Association of a gene-expression subtype to outcome and treatment response in patients with recurrent/metastatic head and neck squamous cell carcinoma treated with nivolumab.

BACKGROUND: Immune checkpoint inhibitors have been approved and currently used in the clinical management of recurrent and metastatic head and neck squamous cell carcinoma (R/M HNSCC) patients. The reported benefit in clinical trials is variable and heterogeneous. Our study aims at exploring and comparing the predictive role of gene-expression signatures with classical biomarkers for immunotherapy-treated R/M HNSCC patients in a multicentric phase IIIb trial. METHODS: Clinical data were prospectively collected in Nivactor tiral (single-arm, open-label, multicenter, phase IIIb clinical trial in platinum-refractory HNSCC treated with nivolumab). Findings were validated in an external independent cohort of immune-treated HNSCC patients, divided in long-term and short-term survivors (overall survival >18 and <6 months since the start of immunotherapy, respectively). Pretreatment tumor tissue specimen from immunotherapy-treated R/M HNSCC patients was used for PD-L1 (Tumor Proportion Score; Combined Positive Score (CPS)) and Tumor Mutational Burden (Oncopanel TSO500) evaluation and gene expression profiling; classical biomarkers and immune signatures (retrieved from literature) were challenged in the NIVACTOR dataset. RESULTS: Cluster-6 (Cl6) stratification of NIVACTOR cases in high score (n=16, 20%) and low score (n=64, 80%) demonstrated a statistically significant and clinically meaningful improvement in overall survival in the high-score cases (p=0.00028; HR=4.34, 95% CI 1.84 to 10.22) and discriminative ability reached area under the curve (AUC)=0.785 (95% CI 0.603 to 0.967). The association of high-score Cl6 with better outcome was also confirmed in: (1) NIVACTOR progression-free survival (p=4.93E-05; HR=3.71, 95% CI 1.92 to 7.18) and objective-response-rate (AUC=0.785; 95% CI 0.603 to 0.967); (2) long survivors versus short survivors (p=0.00544). In multivariate Cox regression analysis, Cl6 was independent from Eastern Cooperative Oncology Group performance status, PDL1-CPS, and primary tumor site. CONCLUSIONS: These data highlight the presence of underlying biological differences able to predict survival and response following treatment with immunotherapy in platinum-refractory R/M HNSCC that could have translational implications improving treatment selection. TRIAL REGISTRATION NUMBER: EudraCT Number: 2017-000562-30.

Tumor molecular landscape of Epstein-Barr virus (EBV) related nasopharyngeal carcinoma in EBV-endemic and non-endemic areas: Implications for improving treatment modalities.

Epstein-Barr virus (EBV) related- nasopharyngeal carcinoma (NPC) is a squamous carcinoma of the nasopharyngeal mucosal lining. Endemic areas (EA) are east and Southeast Asia, were NPC was recorded with higher incidence and longer estimated survival than in non-endemic area (NEA) such as Europe, We analyzed the gene expression and microenvironment properties of NPC in both areas to identify molecular subtypes and assess biological and clinical correlates that might explain the differences in incidence and outcome between EA- and NEA-NPCs. Six EA-NPC transcriptomic datasets, including tumor and normal samples, were integrated in a meta-analysis to identify molecular subtypes using a ConsensusClusterPlus bioinformatic approach. Based on the biological/functional characterization of four identified clusters were identified: Cl1, Immune-active; Cl2, defense-response; Cl3, proliferation; Cl4, perineural-interaction/EBV-exhaustion. Kaplan-Meier survival analysis, applied to the single dataset with available disease-free survival indicated Cl3 as the cluster with the worst prognosis (P = 0.0476), confirmed when applying four previously published prognostic signatures. A Cl3 classifier signature was generated and its prognostic performance was confirmed (P = 0.0368) on a validation dataset. Prediction of treatment response suggested better responses to: radiotherapy and immune checkpoint inhibitors immune-active and defense-response clusters; chemotherapy proliferation cluster; cisplatin perineural-interaction/EBV-exhaustion cluster. RNA sequencing for gene expression profiling was performed on 50 NEA-NPC Italian samples. In the NEA cohort, Cl1, Cl2 and Cl3 were represented, while perineural-interaction/EBV-exhaustion was almost absent. The immune/biological characterization and treatment-response prediction analyses of NEA-NPC partially replicated the EA-NPC results. Well characterized EA- and NEA-NPC retrospective and prospective cohorts are needed to validate the obtained results and can help designing future clinical studies.

Safety and anti-tumour activity of the IgE antibody MOv18 in patients with advanced solid tumours expressing folate receptor-alpha: a phase I trial.

All antibodies approved for cancer therapy are monoclonal IgGs but the biology of IgE, supported by comparative preclinical data, offers the potential for enhanced effector cell potency. Here we report a Phase I dose escalation trial (NCT02546921) with the primary objective of exploring the safety and tolerability of MOv18 IgE, a chimeric first-in-class IgE antibody, in patients with tumours expressing the relevant antigen, folate receptor-alpha. The trial incorporated skin prick and basophil activation tests (BAT) to select patients at lowest risk of allergic toxicity. Secondary objectives were exploration of anti-tumour activity, recommended Phase II dose, and pharmacokinetics. Dose escalation ranged from 70 µg-12 mg. The most common toxicity of MOv18 IgE is transient urticaria. A single patient experienced anaphylaxis, likely explained by detection of circulating basophils at baseline that could be activated by MOv18 IgE. The BAT assay was used to avoid enrolling further patients with reactive basophils. The safety profile is tolerable and maximum tolerated dose has not been reached, with evidence of anti-tumour activity observed in a patient with ovarian cancer. These results demonstrate the potential of IgE therapy for cancer.

Folate receptor alpha in ovarian cancer tissue and patient serum is associated with disease burden and treatment outcomes.

BACKGROUND: Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker. METHODS: We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay. RESULTS: Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses. CONCLUSIONS: sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments.

Dietary intervention for tertiary prevention in head and neck squamous cell carcinoma survivors: clinical and translational results of a randomized phase II trial.

Background: There is a strong need for preventive approaches to reduce the incidence of recurrence, second cancers, and late toxicities in head and neck squamous cell carcinoma (HNSCC) survivors. We conducted a randomized controlled trial (RCT) to assess a dietary intervention as a non-expensive and non-toxic method of tertiary prevention in HNSCC survivors. Methods: Eligible participants were disease-free patients with HNSCC in follow-up after curative treatments. Subjects were randomized 1:1 to receive a highly monitored dietary intervention plus the Word Cancer Research Fund/American Institute for Cancer Research recommendations for cancer prevention (intervention arm) or standard-of-care recommendations (control arm). The planned sample size for the event-free survival evaluation (primary endpoint) was not reached, and the protocol was amended in order to investigate the clinical (nutritional and quality-of-life questionnaires) and translational study [plasma-circulating food-related microRNAs (miRNAs)] as main endpoints, the results of which are reported herein. Results: One hundred patients were screened, 94 were randomized, and 89 were eligible for intention-to-treat analysis. Median event-free survival was not reached in both arms. After 18 months, nutritional questionnaires showed a significant increase in Recommended Food Score (p = 0.04) in the intervention arm vs. control arm. The frequency of patients with and without a clinically meaningful deterioration or improvement of the C30 global health status in the two study arms was similar. Food-derived circulating miRNAs were identified in plasma samples at baseline, with a significant difference among countries. Conclusion: This RCT represented the first proof-of-principle study, indicating the feasibility of a clinical study based on nutritional and lifestyle interventions in HNSCC survivors. Subjects receiving specific counseling increased the consumption of the recommended foods, but no relevant changes in quality of life were recorded between the two study arms. Food-derived plasma miRNA might be considered promising circulating dietary biomarkers.

An Inflammatory Signature to Predict the Clinical Benefit of First-Line Cetuximab Plus Platinum-Based Chemotherapy in Recurrent/Metastatic Head and Neck Cancer.

Epidermal growth factor receptor (EGFR) pathway has been shown to play a crucial role in several inflammatory conditions and host immune-inflammation status is related to tumor prognosis. This study aims to evaluate the prognostic significance of a four-gene inflammatory signature in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients treated with the EGFR inhibitor cetuximab plus chemotherapy. The inflammatory signature was assessed on 123 R/M HNSCC patients, enrolled in the multicenter trial B490 receiving first-line cetuximab plus platinum-based chemotherapy. The primary endpoint of the study was progression free survival (PFS), while secondary endpoints were overall survival (OS) and objective response rate (ORR). The patient population was subdivided into 3 groups according to the signature score groups. The four-genes-signature proved a significant prognostic value, resulting in a median PFS of 9.2 months in patients with high vs. 6.2 months for intermediate vs. 3.9 months for low values (p = 0.0016). The same findings were confirmed for OS, with median time of 18.4, 13.4, and 7.5 months for high, intermediate, and low values of the score, respectively (p = 0.0001). When ORR was considered, the signature was significantly higher in responders than in non-responders (p = 0.0092), reaching an area under the curve (AUC) of 0.65 (95% CI: 0.55-0.75). Our findings highlight the role of inflammation in the response to cetuximab and chemotherapy in R/M-HNSCC and may have translational implications for improving treatment selection.

Validity of Anti-PSMA ScFvD2B as a Theranostic Tool: A Narrative-Focused Review.

Prostate cancer (PCa) is the second leading cause of cancer among men, and its diagnosis and adequate staging are fundamental. Among the biomarkers identified in recent years for PCa management, prostate-specific-membrane-antigen (PSMA), physiologically expressed at a low level on healthy prostate and in other normal tissues and highly overexpressed in PCa, represents a reliable marker ideal for imaging and therapy. The development of anti-PSMA antibodies, such as D2B, demonstrated slow clearance of intact antibodies compared with fragments resulting in low tumor-to-blood ratios; however, the modular structural and functional nature of antibodies allowed the generation of smaller fragments, such as scFvs. In this review of the anti-PSMA antibody fragment scFvD2B, we combined further characterization of its biomolecular and tissue cross-reactivity characteristics with a comprehensive summary of what has already been performed in preclinical models to evaluate imaging and therapeutic activities. A molecular dynamics study was performed, and ScFvD2B occupied a limited conformational space, characterized by low-energy conformational basins, confirming the high stability of the protein structure. In the cross-reactivity study, the weak/absent immunoreactivity in non-tumor tissues was comparable to the PSMA expression reported in the literature. Biodistribution studies and therapeutic treatments were conducted in different animal models obtained by subcutaneous or locoregional injection of PSMA-positive-versus-negative xenografts. The maximum tumor uptake was observed for 123I(SPECT), 124I(PET), and optical imaging, which avoids kidney accumulation (compared with radiometals) and leads to an optimal tumor-to-kidney and tumor-to-background ratios. Regarding its possible use in therapy, experimental data suggested a strong and specific antitumor activity, in vitro and in vivo, obtained using CAR-T or NK-92/CAR cells expressing scFvD2B. Based on presented/reviewed data, we consider that scFvD2B, due to its versatility and robustness, seems to: (i) overcome some problems observed in other studied scFvs, very often relatively unstable and prone to form aggregates; (ii) have sufficient tumor-to-background ratios for targeting and imaging PSMA-expressing cancer; (iii) significantly redirect immune killing cells to PSMA-positive tumors when inserted in second-generation CAR-T or NK-92/CAR cells. These data suggest that our product can be considered the right reagent to fill the gap that still exists in PCa diagnosis and treatment.

Clinical Validity of a Prognostic Gene Expression Cluster-Based Model in Human Papillomavirus-Positive Oropharyngeal Carcinoma.

Under common therapeutic regimens, the prognosis of human papillomavirus (HPV)-positive squamous oropharyngeal carcinomas (OPCs) is more favorable than HPV-negative OPCs. However, the prognosis of some tumors is dismal, and validated prognostic factors are missing in clinical practice. The present work aimed to validate the prognostic significance of our published three-cluster model and to compare its prognostic value with those of the 8th edition of the tumor-node-metastasis staging system (TNM8) and published signatures and clustering models. METHODS: Patients with HPV DNA-positive OPCs with locoregionally advanced nonmetastatic disease treated with curative intent (BD2Decide observational study, NCT02832102) were considered as validation cohort. Patients were treated in seven European centers, with expertise in the multidisciplinary management of patients with head and neck cancer. The median follow-up was 46.2 months (95% CI, 41.2 to 50), and data collection was concluded in September 2019. The primary end point of this study was overall survival (OS). Three-clustering models and seven prognostic signatures were compared with our three-cluster model. RESULTS: The study population consisted of 235 patients. The three-cluster model confirmed its prognostic value. Two-year OS in each cluster was 100% in the low-risk cluster, 96.6% in the intermediate-risk cluster, and 86.3% in the high-risk cluster (P = .00074). For the high-risk cluster, we observed an area under the curve = 0.832 for 2-year OS, significantly outperforming TNM 8th edition (area under the curve = 0.596), and functional and biological differences were identified for each cluster. CONCLUSION: The rigorous clinical selection of the cases included in this study confirmed the robustness of our three-cluster model in HPV-positive OPCs. The prognostic value was found to be independent and superior compared with TNM8. The next step includes the translation of the three-cluster model in clinical practice. This could open the way to future exploration of already available therapies in HPV-positive OPCs tailoring de-escalation or intensification according to the three-cluster model.

Development of a multiomics database for personalized prognostic forecasting in head and neck cancer: The Big Data to Decide EU Project.

BACKGROUND: Despite advances in treatments, 30% to 50% of stage III-IV head and neck squamous cell carcinoma (HNSCC) patients relapse within 2 years after treatment. The Big Data to Decide (BD2Decide) project aimed to build a database for prognostic prediction modeling. METHODS: Stage III-IV HNSCC patients with locoregionally advanced HNSCC treated with curative intent (1537) were included. Whole transcriptomics and radiomics analyses were performed using pretreatment tumor samples and computed tomography/magnetic resonance imaging scans, respectively. RESULTS: The entire cohort was composed of 71% male (1097)and 29% female (440): oral cavity (429, 28%), oropharynx (624, 41%), larynx (314, 20%), and hypopharynx (170, 11%); median follow-up 50.5 months. Transcriptomics and imaging data were available for 1284 (83%) and 1239 (80%) cases, respectively; 1047 (68%) patients shared both. CONCLUSIONS: This annotated database represents the HNSCC largest available repository and will enable to develop/validate a decision support system integrating multiscale data to explore through classical and machine learning models their prognostic role.

Complete Response to Nivolumab in Recurrent/Metastatic HPV-Positive Head and Neck Squamous Cell Carcinoma Patient After Progressive Multifocal Leukoencephalopathy: A Case Report.

In an immune-competent context nivolumab showed long-term benefit in overall survival in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC); however, in special cancer population such as these patients with immunodeficiency and viral infections, data on checkpoint inhibitors (ICI) activity are scant. Herein, we report a patient with a Human papilloma virus (HPV)-related oropharyngeal cancer (OPC) and CD4 lymphocytopenia. After a first-line treatment complete remission, the patient experienced Human Polyomavirus (JCV) infection in the brain. Consequently, to the recovery from progressive multifocal leukoencephalopathy (PML) the patient metastasized and was enrolled in a single-arm trial with nivolumab (EudraCT number: 2017-000562-30). A complete and durable response (more 3 years) was observed after 10 nivolumab injections Q2wks, interrupted for persistent drug related G2 diarrhea and a syndrome of inappropriate antidiuretic hormone secretion. We describe the circulating immune profile (before-, during-, and after nivolumab), consistent with the clinical history. Moreover, during nivolumab treatment, brain MRI evidenced the presence of small punctuate areas of contrast enhancement, reflecting a mild immune response in perivascular spaces. By cytofluorimetry, we observed that during JCV infection the CD4/CD8 ratio of the patient was under the normal values. After JCV infection recovery and before nivolumab treatment, CD4/CD8 ratio reached the normality threshold, even if the CD4+ T cell count remained largely under the normal values. During ICI, gene expression xCell analyses of circulating immune cells of the patient, showed a progressive normalization of the total immune profile, with significant boost in CD4+ and CD8+ T cells and a reduction in NK T, comparable to the circulating immune profile of reference tumor-free HNSCC patients. The present case supports the activity of ICI in a population of special cancer patients; whether JCV and HPV infections (alone or together) might have a possible role as immune booster(s), require further investigations.

Ovarian Cancer Translational Activity of the Multicenter Italian Trial in Ovarian Cancer (MITO) Group: Lessons Learned in 10 Years of Experience.

Ovarian cancer is the most lethal gynecological cancer, and despite years of research, with the exception of a BRCA mutation driving the use of PARP inhibitors, no new prognostic/predictive biomarkers are clinically available. Improvement in biomarker selection and validation may derive from the systematic inclusion of translational analyses into the design of clinical trials. In the era of personalized medicine, the prospective centralized collection of high-quality biological material, expert pathological revision, and association to well-controlled clinical data are important or even essential added values to clinical trials. Here, we present the academic experience of the MITO (Multicenter Italian Trial in Ovarian Cancer) group, including gynecologists, pathologists, oncologists, biostatisticians, and translational researchers, whose effort is dedicated to the care and basic/translational research of gynecologic cancer. In our ten years of experience, we have been able to collect and process, for translational analyses, formalin-fixed, paraffin-embedded blocks from more than one thousand ovarian cancer patients. Standard operating procedures for collection, shipping, and processing were developed and made available to MITO researchers through the coordinating center's web-based platform. Clinical data were collected through dedicated electronic case report forms hosted in a web-based electronic platform and stored in a central database at the trial's coordinating center, which performed all the analyses related to the proposed translational researches. During this time, we improved our strategies of block management from retrospective to prospective collection, up to the design of a prospective collection with a quality check for sample eligibility before patients' accrual. The final aim of our work is to share our experience by suggesting a guideline for the process of centralized collection, revision processing, and storing of formalin-fixed, paraffin-embedded blocks for translational purposes.

Mining of Self-Organizing Map Gene-Expression Portraits Reveals Prognostic Stratification of HPV-Positive Head and Neck Squamous Cell Carcinoma.

Patients (pts) with head and neck squamous cell carcinoma (HNSCC) have different epidemiologic, clinical, and outcome behaviors in relation to human papillomavirus (HPV) infection status, with HPV-positive patients having a 70% reduction in their risk of death. Little is known about the molecular heterogeneity in HPV-related cases. In the present study, we aim to disclose the molecular subtypes with potential biological and clinical relevance. Through a literature review, 11 studies were retrieved with a total of 346 gene-expression data points from HPV-positive HNSCC pts. Meta-analysis and self-organizing map (SOM) approaches were used to disclose relevant meta-gene portraits. Unsupervised consensus clustering provided evidence of three biological subtypes in HPV-positive HNSCC: Cl1, immune-related; Cl2, epithelial-mesenchymal transition-related; Cl3, proliferation-related. This stratification has a prognostic relevance, with Cl1 having the best outcome, Cl2 the worst, and Cl3 an intermediate survival rate. Compared to recent literature, which identified immune and keratinocyte subtypes in HPV-related HNSCC, we confirmed the former and we separated the latter into two clusters with different biological and prognostic characteristics. At present, this paper reports the largest meta-analysis of HPV-positive HNSCC studies and offers a promising molecular subtype classification. Upon further validation, this stratification could improve patient selection and pave the way for the development of a precision medicine therapeutic approach.

A functional gene expression analysis in epithelial sinonasal cancer: Biology and clinical relevance behind three histological subtypes.

Epithelial sinonasal cancers (SNCs) are rare diseases with overlapping morphological features and a dismal prognosis. We aimed to investigate the expression differences among the histological subtypes for discerning their molecular characteristics. We selected 47 SNCs: (i) 21 nonkeratinizing squamous cell carcinomas (NKSCCs), (ii) 13 sinonasal neuroendocrine cancers (SNECs), and (iii) 13 sinonasal undifferentiated cancers (SNUCs). Gene expression profiling was performed by DASL (cDNA-mediated annealing, selection, extension, and ligation) microarray analysis with internal validation by quantitative RT-PCR (RT-qPCR). Relevant molecular patterns were uncovered by sparse partial-least squares discriminant analysis (sPLS-DA), microenvironment cell type (xCell), CIBERSORT, and gene set enrichment (GSEA) analyses. The first two sPLS-DA components stratified samples by histological subtypes. xCell highlighted increased expression of immune components (CD8+ effector memory cells, in SNUC) and "other cells": keratinocytes and neurons in NKSCC and SNEC, respectively. Pathway enrichment was observed in NKSCC (six gene sets, proliferation related), SNEC (one gene set, pancreatic ß-cells), and SNUC (twenty gene sets, some of them immune-system related). Major neuroendocrine involvement was observed in all the SNEC samples. Our high-throughput analysis revealed a good diagnostic ability to differentiate NKSCC, SNEC, and SNUC, but indicated that the neuroendocrine pathway, typical and pathognomonic of SNEC is also present at lower expression levels in the other two histological subtypes. The different and specific profiles may be exploited for elucidating their biology and could help to identify prognostic and therapeutic opportunities.

Integration of MRI and MRS approaches to monitor molecular imaging and metabolomic effects of trabectedin on a preclinical ovarian cancer model.

Although several drugs are available to treat recurrences of human epithelial ovarian cancer (EOC), clinical responses often remain short lived and lead to only marginal improvements in patients' survival. One of the new drugs proposed for recurrent platinum-resistant EOC patients is trabectedin (Trab), a marine-derived antitumor agent initially isolated from the tunicate Ecteinascidia turbinata and currently produced synthetically. Predictive biomarkers of therapy response to this drug and the potential use of non-invasive functional MRI and MRS approaches for an early assessment of Trab efficacy have not yet been evaluated, although they might be relevant for improving the clinical management of EOC patients. In the present work we combined functional and spectroscopic magnetic resonance technologies, such as in vivo diffusion-weighted MRI and 1 H MRS, with ex vivo high resolution MRS (HR-MRS) metabolomic analyses, with the aim of identifying new pharmacodynamic markers of Trab effectiveness on well characterized, highly aggressive human SKOV3.ip (a HER2-enriched cell variant derived from SKOV3 cells) EOC xenografts. In vivo treatment with Trab (three consecutive weekly 0.2 mg/kg i.v. injections) resulted in the following: (1) a significant reduction of in vivo tumor growth, along with the formation in cancer lesions of diffuse hyper-intense areas detected by T2 -weighted MRI and attributed to necrosis, in agreement with histopathology findings; (2) significant increases in the apparent diffusion coefficient mean and median values versus saline-treated control tumors; and (3) a significant reduction in the choline-containing metabolites' signal detected by quantitative in vivo MRS. Multivariate and quantitative HR-MRS analyses on ex vivo tissue samples revealed Trab-induced alterations in phospholipid and glucose metabolism identified as a decrease in phosphocholine and an increase in lactate. Collectively, these data identify Trab-induced functional MRI and MRS alterations in EOC models as a possible basis for further developments of these non-invasive imaging methods to improve the clinical management of EOC patients.

The genomics of desmoplastic small round cell tumor reveals the deregulation of genes related to DNA damage response, epithelial-mesenchymal transition, and immune response.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive, and poorly investigated simple sarcoma with a low frequency of genetic deregulation other than an Ewing sarcoma RNA binding protein 1 (EWSR1)-Wilm's tumor suppressor (WT1) translocation. We used whole-exome sequencing to interrogate six consecutive pre-treated DSRCTs whose gene expression was previously investigated. METHODS: DNA libraries were prepared from formalin-fixed, paraffin-embedded archival tissue specimens following the Agilent SureSelectXT2 target enrichment protocol and sequenced on Illumina NextSeq 500. Raw sequence data were aligned to the reference genome with Burrows-Wheeler Aligner algorithm. Somatic mutations and copy number alterations (CNAs) were identified using MuTect2 and EXCAVATOR2, respectively. Biological functions associated with altered genes were investigated through Ingenuity Pathway Analysis (IPA) software. RESULTS: A total of 137 unique somatic mutations were identified: 133 mutated genes were case-specific, and 2 were mutated in two cases but in different positions. Among the 135 mutated genes, 27% were related to two biological categories: DNA damage-response (DDR) network that was also identified through IPA and mesenchymal-epithelial reverse transition (MErT)/epithelial-mesenchymal transition (EMT) already demonstrated to be relevant in DSRCT. The mutated genes in the DDR network were involved in various steps of transcription and particularly affected pre-mRNA. Half of these genes encoded RNA-binding proteins or DNA/RNA-binding proteins, which were recently recognized as a new class of DDR players. CNAs in genes/gene families, involved in MErT/EMT and DDR, were recurrent across patients and mostly segregated in the MErT/EMT category. In addition, recurrent gains of regions in chromosome 1 involving many MErT/EMT gene families and loss of one arm or the entire chromosome 6 affecting relevant immune-regulatory genes were recorded. CONCLUSIONS: The emerging picture is an extreme inter-tumor heterogeneity, characterized by the concurrent deregulation of the DDR and MErT/EMT dynamic and plastic programs that could favour genomic instability and explain the refractory DSRCT profile.

Anti-Folate Receptor Alpha-Directed Antibody Therapies Restrict the Growth of Triple-negative Breast Cancer.

Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy.Experimental Design: We evaluated FRα expression in breast cancers by genomic (n = 3,414) and IHC (n = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro, and in human TNBC xenograft models.Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth.Conclusions: FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. Clin Cancer Res; 24(20); 5098-111. ©2018 AACR.

Simultaneous E-cadherin and PLEKHA7 expression negatively affects E-cadherin/EGFR mediated ovarian cancer cell growth.

BACKGROUND: The disruption of E-cadherin-mediated adhesion is considered an important driver of tumor progression. Nevertheless, numerous studies have demonstrated that E-cadherin promotes growth- or invasion-related signaling, contrary to the prevailing notion. During tumor progression, epithelial ovarian cancer (EOC) maintains E-cadherin expression and can positively affect EOC cell growth by contributing to PI3K/AKT activation. In polarized epithelia PLEKHA7, a regulator of the zonula adherens integrity, impinges E-cadherin functionality, but its role in EOCs has been never studied. METHODS: Ex-vivo EOC cells and cell lines were used to study E-cadherin contribution to growth and EGFR activation. The expression of the proteins involved was assessed by real time RT-PCR, immunohistochemistry and western blotting. Cells growth and drug susceptibility was monitored in different 3-dimensional (3D) systems. Recombinant lentivirus-mediated gene expression, western blotting, immunoprecipitation and confocal microscopy were applied to investigate the biological impact of PLEKHA7 on E-cadherin behaviour. The clinical impact of PLEKHA7 was determined in publicly available datasets. RESULTS: We show that E-cadherin expression contributes to growth of EOC cells and forms a complex with EGFR thus positively affecting ligand-dependent EGFR/CDK5 signaling. Accordingly, 3D cultures of E-cadherin-expressing EOC cells are sensitive to the CDK5 inhibitor roscovitine combined with cisplatin. We determined that PLEKHA7 overexpression reduces the formation of E-cadherin-EGFR complex, EGFR activation and cell tumorigenicity. Clinically, PLEKHA7 mRNA is statistically decreased in high grade EOCs respect to low malignant potential and low grade EOCs and correlates with better EOC patient outcome. CONCLUSIONS: These data represent a significant step towards untangling the role of E-cadherin in EOCs by assessing its positive effects on EGFR/CDK5 signaling and its contribution to cell growth. Hence, the inhibition of this signaling using a CDK5 inhibitor exerts a synergistic effect with cisplatin prompting on the design of new therapeutic strategies to inhibit growth of EOC cells. We assessed for the first time in EOC cells that PLEKHA7 induces changes in the asset of E-cadherin-containing cell-cell contacts thus inhibiting E-cadherin/EGFR crosstalk and leading to a less aggressive tumor phenotype. Accordingly, PLEKHA7 levels are lower in high grade EOC patient tumors and EOC patients with better outcomes display higher PLEKHA7 levels.

Design, selection and optimization of an anti-TRAIL-R2/anti-CD3 bispecific antibody able to educate T cells to recognize and destroy cancer cells.

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or TRAIL-receptor agonistic monoclonal antibodies promote apoptosis in most cancer cells, and the differential expression of TRAIL-R2 between tumor and normal tissues allows its exploitation as a tumor-associated antigen. The use of these antibodies as anticancer agents has been extensively studied, but the results of clinical trials were disappointing. The observed lack of anticancer activity could be attributed to intrinsic or acquired resistance of tumor cells to this type of treatment. A possible strategy to circumvent drug resistance would be to strike tumor cells with a second modality based on a different mechanism of action. We therefore set out to generate and optimize a bispecific antibody targeting TRAIL-R2 and CD3. After the construction of different bispecific antibodies in tandem-scFv or single-chain diabody formats to reduce possible immunogenicity, we selected a humanized bispecific antibody with very low aggregates and long-term high stability and functionality. This antibody triggered TRAIL-R2 in an agonistic manner and its anticancer activity proved dramatically potentiated by the redirection of cytotoxic T cells against both sensitive and resistant melanoma cells. The results of our study show that combining the TRAIL-based antitumor strategy with an immunotherapeutic approach in a single molecule could be an effective addition to the anticancer armamentarium.

Gene signatures and expression of miRNAs associated with efficacy of panitumumab in a head and neck cancer phase II trial.

OBJECTIVE: Platinum-based chemotherapy plus the anti-EGFR monoclonal antibody (mAb) cetuximab is used to treat recurrent/metastatic (RM) head-neck squamous cell carcinoma (HNSCC). Recently, we defined Cluster3 gene-expression signature as a potential predictor of favorable progression-free survival (PFS) in cetuximab-treated RM-HNSCC patients and predictor of partial metabolic FDG-PET response in an afatinib window-of-opportunity trial. Another anti-EGFR-mAb (panitumumab) was used as the treatment agent in RM-HNSCC patients in the phase II PANI01trial. PANI01 tumor samples were analyzed using functional genomics to explore response predictors to anti-EGFR therapy. MATERIALS AND METHODS: Whole-gene expression and real-time PCR analyses were applied to pre-treatment samples from 25 PANI01 patients. Three gene signatures (Cluster3 score, RAS onco-signature, microenvironment score) and seven selected miRNAs were separately analyzed for association with panitumumab efficacy. RESULTS: Cluster3 expression levels had a profile with a significant bimodal separation of samples (P =  3.08 E-13). Higher RAS activation, microenvironment score, and miRNA expression were associated with low-Cluster3 patients. The same biomarkers were separately associated with PFS. Patients with high-Cluster3 had significantly longer PFS than patients with low-Cluster3 (median PFS: 174 versus 51 days; log-rank P = 0.0021). ROC analysis demonstrated accuracy in predicting PFS (AUC = 0.877). CONCLUSIONS: Despite differences in clinical settings and anti-EGFR inhibitors used for treatment, response prediction by the Cluster3 signature and selected miRNAs was essentially the same. Translation into a useful clinical assay requires validation in a broader setting.

Are Fusion Transcripts in Relapsed/Metastatic Head and Neck Cancer Patients Predictive of Response to Anti-EGFR Therapies?

Prediction of benefit from combined chemotherapy and the antiepidermal growth factor receptor cetuximab is a not yet solved question in head and neck squamous cell carcinoma (HNSCC). In a selected series of 14 long progression-free survival (PFS) and 26 short PFS patients by whole gene and microRNA expression analysis, we developed a model potentially predictive of cetuximab sensitivity. To better decipher the "omics" profile of our patients, we detected transcript fusions by RNA-seq through a Pan-Cancer panel targeting 1385 cancer genes. Twenty-seven different fusion transcripts, involving mRNA and long noncoding RNA (lncRNA), were identified. The majority of fusions (81%) were intrachromosomal, and 24 patients (60%) harbor at least one of them. The presence/absence of fusions and the presence of more than one fusion were not related to outcome, while the lncRNA-containing fusions resulted enriched in long PFS patients (P = 0.0027). The CD274-PDCD1LG2 fusion was present in 7/14 short PFS patients harboring fusions and was absent in long PFS patients (P = 0.0188). Among the short PFS patients, those harboring this fusion had the worst outcome (P = 0.0172) and increased K-RAS activation (P = 0.00147). The associations between HNSCC patient's outcome following cetuximab treatment and lncRNA-containing fusions or the CD274-PDCD1LG2 fusion deserve validation in prospective clinical trials.