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Abstract The quest for targeted therapies is critical in the battle against cancer. The RAS/MAP kinase pathway is frequently implicated in neoplasia, with ERK playing a crucial role as the most distal kinase in the RAS signaling cascade. Our previous research demonstrated that the interaction between ERK and MYD88, an adaptor protein in innate immunity, is crucial for RAS-dependent transformation and cancer cell survival. In this study, we examine the biological consequences of disrupting the ERK-MYD88 interaction through the ERK D-recruitment site (DRS), while preserving ERK’s kinase activity. Our results indicate that EI-52, a small-molecule benzimidazole targeting ERK-MYD88 interaction induces an HRI-mediated integrated stress response (ISR), resulting in immunogenic apoptosis specific to cancer cells. Additionally, EI-52 exhibits anti-tumor efficacy in patient-derived tumors and induces an anti-tumor T cell response in mice in vivo. These findings suggest that inhibiting the ERK-MYD88 interaction may be a promising therapeutic approach in cancer treatment.

Abstract Introduction The objective was to determine the added value of comprehensive molecular profile by whole‐exome and RNA sequencing (WES/RNA‐Seq) in advanced and refractory cancer patients who had no molecular‐based treatment recommendation (MBTR) based on a more limited targeted gene panel (TGP) plus array‐based comparative genomic hybridization (aCGH). Materials and Methods In this retrospective analysis, we selected 50 patients previously included in the PROFILER trial (NCT01774409) for which no MBT could be recommended 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). Results After exclusion of variants of unknown significance, a total of 167 somatic molecular alterations were identified in 50 patients (median: 3 [1–10]). Out of these 167 relevant molecular alterations, 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 two fusion transcripts in two patients. A MBTR was provided in 4/50 (8%) patients using the information from TGP/aCGH versus 9/50 (18%) patients using WES/RNA‐Seq findings. Three patients had similar recommendations based on TGP/aCGH and WES/RNA‐Seq. Conclusions In advanced and refractory cancer patients in whom no MBTR 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 MBTR.

Abstract Background TGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFβ but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFβ-induced senescence escape. Methods In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-β-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections. Results A screen with active kinases in HMECs upon TGFβ treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFβ-induced senescence. Interestingly, RSK3 expression decreased in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFβ-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFβ-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland. Conclusions We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFβ signaling.

IntroductionResidential exposure is estimated to be responsible for nearly 10% of lung cancers in 2015 in France, making it the second leading cause, after tobacco. The Auvergne-Rhône-Alpes region, in the southwest of France, is particularly affected by this exposure as 30% of the population lives in areas with medium or high radon potential. This study aimed to investigate the impact of radon exposure on the survival of lung cancer patients.MethodsIn this single-center study, patients with a histologically confirmed diagnosis of lung cancer, and newly managed, were prospectively included between 2014 and 2020. Univariate and multivariate survival analyses were carried out using a non-proportional risk survival model to consider variations in risk over time.ResultsA total of 1,477 patients were included in the analysis. In the multivariate analysis and after adjustment for covariates, radon exposure was not statistically associated with survival of bronchopulmonary cancers (HR = 0.82 [0.54–1.23], HR = 0.92 [0.72–1.18], HR = 0.95 [0.76–1.19] at 1, 3, and 5 years, respectively, for patients residing in category 2 municipalities; HR = 0.87 [0.66–1.16], HR = 0.92 [0.76–1.10], and HR = 0.89 [0.75–1.06] at 1, 3, and 5 years, respectively, for patients residing in category 3 municipalities).DiscussionAlthough radon exposure is known to increase the risk of lung cancer, in the present study, no significant association was found between radon exposure and survival of bronchopulmonary cancers.

A prevalence of around 26% of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has been previously reported. HPV induced oncogenesis mainly involving E6 and E7 viral oncoproteins. In some cases, HPV viral DNA has been detected to integrate with the host genome and possibly contributes to carcinogenesis by affecting the gene expression. We retrospectively assessed HPV integration sites and signatures in 80 HPV positive patients with HNSCC, by using a double capture‐HPV method followed by next‐generation Sequencing. We detected HPV16 in 90% of the analyzed cohort and confirmed five previously described mechanistic signatures of HPV integration [episomal (EPI), integrated in a truncated form revealing two HPV‐chromosomal junctions colinear (2J‐COL) or nonlinear (2J‐NL), multiple hybrid junctions clustering in a single chromosomal region (MJ‐CL) or scattered over different chromosomal regions (MJ‐SC) of the human genome]. Our results suggested that HPV remained episomal in 38.8% of the cases or was integrated/mixed in the remaining 61.2% of patients with HNSCC. We showed a lack of association of HPV genomic signatures to tumour and patient characteristics, as well as patient survival. Similar to other HPV associated cancers, low HPV copy number was associated with worse prognosis. We identified 267 HPV‐human junctions scattered on most chromosomes. Remarkably, we observed four recurrent integration regions: PDL1/PDL2/PLGRKT (8.2%), MYC/PVT1 (6.1%), MACROD2 (4.1%) and KLF5/KLF12 regions (4.1%). We detected the overexpression of PDL1 and MYC upon integration by gene expression analysis. In conclusion, we identified recurrent targeting of several cancer genes such as PDL1 and MYC upon HPV integration, suggesting a role of altered gene expression by HPV integration during HNSCC carcinogenesis.

Abstract Background The Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) to better define the cancer molecular milieu based on genomics/transcriptomics from tumor and analogous normal tissue biopsies. SPRING is the first trial to assess a SIMS‐based tri‐therapy regimen in advanced non‐small cell lung cancer (NSCLC). Methods Patients with advanced NSCLC (no EGFR, ALK, or ROS1 alterations; PD‐L1 unrestricted; ≤2 prior therapy lines) received avelumab, axitinib, and palbociclib (3 + 3 dose escalation design). Results Fifteen patients were treated (five centers, four countries): six at each of dose levels 1 (DL1) and DL2; three at DL3. The most common ≥Grade 3 adverse events were neutropenia, hypertension, and fatigue. The recommended Phase II dose (RP2D) was DL1: avelumab 10 mg/kg IV q2weeks, axitinib 3 mg po bid, and palbociclib 75 mg po daily (7 days off/21 days on). Four patients (27%) achieved a partial response (PR) (progression‐free survival [PFS]: 14, 24, 25 and 144+ weeks), including two after progression on pembrolizumab. Four patients attained stable disease (SD) that lasted ≥24 weeks: 24, 27, 29, and 64 weeks. At DL1 (RP2D), four of six patients (66%) achieved stable disease (SD) ≥6 months/PR (2 each). Responders included patients with no detectable PD‐L1 expression and low tumor mutational burden. Conclusions Overall, eight of 15 patients (53%) achieved clinical benefit (SD ≥ 24 weeks/PR) on the avelumab, axitinib, and palbociclib combination. This triplet showed antitumor activity in NSCLC, including in tumors post‐pembrolizumab progression, and was active at the RP2D, which was well tolerated. NCT03386929 clinicaltrial.gov

IntroductionIn this study, we aim to build radiomics and multiomics models based on transcriptomics and radiomics to predict the response from patients treated with the PD-L1 inhibitor.Materials and methodsOne hundred and ninety-five patients treated with PD-1/PD-L1 inhibitors were included. For all patients, 342 radiomic features were extracted from pretreatment computed tomography scans. The training set was built with 110 patients treated at the Léon Bérard Cancer Center. An independent validation cohort was built with the 85 patients treated in Dijon. The two sets were dichotomized into two classes, patients with disease control and those considered non-responders, in order to predict the disease control at 3 months. Various models were trained with different feature selection methods, and different classifiers were evaluated to build the models. In a second exploratory step, we used transcriptomics to enrich the database and develop a multiomic signature of response to immunotherapy in a 54-patient subgroup. Finally, we considered the HOT/COLD status. We first trained a radiomic model to predict the HOT/COLD status and then prototyped a hybrid model integrating radiomics and the HOT/COLD status to predict the response to immunotherapy.ResultsRadiomic signature for 3 months’ progression-free survival (PFS) classification: The most predictive model had an area under the receiver operating characteristic curve (AUROC) of 0.94 on the training set and 0.65 on the external validation set. This model was obtained with the t-test selection method and with a support vector machine (SVM) classifier. Multiomic signature for PFS classification: The most predictive model had an AUROC of 0.95 on the training set and 0.99 on the validation set. Radiomic model to predict the HOT/COLD status: the most predictive model had an AUROC of 0.93 on the training set and 0.86 on the validation set. HOT/COLD radiomic hybrid model for PFS classification: the most predictive model had an AUROC of 0.93 on the training set and 0.90 on the validation set.ConclusionIn conclusion, radiomics could be used to predict the response to immunotherapy in non-small-cell lung cancer patients. The use of transcriptomics or the HOT/COLD status, together with radiomics, may improve the working of the prediction models.

Identification of tumors harboring an overall active immune phenotype may help for selecting patients with advanced head and neck squamous cell carcinomas (HNSCC) and non-small cell lung cancer (NSCLC) who may benefit from immunotherapies. In this context, we generated targeted gene expression profiles in three and two independent cohorts of patients with HNSCC or NSCLC respectively, treated or not by PD-1/PD-L1 inhibitors. Notably, we generated two datasets including 102 and 82 patients with HNSCC or NSCLC treated with PD-1/PD-L1 inhibitors. Clinical information, including detailed survival raw data, is available for each patient, allowing to test association between gene expression data and patient survival (overall and progression-free survival). Moreover, we also generated gene expression datasets of 27 paired HNSCC samples from diagnostic biopsies and versus surgically resected specimens as well as 33 paired HNSCC samples at initial diagnosis (untreated) and at recurrence. Those datasets may allow to test the stability of a given biomarker across paired samples.

Abstract Mammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

Abstract The navigation cue netrin‐1 is well‐documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin‐1. Interestingly, the epitope recognized by NP137 in netrin‐1 shares 90% homology with its counterpart in netrin‐3, the closest member to netrin‐1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin‐3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin‐3 and netrin‐1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL‐1 or NeuroD1 transcription factors in SCLC. Netrin‐3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin‐3 for netrin‐1 receptors and we demonstrated that netrin‐3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin‐3 in NB and SCLC.

Introduction Patients with metastatic non-small cell lung cancer (mNSCLC) suffer from numerous symptoms linked to disease and treatment which may further impair the patient’s overall condition. In addition to its benefits on quality of life and fatigue, physical exercise may improve treatment response, notably due to its known effects on the immune system. The ERICA study is designed to assess the feasibility of a supervised acute physical exercise therapy realised immediately prior immune-chemotherapy infusion in patients with mNSCLC. Secondary objectives will examine the effects of acute exercise combined with an unsupervised home-walking programme on clinical, physical, psychosocial and biological parameters.Methods and analysis ERICA is a prospective, monocentric, randomised controlled, open-label feasibility study conducted at the Centre Léon Bérard Comprehensive Cancer Center (France). Thirty patients newly diagnosed with mNSCLC will be randomised (2:1 ratio) to the ‘exercise’ or the ‘control’ group. At baseline and during the last treatment cycle, participants in both groups will receive Physical Activity recommendations, and two nutritional assessments. In the exercise group, participants will receive a 3-month programme consisting of a supervised acute physical exercise session prior to immune-chemotherapy infusion, and an unsupervised home-based walking programme with an activity tracker. The acute exercise consists of 35 min interval training at submaximal intensity scheduled to terminate 15 min prior to infusion. Clinical, physical, biological and psychosocial parameters will be assessed at baseline, 3 and 6 months after inclusion. Biological measures will include immune, inflammatory, metabolic, oxidative stress biomarkers and molecular profiling.Ethics and dissemination The study protocol was approved by the French ethics committee (Comité de protection des personnes Ile de France II, N°ID-RCB 20.09.04.65226, 8 December 2020). The study is registered on ClinicalTrials.gov (NCT number:NCT04676009) and is at the pre-results stage. All participants will sign an informed consent form. The findings will be disseminated in peer-reviewed journals and academic conferences.

Background Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance.Methods Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8+ T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment.Results Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8+ T cells. Activation of CD8+ T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R+CD11c+ MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME.Conclusion Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor–host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most incident cancer worldwide. More than half of HNSCC patients experience locoregional or distant relapse to treatment despite aggressive multimodal therapeutic approaches that include surgical resection, radiation therapy, and adjuvant chemotherapy. Before the arrival of immunotherapy, systemic chemotherapy was previously employed as the standard first-line protocol with an association of cisplatin or carboplatin plus 5-fluorouracil plus cetuximab (anti-EFGR antibody). Unfortunately, acquisition of therapy resistance is common in patients with HNSCC and often results in local and distant failure. Despite our better understanding of HNSCC biology, no other molecular-targeted agent has been approved for HNSCC. In this review, we outline the mechanisms of resistance to the therapeutic strategies currently used in HNSCC, discuss combination treatment strategies to overcome them, and summarize the therapeutic regimens that are presently being evaluated in early- and late-phase clinical trials.

Keratin 8 (K8) expressed at the surface of cancer cells, referred as externalized K8 (eK8), has been observed in a variety of carcinoma cell lines. K8 has been previously reported to be expressed in poorly differentiated head and neck squamous cell carcinoma (HNSCC); however, its role during the invasive phase of upper aerodigestive tract tumorigenesis is unknown. Cohorts of HNSCC tumors for protein and mRNA expression and panel of cell lines were used for investigation. K8 was found to be externalized in a majority of HNSCC cell lines. Among the two main K8 protein isoforms only the 54 kDa was found to be present at the plasma membrane of HNSCC cells. The plasminogen-induced invasion of HNSCC cells was inhibited by the anti-eK8 D-A10 antagonist monoclonal antibody. Overexpression of K8 mRNA and protein were both correlated with tumor aggressive features and poor outcome. The effect of eK8 neutralization on invasion, its presence exclusively in cancer cells and the association of K8 expression with aggressive features and poor clinical outcome in HNSCC unravel eK8 as key player in invasion and a promising therapeutic target in HNSCC.

Understanding the dynamics of the immune microenvironment is critical to the development of immuno-based strategies for the prevention of oral potentially malignant disorders transformation to oral squamous cell carcinoma (OSCC). We used laser capture microdissection and RNA-sequencing to profile the expression of 13 matched pairs of epithelial versus stromal compartments from normal mucosa, hyperplasia, dysplasia, and invasive tumors in the 4-nitroquinolein (4-NQO) murine model of oral carcinogenesis. Genes differentially expressed at each step of transformation were defined. Immune cell deconvolution and enrichment scores of various biological processes including immune-related ones were computed. Immunohistochemistry was also performed to characterize the immune infiltrates by T-cells (T-cells CD3+, helper CD4+, cytotoxic CD8+, regulatory FoxP3+), B-cells (B220+), and macrophages (M1 iNOS+, M2 CD163+) at each histological step. Enrichment of three independent M2 macrophages signatures were computed in 86 oral leukoplakia with available clinical outcome. Most gene expression changes were observed in the stromal compartment and related to immune biological processes. Immune cell deconvolution identified infiltration by the macrophage population as the most important quantitatively especially at the stage of dysplasia. In 86 patients with oral leukoplakia, three M2 macrophages signatures were independently associated with improved oral cancer-free survival. This study provides a better understanding of the dynamics of the immune microenvironment during oral carcinogenesis and highlights an unexpected association of M2 macrophages gene expression signatures with oral cancer free survival in patients with oral leukoplakia.

Summary: Epigenetic deregulation of gene transcription is central to cancer cell plasticity and malignant progression but remains poorly understood. We found that the uncharacterized epigenetic factor chromodomain on Y-like 2 (CDYL2) is commonly over-expressed in breast cancer, and that high CDYL2 levels correlate with poor prognosis. Supporting a functional role for CDYL2 in malignancy, it positively regulated breast cancer cell migration, invasion, stem-like phenotypes, and epithelial-to-mesenchymal transition. CDYL2 regulation of these plasticity-associated processes depended on signaling via p65/NF-κB and STAT3. This, in turn, was downstream of CDYL2 regulation of MIR124 gene transcription. CDYL2 co-immunoprecipitated with G9a/EHMT2 and GLP/EHMT1 and regulated the chromatin enrichment of G9a and EZH2 at MIR124 genes. We propose that CDYL2 contributes to poor prognosis in breast cancer by recruiting G9a and EZH2 to epigenetically repress MIR124 genes, thereby promoting NF-κB and STAT3 signaling, as well as downstream cancer cell plasticity and malignant progression.

In some western countries, an increasing incidence of oral squamous cell carcinoma (OSCC) has been observed in non-smoker non-drinker patients (NSND), mostly in women with HPV-negative OSCC. In the context of the unknown etiology and mechanisms of tumorigenesis of OSCC in NSND, we discuss data supporting the hypothesis of a viral origin not related to HPV. OSCC from NSND are characterized by an antiviral DNA methylation and gene expression signature. Based on the similar increasing incidence of oral tongue SCC (OTSCC) and oropharyngeal SCC (OPSCC) in young women and men respectively, we hypothesize that changes in sexual behaviors may lead to an increasing incidence of herpesvirus in the oral cavity, especially HSV-2, similarly to what has already been described in HPV-positive OPSCC. Because viral genome integration has not been detected in OSCC from NSND, a “hit and run” viral mechanism involving epigenome deregulation could therefore play a key role at early steps of oral carcinogenesis in this population of patients. In conclusion, epidemiological, clinical and molecular data supports a “hit and run” viral origin of OSCC from NSND.

Circulating tumor DNA (ctDNA)-based molecular profiling is rapidly gaining traction in clinical practice of advanced cancer patients with multi-gene next-generation sequencing (NGS) panels. However, clinical outcomes remain poorly described and deserve further validation with personalized treatment of patients with genomic alterations detected in plasma ctDNA. Here, we describe the outcomes, disease control rate (DCR) at 3 months and progression-free survival (PFS) in oncogenic-addicted advanced NSCLC patients with actionable alterations identified in plasma by ctDNA liquid biopsy assay, InVisionFirst®-Lung. A pooled retrospective analysis was completed of 81 advanced NSCLC patients with all classes of alterations predicting response to current FDA approved drugs: sensitizing common EGFR mutations (78%, n = 63) with T790M (73%, 46/63), ALK / ROS1 gene fusions (17%, n = 14) and BRAF V600E mutations (5%, n = 4). Actionable driver alterations detected in liquid biopsy were confirmed by prior tissue genomic profiling in all patients, and all patients received personalized treatment. Of 82 patients treated with matched targeted therapies, 10% were at first-line, 41% at second-line, and 49% beyond second-line. Acquired T790M at TKI relapse was detected in 73% (46/63) of patients, and all prospective patients (34/46) initiated osimertinib treatment based on ctDNA results. The 3-month DCR was 86% in 81 evaluable patients. The median PFS was of 14.8 months (12.1-22.9m). Baseline ctDNA allelic fraction of genomic driver did not correlate with the response rate of personalized treatment (p = 0.29). ctDNA molecular profiling is an accurate and reliable tool for the detection of clinically relevant molecular alterations in advanced NSCLC patients. Clinical outcomes with targeted therapies endorse the use of liquid biopsy by amplicon-based NGS ctDNA analysis in first line and relapse testing for advanced NSCLC patients.

Abstract Background Radiotherapy for head and neck squamous cell carcinomas (HNSCC) is associated with a substantial morbidity and inconsistent efficacy. Human papillomavirus (HPV)-positive status is recognized as a marker of increased radiosensitivity. Our goal was to identify molecular markers associated with benefit to radiotherapy in patients with HPV-negative disease. Methods Gene expression profiles from public repositories were downloaded for data mining. Training sets included 421 HPV-negative HNSCC tumors from The Cancer Genome Atlas (TCGA) and 32 HNSCC cell lines with available radiosensitivity data (GSE79368). A radioresistance (RadR) score was computed using the single sample Gene Set Enrichment Analysis tool. The validation sets included two panels of cell lines (NCI-60 and GSE21644) and HPV-negative HNSCC tumor datasets, including 44 (GSE6631), 82 (GSE39366), and 179 (GSE65858) patients, respectively. We finally performed an integrated analysis of the RadR score with known recurrent genomic alterations in HNSCC, patterns of protein expression, biological hallmarks, and patterns of drug sensitivity using TCGA and the E-MTAB-3610 dataset (659 pancancer cell lines, 140 drugs). Results We identified 13 genes differentially expressed between tumor and normal head and neck mucosa that were associated with radioresistance in vitro and in patients. The 13-gene expression-based RadR score was associated with recurrence in patients treated with surgery and adjuvant radiotherapy but not with surgery alone. It was significantly different among different molecular subtypes of HPV-negative HNSCC and was significantly lower in the “atypical” molecular subtype. An integrated analysis of RadR score with genomic alterations, protein expression, biological hallmarks and patterns of drug sensitivity showed a significant association with CCND1 amplification, fibronectin expression, seven hallmarks (including epithelial-to-mesenchymal transition and unfolded protein response), and increased sensitivity to elesclomol, an HSP90 inhibitor. Conclusions Our study highlights the clinical relevance of the molecular classification of HNSCC and the RadR score to refine radiation strategies in HPV-negative disease.

Notch signalling is deregulated in several cancers; therefore, strategies targeting this pathway are currently being explored. Here the authors report a pro-apoptotic function of Notch3 in endothelial cells; consequently, when Notch3 is silenced in stroma cells, tumour growth and angiogenesis are increased.

Breast cancer is separated into multiple subtypes based on the expression of HER2 and hormone receptors. Here, the authors report the whole genome sequence of 64 HER2 positive tumours and show that these can be further separated into four groups with different gene expression profiles and genomic features.

Background: Oral premalignant lesions (OPLs) represent the most common oral precancerous conditions. One of the major challenges in this field is the identification of OPLs at higher risk for oral squamous cell cancer (OSCC) development, by discovering molecular pathways deregulated in the early steps of malignant transformation. Analysis of deregulated levels of single genes and pathways has been successfully applied to head and neck squamous cell cancers (HNSCC) and OSCC with prognostic/predictive implications. Exploiting the availability of gene expression profile and clinical follow-up information of a well-characterized cohort of OPL patients, we aim to dissect tissue OPL gene expression to identify molecular clusters/signatures associated with oral cancer free survival (OCFS). Materials and methods: The gene expression data of 86 OPL patients were challenged with: an HNSCC specific 6 molecular subtypes model (Immune related: HPV related, Defense Response and Immunoreactive; Mesenchymal, Hypoxia and Classical); one OSCC-specific signature (13 genes); two metabolism-related signatures (3 genes and signatures raised from 6 metabolic pathways associated with prognosis in HNSCC and OSCC, respectively); a hypoxia gene signature. The molecular stratification and high versus low expression of the signatures were correlated with OCFS by Kaplan–Meier analyses. The association of gene expression profiles among the tested biological models and clinical covariates was tested through variance partition analysis. Results: Patients with Mesenchymal, Hypoxia and Classical clusters showed an higher risk of malignant transformation in comparison with immune-related ones (log-rank test, p = 0.0052) and they expressed four enriched hallmarks: “TGF beta signaling” “angiogenesis”, “unfolded protein response”, “apical junction”. Overall, 54 cases entered in the immune related clusters, while the remaining 32 cases belonged to the other clusters. No other signatures showed association with OCFS. Our variance partition analysis proved that clinical and molecular features are able to explain only 21% of gene expression data variability, while the remaining 79% refers to residuals independent of known parameters. Conclusions: Applying the existing signatures derived from HNSCC to OPL, we identified only a protective effect for immune-related signatures. Other gene expression profiles derived from overt cancers were not able to identify the risk of malignant transformation, possibly because they are linked to later stages of cancer progression. The availability of a new well-characterized set of OPL patients and further research is needed to improve the identification of adequate prognosticators in OPLs.

The neurotrophin-3 (NT-3) receptor tropomyosin receptor kinase C (TrkC/NTRK3) has been described as a dependence receptor and, as such, triggers apoptosis in the absence of its ligand NT-3. This proapoptotic activity has been proposed to confer a tumor suppressor activity to this classic tyrosine kinase receptor (RTK). By investigating interacting partners that might facilitate TrkC-induced cell death, we have identified the basic helix-loop-helix (bHLH) transcription factor Hey1 and importin-α3 (karyopherin alpha 4 [KPNA4]) as direct interactors of TrkC intracellular domain, and we show that Hey1 is required for TrkC-induced apoptosis. We propose here that the cleaved proapoptotic portion of TrkC intracellular domain (called TrkC killer-fragment [TrkC-KF]) is translocated to the nucleus by importins and interacts there with Hey1. We also demonstrate that Hey1 and TrkC-KF transcriptionally silence mouse double minute 2 homolog (MDM2), thus contributing to p53 stabilization. p53 transcriptionally regulates the expression of TrkC-KF cytoplasmic and mitochondrial interactors cofactor of breast cancer 1 (COBRA1) and B cell lymphoma 2-associated X (BAX), which will subsequently trigger the intrinsic pathway of apoptosis. Of interest, TrkC was proposed to constrain tumor progression in neuroblastoma (NB), and we demonstrate in an avian model that TrkC tumor suppressor activity requires Hey1 and p53.

Nivolumab, an anti PD-1 checkpoint inhibitor has demonstrated efficacy in metastatic non-small-cell lung cancer (NSCLC) patients after failure to standard chemotherapy. Standard chemotherapy agents could promote antitumor immune response. We thus examined whether the response to first line chemotherapy could impact on nivolumab benefit. One hundred and 15 patients with NSCLC were included in this retrospective study from 4 different French centers. Forty-three squamous cell carcinomas (SCC), and 72 non-SCC received nivolumab between 2015 and 2016 (3 mg/kg IV Q2W). Response to first-line chemotherapy and to nivolumab was retrospectively assessed on CT-scan by central review. The association between RECIST response to first-line chemotherapy and nivolumab efficacy were determined using Fisher's exact test and Cox proportional hazard model. Respectively 46 (40%), 44 (38%) and 25 (22%) patients experienced partial response (PR), stable disease (SD), or progressive disease (PD) in response to first-line platinum- based chemotherapy. Twenty 5 (21%), 34 (30%), 56 (49%) respectively experienced PR, SD and PD in response to nivolumab. 60% (54/90) of patients who experienced clinical benefit (PR + SD) after first-line chemotherapy also had clinical benefit after nivolumab, while only 20% (5/25) of patients with initial PD subsequently experienced clinical benefit with nivolumab (Fisher's exact test, P = 0.001). The type of first-line doublet chemotherapy did not influence the response rate to nivolumab. Univariate and multivariate analyses showed that patients with clinical benefit from first-line chemotherapy had higher second-line PFS (P = 0.003) (median PFS on nivolumab of 5, 3.3 and 1.9 months for patients with PR, SD and PD in response to first-line therapy, respectively). Similar results were obtained for OS. Thus this study suggests that the efficacy of first-line chemotherapy may be a valuable surrogate marker of the benefit of nivolumab in terms of PFS and OS.

Acute myeloid leukemia (AML) is a heterogeneous disease. Prognosis is mainly influenced by patient age at diagnosis and cytogenetic alterations, two of the main factors currently used in AML patient risk stratification. However, additional criteria are required to improve the current risk classification and better adapt patient care. In neoplastic cells, ribosome biogenesis is increased to sustain the high proliferation rate and ribosome composition is altered to modulate specific gene expression driving tumorigenesis. Here, we investigated the usage of ribosome biogenesis factors as clinical markers in adult patients with AML. We showed that nucleoli, the nucleus compartments where ribosome production takes place, are modified in AML by analyzing a panel of AML and healthy donor cells using immunofluorescence staining. Using four AML series, including the TCGA dataset, altogether representing a total of about 270 samples, we showed that not all factors involved in ribosome biogenesis have clinical values although ribosome biogenesis is increased in AML. Interestingly, we identified the regulator of ribosome production nucleolin (NCL) as over-expressed in AML blasts. Moreover, we found in two series that high NCL mRNA expression level was associated with a poor overall survival, particular in elderly patients. Multivariate analyses taking into account age and cytogenetic risk indicated that NCL expression in blast cells is an independent marker of reduced survival. Our study identifies NCL as a potential novel prognostic factor in AML. Altogether, our results suggest that the ribosome biogenesis pathway may be of interest as clinical markers in AML.

The mammalian target of rapamycin (mTOR) signalling pathway is a central regulator of metabolism in all cells. It senses intracellular and extracellular signals and nutrient levels, and coordinates the metabolic requirements for cell growth, survival, and proliferation. Genetic alterations that deregulate mTOR signalling lead to metabolic reprogramming, resulting in the development of several cancers including those of the head and neck. Gain-of-function mutations in EGFR, PIK3CA, and HRAS, or loss-of-function in p53 and PTEN are often associated with mTOR hyperactivation, whereas mutations identified from The Cancer Genome Atlas (TCGA) dataset that potentially lead to aberrant mTOR signalling are found in the EIF4G1, PLD1, RAC1, and SZT2 genes. In this review, we discuss how these mutant genes could affect mTOR signalling and highlight their impact on metabolic processes, as well as suggest potential targets for therapeutic intervention, primarily in head and neck cancer.

Epigenetic silencing is a common mechanism to inactivate tumor suppressor genes during carcinogenesis. Enhancer of Zeste 2 (EZH2) is the histone methyltransferase subunit in polycomb repressive complex 2 which mediates transcriptional repression through histone methylation. EZH2 overexpression has been linked to aggressive phenotypes of certain cancers. However, the mechanism that EZH2 played in promoting malignancy in non-small cell lung cancer (NSCLC) remains unclear. In addition, the correlation of EZH2 overexpression and the prognosis of NSCLC patients in non-Asian cohort need to be determined.Up-regulation of EZH2 was found in NSCLC cells compared with normal human bronchial epithelial cells by western blot assay. Upon EZH2 knockdown using small interfering RNA (siRNA), the proliferation, anchorage-independent growth and invasion of NSCLC cells were remarkably suppressed with profound induction of G1 arrest. Furthermore, the expression of cyclin D1 was notably reduced whereas p15(INK4B), p21(Waf1/Cip1) and p27(Kip1) were increased in NSCLC cells after EZH2-siRNA delivery. To determine whether EZH2 expression contributes to disease progression in patients with NSCLC, Taqman quantitative real-time RT-PCR was used to measure the expression of EZH2 in paired tumor and normal samples. Univariate analysis revealed that patients with NSCLC whose tumors had a higher EZH2 expression had significantly inferior overall, disease-specific, and disease-free survivals compared to those whose tumors expressed lower EZH2 (P = 0.005, P = 0.001 and P = 0.003, respectively). In multivariate analysis, EZH2 expression was an independent predictor of disease-free survival (hazard ratio = 0.450, 95% CI: 0.270 to 0.750, P = 0.002).Our results demonstrate that EZH2 overexpression is critical for NSCLC progression. EZH2 mRNA levels may serve as a prognostic predictor for patients with NSCLC.