ctDNA whole exome sequencing in pancreatic ductal adenocarcinoma unveils organ-dependent metastatic mechanisms and identifies actionable alterations in fast progressing patients.

Understanding progression mechanisms and developing new targeted therapies is imperative in pancreatic ductal adenocarcinoma (PDAC). In this study, 80 metastatic PDAC patients were prospectively recruited and divided into discovery (n=37) and validation (n=43) cohorts. Tumor and plasma samples taken at diagnosis were pair analyzed using whole exome sequencing (WES) in patients belonging to the discovery cohort alone. The variant allele frequency (VAF) of KRAS mutations was measured by ddPCR in plasma at baseline and response assessment in all patients. Plasma WES identified at least one pathogenic variant across the cohort, uncovering oncogenic mechanisms, DNA repair, microsatellite instability, and alterations in the TGFb pathway. Interestingly, actionable mutations were mostly found in plasma rather than tissue. Patients with shorter survival showed enrichment in cellular organization regulatory pathways. Through WES we could identify a specific molecular profile of patients with liver metastasis, which exhibited exclusive mutations in genes related to the adaptive immune response pathway, highlighting the importance of the immune system in liver metastasis development. Moreover, KRAS mutations in plasma (both at diagnosis and persistent at follow-up) correlated with shorter progression free survival (PFS). Patients presenting a reduction of over 84.75% in KRAS VAF at response assessment had similar PFS to KRAS-negative patients. Overall, plasma WES reveals molecular profiles indicative of rapid progression, potentially actionable targets, and associations between adaptive immune response pathway alterations and liver tropism.

The percentage of mesorectal infiltration as a prognostic factor after curative surgery for pT3 rectal cancer.

AIM: The aim of this study is to evaluate the prognostic value of a novel variable - the percentage of mesorectal infiltration (PMI) - in pT3 rectal cancer. METHOD: A cohort of 241 patients with pT3 rectal adenocarcinoma, operated on between February 2002 and May 2019, was selected for the analysis. Data concerning patient, treatment and tumour characteristics were collected. The depth of mesorectal infiltration (DMI) and the distance between the deepest invasion and the circumferential resection margin (CRM) were measured. The PMI was calculated using a formula combining these parameters. RESULTS: Neoadjuvant therapy was administered in 33.2% of cases. A complete mesorectal excision was achieved in 74% of patients. The CRM was affected in 24 patients (9.9%). The 5-year actuarial local recurrence (LR), overall recurrence (OR) and overall survival (OS) rates were 7.5%, 22.9% and 72.4%, respectively. The PMI was significantly associated with worse oncological outcomes regarding LR (p = 0.009), OR (p = 0.001) and OS (p = 0.016) rates. A cut-off value of PMI >60% had the highest specificity (80%) for LR (p = 0.026), OR (p = 0.04) and OS (p = 0.07). CONCLUSION: The PMI has an adverse prognostic impact on the oncological results following surgery for pT3 rectal cancer. It allows prediction of the risk of both LR and distant recurrence with higher accuracy than the DMI or the distance to the CRM. A PMI >60% may be used as a cut off value while subclassifying pT3 rectal tumours. It may influence decision-making while establishing adjuvant treatment and the follow-up schedule.

"Proteotranscriptomic analysis of advanced colorectal cancer patient derived organoids for drug sensitivity prediction".

BACKGROUND: Patient-derived organoids (PDOs) from advanced colorectal cancer (CRC) patients could be a key platform to predict drug response and discover new biomarkers. We aimed to integrate PDO drug response with multi-omics characterization beyond genomics. METHODS: We generated 29 PDO lines from 22 advanced CRC patients and provided a morphologic, genomic, and transcriptomic characterization. We performed drug sensitivity assays with a panel of both standard and non-standard agents in five long-term cultures, and integrated drug response with a baseline proteomic and transcriptomic characterization by SWATH-MS and RNA-seq analysis, respectively. RESULTS: PDOs were successfully generated from heavily pre-treated patients, including a paired model of advanced MSI high CRC deriving from pre- and post-chemotherapy liver metastasis. Our PDOs faithfully reproduced genomic and phenotypic features of original tissue. Drug panel testing identified differential response among PDOs, particularly to oxaliplatin and palbociclib. Proteotranscriptomic analyses revealed that oxaliplatin non-responder PDOs present enrichment of the t-RNA aminoacylation process and showed a shift towards oxidative phosphorylation pathway dependence, while an exceptional response to palbociclib was detected in a PDO with activation of MYC and enrichment of chaperonin T-complex protein Ring Complex (TRiC), involved in proteome integrity. Proteotranscriptomic data fusion confirmed these results within a highly integrated network of functional processes involved in differential response to drugs. CONCLUSIONS: Our strategy of integrating PDOs drug sensitivity with SWATH-mass spectrometry and RNA-seq allowed us to identify different baseline proteins and gene expression profiles with the potential to predict treatment response/resistance and to help in the development of effective and personalized cancer therapeutics.

Integrative immune transcriptomic classification improves patient selection for precision immunotherapy in advanced gastro-oesophageal adenocarcinoma.

BACKGROUND: Advanced gastro-oesophageal cancer (GEA) treatment has been improved by the introduction of immune checkpoint inhibitors (CPIs), yet identifying predictive biomarkers remains a priority, particularly in patients with a combined positive score (CPS) < 5, where the benefit is less clear. Our study assesses certain immune microenvironment features related to sensitivity or resistance to CPIs with the aim of implementing a personalised approach across CPS < 5 GEA. DESIGN: Through integrative transcriptomic and clinicopathological analyses, we studied in both a retrospective and a prospective cohort, the immune tumour microenvironment features. We analysed the cell types composing the immune infiltrate highlighting their functional activity. RESULTS: This integrative study allowed the identification of four different groups across our patients. Among them, we identified a cluster whose tumours expressed the most gene signatures related to immunomodulatory pathways and immunotherapy response. These tumours presented an enriched immune infiltrate showing high immune function activity that could potentially achieve the best benefit from CPIs. Finally, our findings were proven in an external CPI-exposed population, where the use of our transcriptomic results combined with CPS helped better identify those patients who could benefit from immunotherapy than using CPS alone (p = 0.043). CONCLUSIONS: This transcriptomic classification could improve precision immunotherapy for GEA.

Circulating Tumor DNA in Stage III Colorectal Cancer, beyond Minimal Residual Disease Detection, toward Assessment of Adjuvant Therapy Efficacy and Clinical Behavior of Recurrences.

PURPOSE: Sensitive methods for risk stratification, monitoring therapeutic efficacy, and early relapse detection may have a major impact on treatment decisions and patient management for stage III colorectal cancer patients. Beyond assessing the predictive power of postoperative ctDNA detection, we explored the added benefits of serial analysis: assessing adjuvant chemotherapy (ACT) efficacy, early relapse detection, and ctDNA growth rates. EXPERIMENTAL DESIGN: We recruited 168 patients with stage III colorectal cancer treated with curative intent at Danish and Spanish hospitals between 2014 and 2019. To quantify ctDNA in plasma samples (n = 1,204), 16 patient-specific somatic single-nucleotide variants were profiled using multiplex-PCR, next-generation sequencing. RESULTS: Detection of ctDNA was a strong recurrence predictor postoperatively [HR = 7.0; 95% confidence interval (CI), 3.7-13.5; P < 0.001] and directly after ACT (HR = 50.76; 95% CI, 15.4-167; P < 0.001). The recurrence rate of postoperative ctDNA-positive patients treated with ACT was 80% (16/20). Only patients who cleared ctDNA permanently during ACT did not relapse. Serial ctDNA assessment after the end of treatment was similarly predictive of recurrence (HR = 50.80; 95% CI, 14.9-172; P < 0.001), and revealed two distinct rates of exponential ctDNA growth, slow (25% ctDNA-increase/month) and fast (143% ctDNA-increase/month; P < 0.001). The ctDNA growth rate was prognostic of survival (HR = 2.7; 95% CI, 1.1-6.7; P = 0.039). Serial ctDNA analysis every 3 months detected recurrence with a median lead-time of 9.8 months compared with standard-of-care computed tomography. CONCLUSIONS: Serial postoperative ctDNA analysis has a strong prognostic value and enables tumor growth rate assessment. The novel combination of ctDNA detection and growth rate assessment provides unique opportunities for guiding decision-making.See related commentary by Morris and George, p. 438.

Molecular profiling of advanced solid tumours. The impact of experimental molecular-matched therapies on cancer patient outcomes in early-phase trials: the MAST study.

INTRODUCTION: Molecular-matched therapies have revolutionized cancer treatment. We evaluated the improvement in clinical outcomes of applying an in-house customized Next Generation Sequencing panel in a single institution. METHODS: Patients with advanced solid tumors were molecularly selected to receive a molecular-matched treatment into early phase clinical trials versus best investigators choice, according to the evaluation of a multidisciplinary molecular tumor board. The primary endpoint was progression-free survival (PFS) assessed by the ratio of patients presenting 1.3-fold longer PFS on matched therapy (PFS2) than with prior therapy (PFS1). RESULTS: Of a total of 231 molecularly screened patients, 87 were eligible for analysis. Patients who received matched therapy had a higher median PFS2 (6.47 months; 95% CI, 2.24-14.43) compared to those who received standard therapy (2.76 months; 95% CI, 2.14-3.91, Log-rank p = 0.022). The proportion of patients with a PFS2/PFS1 ratio over 1.3 was significantly higher in the experimental arm (0.33 vs 0.08; p = 0.008). DISCUSSION: We demonstrate the pivotal role of the institutional molecular tumor board in evaluating the results of a customized NGS panel. This process optimizes the selection of available therapies, improving disease control. Prospective randomized trials are needed to confirm this approach and open the door to expanded drug access.

Circulating Tumor DNA Detection by Digital-Droplet PCR in Pancreatic Ductal Adenocarcinoma: A Systematic Review.

Pancreatic cancer (PC) is one of the most devastating malignant tumors, being the seventh leading cause of cancer-related death worldwide. Researchers and clinicians are endeavoring to develop strategies for the early detection of the disease and the improvement of treatment results. Adequate biopsy is still challenging because of the pancreas's poor anatomic location. Recently, circulating tumor DNA (ctDNA) could be identified as a liquid biopsy tool with huge potential as a non-invasive biomarker in early diagnosis, prognosis and management of PC. ctDNA is released from apoptotic and necrotic cancer cells, as well as from living tumor cells and even circulating tumor cells, and it can reveal genetic and epigenetic alterations with tumor-specific and individual mutation and methylation profiles. However, ctDNA sensibility remains a limitation and the accuracy of ctDNA as a biomarker for PC is relatively low and cannot be currently used as a screening or diagnostic tool. Increasing evidence suggests that ctDNA is an interesting biomarker for predictive or prognosis studies, evaluating minimal residual disease, longitudinal follow-up and treatment management. Promising results have been published and therefore the objective of our review is to understand the current role and the future perspectives of ctDNA in PC.

Neoadjuvant Chemotherapy in Locally Advanced Rectal Cancer.

Most clinical practice guidelines recommend a selective approach for rectal cancer after clinical staging. In low-risk patients, upfront surgery may be an appropriate option. However, in patients with MRI-defined high-risk features such as extramural vascular invasion, multiple nodal involvement or T4 and/or tumors close to or invading the mesorectal fascia, a more intensive preoperative approach is recommended, which may include neoadjuvant or preoperative chemotherapy. The potential benefits include better compliance than postoperative chemotherapy, a higher pathological complete remission rate, which facilitates a non-surgical approach, and earlier treatment of micrometastatic disease with improved disease-free survival compared to standard preoperative chemoradiation or short-course radiation. Two recently reported phase III randomized trials, RAPIDO and PRODIGE 23, show that adding neoadjuvant chemotherapy to either standard short-course radiation or standard long-course chemoradiation in locally advanced rectal cancer patients reduces the risk of metastasis and significantly prolongs disease-related treatment failure and disease-free survival. This review discusses these potentially practice-changing trials and how they may affect our current understanding of treating locally advanced rectal cancers.

Neoadjuvant treatment for locally advanced unresectable and borderline resectable pancreatic cancer: oncological outcomes at a single academic centre.

INTRODUCTION: Pancreatic cancer (PC), even in the absence of metastatic disease, has a dismal prognosis. One-third of them are borderline resectable (BRPC) or locally advanced unresectable PC (LAUPC) at diagnosis. There are limited prospective data supporting the best approach on these tumours. Neoadjuvant chemotherapy (ChT) is being increasingly used in this setting. METHODS: This is a retrospective series of consecutive patients staged as BRPC or LAUPC after discussion in the multidisciplinary board (MDB) at an academic centre. All received neoadjuvant ChT, followed by chemoradiation (ChRT) in some cases, and those achieving enough downstaging had a curative-intent surgery. Descriptive data about patient's characteristics, neoadjuvant treatments, toxicities, curative resections, postoperative complications, pathology reports and adjuvant treatment were collected. Overall survival (OS) and progression-free survival was calculated with Kaplan-Meier method and log-rank test. RESULTS: Between August 2011 and July 2019, 49 patients fulfilled the inclusion criteria, and all of them received neoadjuvant ChT. Fluorouracil+folinic acid, irinotecan and oxaliplatin was the most frequently used scheme (77%). The most prevalent grade 3 or 4 toxicities were neutropenia (26.5%), neurotoxicity (12.2%), diarrhoea (8.2%) and nausea (8.2%). 18 patients (36.7%) received ChRT thereafter. In total, 22 patients (44,9%) became potentially resectable and 19 of them had an R0 or R1 pancreatic resection. One was found to be unresectable at surgery and two refused surgery. A vascular resection was required in 7 (35%). No postoperative deaths were observed. Postoperative ChT was given to 12 (66.7%) of resected patients. Median OS of the whole cohort was 24,9 months (95% CI 14.1 to 35.7), with 30.6 months for resected and 13.1 months for non-resected patients, respectively (p<0.001). CONCLUSION: A neoadjuvant approach in BRPC and LAUPC was well tolerated and allowed a curative resection in 38.8% of them with a potential improvement on OS.

Detection of postoperative plasma circulating tumour DNA and lack of CDX2 expression as markers of recurrence in patients with localised colon cancer.

BACKGROUND: Colon cancer (CC) is a heterogeneous disease. Novel prognostic factors beyond pathological staging are required to accurately identify patients at higher risk of relapse. Integrating these new biological factors, such as plasma circulating tumour DNA (ctDNA), CDX2 staining, inflammation-associated cytokines and transcriptomic consensus molecular subtypes (CMS) classification, into a multimodal approach may improve our accuracy in determining risk of recurrence. METHODS: One hundred and fifty patients consecutively diagnosed with localised CC were prospectively enrolled in our study. ctDNA was tracked to detect minimal residual disease by droplet digital PCR. CDX2 expression was analysed by immunostaining. Plasma levels of cytokines potentially involved in disease progression were measured using ELISAs. A 96 custom gene panel for nCounter assay was used to classify CC into colorectal cancer assigner and CMS. RESULTS: Most patients were classified into CMS4 (37%) and CMS2 (28%), followed by CMS1 (20%) and CMS3 (15%) groups. CDX2-negative tumours were enriched in CMS1 and CMS4 subtypes. In univariable analysis, prognosis was influenced by primary tumour location, stage, vascular and perineural invasion together with high interleukin-6 plasma levels at baseline, tumours belonging to CMS 1 vs CMS2 +CMS3, ctDNA presence in plasma and CDX2 loss. However, only positive ctDNA in plasma samples (HR 13.64; p=0.002) and lack of CDX2 expression (HR 23.12; p=0.001) were found to be independent prognostic factors for disease-free survival in the multivariable model. CONCLUSIONS: ctDNA detection after surgery and lack of CDX2 expression identified patients at very high risk of recurrence in localised CC.

Precision Medicine to Treat Advanced Gastroesophageal Adenocarcinoma: A Work in Progress.

Gastroesophageal adenocarcinoma (GEA) represents a heterogeneous disease and, when diagnosed as locally advanced or metastatic, it is characterized by poor prognosis. During the last few years, several molecular classifications have been proposed to try to personalize treatment for those patients diagnosed with advanced disease. Nevertheless, despite the great effort, precision medicine is still far from being a reality. The improvement in the molecular analysis due to the application of high throughput technologies based on DNA and RNA sequencing has opened a novel scenario leading to the personalization of treatment. The possibility to target epidermal growth factor receptor (HER)2, Claudine, Fibroblast Growth Factor Receptors (FGFR), and other alterations with a molecular matched therapy could significantly improve clinical outcomes over advanced gastric cancer patients. On the other hand, the development of immunotherapy could also represent a promising strategy in a selected population. In this review, we sought to describe the novel pathways implicated in GEA progression and the results of the molecular matched therapies.

How we treat metastatic colorectal cancer.

Colorectal cancer is the second leading cause of cancer-related death worldwide. About 20% of patients suffer from metastatic disease at diagnosis, while about one-third of patients treated with curative intent relapsed. In these patients, an accurate staging allows to plan a treatment strategy within a multidisciplinary team in order to achieve predefined goals. Patient's clinical features, tumour characteristics and molecular profile (RAS/BRAF and microsatellite instability (MSI) status) should be considered during the treatment choice. Combination of chemotherapy (fluoropyrimidines, oxaliplatin and irinotecan) plus biological agents (antiepidermal growth factor receptor or antiangiogenic drugs) in addition to surgery, could give a chance of cure in resectable or potentially resectable tumours. However, in never resectable tumours, disease control and prolonging survival should be the goal to achieve simultaneously with control of symptoms. In addition to standard therapies, especially in case of unresectable oligometastatic disease, several local ablative treatment are available. In later lines, when improving quality of life become predominant, regorafenib and trifluridine/tipiracil demonstrated survival benefit, while re-challenge therapies represent an option only in selected patients. In patients with BRAFV600E-mutant tumour or with MSI, new therapies showed survival gain and probably will be a new piece in the treatment algorithm.

Personalized Medicine: Recent Progress in Cancer Therapy.

Translational research has revolutionized how we develop new treatments for cancer patients. The change from an organ-centric concept guiding treatment choice towards deep molecular analysis, driving a personalized approach, is one of the most important advances of modern oncology. Several tools such as next generation sequencing and RNA sequencing have greatly improved the capacity to detect predictive and prognostic molecular alterations. Detection of gene mutations, amplifications, and fusions has therefore altered the history of several diseases in both a localized and metastatic setting. This shift in perspective, in which attention is focused on the specific molecular alterations of the tumor, has opened the door to personalized treatment. This situation is reflected in the increasing number of basket trials selecting specific molecular targets. Nonetheless, some weaknesses need to be addressed. The complexity of cancer cells enriched with concomitant molecular alterations complicates identification of the driver. Moreover, tumor heterogeneity could be responsible for the lack of benefit when targeted agents are used. In light of this, there is growing interest in the role of multidisciplinary committees or molecular tumor boards to try to enhance selection. The aim of this review is to critically analyze the evolution of cancer treatment towards a precision approach, underlining some recent successes and unexpected failures.

Impact of perioperative transfusions and sepsis on long-term oncologic outcomes after curative colon cancer resection. A retrospective analysis of a prospective database / Impacto de las trasfusiones y de las sepsis perioperatorias en los resultados oncológicos de largo plazo tras cirugía curativa de cáncer de colon. Análisis retrospectivo de una base prospectiva

Objective: Intra-abdominal septic complications (IASC) affect short-term outcomes after surgery for colon cancer. Blood transfusions have been associated with worse short-term results. The role of IASC and blood transfusions on long-term oncologic results is still debated. This study aims to assess the impact of these two variables on survival after curative colon cancer resection. Patients and methods: Retrospective analysis of a prospectively maintained database of patients who underwent curative surgery for colon cancer at a university hospital, between 1993 and 2010. Cox regression was used to identify the role of IASC and transfusions (alone and combined) on local recurrence (LR), disease-free survival (DFS), and cancer-specific survival (CSS). Results: Out of the 1686 patients analyzed, 1277 fit in the inclusion criteria. Colorectal surgeons performed the procedure in 82.2% of the patients. Blood transfusions were administered to 25.8% of the patients. Thirty-day complication and mortality rates were 34.5% and 6.1%. IASC occurred in 9.9%. The mean follow-up was 66 months. The 5-year rates of LR, DFS, and CSS were 7%, 79.8%, and 85.1%. The year of surgery and pT (Hazard ratio 9.35, 95% CI 1.23-70.9, for T4) and pN (Hazard ratio 2.57, 95% CI 1.39-4.72, for N2) stages were independent risk factors for LR. The same variables, bowel obstruction and surgeries performed by surgeons not specialized in colorectal surgery, were also associated with worse DFS and CSS. IASC and blood transfusions were not associated with LR, DFS, and CSS, whether alone or combined. Conclusions: IASC and transfusions were not associated with worse oncological outcomes after curative colon cancer surgery per se. Other factors were more important predictors of survival

Objetivos: Las complicaciones sépticas intra-abdominales(CSIA) empeoran los resultados a corto plazo después de cirugía por cáncer de colon. Las trasfusiones de sangre también han sido relacionadas con peores resultados a corto plazo. El impacto de la CSIA y de las transfusiones en los resultados oncológicos es todavía debatido. Objetivo del presente estudio fue valorar el impacto de estas dos variables en la supervivencia de pacientes intervenidos por cáncer de colon. Pacientes y métodos: Análisis retrospectivo de una base prospectiva de pacientes sometidos a cirugía curativa por cáncer de colon en un hospital universitario(1993-2010). Se utilizó regresión de Cox para valorar el efecto de CSIA y trasfusiones(aislados o en combinación) sobre recidiva local(RL), supervivencia libre de enfermedad(SLE) y supervivencia cáncer-especifica(SCE). Resultados: De los 1686 pacientes analizados, se incluyeron 1277. La cirugía fue realizada por cirujanos colorrectales en el 82,2% de los pacientes. El 25,8% recibió transfusiones. Las tasas de complicaciones y mortalidad a los 30 días fueron del 34,5% y 6,1%. La frecuencia de CSIA fue del 9,9%. El seguimiento mediano fue de 66 meses. Las tasas a los 5 años de RL,SLE y SCE fueron 7%, 79,8% y 85,1%. El año de tratamiento, los estadios pT(Cociente de riesgo 9,35,IC95% 1,23-70,9,en T4)y pN(Cociente de riesgo 2,57,IC95% 1,39-4,72,en N2)resultaron como factores de riesgo para RL. Las mismas variables, la obstrucción intestinal y la cirugía realizada por cirujanos no colorrectales se asociaron también a peor SLE y SCE. CSIA y trasfusiones no resultaron asociadas con RL, SLE y SCE, ni de forma aislada ni combinadas. Conclusiones: Las CSIA y trasfusiones no afectaron per se los resultados oncológicos de la cirugía de cáncer de colon. Otros factores resultaron más importantes predictores de supervivencia

Impact of perioperative transfusions and sepsis on long-term oncologic outcomes after curative colon cancer resection. A retrospective analysis of a prospective database.

OBJECTIVE: Intra-abdominal septic complications (IASC) affect short-term outcomes after surgery for colon cancer. Blood transfusions have been associated with worse short-term results. The role of IASC and blood transfusions on long-term oncologic results is still debated. This study aims to assess the impact of these two variables on survival after curative colon cancer resection. PATIENTS AND METHODS: Retrospective analysis of a prospectively maintained database of patients who underwent curative surgery for colon cancer at a university hospital, between 1993 and 2010. Cox regression was used to identify the role of IASC and transfusions (alone and combined) on local recurrence (LR), disease-free survival (DFS), and cancer-specific survival (CSS). RESULTS: Out of the 1686 patients analyzed, 1277 fit in the inclusion criteria. Colorectal surgeons performed the procedure in 82.2% of the patients. Blood transfusions were administered to 25.8% of the patients. Thirty-day complication and mortality rates were 34.5% and 6.1%. IASC occurred in 9.9%. The mean follow-up was 66 months. The 5-year rates of LR, DFS, and CSS were 7%, 79.8%, and 85.1%. The year of surgery and pT (Hazard ratio 9.35, 95% CI 1.23-70.9, for T4) and pN (Hazard ratio 2.57, 95% CI 1.39-4.72, for N2) stages were independent risk factors for LR. The same variables, bowel obstruction and surgeries performed by surgeons not specialized in colorectal surgery, were also associated with worse DFS and CSS. IASC and blood transfusions were not associated with LR, DFS, and CSS, whether alone or combined. CONCLUSIONS: IASC and transfusions were not associated with worse oncological outcomes after curative colon cancer surgery per se. Other factors were more important predictors of survival.

Analysis of KRAS, NRAS, BRAF, PIK3CA and TP53 mutations in a large prospective series of locally advanced rectal cancer patients.

Little information is available on the clinical significance of cancer-related genes such as KRAS, NRAS, BRAF, PIK3CA and TP53 in nonmetastatic rectal cancer. We investigated mutations of these genes in a large prospective series of locally advanced rectal cancer (LARC) patients who were recruited into two phase II trials. Mutational analyses were performed with diagnostically validated methods including polymerase chain reaction, capillary electrophoresis single-strand conformational analysis, Sanger sequencing and next-generation sequencing. Associations between single or multiple gene mutations and clinicopathological characteristics and treatment outcomes were explored. Of these 269, 210 (78%) patients were assessable. Mutations of KRAS, NRAS, BRAF, PIK3CA and TP53 occurred in 43, 9, 4, 9 and 60% of patients, respectively. Concordance between paired biopsy and resection specimens was 82% for KRAS, 95% for NRAS, 99% for BRAF, 96% for PIK3CA and 63% for TP53. TP53 mutations were associated with extramural venous invasion on baseline MRI (78% vs. 65%, p = 0.04), poor pathological tumour regression (23% vs. 36%, p = 0.05) and a trend toward a worse 5-year progression-free survival (PFS; 60% vs. 74%, HR 1.59, p = 0.06). Patients with tumours harbouring mutation of TP53 and either KRAS or NRAS (32%) had a worse 5-year PFS than those with TP53/KRAS/NRAS wild-type tumours (54% vs. 72%, HR 1.75, p = 0.02). In univariate analysis, BRAF mutation predicted poor 5-year overall survival only among patients treated without cetuximab (20% vs. 73%, HR 3.29, p = 0.03). This is one of the largest biomarker studies in a prospective, largely homogeneous, LARC population. Our findings are hypothesis generating and require validation in independent series.

Assessing molecular subtypes of gastric cancer: microsatellite unstable and Epstein-Barr virus subtypes. Methods for detection and clinical and pathological implications.

BACKGROUND: The molecular classification of gastric cancer recognises two subtypes prone to immune checkpoint blockade: the microsatellite unstable and the Epstein-Barr virus (EBV)-related tumours. We aim to assess the concordance between immunohistochemistry and PCR for microsatellite status evaluation, and explore the value of microsatellite instability (MSI) and EBV as predictive survival factors. MATERIAL AND METHODS: We collected 246 consecutively diagnosed gastric cancer cases in all stages and evaluated the microsatellite status using immunohistochemistry for mismatched repair (MMR) proteins and PCR. EBV expression was studied through in situ hybridisation. RESULTS: Forty-five (18%) cases presented MSI and 13 (6%) were positive for EBV. MSI was associated with female sex, older age, distal location and distal non-diffuse type of the modified Lauren classification. EBV expression was most frequent in proximal location and proximal non-diffuse type. The sensitivity, specificity, positive predictive value and negative predictive value of immunohistochemistry for the microsatellite study were 91%, 98%, 91% and 98%, respectively. In the multivariate analysis, MSI was an independent predictor of favourable tumour-specific survival (TSS) in stages I-III (MSI: HR: 0.37, 95% CI 0.12 to 0.95, p=0.04). CONCLUSIONS: The MSI status and the EBV expression should be incorporated in routine pathological report for two reasons. First, MSI defines a different pathological entity with a better outcome. Second, MSI and EBV may be useful biomarkers to identify patients who will respond to immune checkpoint blockade inhibitors. For this purpose, immunohistochemical study for MMR proteins and in situ hybridisation study for EBV evaluation are feasible and cost-effective methods.

Improving tumour budding evaluation in colon cancer by extending the assessment area in colectomy specimens.

AIMS: It is recommended that tumour budding in colon cancer be counted on haematoxylin and eosin-stained sections in a hotspot area of 0.785 mm2 with a ×20 microscope objective. However, tumour buds may be difficult to visualise on haematoxylin and eosin-stained sections, and counting in such a limited area may result in overestimation in cases with focal budding. The aim of this study was to assess the contributions of various factors to improving tumour budding risk stratification: increasing the number of fields counted, using cytokeratin immunostaining, and recording proliferation, the apoptotic index and the emperipoletic index in tumour buds. METHODS AND RESULTS: We created an exploratory series composed of 172 cases of colon cancer in all stages, and we analysed the survival probability in a second cohort of 158 stage I-II patients. According to our results, counting of budding in 10 fields was the only factor that was significantly correlated with disease-free survival probability in stage I-II patients [hazard ratio (HR) for high versus low grade of 7.64, 95% confidence interval (CI) 5.54-27.92, P = 0.01; HR for intermediate versus low grade of 3.02, 95% CI 1.54-26.72, P = 0.04). Emperipolesis was frequently observed in tumour buds, whereas the mitotic index and the apoptotic index were extremely low. Although cytokeratin immunostaining increased interobserver concordance, it did not improve the accuracy of tumour budding grading. CONCLUSIONS: According to our results, counting in 10 fields significantly enhanced the budding grade risk stratification in colon cancer patients, and cytokeratin immunostaining could be reserved as a complementary technique for challenging cases with an inflammatory infiltrate and/or striking fibrosis.

Prognostic Impact of pT Stage and Peritoneal Invasion in Locally Advanced Colon Cancer.

BACKGROUND: TNM stage has been identified as an independent variable for local recurrence and survival after colon cancer resection. It is still unclear whether peritoneal invasion (pT4a) is a risk factor for adverse oncologic outcome or whether these patients have better results compared with contiguous organs infiltration (pT4b), independent from nodal status (pN). OBJECTIVE: The purpose of this study was to analyze whether peritoneal invasion is an independent risk factor for worse oncologic outcome after curative colon cancer resection. DESIGN: This was a retrospective analysis with multivariate regression of a prospective database, according to Strengthening the Reporting of Observational Studies in Epidemiology Statement. SETTINGS: The study was conducted in a specialized colorectal unit of a tertiary hospital. PATIENTS: A consecutive series of pT3-pT4a-pT4b patients with colon cancer who underwent curative surgery (1993-2010) were included, and patients with metastasis were excluded. MAIN OUTCOME MEASURES: A multivariate Cox regression analysis was performed to assess independent risk factors for 5-year local recurrence, peritoneal carcinomatosis-like recurrence, disease-free survival, and cancer-specific survival. RESULTS: A total of 1010 patients were analyzed (79.3% pT3, 9.9% pT4a, and 10.8% pT4b). At diagnosis, 22.0% had obstructive symptoms, and 10.5% had bowel perforation. A total of 72.2% of the surgeries were elective, and in 15.6% en bloc resection of contiguous organs was performed. Median follow-up was 62 months (38-100 mo). For the whole group, 5-year actuarial rates were 8.8% for local recurrence, 2.5% for peritoneal carcinomatosis, 75.5% for disease-free survival, and 81.8% for cancer-specific survival. At multivariate analysis, pT4a stage was an independent risk factor for local recurrence (p = 0.002; HR = 3.1), peritoneal carcinomatosis (p = 0.02; HR = 4.9), worse disease-free survival (p = 0.002; HR = 1.9), and cancer-specific survival (p = 0.001; HR = 2.2). When considering only the 566 patients with ≥12 nodes identified, T stage was still associated with higher local recurrence (p = 0.04) and carcinomatosis rate (p = 0.04), as well as worse disease-free (p = 0.009) and cancer-specific survival (p = 0.014). LIMITATIONS: This was a retrospective, single-center study. CONCLUSIONS: pT4a stage is an independent risk factor for worse oncologic outcome after curative colon cancer resection compared with pT3 and pT4b stages. The current pT4a-pT4b classification should be reconsidered. Of note, even in pT4a patients, 5-year carcinomatosis rate does not exceed 6%. See Video Abstract at http://links.lww.com/DCR/A926.

Low miR200c expression in tumor budding of invasive front predicts worse survival in patients with localized colon cancer and is related to PD-L1 overexpression.

At the histological level, tumor budding in colon cancer is the result of cells undergoing at least partial epithelial-to-mesenchymal transition. The microRNA 200 family is an important epigenetic driver of this process, mainly by downregulating zinc-finger E-box binding homeobox (ZEB) and transforming growth factor beta (TGF-ß) expression. We retrospectively explored the expression of the miR200 family, and ZEB1 and ZEB2, and their relationship with immune resistance mediated through PD-L1 overexpression. For this purpose, we analyzed a series of 125 colon cancer cases and took samples from two different tumor sites: the area of tumor budding at the invasive front and from the tumor center. We found significant ZEB overexpression and a reduction in miR200 in budding areas, a profile compatible with epithelial-to-mesenchymal transition. In multivariate analysis of the cases with localized disease, low miR200c expression in budding areas, but not at the tumor center, was an adverse tumor-specific survival factor (hazard ratio: 0.12; 95% confidence interval: 0.03-0.81; p = 0.02) independent of the clinical stage of the disease. PD-L1 was significantly overexpressed in the budding areas and its levels correlated with a mesenchymal transition profile. These results highlight the importance of including budding areas among the samples used for biomarker evaluation and provides relevant data on the influence of mesenchymal transition in the immune resistance mediated by PD-L1 overexpression.