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Functional analyses are the main method to classify mismatch repair (MMR) gene variants of uncertain significance (VUSs). However, the pathogenicity remains unclear for many variants because of conflicting results between clinical, molecular, and functional data. In this study, we evaluated whether whole exome sequencing (WES) could add another layer of evidence to elucidate the pathogenicity of MMR variants with conflicting interpretations. WES was performed on formalin-fixed paraffin-embedded tumor tissue of eight patients with a constitutional MMR VUS (seven families), including eight colorectal and two endometrial carcinomas and one ovarian carcinoma. Cell-free CIMRA assays were performed to assign Odds of Pathogenicity to these VUSs. In four families, seven tumors showed MMR deficiency-associated mutational signatures, supporting the pathogenicity of the VUS. Moreover, somatic (second) MMR hits identified in the WES data were found to explain MMR staining patterns when the MMR staining was discordant with the reported germline MMR gene variant. In conclusion, WES did not significantly reclassify VUS in these cases but clarified some phenotypic aspects such as age of onset and explanations in case of discordant MMR stainings., Competing Interests: Declaration of competing interest On behalf of myself and the other authors, I declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39-56.75 years], 39 years (IQR, 29-51 years), 58 years (IQR, 53.5-67 years), and 68 years (IQR, 65.6-68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1 gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Lynch syndrome (LS) is caused by a pathogenic heterozygous germline variant in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6 or PMS2. LS-associated colorectal carcinomas (CRCs) are characterized by MMR deficiency and by accumulation of multiple insertions/deletions at coding microsatellites (cMS). MMR deficiency-induced variants at defined cMS loci have a driver function and promote tumorigenesis. Notably, PMS2 variant carriers face only a slightly increased risk of developing CRC. Here, we investigate whether this lower penetrance is also reflected by differences in molecular features and cMS variant patterns. Tumor DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue cores or sections (n = 90). Tumors originated from genetically proven germline pathogenic MMR variant carriers (including 14 PMS2-deficient tumors). The mutational spectrum was analyzed using fluorescently labeled primers specific for 18 cMS previously described as mutational targets in MMR-deficient tumors. Immune cell infiltration was analyzed by immunohistochemical detection of T-cells on FFPE tissue sections. The cMS spectrum of PMS2-deficient CRCs did not show any significant differences from MLH1/MSH2-deficient CRCs. PMS2-deficient tumors, however, displayed lower CD3-positive T-cell infiltration compared to other MMR-deficient cancers (28.00 vs. 55.00 per 0.1 mm 2 , p = 0.0025). Our study demonstrates that the spectrum of potentially immunogenic cMS variants in CRCs from PMS2 gene variant carriers is similar to that observed in CRCs from other MMR gene variant carriers. Lower immune cell infiltration observed in PMS2-deficient CRCs could be the result of alternative mechanisms of immune evasion or immune cell exclusion, similar to those seen in MMR-proficient tumors., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Metabolomics analysis of body fluids as well as cells is depended on many factors. While several well-accepted standard operating procedures for the analysis of body fluids are available, the NMR based quantitative analysis of cellular metabolites is less well standardized. Experimental designs depend on the cell type, the quenching protocol and the applied post-acquisition workflow. Here, we provide a tutorial for the quantitative description of the metabolic phenotype of mammalian cells using NMR spectroscopy. We discuss all key steps of the process, starting from the selection of the appropriate culture medium, quenching techniques to arrest metabolism in a reproducible manner, the extraction of the intracellular components and the profiling of the culture medium. NMR data acquisition and methods for both qualitative and quantitative analysis are also provided. The suggested methods cover experiments for adherent cells and cells in suspension. We ultimately describe the application of the discussed workflow to a thyroid cancer cell line. Although this tutorial focuses on mammalian cells, the given guidelines and procedures may be adjusted for the analysis of other cell types., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Mismatch repair (MMR)-deficiency analysis is increasingly recommended for all endometrial cancers, as it identifies Lynch syndrome patients, and is emerging as a prognostic classifier to guide adjuvant treatment. The aim of this study was to define the optimal approach for MMR-deficiency testing and to clarify discrepancies between microsatellite instability (MSI) analysis and immunohistochemical (IHC) analysis of MMR protein expression., Patients and Methods: Six hundred ninety- six endometrial cancers were analyzed for MSI (pentaplex panel) and MMR protein expression (IHC). Agreement between methodologies was calculated using Cohen's Kappa. MLH1 promoter hypermethylation, dinucleotide microsatellite markers and somatic MMR and POLE exonuclease domain (EDM) gene variants (using next-generation/Sanger sequencing) were analyzed in discordant cases., Results: MSI was found in 180 patients. Complete loss of expression of one or more MMR proteins was observed in 196 cases. A PMS2- and MSH6-antibody panel detected all cases with loss of MMR protein expression. The results of MSI and MMR protein expression were concordant in 655/696 cases (kappa = 0.854, P < 0.001). Ambiguous cases (n = 41, 6%) included: subclonal loss of MMR protein expression (n = 18), microsatellite stable or MSI-low cases with loss of MMR protein expression (n = 20), and MSI-low or MSI-high cases with retained MMR protein expression (n = 3). Most of these cases could be explained by MLH1 promoter hypermethylation. Five of seven cases with solitary loss of PMS2 or MSH6 protein expression carried somatic gene variants. Two MSI-high cases with retained MMR protein expression carried a POLE-EDM variant., Conclusion: MSI and IHC analysis are highly concordant in endometrial cancer. This holds true for cases with subclonal loss of MMR protein expression. Discordant MMR-proficient/MSI-high cases (<1%), may be explained by POLE-EDM variants., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: Lynch syndrome (LS), a heritable disorder with an increased risk of primarily colorectal cancer (CRC) and endometrial cancer (EC), can be caused by mutations in the PMS2 gene. We wished to establish whether genotype and/or parent-of-origin effects (POE) explain (part of) the reported variability in severity of the phenotype., Methods: European PMS2 mutation carriers (n = 381) were grouped and compared based on RNA expression and whether the mutation was inherited paternally or maternally., Results: Mutation carriers with loss of RNA expression (group 1) had a significantly lower age at CRC diagnosis (51.1 years vs. 60.0 years, P = 0.035) and a lower age at EC diagnosis (55.8 years vs. 61.0 years, P = 0.2, nonsignificant) compared with group 2 (retention of RNA expression). Furthermore, group 1 showed slightly higher, but nonsignificant, hazard ratios (HRs) for both CRC (HR: 1.31, P = 0.38) and EC (HR: 1.22, P = 0.72). No evidence for a significant parent-of-origin effect was found for either CRC or EC., Conclusions: PMS2 mutation carriers with retention of RNA expression developed CRC 9 years later than those with loss of RNA expression. If confirmed, this finding would justify a delay in surveillance for these cases. Cancer risk was not influenced by a parent-of-origin effect.Genet Med 18 4, 405-409.

Aging is one of the prime risk factors for the development of cancer. Expression patterns of epigenetic regulators, including histone modification levels, are altered during aging of normal cells, a phenomenon referred to as epigenetic drift. Furthermore, it is known that epigenetic mechanisms are involved in the development of cancer. We hypothesized that expression of histone modifications, acetylation of histone 3 lysine 9 (H3K9Ac) and trimethylation of histone 3 lysine 27 (H3K27me3), with reported normal age-related expression patterns might show an age-dependent prognostic value in colorectal cancer (CRC). To quantify expression, we performed immunohistochemical staining of these histone modifications on a tissue microarray containing colorectal tissues of the 254 patients with TNM stage I-III CRC. Stratification of patients according to survival status revealed age-related tumor expression patterns of both H3K9Ac and H3K27me3. Decreased expression with advancing age was observed in patients who were alive after follow-up (no-event group), whereas increased expression with advancing age was observed in patients who presented with a recurrence or death in follow-up (event group). These opposite expression patterns translated into an age-dependent prognostic value in CRC for the individual histone modifications and their combination. The prognostic value reverses with advancing age, high nuclear expression associated with good clinical outcome in young adults, and, in contrast, with worse clinical outcome in elderly patients. In conclusion, for the first time, we demonstrated prognostic impact of epigenetic biomarkers that reverses with advancing age. This new insight supports the hypothesis that CRC biology is different in young vs elderly patients and emphasizes the importance of focusing on age-related effects in CRC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Differentiating primary endometrioid or mucinous ovarian tumors from secondary ovarian tumors can be challenging. We compared somatic mutation profiles of primary and secondary ovarian cancers to investigate if these profiles can help diagnose ovarian tumors. Cancer-related genes (n = 115) were screened by target-enriched next-generation sequencing in formalin-fixed, paraffin-embedded tumor tissue from 43 primary endometrioid and mucinous ovarian carcinomas and 28 proven colorectal cancer metastases to the ovary. Results were validated by high-resolution melting curve analysis and Sanger sequencing. TP53, NOTCH1, PIK3CA, and FAT4 versus APC, TP53, KRAS, and FAT4 mutations were the most common in the primary ovarian tumors and ovarian colorectal cancer metastases, respectively. An inactivating APC mutation was found in 4.7% of primary ovarian tumors (2 of 43; 95% CI, 1.6%-10.9%). In contrast, inactivating APC mutations were identified in 71% of colorectal cancer metastases (20 of 28; 95% CI, 55%-88%) (P < 0.001; sensitivity: 71.4%, 95% CI, 51.1%-86.0%; specificity: 95.4%, 95% CI, 82.9%-99.1%). Loss of heterozygosity and APC promoter hypermethylation did not differ significantly between the primary and secondary ovarian tumors. NOTCH1 mutations were observed specifically in primary ovarian tumors, although at a low frequency, but not in metastases (6 of 41; 14.6%; 95% CI, 3.8%-25.4%). APC mutation analysis can be used to differentiate primary endometrioid and mucinous ovarian tumors from colorectal cancer metastases to the ovary., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Preoperative biopsies or imbedded cytological cells will become more and more a primary source of tissue for molecular diagnostic analyses as a result of novel neo-adjuvant treatment regimens for several cancer types. Furthermore there is a growing need to examine metastatic cancer tissue. Hence, nucleic acids need to be reliably isolated and analyzed from small amounts of formalin-fixed and paraffin-embedded (FFPE) tissue. The limited numbers of (tumor) cells in these samples make high quality and sensitive DNA isolation challenging. Also demands for faster turnaround times are growing. Therefore, we evaluated a fully automated DNA/RNA isolation system and compared this with a manual, classical routine molecular pathology method. We compared the quality of the isolates from both tissue cores and micro-dissection for detection of hotspot mutations in KRAS, BRAF applying hydrolysis probe assays. In addition we determined whether the automated method decreases the hands-on-time and turnaround times in routine molecular pathology workflow. In conclusion, the automated method delivers high quality DNA from both small FFPE tissue cores and micro-dissected tissue material. In comparison to classical methods, less than 50% of starting tissue was sufficient as input for micro-dissection. Turnaround times decreased significantly and 50% less hands-on time was needed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Thyroid cancer is a heterogeneous disease that is classified into differentiated thyroid carcinoma (DTC), undifferentiated/anaplastic thyroid carcinoma (ATC) and medullary thyroid carcinoma. Results of conventional treatment modalities in advanced thyroid cancer have been disappointing and therefore, new therapies are needed., Methods: We searched PubMed, The Cochrane Library, Medline and EMBASE databases and abstracts published in annual proceedings for new treatment modalities in advanced thyroid cancer. We also searched for ongoing trials in www.clinicaltrials.gov., Results: Six phase I, 17 phase II and 1 phase III trials with tyrosine kinase inhibitors were carried out. We found 2 pilot studies and 11 phase II trials with redifferentiation therapies, mainly in DTC. For antiproliferative approaches, three phase I and four phase II trials were found. Immunomodulatory gene therapy was tested in a pilot study in ATC patients. Two phase II trials were carried out with immunotherapy. One phase I and nine phase II trials were found with radionucleotide therapy in patients with DTC., Conclusion: The developments in the treatment of advanced thyroid cancer are intriguing. Future trials should aim at combinations of targeted agents with or without other treatment modalities, and will hopefully contribute to further improvement of outcomes.

Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P=0.02) and fivefold (P=0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors.

Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.

Expression profiling is a well established tool for the genome-wide analysis of human cancers. However, the high sensitivity of this approach combined with the well known cellular and molecular heterogeneity of cancer often result in extremely complex expression signatures that are difficult to interpret functionally. The majority of sporadic colorectal cancers are triggered by mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, leading to the constitutive activation of the Wnt/beta-catenin signaling pathway and formation of adenomas. Despite this common genetic basis, colorectal cancers are very heterogeneous in their degree of differentiation, growth rate, and malignancy potential. Here, we applied a cross-species comparison of expression profiles of intestinal polyps derived from hereditary colorectal cancer patients carrying APC germline mutations and from mice carrying a targeted inactivating mutation in the mouse homologue Apc. This comparative approach resulted in the establishment of a conserved signature of 166 genes that were differentially expressed between adenomas and normal intestinal mucosa in both species. Functional analyses of the conserved genes revealed a general increase in cell proliferation and the activation of the Wnt/beta-catenin signaling pathway. Moreover, the conserved signature was able to resolve expression profiles from hereditary polyposis patients carrying APC germline mutations from those with bi-allelic inactivation of the MYH gene, supporting the usefulness of such comparisons to discriminate among patients with distinct genetic defects.

Aims: Presently, in Europe the treatment of node-negative colorectal cancer (CRC) patients consists of surgical resection of the primary tumour without adjuvant systemic therapy. However, up to 30% of these patients will develop disease recurrence. These high-risk patients are possibly identified by occult tumour cell (OTC) assessment in lymph nodes. In this paper, studies on the clinical relevance of OTC in lymph nodes are reviewed., Methods: A literature search was conducted in the National Library of Medicine by using the keywords colonic, rectal, colorectal, neoplasm, adenocarcinoma, cancer, lymph node, polymerase chain reaction, mRNA, immunohistochemistry, micrometastases and isolated tumour cells. Additional articles were identified by cross-referencing from papers retrieved in the initial search., Results: The upstaging percentages through OTC assessment and the prognostic relevance of OTC in lymph nodes vary among studies, which is related to differences in techniques used to detect OTC., Conclusions: We conclude that OTC examination techniques should be standardized to illuminate whether OTC in lymph nodes can reliably identify high-risk node-negative patients.

As the number of identified single-nucleotide polymorphisms (SNPs) increases, high-throughput methods are required to characterize the informative loci in large patient series. We investigated the feasibility of MassEXTEND LOH analysis using Sequenom's MassArray RT software, a mass spectrometry method, as an alternative to determine loss of heterozygosity (LOH). For this purpose, we studied the c.827A>C SNP (1176A>C p.Gln276Pro) in protein tyrosine phosphatase receptor type-J (PTPRJ), which is frequently deleted in human cancers. In sporadic colorectal cancer (CRC), c.827A>C showed allele-specific LOH of the c.827A allele, which is important because LOH of PTPRJ may be an early event during sporadic CRC. To elucidate the impact of this low-penetrance gene on familial CRC, we studied c.827A>C in 222 familial CRC cases and 156 controls. In 6.2% of the A/C genotyped CRC samples, LOH of c.827A was observed with MassEXTEND LOH analysis and confirmed by conventional sequencing. Furthermore, a case with LOH of c.827A showed no LOH in 22 synchronously detected adenomas, including one with malignant transformation. The importance of the PTPRJ- c.827A>C SNP appears to be limited in familial CRC. We conclude that MassEXTEND LOH analysis (using Sequenom's MassARRAY RT software) is a sensitive, high-throughput, and cost-effective method to screen SNP loci for LOH in formalin-fixed paraffin-embedded tissue.

Aims: Sentinel node mapping (SNM) has been introduced in colorectal cancer (CRC) to improve staging by facilitating occult tumour cell (OTC) assessment in lymph nodes that are most likely to be tumour-positive. In this paper, studies on the feasibility and reliability of SNM in CRC are reviewed., Methods: A literature search was conducted in the National Library of Medicine by using the keywords colonic, rectal, colorectal, neoplasm, adenocarcinoma, cancer and sentinel. Additional articles were identified by cross-referencing from papers retrieved in the initial search., Results: There is a large variation in identification rates and false-negative rates mainly due to the learning curve effect, differences in SNM technique and tumour stage., Conclusions: We conclude that SNM in CRC is technically feasible. Standardization of SNM procedures is mandatory to resolve the debate on the reliability of sentinel node status for predicting the tumour status of all lymph nodes. Only then can adjuvant treatment of patients upstaged by OTC detection in sentinel nodes be justified.

At present, limiting factors in the use of tissue microarrays (TMAs) for high-throughput analysis relate to the visual evaluation of the staining patterns of each of the individual cores in the array and to the subsequent input of the results into a database. Such a database is essential to correlate the data with tumor type and outcome, and to evaluate the performance against other markers achieved in separate experiments. So far, these steps are mostly performed by hand, and consequently are time-consuming and potentially prone to bias and errors, respectively. This paper describes the use of a high-resolution flat-bed scanner for digitization of TMAs with a resolution of about 5 x 5 micro m(2). The arrays are acquired, the positions of the tissue cores are automatically determined, and measurement data including the images of the individual cores are archived. The program provides digital zooming of arrays for interactive verification of the results and rapid linkage of individual core images to data sets of other markers derived from the same array. Performance of the system was compared to manual classification for a representative set of arrays containing colorectal tumors stained with different markers.

Immunohistochemistry (IHC) of mismatch repair (MMR) proteins in colorectal tumors together with microsatellite analysis (MSI) can be helpful in identifying families eligible for mutation analysis. The aims were to determine sensitivity of IHC for MLH1, MSH2, and MSH6 and MSI analysis in tumors from known MMR gene mutation carriers; and to evaluate the use of tissue microarrays for IHC (IHC-TMA) of colon tumors in its ability to identify potential carriers of MMR gene mutations, and compare it with IHC on whole slides. IHC on whole slides was performed in colorectal tumors from 45 carriers of a germline mutation in one of the MMR genes. The TMA cohort consisted of 129 colon tumors from (suspected) hereditary nonpolyposis colorectal cancer (HNPCC) patients. Whole slide IHC analysis had a sensitivity of 89% in detecting MMR deficiency in carriers of a pathogenic MMR mutation. Sensitivity by MSI analysis was 93%. IHC can also be used to predict which gene is expected to harbor the mutation: for MLH1, MSH2, and MSH6, IHC on whole slides would have correctly predicted the mutation in 48%, 92%, and 75% of the cases, respectively. We propose a scheme for the diagnostic approach of families with (suspected) HNPCC. Comparison of the IHC results based on whole slides versus TMA, showed a concordance of 85%, 95%, and 75% for MLH, MSH2, and MSH6, respectively. This study therefore shows that IHC-TMA can be reliably used to simultaneously screen a large number of tumors from (suspected) HNPCC patients, at first in a research setting.