IMMUNOREACT 7: Regular aspirin use is associated with immune surveillance activation in colorectal cancer.

BACKGROUND: Long-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. METHODS: Patients 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. RESULTS: In 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). CONCLUSIONS: These data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC.

IMMUNOREACT 6: weak immune surveillance characterizes early-onset rectal cancer.

BACKGROUND: Colon cancer in young patients is often associated with hereditary syndromes; however, in early-onset rectal cancer, mutations of these genes are rarely observed. The aim of this study was to analyse the features of the local immune microenvironment and the mutational pattern in early-onset rectal cancer. METHODS: Commonly mutated genes were analysed within a rectal cancer series from the University Hospital of Padova. Mutation frequency and immune gene expression in a cohort from The Cancer Genome Atlas ('TCGA') were compared and immune-cell infiltration levels in the healthy rectal mucosa adjacent to rectal cancers were evaluated in the IMMUNOlogical microenvironment in REctal AdenoCarcinoma Treatment 1 and 2 ('IMMUNOREACT') series. RESULTS: In the authors' series, the mutation frequency of BRAF, KRAS, and NRAS, as well as microsatellite instability frequency, were not different between early- and late-onset rectal cancer. In The Cancer Genome Atlas series, among the genes with the most considerable difference in mutation frequency between young and older patients, seven genes are involved in the immune response and CD69, CD3, and CD8ß expression was lower in early-onset rectal cancer. In the IMMUNOlogical microenvironment in REctal AdenoCarcinoma Treatment 1 and 2 series, young patients had a lower rate of CD4+ T cells, but higher T regulator infiltration in the rectal mucosa. CONCLUSION: Early-onset rectal cancer is rarely associated with common hereditary syndromes. The tumour microenvironment is characterized by a high frequency of mutations impairing the local immune surveillance mechanisms and low expression of immune editing-related genes. A constitutively low number of CD4 T cells associated with a high number of T regulators indicates an imbalance in the immune surveillance mechanisms.

IMMUNOREACT 5: female patients with rectal cancer have better immune editing mechanisms than male patients - a cohort study.

BACKGROUND: Studies evaluating sex differences in colorectal cancer (CRC) tumor microenvironment are limited, and no previous study has focused on rectal cancer patients' constitutive immune surveillance mechanisms. The authors aimed to assess gender-related differences in the immune microenvironment of rectal cancer patients. METHODS: A systematic review and meta-analysis were conducted up to 31 May 2021, including studies focusing on gender-related differences in the CRC tumor microenvironment. Data on the mutational profile of rectal cancer were extracted from the Cancer Genome Atlas (TCGA). A subanalysis of the two IMMUNOREACT trials (NCT04915326 and NCT04917263) was performed, aiming to detect gender-related differences in the immune microenvironment of the healthy mucosa in patients with early (IMMUNOREACT 1 cohort) and locally advanced rectal cancer following neoadjuvant therapy (IMMUNOREACT 2 cohort). In the retrospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 442 patients (177 female and 265 male), while in the retrospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), we enrolled 264 patients (80 female and 184 male). In the prospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 72 patients (26 female and 46 male), while in the prospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), the authors enrolled 105 patients (42 female and 63 male). RESULTS: Seven studies reported PD-L1 expression in the CRC microenvironment, but no significant difference could be identified between the sexes. In the TGCA series, mutations of SYNE1 and RYR2 were significantly more frequent in male patients with rectal cancer. In the IMMUNOREACT 1 cohort, male patients had a higher expression of epithelial cells expressing HLA class I, while female patients had a higher number of activated CD4+Th1 cells. Female patients in the IMMUNOREACT 2 cohort showed a higher infiltration of epithelial cells expressing CD86 and activated cytotoxic T cells (P=0.01). CONCLUSIONS: Male patients have more frequent oncogene mutations associated with a lower expression of T-cell activation genes. In the healthy mucosa of female patients, more Th1 cells and cytotoxic T cells suggest a potentially better immune response to the tumor. Sex should be considered when defining the treatment strategy for rectal cancer patients or designing prognostic scores.

Impact of DNA mismatch repair proteins deficiency on number and ratio of lymph nodal metastases in colorectal adenocarcinoma.

BACKGROUND: Approximately 15 % of colorectal adenocarcinomas (CRCs) are characterized by an altered expression of DNA mismatch repair (MMR) proteins (i.e. MMR deficiency [MMRd]). Lymph node ratio (LNR) represents one of the most important prognostic markers in non-advanced CRCs. No significant data are available regarding LNR distribution depending on MMR status. PURPOSE OF THE STUDY: The aim of the present work was to compare pathological and clinical characteristics of MMRd tumors versus MMR proficient (MMRp) cases. Particular attention was paid to how these molecular sub-groups relate to the LNR. MATERIALS AND METHODS: A mono-Institutional series of 1037 consecutive surgically treated stage I-IV CRCs were retrospectively selected and data were obtained from pathological reports. Cases were characterized for MMR/MSI status by means of immunohistochemistry or for microsatellite instability (MSI) analysis. RESULTS: MMRd/MSI tumors (n = 194; 18.7 %) showed significant differences in comparison to MMRp lesions for sex (female prevalence 50.5 % vs 40.7 %; p = 0.013), age (74.2 vs 69.2; p < 0.001), location (right side; p < 0.001), diameter (larger than MMRp; p < 0.001), growth pattern (expansive pattern of growth; p < 0.001), peri- (p = 0.0002) and intra-neoplastic (p = 0.0018) inflammatory infiltrate, presence of perineural invasion (p < 0.001), stage (lower stage at presentation; p < 0.001), grade (higher prevalence of high-grade tumors; p < 0.001), and LNR (lower; p < 0.001). CONCLUSIONS: MMRd/MSI tumors are a distinct molecular CRC subtype characterized by a significantly lower LNR in comparison to MMRp lesions. These data further support the prognostic impact of MMRd/MSI status in early-stage CRCs.

Prognostic impact of FGFR2/3 alterations in patients with biliary tract cancers receiving systemic chemotherapy: the BITCOIN study.

AIM: FGFR2 rearrangements have been identified as a novel therapeutic target of biliary tract cancer (BTC). However, reliable prevalence estimates of this molecular alteration and its prognostic role have not been fully elucidated. METHODS: A retrospective mono-institutional series of 286 patients affected by locally advanced or metastatic BTC (183 intrahepatic cholangiocarcinomas, 67 extrahepatic cholangiocarcinomas, 36 gallbladder carcinomas) was profiled by means of targeted DNA/RNA next-generation sequencing, immunohistochemistry and fluorescence in situ hybridisation for FGFR2/3, ERBB2, NTRK alterations, IDH1/2 and BRAF mutations and DNA mismatch repair complex proteins alterations/microsatellite instability. RESULTS: FGFR2 rearrangements, amplifications and point mutations were detected in 15 (5.2%), 1 and 3 cases, respectively. FGFR3 alterations were observed in 5 (1.7%) cases. IDH1/2 were mutated in 35/223 cases (15.7%). A total of 9/258 (3.5%) and 6/260 (2.3%) BTCs had ERBB2 and BRAF gene alterations, respectively. Two cases (2/242; 0.8%) had NTRK1 amplifications but no rearrangement was found. A deficit of mismatch repair protein expression was identified in 9/237 cases (3.8%). At multivariate analysis, age, ECOG performance status, number of metastatic sites, tumour stage, FGFR2/3 alterations and IDH1/2 mutations were prognostic factors of overall survival. CONCLUSIONS: These data provide a strong proof - challenged with a robust and detailed multivariate model - that FGFR2/3 aberrations (including FGFR2 rearrangements) and IDH1/2 mutations can be prognostic for better survival in patients with BTC . The recognition and the measurement of their prognostic impact could be of primary importance for the correct interpretation of currently available data and in the design of new therapeutic trials.

Association of CLDN18 Protein Expression with Clinicopathological Features and Prognosis in Advanced Gastric and Gastroesophageal Junction Adenocarcinomas.

The tight junction protein claudin-18 (CLDN18), is often expressed in various cancer types including gastric (GC) and gastroesophageal adenocarcinomas (GECs). In the last years, the isoform CLDN18.2 emerged as a potential drug target in metastatic GCs, leading to the development of monoclonal antibodies against this protein. CLDN18.2 is the dominant isoform of CLDN18 in normal gastric and gastric cancer tissues. In this work, we evaluated the immunohistochemical (IHC) profile of CLDN18 and its correlation with clinical and histopathological features including p53, E-cadherin, MSH2, MSH6, MLH1, PMS2, HER2, EBER and PD-L1 combined positive score, in a large real-world and mono-institutional series of advanced GCs (n = 280) and GECs (n = 70). The association of IHC results with survival outcomes was also investigated. High membranous CLDN18 expression (2+ and 3+ intensity ≥75%) was found in 117/350 (33.4%) samples analyzed. CLDN18 expression correlated with age <70 (p = 0.0035), positive EBV status (p = 0.002), high stage (III, IV) at diagnosis (p = 0.003), peritoneal involvement (p < 0.001) and lower incidence of liver metastases (p = 0.013). CLDN18 did not correlate with overall survival. The predictive value of response of CLDN18 to targeted agents is under investigation in several clinical trials and further studies will be needed to select patients who could benefit from these therapies.

Circulating microRNA expression profiling revealed miR-92a-3p as a novel biomarker of Barrett's carcinogenesis.

The main intent of secondary prevention strategies for Barrett's esophagus (BE) patients relies in the prompt identification of patients with dysplasia (or intra-epithelial neoplasia; IEN) and early-stage adenocarcinoma (Barrett's adenocarcinoma; BAc). Despite the adequate characterization of the molecular landscape characterizing Barrett's carcinogenesis, no tissue and/or circulating biomarker has been approved for clinical use. A series of 25 serum samples (12 BE, 5 HG-IEN and 8 BAc) were analyzed for comprehensive miRNA profiling and ten miRNAs were found to be significantly dysregulated. In particular seven were upregulated (i.e. miR-92a-3p, miR-151a-5p, miR-362-3p, miR-345-3p, miR-619-3p, miR-1260b, and miR-1276) and three downregulated (i.e. miR-381-3p, miR-502-3p, and miR-3615) in HG-IEN/BAc samples in comparison to non-dysplastic BE. All the identified miRNAs showed significant ROC curves in discriminating among groups with AUC values range of 0.75-0.83. Validation of the results were performed by droplet digital PCR in two out of three tested miRNAs. To understand the cellular source of circulating miR-92a-3p, we analyzed its expression in endoscopy biopsy samples by both qRT-PCR and ISH analyses. As observed in serum samples, miR-92a-3p was over-expressed in HG-IEN/BAc samples in comparison to naïve esophageal squamous mucosa and BE and was mainly localized within the epithelial cells, supporting neoplastic cells as the main source of the circulating miRNA. Our data further demonstrated that circulating miRNAs are a promising mini-invasive diagnostic tool in the secondary follow-up and management of BE patients. Larger multi-Institutional studies should validate and investigate the most adequate miRNAs profile in discriminating BE patients in specific risk classes.

The heterogeneous clinical and pathological landscapes of metastatic Braf-mutated colorectal cancer.

Colorectal cancer (CRC) is a complex and molecularly heterogeneous disease representing one of the most frequent causes of cancer-related death worldwide. About 8-15% of CRCs harbor a mutation in BRAF gene, a proto-oncogene involved in cell proliferation, differentiation and survival through the MAPK signaling cascade. The acquisition of BRAF mutation is an early event in the "serrated" CRC carcinogenetic pathway and is associated with specific and aggressive clinico-pathological and molecular features. Despite that the presence of BRAF mutation is a well-recognized negative prognostic biomarker in metastatic CRC (mCRC), a great heterogeneity in survival outcome characterizes these patients, due to the complex, and still not completely fully elucidated, interactions between the clinical, genetic and epigenetic landscape of BRAF mutations. Because of the great aggressiveness of BRAF-mutated mCRCs, only 60% of patients can receive a second-line chemotherapy; so intensive combined and tailored first-line approach could be a potentially effective strategy, but to minimize the selective pressure of resistant clones and to reduce side effects, a better stratification of patients bearing BRAF mutations is needed.

PD-L1 expression in gastroesophageal dysplastic lesions.

Immunotherapy has been recently approved for gastric (GC) and gastroesophageal-junction adenocarcinomas (GEC), and PD-L1 immunohistochemical evaluation represents a promising predictive biomarker in this oncological setting. A series of 125 gastroesophageal dysplastic lesions (52 low-grade, 73 high-grade) was investigated for PD-L1 and DNA mismatch repair proteins status. PD-L1 was positive (combined positive score (CPS) ≥ 1) in 48 (31.0%) dysplastic lesions. A higher prevalence of PD-L1-positive cases was observed among esophageal specimens compared with gastric ones (p = 0.0003), in high-grade and adenocarcinoma samples in comparison with low-grade dysplasia (p < 0.0001), and in lesions with mismatch repair deficiency (p = 0.028). For 30 dysplastic samples, a synchronous matched invasive lesion (GC = 15, GEC = 15) was available and tested for PD-L1 expression; a discordant PD-L1 status was observed in 12/30 (40%) cases. A relatively high prevalence in PD-L1 positivity was observed among gastroesophageal dysplastic lesions and this should be taken into consideration for future therapeutic strategies based on this biomarker.

Targeted therapies in metastatic gastric cancer: Current knowledge and future perspectives.

Gastric cancer (GC) represents a leading cause of cancer related morbidity and mortality worldwide accounting for more than 1 million of newly diagnosed cases and thousands of deaths every year. In the last decade, the development of targeted therapies and the optimization of already available chemotherapeutic drugs has expanded the available treatment options for advanced GC and granted better survival expectations to the patients. At the same time, global efforts have been undertaken to investigate in detail the genomic and epigenomic heterogeneity of this disease, resulting in the identification of new specific and sensitive predictive and prognostic biomarkers and in innovative molecular classifications based on gene expression profiling. Nonetheless, several randomized studies aimed at exploring new innovative agents, such as immune checkpoint inhibitors, failed to demonstrate clinically meaningful survival advantages. Therefore, it is essential to further improve the molecular characterization of GC subgroups in order to provide researchers and medical oncologists with new tools for patients' selection and stratification in future clinical development programs and subsequent trials. The aim of the present manuscript is to provide a global overview of the recent molecular classifications from The Cancer Genome Atlas and the Asian Cancer Research Group and to present key promising developments in the field of immunotherapy and targeted therapies in metastatic GC.

Claudin-18 expression in oesophagogastric adenocarcinomas: a tissue microarray study of 523 molecularly profiled cases.

BACKGROUND: Claudin-18 (CLDN18) is a highly specific tight junction protein of the gastric mucosa. An isoform of CLDN18, the Claudin 18.2, has recently emerged as an innovative drug target for metastatic gastric cancer. METHODS: We investigated the immunohistochemical profile of CLDN18, p53, p16, E-cadherin, MSH2, MSH6, MLH1, PSM2, HER2, and PDL-1 in a large series of 523 primary gastric carcinomas (GCs; n = 408) and gastro-oesophageal carcinomas (GECs; n = 115) and 135 matched and synchronous nodal metastases. The status of HER2 and EBER by means of chromogenic in situ hybridisation (CISH) was also evaluated. RESULTS: High membranous CLDN18 expression was present in 150/510 (29.4%) primary cases and in 45/132 (34.1%) metastases. An abnormal expression (i.e. nuclear and/or cytoplasmic) was observed in 115 (22.5%) primary cases and in 33 (25.0%) metastases. A 38.8% of the cases showed significant CLDN18 intratumoural variability among the different tissue microarray cores obtained from the same tumour. Positive membrane CLDN18 expression was statistically associated with non-antral GCs (p = 0.016), Lauren diffuse type (p = 0.009), and with EBV-associated cancers (p < 0.001). CONCLUSIONS: CLDN18 is frequently expressed in gastric and gastro-oesophageal cancers; further studies should investigate the prognostic significance of CLDN18 heterogeneity in order to implement its test into clinical practice.

Benign TdT-positive cells in pediatric and adult lymph nodes: a potential diagnostic pitfall.

Benign terminal deoxynucleotidyl transferase (TdT)-positive cells have been documented in a variety of nonhematopoietic tissues. Scant data are, however, available on their presence in nonneoplastic lymph nodes. This study is aimed to (1) characterize the presence/distribution of benign TdT-positive cells in pediatric and adult reactive lymph nodes and (2) define the phenotype and nature of such elements. This retrospective study considered 141 reactive lymph nodes from pediatric and adult patients without history of neoplastic disease. TdT-positive cells were characterized by immunohistochemical and morphometric analyses, and their presence was correlated with the clinical-pathological features. The nature of TdT-positive cells was investigated by (1) double immunostaining for early lymphoid cell markers and (2) assessment of TdT expression in fetal lymph nodes. Sparse TdT-positive cells were documented in all pediatric cases and in most (76%) adult lymph nodes. TdT-positive cell density was higher in children than adults (15.9/mm2 versus 8.6/mm2; P < .05). TdT positivity did not correlate with any clinical or histological parameter, and double immunostaining disclosed a phenotype compatible with early lymphoid precursors (positivity for CD34 and CD10, and variable expression of CD7). A very high TdT-positive cell density (802.4/mm2) was reported in all fetal lymph nodes. In conclusion, TdT-positive cells are a common finding in pediatric and adult lymph nodes. The interstitial distribution and low number of such cells allow for the differential diagnosis with precursor lymphoid neoplasms. The high density in fetal lymph nodes and the phenotype of such cells suggest their belonging to an immature lymphoid subset gradually decreasing with age.

Long-Standing Ulcerative Colitis May Trigger a Multilineage Cancerization Field.

Longstanding/relapsing inflammation characterizing ulcerative colitis (UC) has been associated to an increased risk of colon mucosa neoplastic transformation. We describe the clinicopathological features of a UC-related poorly-differentiated neuroendocrine carcinoma coexisting with a conventional adenocarcinoma. This case supports UC as a multilineage cancerization field.