Tumor heterogeneity in Esophageal Adenocarcinoma: a genomic approach

INTRODUCTION: Esophageal adenocarcinoma (EAC) is a severe malignancy in terms of prognosis and mortality rate. Because its great genetic heterogeneity, disputes regarding classification, prevention and treatments are still unsolved. AIM: We investigated intra- and inter-EAC heterogeneity by defining EAC’s somatic mutational profile and the role of candidate microRNAs, to correlate the molecular profile of tumors to clinical outcomes and to identify biomarkers for classification. METHODS: 38 EAC cases were analyzed via high-throughput cell sorting technology combined with targeted sequencing and whole genome low-pass sequencing. Targeted sequencing of further 169 cases was performed to widen the study. miR221 and miR483-3p expression was profiled via qPCR in 112 EACs and correlation with clinical outcomes was investigated. RESULTS: 35/38 EACs carried at least one somatic mutation absent in stromal cells. TP53 was found mutated in 73.7% of cases. Selective sorting revealed tumor subclones with different mutational loads and copy number alterations, confirming the high intra-tumor heterogeneity of EAC. Mutations were in most cases at homozygous state, and we identified alterations that were missed with the whole-tumor analysis. Mutations in HNF1A gene, not previously associated with EAC, were identified in both cohorts. Higher expression of miR483-3p and miR221 was associated with poorer cancer specific survival (P=0.0293 and P=0.0059), and recurrence in the Lauren intestinal subtype (P=0.0459 and P=0.0002). Median expression levels of miRNAs were higher in patients with advanced tumor stages. The loss of SMAD4 immunoreactivity was significantly associated with poorer cancer specific survival and recurrence (P=0.0452; P=0.022 respectively). CONCLUSION: Combining selective sorting technology and next generation sequencing allowed to better define EAC inter- and intra-tumor heterogeneity. We identified HNF1A as a new mutated gene associated to EAC that could be involved in tumor progression and promising biomarkers such as SMAD4, miR221 and miR483-3p to identify patients at higher risk for more aggressive tumors.

Specificità, efficacia e risposte adattative mediate da mutazioni dell'oncosoppressore TP53 degli inibitori del Complesso I mitocondriale nella terapia oncologica personalizzata: dai meccanismi molecolari allo sviluppo di modelli preclinici avanzati

L'inibizione del complesso respiratorio I (CI) è una strategia antitumorale emergente, sebbene la specificità e l’efficacia di nuovi farmaci restino poco investigate. La generazione di modelli cellulari tumorali nulli per il CI rivela la specificità di EVP 4593 e BAY 872243 nell’indurre gli effetti antiproliferativi non associati all’apoptosi, selettivamente via CI, riducendo eventuali effetti collaterali. Studi preliminari in vivo evidenziano un rallentamento della crescita tumorale negli animali trattati con EVP 4593, il quale emerge come l’inibitore più potente. Per il suo ruolo nella riprogrammazione metabolica, e la sua elevata frequenza di mutazioni nelle neoplasie umane, sono stati investigati i potenziali meccanismi di adattamento alla terapia anti-CI sulla base dello stato mutazionale di TP53. L’auxotrofia da aspartato, un hallmark metabolico delle cellule tumorali con un danno al CI, causa un blocco della sintesi proteica mTORC1-dipendente nelle linee cellulari con una p53 mutata o nulla, inducendo un collasso metabolico. Viceversa, l'attivazione del sensore energetico AMPK promuove un recupero parziale della sintesi di aspartato in linee cellulari con la forma wild type di P53, che è in grado di sostenere una migliore anaplerosi attraverso SCO2, fattore di assemblaggio del complesso respiratorio IV. Al fine di traslare questi risultati in un modello preclinico, si è ottimizzato l’ottenimento di colture di tumori umani espiantati tramite il bioreattore U-CUP. Il modello scelto è stato quello di carcinoma sieroso ad alto grado dell’ovaio (HGSOC), a partire da tessuto congelato, per l’elevata frequenza di mutazioni driver in TP53. I tessuti congelati preservano l'eterogeneità delle componenti cellulari del tessuto di origine e sono caratterizzati da cellule in attiva proliferazione senza attivazione di apoptosi. Dati preliminari mostrano un trend di riduzione dell’area tumorale nei tessuti trattati con EVP 4593 e supportano l’utilizzo del modello preclinico nello studio di nuovi inibitori del CI sfruttando materiale primario di pazienti oncologici.

Identification of molecular biomarkers in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) is a severe cancer that has been on the rise in Western nations over the past few decades. It has a high mortality rate and the 5-year survival rate is only 35%–45%. EAC has been included in a group of tumors with one of the highest rates of copy number alterations (CNAs), somatic structural rearrangements, high mutation frequency, with different mutational signatures, and with epigenetic mechanisms. The vast heterogeneity of EAC mutations makes it challenging to comprehend the biology that underlies tumor onset and development, identify prognostic biomarkers, and define a molecular classification to stratify patients. The only way to resolve the current disagreements is through an exhaustive molecular analysis of EAC. We examined the genetic profile of 164 patients' esophageal adenocarcinoma samples (without chemo-radiotherapy). The included patients did not receive neoadjuvant therapies, which can change the genetic and molecular composition of the tumor. Using next-generation sequencing technologies (NGS) at high coverage, we examined a custom panel of 26 cancer-related genes. Over the entire cohort, 337 variants were found, with the TP53 gene showing the most frequent alteration (67.27%). Poorer cancer-specific survival was associated with missense mutations in the TP53 gene (Log Rank P=0.0197). We discovered HNF1alpha gene disruptive mutations in 7 cases that were also affected by other gene changes. We started to investigate its role in EAC cell lines by silencing HNF1alpha to mimic our EAC cohort and we use Seahorse technique to analyze its role in the metabolism in esophageal cell. No significant changes were found in transfected cell lines. We conclude by finding that a particular class of TP53 mutations (missense changes) adversely impacted cancer-specific survival in EAC. HNF1alpha, a new EAC-mutated gene, was found, but more research is required to fully understand its function as a tumor suppressor gene.

Understanding molecular mechanisms of resistance to immune checkpoint inhibitors in advanced non-small cell lung cancer (NSCLC)

Immune checkpoint inhibitors (ICI) that target PD-1/PD-L1 have recently emerged as an integral component of front-line treatment in metastatic NSCLC patients. The PD-1 inhibitor pembrolizumab is approved as monotherapy for advanced NSCLC with a PD-L1 tumor proportion score (TPS) of ≥1% and in combination with platinum doublet chemotherapy regardless of PD-L1 expression level. However, responses to either regimen occur in only a minority of cases, and PD-L1 TPS is limited as a biomarker in predicting whether a cancer will respond to PD-1 inhibition alone or would be more likely to benefit from PD-1 inhibition plus chemotherapy. Additional biomarkers of immunotherapy efficacy, such as tumor mutational burden (TMB), have not been incorporated into routine clinical practice for treatment selection. The identification of patients who have the greatest likelihood of responding to immunotherapies is critical for guiding treatment decisions. IN addition, early indicators of response could theoretically prevent patients from staying on an ineffective therapy where they might experience complications due to disease progression or develop toxicities from unnecessary exposure to an inactive agent. The aim of this research project is to investigate the clinicopathologic and molecular determinant of response/resistance to the currently available immune checkpoint inhibitors, in order to identify therapeutic vulnerabilities that can be exploited to improve the clinical outcomes of patients with advanced NSCLC.

Liquid biopsy for prostate cancer molecular characterization: homologous recombination system and genomic profile

Pathogenic aberrations in homologous recombination DNA repair (HRR) genes occur in approximately 1 to 4 men with advanced prostate cancer (PCa). Treatment with PARP inhibitors (PARPi) has recently been introduced for metastatic castration-resistant PCa patients, increasing clinicians' interest in the molecular characterization of all PCa patients. The limitations of using old, low-quality tumor tissue for genetic analysis, which is very common for PCa, can be overcome by using liquid biopsy as an alternative biomarker source. In this study, we aimed to evaluate the detection of molecular alterations in HRR genes on liquid biopsy compared with tumor tissue from PCa patients. Secondarily, we explored the genomic instability score (GIS), and a broader range of gene alterations for in-depth characterization of the PCa cohort. Plasma samples were collected from 63 patients with PCa. Sophia Homologous Recombination Solution (targeting 16 HRR genes) and shallow whole genome sequencing (sWGS) were used for genomic analysis of tissue DNA and circulating tumor DNA (ct). A total of 33 alterations (mainly on TP53, ATM, CHEK2, CDK12, and BRCA1/2) were identified in 28,5% of PCa plasma patients. By integrating the mutational and sWGS data, the HRR status of PCa patients was determined and a concordance agreement of 85,7% was identified with tumor tissue. A median GIS of 15 was obtained, reaching a score of 63 in 2 samples with double alterations, BRCA1 and TP53. We explored the PCa mutation landscape, and the most significant enriched pathways identified were the sphingosine 1-phosphate (S1P) receptor signaling and the PI3K-AKT-mTOR pathway. HRR analysis on FFPE and liquid biopsy samples show high concordance, demonstrating that the noninvasive ctDNA-enriched plasma can be an optimal alternative source for molecular SNV and CNV analysis. In addition, the evaluation of GIS and pathway interaction should be considered for more comprehensive molecular characterization in PCa patients.