Immunotherapy and Cancer: The Multi-Omics Perspective.

Immunotherapies have revolutionized cancer treatment approaches. Because not all patients respond positively to immune therapeutic agents, it represents a challenge for scientists who strive to understand the mechanisms behind such resistance. In-depth exploration of tumor biology, using novel technologies such as omics science, can help decode the role of the tumor immune microenvironment (TIME) in producing a response to the immune blockade strategies. It can also help to identify biomarkers for patient stratification and personalized treatment. This review aims to explore these new models and highlight their possible pivotal role in changing clinical practice.

Mismatch Repair system protein deficiency as a resistance factor for locally advanced rectal adenocarcinoma patients receiving neoadjuvant chemo-radiotherapy.

BACKGROUND: Available data on Mismatch Repair system (MMR) deficiency are conflicting and derived from small studies. Our study aimed to evaluate the therapeutic implications of MMR status in patients with locally advanced rectal cancer (LARC). METHODS: We retrospectively collected data from 318 patients affected by LARC treated in Italy at the Medical Oncology Units of the University Hospital of Cagliari, Istituto Nazionale dei Tumori Milan, and AOU Ospedali Riuniti Ancona. All patients underwent neoadjuvant chemoradiotherapy. The primary objective was major TRG while secondary objectives were pathological complete response, disease-free survival (DFS) and overall survival (OS). RESULTS: One hundred sixty patients (148 pMMR and 12 dMMR) were included in the exploratory cohort and 158 (146 pMMR and 12 dMMR) were included in the validation cohort. A major TRG has been shown in 42.6% and 43.1% patients with pMMR in exploratory and validation cohort, respectively; while no major TRG have been shown in dMMR patients in both cohorts. Exploratory and validation cohorts showed a statistically significant higher mDFS in pMMR patients compared to dMMR: NR vs. 14 months and NR vs. 17 months, respectively. CONCLUSION: Our results indicated an association between dMMR and poor response to preoperative chemoradiotherapy and they represent a hypothesis-generating data for new neoadjuvant strategies.

HOXD8 hypermethylation as a fully sensitive and specific biomarker for biliary tract cancer detectable in tissue and bile samples.

BACKGROUND: Biliary tract cancers (BTC) are rare but highly aggressive tumours with poor prognosis, usually detected at advanced stages. Herein, we aimed at identifying BTC-specific DNA methylation alterations. METHODS: Study design included statistical power and sample size estimation. A genome-wide methylation study of an explorative cohort (50 BTC and ten matched non-tumoral tissue samples) has been performed. BTC-specific altered CpG islands were validated in over 180 samples (174 BTCs and 13 non-tumoral controls). The final biomarkers, selected by a machine-learning approach, were validated in independent tissue (18 BTCs, 14 matched non-tumoral samples) and bile (24 BTCs, five non-tumoral samples) replication series, using droplet digital PCR. RESULTS: We identified and successfully validated BTC-specific DNA methylation alterations in over 200 BTC samples. The two-biomarker panel, selected by an in-house algorithm, showed an AUC > 0.97. The best-performing biomarker (chr2:176993479-176995557), associated with HOXD8, a pivotal gene in cancer-related pathways, achieved 100% sensitivity and specificity in a new series of tissue and bile samples. CONCLUSIONS: We identified a novel fully efficient BTC biomarker, associated with HOXD8 gene, detectable both in tissue and bile by a standardised assay ready-to-use in clinical trials also including samples from non-invasive matrices.

BRCA-mutant pancreatic ductal adenocarcinoma.

Despite continued research, pancreatic ductal adenocarcinoma (PDAC) remains one of the main causes of cancer death. Interest is growing in the role of the tumour suppressors breast cancer 1 (BRCA1) and BRCA2-typically associated with breast and ovarian cancer-in the pathogenesis of PDAC. Indeed, both germline and sporadic mutations in BRCA1/2 have been found to play a role in the development of PDAC. However, data regarding BRCA1/2-mutant PDAC are lacking. In this review, we aim to outline the specific landscape of BRCA-mutant PDAC, focusing on heritability, clinical features, differences between BRCA1 and 2 mutations and between germline and sporadic alterations, as well as established therapeutic strategies and those that are still under evaluation.

Colorectal Cancer Early Detection in Stool Samples Tracing CpG Islands Methylation Alterations Affecting Gene Expression.

Colorectal cancer (CRC) is a major cause of cancer mortality. Early diagnosis is relevant for its prevention and treatment. Since DNA methylation alterations are early events in tumourigenesis and can be detected in cell-free DNA, they represent promising biomarkers for early CRC diagnosis through non-invasive methods. In our previous work, we identified 74 early altered CpG islands (CGIs) associated with genes involved in cell cross-talking and cell signalling pathways. The aim of this work was to test whether methylation-based biomarkers could be detected in non-invasive matrices. Our results confirmed methylation alterations of GRIA4 and VIPR2 in CRC tissues, using MethyLight, as well as in stool samples, using a much more sensitive technique as droplet digital PCR. Furthermore, we analysed expression levels of selected genes whose promoter CGIs were hypermethylated in CRC, detecting downregulation at mRNA and protein levels in CRC tissue for GRIA4, VIPR2, SPOCK1 and SLC6A3. Most of these genes were already lowly expressed in colon normal tissues supporting the idea that cancer DNA methylation targets genes already barely expressed in the matched normal tissues. Our study suggests GRIA4 and VIPR2 as biomarkers for early CRC diagnosis using stool samples and confirms downregulation of genes hypermethylated in CRC.

The role of primary tumour sidedness, EGFR gene copy number and EGFR promoter methylation in RAS/BRAF wild-type colorectal cancer patients receiving irinotecan/cetuximab.

BACKGROUND: The data from randomised trials suggested that primary tumour sidedness could represent a prognostic and predictive factor in colorectal cancer (CRC) patients, particularly during treatment with anti-epidermal growth factor receptor (EGFR) therapy. However, an in-deep molecular selection might overcome the predictive role of primary tumour location in this setting. METHODS: We conducted a retrospective analysis in which tumour samples from RAS/BRAF wild-type (WT) metastatic CRC patients treated with second-third-line irinotecan/cetuximab were analysed for EGFR gene copy number (GCN) and promoter methylation. Study objective was to evaluate the correlation of tumour sidedness, EGFR promoter methylation and EGFR GCN with clinical outcome. Median follow-up duration was 14.3 months. RESULTS: Eighty-eight patients were included in the study, 27.3% had right-sided CRC, 72.7% had left-sided CRC; 36.4% had EGFR GCN<2.12 tumour, 63.6% had EGFR GCN⩾2.12 tumour; 50% had EGFR promoter-methylated tumour. Right-sided colorectal cancer (RSCRC) were associated with reduced overall response rate (ORR) (4.2% for RSCRC vs 35.9% for left sided colorectal cancer (LSCRC), P=0.0030), shorter progression-free survival (PFS) (3.0 vs 6.75 months, P<0.0001) and shorter overall survival (OS) (8 vs 13.6 months, P<0.0001). EGFR GCN<2.12 tumours were associated with reduced ORR (6.2% for EGFR GCN<2.12 vs 39.3% for EGFR GCN⩾2.12 tumours, P=0.0009), shorter PFS (3.5 vs 6.5 months, P=0.0006) and shorter OS (8.5 vs 14.0 months, P<0.0001). Epidermal growth factor receptor-methylated tumours were associated with reduced ORR (9.1% for methylated vs 45.5% for unmethylated, P=0.0001), shorter PFS (3 vs 7.67 months, P<0.0001) and shorter OS (8 vs 17 months, P<0.0001). At multivariate analysis, EGFR GCN and EGFR promoter methylation maintained their independent role for ORR (respectively P=0.0082 and 0.0025), PFS (respectively P=0.0048 and<0.0001) and OS (respectively P=0.0001 and<0.0001). CONCLUSIONS: In our study, an accurate molecular selection based on an all RAS and BRAF analysis along with EGFR GCN and EGFR promoter methylation status seems to be more relevant than primary tumour sidedness in the prediction of clinical outcome during cetuximab/irinotecan therapy. However, these data need to be validated with future prospective and translational studies.

CD44: A New Prognostic Marker in Colorectal Cancer?

Cluster of differentiation 44 (CD44) is a non-kinase cell surface glycoprotein. It is overexpressed in several cell types, including cancer stem cells (CSCs). Cells overexpressing CD44 exhibit several CSC traits, such as self-renewal, epithelial-mesenchymal transition (EMT) capability, and resistance to chemo- and radiotherapy. The role of CD44 in maintaining stemness and the CSC function in tumor progression is accomplished by binding to its main ligand, hyaluronan (HA). The HA-CD44 complex activates several signaling pathways that lead to cell proliferation, adhesion, migration, and invasion. The CD44 gene regularly undergoes alternative splicing, resulting in the standard (CD44s) and variant (CD44v) isoforms. The different functional roles of CD44s and specific CD44v isoforms still need to be fully understood. The clinicopathological impact of CD44 and its isoforms in promoting tumorigenesis suggests that CD44 could be a molecular target for cancer therapy. Furthermore, the recent association observed between CD44 and KRAS-dependent carcinomas and the potential correlations between CD44 and tumor mutational burden (TMB) and microsatellite instability (MSI) open new research scenarios for developing new strategies in cancer treatment. This review summarises current research regarding the different CD44 isoform structures, their roles, and functions in supporting tumorigenesis and discusses its therapeutic implications.

Prediction of Response to Anti-Angiogenic Treatment for Advanced Colorectal Cancer Patients: From Biological Factors to Functional Imaging.

Colorectal cancer (CRC) is a leading tumor worldwide. In CRC, the angiogenic pathway plays a crucial role in cancer development and the process of metastasis. Thus, anti-angiogenic drugs represent a milestone for metastatic CRC (mCRC) treatment and lead to significant improvement of clinical outcomes. Nevertheless, not all patients respond to treatment and some develop resistance. Therefore, the identification of predictive factors able to predict response to angiogenesis pathway blockade is required in order to identify the best candidates to receive these agents. Unfortunately, no predictive biomarkers have been prospectively validated to date. Over the years, research has focused on biologic factors such as genetic polymorphisms, circulating biomarkers, circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and microRNA. Moreover, research efforts have evaluated the potential correlation of molecular biomarkers with imaging techniques used for tumor assessment as well as the application of imaging tools in clinical practice. In addition to functional imaging, radiomics, a relatively newer technique, shows real promise in the setting of correlating molecular medicine to radiological phenotypes.

Influence of antidiabetic drugs on glucose metabolism and immune response in patients with metastatic pancreatic ductal adenocarcinoma receiving gemcitabine plus nab-paclitaxel as first-line treatment.

BACKGROUND: Association between pancreatic ductal adenocarcinoma (PDAC) and type 2 diabetes mellitus (DM2) has long been evaluated. Indeed, DM2 can be both an epiphenomenon of PDAC and a risk factor. The present study aimed to investigate the correlation between overall survival (OS) and antidiabetic drugs in patients with metastatic pancreatic ductal adenocarcinoma and DM2. METHOD: Data from 232 patients were collected retrospectively from 2014 to 2021. 174 from AOU Cagliari Medical Oncology and 58 from AOU Ancona Medical Oncology. All patients received gemcitabine plus nab-paclitaxel first-line chemotherapy. We aimed to evaluate the correlation between DM2, anti-diabetic medications and overall survival. Survival distribution was assessed by Kaplan-Meier curves. RESULTS: Median age was 68±9, 127 (55%) were male. 138/232 (59%) patients were not affected by DM2, 94/232 (41%) were affected by DM2. 57 were insulin-treated and 37 were metformin-treated. DM2 treated patients showed an higher median overall survival (26 vs 12 months, p = 0,0002). Among DM2 patients insulin-treated and metformin-treated showed an mOS of 21 months and 33 months, respectively. CONCLUSIONS: Results showed a correlation between treated DM2 and higher mOS in patients with mPDAC. Limitations due to retrospective data collection must be considered. Further studies in this setting are needed.

New Horizons in Metastatic Colorectal Cancer: Prognostic Role of CD44 Expression.

BACKGROUND: The transmembrane glycoprotein CD44, the major hyaluronan (HA) receptor, has been proven to regulate cell growth, survival, differentiation, and migration. It is therefore widely considered to be involved in carcinogenesis. Its role as a new therapeutic target in solid tumors is under evaluation in clinical trials. The prognostic value remains controversial. Here, we aimed to investigate the correlation between CD44 expression and the clinicopathological features and survival in metastatic colorectal cancer (mCRC) patients. METHODS: Data from 65 mCRC patients of the Medical Oncology Unit, University Hospital and University of Cagliari were retrospectively collected from 2008 to 2021. Immunohistochemical analysis was performed at the Pathology Division, University Hospital of Cagliari on 3 µm thick sections obtained from paraffin blocks. The intensity of immunohistochemical staining was subclassified into four groups: score 0 if negative or weak membrane staining in less than 10% of tumor cells; score 1+ if weak membrane staining in at least 10% of tumor cells or moderate membrane staining in less than 10% of tumor cells; score 2+ if moderate membrane staining in at least 10% of tumor cells or intensive membrane staining in less than 10% of tumor cells; score 3+ if intense membrane staining in at least 10% of tumor cells. Based on this score, we distinguished patients into low CD44 expression (score 0, 1+, 2+) and high CD44 expression (score 3+). Statistical analysis was performed with MedCalc (survival distribution: Kaplan-Meier; survival comparison: log-rank test; association between categorical variables: Fisher's exact test). RESULTS: Patients' median age was 66 years (range 49-85). Regarding CD44 expression, score was 0 in 18 patients, 1+ in 15 patients, 2+ in 18 patients, and 3+ in 14 patients. Median overall survival (mOS) was 28.1 months (95%CI: 21.3-101). CD44 overexpression (3+) was correlated with poor prognosis (p = 0.0011; HR = 0.2), with a mOS of 14.5 months (95%CI 11.7 to 35.9) versus 30.7 months (95%CI 27.8 to 101) in lower CD44 expression. Higher CD44 expression was associated with clinically poor prognostic features: age ≥ 70 years (p = 0.0166); inoperable disease (p = 0.0008); stage IV at diagnosis (p = 0.0241); BRAF mutated (p = 0.0111), high-grade tumor (p = 0.0084). CONCLUSIONS: CD44 markedly correlated with aggressive tumor behavior and contributed to the earlier progression of disease, thus suggesting its role as a novel prognostic marker and potential therapeutic target for mCRC patients.

Prognostic Value of Body Mass Index in Stage II/III Colon Cancer: Posthoc Analysis From the TOSCA Trial.

BACKGROUND: High body mass index (BMI) plays a key role in the development of colon cancer (CC). Our post-hoc analysis from the TOSCA trial analyzed the association between BMI and survival outcomes in terms of relapse-free survival (RFS) and overall survival (OS) in stage II/III CC patients. PATIENTS AND METHODS: Patients enrolled in the TOSCA trial between 2007-2013 with BMI data entered the study. The prognostic impact of BMI on survival outcomes was investigated through uni- and multivariable Cox regression analyses. RESULTS: Overall, 1455 patients with stage II/III CC patients were included. The median follow-up was of 61.5 months; 16.1% of patients relapsed, 11.2% died and 19.5% patients relapsed or died. No impact of BMI on RFS was detected at univariate or multivariable analyses. By univariate analysis for OS, a significantly impact of a BMI > 30 kg/m2 was reported (HR [>30 vs <25] 1.57, 95% CI 1.00-2.47, p = 0.049; HR [>30 vs <30] 1.55, 95% CI 1.01-2.37, p = 0.045). Multivariable analyses did not confirm this data. In the subgroup of stage III patients, a negative survival impact of BMI was found in univariate and multivariable models both for RFS and for OS. CONCLUSIONS: In our study, obesity with BMI > 30 kg/m2 was an independent prognostic factor for RFS and OS in CC patients treated with adjuvant chemotherapy, regardless of its duration (3 or 6 months). However, the prognostic impact of adiposity and body composition measurement should be considered to better classify patients with high visceral fat and refine their risk assessment.

CDX-2 expression correlates with clinical outcomes in MSI-H metastatic colorectal cancer patients receiving immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) showed efficacy in metastatic colorectal cancer (mCRC) with mismatch-repair deficiency or high microsatellite instability (dMMR-MSI-H). Unfortunately, a patient's subgroup did not benefit from immunotherapy. Caudal-related homeobox transcription factor 2 (CDX-2) would seem to influence immunotherapy's sensitivity, promoting the chemokine (C-X-C motif) ligand 14 (CXCL14) expression. Therefore, we investigated CDX-2 role as a prognostic-predictive marker in patients with mCRC MSI-H. We retrospectively collected data from 14 MSI-H mCRC patients treated with ICIs between 2019 and 2021. The primary endpoint was the 12-month progression-free-survival (PFS) rate. The secondary endpoints were overall survival (OS), PFS, objective response rate (ORR), and disease control rate (DCR). The PFS rate at 12 months was 81% in CDX-2 positive patients vs 0% in CDX-2 negative patients (p = 0.0011). The median PFS was not reached (NR) in the CDX-2 positive group versus 2.07 months (95%CI 2.07-10.8) in CDX-2 negative patients (p = 0.0011). Median OS was NR in CDX-2-positive patients versus 2.17 months (95% Confidence Interval [CI] 2.17-18.7) in CDX2-negative patients (p = 0.026). All CDX-2-positive patients achieved a disease response, one of them a complete response. Among CDX-2-negative patients, one achieved stable disease, while the other progressed rapidly (ORR: 100% vs 0%, p = 0.0005; DCR: 100% vs 50%, p = 0.02). Twelve patients received 1st-line pembrolizumab (11 CDX-2 positive and 1 CDX-2 negative) not reaching median PFS, while two patients (1 CDX-2 positive and 1 CDX-2 negative) received 3rd-line pembrolizumab reaching a median PFS of 10.8 months (95% CI, 10.8-12.1; p = 0.036). Although our study reports results on a small population, the prognostic role of CDX-2 in CRC seems confirmed and could drive a promising predictive role in defining the population more sensitive to immunotherapy treatment. Modulating the CDX-2/CXCL14 axis in CDX-2-negative patients could help overcome primary resistance to immunotherapy.

Clinical score for colorectal cancer patients with lung-limited metastases undergoing surgical resection: Meta-Lung Score.

BACKGROUND: Radical resection of isolated lung metastases (LM) from colorectal cancer (CRC) is debated. Like Fong's criteria in liver metastases, our study was meant to assign a clinical prognostic score in patients with LM from CRC, aiming for better surgery selection. METHODS: We retrospectively analyzed data from 260 CRC patients who underwent curative LM resection from December 2002 to January 2022, verifying the impact of different clinicopathological features on the overall survival (OS). RESULTS: At the univariate analysis: higher baseline CEA levels (p = 0.0001), disease-free survival less than or equal to 12 months (m) (p = 0.0043), LM size larger than 2 cm (p = 0.0187), multiple resectable nodules (p = 0.0083), and positive nodal status of the primary tumor (p = 0.0011) were associated with worse prognosis. In a Cox regression model, these characteristics retained their independent role for OS (p < 0.0001) and were chosen as criteria to be assigned one point each for clinical risk score. The 5-year survival rate in patients with 0 points was 88%, while no patients with a 5-point score survived at 2 years. Based on the 0-1 vs. 2-5 score range, we obtained a significant difference in median OS: not reached vs. 40.8 months (95 %CI 36 to 87.5), respectively (p < 0.0001) stratifying patients into good and poor prognosis. The prognostic role of the score was also confirmed in terms of median RFS: not reached in 0-1 scored patients vs. 30.5 months (95 %CI 19.4 to 42) in patients with 2-5 scores (p = 0.0006). CONCLUSIONS: When LM from CRC is resectable, the Meta-Lung Score provides valuable prognostic information. Indeed, while upfront surgery should be considered in patients with scores of 0 to 1, it should be cautiously suggested in patients with scores of 2 to 5, for whom a prognosis comparison between preventive surgery and other treatments should be investigated in prospective randomized clinical trials.

Circulating tumour DNA in gastrointestinal cancer in clinical practice: Just a dream or maybe not?

The evaluation of circulating tumor DNA (ctDNA) is increasingly integrated into the management of diagnosis and treatment of gastrointestinal cancer as it represents an innovative and minimally invasive biomarker that could allow us to reach clinical needs not met yet in randomized clinical trials. Recent research provided an interesting overview of the role of circulating tumor DNA in gastric, biliary, liver, pancreatic, and colorectal cancer. Data regarding upper gastrointestinal tumors are currently not practice changing. Tumor detection rates are low in the early stages, while in advanced stages ctDNA is useful for molecular tracking evaluation. Most of the evidence comes from colorectal cancer studies, where ctDNA was evaluated both in the early and advanced stages with the post-surgery minimal residual disease assessment and the response assessment, respectively. ctDNA qualifies as a promising tool in the era of precision medicine, with potential applications in the entire management of gastrointestinal cancer patients. Further evidence is needed to establish which setting may be influenced greatly by liquid biopsy in clinical practice.

How to improve metastatic pancreatic ductal adenocarcinoma patients' selection: Between clinical trials and the real-world.

As underlined in the minireview by Blomstrand et al, given the poor prognosis and the paucity of data on a therapeutic sequence in pancreatic ductal adenocarcinoma (PDAC), additional randomized controlled trials and real-world evidence studies addressing current and novel regimens are needed. The real-world outcomes of first-line chemotherapy regimens such as FOLFIRINOX and gemcitabine/nab-paclitaxel are thoroughly reviewed and seem to be largely generalizable in a real-world context. Regarding second-line chemotherapy, the key question about the optimal sequence of regimens remains uncertain. Precisely in this setting, it is therefore useful to encourage the implementation of clinical studies that may contribute to the scarcity of data available up to now. We report our experience with a small group of patients treated with second-line liposomal irinotecan (nal-IRI) plus 5-fluorouracil and leucovorin. To improve the treatment of patients affected by PDAC, it is useful to identify subgroups of patients who may benefit from target treatments (e.g., BRCA mutant) and it is also important to focus on any prognostic factors that may affect the survival and treatment of these patients.

The role of immune checkpoint inhibitors in the treatment sequence of advanced gastric or gastro-esophageal junction cancer: A systematic review and meta-analysis of randomized trials.

PURPOSE: This study aimed to clarify the current knowledge on the use of immunotherapy in patients with advanced gastric(G)/gastroesophageal(GEJ) cancers. MATERIALS AND METHODS: A meta-analysis was done to evaluate the efficacy of immune checkpoint inhibitors(ICIs) in G/GEJ cancer both in the unselected population and in that stratified for combined positive score (CPS)≥ 1 and ≥ 10 patients. RESULTS: In the unselected population the result showed 21%(P < 0.00001), and 29%(P < 0.00001) reduction of death and progression risk for patients treated with ICIs compared without ICIs, while in CPS≥ 1 and≥ 10 populations, the result showed a death reduction of 19%(P = 0.001) and 33%(P < 0.00001) respectively, and, and 23% (P < 0.00001) and 43% (P < 0.00001) progression risk reduction respectively, for patients treated with and without ICIs. CONCLUSION: overall survival(OS) and progression free survival(PFS) data obtained in our meta-analysis, are in favor to use ICIs in association with standard first line chemotherapy for G/GEJ cancer patients regardless to CPS status.

Hepatocellular carcinoma and microbiota: Implications for clinical management and treatment.

Gut microbiota plays an essential role in host homeostasis. It is involved in several physiological processes such as nutrients digestion and absorption, maintenance of intestinal epithelial barrier integrity and immune system self-tolerance. Especially the gut microbiota is assumed to play a crucial role in many gastrointestinal, pancreatic and liver disorders. Its role in hepatic carcinogenesis is also gaining increasing interest, especially regarding the development of therapeutic strategies. Different studies are highlighting a link between some bacterial strains and liver disease, including hepatocellular carcinoma (HCC). Indeed, HCC represents an interesting field of research in this perspective, due to the gut-liver axis, to the implication of microbiota in the immune system and to the increasing number of immunotherapy agents investigated in this tumour. Thus, the assessment of the role of microbiota in influencing clinical outcome for patients treated with these drugs is becoming of increasing importance. Our review aims to give an overview on the relationship between microbiota and HCC development/progression and treatment. We focus on potential implications on the available treatment strategies and those under study in the various stages of disease. We highlight the pathogenic mechanisms and investigate the underlying molecular pathways involved. Moreover, we investigate the potential prognostic and/or predictive role of microbiota for target therapies, immune checkpoint inhibitors and loco-regional treatment. Finally, given the limitation of current treatments, we analyze the gut microbiota-mediated therapies and its potential options for HCC treatment focusing on fecal microbiota transplantation.

Colorectal cancer promoter methylation alteration affects the expression of glutamate ionotropic receptor AMPA type subunit 4 alternative isoforms potentially relevant in colon tissue.

DNA methylation alterations are early events during tumourigenesis, affecting genes involved in the crosstalk between cells and surroundings in colorectal cancer (CRC). Among these genes, GRIA4, Glutamate Ionotropic Receptor AMPA Type Subunit 4, displays hypermethylation in the promoter region, and is an early diagnostic biomarker. It is well known that methylation can also affect alternative transcription. The purpose of this study is to evaluate the expression, at transcript and protein level, of GRIA4 main isoforms (the canonical one and a short variant) in 23 CRC and matched normal samples, of which we previously verified the methylation status. We further predicted miRNA/transcript target interactions as a possible post-transcriptional regulation using bioinformatics tools. As expected, downregulation of both variants has been observed in tumours. Interestingly, in contrast to what observed at transcriptional level, the GluR4 protein short isoform displayed higher expression than the canonical one either in normal or tumoural tissues. This may be explained by miRNA specifically targeting the canonical isoform. Our study is the first one that shows the expression of both isoforms in colon tissues. To note, the evident expression of the short isoform suggests a functional role in intestinal cell biology.

Liquid Biopsy-Driven Cetuximab Rechallenge Strategy in Molecularly Selected Metastatic Colorectal Cancer Patients.

Background: Rechallenge with EGFR inhibitors represents a promising strategy for patients with RAS wild type (WT) colorectal cancer (CRC) but definitive selection criteria are lacking. Recently, the RAS WT status on circulating tumor DNA (ct-DNA) emerged as a potential watershed for this strategy. Our study explored the liquid biopsy-driven cetuximab rechallenge in a RAS and BRAF WT selected population. Methods: CRC patients with RAS and BRAF WT both on tumor tissue and on ct-DNA at baseline receiving rechallenge with cetuximab were eligible for our analysis. Ct-DNA was analyzed for RAS-BRAF mutations with pyro-sequencing and nucleotide sequencing assays. Real-time PCR and droplet digital PCR were performed to confirm the RAS-BRAF mutational status. Results: A total of 26 patients were included in our analysis. In the global population, RR was 25.0%, median overall survival (mOS) was 5.0 months, and median progression-free survival (mPFS) was 3.5 months. Previous response to anti-EGFR was associated with improved mPFS (5.0 vs. 2.0 months, HR: 0.26, p = 0.048); anti-EGFR free interval > 14 months and anti-EGFR free interval > 16 months were associated with improved mPFS (respectively 7.0 vs. 3.0 months, HR: 0.27, p = 0.013 and not reached vs. 3.0 months, HR: 0.20, p = 0.002) and with improved mOS (respectively 13.0 vs. 5.0 months, HR: 0.27, p = 0.013 and 13.0 vs. 5.0 months, HR: 0.20, p = 0.002). Previous lines >2 were correlated with improved mPFS (4.0 vs. 1.0 month, HR: 0.05, p = 0.041) and with improved mOS (7.0 vs. 1.0 month, HR: 0.045, p = 0.034). In a multiple logistic regression model, only the anti-EGFR free interval was confirmed to be a significant predictor for mOS and mPFS. Conclusions: Liquid biopsy-driven cetuximab rechallenge was confirmed to be effective. The clinical outcome was consistent with available results from phase II studies. In addition to the molecular selection through the analysis of ct-DNA for RAS, the long anti-EGFR free interval is confirmed as a prospective selection criterion for this therapeutic option.