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BACKGROUND Awareness of the potential global overtreatment of patients with appendiceal neuroendocrine tumours (NETs) of 1-2 cm in size by performing oncological resections is increasing, but the rarity of this tumour has impeded clear recommendations to date. We aimed to assess the malignant potential of appendiceal NETs of 1-2 cm in size in patients with or without right-sided hemicolectomy. METHODS In this retrospective cohort study, we pooled data from 40 hospitals in 15 European countries for patients of any age and Eastern Cooperative Oncology Group performance status with a histopathologically confirmed appendiceal NET of 1-2 cm in size who had a complete resection of the primary tumour between Jan 1, 2000, and Dec 31, 2010. Patients either had an appendectomy only or an appendectomy with oncological right-sided hemicolectomy or ileocecal resection. Predefined primary outcomes were the frequency of distant metastases and tumour-related mortality. Secondary outcomes included the frequency of regional lymph node metastases, the association between regional lymph node metastases and histopathological risk factors, and overall survival with or without right-sided hemicolectomy. Cox proportional hazards regression was used to estimate the relative all-cause mortality hazard associated with right-sided hemicolectomy compared with appendectomy alone. This study is registered with ClinicalTrials.gov, NCT03852693. FINDINGS 282 patients with suspected appendiceal tumours were identified, of whom 278 with an appendiceal NET of 1-2 cm in size were included. 163 (59%) had an appendectomy and 115 (41%) had a right-sided hemicolectomy, 110 (40%) were men, 168 (60%) were women, and mean age at initial surgery was 36·0 years (SD 18·2). Median follow-up was 13·0 years (IQR 11·0-15·6). After centralised histopathological review, appendiceal NETs were classified as a possible or probable primary tumour in two (1%) of 278 patients with distant peritoneal metastases and in two (1%) 278 patients with distant metastases in the liver. All metastases were diagnosed synchronously with no tumour-related deaths during follow-up. Regional lymph node metastases were found in 22 (20%) of 112 patients with right-sided hemicolectomy with available data. On the basis of histopathological risk factors, we estimated that 12·8% (95% CI 6·5 -21·1) of patients undergoing appendectomy probably had residual regional lymph node metastases. Overall survival was similar between patients with appendectomy and right-sided hemicolectomy (adjusted hazard ratio 0·88 [95% CI 0·36-2·17]; p=0·71). INTERPRETATION This study provides evidence that right-sided hemicolectomy is not indicated after complete resection of an appendiceal NET of 1-2 cm in size by appendectomy, that regional lymph node metastases of appendiceal NETs are clinically irrelevant, and that an additional postoperative exclusion of metastases and histopathological evaluation of risk factors is not supported by the presented results. These findings should inform consensus best practice guidelines for this patient cohort. FUNDING Swiss Cancer Research foundation.

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field.In the present study, we tested an innovative fixative, Glyoxal Acid-Free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard Phosphate Buffered Formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local centre reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ±0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ±0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organ tested. The present clinical performance study, performed following the international standard for performance evaluation ofin vitrodiagnostic medical devices, highlights the capability of GAF to ensure both, structural preservation and diagnostic value of the preparations.

PDF file - 674K, Histological characterization of patients' carcinomas and the correspondent xenograft tumors.

Supplementary Figures S1-S11. Figure S1. Apoptosis of CRC patient-derived cells upon treatment with API-2 or NVP-BKM120 in combination with XAV939. Figure S2. Evaluation of apoptosis in primary sphere cell cultures by immunofluorescent quantification of cleaved-CASPASE3. Figure S3. Frequent mutations in CRC do not determine the response to API2, NVP-BKM120 or NVP-TNKS656. Figure S4. NVP-TNKS656 is not toxic and reduces nuclear β-catenin in the skin and small intestine in NOD-SCID mice. Figure S5. Analyses of pharmacodynamic markers at endpoint of in vivo experiments with API2 and NVP-TNKS656. Figure S6. Nuclear FOXO3A content is preserved in PDX and primary sphere cultures. Figure S7. Nuclear FOXO3A in paired primary tumors and liver metastases. Figure S8. Immunohistochemical staining of β-catenin in CRC tumors. Figure S9. β-catenin in single-agent treatments, relevant mutations, TNM stage or tumor site do not associate with a differential response to PI3K/AKT/mTOR inhibitors. Figure S10. Gene expression clustering and mutational status of baseline tumor samples from patients treated with PI3K/AKT/mTOR inhibitors. Figure S11. Molecular prescreening scheme to select CRC patients eligible for the treatment with PI3K/AKT and/or Wnt/β-catenin inhibitory drugs.

PDF file - 115K, 18-FDG is the best radiotracer for tumor growth evaluation.

Supplementary Tables S1-S15. Table S1. Genes and positions analyzed by amplicon sequencing. Table S2. Genes sequenced by Haloplex Target Enrichment System. Table S3. Antibodies. Table S4. Treatment-induced apoptosis in 10 primary patient-derived sphere cell cultures. Table S5. Genotype of patient-derived colorectal cancer models. Table S6. Exome sequencing of colorectal cancer patient-derived models. Table S7. Genes regulated by NVP-TNKS656 in PDX-P2. Table S8. Genes regulated by NVP-TNKS656 in PDX-P30. Table S9. Genes regulated by NVP-TNKS656 in PDX-P5. Table S10. Intestinal β-catenin/TCF signature in human CRC. Table S11. Genes regulated by β-catenin/FOXO3A signaling in human CRC. Table S12. Molecular profiling of 130 CRC tumors. Table S13. Clinicopathological data of 40 CRC patients treated with PI3K/AKT/mTOR inhibitors and their corresponding baseline tumor samples analyzed for mutations and nuclear β-catenin content. Table S14. Nuclear β-catenin content but not relevant mutations or TNM stage determine response of CRC patients to PI3K/AKT/mTOR inhibitors. Table S15. Multivariate analysis of baseline tumors from patients treated with PI3K/AKT/mTOR inhibitors.

Purpose: Oncogenic mutations in the KRAS/PI3K/AKT pathway are one of the most frequent alterations in cancer. Although PI3K or AKT inhibitors show promising results in clinical trials, drug resistance frequently emerges. We previously revealed Wnt/β-catenin signaling hyperactivation as responsible for such resistance in colorectal cancer. Here we investigate Wnt-mediated resistance in patients treated with PI3K or AKT inhibitors in clinical trials and evaluate the efficacy of a new Wnt/tankyrase inhibitor, NVP-TNKS656, to overcome such resistance.Experimental Design: Colorectal cancer patient-derived sphere cultures and mouse tumor xenografts were treated with NVP-TNKS656, in combination with PI3K or AKT inhibitors.We analyzed progression-free survival of patients treated with different PI3K/AKT/mTOR inhibitors in correlation with Wnt/β-catenin pathway activation, oncogenic mutations, clinicopathological traits, and gene expression patterns in 40 colorectal cancer baseline tumors.Results: Combination with NVP-TNKS656 promoted apoptosis in PI3K or AKT inhibitor-resistant cells with high nuclear β-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear β-catenin content and progressed earlier upon PI3K/AKT/mTOR inhibition. Nuclear β-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not.Conclusions: High nuclear β-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear β-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/β-catenin inhibition and together with β-catenin may be predictive biomarkers of drug response providing a rationale to stratify colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/β-catenin inhibitors. Clin Cancer Res; 22(3); 644–56. ©2015 AACR.

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Purpose: Within the aim of advancing precision oncology, we have generated a collection of patient-derived xenografts (PDX) characterized at the molecular level, and a preclinical model of colon cancer metastasis to evaluate drug-response and tumor progression.Experimental Design: We derived cells from 32 primary colorectal carcinomas and eight liver metastases and generated PDX annotated for their clinical data, gene expression, mutational, and histopathological traits. Six models were injected orthotopically into the cecum wall of NOD-SCID mice in order to evaluate metastasis. Three of them were treated with chemotherapy (oxaliplatin) and three with API2 to target AKT activity. Tumor growth and metastasis progression were analyzed by positron emission tomography (PET).Results: Patient-derived cells generated tumor xenografts that recapitulated the same histopathological and genetic features as the original patients' carcinomas. We show an 87.5% tumor take rate that is one of the highest described for implanted cells derived from colorectal cancer patients. Cecal injection generated primary carcinomas and distant metastases. Oxaliplatin treatment prevented metastasis and API2 reduced tumor growth as evaluated by PET.Conclusions: Our improved protocol for cancer cell engraftment has allowed us to build a rapidly expanding collection of colorectal PDX, annotated for their clinical data, gene expression, mutational, and histopathological statuses. We have also established a mouse model for metastatic colon cancer with patient-derived cells in order to monitor tumor growth, metastasis evolution, and response to treatment by PET. Our PDX models could become the best preclinical approach through which to validate new biomarkers or investigate the metastatic potential and drug-response of individual patients. Clin Cancer Res; 19(24); 6787–801. ©2013 AACR.

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Supplementary Data from Aprataxin Tumor Levels Predict Response of Colorectal Cancer Patients to Irinotecan-based Treatment

PDF file - 1113K, Patient-derived cells preserve pluripotency.

Supplementary Figure 1

XLSX file - 768K, Nanostring CRC codeset.

Supplementary Figure Legends

Supporting Information. Supplementary methods to complete the information for gene expression, genotyping, immunohistochemistry evaluation criteria and protein extraction. The file also contains legends for supplementary figures S1-S11 and supplementary tables S1-S15.

PDF file - 248K, Distant metastasis generated by cells derived from patient 5.

Purpose: Irinotecan (CPT11) treatment significantly improves the survival of colorectal cancer patients and is routinely used for the treatment of these patients, alone or in combination with other agents. However, only 20% to 30% of patients show an objective response to irinotecan, and there is great need for new molecular markers capable of identifying the subset of patients who are unlikely to respond.Experimental Design: Here we used microarray analysis of a panel of 30 colorectal cancer cell lines and immunohistochemistry to identify and validate a new biomarker of response to irinotecan.Results: A good correlation was observed between irinotecan sensitivity and the expression of aprataxin (APTX), a histidine triad domain superfamily protein involved in DNA repair. Moreover, using an isogenic in vitro system deficient in APTX, we show that aprataxin directly regulates the cellular sensitivity to camptothecin, suggesting that it could be used to predict patient response to irinotecan. We constructed a tissue microarray containing duplicate tumor samples from 135 patients that received irinotecan for the treatment of advanced colorectal cancer. Immunohistochemical assessment of the tumor levels of aprataxin showed a significant association with treatment response and patient survival. Patients with low aprataxin had longer progression-free (9.2 versus 5.5 months; P = 0.03) and overall survival (36.7 versus 19.0 months; P = 0.008) than patients with high tumor aprataxin. No associations were found between coding APTX variants and aprataxin levels or camptothecin sensitivity.Conclusions: These results show that aprataxin tumor levels can be used to identify patients with low probability of response to irinotecan-based therapy who are ideal candidates to receive treatment with alternative agents in an attempt to improve patient survival. Clin Cancer Res; 16(8); 2375–82. ©2010 AACR.

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Purpose:The limited knowledge of the molecular alterations that characterize poorly differentiated neuroendocrine carcinomas has limited the clinical development of targeted agents directed to driver mutations. Here we aim to identify new molecular targets in colon neuroendocrine carcinomas (co-NEC) and proof the efficacy of matching drugs.Experimental Design:We performed a multi-omic analysis of co-NEC to identify genetic or epigenetic alterations that could be exploited as effective drug targets. We compared co-NEC samples with colorectal carcinomas (CRC) to identify neuroendocrine-specific traits. Patients with co-NEC and patient-derived xenografts were treated with a BRAFV600E-blocking drug to demonstrate sensitivity.Results:co-NEC and CRC are similar in their mutational repertoire, although co-NECs are particularly enriched in BRAFV600E mutations. We report for the first time that V600EBRAF-mutant co-NECs may benefit from BRAF inhibition in monotherapy and how EGFR status is essential to predict innate sensitivity and acquired resistance by a differential methylation of its gene regulatory regions.Conclusions:The identification of V600E BRAF mutations in high-grade co-NECs has allowed the description of radiological responses to combination therapy of BRAF and MEK inhibitors in basket clinical trials. However, the molecular rationale for this treatment combination was based on the presence of the BRAF mutation and the efficacy observed in other cancer types such as melanoma. Future drug development in this setting should test BRAF inhibitors upfront and the addition of anti-EGFR antibodies instead of MEK inhibitors for an efficient blockade of acquired resistance.

PDF file - 290K, Histopathological and genetic traits of CRC PDX.

Supplementary Figures 1-6, Tables 1-6 from The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis

Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.

Stage II colorectal cancer (CRC) recurrence remains a clinical problem. Some of these patients are true stage III CRC with a pN0 pathology stage. This large prospective multicentre cohort study aimed at evaluating the diagnostic ability of lymph node (LN) cytology smears to perform the pN stage and compare it with the conventional haematoxylin and eosin (H&E) pathology pN stage. Additionally, we used the One-Step Nucleic Acid Amplification (OSNA), a high-sensitive molecular method of LN staging. A total of 3936 fresh LNs from 217 CRC surgical specimens were examined by three methods, H&E, LN cytology smears, and OSNA. H&E detected 29% of patients with positive LNs, cytology smears 35%, and OSNA 33.2% (p < 0.0001). H&E and cytology concordantly classified 92.2% of tumours, and 88.5% between OSNA and HΕ Cytology had 96.8% sensitivity and 90.3% specificity to discriminate positive/negative patients compared to H&E (p = 0.004), and 87.3% sensitivity and 89% specificity when compared to OSNA (p = 0.56). Patients with positive LNs detected by any of the three methods had significantly worse disease-free and overall survival. We conclude that pN stage accuracy for detecting positive LNs is superior with LN cytological smears than with conventional H&E, which would enable a better pN stage and management of early-stage CRC patients. This research was funded by Fondo de Investigación Sanitaria grant number PI17/01304, PI20/00863, awarded to MC and JC. We acknowledge the Agència de Gestió d’Ajuts Universitaris i de Recerca (Generalitat de Catalunya, GRC 2017SGR653,). This article is based upon work from COST Action CA17118, supported by COST (European Cooperation in Science and Technology). www.cost.eu. SL holds a PFIS grand from Instituto de Salud Carlos iii and co-funded by the European Regional Development Fund (ERDF) (FI18/00221).

Single immune checkpoint blockade in advanced neuroendocrine neoplasms (NENs) shows limited efficacy; dual checkpoint blockade may improve treatment activity. Dune is a non-randomized controlled multicohort phase II clinical trial evaluating durvalumab plus tremelimumab activity and safety in advanced NENs. This study included 123 patients presenting between 2017 and 2019 with typical/atypical lung carcinoids (Cohort (C) 1), G1/2 gastrointestinal (C2), G1/2 pancreatic (C3) and G3 gastroenteropancreatic (GEP) (C4) NENs; who progressed to standard therapies. Patients received 1500 mg durvalumab and 75 mg tremelimumab for up to 13 and 4 cycles (every 4 weeks), respectively. The primary objective was the 9-month (m) disease control rate (DCR) for C1-3 and 9-m overall survival (OS) rate for C4. Median follow-up was 16.5 m; 9-m DCR was 25.9%/35.5%/25% for C1, 2, and 3 respectively. The 9-m OS rate for C4 was 36.1%. Benefit in C4 was observed regardless of differentiation and Ki67 levels. PD-L1 combined scores did not correlate with treatment activity. Safety profile was consistent with that of prior studies. In conclusion, durvalumab plus tremelimumab was safe in NENs and showed modest survival benefit in G3 GEP-NENs; with one-third of these patients experiencing a prolonged OS.

There is no standardized treatment for grade 3 neuroendocrine tumors (G3 NETs). We aimed to describe the treatments received in patients with advanced G3 NETs and compare their efficacy. Patients with advanced digestive G3 NETs treated between 2010 and 2018 in seven expert centers were retrospectively studied. Pathological samples were centrally reviewed, and radiological data were locally reviewed. We analyzed RECIST-defined objective response (OR), tumor growth rate (TGR) and progression-free survival (PFS) obtained with first- (L1) or second-line (L2) treatments. We included 74 patients with advanced G3 NETs, mostly from the duodenal or pancreatic origin (71.6%), with median Ki-67 of 30%. The 126 treatments (L1 = 74; L2 = 52) included alkylating-based (n = 32), etoposide-platinum (n = 22) or adenocarcinoma-like (n = 20) chemotherapy, somatostatin analogs (n = 21), targeted therapies (n = 22) and liver-directed therapies (n = 7). Alkylating-based chemotherapy achieved the highest OR rate (37.9%) compared to other treatments (multivariable OR 4.22, 95% CI (1.5–12.2); P = 0.008). Adenocarcinoma-like and alkylating-based chemotherapies showed the highest reductions in 3-month TGR (P < 0.001 and P = 0.008, respectively). The longest median PFS was obtained with adenocarcinoma-like chemotherapy (16.5 months (9.0–24.0)) and targeted therapies (12.0 months (8.2–15.8)), while the shortest PFS was observed with somatostatin analogs (6.2 months (3.8–8.5)) and etoposide-platinum chemotherapy (7.2 months (5.2–9.1)). Etoposide-platinum CT achieved shorter PFS than adenocarcinoma-like (multivariable HR 3.69 (1.61–8.44), P = 0.002) and alkylating-based chemotherapies (multivariable HR 1.95 (1.01–3.78), P = 0.049). Overall, adenocarcinoma-like and alkylating-based chemotherapies may be the most effective treatments for patients with advanced G3 NETs regarding OR and PFS. Etoposide-platinum chemotherapy has poor efficacy in this setting.

Resumen En esta actualizacion del consenso de la Sociedad Espanola de Oncologia Medica (SEOM) y la Sociedad Espanola de Anatomia Patologica (SEAP) se revisan los avances producidos en el analisis de biomarcadores en cancer colorrectal (CCR) avanzado, asi como en los marcadores de susceptibilidad del CCR hereditario y los biomarcadores moleculares del CCR localizado. Tambien se evaluan la informacion publicada recientemente sobre la determinacion imprescindible de las mutaciones de KRAS, NRAS y BRAF y la conveniencia de determinar la amplificacion del receptor del factor de crecimiento epidermico 2 (HER2), la expresion de las proteinas de la via reparadora de ADN y el estudio de las fusiones de NTRK. Desde el punto de vista anatomopatologico, se revisa la importancia de analizar la presencia de celulas tumorales aisladas o en pequenos grupos de menos de 5 en el frente invasivo tumoral del CCR y su valor pronostico en el CCR. Tambien se revisa la incorporacion de tecnologias pangenomicas, como la secuenciacion de nueva generacion (next-generation sequencing [NGS]) y la biopsia liquida, en el manejo clinico del paciente con CCR. Todos estos aspectos se desarrollan en la presente guia que, como la anterior, permanecera abierta a cualquier revision necesaria en el futuro.

Summary The diagnosis of microscopic colitis (MC) relies on specific histopathological findings in colon biopsies. The number of biopsies needed to diagnose MC remains disputed. The aim of the study was to determine the number and site of biopsies necessary for the diagnosis and the effect of perpendicular orientation when embedding the biopsies. This retrospective multicenter European study included 42 patients with a consensus diagnosis of collagenous colitis (CC), 51 patients with lymphocytic colitis (LC), and three patients with incomplete LC (LCi). The number of individual diagnostic biopsies from each patient was determined. The diagnostic rate of 744 individual biopsies from 96 patients with MC was 69.5% for the specific MC subgroup, 79.4% for MC and 93.4% for MC plus incomplete MC (MCi). The risk of missing a diagnosis of the specific subgroup of MC when analyzing four biopsies was 0.87%, decreasing to 0.18% for MC and 0.0019% for MC plus MCi. More biopsies from the right colon were diagnostic of the specific MC subgroup (76.3% vs. 64.0%, p = 0.0014). Perpendicular orientation of biopsies increased the diagnostic rate of the specific MC subgroup (73.1% vs. 65.0%, p = 0.0201). Histological changes diagnostic of MC were present in almost all biopsies from the right colon, with orientated biopsies more often being diagnostic of the specific MC subgroup. The results of this study indicate that four biopsies from the colon, rectum excluded, are sufficient to diagnose MC.

Purpose: The limited knowledge of the molecular alterations that characterize poorly differentiated neuroendocrine carcinomas has limited the clinical development of targeted agents directed to driver mutations. Here we aim to identify new molecular targets in colon neuroendocrine carcinomas (co-NEC) and proof the efficacy of matching drugs. Experimental Design: We performed a multi-omic analysis of co-NEC to identify genetic or epigenetic alterations that could be exploited as effective drug targets. We compared co-NEC samples with colorectal carcinomas (CRC) to identify neuroendocrine-specific traits. Patients with co-NEC and patient-derived xenografts were treated with a BRAFV600E-blocking drug to demonstrate sensitivity. Results: co-NEC and CRC are similar in their mutational repertoire, although co-NECs are particularly enriched in BRAFV600E mutations. We report for the first time that V600EBRAF-mutant co-NECs may benefit from BRAF inhibition in monotherapy and how EGFR status is essential to predict innate sensitivity and acquired resistance by a differential methylation of its gene regulatory regions. Conclusions: The identification of V600E BRAF mutations in high-grade co-NECs has allowed the description of radiological responses to combination therapy of BRAF and MEK inhibitors in basket clinical trials. However, the molecular rationale for this treatment combination was based on the presence of the BRAF mutation and the efficacy observed in other cancer types such as melanoma. Future drug development in this setting should test BRAF inhibitors upfront and the addition of anti-EGFR antibodies instead of MEK inhibitors for an efficient blockade of acquired resistance.

Biomarcadores del cáncer; Cáncer metástico colorrectal; Terapias experimentales Cancer biomarkers; Colorectal metastatic cancer; Experimental therapies Biomarcadors del càncer; Càncer metastàtic colorrectal; Teràpies experimentals Protein biomarkers are widely used in cancer diagnosis, prognosis, and prediction of treatment response. Here we introduce the use of targeted multiplex proteomics (TMP) as a tool to simultaneously measure a panel of 54 proteins involved in oncogenic, tumour suppression, drug metabolism and resistance, in patients with metastatic colorectal cancer (mCRC). TMP provided valuable diagnostic information by unmasking an occult neuroendocrine differentiation and identifying a misclassified case based on abnormal proteins phenotype. No significant differences in protein levels between unpaired primary and metastatic samples were observed. Four proteins were found differentially expressed in KRAS-mutant as compared to wild-type tumours (overexpressed in mutant: KRAS, EGFR; overexpressed in wild-type: TOPO1, TOP2A). Survival analyses revealed the association between mesothelin expression and poor overall survival, whereas lack of PTEN protein expression associated with lower progression-free survival with anti-EGFR-based therapy in the first-line setting for patients with RAS wild-type tumour. Finally, outlier analysis identified putative targetable proteins in 65% of patients lacking a targetable genomic alteration. Our data show that TMP constitutes a promising, novel molecular prescreening tool in mCRC to identify protein expression alterations that may impact on patient outcomes and more precisely guide patient eligibility to clinical trials with novel targeted experimental therapies.

Gastric cancer constitutes the third leading cause of cancer mortality worldwide and tumors with diffuse histology have poor prognosis and low response rates to therapy. Missense mutations in the RHOA (Ras homolog family member A) GTPase have recently been identified in 24% of patients with diffuse gastric cancer. RHOA mutations are not randomly distributed along the coding sequence, but enriched in specific and recurrent hotspots. Substitution in codon 42 (Y42C) occurs in approximately 50% of the patients in which RHOA is mutated. RHOA orchestrates cellular processes such as proliferation, differentiation, migration and invasion, whose deregulation is essential for the onset, maintenance and progression of tumors. However, the functional role of wild type RHOA and RHOA mutants remains poorly characterized in gastric cancer. We used isogenic cell lines systems and animal models to study the impact of RHOA in the gastric tumorigenesis. RHOA loss strongly increased the proliferation, migration and invasion of diffuse gastric cancer cells in vitro, but also when growing as subcutaneous xenografts in immunodeficient mice, and in experimental mouse models of peritoneal and lung metastasis. Stomach tumor burden was also increased in transgenic mice conditionally expressing a dominant negative form of RHOA in the gastrointestinal tract (RHOA T19N). Moreover, cells overexpressing wild-type RHOA or G14V RHOA (a constitutive active form of the GTPase) exhibited reduced tumorigenic features both in vitro and in vivo. Conversely, diffuse gastric cancer cells engineered for overexpressing RHOA Y42C mutant, displayed enhanced growth and invasion in vitro. Importantly, stomach cancer tumors formed more efficiently in a transgenic mouse model in which we targeted the expression of Y42C RHOA mutant to the gastric epithelium. Collectively, these results demonstrate that RHOA has tumor-suppressive activity in diffuse gastric cancer, while the recurrent Y42C hotspot RHOA mutation found in these tumors is oncogenic. Citation Format: Agueda Martinez-Barriocanal, Higinio Dopeso, Lizbeth M. Jiménez-Flores, Juliana C. Santos, Estefania Anguita, Josipa Bilic, Rocio Nieto, Elia García-Vidal, Manuel Sánchez Martín, Stefania Landolfi, Kazuto Kobayashi, Javier Hernandez-Losa, Simo Schwartz, Santiago Ramón y Cajal, Diego Arango. Role of RHOA in diffuse gastric cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 847.

BACKGROUND Trichloroacetic acid (TCA) and electrocautery ablation (ECA) are 2 of the main treatment options for anal high-grade squamous intraepithelial lesion (HSIL). Our aim was to compare the efficacy and tolerance of TCA vs. ECA for HSIL. METHODS Retrospective uncontrolled study of HIV-infected men who have sex with men who had an anal HSIL treated with TCA or ECA. On-treatment effectiveness was evaluated at 6-8 weeks after treatment. A complete response was defined as resolution of HSIL, a partial response as regression to low-grade lesion, and recurrence as biopsy-proven HSIL during follow-up. A propensity-score analysis was used to adjust efficacy to potential confounding. RESULTS From May 2009 to March 2018, 182 and 56 cases of anal HSIL were treated with ECA and TCA, respectively. Comparing ECA with TCA, a complete response was observed in 33.5% (95% confidence interval: 25.8 to 41.6) vs. 60.7% (50.0 to 74.8) and a partial response in 28.0% (20.3 to 36.0) vs. 23.2% (12.5 to 37.3), respectively (P < 0.001). These differences were maintained in the propensity-score analyses. Side effects were common in both treatment, but tolerance was reported as good in 80.6% (74.2 to 89.2) and 82.6% (73.9 to 93.9) of cases treated with ECA and TCA, respectively, and no serious events were described. Recurrence cumulative incidence for the first 12 months was 14.6% (9.1 to 23.1) for ECA episodes and 27.6% (11.5 to 57.7) for TCA (P = 0.183). CONCLUSIONS Our study showed a higher efficacy of TCA than ECA with similar rates of side effects. In our opinion, considering the benefits of TCA, it should be considered as a first-line therapy for most anal HSIL management.

Fusobacterium nucleatum; Càncer de recte avançat local; Quimioteràpia preoperatòria Fusobacterium nucleatum; Cáncer de recto avanzado local; Quimiorradioterapia preoperatoria Fusobacterium nucleatum; Locally advanced rectal cancer; Preoperative chemoradiotherapy Background Accumulating evidence has identified Fusobacterium as an important pathogenic gut bacterium associated with colorectal cancer. Nevertheless, only limited data exist about the role of this bacterium in locally advanced rectal cancer (LARC). In this study, we quantified Fusobacterium nucleatum in untreated and post-neoadjuvant chemoradiotherapy (nCRT) samples from LARC patients and investigated its association with therapy response and survival. Patients and methods A total of 254 samples from 143 patients with rectal adenocarcinomas were analyzed for the presence and abundance of F. nucleatum using RNA in situ hybridization and digital image analysis. Assay accuracy was determined using infected cell lines and tumor samples with available quantitative PCR data. We studied the impact of F. nucleatum load on pathologic complete response and relapse-free survival. Treatment-induced changes were evaluated in paired pre- and post-nCRT samples (n = 71). Finally, tumor microenvironment changes during nCRT were assessed in paired samples (n = 45) by immune contexture analysis. Results F. nucleatum tissue levels by RNA in situ hybridization strongly correlated with quantitative PCR (r = 0.804, P < 0.001). F. nucleatum abundance was higher in untreated [median, 7.4; 95% confidence interval (3.7–16.2)] compared with treated [median, 1.6; 95% confidence interval (1.3–2.4)] tumors (P

[Background] Gastrointestinal stromal tumor (GIST) initiation and evolution is commonly framed by KIT/PDGFRA oncogenic activation, and in later stages by the polyclonal expansion of resistant subpopulations harboring KIT secondary mutations after the onset of imatinib resistance. Thus, circulating tumor (ct)DNA determination is expected to be an informative non-invasive dynamic biomarker in GIST patients., [Methods] We performed amplicon-based next-generation sequencing (NGS) across 60 clinically relevant genes in 37 plasma samples from 18 GIST patients collected prospectively. ctDNA alterations were compared with NGS of matched tumor tissue samples (obtained either simultaneously or at the time of diagnosis) and cross-validated with droplet digital PCR (ddPCR)., [Results] We were able to identify cfDNA mutations in five out of 18 patients had detectable in at least one timepoint. Overall, NGS sensitivity for detection of cell-free (cf)DNA mutations in plasma was 28.6%, showing high concordance with ddPCR confirmation. We found that GIST had relatively low ctDNA shedding, and mutations were at low allele frequencies. ctDNA was detected only in GIST patients with advanced disease after imatinib failure, predicting tumor dynamics in serial monitoring. KIT secondary mutations were the only mechanism of resistance found across 10 imatinib-resistant GIST patients progressing to sunitinib or regorafenib., [Conclusions] ctDNA evaluation with amplicon-based NGS detects KIT primary and secondary mutations in metastatic GIST patients, particularly after imatinib progression. GIST exhibits low ctDNA shedding, but ctDNA monitoring, when positive, reflects tumor dynamics., This research is supported by a Fero Fellowship Award (C.S.), Asociación Española Contra el Cáncer (J.P. Barcelona) (C.S.), and ISCIII PI16/01371 (C.S.). C.S. and A.V. acknowledge to the Cellex Foundation for providing facilities and equipment.

Microscopic colitis (MC) comprising lymphocytic colitis (LC), collagenous colitis (CC) and the incomplete forms of microscopic colitis (MCi) are frequent causes of chronic watery diarrhea. The diagnosis is based on specific histological features in colonic biopsies. Especially regarding MCi, the histological features may be subtle. The PRO-MC collaboration was established in 2016 with the aims to systematically describe the disease course and to validate the diagnostic criteria of MC. In the present study, we analysed pathologists' initial approach to diagnose MC. Five pathologists with expertise in gastro-intestinal pathology reviewed the first 10 cases enrolled in the PRO-MC registry in six of the participating centres. Despite considerable differences in strategies in biopsy sampling, in choice of stains and in minimum number of biopsies and segments required for diagnosing MC, inter-observer agreement between the participating centres and expert pathologists as well as among the latter was substantial. Disagreed cases most often related to difficulties in distinguishing between MC subgroups. We recommend that pathologists as well as clinicians reach consensus in their diagnostic approach to MC, which is a prerequisite to compare MC cohorts internationally and to facilitate clinical MC trials and follow-up studies.

Introduction Microscopic colitis is a chronic inflammatory bowel disease characterised by normal or almost normal endoscopic appearance of the colon, chronic watery, non-bloody diarrhoea and distinct histological abnormalities, which identify three histological subtypes, the collagenous colitis, the lymphocytic colitis and the incomplete microscopic colitis. With ongoing uncertainties and new developments in the clinical management of microscopic colitis, there is a need for evidence-based guidelines to improve the medical care of patients suffering from this disorder. Methods Guidelines were developed by members from the European Microscopic Colitis Group and United European Gastroenterology in accordance with the Appraisal of Guidelines for Research and Evaluation II instrument. Following a systematic literature review, the Grading of Recommendations Assessment, Development and Evaluation methodology was used to assess the certainty of the evidence. Statements and recommendations were developed by working groups consisting of gastroenterologists, pathologists and basic scientists, and voted upon using the Delphi method. Results These guidelines provide information on epidemiology and risk factors of microscopic colitis, as well as evidence-based statements and recommendations on diagnostic criteria and treatment options, including oral budesonide, bile acid binders, immunomodulators and biologics. Recommendations on the clinical management of microscopic colitis are provided based on evidence, expert opinion and best clinical practice. Conclusion These guidelines may support clinicians worldwide to improve the clinical management of patients with microscopic colitis. Funding Agencies|UEG Activity Grant