Methylation Analysis of Colitis-Associated Colorectal Carcinomas.

BACKGROUND: Ulcerative colitis is a well-known inflammatory bowel disease. Patients have an increased risk of developing colitis associated carcinoma (CAC). It is important for patient management to be able to distinguish between ulcerative colitis associated carcinoma and sporadic carcinoma (sCRC). However, this distinction is frequently very challenging. It is not readily possible to differentiate this histologically. However, the diagnosis is crucial for the patient's further treatment and follow-up. An attempt was therefore made to develop a diagnostic regime that would enable a reliable distinction between sCRC and CAC. METHODS: We screened 96 patients analyzing more than 850,000 methylation hotspots, to detect distinct epigenetic patterns between both types of carcinomas. Patients with sporadic carcinoma and colitis-associated carcinoma as well as patients with normal colon and patients with confirmed ulcerative colitis without neoplasia were used for the analysis. By extensively filtering the results, methylation sites relevant to distinguish between CAC and sCRC were identified. RESULTS: After the results were filtered, three methylation sites relevant to distinguish between CAC and sCRC were identified. For this purpose, methylation limit values were defined, which favor the samples as CAC or sCRC up to a certain methylation value of the methylation sites. The combination of three methylation sites allows a correct assignment to CAC or sCRC in 94.5% of the cases. CONCLUSION: The results show that these three methylation sites are promising markers in the diagnosis of CAC vs sCRC. Nevertheless, the diagnosis should always be made in conjunction with histomorphological analyses.

Detection of Fusobacterium nucleatum in Patients with Colitis-Associated Colorectal Cancer.

Fusobacterium nucleatum is supposed to play a critical role in the development of colorectal cancer. The species has also been associated with ulcerative colitis (UC) that can progress into colorectal cancer, however, the involvement of bacteria in this process remains unclear. We analysed 177 colon biopsies obtained from patients during screening, including 20 healthy controls, 56 UC cases and 69 cases at different stages of progression to colitis-associated cancer (CAC); 32 samples of sporadic colorectal carcinoma (sCRC) were also included. The presence of F. nucleatum was detected by quantitative real-time PCR (qPCR). Our data show an association between the presence of the bacteria and the progression of carcinogenesis in UC patients. In 39.5% of CAC samples F. nucleatum was detected, compared to only 1.8% in UC cases. The bacteria were detected in 6.3% of samples with initial neoplastic transformation, so-called low-grade dysplasia (LGD), whereas high-grade dysplasia (HGD) resulted in 33.3% of samples positive for F. nucleatum. The fraction of F. nucleatum-positive samples from sCRC cases was 56.3%, which was not significantly different to the CAC group. We conclude that F. nucleatum is associated with the occurrence and progression of colon carcinogenesis, rather than with UC itself.

Frequency of Her2-low in colorectal cancer and its relations with the tumor microenvironment.

BACKGROUND: To date, little is known regarding human epithelial growth factor receptor (HER2) low-expressing colorectal cancer (CRC). Due to promising rising therapies with HER2-antibody-drug conjugates we aimed to analyze the frequency of HER2-low in patients with CRC. Additionally we characterized the clinicopathologic background of this group and its potential relationship with the tumor microenvironment represented by budding and tumor infiltrating lymphocytes (TILs). METHODS: 319 patients with CRC, stages I-IV, were enrolled. HER2-immunohistochemistry (IHC) as well as fluorescence in situ hybridization (FISH) were performed on tissue microarrays. IHC was evaluated semiquantitatively and software-assisted using the HERACLES Diagnostic Criteria for CRC. HER2-low was defined as IHC 1 + or 2 +/FISH negative. HER2-IHC results were compared with budding, TILs and their combinations. RESULTS: The HER2 low-expressing subset represented almost one half of all CRC (47.1 %). Assessment was highly reproducible with different methods. HER2-low cases were significantly more often lower T-, N-, and tumor stage and had less L1 compared with HER2-0. Additionally, they showed more often TILs > 5 % (p = 0.001). The difference between HER2-0 and HER2-low was highly significant between the four budding/TILs-groups (p < 0.001). Cases with low budding/high TILs were more often HER2-low. The highest difference was seen between the low budding/high TILs-group and the low budding/low TILs-group (p < 0.001). CONCLUSIONS: HER2-low expression in CRC is frequent and involves nearly one half of all patients. We could show a relationsship between HER2-low expression and the tumor microenvironment. Special attention should be paid to the low budding/high TILs group in future research.

PU.1-driven Th9 Cells Promote Colorectal Cancer in Experimental Colitis Models Through Il-6 Effects in Intestinal Epithelial Cells.

BACKGROUND AND AIMS: Colorectal cancer [CRC] is one of the most frequent malignancies, but the molecular mechanisms driving cancer growth are incompletely understood. We characterised the roles of the cytokine IL-9 and Th9 cells in regulating CRC development. METHODS: CRC patient samples and samples from AOM/DSS treated mice were analysed for expression of IL-9, CD3, and PU.1 by FACS analysis and immunohistochemistry. IL-9 citrine reporter mice, IL-9 knockout mice, and PU.1 and GATA3 CD4-Cre conditional knockout mice were studied in the AOM/DSS model. DNA minicircles or hyper-IL-6 were used for overexpression of cytokines in vivo. Effects of IL-6 and IL-9 were determined in organoid and T cell cultures. Claudin2/3 expression was studied by western blotting and bacterial translocation by FISH. RESULTS: We uncovered a significant expansion of IL-9- and PU.1-expressing mucosal Th9 cells in CRC patients, with particularly high levels in patients with colitis-associated neoplasias. PU.1+ Th9 cells accumulated in experimental colorectal neoplasias. Deficiency of IL-9 or inactivation of PU.1 in T cells led to impaired tumour growth in vivo, suggesting a protumoral role of Th9 cells. In contrast, GATA3 inactivation did not affect Th9-mediated tumour growth. Mechanistically, IL-9 controls claudin2/3 expression and T cell-derived IL-6 production in colorectal tumours. IL-6 abrogated the anti-proliferative effects of IL-9 in epithelial organoids in vivo. IL-9-producing Th9 cells expand in CRC and control IL-6 production by T cells. CONCLUSIONS: IL-9 is a crucial regulator of tumour growth in colitis-associated neoplasias and emerges as potential target for therapy.

Programmed death ligand 1 (PD-L1) in colon cancer and its interaction with budding and tumor-infiltrating lymphocytes (TILs) as tumor-host antagonists.

PURPOSE: To analyze the role of programmed death ligand 1 (PD-L1) immunohistochemisty in the context of tumor microenvironment in colon cancer (CC) with focus on the interaction between tumor budding and tumor-infiltrating lymphocytes (TILs) and to elucidate its potential value for immunooncologic treatment decisions. METHODS: Three hundred forty seven patients with CC, stages I to IV, were enrolled. PD-L1 immunohistochemistry was performed using two different antibodies (clone 22C3 pharmDx, Agilent and clone QR1, Quartett). Tumor proportion score (TPS) as well as immune cell score (IC) was assessed. Budding and TILs were assessed according to the criteria of the International Tumor Budding Consensus Conference (ITBCC) and International TILs Working Group (ITWG). Correlation analyses as well as survival analyses were performed. RESULTS: PD-L1 positivity significantly correlated with TILs > 5% and MMR deficiency, and PD-L1-positive cases (overall and IC) showed significantly longer overall survival (OS) with both antibodies.The parameters "high grade," "right-sidedness," and "TILS > 5% regardless of MMR status" evolved as potential parameters for additional immunological treatment decisions. Additionally, TPS positivity correlated with low budding. More PD-L1-positive cases were seen in both high TIL groups. The low budding/high TIL group showed longer disease-free survival and longer OS in PD-L1-positive cases. CONCLUSION: Overall, PD-L1 positivity correlated with markers of good prognosis. PD-L1 immunohistochemistry was able to identify parameters as additional potential candidates for immune therapy. Furthermore, it was able to stratify patients within the low budding/high TIL group with significant prognostic impact.

Adjuvant chemotherapy in stage II and III colon cancer: the role of the "budding and TILs-(tumor-infiltrating lymphocytes) combination" as tumor-host antagonists.

PURPOSE: To analyze the influence of adjuvant chemotherapy on the combination of tumor budding and tumor-infiltrating lymphocytes (TILs) in stage II and III colon cancer and to elucidate its potential value for adjuvant treatment decisions. METHODS: 306 patients with stage II and 205 patients with stage III colon cancer diagnosed between 2005 and 2016 who had undergone surgery in a curative setting were enrolled. Budding and TILs were assessed according to the criteria of the International Tumor Budding Consensus Conference (ITBCC) and the criteria of the International TILs Working Group (ITWG). Combinations of budding and TILs were analyzed, and the influence of adjuvant chemotherapy was assessed. RESULTS: In stage II colon cancer, stratification into the four budding/TILs groups showed no significant differences in overall survival (OS) between the chemotherapy and the surgery-alone group, not even in cases with high-risk features. In stage III colon cancer, patients with low budding/high TILs benefited significantly from chemotherapy (p=0.005). Patients with high budding/low TILs as well as high budding/high TILs showed a trend to benefit from adjuvant treatment. However, no chemotherapy benefit was seen for the low budding/low TIL group. CONCLUSIONS: The budding/TIL combination identified subgroups in stage II and III colon cancer with and without benefit from adjuvant treatment. The results this study suggest that the combination of budding and TILs as tumor-host antagonists might be an additional helpful tool in adjuvant treatment decisions in stage II and III colon cancer.

Validation of the 'Inflammatory Bowel Disease-Distribution, Chronicity, Activity [IBD-DCA] Score' for Ulcerative Colitis and Crohn´s Disease.

BACKGROUND AND AIMS: Histological scoring plays a key role in the assessment of disease activity in ulcerative colitis [UC] and is also important in Crohn´s disease [CD]. Currently, there is no common scoring available for UC and CD. We aimed to validate the Inflammatory Bowel Disease [IBD]-Distribution [D], Chronicity [C], Activity [A] score [IBD-DCA score] for histological disease activity assessment in IBD. METHODS: Inter- and intra-rater reliability were assessed by 16 observers on biopsy specimens from 59 patients with UC and 25 patients with CD. Construct validity and responsiveness to treatment were retrospectively evaluated in a second cohort of 30 patients. RESULTS: Inter-rater reliability was moderate to good for the UC cohort (intraclass correlation coefficients [ICCs] = 0.645, 0.623, 0.767 for D, C, and A, respectively) and at best moderate for the CD cohort [ICC = 0.690, 0.303, 0.733 for D, C, and A, respectively]. Intra-rater agreement ranged from good to excellent in both cohorts. Correlation with the Nancy Histological Index [NHI] was moderate and strong with the Simplified Geboes Score [SGS] and a Visual Analogue Scale [VAS], respectively. Large effect sizes were obtained for all three parameters. External responsiveness analysis revealed correlated changes between IBD-DCA score and NHI, SGS and VAS. CONCLUSIONS: The IBD-DCA score is a simple histological activity score for UC and CD, agreed and validated by a large group of IBD specialists. It provides reliable information on treatment response. Therefore, it has potential value for use in routine diagnostics as well as clinical studies.

Sporadic adenoma or ulcerative colitis associated neoplasia? The endoscopist's information has an impact on diagnosis and patient management.

BACKGROUND: Diagnosing low grade intraepithelial neoplasia (LGIN) in patients with ulcerative colitis (UC) is difficult. Distinguishing between sporadic adenoma (SA) and UC associated LGIN is even more challenging but has clinical impact. We aimed to examine, if the morphological distinction between both entities is reliably possible, how it influences patient's outcome and the role of the endoscopist in this decision with respect to current endoscopy classification schemes. METHODS: Seven pathologists retrospectively reevaluated 425 cases of LGIN in UC patients, diagnosed between 2009 and 2017 with preceding expert consensus and follow up in two separate readings, based on published morphological differentiation criteria. In the first evaluation, the observers were blinded to any clinical data. In the second evaluation, they knew patients' age as well as endoscopic features. They also rated their subjective diagnostic certainty. RESULTS: Diagnostic correctness improved significantly in the second assessment as did the pathologists' confidence in their diagnoses (p < 0.001 - p = 0.019). Knowledge of clinical and endoscopical data led to a higher percentage of SA (71.8% vs. 85.6%). UC associated LGIN showed significant earlier LGIN relapse as well as more high grade intraepithelial neoplasia and carcinoma during follow up (p < 0.001, p < 0.001, p = 0.005). CONCLUSIONS: Distinction between SA and UC associated LGIN is important as it has an impact on patients' follow up and treatment. Morphological distinction remains difficult with moderate interobserver variability. Adequate clinical information significantly improves pathologists' diagnoses as well as their confidence in their diagnoses.