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In a previous study on esophageal adenocarcinoma (EAC) cases submitted to surgery (without neoadjuvant treatment), an algorithm (EACGSE classification) 1 based on morphologic distinctions provided significant prognostic impact. Tumor budding is a specific type of invasive growth pattern and was found to be an important prognostic parameter in gastrointestinal cancers. 2–3 We aimed to evaluate the prevalence of budding in glandular adenocarcinomas and its impact on survival. The EAC cohort classified according to EACGSE 1 was included in the study: among the different histotypes, 215 cases, classified as well (WD) and poorly (PD) differentiated glandular adenocarcinomas, were considered. Tumor budding was defined as single cells or clusters of up to four cells at the invasive margin. Budding was grouped according to ITBCC 2 into Bd1 (0-4 buds), Bd2 (5-9 buds), and Bd3 (10 or more buds). Furthermore, the most prognostically relevant cut off for bud number in EAC was defined by ROC analysis. Cancer specific survival analyses were performed using the Kaplan–Meier method and Cox regression analyses. Tumor budding was assessed in one hotspot (a field measuring 0.785 mm2) at the invasive front and ROC analysis identified 8 buds per field cut off as the most informative in relation to survival. By using this cut off, frequency of budding was higher in glandular PD (84/124,68%) cases than in WD ones (7/91,8%, p Tumor budding showed a prognostically negative impact in glandular EACs. The 8 buds per field cut off appears to provide the best prognostic discriminant. Tumor budding assessment should be added to the identification of other prognostic factors already described in the same series (stage, histotype and vascular invasion). 1

The incidence of esophageal adenocarcinoma (EAC) has constantly risen in western countries over the past decades, often diagnosed at advanced stage and with a five-year survival around 20%1. In a previous study on EAC cases submitted to surgery (without neoadjuvant treatment) an algorithm (EACGSE classification)2 based on morphologic distinctions provided a significant prognostic impact. We aimed to evaluate the molecular basis underlying these differences, to improve patient management. The EAC cohort classified according to EACGSE2 was included in the study. Genomic DNA was available from formalin fixed paraffin embedded surgical specimens for 207 cases. The cases were sequenced for 26 cancer-related genes (panel #226722257; IDT) with high coverage on NextSeq500 (Illumina). Data analysis was performed using a dedicated pipeline3. 245 cases were analyzed for SMAD4 immunostaining (IHC). Loss of SMAD4 immunostaining was reported as % of negative tumor cells (at least 35% of neoplastic cells). TP53 was the most frequently altered gene (134/207 cases with at least one mutation). The presence of TP53 missense variants was associated to a poor cancer-specific survival (CSS) in the high-risk cases (glandular poorly differentiated, mucinous invasive, diffuse anaplastic, mixed) according to EACGSE2 (P=0.0005). A significant correlation was observed for TP53 truncative variants and loss of SMAD4 staining (P=0.008). Actually, SMAD4 loss was observed in 85/245 (35%) of all EAC cases analyzed via immunostaining. SMAD4 loss correlated with poor CCS (P=0.007) and disease-free survival (P=0.002) in EACGSE high-risk cases. TP53 missense mutations correlated to poor outcomes (CSS) in high-risk cases, TP53 truncative mutations were associated to SMAD4 loss. SMAD4 loss itself resulted a frequent event in EAC and correlated with lower CCS and disease-free survival in high-risk cases. Therefore, we were able to correlate EAC histological classification, clinical outcomes and molecular phenotypes. Validation in independent samples is warranted to corroborate these findings. References 1. Coleman et al. Gastroenterology2018;154:390–405. 2. Fiocca et al. Cancers 2021;13:5211. 3. Isidori et al. CTG 202011:e00202.

Many hereditary cancer syndromes are associated with an increased risk of small and large intestinal adenocarcinomas. However, conditions bearing a high risk to both adenocarcinomas and neuroendocrine tumors are yet to be described. We studied a family with 16 individuals in four generations affected by a wide spectrum of intestinal tumors, including hyperplastic polyps, adenomas, small intestinal neuroendocrine tumors, and colorectal and small intestinal adenocarcinomas. To assess the genetic susceptibility and understand the novel phenotype, we utilized multiple molecular methods, including whole genome sequencing, RNA sequencing, single cell sequencing, RNA in situ hybridization and organoid culture. We detected a heterozygous deletion at the cystic fibrosis locus (7q31.2) perfectly segregating with the intestinal tumor predisposition in the family. The deletion removes a topologically associating domain border between CFTR and WNT2, aberrantly activating WNT2 in the intestinal epithelium. These consequences suggest that the deletion predisposes to small intestinal neuroendocrine tumors and small and large intestinal adenocarcinomas, and reveals the broad tumorigenic effects of aberrant WNT activation in the human intestine.

The cyclooxygenase-2 (COX-2) enzyme, which is induced by inflammatory and mitogenic stimuli, plays a protumorigenic role in several human cancers. COX-2 is overexpressed in invasive and in situ breast cancers. Atypical hyperplasia in breast tissue, although benign, is associated with a high risk of breast cancer. We investigated whether COX-2 overexpression in atypical hyperplasia is associated with the risk of subsequent breast cancer.COX-2 expression was assessed immunohistochemically in archival sections from 235 women with atypia whose biopsy specimens were obtained at the Mayo Clinic from January 1, 1967, through December 31, 1991. COX-2 expression was scored as 0 (negative), 1+ (weak), 2+ (moderate), or 3+ (strong). Risk factor information and follow-up for breast cancer events were obtained via a study questionnaire and the medical records. All statistical tests were two-sided.Forty-one (17%) of the 235 women developed breast cancer during a median follow-up of 15 years. Moderate (category 2+) or strong (category 3+) COX-2 expression was identified in 71 (30%) and 34 (14%) of the 235 samples, respectively. The risk for developing breast cancer, relative to a control population (the Iowa Surveillance, Epidemiology, and End Results registry), increased with increasing COX-2 expression (relative risk [RR] = 2.63, 95% confidence interval [CI] = 1.56 to 4.15, for those with negative or weak COX-2 expression; RR = 3.56, 95% CI = 1.94 to 5.97, for those with moderate expression; and RR = 5.66, 95% CI = 2.59 to 10.75, for those with strong expression; P = .07). Overexpression of COX-2 was statistically significantly associated with the type of atypia (lobular vs ductal, P.001), number of foci of atypia in the biopsy (P = .02), and older age at time of biopsy (45 years, P = .01).COX-2 appears to be a biomarker that further stratifies breast cancer risk among women with atypia and may be a relevant target for chemoprevention strategies.