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.

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.

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.

[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.

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.

[Detection of BRAF V600E mutation in metastatic colorectal carcinoma : A QuIP round robin test]. / Nachweis der BRAF-V600E-Mutation beim metastasierten kolorektalen Karzinom : Ein QuIP-Ringversuch.

Round robin testing is an important instrument for quality assurance. Increasingly, this also applies to the results of molecular diagnostics in pathology, which directly influence therapy decisions in precision oncology. In metastatic colorectal carcinoma (mCRC), the focus has been on detecting KRAS and NRAS mutations, whose absence allows therapy with EGFR blocking antibodies. Recently, BRAF has been added as another predictive marker, since mCRC patients with BRAF V600E mutation benefit significantly from treatment with encorafenib (a BRAF inhibitor) in combination with cetuximab (anti-EGFR antibody) after systemic therapy. Due to the approval of this treatment in 2020, it is a pre-requisite that BRAF V600E mutation detection in diagnostic pathologies is reliably performed. Therefore, this round robin test with BRAF V600E testing either by immunohistochemistry or molecular methods was performed. The round robin test results demonstrate that molecular BRAF V600E detection is currently clearly superior to immunohistochemical detection.

The Different Immune Profiles of Normal Colonic Mucosa in Cancer-Free Lynch Syndrome Carriers and Lynch Syndrome Colorectal Cancer Patients.

BACKGROUND & AIMS: Owing to the high load of immunogenic frameshift neoantigens, tumors arising in individuals with Lynch syndrome (LS), the most common inherited colorectal cancer (CRC) syndrome, are characterized by a pronounced immune infiltration. However, the immune status of normal colorectal mucosa in LS is not well characterized. We assessed the immune infiltrate in tumor-distant normal colorectal mucosa from LS CRC patients, sporadic microsatellite-unstable (MSI) and microsatellite-stable (MSS) CRC patients, and cancer-free LS carriers. METHODS: CD3-positive, FOXP3-positive, and CD8-positive T cells were quantified in, respectively, 219, 233, and 201 formalin-fixed paraffin-embedded (FFPE) normal colonic mucosa tissue sections from CRC patients and cancer-free LS carriers and 26, 22, and 19 LS CRCs. CD3-positive T cells were also quantified in an independent cohort of 97 FFPE normal rectal mucosa tissue sections from LS carriers enrolled in the CAPP2 clinical trial. The expression of 770 immune-relevant genes was analyzed in a subset of samples with the use of the NanoString nCounter platform. RESULTS: LS normal mucosa specimens showed significantly elevated CD3-, FOXP3-, and CD8-positive T-cell densities compared with non-LS control specimens. Gene expression profiling and cluster analysis revealed distinct immune profiles in LS carrier mucosa with and without cancer manifestation. Long-term follow-up of LS carriers within the CAPP2 trial found a correlation between mucosal T-cell infiltrate and time to subsequent tumor occurrence. CONCLUSIONS: LS carriers show elevated mucosal T-cell infiltration even in the absence of cancer. The normal mucosa immune profile may be a temporary or permanent tumor risk modifier in LS carriers.

The challenge to differentiate between sarcoma or adrenal carcinoma-an observational study.

BACKGROUND: Adrenal sarcomas are rare malignant tumors with structural and clinical similarities to sarcomatoid adrenocortical carcinoma. Preoperative diagnosis of tumors of the adrenal gland can be challenging and often misleading thus detaining patients from appropriate oncological strategies. OBJECTIVE: This analysis of a case series evaluated the predictive capability of the primary clinical diagnosis in case of malignancies of the adrenal gland. METHODS: Thirty two patients were treated from 2009 to 2015 at our clinic and analyzed retrospectively. All patients had computed tomography and/or magnet resonance imaging and a primary histopathological examination at our institution after surgery. Ten questionable cases were surveyed by a reference pathologist. RESULTS: Twelve out of 32 diagnoses had to be revised (37.5%). Only 15 out of 24 tumors primarily classified as adrenocortical carcinoma were finally described as primary adrenal cancer. We found two leiomyosarcomas, one liposarcoma, one sarcomatoid adrenocortical carcinoma, and one epitheloid angiosarcoma among 12 misleading diagnoses. Other tumors turned out to be metastases of lung, hepatocellular, and neuroendocrine tumors. Larger tumors were significantly more often correctly diagnosed compared to smaller tumors. Four patients of the group of revised diagnoses died whereas all patients with confirmed diagnoses survived during the follow-up. CONCLUSION: Preoperative assessment of tumors of the adrenal gland is still challenging. In case of wrong primary diagnosis, the prognosis could be impaired due to inadequate surgical procedures or insufficient preoperative oncological treatment.

FGF-Receptors and PD-L1 in Anaplastic and Poorly Differentiated Thyroid Cancer: Evaluation of the Preclinical Rationale.

Background: Treatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising. Materials and Methods: Primary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable. Results: PD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p<0.01) and NT (0%, p<0.001). 53% of PDTC samples had PD-L1 expression ≤5%. FGFR mRNA expression was generally low in all samples but combined FGFR1-4 expression was significantly higher in PDTC and ATC compared to NT (each p<0.001). No impact of PD-L1 and FGFR 1-4 expression was observed on DSS. Conclusion: High tumoral expression of PD-L1 in a large proportion of ATCs and a subgroup of PDTCs provides a rationale for immune checkpoint inhibition. FGFR expression is low thyroid tumor cells. The clinically observed synergism of PEM with LEN may be caused by immune modulation.

MSI testing : What's new? What should be considered?

Based on new trial data regarding immune checkpoint inhibitors (ICIs), the detection of high-grade microsatellite instability (MSI-H) or underlying deficient mismatch repair protein (dMMR) is now becoming increasingly important for predicting treatment response. For the first time, a PD­1 ICI (pembrolizumab) has been approved by the European Medicines Agency (EMA) for first-line treatment of advanced (stage IV) dMMR/MSI­H colorectal cancer (CRC). Further indications, such as dMMR/MSI­H endometrial carcinoma (EC), have already succeeded (Dostarlimab, 2nd line treatment) and others are expected to follow before the end of 2021. The question of optimal testing in routine diagnostics should therefore be re-evaluated. Based on a consideration of the strengths and weaknesses of the widely available methods (immunohistochemistry and PCR), a test algorithm is proposed that allows quality assured, reliable, and cost-effective dMMR/MSI­H testing. For CRC and EC, testing is therefore already possible at the primary diagnosis stage, in line with international recommendations (NICE, NCCN). The clinician is therefore enabled from the outset to consider not only the predictive but also the prognostic and predispositional implications of such a test when counseling patients and formulating treatment recommendations. As a basis for quality assurance, participation in interlaboratory comparisons and continuous documentation of results (e.g., QuIP Monitor) are strongly recommended.

Distinct Mutational Profile of Lynch Syndrome Colorectal Cancers Diagnosed under Regular Colonoscopy Surveillance.

Regular colonoscopy even with short intervals does not prevent all colorectal cancers (CRC) in Lynch syndrome (LS). In the present study, we asked whether cancers detected under regular colonoscopy surveillance (incident cancers) are phenotypically different from cancers detected at first colonoscopy (prevalent cancers). We analyzed clinical, histological, immunological and mutational characteristics, including panel sequencing and high-throughput coding microsatellite (cMS) analysis, in 28 incident and 67 prevalent LS CRCs (n total = 95). Incident cancers presented with lower UICC and T stage compared to prevalent cancers (p < 0.0005). The majority of incident cancers (21/28) were detected after previous colonoscopy without any pathological findings. On the molecular level, incident cancers presented with a significantly lower KRAS codon 12/13 (1/23, 4.3% vs. 11/21, 52%; p = 0.0005) and pathogenic TP53 mutation frequency (0/17, 0% vs. 7/21, 33.3%; p = 0.0108,) compared to prevalent cancers; 10/17 (58.8%) incident cancers harbored one or more truncating APC mutations, all showing mutational signatures of mismatch repair (MMR) deficiency. The proportion of MMR deficiency-related mutational events was significantly higher in incident compared to prevalent CRC (p = 0.018). In conclusion, our study identifies a set of features indicative of biological differences between incident and prevalent cancers in LS, which should further be monitored in prospective LS screening studies to guide towards optimized prevention protocols.