Annegret Müller

Microsatellite analysis of hereditary nonpolyposis colorectal cancer-associated colorectal adenomas by laser-assisted microdissection: Correlation with mismatch repair protein expression provides new insights in early steps of tumorigenesis

Although microsatellite instability (MSI) testing is a useful tool for molecular screening of hereditary nonpolyposis colorectal cancer (HNPCC) carcinomas, conflicting results have been obtained in colorectal adenomas. This might result from different techniques of tissue sampling and MSI analysis. Alternatively, some HNPCC-associated adenomas may follow a molecular route that differs from the MSI pathway. In the present study we examined the MSI status of 18 adenomas from 17 HNPCC patients by comparing manual adenoma dissection under gross visual control with laser microdissection of single adenoma crypts. After manual gross dissection, 50% (9 of 18) and 11.1% (2 of 18) of the adenomas displayed high-level (MSI-H) and low-level (MSI-L) MSI, respectively. The same set of adenomas split into 83.3% (15 of 18) MSI-H and 5.6% (1 of 18) MSI-L after laser microdissection. The expression pattern of mismatch repair (MMR) proteins showed a higher concordance rate with the MSI status in laser-dissected (94%) than gross-dissected (47%) adenomas. Whereas two adenomas remained microsatellite stable (MSS) and MMR proficient even after laser-assisted dissection, two MSI-H cases showed either rare instabilities at coding microsatellites or intratumoral heterogeneity of MSI with and without MSH2 expression. This suggests that in some adenomas development of MMR dysfunction occurs stepwise with MSI, arising before complete loss of MMR gene expression, whereas other HNPCC-associated adenomas might develop independently of MMR deficiency.

A novel MSH2 germline mutation in homozygous state in two brothers with colorectal cancers diagnosed at the age of 11 and 12 years

Hereditary non‐polyposis colorectal cancer (HNPCC) syndrome is caused by heterozygous germline mutations in DNA mismatch repair genes (MMR), (MSH2, MLH1, MSH6, and PMS2) and it is inherited in an autosomal dominant pattern with high penetrance. Several patients have been reported carrying bi‐allelic MMR gene mutations and whose phenotype resembled a syndrome with childhood malignancies including hematological malignancies, brain, and colorectal tumors. This phenotype is similar to the tumor spectrum of MMR knockout mice. Herein we describe two brothers of healthy consanguineous parents from Pakistan, who had developed two and three colorectal cancers at the ages of 11 and 12 years, respectively, and less than 30 polyps. Tumor specimens were microsatellite instable (MSI‐H), and expression of MSH2 and MSH6 was lost. Mutation analyses of DNA samples from both patients revealed a novel homozygous c.2006‐5T > A mutation in intron 12 of the MSH2 gene. This phenotype of the brothers is unusual as they neither develop hematological malignancies nor brain tumors at an older age of presentation than other patients with homozygous MSH2 mutations. The milder phenotype may be due to the expression of low amounts of MSH2 protein with reduced activity.

MSI-Testing in Hereditary Non-Polyposis Colorectal Carcinoma (HNPCC)

Genomic instability at simple repeated sequences, termed microsatellite instability (MSI), plays an important role in the analysis of sporadic and hereditary colon cancers. In hereditary non-polyposis colorectal cancer syndrome (HNPCC) more than 90% of cases show MSI, whereas only 10–15% of sporadic colorectal cancers do so. Thus, microsatellite analysis is commonly used as the first diagnostic screening test for HNPCC. In 1997, an international collaborative workshop sponsored by the National Cancer Institute (NCI) proposed a set of guidelines for MSI-testing to improve reliability and reproducibility of the analysis as well to allow comparisons between different studies and different laboratories. In this review we assess the value of current protocols forMSI-testing and discuss some diagnostic pitfalls. Our findings support continued use of the MSI marker panel recommended in 1997. Additionally, MSI-testing should be improved by use of microdissection, which helps to identify additional patients with MSI due to enrichment of tumor cells and therefore increased sensitivity. In our view, immunohistochemical staining for mismatch repair protein expression is not a substitute for MSI-analysis but complements MSI screening and helps direct further testing. In summary, MSI-analysis is a highly sensitive and reliable screening method for HNPCC, that requires a well-equipped laboratory as well as an experienced pathologist. Integration of family history and histo-pathological features is also critical.

Laser microdissection of small tissue samples--application to chronic pancreatitis tissues.

Laser microdissection is considered to be the gold standard of tissue sampling, especially if a defined small tissue area consisting of single or few cells within a heterogeneous tissue compartment is of interest. This sophisticated technique offers the opportunity of rapid and contamination-free tissue sampling for RNA- or DNA-based molecular genetic studies. We have applied laser microdissection to a molecular genetic study of pancreatic intraductal lesions (PanINs) in tissues of chronic pancreatitis, where an exact microdissection of small ducts within a dense fibrous tissue is of paramount importance for following analysis. From nine patients suffering from chronic pancreatitis, formalin-fixed, paraffin-embedded tissue specimens were laser microdissected, and a total of 202 normal ducts and PanINs of grade PanIN-1A to grade PanIN-2 were harvested. After whole genome amplification by improved primer extension and preamplification PCR (I-PEP-PCR), microsatellite-PCR based loss of heterozygosity analysis (LOH) of the tumor suppressor gene loci TP53, p16INK4, and DPC4 was performed. One of 85 informative duct lesions (1.2%) had LOH of TP53, 1 of 76 duct lesions (1.3%) had LOH of DPC4, and 2/29 duct lesions (6.9%) showed LOH of p16INK4. Microsatellite instability (MSI) was seen in 2 of 178 duct lesions (1.1%). Immunohistochemical staining of p53 protein and DPC4 protein revealed no aberrant expression. These preliminary data indicate that LOH of tumor suppressor genes, important in pancreatic cancer genesis or MSI, can be found in chronic pancreatitis tissues, but their incidence is low.

Implantation of a colorectal stent as a therapeutic approach in the treatment of esophageal leakage

BackgroundWhile the mortality of esophageal surgery has decreased due to technological advancements, there is still a complication rate of about 30%. One of the main complications is the anastomotic leakage associated with a significant rate of morbidity and mortality. To close the leakage the efficacy of self-expanding stents (SES) has been shown in different studies. However, the high rate of stent migration limits the use of commercial available stents. In our case we were faced with the problem that the diameter of all available stents was too small to attach tightly to the mucosal wall of the esophagogastric anastomosis.Case presentationWe used, for the first time to our knowledge, a metal stent designed for colorectal application in an extensive anastomotic leak after esophageal resection in a patient with an esophageal cancer. After primary surgery with subtotal esohagectomy the anastomotic leak was stented endoscopically with a Polyflex self-expanding covered plastic stent after no response to intensive conventional management. Even though the stent was placed correctly, the diameter of the Polyflex stent was too small to attach onto the wall of the esophagogastric anastomosis. Again surgery was performed with a thoracal resection of the esophageal remnant and a hand made anastomosis. Unfortunately, again an anastomotic leak was detected soon after. To close the leak we decided to use a covered colorectal stent (Hanarostent) with an inner diameter of 30 mm. Sixteen weeks later the stent was extracted and complete mucosal healing of the esophageal leak was observed.ConclusionThe stent implantation with a large wide diameter offers a good chance to close more extensive leaks and prevent stent migration.

Reduced mRNA expression in paraffin-embedded tissue identifies MLH1- and MSH2-deficient colorectal tumours and potential mutation carriers

Based on the principle of nonsense-mediated mRNA decay, we sought to identify MLH1 or MSH2-deficient colorectal tumours through relative quantification of mRNA expression with real-time PCR (RT-PCR) analysis. MLH1 and MSH2 mRNAs were almost equally expressed as defined by MLH1 to MSH2 transcript ratio (mean 1.41) in microsatellite stable, mismatch repair (MMR) proficient tumours (n = 16). A close correlation between loss of protein expression and MMR–mRNA levels was found in highly microsatellite instable (MSI-H) tumours deficient of MLH1 or MSH2. MLH1/MSH2 ratio was low in 11 sporadic and nine hereditary MLH1-deficient carcinomas (mean 0.51), whereas the ratio was high in 17 MSH2-deficient hereditary non-polyposis colorectal cancer (HNPCC) associated carcinomas (mean 6.8). Notably, in the normal tissues of HNPCC patients with MSH2 mutations, the MLH1/MSH2 transcript ratios were significantly elevated (ratio > 2.0) as compared to the ratios of normal mucosa in patients with MMR-proficient tumours (27 of 32 ratio < 2.0; p = 0.00113). Analysis of B-lymphocytes of HNPCC patients with proven MMR gene mutation confirmed these findings. In conclusion, RT-PCR allows relative quantification of MMR gene mRNA expression in formalin-fixed and paraffin-embedded tissue. Furthermore, this approach enables quantification of haploinsufficiency due to nonsense-mediated mRNA decay in normal tissue and B-lymphocytes from patients carrying MSH2 germline mutations and may be useful for identification of asymptomatic carriers of pathogenic germline mutations.

Genotype and phenotype of a new 2-bp deletion of hMSH2 at codon 233

Abstract. Germline mutations within mismatch repair genes, such as hMSH2, hMLH1, and hMSH6, have been shown to be the hallmark of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. The spectrum of tumors associated with mismatch repair gene defects and the possible relationship between genotype and phenotype are still unclear. Therefore, the spectrum of tumors and the possible genotype–phenotype relationship are still under discussion. Here, we report on a family with a new germline mutation in the hMSH2 gene with a 2-bp deletion at codons 232 and 233 leading to a frame shift and a stop at codon 254. Accordingly, immunohistochemistry revealed loss of hMSH2 expression in colorectal carcinomas of three affected family members. In this one family, there was a high penetrance. Interestingly, mutational screening of the family revealed a high penetrance of the mutation affecting four of five tested people at risk, with a high mortality rate and a trend toward lower age of onset in subsequent generations. Finally, a metachronous breast cancer in one patient turned out to be a tumor unrelated to microsatellite instability phenocopy, i.e., a sporadic tumor unrelated to HNPCC that expressed the hMSH2 gene and did not show any microsatellite instability.

Die Sequenzvarianten Arg72Pro des Tumorsuppressorgens p53 und Arg462Gln des Prostatakarzinom-Suszeptibilitätsgens RNASEL haben einen additiven Effekt auf das Erkrankungsalter von HNPCC-Patienten

p53 and the prostate-cancer-susceptibility gene RNASEL are tumour suppressor genes involved in apoptosis. We have previously reported that the common, functionally different variants Arg72Pro in p53 and Arg462Gln in RNASEL are associated with the age of disease onset of colorectal cancer in Lynch syndrome patients. To assess the combined effect of both variants, we screened 246 unrelated Lynch syndrome patients with a pathogenic germline mutation either in MSH2 (n = 138) or in MLH1 (n = 108) and colorectal cancer as first tumour, and 245 healthy controls. The global log rank test revealed significant differences in the age of disease onset for the genotypes of each variant (p = 0.0176 for p53 and p = 0.0358 for RNASEL) and for the combined genotypes of both variants (p = 0.0174). The highest difference in median age of disease onset was seen between homozygotes for the wildtypes in both genes (42 years [range 22–75]) and homozygotes for the variant alleles in both genes (30 years [range 26–47]). A multivariate Cox regression model indicated that only the p53 and RNASEL genotypes had a significant influence on age of disease onset (p = 0.016 for p53 and p = 0.014 for RNASEL) in an additive mode of inheritance, and that the effects of both variants are purely additive, which supports the notion that the p53 and RNase L pathways do not interact. These findings may be relevant for preventive strategies in Lynch syndrome.

Eine neue homozygote MSH2-Keimbahnmutation als Ursache für kolorektale Karzinome bei zwei 11 und 12 Jahre alten Brüdern

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is inherited in an autosomal dominant manner with high penetrance. Heterozygous germline mutations in DNA mismatch repair genes (MMR), (MSH2, MLH1, MSH6 and PMS2) cause HNPCC. Here, we describe two brothers of healthy consanguineous parents, who had developed two and three colorectal cancers at the ages of 11 and 12 years, respectively, and less than 30 polyps. Mutation analyses of DNA samples from both patients revealed a novel homozygous splice site mutation in intron 12 of the MSH2 gene which results in the insertion of one additional amino acid into the MSH2 protein. This phenotype of the brothers is unusual as they did not develop hematological malignancies nor brain tumors at an older age of presentation than other patients with homozygous MSH2 mutations. The milder phenotype may be due to the expression of low amounts of MSH2 protein with reduced activity. Einleitung Zu den haufigsten Formen des erblichen Dickdarmkrebses gehort das HNPCC-Syndrom (hereditares nicht-Polyposis-assoziiertes kolorektales Karzinomsyndrom) und die FAP (familiare adenomatose Polyposis) [1]. Das kolorektale Karzinom im Kindesalter ist ausgesprochen selten. Weltweit sind wenige Familien bekannt, bei denen sich unterschiedliche Tumoren bereits im fruhesten Kindesalter auf der Basis biallelischer Keimbahn-Mutationen in einem der vier Mismatch Repair Gene (MMR) MSH2, MLH1, MSH6 oder PMS2 entwickelt haben [2]. Das Tumorspektrum ist dem der MMRKnockout-Mause mit hamatologischen Malignomen, Hirntumoren und gastrointestinalen Karzinomen sehr ahnlich [3]. Methodik Ein 11-jahriger Junge wurde bei Anamie und Nachweis von Blut im Stuhl koloskopiert. Neben mehreren Polypen fand sich ein Karzinom an der linken Kolonflexur. Daneben wies der Junge Cafeau-lait Flecken auf. Die Untersuchung der ubrigen Familie entdeckte auch bei dem 12-jahrige Bruder drei synchrone kolorektale Karzinome neben einigen Polypen. Die Untersuchungen weiterer Bruder (1 Jahr und 6 Jahre alt) sowie beider Eltern waren ohne pathologischen Befund. Die Familienanamnese ergab eine Blutsverwandtschaft der Eltern. Bei Verdacht auf Vorliegen eines HNPCC-Syndroms und nach Ausschluss einer FAP wurde die molekulare Diagnostik eingeleitet.

Die Bedeutung der Untersuchung von Glioblastomen im Rahmen des HNPCC-Screenings

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common forms of inherited colorectal cancer. Besides the clustering of colorectal carcinomas in one family, there is an excess of extracolonic cancer in the endometrium, ovary, stomach and urinary tract. Lacking of clinical features, diagnosis is based on family history, followed by microsatellite analysis. Furthermore the potential mutated genes can be revealed by immunohistochemical staining. Diagnosis is confirmed by the detection of a mutation in one of the known six DNA-Mismatch-Repair (MMR) genes (MSH2, MHL1, MSH6, PMS1, PMS2 and MLH3).