Mutational landscape of inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most pro-metastatic form of BC. Better understanding of its enigmatic pathophysiology is crucial. We report here the largest whole-exome sequencing (WES) study of clinical IBC samples. METHODS: We retrospectively applied WES to 54 untreated IBC primary tumor samples and matched normal DNA. The comparator samples were 102 stage-matched non-IBC samples from TCGA. We compared the somatic mutational profiles, spectra and signatures, copy number alterations (CNAs), HRD and heterogeneity scores, and frequencies of actionable genomic alterations (AGAs) between IBCs and non-IBCs. The comparisons were adjusted for the molecular subtypes. RESULTS: The number of somatic mutations, TMB, and mutational spectra were not different between IBCs and non-IBCs, and no gene was differentially mutated or showed differential frequency of CNAs. Among the COSMIC signatures, only the age-related signature was more frequent in non-IBCs than in IBCs. We also identified in IBCs two new mutational signatures not associated with any environmental exposure, one of them having been previously related to HIF pathway activation. Overall, the HRD score was not different between both groups, but was higher in TN IBCs than TN non-IBCs. IBCs were less frequently classified as heterogeneous according to heterogeneity H-index than non-IBCs (21% vs 33%), and clonal mutations were more frequent and subclonal mutations less frequent in IBCs. More than 50% of patients with IBC harbored at least one high-level of evidence (LOE) AGA (OncoKB LOE 1-2, ESCAT LOE I-II), similarly to patients with non-IBC. CONCLUSIONS: We provide the largest mutational landscape of IBC. Only a few subtle differences were identified with non-IBCs. The most clinically relevant one was the higher HRD score in TN IBCs than in TN non-IBCs, whereas the most intriguing one was the smaller intratumor heterogeneity of IBCs.

Macrophages reprogramming driven by cancer-associated fibroblasts under FOLFIRINOX treatment correlates with shorter survival in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a clinically challenging cancer, mainly due to limited therapeutic options and the presence of a highly prominent tumor microenvironment (TME), facilitating tumor progression. The TME is predominated by heterogeneous populations of cancer-associated fibroblasts (CAFs) and tumor associated macrophages (TAMs), in constant communication with each other and with tumor cells, influencing many tumoral abilities such as therapeutic resistance. However how the crosstalk between CAFs and macrophages evolves following chemotherapeutic treatment remains poorly understood, limiting our capacity to halt therapeutic resistance. METHODS: We combined biological characterization of macrophages indirectly cocultured with human PDAC CAFs, under FOLFIRINOX treatment, with mRNAseq analyses of such macrophages and evaluated the relevance of the specific gene expression signature in a large series of primary PDAC patients to search for correlation with overall survival (OS) after FOLFIRINOX chemotherapy. RESULTS: Firstly, we demonstrated that CAFs polarize naïve and M1 macrophages towards an M2-like phenotype with a specific increase of CD200R and CD209 M2 markers. Then, we demonstrated that CAFs counteract the pro-inflammatory phenotype induced by the FOLFIRINOX on Macrophages. Indeed, we highlighted that, under FOLFIRINOX, CAFs limit the FOLFIRINOX-induced cell death of macrophages and further reinforce their M2 phenotype as well as their immunosuppressive impact through specific chemokines production. Finally, we revealed that under FOLFIRINOX CAFs drive a specific macrophage gene expression signature involving SELENOP and GOS2 that correlates with shortened OS in FOLFIRINOX-treated PDAC patients. CONCLUSION: Our study provides insight into the complex interactions between TME cells under FOLFIRINOX treatment. It suggests potential novel candidates that could be used as therapeutic targets in combination with FOLFIRINOX to prevent and alleviate TME influx on therapeutic resistance as well as biomarkers to predict FOLFIRINOX response in PDAC patients. Video Abstract.

The coenzyme A precursor pantethine enhances antitumor immunity in sarcoma.

The tumor microenvironment is a dynamic network of stromal, cancer, and immune cells that interact and compete for resources. We have previously identified the Vanin1 pathway as a tumor suppressor of sarcoma development via vitamin B5 and coenzyme A regeneration. Using an aggressive sarcoma cell line that lacks Vnn1 expression, we showed that the administration of pantethine, a vitamin B5 precursor, attenuates tumor growth in immunocompetent but not nude mice. Pantethine boosts antitumor immunity, including the polarization of myeloid and dendritic cells towards enhanced IFNγ-driven antigen presentation pathways and improved the development of hypermetabolic effector CD8+ T cells endowed with potential antitumor activity. At later stages of treatment, the effect of pantethine was limited by the development of immune cell exhaustion. Nevertheless, its activity was comparable with that of anti-PD1 treatment in sensitive tumors. In humans, VNN1 expression correlates with improved survival and immune cell infiltration in soft-tissue sarcomas, but not in osteosarcomas. Pantethine could be a potential therapeutic immunoadjuvant for the development of antitumor immunity.

Immunologic constant of rejection as a predictive biomarker of immune checkpoint inhibitors efficacy in non-small cell lung cancer.

BACKGROUND: Anti-PD1/PDL1 immune checkpoint inhibitors (ICI) transformed the prognosis of patients with advanced non-small cell lung cancer (NSCLC). However, the response rate remains disappointing and toxicity may be life-threatening, making urgent identification of biomarkers predictive for efficacy. Immunologic Constant of Rejection signature (ICR) is a 20-gene expression signature of cytotoxic immune response with prognostic value in some solid cancers. Our objective was to assess its predictive value for benefit from anti-PD1/PDL1 in patients with advanced NSCLC. METHODS: We retrospectively profiled 44 primary tumors derived from NSCLC patients treated with ICI as single-agent in at least the second-line metastatic setting. Transcriptomic analysis was performed using the nCounter® analysis system and the PanCancer Immune Profiling Panel. We then pooled our data with clinico-biological data from four public gene expression data sets, leading to a total of 162 NSCLC patients treated with single-agent anti-PD1/PDL1. ICR was applied to all samples and correlation was searched between ICR classes and the Durable Clinical Benefit (DCB), defined as stable disease or objective response according to RECIST 1.1 for a minimum of 6 months after the start of ICI. RESULTS: The DCB rate was 29%; 22% of samples were classified as ICR1, 30% ICR2, 22% ICR3, and 26% ICR4. These classes were not associated with the clinico-pathological variables, but showed enrichment from ICR1 to ICR4 in quantitative/qualitative markers of immune response. ICR2-4 class was associated with a 5.65-fold DCB rate when compared with ICR1 class. In multivariate analysis, ICR classification remained associated with DCB, independently from PDL1 expression and other predictive immune signatures. By contrast, it was not associated with disease-free survival in 556 NSCLC TCGA patients untreated with ICI. CONCLUSION: The 20-gene ICR signature was independently associated with benefit from anti-PD1/PDL1 ICI in patients with advanced NSCLC. Validation in larger retrospective and prospective series is warranted.

ZNF703 mRNA-Targeting Antisense Oligonucleotide Blocks Cell Proliferation and Induces Apoptosis in Breast Cancer Cell Lines.

The luminal B molecular subtype of breast cancers (BC) accounts for more than a third of BCs and is associated with aggressive clinical behavior and poor prognosis. The use of endocrine therapy in BC treatment has significantly contributed to the decrease in the number of deaths in recent years. However, most BC patients with prolonged exposure to estrogen receptor (ER) selective modulators such as tamoxifen develop resistance and become non-responsive over time. Recent studies have implicated overexpression of the ZNF703 gene in BC resistance to endocrine drugs, thereby highlighting ZNF703 inhibition as an attractive modality in BC treatment, especially luminal B BCs. However, there is no known inhibitor of ZNF703 due to its nuclear association and non-enzymatic activity. Here, we have developed an antisense oligonucleotide (ASO) against ZNF703 mRNA and shown that it downregulates ZNF703 protein expression. ZNF703 inhibition decreased cell proliferation and induced apoptosis. Combined with cisplatin, the anti-cancer effects of ZNF703-ASO9 were improved. Moreover, our work shows that ASO technology may be used to increase the number of targetable cancer genes.

Clinical, pathological, and comprehensive molecular analysis of the uterine clear cell carcinoma: a retrospective national study from TMRG and GINECO network.

BACKGROUND: Uterine clear cell carcinomas (CCC) represent less than 5% of uterine cancers. Their biological characteristics and clinical management remain uncertain. A multicenter study to explore both clinical and molecular features of these rare tumors was conducted. METHODS: This multicenter retrospective national study was performed within the French TMRG (Rare Gynecologic Malignant Tumors) network. Clinical data and, when available, FFPE blocks were collected. Clinical features, treatments, and outcome (progression-free survival (PFS) and overall survival (OS)) were analyzed and correlated to the protein (tissue micro-array), RNA (Nanostring nCounter® technology), and DNA (array-Comparative Genomic hybridization and target-next generation sequencing) levels using the tumor samples available. RESULTS: Sixty-eight patients with uterine CCC were enrolled, 61 from endometrial localization and 5 with cervix localization. Median age at diagnosis was 68.9 years old (range 19-89.7). Most tumors were diagnosed at an early stage (78% FIGO stage I-II). Hysterectomy (performed in 90%) and lymph node dissection (80%) were the most frequent surgical treatment. More than 70% of patients received external beam radiotherapy and 57% received brachytherapy. Nearly half (46%) of the patients received chemotherapy. After a median follow-up of 24.7 months, median PFS was 64.8 months (95 CI [5.3-124.4]) and median OS was 79.7 (IC95 [31.0-128.4]). Low hormone receptor expression (13% estrogen-receptor positive), frequent PI3K pathway alterations (58% PTEN loss, 50% PIK3CA mutations), and P53 abnormalities (41%) were observed. Mismatch repair deficiency was identified in 20%. P16 expression was associated with shorter PFS (HR = 5.88, 95 CI [1.56-25], p = 0.009). Transcriptomic analyzes revealed a specific transcriptomic profile notably with a high expression of immune response-associated genes in uterine CCC displaying a very good overall prognosis. CONCLUSIONS: Uterine CCC reported to be potentially MSI high, hormone receptors negative, and sometimes TP53 mutated. However, some patients with immune response-associated features and better prognosis may be candidate to treatment de-escalation and immunotherapy.

An OMA1 redox site controls mitochondrial homeostasis, sarcoma growth, and immunogenicity.

Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell-deficient, mice. These cells prime CD8+ lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity.

SK2 channels set a signalling hub bolstering CAF-triggered tumourigenic processes in pancreatic cancer.

OBJECTIVE: Intercellular communication within pancreatic ductal adenocarcinoma (PDAC) dramatically contributes to metastatic processes. The underlying mechanisms are poorly understood, resulting in a lack of targeted therapy to counteract stromal-induced cancer cell aggressiveness. Here, we investigated whether ion channels, which remain understudied in cancer biology, contribute to intercellular communication in PDAC. DESIGN: We evaluated the effects of conditioned media from patient-derived cancer-associated fibroblasts (CAFs) on electrical features of pancreatic cancer cells (PCC). The molecular mechanisms were deciphered using a combination of electrophysiology, bioinformatics, molecular and biochemistry techniques in cell lines and human samples. An orthotropic mouse model where CAF and PCC were co-injected was used to evaluate tumour growth and metastasis dissemination. Pharmacological studies were carried out in the Pdx1-Cre, Ink4afl/fl LSL-KrasG12D (KICpdx1) mouse model. RESULTS: We report that the K+ channel SK2 expressed in PCC is stimulated by CAF-secreted cues (8.84 vs 2.49 pA/pF) promoting the phosphorylation of the channel through an integrin-epidermal growth factor receptor (EGFR)-AKT (Protein kinase B) axis. SK2 stimulation sets a positive feedback on the signalling pathway, increasing invasiveness in vitro (threefold) and metastasis formation in vivo. The CAF-dependent formation of the signalling hub associating SK2 and AKT requires the sigma-1 receptor chaperone. The pharmacological targeting of Sig-1R abolished CAF-induced activation of SK2, reduced tumour progression and extended the overall survival in mice (11.7 weeks vs 9.5 weeks). CONCLUSION: We establish a new paradigm in which an ion channel shifts the activation level of a signalling pathway in response to stromal cues, opening a new therapeutic window targeting the formation of ion channel-dependent signalling hubs.

Prognostic and Predictive Value of LIV1 Expression in Early Breast Cancer and by Molecular Subtype.

BACKGROUND: LIV1 is a transmembrane protein that may become a new therapeutic target through the development of antibody-drug conjugates (ADCs). Few studies are available regarding the assessment of LIV1 expression in clinical breast cancer (BC) samples. METHODS: We analyzed LIV1 mRNA expression in 8982 primary BC. We searched for correlations between LIV1 expression and clinicopathological data, including disease-free survival (DFS), overall survival (OS), pathological complete response to chemotherapy (pCR), and potential vulnerability and actionability to anti-cancer drugs used or under development in BC. Analyses were performed in the whole population and each molecular subtype separately. RESULTS: LIV1 expression was associated with good-prognosis features and with longer DFS and OS in multivariate analysis. However, patients with high LIV1 expression displayed a lower pCR rate than patients with low expression after anthracycline-based neoadjuvant chemotherapy, including in multivariate analysis adjusted on grade and molecular subtypes. LIV1-high tumors were associated with higher probabilities of sensitivity to hormone therapy and CDK4/6 inhibitors and lower probabilities of sensitivity to immune-checkpoint inhibitors and PARP inhibitors. These observations were different according to the molecular subtypes when analyzed separately. CONCLUSIONS: These results may provide novel insights into the clinical development and use of LIV1-targeted ADCs by identifying prognostic and predictive value of LIV1 expression in each molecular subtype and associated vulnerability to other systemic therapies.

Mechanistic modeling of metastatic relapse in early breast cancer to investigate the biological impact of prognostic biomarkers.

BACKGROUND AND OBJECTIVE: Estimating the risk of metastatic relapse is a major challenge to decide adjuvant treatment options in early-stage breast cancer (eBC). To date, distant metastasis-free survival (DMFS) analysis mainly relies on classical, agnostic, statistical models (e.g., Cox regression). Instead, we propose here to derive mechanistic models of DMFS. METHODS: The present series consisted of eBC patients who did not receive adjuvant systemic therapy from three datasets, composed respectively of 692 (Bergonié Institute), 591 (Paoli-Calmettes Institute, IPC), and 163 (Public Hospital Marseille, AP-HM) patients with routine clinical annotations. The last dataset also contained expression of three non-routine biomarkers. Our mechanistic model of DMFS relies on two mathematical parameters that represent growth (α) and dissemination (µ). We identified their population distributions using mixed-effects modeling. Critically, we propose a novel variable selection procedure allowing to: (i) identify the association of biological parameters with either α, µ or both, and (ii) generate an optimal candidate model for DMFS prediction. RESULTS: We found that Ki67 and Thymidine Kinase-1 were associated with α, and nodal status and Plasminogen Activator Inhibitor-1 with µ. The predictive performances of the model were excellent in calibration but moderate in discrimination, with c-indices of 0.72 (95% CI [0.48, 0.95], AP-HM), 0.63 ([0.44, 0.83], Bergonié) and 0.60 (95% CI [0.54, 0.80], IPC). CONCLUSIONS: Overall, we demonstrate that our novel method combining mechanistic and advanced statistical modeling is able to unravel the biological roles of clinicopathological parameters from DMFS data.

PVRIG Expression Is an Independent Prognostic Factor and a New Potential Target for Immunotherapy in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a frequent and deadly cancer in need of new treatments. Immunotherapy has shown promising results in several solid tumors. The TIGIT/DNAM-1 axis gathers targets for new immune checkpoint inhibitors (ICIs). Here, we aimed at highlighting the potential of this axis as a new therapeutic option for HCC. For this, we built a large transcriptomic database of 683 HCC samples, clinically annotated, and 319 normal liver tissues. We interrogated this database for the transcriptomic expression of each member of the TIGIT/DNAM-1 axis and tested their prognostic value for survival. We then focused on the most discriminant one for these criteria, i.e., PVRIG, and analyzed the clinical characteristics, the disease-free and overall survivals, and biological pathways associated with PVRIG High tumors. Among all members of the TIGIT/DNAM-1 axis, PVRIG expression was higher in tumors than in normal liver, was heterogeneous across tumors, and was the only member with independent prognostic value for better survival. PVRIG High tumors were characterized by a higher lymphocytic infiltrate and enriched for signatures associated with tertiary lymphoid structures and better anti-tumor immune response. These results suggest that patients with PVRIG High tumors might be good candidates for immune therapy involving ICIs, notably ICIs targeting the TIGIT/DNAM-1 axis. Further functional and clinical validation is urgently required.

Axin1 Protects Colon Carcinogenesis by an Immune-Mediated Effect.

BACKGROUND & AIMS: Axin1 is a negative regulator of wingless-type MMTV integration site family, member 1 (Wnt)/ß-catenin signaling with tumor-suppressor function. The Wnt pathway has a critical role in the intestine, both during homeostasis and cancer, but the role of Axin1 remains elusive. METHODS: We assessed the role of Axin1 in normal intestinal homeostasis, with control, epithelial-specific, Axin1-knockout mice (Axin1ΔIEC) and Axin2-knockout mice. We evaluated the tumor-suppressor function of Axin1 during chemically induced colorectal tumorigenesis and dextran sulfate sodium-induced colitis, and performed comparative gene expression profiling by whole-genome RNA sequencing. The clinical relevance of the Axin1-dependent gene expression signature then was tested in a database of 2239 clinical colorectal cancer (CRC) samples. RESULTS: We found that Axin1 was dispensable for normal intestinal homeostasis and redundant with Axin2 for Wnt pathway down-regulation. Axin1 deficiency in intestinal epithelial cells rendered mice more susceptible to chemically induced colon carcinogenesis, but reduced dextran sulfate sodium-induced colitis by attenuating the induction of a proinflammatory program. RNA-seq analyses identified an interferon γ/T-helper1 immune program controlled by Axin1 that enhances the inflammatory response and protects against CRC. The Axin1-dependent gene expression signature was applied to human CRC samples and identified a group of patients with potential vulnerability to immune checkpoint blockade therapies. CONCLUSIONS: Our study establishes, in vivo, that Axin1 has redundant function with Axin2 for Wnt down-regulation and infers a new role for Axin1. Physiologically, Axin1 stimulates gut inflammation via an interferon γ/Th1 program that prevents tumor growth. Linked to its T-cell-mediated effect, the colonic Axin1 signature offers therapeutic perspectives for CRC.

DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer.

The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

EGFR is a master switch between immunosuppressive and immunoactive tumor microenvironment in inflammatory breast cancer.

Inflammatory breast cancer (IBC), the most aggressive breast cancer subtype, is driven by an immunosuppressive tumor microenvironment (TME). Current treatments for IBC have limited efficacy. In a clinical trial (NCT01036087), an anti-EGFR antibody combined with neoadjuvant chemotherapy produced the highest pathological complete response rate ever reported in patients with IBC having triple-negative receptor status. We determined the molecular and immunological mechanisms behind this superior clinical outcome. Using novel humanized IBC mouse models, we discovered that EGFR-targeted therapy remodels the IBC TME by increasing cytotoxic T cells and reducing immunosuppressive regulatory T cells and M2 macrophages. These changes were due to diminishing immunosuppressive chemokine expression regulated by transcription factor EGR1. We also showed that induction of an immunoactive IBC TME by an anti-EGFR antibody improved the antitumor efficacy of an anti-PD-L1 antibody. Our findings lay the foundation for clinical trials evaluating EGFR-targeted therapy combined with immune checkpoint inhibitors in patients with cancer.

CSPG4 expression in soft tissue sarcomas is associated with poor prognosis and low cytotoxic immune response.

BACKGROUND: Soft tissue sarcomas (STS) are heterogeneous and pro-metastatic tumors. Identification of accurate prognostic factors and novel therapeutic targets are crucial. CSPG4 is a cell surface proteoglycan with oncogenic functions. It recently emerged as a potential target for immunotherapy, including cell therapy based on CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in STS. However, expression of CSPG4 is poorly known in STS so far. METHODS: We analyzed CSPG4 gene expression in 1378 localized STS clinical samples, and searched for correlations with clinicopathological data, including disease-free survival (DFS), and with tumor immune features. RESULTS: CSPG4 expression was heterogeneous across samples. High expression was associated with younger patients' age, more frequent undifferentiated pleomorphic sarcoma and myxofibrosarcoma pathological subtypes, more frequent internal trunk tumor site, and more CINSARC high-risk samples. No correlation existed with pathological tumor size and grade, and tumor depth. Patients with high CSPG4 expression displayed 49% (95% CI 42-57) 5-year DFS versus 61% (95% CI 56-68) in patients with low expression (p = 3.17E-03), representing a 49% increased risk of event in the "CSPG4-high" group (HR = 1.49, 95% CI 1.14-1.94). This unfavorable prognostic value persisted in multivariate analysis, independently from other variables. There were significant differences in immune variables between "CSPG4-high" and "CSPG4-low" tumors. The "CSPG4-low" tumors displayed profiles suggesting higher anti-tumor cytotoxic immune response and higher potential vulnerability to immune checkpoint inhibitors (ICI). By contrast, the "CSPG4-high" tumors displayed profiles implying an immune-excluded tumor microenvironment, potentially induced by hypoxia, resulting from an immature chaotic microvasculature, and/or the presence of contractile myofibroblasts. CONCLUSIONS: Patients with "CSPG4-high" STS, theoretically candidate for CAR.CIKs, display shorter DFS and an immune environment unfavorable to vulnerability to CAR.CIKs, which could be improved by combining anti-angiogenic drugs able to normalize the tumor vasculature. By contrast, "CSPG4-low" STS are better candidates for immune therapy involving ICI.