Large-scale external validation and meta-analysis of gene methylation biomarkers in tumor tissue for colorectal cancer prognosis.

BACKGROUND: DNA methylation biomarkers in colorectal cancer (CRC) tissue hold potential as prognostic indicators. However, individual studies have yielded heterogeneous results, and external validation is largely absent. We conducted a comprehensive external validation and meta-analysis of previously suggested gene methylation biomarkers for CRC prognosis. METHODS: We performed a systematic search to identify relevant studies investigating gene methylation biomarkers for CRC prognosis until March 2024. Our external validation cohort with long-term follow-up included 2303 patients with CRC from 22 hospitals in southwest Germany. We used Cox regression analyses to assess associations between previously suggested gene methylation biomarkers and prognosis, adjusting for clinical variables. We calculated pooled hazard ratios (HRs) and their 95% confidence intervals (CIs) using random-effects models. FINDINGS: Of 151 single gene and 29 multiple gene methylation biomarkers identified from 121 studies, 37 single gene and seven multiple gene biomarkers were significantly associated with CRC prognosis after adjustment for clinical variables. Moreover, the directions of these associations with prognosis remained consistent between the original studies and our validation analyses. Seven single biomarkers and two multi-biomarker signatures were significantly associated with CRC prognosis in the meta-analysis, with a relatively strong level of evidence for CDKN2A, WNT5A, MLH1, and EVL. INTERPRETATION: In a comprehensive evaluation of the so far identified gene methylation biomarkers for CRC prognosis, we identified candidates with potential clinical relevance for further investigation. FUNDING: The German Research Council, the Interdisciplinary Research Program of the National Center for Tumor Diseases (NCT), Germany, the German Federal Ministry of Education and Research.

CpG-biomarkers in tumor tissue and prediction models for the survival of colorectal cancer: A systematic review and external validation study.

The research aimed to identify previously published CpG-methylation-based prognostic biomarkers and prediction models for colorectal cancer (CRC) prognosis and validate them in a large external cohort. A systematic search was conducted, analyzing 298 unique CpGs and 12 CpG-based prognostic models from 28 studies. After adjustment for clinical variables, 48 CpGs and five prognostic models were confirmed to be associated with survival. However, the discrimination ability of the models was insufficient, with area under the receiver operating characteristic curves ranging from 0.53 to 0.62. Calibration accuracy was mostly poor, and no significant added prognostic value beyond traditional clinical variables was observed. All prognostic models were rated at high risk of bias. While a fraction of CpGs showed potential clinical utility and generalizability, the CpG-based prognostic models performed poorly and lacked clinical relevance.

Sex, Genotype, and Liver Volume Progression as Risk of Hospitalization Determinants in Autosomal Dominant Polycystic Liver Disease.

BACKGROUND & AIMS: Autosomal dominant polycystic liver disease is a rare condition with a female preponderance, based mainly on pathogenic variants in 2 genes, PRKCSH and SEC63. Clinically, autosomal dominant polycystic liver disease is characterized by vast heterogeneity, ranging from asymptomatic to highly symptomatic hepatomegaly. To date, little is known about the prediction of disease progression at early stages, hindering clinical management, genetic counseling, and the design of randomized controlled trials. To improve disease prognostication, we built a consortium of European and US centers to recruit the largest cohort of patients with PRKCSH and SEC63 liver disease. METHODS: We analyzed an international multicenter cohort of 265 patients with autosomal dominant polycystic liver disease harboring pathogenic variants in PRKCSH or SEC63 for genotype-phenotype correlations, including normalized age-adjusted total liver volumes and polycystic liver disease-related hospitalization (liver event) as primary clinical end points. RESULTS: Classifying individual total liver volumes into predefined progression groups yielded predictive risk discrimination for future liver events independent of sex and underlying genetic defects. In addition, disease severity, defined by age at first liver event, was considerably more pronounced in female patients and patients with PRKCSH variants than in those with SEC63 variants. A newly developed sex-gene score was effective in distinguishing mild, moderate, and severe disease, in addition to imaging-based prognostication. CONCLUSIONS: Both imaging and clinical genetic scoring have the potential to inform patients about the risk of developing symptomatic disease throughout their lives. The combination of female sex, germline PRKCSH alteration, and rapid total liver volume progression is associated with the greatest odds of polycystic liver disease-related hospitalization.

Testing for deficient mismatch repair and microsatellite instability : A focused update.

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021).Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education.

Comparative bioinformatic analysis of KRAS, STK11 and KEAP1 (co-)mutations in non-small cell lung cancer with a special focus on KRAS G12C.

OBJECTIVES: Mutations in STK11 (STK11MUT) and KEAP1 (KEAP1MUT) occur frequently in non-small cell lung cancer (NSCLC) and are often co-mutated with KRAS. Several studies linked the co-occurrence of KRASMUT + STK11MUT, as well as KRASMUT + KEAP1MUT to reduced response to immune checkpoint inhibitors (ICI) and even a negative impact on survival. Data focusing STK11 + KEAP1 co-mutations or the triple mutation (KRAS + STK11 + KEAP1) are scarce. The recent availability of KRAS-G12C inhibitors increases the clinical relevance of those co-mutations in KRAS-mutated NSCLC. MATERIALS AND METHODS: We present a comprehensive bioinformatic analysis encompassing six datasets retrieved from cBioPortal. RESULTS: Independent of the treatment, triple mutations and STK11MUT + KEAP1MUT were significantly associated with a reduced overall survival (OS). Across treatments, OS of patients with a KRAS G12C triple mutation was significantly reduced compared to patients with KRAS G12C-only. Under ICI-therapy, there was no significant difference in OS between patients harboring the KRAS G12C-only and patients with the KRAS G12C triple mutation, but a significant difference between patients harboring KRAS non-G12C and KRAS non-G12C triple mutations. Triple mutated primary tumors showed a significantly increased frequency of distant metastases to bone and adrenal glands compared to KRAS-only mutated tumors. Additionally, our drug response analysis in cancer cell lines harboring the triple mutations revealed the WNT pathway inhibitor XAV-939 as a potential future drug candidate for this mutational situation. CONCLUSION: The triple mutation status may serve as a negative prognostic and predictive factor across treatments compared to KRASMUT-only. KRAS G12C generally seems to be a negative predictive marker for ICI-therapy.

[Testing deficient mismatch repair and microsatellite instability : A focused update. German version]. / Testung auf Mismatch-Reparatur-Defizienz und Mikrosatelliteninstabilität : Eine fokussierte Aktualisierung.

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021).Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education.

[Hereditary colorectal carcinogenesis]. / Hereditäre kolorektale Karzinogenese.

Hereditary cancer is characterized by the development of certain cancer types in combination with pathogenic germline mutations in genes known to predispose to these cancer types. Familial cancer differs from hereditary cancer in that no predisposing germline mutation is detected in affected families. However, familial cancer may have a genetic background of as yet unknown origin. Colorectal cancer is unique among human tumors since almost all cancers derive from macroscopically visible benign polypoid precursors. Molecular mechanisms of precursor development differ from that of malignant transformation. Hereditary colorectal cancer can be categorized into polypous and non-polypous predispositions. While the former elevate cancer risk by increasing the number of cancer precursors, the latter elevate cancer risk by increasing the likeliness of malignant transformation. It is the pathologist's responsibility to use morphologic criteria in combination with clinical data in order to raise suspicion of hereditary tumorigenesis and recommend genetic counselling. This article summarizes the current knowledge on hereditary colorectal cancer.

Ocrelizumab-induced colitis and cytomegalovirus infection and their disadvantageous interaction with underlying multiple sclerosis.

Ocrelizumab is a humanized monoclonal antibody against the B-lymphocyte antigen CD20 and the only approved treatment option in primary progressive multiple sclerosis. Herpesvirus-related infections like cytomegalovirus (CMV) infections are common in patients receiving ocrelizumab, whereas gastrointestinal side effects with inflammatory bowel disease (IBD) like esophagitis or colitis are very rare. This case report describes the challenging clinical, endoscopic, and histologic features of an ocrelizumab-induced colitis overlapping with CMV infection and their disadvantageous interaction with the underlying multiple sclerosis.

Guideline adherence and implementation of tumor board therapy recommendations for patients with gastrointestinal cancer.

PURPOSE: Although participation in multidisciplinary tumor boards (MTBs) is an obligatory quality criterion for certification, there is scarce evidence, whether MTB recommendations are consistent with consensus guidelines and whether they are followed in clinical practice. Reasons of guideline and tumor board deviations are poorly understood so far. METHODS: MTB's recommendations from the weekly MTB for gastrointestinal cancers at the University Cancer Center Leipzig/Germany (UCCL) in 2020 were analyzed for their adherence to therapy recommendations as stated in National German guidelines and implementation within an observation period of 3 months. To assess adherence, an objective classification system was developed assigning a degree of guideline and tumor board adherence to each MTB case. For cases with deviations, underlying causes and influencing factors were investigated and categorized. RESULTS: 76% of MTBs were fully adherent to guidelines, with 16% showing deviations, mainly due to study inclusions and patient comorbidities. Guideline adherence in 8% of case discussions could not be determined, especially because there was no underlying guideline recommendation for the specific topic. Full implementation of the MTBs treatment recommendation occurred in 64% of all cases, while 21% showed deviations with primarily reasons of comorbidities and differing patient wishes. Significantly lower guideline and tumor board adherences were demonstrated in patients with reduced performance status (ECOG-PS ≥ 2) and for palliative intended therapy (p = 0.002/0.007). CONCLUSIONS: The assessment of guideline deviations and adherence to MTB decisions by a systematic and objective quality assessment tool could become a meaningful quality criterion for cancer centers in Germany.

Deviating HER2 test results in gastric cancer: analysis from the prospective multicenter VARIANZ study.

PURPOSE: The prospective multicenter VARIANZ study aimed to identify resistance biomarkers for HER2-targeted treatment in advanced gastric and esophago-gastric junction cancer (GC, EGJC). HER2 test deviations were found in 90 (22.3%) of 404 cases (central versus local testing) and were associated with negative impact on survival for trastuzumab-treated patients. Here, we investigated methodological and biological variables that may promote deviating HER2 test results. METHODS: We analyzed HER2 testing procedures and participation in quality assurance programs of 105 participating local pathology laboratories. Furthermore, tumor localization and histological subtypes were compared between patients with centrally confirmed (central HER2 + /local HER2 + , n = 68) and unconfirmed HER2 status (central HER2 -/local HER2 + , n = 68). RESULTS: For central HER2 testing, concordance between in situ hybridization (ISH) and immunohistochemistry (IHC) was 98.3%, with IHC sensitivity of 93.3% (84 IHC + of 90 ISH +), specificity of 99.5% (389 IHC- of 391 ISH-), and a positive diagnosis rate of 97.7%. Central confirmation of the local HER2 IHC scores were seen for the majority of locally HER2- IHC 0/1 (172/178; 96.6%), but less frequently for locally IHC3 + (57/124; 46.0%) cases. Deviation rate was not associated with IHC antibody platform used in the local pathology institute neither with participation in quality-assuring tests. Regarding tumor characteristics, deviating test results were more frequently found in GC vs. EGJC (69.1% vs. 39.7%; p = 0.001) and in Laurén diffuse vs. intestinal subtype (23.5% vs. 5.9%, p = 0.004). CONCLUSION: Tumor localization and histological subtype have an impact on HER2 test deviation rates. Assessment of HER2 remains challenging for GC and EGJC.

[What is confirmed in the treatment of colon cancer?] / Was ist gesichert in der Therapie des Kolonkarzinoms.

Colorectal cancer is the second most common cancer diagnosed in Germany and is the third most frequent cause of cancer-related death in both males and females. The majority of colorectal cancers occur via the adenoma-carcinoma sequence of origin. This means that colorectal cancers can be endoscopically detected in premalignant stages and can be curatively treated within the framework of early detection. Screening colonoscopy and, to a lesser extent, fecal occult blood testing, have led to a reduction in the colon cancer-related incidence and mortality. The acceptance and the use of screening colonoscopy should therefore be developed further. Treatment strategies for colorectal cancer are based on TNM staging, supplemented by anatomical and histopathological risk features as well as individual patient characteristics and treatment preferences. The molecular tumor profile is increasingly used to complement decision-making in the surgical, adjuvant and palliative treatment of colorectal cancer. Colon and rectal cancer have many similarities; however, they differ in the preoperative, surgical and adjuvant treatment strategies. This article focuses on colon cancer.

DNA methylation-based classification of sinonasal tumors.

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs.

Improving one-step scarless genome editing in Drosophila melanogaster by combining ovoD co-CRISPR selection with sgRNA target site masking.

The precise and rapid construction of alleles through CRISPR/Cas9-mediated genome engineering renders Drosophila melanogaster a powerful animal system for molecular structure-function analyses and human disease models. Application of the ovoD co-selection method offers expedited generation and enrichment of scarlessly edited alleles without the need for linked transformation markers, which specifically in the case of exon editing can impact allele usability. However, we found that knockin procedures by homology-directed repair (HDR) under ovoD co-selection resulted in low transformation efficiency. This is likely due to repeated rounds of Cas9 cleavage of HDR donor and/or engineered genomic locus DNA, as noted for other CRISPR/Cas9 editing strategies before, impeding the recovery of correctly edited alleles. Here we provide a one-step protocol to improve the generation of scarless alleles by ovoD -co-selection with single-guide RNA (sgRNA) binding site masking. Using this workflow, we constructed human disease alleles for two Drosophila genes, unc-13/CG2999 and armadillo/CG11579. We show and quantify how a known countermeasure, the insertion of silent point mutations into protospacer adjacent motif (PAM) or sgRNA homology regions, can potently suppress unintended sequence modifications during CRISPR/Cas9 genome editing of D. melanogaster under ovoD co-selection. This strongly increased the recovery frequency of disease alleles.