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Abstract Background The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma. Methods 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting. Results The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan–Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints. Conclusions The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research.

The introduction of targeted therapies represented one of the most significant advances in the treatment of BRAFV600E melanoma. However, the onset of acquired resistance remains a challenge. Previously, we showed in mouse xenografts that vascular endothelial growth factor (VEGFA) removal enhanced the antitumor effect of BRAF inhibition through the recruitment of M1 macrophages. In this work, we explored the strategy of VEGFA/BRAF inhibition in immunocompetent melanoma murine models. In BRAF mutant D4M melanoma tumors, VEGFA/BRAF targeting reshaped the tumor microenvironment, largely by stimulating infiltration of M1 macrophages and CD8+ T cells, and sensitized tumors to immune checkpoint blockade (ICB). Furthermore, we reported that the association of VEGFA/BRAF targeting with anti‐PD‐1 antibody (triple therapy) resulted in a durable response and enabled complete tumor eradication in 50% of the mice, establishing immunological memory. Neutralization and CRISPR‐Cas‐mediated editing of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) abrogated antitumor response prompted by triple therapy and identified GM‐CSF as the cytokine instrumental in M1‐macrophage recruitment. Our data suggest that VEGFA/BRAF targeting in melanoma induces the activation of innate and adaptive immunity and prepares tumors for ICB. Our study contributes to understanding the tumor biology of BRAFV600E melanoma and suggests VEGFA as therapeutic target.

Abstract Background Transcriptional classification has been used to stratify colorectal cancer (CRC) into molecular subtypes with distinct biological and clinical features. However, it is not clear whether such subtypes represent discrete, mutually exclusive entities or molecular/phenotypic states with potential overlap. Therefore, we focused on the CRC Intrinsic Subtype (CRIS) classifier and evaluated whether assigning multiple CRIS subtypes to the same sample provides additional clinically and biologically relevant information. Methods A multi-label version of the CRIS classifier (multiCRIS) was applied to newly generated RNA-seq profiles from 606 CRC patient-derived xenografts (PDXs), together with human CRC bulk and single-cell RNA-seq datasets. Biological and clinical associations of single- and multi-label CRIS were compared. Finally, a machine learning-based multi-label CRIS predictor (ML2CRIS) was developed for single-sample classification. Results Surprisingly, about half of the CRC cases could be significantly assigned to more than one CRIS subtype. Single-cell RNA-seq analysis revealed that multiple CRIS membership can be a consequence of the concomitant presence of cells of different CRIS class or, less frequently, of cells with hybrid phenotype. Multi-label assignments were found to improve prediction of CRC prognosis and response to treatment. Finally, the ML2CRIS classifier was validated for retaining the same biological and clinical associations also in the context of single-sample classification. Conclusions These results show that CRIS subtypes retain their biological and clinical features even when concomitantly assigned to the same CRC sample. This approach could be potentially extended to other cancer types and classification systems.

Abstract Background Colorectal cancer (CRC) remains largely incurable when diagnosed at the metastatic stage. Despite some advances in precision medicine for this disease in recent years, new molecular targets, as well as prognostic/predictive markers, are highly needed. Neuroligin 1 (NLGN1) is a transmembrane protein that interacts at the synapse with the tumor suppressor adenomatous polyposis Coli (APC), which is heavily involved in the pathogenesis of CRC and is a key player in the WNT/β-catenin pathway. Methods After performing expression studies of NLGN1 on human CRC samples, in this paper we used in vitro and in vivo approaches to study CRC cells extravasation and metastasis formation capabilities. At the molecular level, the functional link between APC and NLGN1 in the cancer context was studied. Results Here we show that NLGN1 is expressed in human colorectal tumors, including clusters of aggressive migrating (budding) single tumor cells and vascular emboli. We found that NLGN1 promotes CRC cells crossing of an endothelial monolayer (i.e. Trans-Endothelial Migration or TEM) in vitro, as well as cell extravasation/lung invasion and differential organ metastatization in two mouse models. Mechanistically, NLGN1 promotes APC localization to the cell membrane and co-immunoprecipitates with some isoforms of this protein stimulates β-catenin translocation to the nucleus, upregulates mesenchymal markers and WNT target genes and induces an “EMT phenotype” in CRC cell lines Conclusions In conclusion, we have uncovered a novel modulator of CRC aggressiveness which impacts on a critical pathogenetic pathway of this disease, and may represent a novel therapeutic target, with the added benefit of carrying over substantial knowledge from the neurobiology field.

High-grade mucinous colorectal cancer (HGM CRC) is particularly aggressive, prone to metastasis and treatment resistance, frequently accompanied by “signet ring” cancer cells. A sizeable fraction of HGM CRCs (20-40%) arises in the context of the Lynch Syndrome, an autosomal hereditary syndrome that predisposes to microsatellite instable (MSI) CRC. Development of patient-derived preclinical models for this challenging subtype of colorectal cancer represents an unmet need in oncology. We describe here successful propagation of preclinical models from a case of early-onset, MSI-positive metastatic colorectal cancer in a male Lynch syndrome patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, surprisingly, also to immunotherapy. Surgical material from a debulking operation was implanted in NOD/SCID mice, successfully yielding one patient-derived xenograft (PDX). PDX explants were subsequently used to generate 2D and 3D cell cultures. Histologically, all models resembled the tumor of origin, displaying a high-grade mucinous phenotype with signet ring cells. For preclinical exploration of alternative treatments, in light of recent findings, we considered inhibition of the proteasome by bortezomib and of the related NEDD8 pathway by pevonedistat. Indeed, sensitivity to bortezomib was observed in mucinous adenocarcinoma of the lung, and we previously found that HGM CRC is preferentially sensitive to pevonedistat in models with low or absent expression of cadherin 17 (CDH17), a differentiation marker. We therefore performed IHC on the tumor and models, and observed no CDH17 expression, suggesting sensitivity to pevonedistat. Both bortezomib and pevonedistat showed strong activity on 2D cells at 72 hours and on 3D organoids at 7 days, thus providing valid options for in vivo testing. Accordingly, three PDX cohorts were treated for four weeks, respectively with vehicle, bortezomib and pevonedistat. Both drugs significantly reduced tumor growth, as compared to the vehicle group. Interestingly, while bortezomib was more effective in vitro, pevonedistat was more effective in vivo. Drug efficacy was further substantiated by a reduction of cellularity and of Ki67-positive cells in the treated tumors. These results highlight proteasome and NEDD8 inhibition as potentially effective therapeutic approaches against Lynch syndrome-associated HGM CRC, also when the disease is refractory to all available treatment options.

Metabolic reprogramming upon SIRT6 loss induces tumour formation in the intestine but the mechanism is unclear. Here, the authors show that loss of SIRT6 leads to the expansion of epithelial cells with high pyruvate dehydrogenase kinase activity resulting in enhanced stem cell activity and tumour-initiating potential

Abstract Background Patient-derived xenografts (PDX) mice models play an important role in preclinical trials and personalized medicine. Sharing data on the models is highly valuable for numerous reasons – ethical, economical, research cross validation etc. The EurOPDX Consortium was established 8 years ago to share such information and avoid duplicating efforts in developing new PDX mice models and unify approaches to support preclinical research. EurOPDX Data Portal is the unified data sharing platform adopted by the Consortium. Main body In this paper we describe the main features of the EurOPDX Data Portal ( https://dataportal.europdx.eu/ ), its architecture and possible utilization by researchers who look for PDX mice models for their research. The Portal offers a catalogue of European models accessible on a cooperative basis. The models are searchable by metadata, and a detailed view provides molecular profiles (gene expression, mutation, copy number alteration) and treatment studies. The Portal displays the data in multiple tools (PDX Finder, cBioPortal, and GenomeCruzer in future), which are populated from a common database displaying strictly mutually consistent views. (Short) Conclusion EurOPDX Data Portal is an entry point to the EurOPDX Research Infrastructure offering PDX mice models for collaborative research, (meta)data describing their features and deep molecular data analysis according to users’ interests.

Summary: Heme is an iron-containing porphyrin of vital importance for cell energetic metabolism. High rates of heme synthesis are commonly observed in proliferating cells. Moreover, the cell-surface heme exporter feline leukemia virus subgroup C receptor 1a (FLVCR1a) is overexpressed in several tumor types. However, the reasons why heme synthesis and export are enhanced in highly proliferating cells remain unknown. Here, we illustrate a functional axis between heme synthesis and heme export: heme efflux through the plasma membrane sustains heme synthesis, and implementation of the two processes down-modulates the tricarboxylic acid (TCA) cycle flux and oxidative phosphorylation. Conversely, inhibition of heme export reduces heme synthesis and promotes the TCA cycle fueling and flux as well as oxidative phosphorylation. These data indicate that the heme synthesis-export system modulates the TCA cycle and oxidative metabolism and provide a mechanistic basis for the observation that both processes are enhanced in cells with high-energy demand.

Abstract Background Neoantigens that arise as a consequence of tumor-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumor types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time. Methods We performed whole exome sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and in vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures, and predicted neoantigens. Results The majority of CRC models showed remarkably stable mutational and neoantigen profiles; however, those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly evolving CRC. Conclusions These results indicate that CRCs carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

Purpose Overexpression of miR-100 in stem cells derived from basal-like breast cancers causes loss of stemness, induction of luminal breast cancer markers and response to endocrine therapy. We, therefore, explored miR-100 as a novel biomarker in patients with luminal breast cancer.Methods miR-100 expression was studied in 90 patients with oestrogen-receptor-positive/human-epidermal growth factor receptor 2-negative breast cancer enrolled in a prospective study of endocrine therapy given either preoperatively, or for the treatment of de novo metastatic disease. Response was defined as a Ki67 ≤2.7% after 21±3 days of treatment. The prognostic role of miR-100 expression was evaluated in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) breast cancer datasets. Additionally, we explored the correlation between miR-100 and the expression its targets reported as being associated with endocrine resistance. Finally, we evaluated whether a signature based on miR-100 and its target genes could predict the luminal A molecular subtype.Results Baseline miR-100 was significantly anticorrelated with baseline and post-treatment Ki67 (p

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer.More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted.Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment.Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

Stromal cells contribute to the gene expression profiles based on which colorectal cancer (CRC) molecular subtypes are classified. Here, patient-derived xenografts enable the authors to obtain cancer cell-specific transcriptomes by excluding transcripts from murine stromal cells, based on which they define CRC intrinsic subtypes (CRIS) and evaluate their prognostic and predictive potential.

Abstract In colorectal cancer (CRC), WNT pathway activation by genetic rearrangements of RSPO3 is emerging as a promising target. However, its low prevalence severely limits availability of preclinical models for in‐depth characterization. Using a pipeline designed to suppress stroma‐derived signal, we find that RSPO3 “outlier” expression in CRC samples highlights translocation and fusion transcript expression. Outlier search in 151 CRC cell lines identified VACO6 and SNU1411 cells as carriers of, respectively, a canonical PTPRK(e1)‐RSPO3(e2) fusion and a novel PTPRK(e13)‐RSPO3(e2) fusion. Both lines displayed marked in vitro and in vivo sensitivity to WNT blockade by the porcupine inhibitor LGK974, associated with transcriptional and morphological evidence of WNT pathway suppression. Long‐term treatment of VACO6 cells with LGK974 led to the emergence of a resistant population carrying two frameshift deletions of the WNT pathway inhibitor AXIN1, with consequent protein loss. Suppression of AXIN1 in parental VACO6 cells by RNA interference conferred marked resistance to LGK974. These results provide the first mechanism of secondary resistance to WNT pathway inhibition.

Systemic anaplastic large cell lymphoma is a category of T-cell non-Hodgkin’s lymphoma which can be further subdivided into two distinct entities (ALK+ and ALK−) based on the presence or absence of ALK gene rearrangements. Among several pathways triggered by ALK signaling, constitutive activation of STAT3 is strictly required for ALK-mediated transformation and survival. Here we performed genome-wide microRNA profiling and identified 48 microRNA concordantly modulated by the inducible knock-down of ALK and STAT3. To evaluate the functional role of differentially expressed miRNA, we forced their expression in ALK+ anaplastic large cell lymphoma cells, and monitored their influence after STAT3 depletion. We found that the expression of the microRNA-17~92 cluster partially rescues STAT3 knock-down by sustaining proliferation and survival of ALK+ cells. Experiments in a xenograft mouse model indicated that forced expression of microRNA-17~92 interferes with STAT3 knock-down in vivo. High expression levels of the microRNA-17~92 cluster resulted in down-regulation of BIM and TGFβRII proteins, suggesting that their targeting might mediate resistance to STAT3 knock-down in anaplastic large cell lymphoma cells. We speculate that the microRNA-17~92 cluster is involved in lymphomagenesis of STAT3+ ALCL and that its inhibition might represent an alternative avenue to interfere with ALK signaling in anaplastic large cell lymphomas.

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.