Background: Transanal excision of rectal cancer can be considered the definitive surgical treatment if the depth spread is T1 or lower, and the lesion is completely included within the resection margin. This study aims to analyze the immune microenvironment in healthy rectal mucosa as a possible predictor of tumor infiltration depth, lateral tumor spread, and recurrence of rectal cancer after transanal local excision. Methods: This study is a subanalysis of data from the IMMUNOREACT 1 and 2 trials (NCT04915326 and NCT04917263, respectively) including all the patients who underwent transanal excision of rectal cancer. This multicentric study collected healthy mucosa surrounding the neoplasms of patients with rectal cancer. A panel of immune markers was investigated at immunohistochemistry: CD3, CD4, CD8, CD8(3, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. Flow cytometry determined the proportion of epithelial cells expressing CD80, CD86, CD40, HLA ABC or HLA DR and the proportion of activated CD8+ T cells, CD4+ Th1 cells, and Treg. Results: Receiver operating characteristic curve analysis for predicting deep tumor spread showed an area under the curve of 0.70 (95% confidence interval: 0.60-0.80) for CD25+FoxP3+ cell rate and 0.74 (95% confidence interval: 0.53-0.92) for CK+CD86+ cell rate. Receiver operating characteristic curve analysis for predicting lateral tumor spread showed an area under the curve of 0.82 (95% confidence interval: 0.61-0.99) for CD8+CD38+ MFI, 0.96(95% confidence interval: 0.85-0.99) for CD8(3 infiltration, and 0.97 (95% confidence interval: 0.87-0.99) for CK+HLAabc+ cell rate. Receiver operating characteristic curve analysis for predicting recurrence showed an area under the curve of 0.93 (95% confidence interval: 0.76-0.99) for CD8+CD38+ MFI and 0.94 (95% confidence interval: 0.78-0.99) for CD8+CD28+ MFI. Low CD8+CD38+ MFI and low CD8+CD28+ MFI were associated with shorter disease-free survival (P = .025 and P = .021, respectively). Conclusion: Our study showed that the association between the high proportion of epithelial cells acting as presenting cells and deep or lateral tumor spread may be explained by the presence of a greater tumor load at the site. Moreover, it showed that weak activation of CD8+ T cells within the rectal mucosa is associated with lateral tumor spread and eventually a higher recurrence rate. The mucosal level of CD8(3 infiltration detected at immunohistochemistry might be tested as a marker of lateral tumor spread and potentially translated into clinical practice. (c) 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a dismal prognosis; this is in part due to its late diagnosis at advanced stages. For many patients, medical treatment is the only practicable therapy. In recent years, the development of new technologies that investigate genomic biomarkers has improved the concept of precision medicine to treat patients with PDAC. Through endoscopic ultrasound–tissue acquisition (EUS-TA), tissue from pancreatic cancers can be collected; thus, it has the potential to advance personalized treatment by allowing the assessment of genomic alterations. In this review, we explore the role of EUS in genomic profiling and its strengths and pitfalls in obtaining samples for next-generation sequencing (NGS).

BackgroundLong-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer.MethodsPatients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry.ResultsIn the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p = .008) and tumor-infiltrating lymphocyte infiltration was higher (p = .02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p = .001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p = .027 and p = .034, respectively).ConclusionsThese data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC. In patients with colorectal cancer, nodal metastasis was significantly less frequent, and tumor-infiltrating lymphocytes were higher among aspirin users. In the colorectal cancer primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment. In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users.

Background: Programmed death-ligand 1 (PD-L1) expression has been recognized as a potential biomarker for various cancers, yet its diagnostic and prognostic significance in urothelial bladder cancer (BCa) requires further investigation. Methods: In this prospective single-center study, we aimed to assess the feasibility and diagnostic adequacy of PD-L1 expression analysis using cytoinclusion in BCa patients. We enrolled consecutive patients undergoing endoscopic transurethral resection of bladder tumor (TURBT), repeat TURBT, or robot-assisted radical cystectomy. Urinary and tissue specimens were collected from these patients for cytoinclusion and histopathological analysis to evaluate PD-L1 expression. Results: Out of 29 patients, PD-L1 expression was detected from cytoinclusion in 42.8% (3 out of 7), 10% (1 out of 10), and 66.8% (8 out of 12) of patients with negative/papilloma, low-grade, and high-grade tumors, respectively. Conversely, histopathological analysis identified PD-L1 expression in 57.2% (4 out of 7), 30% (3 out of 10), and 83.3% (10 out of 12) of patients with negative/papilloma, low-grade, and high-grade tumors, respectively. The diagnostic concordance between cytoinclusion and histopathology was 85.7%, 80%, and 83.3% in patients with negative/papilloma, low-grade, and high-grade tumors, respectively. Conclusions: Our study underscores the promise of cytoinclusion as a minimally invasive method for quantifying urinary PD-L1 percentages. This approach could serve as both a potential prognostic and diagnostic indicator, easily obtainable from urine samples. Standardizing this technique could facilitate its widespread use as a valuable tool.

Purpose Small-cell lung cancer (SCLC) is an aggressive disease with a dismal prognosis. The addition of immune checkpoints inhibitors to standard platinum-based chemotherapy in first-line setting achieves a durable benefit only in a patient subgroup. Thus, the identification of predictive biomarkers is an urgent unmet medical need.Experimental design Tumor samples from naive extensive-stage (ES) SCLC patients receiving atezolizumab plus carboplatin–etoposide were analyzed by gene expression profiling and two 9-color multiplex immunofluorescence panels, to characterize the immune infiltrate and SCLC subtypes. Associations of tissue biomarkers with time-to-treatment failure (TTF), progression-free survival (PFS) and overall survival (OS), were assessed.Results 42 patients were included. Higher expression of exhausted CD8-related genes was independently associated with a longer TTF and PFS while increased density of B lymphocytes correlated with longer TTF and OS. Higher percentage of M2-like macrophages close to tumor cells and of CD8+T cells close to CD4+T lymphocytes correlated with increased risk of TF and longer survival, respectively. A lower risk of TF, disease progression and death was associated with a higher density of ASCL1+tumor cells while the expression of POU2F3 correlated with a shorter survival. A composite score combining the expression of exhausted CD8-related genes, B lymphocyte density, ASCL1 tumor expression and quantification of CD163+macrophages close to tumor cells, was able to stratify patients into high-risk and low-risk groups.Conclusions In conclusion, we identified tissue biomarkers and a combined score that can predict a higher benefit from chemoimmunotherapy in ES-SCLC patients.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited survival. Curative opportunities are only available for patients with resectable cancer. Palliative chemotherapy is the current standard of care for unresectable tumors. Numerous efforts have been made to investigate new therapeutic strategies for PDAC. Immunotherapy has been found to be effective in treating tumors with high microsatellite instability (MSI-H), including PDAC. The ability of the Endoscopic Ultrasound Fine Needle Biopsy (EUS-FNB) to reliably collect tissue could enhance new personalized treatment by permitting genomic alterations analysis. The aim of this study was to investigate the feasibility of obtaining adequate DNA for molecular analysis from EUS-FNB formalin-fixed-paraffin-embedded (FFPE) specimens. For this purpose, FFPE-DNA obtained from 43 PDAC archival samples was evaluated to verify adequacy in terms of quantity and quality and was tested to evaluate MSI-H status by droplet digital PCR (ddPCR). All samples were suitable for ddPCR analysis. Unlike the 1-2% MSI-H frequency found with traditional techniques, ddPCR detected this phenotype in 16.28% of cases. This study suggests the ddPCR ability to identify MSI-H phenotype, with the possibility of improving the selection of patients who may benefit from immunotherapy and who would be excluded by performing traditional diagnostic methods.

Intrabdominal dissemination of malignant mesothelioma (MM) and pseudomyxoma peritonei (PMP) is poorly characterized with respect to the stemness window which malignant cells activate during their reshaping on the epithelial-mesenchymal (E/M) axis. To gain insights into stemness properties and their prognostic significance in these rarer forms of peritoneal metastases (PM), primary tumor cultures from 55 patients selected for cytoreductive surgery with hyperthermic intraperitoneal chemotherapy were analyzed for cancer stem cells (CSC) by aldehyde dehydrogenase 1 (ALDH1) and spheroid formation assays, and for expression of a set of plasticity-related genes to measure E/M transition (EMT) score. Intratumor heterogeneity was also analyzed. Samples from PM of colorectal cancer were included for comparison. Molecular data were confirmed using principal component and cluster analyses. Associations with survival were evaluated using Kaplan-Meier and Cox regression models. The activity of acetylsalicylic acid (ASA), a stemness modifier, was tested in five cultures. Significantly increased amounts of ALDH1bright-cells identified high-grade PMP, and discriminated solid masses from ascitic/mucin-embedded tumor cells in both forms of PM. Epithelial/early hybrid EMT scores and an early hybrid expression pattern correlated with pluripotency factors were significantly associated with early peritoneal progression (p = .0343 and p = .0339, respectively, log-rank test) in multivariable models. ASA impaired spheroid formation and increased cisplatin sensitivity in all five cultures. These data suggest that CSC subpopulations and hybrid E/M states may guide peritoneal spread of MM and PMP. Stemness could be exploited as targetable vulnerability to increase chemosensitivity and improve patient outcomes. Additional research is needed to confirm these preliminary data.

Lynch syndrome is rarely associated with rectal cancer (RC) and thus, metachronous RC has been scarcely investigated. This study aimed to analyze the mucosal immune microenvironment in sporadic and metachronous RC. We analyzed the mucosal immune microenvironment in the 25 metachronous RCs present in the IMMUNOREACT 1 and 2 multicentre observational studies (624 patients). A panel of immune markers was retrospectively investigated at immunohistochemistry: CD3, CD4, CD8, CD8b, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. Single-cell suspensions were subjected to flow-cytometry to determine the proportion of epithelial cells (pan-cytokeratin) acting as antigen-presenting cells (expressing CD80, CD86, HLA-ABC) and the proportion of activated CD8 + T cells (CD8 + positive for CD28, CD38), inhibitory T cells (CD3 + CTLA-4+) of activated CD4 + T helper cells (CD4 + CD25+) and activated T regulatory cells (CD4 + CD25 + FoxP3+). No mismatch repair gene deficiencies were observed in the patients. The previous history of colorectal adenoma was significantly more frequent in metachronous RC. In healthy epithelial cells, HLA-ABC expression was significantly higher in patients with metachronous RC. In therapy-na & iuml;ve metachronous RC patients, a significantly lower level of circulating lymphocytes and CD3 + T-cell infiltration in the healthy mucosa surrounding the RC was observed compared to patients with non-metachronous cancer. Our study supports the hypothesis that metachronous RC can occur in a cancerization field in patients with weak systemic and local immune systems. The peculiar site of RC makes the mismatch-repair genes deficiency in metachronous cancer onset less relevant.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly immunosuppressive tumor microenvironment (TME). The aim of this study is to determine the potential significant TME immune markers of long-term survival. Methods: We retrospectively included patients with a diagnosis of resectable PDAC having undergone upfront surgery. Immunohistochemical (IHC) staining using tissue microarray for PD-L1, CD3, CD4, CD8, FOXP3, CD20, iNOS and CD163 was performed in order to characterize the TME. The primary endpoint was long-term survival, defined as the Overall Survival > 24 months from surgery. Results: A total of 38 consecutive patients were included, and 14 (36%) of them were long-term survivors. Long-term survivors showed a higher density of CD8+ lymphocytes intra- and peri-acinar (p = 0.08), and a higher CD8/FOXP3 intra- and peri-tumoral ratio (p = 0.05). A low density of intra- and peri-tumoral FOXP3 infiltration is a good predictor of long-term survival (p = 0.04). A significant association of the low density of intra- and peri-tumoral tumor-associated macrophages (TAMs) iNOS+ with long-term survival was detected (p = 0.04). Conclusions: Despite the retrospective nature and small sample size, our study showed that the high infiltration of CD8+ lymphocytes and low infiltration of FOXP3+ and TAMs iNOS+ are predictors of good prognosis. A preoperative assessment of these potential immune markers could be useful and determinant in the staging process and in PDAC management.

BACKGROUND:The DNA mismatch repair (MMR) system is a highly preserved protein complex recognizing short insertions, short deletions, and single base mismatches during DNA replication and recombination. MMR protein status is identified using immunohistochemistry. Deficit in one or more MMR proteins, configuring deficient MMR status (dMMR), leads to frameshift mutations particularly clustered in microsatellite repeats. Thus, microsatellite instability (MSI) is the epiphenomenon of dMMR. In colorectal cancer (CRC), MMR/MSI status is a biomarker with prognostic and predictive value of resistance to 5-fluorouracil and response to immune checkpoint inhibitor therapy.SUMMARY:In this Review, we describe the challenges the practicing pathologist may face in relation to the assessment of MMR/MSI status and any open issues which still need to be addressed, focusing on pre-analytic issues, pitfalls in the interpretation, and technical aspects of the different assays.KEY MESSAGES:The current methods of detecting dMMR/MSI status have been optimized for CRCs, and whether these techniques can be applied to all tumor and specimen types is still not fully understood. Following the Food and Drug Administration (FDA), tissue/site agnostic drug approval of pembrolizumab for advanced/metastatic MSI tumors, MMR/MSI status in gastrointestinal tract is a common request from the oncologist. In this setting, several issues still need to be addressed, including criteria for sample adequacy.

IntroductionRearranged during transfection (RET) gene rearrangements occur in 1%-2% of non-small cell lung cancer (NSCLC). Because of the results of the study LIBRETTO-001, selpercatinib has been approved as the first-line treatment for patients with RET fusion-positive advanced NSCLC. Selpercatinib demonstrated to be well tolerated. Despite this, gastrointestinal adverse events (AEs) are frequently reported, and no clinical-radiological and endoscopic features and their impact in terms of treatment discontinuations, interruptions, and dose reductions have been described so far. Case reportA 37-year-old never-smoker woman was treated in our institution with selpercatinib for a RET fusion-positive NSCLC. After 9 months of treatment, the patient referred abdominal pain of grade (G) 2, associated with nausea of G2, bilious vomiting of G3, and weight loss of G1. At computed tomography scan, the presence of important bowel wall thickening, free ascitic fluid, mesenteric congestion, and stranding was detected. The patient underwent an anterograde enteroscopy extended to jejunum with detection of lymphocytic duodenitis with sub-mucosal edema. Selpercatinib treatment was temporary interrupted with complete resolution of the symptoms and then re-administered with dose reduction, without relapsed of the gastrointestinal toxicity after 120 days. ConclusionTo our knowledge, this is the first case report of a patient with NSCLC treated with selpercatinib outside a clinical study who developed severe gastrointestinal toxicity characterized by small bowel edema and lymphocytic duodenitis, leading to treatment interruption and dose reduction. The gastrointestinal AE has been described by a radiological, endoscopic, and histopathological point of view. Further investigations are needed to better identify pathological mechanisms of gastrointestinal toxicity for an appropriate AE management.