Investigating intra-tumor heterogeneity and expression gradients of miR-21, miR-92a and miR-200c and their potential of predicting lymph node metastases in early colorectal cancer.

INTRODUCTION: miR-21, miR-92a and miR-200c are regulators of pathways involved in migration, intravasation and metastasis, and their tumor expression levels have been proposed as potential prognostic markers in colorectal cancer (CRC). In two parallel cohorts we examine intra-tumor expression levels in early stage CRC tissue in order to determine intra-tumor heterogeneity, potential systematic intra-tumor expression gradients of the miRNAs and to investigate the association to metastatic disease in early stage CRC. MATERIAL AND METHODS: Two parallel studies on archived formalin-fixed paraffin-embedded (FFPE) CRC tissue. Intra-tumor and inter-patient variances were analyzed in 9 early metastatic CRCs by measuring expression levels by qRT-PCR on isolated tissue samples from luminal, central and invasive border zones. Associations between miRNA expression levels and early metastasizing tumors was investigated in FFPE tissue from invasive border and central tumor zones from 47 early metastatic CRCs matched with 47 non-metastatic CRCs. Intra-tumor expression gradients were analyzed on both cohorts. RESULTS: Mean intra-tumor coefficient of variation in the heterogeneity cohort was 38.5% (range: 33.1-49.0%) only slightly less than variation between patients (45.1%, range 37.0-49.5%). We demonstrated systematic expression gradients between tumor zones equal to a 3.23 (p=0.003) and 1.36 (p=0.014) fold lower expression in invasive areas for miR-200c, 1.52 (p<0.001) and 1.27 (p=0.021) fold lower expression in invasive areas for miR-92a. For miR-21 we found a 1.75 (p<0.001) and 1.21 (p=0.064) fold higher expression in invasive areas compared to luminal and central zones, respectively. No significant difference in expression levels between metastatic and non-metastatic tumors was demonstrated, nor a difference in intra-tumor gradients between metastatic and non-metastatic tumors. CONCLUSION: This study provides evidence for moderate intra-tumor and inter-patient heterogeneities of three well-described potential prognostic markers in CRC. We demonstrate intra-tumor expression gradients indicating a differentiated expression of the target miRNAs between functional tumor zones, but the potential role as markers of early metastatic disease is still not fully clarified.

Intra-tumor heterogeneity of microRNA-92a, microRNA-375 and microRNA-424 in colorectal cancer.

UNLABELLED: Various microRNAs (miRNAs) have been investigated in order to improve diagnostics and risk assessment in colorectal cancer (CRC). To clarify the potential of miRNA profiling in CRC, knowledge of intra-tumor heterogeneity in expression levels is crucial. The study aim was to estimate the intra-tumor variance of three selected miRNAs: miR-92a, miR-375 and miR-424 in CRC tissue. MATERIAL AND METHODS: A retrospective study on archived formalin-fixed paraffin embedded tissue from 9 patients with CRC. miRNA tissue expression levels were analyzed by qRT-PCR on tissue representing luminal, central and invasive border zones. Variance components were estimated based on ∆∆Cp values using mixed modeling and presented as coefficients of variation (CV). RESULTS: Intra-tumor variance was approximately half of the variance observed between patients with a mean intra-tumor CV of 56.4% (range 33.1-77.1%) and a mean inter-patient CV of 101.7% (range 48.8-152.7%). Furthermore we found a significant systematic difference in expression levels between tumor zones for miR-92a and miR-375 with a luminal-invasive difference equal to 0.60 Cp (95% CI: 0.30-0.89, p=0.0003) for miR-92a and a luminal-invasive difference equal to 0.78 Cp (95% CI: 0.10-1.46, p=0.027) for miR-375. Conclusion While the intra-tumor variance of miR-92a, miR-375 and miR-424 is substantial, it only constitutes approximately 30% of the total variation. Functional deregulation between tumor zones might contribute to variations in measured expression levels, and thus knowledge of specific intra-tumor expression patterns is crucial in tissue sampling for research as well as in future diagnostics.

Predictors for dMMR colorectal cancer in patients with serrated lesions and polyps - A register-based cohort study.

BACKGROUND: Serrated lesions and polyps (SP) are precursors of up to 30 % of colorectal cancers (CRC) through the serrated pathway. This often entails early BRAF mutations and MLH1 hypermethylation leading to mismatch repair deficient (dMMR) CRC. We investigated predictors of dMMR CRC among patients with co-occurrence of CRC and SP to increase our knowledge on the serrated pathway. METHODS: We used data from The Danish Pathology Registry and Danish Colorectal Cancer Groups Database from the period 2010-2021 to investigate risk factors for development of dMMR CRC. We used logistic regression models to identify difference in risk factors of developing dMMR CRC in comparison to CRC with proficient MMR (pMMR). RESULTS: We included 3273 patients with a median age of 70.7 years [64.3,76.4] of which 1850 (56.5 %) were male. dMMR CRC was present in 592 patients (18.1 %), with loss of MLH1/PMS2 being most common. The risk of dMMR CRC was significantly higher in females OR 3.47 [2.87;4.20]. When adjusting for age, SP subtype, conventional adenomas (CA), anatomical location and lifestyle factors, female sex remained the strongest predictor OR 2.84 [2.27;3.56]. The presence of sessile serrated lesions with or without dysplasia was related to higher risk OR 1.60 [1.11;2.31] and OR 1.42 [1.11;1.82] respectively, while conventional adenomas constituted a lower risk OR 0.68 [0.55;0.84]. CONCLUSION: In conclusion we found several predictors of whom female sex had the strongest correlation with dMMR CRC in patients with SP.

Colorectal serrated lesions and polyps in the Danish population: A large nationwide register-based cohort study.

Background and study aims Colorectal serrated lesions and polyps (SPs) include hyperplastic polyps (HP), sessile serrated lesions-/+dysplasia (SSL/SSL-D), and traditional serrated adenomas (TSA). From 20% to 30% of colorectal cancers (CRC) develop from SP. We present incidence and baseline characteristics of SP in a Danish cohort. Patients and methods We used The Danish Pathology Registry to include all SPs in the Danish population from January 1, 2000 to December 31, 2021. Based on the unique Danish personal identification number and SNOMED-codes, combined with the age and sex of patients, and date of procedure, we determined the incidence of the SP subtypes, anatomical location, and changes over time. Results During the period from 2000 to 2021, a total of 292,761 SPs were removed from 163,840 patients: 51,649 SSLs, 5959 SSL-Ds, 224,860 HDs, and 10,293 TSAs. The median age of patients was 64.1 years (range 55.2-71.6) and 53.3% were male. We found a general increase in SPs from 3525 in 2000 to 25,853 in 2021 and a rise in the SSL proportion from 1.7% in 2006 to 38% in 2021. Half of all patients had more than one lesion at endoscopy with conventional adenomas being the most common. CRC was found along with SPs in 3.3% of procedures, while 1% to 2.5% of the patients developed metachronous CRC. Conclusions We found an increasing number of SPs, especially SSLs. From 2019 to 2021 the number of SPs seem to stabilize, while the proportion of SSLs keeps rising. Synchronous lesions were common along all subtypes of SP.

Upstaging of early colorectal cancers following improved lymph node yield after methylene blue injection.

AIMS: To evaluate whether the use of intra-arterial methylene blue injection improves lymph node yield, and to determine whether a higher lymph node count results in upstaging in colorectal cancer. METHOD AND RESULTS: We performed a retrospective study of colorectal cancer specimens (n = 234) 1 year after implementation of the method. All colorectal cancer specimens from the previous year served as our control group. Data concerning tumour characteristics, lymph node count, number of positive lymph nodes and success of methylene injection had been prospectively collected in accordance with the department's ongoing registration. The method was easy to implement and perform with a high rate of success (86%). The number of identified lymph nodes was highly significantly improved in the study group (P < 0.0001). In resections with pT1/T2 tumours, we demonstrated a significant increase in the number of resection specimens containing positive lymph nodes, with an increase in pN1 resections from 9.4% in the control group to 26.7% in the study group (P = 0.04). CONCLUSIONS: THE methylene blue technique significantly improves lymph node identification in colorectal cancer specimens, and the improved lymph node identification leads to upstaging of International Union Against Cancer (UICC) pT1/pT2 cancers.

Automated Quantification of sTIL Density with H&E-Based Digital Image Analysis Has Prognostic Potential in Triple-Negative Breast Cancers.

Triple-negative breast cancer (TNBC) is an aggressive and difficult-to-treat cancer type that represents approximately 15% of all breast cancers. Recently, stromal tumor-infiltrating lymphocytes (sTIL) resurfaced as a strong prognostic biomarker for overall survival (OS) for TNBC patients. Manual assessment has innate limitations that hinder clinical adoption, and the International Immuno-Oncology Biomarker Working Group (TIL-WG) has therefore envisioned that computational assessment of sTIL could overcome these limitations and recommended that any algorithm should follow the manual guidelines where appropriate. However, no existing studies capture all the concepts of the guideline or have shown the same prognostic evidence as manual assessment. In this study, we present a fully automated digital image analysis pipeline and demonstrate that our hematoxylin and eosin (H&E)-based pipeline can provide a quantitative and interpretable score that correlates with the manual pathologist-derived sTIL status, and importantly, can stratify a retrospective cohort into two significant distinct prognostic groups. We found our score to be prognostic for OS (HR: 0.81 CI: 0.72-0.92 p = 0.001) independent of age, tumor size, nodal status, and tumor type in statistical modeling. While prior studies have followed fragments of the TIL-WG guideline, our approach is the first to follow all complex aspects, where appropriate, supporting the TIL-WG vision of computational assessment of sTIL in the future clinical setting.

Digital image analysis of pan-cytokeratin stained tumor slides for evaluation of tumor budding in pT1/pT2 colorectal cancer: Results of a feasibility study.

Tumor budding is an independent prognostic factor in colorectal cancer. However, varying degrees of interobserver agreement and reproducibility challenges the use of tumor budding in diagnostics. Immunohistochemical staining of tumor slides with pan-cytokeratin visualizes the budding tumor cells and has been suggested to improve reproducibility. Here we demonstrate the methodology of tumor budding assessment using digital image analysis based on tumor slides stained for pan-cytokeratin, and investigate interobserver agreement, agreement between manual and digital assessment methods and digital reproducibility between users. Tumor slides from 126 patients with pT1/pT2 colorectal cancer were stained with pan-cytokeratin and tumor budding at the invasive tumor front was assessed by conventional manual microscopy. A digital image analysis algorithm for identification and quantification of budding tumor cells was developed and tested on the pan-cytokeratin stained slides. Manual assessment of tumor budding using pan-cytokeratin stained tumor slides exhibited high correlations (Spearman Rank 0.84-0.89, p < 0.001),excellent agreement between observers (Intra-class correlation coefficient (ICC): 0.86 -0.87) and 2.20 higher odds for regional metastases with increasing budding counts (p = 0.017). Digital image analysis correlated well to manual assessment (Spearman Rank 0.71-0.88) and agreement between the two methods was good (ICC 0.62-0.82). However, only a trend towards increased odds for metastatic progression was found for the adjusted digital estimates (p = 0.076). Digital estimates were higher than manual estimates, demonstrated by a systematic median difference of 3-4.5 buds. Image analysis was highly reproducible between users of the algorithm (ICC 0.98). In conclusion, assessment of tumor budding using pan-cytokeratin stained tumor slides is a method with high correlation and agreement between observers. Digital image analysis quantifies budding tumor cells in high agreement with manual estimates, but approval of the digital slides by a pathologist is mandatory. The method qualifies for further investigation.

Early metastatic colorectal cancers show increased tissue expression of miR-17/92 cluster members in the invasive tumor front.

Accurate prediction of regional lymph node metastases (LNM) in endoscopically resected pT1 colorectal cancer (CRC) is crucial in treatment stratification for subsequent radical surgery. Several miRNAs have been linked to CRC invasion and metastasis, including the oncogenic miR-17/92 cluster, and expression levels might have predictive value in the risk assessment of early metastatic progression in CRC. We performed global miRNA microarray using tissue samples from the invasive front of pT1 CRC and investigated associations of the miR-17/92 cluster and presence of LNM. In total, 56 matched pT1 CRCs were thoroughly clinicopathologically characterized, and miRNA microarrays were performed on invasive front tissue samples. Global miRNA intensities were screened using paired t-tests between pT1pN+ and pT1pN0. Associations between miR-17/92 and histopathological features were analyzed using general linear models and tumor cell adjusted expression intensities. miR-17-3p and miR-92a were significantly higher expressed in the invasive front of tumors with LNM compared to those without, corresponding to 1.53-fold higher expression of miR-17-3p (95%CI: 1.04-2.24, P = .030) and 1.28-fold higher expression of miR-92a (95%CI: 1.01-1.68, P = .042). An inverse association between miR-19a and presence of high-grade tumor budding was observed (1.55-fold, 95%CI: 1.13-2.12, P = .008). We provide evidence for associations between early regional LNM and high expression levels of the miR-17/92 cluster members: miR-17-3p and miR-92a, in the invasive front of CRC. Our results support a role for the miR-17/92 cluster in early metastatic progression of CRC and calls for further investigation.

Leser­Trélat syndrome in malignant mesothelioma and pulmonary adenocarcinoma: is the EGFR pathway part of the syndrome?

The syndrome of Leser­Trélat (LT) is characterized by the sudden appearance of multiple seborrhoeic keratoses (SKs) in association with internal occult malignancy. Usually, the syndrome has been associated with adenocarcinoma, most frequently of the gastrointestinal tract and breast. The pathogenesis is unclear but might be explained by circulating tumor-associated growth factors. We present two thoracic malignancies associated with LT: adenocarcinoma of the lung (ACL) and pleural malignant mesothelioma (MM). Both malignant tumors expressed high levels of epidermal growth factor receptors (EGFR) detected by immunohistochemistry (IHC), with membranous staining on the majority of malignant cells corresponding to maximum IHC scores of 290 and 300, respectively, for the MM and the ACL. SKs revealed a universal membranous staining throughout the entire epithelium with no difference in EGFR expression between the two cases and two controls with no malignant history. By fluorescence in situ hybridization, no amplification of the EGFR gene in malignant tumors as well as in SK lesions was observed. Further investigations are needed to see whether tumor-associated EGFR ligands/EGFR autocrine loops in malignant cells expressing high levels of EGFR protein on the surface might play a role for the development of SKs, as well as for the growth of malignant tumors in LT.