunotherapy and antiangiogenics by testing the combination of pembrolizumab, an anti–PD-1 antibody, and nintedanib, a pan-antiangiogenic tyrosine kinase inhibitor, in the multicenter PEMBIB trial (NCT02856425). Thirty patients with advanced malignant pleural mesothelioma were treated and explored. Unexpectedly, we found that refractory patients were actively recruiting CD3+CD8+ cytotoxic T cells in their tumors through CXCL9 tumor release upon treatment. However, these patients displayed high levels of somatic copy-number alterations in their tumors that correlated with high blood and tumor levels of IL6 and CXCL8. Those proinflammatory cytokines resulted in higher tumor secretion of VEGF and tumor enrichment in regulatory T cells. Advanced mesothelioma should further benefit from stratified combination therapies adapted to their tumor biology.Significance:Sequential explorations of fresh tumor biopsies demonstrated that mesothelioma resistance to anti–PD-1 + antiangiogenics is not due to a lack of tumor T-cell infiltration but rather due to adaptive immunosuppressive pathways by tumors, involving molecules (e.g., IL6, CXCL8, VEGF, and CTLA4) that are amenable to targeted therapies.This article is highlighted in the In This Issue feature, p. 799

Purpose: This single-institutional feasibility study prospectively characterized genomic alterations in recurrent or refractory solid tumors of pediatric patients to select a targeted therapy.Experimental Design: Following treatment failure, patients with signed consent and ages above 6 months, underwent tumor biopsy or surgical resection of primary or metastatic tumor site. These newly acquired samples were analyzed by comparative genomic hybridization array, next-generation sequencing for 75 target genes, whole-exome and RNA sequencing. Biological significance of the alterations and suggestion of most relevant targeted therapies available were discussed in a multidisciplinary tumor board.Results: From December 2012 to January 2016, 75 patients were included, 73 patients underwent 79 interventions, 56 of which were research biopsies with a low complication rate. All patients were pretreated, 37.0% had a brain tumor, and 63.0% had an extra-cranial solid tumor. Median tumor cell content was 70% (range, 0%–100%). Successful molecular analysis in 69 patients detected in 60.9% of patients an actionable alteration in various oncogenic pathways (42.4% with copy-number change, 33.3% with mutation, 2.1% with fusion), and change in diagnosis in three patients. Fourteen patients received 17 targeted therapies; two had received a matched treatment before inclusion.Conclusions: Research biopsies are feasible in advanced pediatric malignancies that exhibit a considerable amount of potentially actionable alterations. Genetic events affecting different cancer hallmarks and limited access to targeted agents within pediatric clinical trials remain the main obstacles that are addressed in our two subsequent precision medicine studies MAPPYACTS and AcSé-ESMART. Clin Cancer Res; 23(20); 6101–12. ©2017 AACR.

Clinical and genomics data on all patients included

Sensitivity Prediction Score ROC Curve of the logistic regression model.

Quality data of the WES and RNAseq analyzes

mutations' allele frequencies in tDNA and cfDNA

Purpose: Liquid biopsies based on circulating cell-free DNA (cfDNA) analysis are described as surrogate samples for molecular analysis. We evaluated the concordance between tumor DNA (tDNA) and cfDNA analysis on a large cohort of patients with advanced or metastatic solid tumor, eligible for phase I trial and with good performance status, enrolled in MOSCATO 01 trial (clinical trial NCT01566019).Experimental Design: Blood samples were collected at inclusion and cfDNA was extracted from plasma for 334 patients. Hotspot mutations were screened using next-generation sequencing for 50 cancer genes.Results: Among the 283 patients with tDNA–cfDNA pairs, 121 had mutation in both, 99 in tumor only, 5 in cfDNA only, and for 58 patients no mutation was detected, leading to a 55.0% estimated sensitivity [95% confidence interval (CI), 48.4%–61.6%] at the patient level. Among the 220 patients with mutations in tDNA, the sensitivity of cfDNA analysis was significantly linked to the number of metastatic sites, albumin level, tumor type, and number of lines of treatment. A sensitivity prediction score could be derived from clinical parameters. Sensitivity is 83% in patients with a high score (≥8). In addition, we analyzed cfDNA for 51 patients without available tissue sample. Mutations were detected for 22 patients, including 19 oncogenic variants and 8 actionable mutations.Conclusions: Detection of somatic mutations in cfDNA is feasible for prescreening phase I candidates with a satisfactory specificity; overall sensitivity can be improved by a sensitivity score allowing to select patients for whom cfDNA constitutes a reliable noninvasive surrogate to screen mutations. Clin Cancer Res; 22(12); 2960–8. ©2016 AACR.

Legends Supplementary Material

Supplementary methods genomics

Metastatic relapse after treatment is the leading cause of cancer mortality, and known resistance mechanisms are missing for most treatments administered to patients. To bridge this gap, we analyze a pan-cancer cohort (META-PRISM) of 1,031 refractory metastatic tumors profiled via whole-exome and transcriptome sequencing. META-PRISM tumors, particularly prostate, bladder, and pancreatic types, displayed the most transformed genomes compared with primary untreated tumors. Standard-of-care resistance biomarkers were identified only in lung and colon cancers—9.6% of META-PRISM tumors, indicating that too few resistance mechanisms have received clinical validation. In contrast, we verified the enrichment of multiple investigational and hypothetical resistance mechanisms in treated compared with nontreated patients, thereby confirming their putative role in treatment resistance. Additionally, we demonstrated that molecular markers improve 6-month survival prediction, particularly in patients with advanced breast cancer. Our analysis establishes the utility of the META-PRISM cohort for investigating resistance mechanisms and performing predictive analyses in cancer. Significance: This study highlights the paucity of standard-of-care markers that explain treatment resistance and the promise of investigational and hypothetical markers awaiting further validation. It also demonstrates the utility of molecular profiling in advanced-stage cancers, particularly breast cancer, to improve the survival prediction and assess eligibility to phase I clinical trials. This article is highlighted in the In This Issue feature, p. 1027

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Trastuzumab deruxtecan (T-DXd) is an anti-HER2 (human epidermal growth factor receptor 2) antibody-drug conjugate which has previously shown efficacy in patients with HER2-overexpressing and HER2-low metastatic breast cancer (mBC). However, the mechanisms of action and resistance of this drug remain partially unclear. DAISY (NCT04132960) is a phase II, open-label study that included patients with mBC whose disease progressed after at least one line of chemotherapy in the metastatic setting. Patients were enrolled in three cohorts according to HER2 expression determined by immunohistochemistry (IHC); cohort 1: HER2-overexpressing (HER2 IHC 3 + or HER2 IHC 2+/ISH+, n = 72), cohort 2: HER2-low (HER2 IHC2+/ISH- or HER2 IHC 1+, n = 74), and cohort 3: HER2 IHC 0 mBC (n = 40). Patients were treated with T-DXd 5.4 mg/kg every 3 weeks until disease progression or unacceptable toxicity. In the full analysis set population (n = 177), the confirmed objective response rate (ORR) was of 70.6% (95% CI: 58.3–81) in cohort 1, 37.5% (95% CI: 26.4–49.7) in cohort 2, and 29.7% (95% CI: 15.9–47) in cohort 3 (p p = 0.007), and cohort 3 with shorter PFS (adjusted HR: 1.96, 95% IC: 1.21–3.15, p = 0.006) as compared to cohort 2. Exploratory analyses showed that HER2 spatial distribution predicted T-DXd response in patients with HER2-overexpressing mBC and that the transcriptomic response to T-DXd was different according to HER2 expression. No quantitative modulation of tumor microenvironment was observed after 6 to 8 weeks of treatment. Finally, recurrent mutations of the DNA repair gene SLX4 were identified in 20% of samples at resistance (4/20) as compared to 2% in baseline samples (2/88), suggesting that SLX4 mutations could mediate secondary resistance to T-DXd. These data suggest that HER2 is a key determinant of T-DXd efficacy. However, an antitumor activity is also observed in a subgroup of patients without detectable HER2 expression and resistance could be partially mediated by payload sensitivity.

Gut dysbiosis has been associated with intestinal and extraintestinal malignancies, but whether and how carcinogenesis drives compositional shifts of the microbiome to its own benefit remains an open conundrum. Here, we show that malignant processes can cause ileal mucosa atrophy, with villous microvascular constriction associated with dominance of sympathetic over cholinergic signaling. The rapid onset of tumorigenesis induced a burst of REG3γ release by ileal cells, and transient epithelial barrier permeability that culminated in overt and long-lasting dysbiosis dominated by Gram-positive Clostridium species. Pharmacologic blockade of β-adrenergic receptors or genetic deficiency in Adrb2 gene, vancomycin, or cohousing of tumor bearers with tumor-free littermates prevented cancer-induced ileopathy, eventually slowing tumor growth kinetics. Patients with cancer harbor distinct hallmarks of this stress ileopathy dominated by Clostridium species. Hence, stress ileopathy is a corollary disease of extraintestinal malignancies requiring specific therapies. Significance: Whether gut dysbiosis promotes tumorigenesis and how it controls tumor progression remain open questions. We show that 50% of transplantable extraintestinal malignancies triggered a β-adrenergic receptor–dependent ileal mucosa atrophy, associated with increased gut permeability, sustained Clostridium spp.–related dysbiosis, and cancer growth. Vancomycin or propranolol prevented cancer-associated stress ileopathy. This article is highlighted in the In This Issue feature, p. 873

Background: AcSé-ESMART is a proof-of-concept, phase I/II platform trial designed to explore targeted agents in a molecularly enriched pediatric population. WEE1 plays a role in DNA repair and cell cycle control and is overexpressed in pediatric cancers. Adavosertib combinations resulted in enhanced antitumor activity compared to single agent in neuroblastoma, rhabdomyosarcoma, medulloblastoma and high-grade glioma in vivo models. The efficacy and safety of the adavosertib-carboplatin combination has been established in adults with focus on TP53 mutated ovarian cancer. Arm C of AcSé-ESMART applied this regimen to children with advanced malignancies enriched for alterations in TP53, DNA repair/replication stress and cell cycle control. Methods: Adavosertib was administered orally, twice daily on Days 1 to 3 and carboplatin intravenously on Day 1 of a 21-day cycle. Dose finding used the continuous reassessment method starting at adavosertib 100 mg/m2/dose and carboplatin AUC 5. Pharmacokinetic (PK) and retrospective molecular bioinformatic analysis was performed. Results: Twenty patients (median age: 14.0 years, range 3.4-23.5) were included, 18 received a total of 69 cycles. Seven dose-limiting toxicities (DLTs) were observed leading to two de-escalations to adavosertib 75 mg/m2/dose and carboplatin AUC 4. All patients with DLT had thrombocytopenia grade 3/4 requiring transfusions for >7 days and/or neutropenia grade 4 for >7 days. Main overall treatment-related toxicities were hematologic and gastrointestinal. Based on the identified DLT risk, no recommended Phase 2 dose was defined. PK analysis demonstrated equivalent adavosertib exposure in children to that in adults and both doses (75 and 100 mg/m2) achieved the cell kill target. Two patients with neuroblastoma achieved partial response (PR), one with medulloblastoma unconfirmed PR, and five had stable disease (SD) >4 cycles. Patients with PR/SD >4 cycles were considered as clinical benefit (CB) for retrospective molecular analysis. There was no correlation between TP53 genomic alteration alone and response. However, 7 of 8 patients with CB but none of the 10 patients without CB had 1 to 3 genomic alterations in the DNA repair (BRCA2 mutation, 11q loss containing ATM, MRE11A, CHEK1), cell cycle control/replication stress (CCNE1 amplification, RB1 mutation/loss, SETD2 mutation/loss) and RAS pathway (KRAS mutation and amplification, NF1 loss, PTPN11 mutation) in their tumor. Conclusions: Adavosertib combined with carboplatin exhibited significant hematologic toxicity. Activity signals and identified potential molecular biomarkers suggest further combination studies with less hematotoxic DNA damaging therapy in molecularly enriched pediatric cancers. Citation Format: Susanne A. Gatz, Anne C. Harttrampf, Caroline Brard, Francisco J. Bautista, Nicolas André, Samuel Abbou, Jonathan Rubino, Windy Rondof, Marc Deloger, Marc Rübsam, Daniel Hübschmann, Lynley V. Marshall, Souad Nebchi, Isabelle Aerts, Estelle Thebaud, Emilie De Carli, Anne-Sophie Defachelles, Xavier Paoletti, Robert Godin, Kowser Miah, Peter G. Mortimer, Gilles Vassal, Birgit Geoerger. Phase I/II study of the WEE1 inhibitor adavosertib in combination with carboplatin in children with advanced malignancies: arm C of the AcSé-ESMART trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT087.

Metastatic relapse after treatment is the primary cause of cancer morbidity and mortality. While genetic mechanisms of primary tumors and, to a lesser extent, metastatic cancers have been studied in large cohorts, refractory metastatic tumors are not yet sufficiently characterized. Markers of aggressiveness and resistance that molecular profiling can extract from these tumors have yet to be identified and incorporated into clinical care. In this study, we present a pan-cancer cohort of 1,031 metastatic tumors (which we refer to as META-PRISM) that are resistant to at least one systemic therapy or with no approved treatment options. We retrieved the complete clinical history of patients and performed whole-exome (n=571) and transcriptome sequencing (n=947) for this cohort. The prevalence of detected cancer biomarkers was assessed and compared to an external cohort of primary tumors. In the META-PRISM cohort, we observed an increase in (i) whole-genome duplication frequency, (ii) tumor mutational burden, (iii) germline cancer-predisposing variants, and (iv) somatic alterations in cancer genes, including KRAS, EGFR, CCND1, MYC, and TP53, as compared to the tumor type-matched primary tumors. The most extensive increase in genomic variation at metastatic stage was observed in prostate bladder, and pancreatic cancer types. We also identified enrichment of standard-of-care resistance biomarkers in most cancer types. However, only 9.3% of tumors harbored at least one such biomarker, indicating that the current understanding of resistance mechanisms remains insufficient. Our cohort demonstrated a significantly improved 6-month survival prediction from models incorporating molecular markers over models with only clinical markers for breast cancer patients and to a lesser extent for other studied tumor types. Overall, our data establish a unique resource for investigating treatment resistance mechanisms and performing predictive analyses in cancer. Citation Format: Yoann Pradat, Julien Viot, Konstantin Gunbin, Andrey Yurchenko, Luigi Cerbone, Marc Deloger, Guillaume Grisay, Loic Verlingue, Véronique Scott, Ismael Padioleau, Leonardo Panunzi, Stefan Michiels, Antoine Hollebecque, Gerome Jules-Clément, Laura Mezquita, Antoine Lainé, Yohann Loriot, Benjamin Besse, Luc Friboulet, Fabrice André, Paul-Henry Cournède, Daniel Gautheret, Sergey Nikolaev. Integrative pan-cancer genomic and transcriptomic analyses of refractory metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6570.

Brain macrophage populations include parenchymal microglia, border-associated macrophages, and recruited monocyte-derived cells; together, they control brain development and homeostasis but are also implicated in aging pathogenesis and neurodegeneration. The phenotypes, localization, and functions of each population in different contexts have yet to be resolved. We generated a murine brain myeloid scRNA-seq integration to systematically delineate brain macrophage populations. We show that the previously identified disease-associated microglia (DAM) population detected in murine Alzheimer's disease models actually comprises two ontogenetically and functionally distinct cell lineages: embryonically derived triggering receptor expressed on myeloid cells 2 (TREM2)-dependent DAM expressing a neuroprotective signature and monocyte-derived TREM2-expressing disease inflammatory macrophages (DIMs) accumulating in the brain during aging. These two distinct populations appear to also be conserved in the human brain. Herein, we generate an ontogeny-resolved model of brain myeloid cell heterogeneity in development, homeostasis, and disease and identify cellular targets for the treatment of neurodegeneration.

Although CD8

Metastatic relapse after treatment is the primary cause of cancer morbidity and mortality. While genetic mechanisms of primary tumors and, to a lesser extent, metastatic cancers have been studied in large cohorts, refractory metastatic tumors are not yet sufficiently characterized. Markers of aggressiveness and resistance that molecular profiling can extract from these tumors have yet to be identified and incorporated into clinical care. In this study, we present a pan-cancer cohort of 1,031 metastatic tumors (which we refer to as META-PRISM) that are resistant to at least one systemic therapy or with no approved treatment options. We retrieved the complete clinical history of patients and performed whole-exome (n=571) and transcriptome sequencing (n=947) for this cohort. The prevalence of detected cancer biomarkers was assessed and compared to an external cohort of primary tumors. In the META-PRISM cohort, we observed an increase in (i) whole-genome duplication frequency, (ii) tumor mutational burden, (iii) germline cancer-predisposing variants, and (iv) somatic alterations in cancer genes, including KRAS, EGFR, CCND1, MYC, and TP53, as compared to the tumor type-matched primary tumors. The most extensive increase in genomic variation at metastatic stage was observed in prostate cancer. We also identified enrichment of standard-of-care resistance biomarkers in most cancer types. However, only 7.6% of tumors harbored at least one such biomarker, indicating that the current understanding of resistance mechanisms remains insufficient. Our cohort demonstrated a significantly improved 6-month survival prediction from models incorporating molecular markers over models with only clinical markers for breast cancer patients and to a lesser extent for other studied tumor types. Overall, our data establish a unique resource for investigating treatment resistance mechanisms and performing predictive analyses in cancer. Citation Format: Yoann Pradat, Julien Viot, Konstantin Gunbin, Andrei Iurchenko, Marc Deloger, Luigi Cerbone, Guillaume Grisay, Loic Verlingue, Veronique Scott, Stefan Michiels, Antoine Hollebecque, Gerome Jules-Clement, Antoine Laine, Luc Friboulet, Laura Mezquita, Yohann Loriot, Benjamin Besse, Fabrice Andre, Paul-Henry Cournede, Daniel Gautheret, Sergey Nikolaev. Integrative pan-cancer genomic and transcriptomic analyses of refractory metastatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR009.

Vaccination against coronavirus disease 2019 (COVID-19) relies on the in-depth understanding of protective immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We characterized the polarity and specificity of memory T cells directed against SARS-CoV-2 viral lysates and peptides to determine correlates with spontaneous, virus-elicited, or vaccine-induced protection against COVID-19 in disease-free and cancer-bearing individuals. A disbalance between type 1 and 2 cytokine release was associated with high susceptibility to COVID-19. Individuals susceptible to infection exhibited a specific deficit in the T helper 1/T cytotoxic 1 (Th1/Tc1) peptide repertoire affecting the receptor binding domain of the spike protein (S1-RBD), a hotspot of viral mutations. Current vaccines triggered Th1/Tc1 responses in only a fraction of all subject categories, more effectively against the original sequence of S1-RBD than that from viral variants. We speculate that the next generation of vaccines should elicit Th1/Tc1 T-cell responses against the S1-RBD domain of emerging viral variants. Significance: This study prospectively analyzed virus-specific T-cell correlates of protection against COVID-19 in healthy and cancer-bearing individuals. A disbalance between Th1/Th2 recall responses conferred susceptibility to COVID-19 in both populations, coinciding with selective defects in Th1 recognition of the receptor binding domain of spike. See related commentary by McGary and Vardhana, p. 892. This article is highlighted in the In This Issue feature, p. 873

SummaryThe expansion of antigen experienced CD4+ T cells is limited by intrinsic factors. Using in vivo genome-wide CRISPR-Cas9 screens, we identified SOCS1 as a non-redundant checkpoint imposing a brake on CD4+ T-cell proliferation upon rechallenge. We show here that SOCS1 is a critical node integrating both IL-2 and IFN-γ signals and blocking multiple signaling pathways to abrogate CD4+ Th1 cell response. In CD8+ T-cell, SOCS1 does not impact the proliferation but rather reduces survival and effector functions. By targeting SOCS1, both murine and human CD4+ T-cell antitumor adoptive therapies exhibit a restored intra-tumor accumulation, proliferation/survival, persistence and polyfunctionality, promoting long term rejection of established tumors. These findings identify SOCS1 as a major intracellular checkpoint inhibitor of primed CD4+ T cells, opening new possibilities to optimize CAR-T cell therapies composition and efficacy.

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.

Hereditary Renal Cell Carcinomas (RCC) are caused by mutations in predisposing genes, the major ones including VHL, FLCN, FH and MET. However, many families with inherited RCC have no germline mutation in these genes. Using Whole Exome Sequencing on germline DNA from a family presenting three different histological renal tumors (an angiomyolipoma, a clear-cell RCC and an oncocytic papillary RCC), we identified a frameshift mutation in the Neighbor of BRCA1 gene 1 (NBR1), segregating with the tumors. NBR1 encodes a cargo receptor protein involved in autophagy. Genetic and functional analyses suggested a pathogenic impact of the mutation. Indeed, functional study performed in renal cell lines showed that the mutation alters NBR1 interactions with some of its partners (such as p62/SQSTM1), leading to a dominant negative effect. This results in an altered autophagic process and an increased proliferative capacity in renal cell lines. Our study suggests that NBR1 may be a new predisposing gene for RCC, however its characterization needs to be further investigated in order to confirm its role in renal carcinogenesis.

Chromatin modifications orchestrate the dynamic regulation of gene expression during development and in disease. Bulk approaches have characterized the wide repertoire of histone modifications across cell types, detailing their role in shaping cell identity. However, these population-based methods do not capture cell-to-cell heterogeneity of chromatin landscapes, limiting our appreciation of the role of chromatin in dynamic biological processes. Recent technological developments enable the mapping of histone marks at single-cell resolution, opening up perspectives to characterize the heterogeneity of chromatin marks in complex biological systems over time. Yet, existing tools used to analyze bulk histone modifications profiles are not fit for the low coverage and sparsity of single-cell epigenomic datasets. Here, we present ChromSCape, a user-friendly interactive Shiny/R application distributed as a Bioconductor package, that processes single-cell epigenomic data to assist the biological interpretation of chromatin landscapes within cell populations. ChromSCape analyses the distribution of repressive and active histone modifications as well as chromatin accessibility landscapes from single-cell datasets. Using ChromSCape, we deconvolve chromatin landscapes within the tumor micro-environment, identifying distinct H3K27me3 landscapes associated with cell identity and breast tumor subtype., Bulk approaches fail to capture the cell-to-cell heterogeneity of chromatin landscapes, while single-cell approaches provide low coverage datasets. Here, the authors present ChromSCape, a user-friendly interactive application that processes single-cell epigenomic data to assist the biological interpretation of chromatin landscapes within cell populations, as demonstrated in the context of cancer.

Summary Blood myeloid cells are known to be dysregulated in the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity, and whether markers of innate immunity discriminate high risk patients. Thus, we performed high dimensional flow cytometry and single cell RNA sequencing of COVID-19 patient peripheral blood cells and detected the disappearance of non-classical CD14LowCD16High monocytes, the accumulation of HLA-DRLow classical monocytes, and the release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immuno-suppressive profile accumulated as well in blood and lungs, suggesting emergency myelopoiesis. We finally showed that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe COVID-19 form, suggesting a predictive value that deserves prospective evaluation., Highlights • Severe patients accumulate HLA-DRLow monocytes and immature neutrophils in blood/lung. • Calprotectin level positively correlates with neutrophil count and disease severity. • Loss of non-classical monocytes could identify high risk of severe COVID-19.

Comprehensive genomic profiling using high-throughput sequencing brings a wealth of information, and its place in the clinical setting has been increasingly prominent. This review emphasizes the utility of whole-exome sequencing (WES) and transcriptome sequencing (RNAseq) in patient care and clinical research, based on published reports as well as our experience with the MOSCATO-01 (MOlecular Screening for CAncer Treatment Optimization) molecular triage trial at Gustave Roussy Cancer Center. In this trial, all contributive samples of patients with advanced solid tumors were analyzed prospectively with targeted gene sequencing (TGS) and comparative genomic hybridization. In addition, 92 consecutive metastatic patients with contributive biopsies were sequenced for WES and RNAseq and compared with TGS and comparative genomic hybridization. Whole-exome sequencing allowed the reporting of additional variants in relevant genes in 38% of patients. Mutation detection sensitivity of WES was 95% compared with TGS. Additional information derived from WES and RNAseq could influence clinical decision, including fusion transcripts, expression levels, allele-specific expression, alternate transcripts, RNA-based pathogen diagnostic, tumor mutation load, mutational signatures, expression signatures, HLA genotyping, and neoepitope prediction. The current challenge is to be able to process the large-scale data from these comprehensive genome-wide technologies in an efficient way.

The role of human papillomavirus (HPV) in anal squamous cell carcinoma (ASCC) carcinogenesis has been clearly established, involving the expression of viral oncoproteins and optional viral DNA integration into the host genome. In this article, we describe the various mechanisms and sites of HPV DNA insertion and assess their prognostic and predictive value in a large series of patients with HPV-positive ASCC with long-term follow-up. We retrospectively analyzed 96 tumor samples from 93 HPV-positive ASCC patients using the Capture-HPV method followed by Next-Generation Sequencing, allowing determination of HPV genotype and identification of the mechanisms and sites of viral genome integration. We identified five different mechanistic signatures of HPV insertions. The distribution of HPV signatures differed from that previously described in HPV-positive cervical carcinoma (p &lt, 0.001). In ASCC samples, the HPV genome more frequently remained in episomal form (45.2%). The most common signature of HPV insertion was MJ-SC (26.9%), i.e., HPV&ndash, chromosomal junctions scattered at different loci. Functionally, HPV integration signatures were not associated with survival or response to treatment, but were associated with viral load (p = 0.022) and PIK3CA mutation (p = 0.0069). High viral load was associated with longer survival in both univariate (p = 0.044) and multivariate (p = 0.011) analyses. Finally, HPV integration occurred on most human chromosomes, but intragenic integration into the NFIX gene was recurrently observed (n = 4/51 tumors). Overall, the distribution of mechanistic signatures of HPV insertions in ASCC was different from that observed in cervical carcinoma and was associated with viral load and PIK3CA mutation. We confirmed recurrent targeting of NFIX by HPV integration, suggesting a role for this gene in ASCC carcinogenesis.

Background: Combination therapy with dabrafenib + trametinib demonstrated robust activity in patients (pts) with BRAFV600E-mutant advanced non-small cell lung cancer (NSCLC), but its resistance mechanisms are poorly known. Liquid biopsy components such as circulating tumor cells (CTCs) and cell-free (cf) tumor DNA can provide a comprehensive genomic picture of tumor content. Molecular profiling of single CTCs from pts with BRAF-V600Emutant NSCLC was performed to carry out a pilot study to identify resistance mutations at failure to dabrafenib + trametinib and to compare the mutations detected on CTCs to the mutations found on cfDNA and tumor biopsies. Patients and Methods: Eight pts with advanced BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib were prospectively enrolled between Jul 2018 and Mar 2019 at Gustave Roussy (IDRCB2008-A00585-50). Bloods samples were collected. Matched tissue-cfDNA and CTCs were available in 3 pts and matched tissue-CTCs for 4 pts. Single CTC isolation strategy included RosetteSep enrichment, immunofluorescent staining (Hoechst/CD45/cytokeratins) and fluorescence activated cell-sorting. The process to identify CTC mutations included Ampli1 whole-genome amplification, quality controls, multiplex targeted PCR with the Ampli1 CHPCustomBeta cancer panel developed by (Menarini Silicon Biosystems) and next-generation sequencing (NGS). The cfDNA was analyzed using InVisionFirst-Lung. Tissue samples were analyzed using targeted NGS in the MATCH-R trial (Recondo G; NPJ Precis Oncol 2020). Results: Single CTCs were isolated from 7 pts. As baseline characteristics, the median age was 66 years, 5 (71%) were smoker; all the pts with adenocarcinoma histology. Most of the pts received dabrafenib + trametinib as 2nd line (86%). The median of CTCs isolated by patient was 20 (8-28). A wide spectrum of mutations in CTCs was observed at treatment failure that were involved in the main cancer pathways, including MAPK (n=1; NRAS), tyrosine kinase receptors (n=5; EGFR, ALK, FLT3, HER2,…), signal transduction (n=4; IDH1, EZH2,⋯), and DNA repair (n=2; AKT1, ATM,⋯). In the same CTC, several mutations were observed in 5/7 patients, commonly involving more than one cancer pathways. A higher degree of mutational diversity was observed in CTCs compared to tumor tissue biopsies and cfDNA. In the 3 patients with an available tumor/liquid biopsy, only 1 shared mutations between CTCs and matched tumor and cfDNA. Conclusion: Single CTC profiling reveals a wide spectrum of therapeutic resistance mutations not detected by other analyses in pts with BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib. Integration of single CTC sequencing to tumor and cfDNA analysis, provides important perspectives to assess heterogeneous resistance mechanisms and to guide precision medicine in BRAFV600E- NSCLC. Citation Format: Laura Mezquita, Marianne Oulhen, Agathe Aberlenc, Marc Deloger, Aurélie Honoré, Marianna Garonzi, Genny Buson, Claudio Forcato, Yann Lecluse, Mihaela Aldea, Maud NgoCamus, Claudio Nicotra, Karen Howarth, Ludovic Lacroix, Luc Friboulet, Benjamin Besse, Nicolò Manaresi, David Planchard, Françoise Farace. Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 598.

Metastatic cancer refractory to treatment has a dismal prognosis. While genetic mechanisms of primary tumors and to a lesser extent of metastatic cancers have been studied on large cohorts, it is still not well understood if metastatic tumors refractory to systemic treatment contain specific signatures at genomic or transcriptional levels. Molecular characterization of metastatic cancers at terminal stage is indispensable for understanding the mechanisms of resistance to treatment and for prediction of tumor aggressiveness at earlier stages. Here, we present a large pan-cancer cohort of 914 tumors resistant to systemic therapies. Comprehensive clinical information is available for all the patients and includes tumor subtype, biopsy site, treatment, age, overall survival, etc. Biopsies of tumor metastases were taken after the diagnosed resistance. For this cohort we performed whole exome sequencing of a subset of 427 tumors with a median depth of 123X and matching blood, and transcriptome sequencing of all 914 tumors. We will discuss the variation in this cohort along 3 axes. Firstly we will describe the somatic mutations and indels by types of driver mutations, mutational burden, level of subclonality and mutational signatures. Additionally we will elaborate on the genomic instability and quantity of somatic copy number alterations (SCNAs). Secondly, we will present the annotation of germline DNA, frequency and classification of pathogenic variants predisposing to cancer. Germline variants are analyzed in the context of somatic genome focusing on the detection of the second hit by a mutation or a SCNA. Thirdly, we will present results of the analysis of differentially expressed genes and transcripts, activation of oncogenic and treatment resistance pathways, and fusion genes reported by multiple tools. The aforementioned genetic parameters are fed into a multi-omics model for identification of the key factors associated with the clinical features in the cohort. Furthermore this cohort is compared to primary tumors and to another cohort of metastatic tumors in order to unravel genetic markers of refractory disease. To this aim, we harmonized DNA- and RNA-derived variables from The Cancer Genome Atlas (TCGA) and MET500 (Robinson et al., Nature 2017). Such integrative analysis unravels genetic hallmarks of aggressive and treatment-resistant metastatic cancers highlighting the importance of multi-level tumor profiling to inform on therapeutic strategies. Citation Format: Julien Viot, Yoann Pradat, Ismael Padioleau, Andrey Yurchenko, Loic Verlingues, Rebecca Clodion, Stefan Michiels, Marc Deloger, Gerome Jules-Clément, Yohann Loriot, Benjamin Besse, Fabrice Andre, Paul-Henry Cournedes, Daniel Gautheret, Sergey Nikolaev. Integrative genomic analysis of refractory metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2230.

BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality worldwide and constitutes the second most common malignancy in women. CC exhibits differences in clinical behavior; infection by high-risk Human Papilloma Virus (HPV) remains an important initiating event in tumorigenesis and the most important risk factors for CC. As HPV integration types may be specific biomarkers for prediction of clinical outcomes, we analyzed the association between the different viral integration signatures and clinic-pathological parameters in CC patients. EXPERIMENTAL DESIGN: Patients included in this study were enrolled in the EU-funded RAIDs Network (Rational Molecular Assessment and Innovative Drug Selection, www.raids-fp7.eu) prospective CC BioRAIDs study [NCT02428842]. HPV double capture method followed by NGS was used to define the different integration signatures. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed in a large series of 272 CC BioRAIDs patients. RESULTS: The distribution of HPV signatures differed from that previously described in HPV-positive anal squamous cell carcinoma (p CONCLUSION: This is the first study assessing the prognostic value of HPV integration in a prospectively annotated CC patient cohort, which reports a hot-spot of HPV integrations at the MACROD2 gene, known to be implicated in impaired PARP1 activity and chromosome instability. Citation Format: Maud Kamal, Sonia Lameiras, Adeline Morel, Charlotte Lecerf, Sophie Vacher, Celia Dupain, Emmanuelle Jeannot, Marc Deloger, Nicolas Servant, Elodie Girard, Sylvain Baulande, Gemma Kenter, Ekaterina Jordanova, Els Berns, Roman Rouzier, Wulfran Cacheux, Christophe Le Tourneau, Alain Nicolas, Suzy Scholl, Ivan Bieche. HPV double capture NGS method in cervical cancer: Identification of MACROD2 gene as HPV hot spot integration site and viral load status as a prognostic factor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4330.

Amoebiasis is the third-most common cause of mortality worldwide from a parasitic disease. Although the primary etiological agent of amoebiasis is the obligate human parasite Entamoeba histolytica, other members of the genus Entamoeba can infect humans and may be pathogenic. Here, we present the first annotated reference genome for Entamoeba moshkovskii, a species that has been associated with human infections, and compare the genomes of E. moshkovskii, E. histolytica, the human commensal Entamoeba dispar, and the nonhuman pathogen Entamoeba invadens. Gene clustering and phylogenetic analyses show differences in expansion and contraction of families of proteins associated with host or bacterial interactions. They intimate the importance to parasitic Entamoeba species of surface-bound proteins involved in adhesion to extracellular membranes, such as the Gal/GalNAc lectin and members of the BspA and Ariel1 families. Furthermore, E. dispar is the only one of the four species to lack a functional copy of the key virulence factor cysteine protease CP-A5, whereas the gene’s presence in E. moshkovskii is consistent with the species’ potentially pathogenic nature. Entamoeba moshkovskii was found to be more diverse than E. histolytica across all sequence classes. The former is ∼200 times more diverse than latter, with the four E. moshkovskii strains tested having a most recent common ancestor nearly 500 times more ancient than the tested E. histolytica strains. A four-haplotype test indicates that these E. moshkovskii strains are not the same species and should be regarded as a species complex.

Background: Small cell ovarian carcinoma of the hypercalcemic type (SCCOHT) is an extremely rare and aggressive tumor that affects mainly young women (median age = 24 years). Prognosis is poor as most patients die within 2 years of diagnosis. Until recently, the literature describing the genomic profile of SCCOHT was scarce. In the last few months, four groups have now confirmed that SMARCA4 mutations are a frequent event in SCCOHT. Methods: We performed an integrated genomic analysis using WES, RNA-seq and CGH approaches to identify other recurrent genomic alteration and potential therapeutic targets. Candidate SNVs identified by WES were validated by Sanger and RNA-Seq. 8 frozen tumors were available, including 6 with matching normal control DNA. In addition 33 tumor samples were available as FFPE. Results: Despite their high grade and aggressive clinical course, SCCOHT tumors display genomic stability, low mutation load (0.53mut.Mb) and few SCNAs. However when present, genomic alterations were recurrent. Integrated WES and CGH analysis revealed a frequent and unusual 19p CN LOH (in 5 of 6 tumors). Although overall mutation rate was low, the mutations that did occur were novel and recurrent in 50%-80% of SCCOHT. In addition to SMARCA4 mutation (M+), we validated 10 novel recurrent M+ (including ABCA7, ANKRD24, CACTIN, EMR1, FBN3, FUT5, GRIN3B, KANK3, KRI1 and PLK5) in 50%-80% of tumors at high allelic frequencies (mean=0.87). Similarly, when present, amplifications were recurrent, common to a third of tumors: such as the mitochondrial redox enzymes (SHMT2, NDUFA4L2) or the transmembrane transporter LRP1 were amplified (Log2>2.3). No common fusion transcripts were identified. Since SCCOHT often display initial chemosensitivity, but rapid progressions, we investigated the differential genomic profiles of treatment naive versus chemotherapy treated tumors to uncover candidate resistance genes. The only alteration significantly enriched in post treatment tumors were M+ in the putative efflux pump, ABCA7. These M+ could not be identified even at low allele fractions in treatment naive tumors, but were identified in all 3 treated tumors with a mean allelic frequency of 0.83. In terms of therapeutic implications, modulating mitochondrial metabolism or targeting the membrane transporter LRP1 or the ABCA7 efflux pump could provide therapeutic venues in the future. However the most promising approaches for now may be targeting cell cycle checkpoint regulators or chromatin remodelling. M+ in the tumor suppressor PLK5 were confirmed in 79% of a cohort of 33 SCCOHT and many were predicted as damaging. Inactivation of PLK5 is associated with a loss of G2/M checkpoint regulation and cell harbouring PLK5 mutations were sensitive to PLK1 inhibitors. More interestingly, SMARCA4-M+ SCCOHT demonstrated complete loss of SMARCA4 expression as well as loss of the only other SWI/SNF catalytic subunit SMARCA2 suggesting that double SMARCA4/SMARCA2 negative SCCOHTs are characterized by a catalytically inactive SWI/SNF complex. This hypothesis was supported by changes in RNA expression levels in the main transcriptional targets for SWI/SNF (RHOA, RB and the E2F family). Importantly, the double SMARCA2/4 negative BIN-67 SCCOHT cell line displaying both a SMARCA4 M+ and loss of SMARCA2 expression was exquisitely sensitive to HDAC and MT inhibitors, while cell lines with either a SMARCB1-M+ or SMARCA4-M+ with retained SMARCA2 expression were not. These data make double SMARCA2/4 negative by IHC a potential predictive biomarker for histone modifying inhibitors in SCCOHT or potentially in other double SMARCA2/4 negative malignancies. Conclusions: We present the 1st integrated genomic analysis of SCCOHT. SCCOHT demonstrate a remarkably homogeneous genomic profile and potentially actionable alterations. In particular, dual null SMARCA2/SMARCA4 SCCOHTs could be selected for clinical trials with EZH2, HDAC or MT inhibitors. Citation Format: Aurélie Auguste, Marc Deloger, Joanna Cyrta, Audrey Le Formal, Catherine Richon, Nathalie Droin, Beatrice Brunn, Olivier Caron, Mojgan Devouassoux, Catherine Lhomme, Patricia Pautier, Catherine Genestie, Alexandra Leary. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) beyond SMARCA4 mutations: A comprehensive genomic analysis. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-27.

Capture of retroviral envelope genes is likely to have played a role in the emergence of placental mammals, with evidence for multiple, reiterated, and independent capture events occurring in mammals, and be responsible for the diversity of present day placental structures. Here, we uncover a full-length endogenous retrovirus envelope protein, dubbed HEMO [human endogenous MER34 (medium-reiteration-frequency-family-34) ORF], with unprecedented characteristics, because it is actively shed in the blood circulation in humans via specific cleavage of the precursor envelope protein upstream of the transmembrane domain. At variance with previously identified retroviral envelope genes, its encoding gene is found to be transcribed from a unique CpG-rich promoter not related to a retroviral LTR, with sites of expression including the placenta as well as other tissues and rather unexpectedly, stem cells as well as reprogrammed induced pluripotent stem cells (iPSCs), where the protein can also be detected. We provide evidence that the associated retroviral capture event most probably occurred >100 Mya before the split of Laurasiatheria and Euarchontoglires, with the identified retroviral envelope gene encoding a full-length protein in all simians under purifying selection and with similar shedding capacity. Finally, a comprehensive screen of the expression of the gene discloses high transcript levels in several tumor tissues, such as germ cell, breast, and ovarian tumors, with in the latter case, evidence for a histotype dependence and specific protein expression in clear-cell carcinoma. Altogether, the identified protein could constitute a "stemness marker" of the normal cell and a possible target for immunotherapeutic approaches in tumors.