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Detection of microsatellite instability from archival, hematoxylin-eosin--stained colorectal cancer specimen.

Germline mutations in several human DNA mismatch repair genes, including MSH2, MLH1, PMS1, PMS2, and MSH6/GTBP, have been identified in patients with hereditary nonpolyposis colorectal cancer (HNPCC). Alterations in MSH2 and MLH1 account for the majority of classically affected kindreds. Mismatch repair deficiency in tumors of patients with HNPCC is characterized by length alterations in noncoding repeat DNA sequences, so-called microsatellites, resulting from insertion or deletion of 1 or more repeat unit(s). This finding, termed microsatellite mutator phenotype or microsatellite instability (MSI), is detectable in more than 90% of colorectal cancer specimens from patients with HNPCC and in 10% to 15% of sporadic colorectal cancer specimens. (1,2) For MSI analysis, a reference panel of microsatellite markers should be investigated in fresh or paraffin-embedded material. (3) Polymerase chain reaction (PCR) amplification from archival paraffin-embedded tissue is usually performed, after DNA extraction using standard techniques, (4) or as previously reported, by direct PCR. (5) Here, we report MSI analysis of 2 patients with suspected HNPCC. Because no paraffin-embedded colorectal cancer tissue was archived, reference hematoxylin-eosin-stained tumor specimen slides were reviewed for potential predictive variables for DNA microsatellite instability and thereafter used for successful MSI analysis.


Case 1

A 47-year-old woman was referred to our clinic for further evaluation of suspected HNPCC. This patient was diagnosed with a colorectal cancer in 1995. At that time, she was 42 years old and presented with abdominal pain; a colonoscopy revealed a tumor of the cecum adjacent to the ileocecal valve. The patient underwent a right-sided hemicolectomy, and the tumor was classified as pT3 N0 M0 G4 R0. Currently, the patient is healthy without evidence of distant metastases or local recurrence. The patient's mother was diagnosed with endometrial carcinoma at the age of 47 years and with colorectal cancer at the age of 67 years. The patient's grandmother died at the age of 27 years of colorectal cancer. This patient has no siblings and her only son is healthy (Figure 1, A).


Case 2

A female patient was diagnosed at the age of 46 years with a moderately differentiated adenocarcinoma of the transverse colon without evidence of distant metastases. She underwent a partial colectomy, and the tumor was classified pT3 N3 M0 G2 R0. At the ages of 50 and 51 years, the patient developed solitary metastases of the liver and spleen, respectively, and subsequently a partial hepatectomy and splenectomy were performed. Postoperatively, she received 7 cycles of adjuvant chemotherapy with high-dose 5-fluorouracil and leucovorin treatment. Currently, she is doing well without evidence of further metastases or local recurrence. The patient's grandmother was diagnosed with endometrial carcinoma at the age of 48 years, and her grandfather died of pancreatic cancer at the age of 55 years. The patient's parents, her only brother, and her daughter are healthy (Figure 1, B). Because of early onset of colorectal cancer before the age of 50 years and the favorable outcome 8 years after partial liver resection and splenectomy for solitary metastases, this patient was referred at the age of 59 years to our clinic for further workup of suspected HNPCC.


Microscopic Evaluation

Slides from both patients were reviewed to analyze potential predictive variables for DNA microsatellite instability, for example, mucinous or poor differentiation, cribriform architecture, expanding tumor margins, peritumoral chronic inflammation with or without Crohn disease-like lymphocytic nodules, tumor-infiltrating lymphocytes, and the presence of contiguous adenoma. (6) The tumor of patient 1 protruded in a cauliflower-like pattern into the bowel lumen adjacent to the ileocecal valve. Microscopically, this tumor demonstrated a cribriform growth pattern infiltrating all layers of the colonic wall and focally invading the pericolonic fatty tissue. Only occasional lumina were present, and tumor-infiltrating lymphocytes were spread throughout the lesion (Figure 2). Peritumoral chronic inflammation, a Crohn disease-like peritumoral lymphocytic reaction, or residues of a preexisting adenoma were absent.


The tumor of patient 2 was located in the transverse colon and caused a subtotal stenosis of the bowel lumen. All layers of the colonic wall were infiltrated by moderately differentiated adenocarcinoma cells, and in addition to pericolonic fatty tissue invasion, lymphangiosis carcinomatosa was noted. Glandular differentiation was present. No tumor-infiltrating lymphocytes, peritumoral chronic inflammation, Crohn disease-like peritumoral lymphocytic reactions, or residues of a preexisting adenoma were noted.

Microsatellite Instability Analysis

After informed consent was obtained, an archival hematoxylin-eosin-stained, 5-[micro]m slide containing colorectal cancer tissue as well as adjacent normal colonic epithelium was retrieved for each patient. These specimens were used for MSI analysis 5 and 10 years (cases 1 and 2, respectively) after the initial diagnoses of colorectal cancer. The cover slide was removed, and areas of 2 to 3 mm (2) containing tumor tissue and normal colonic epithelium were microdissected and transferred into separate PCR tubes containing 20 [micro]L of 1% Triton X-100 (Sigma, Deisenhofen, Germany). Polymerase chain reaction was carried out as previously described (5) using published sequences of 3 different mononucleotide (BAT-25, BAT-26, BAT-40) and 4 dinucleotide repeat loci (D2S123, D3S1266, D5S346, D17S250) on chromosomes 4q12, 2p21-22, 1p13.1, 2p16, 3p21, 5q21-22, and 17q11.2-12, respectively. (3) Polymerase chain reaction products were mixed with 12 [micro]L formamide and 1 [micro]L TAMRA size standard, and electrophoresis was carried out on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany). All 7 microsatellite markers were successfully PCR amplified and simultaneously electrophoresed. The time from dissection to final analysis was less than 5 hours. Polymerase chain reaction products of the mononucleotide markers BAT-25, BAT-26, and BAT-40 of patient 1 clearly displayed an MSI pattern with additional alleles of shorter size for tumor DNA (Figure 3, A). In the tumor of this patient, distinct microsatellite instability with a shift toward shorter peaks of the amplified dinucleotide microsatellite markers D2S123 and D17S250 was also noted, while the markers D3S1266 and D5S346 did not display additional alleles. Because 5 of 7 analyzed loci demonstrated MSI, the tumor was classified as MSI-high. (3) No MSI was observed for any of the amplified microsatellite markers from tumor DNA of patient 2 (Figure 3, B). The amplification efficiency of the microsatellite markers BAT-25, BAT-26, BAT-40, and D5S346, comprising 110 to 130 base pairs (bp), was higher compared with the microsatellite markers D17S250 and D2S123, which are 150 to 210 bp in size. The lowest amplification efficiency was noted for the PCR product of the microsatellite marker D3S1266, which is 280 to 310 bp.



Hereditary nonpolyposis colorectal cancer accounts for approximately 2% to 5% of colorectal cancers. Identification of a specific germline DNA mismatch repair gene mutation is successful in 50% to 70% of families with classical HNPCC and allows predictive genetic testing of relatives at risk. (1) Recently, the finding of tumor MSI in patients with suspected HNPCC was demonstrated to be the best predictor of a germline DNA mismatch repair gene mutation. (7) Therefore, MSI analysis prior to germline mutation screening can help optimize costs and efforts in the molecular diagnosis of HNPCC. The presence of clinical and histopathologic findings, for example, young age, right-sided location, mucinous or poor differentiation, and/or tumor-infiltrating lymphocytes, indicates colorectal cancer patients who should be tested for MSI. (2,6) Because of limited archival space, paraffin-embedded tissue blocks are stored in many institutions only for a limited period of time, for example, 2 to 5 years. In the 2 patients discussed in this report, no paraffin-embedded tumor specimens were available 5 and 10 years (cases 1 and 2, respectively) after the initial diagnoses of colorectal cancer Therefore, we performed MSI analysis from hematoxylin-eosin-stained archival slides. After lysis of microdissected tumor and normal tissue, fluorescence PCR amplification was successful. Microsatellite instability was demonstrated in 1 patient with early-onset colorectal cancer and a family history highly suggestive of HNPCC.

Conflicting data concerning the reproducibility of PCR amplification from stained tissue have been reported. Burton et al (8) showed that PCR amplification of DNA extracted from hematoxylin-eosin-stained tissue is often unsuccessful. In contrast, Banaschak et al (9) demonstrated that PCR amplification of short tandem repeat loci is consistently successful using hematoxylin-eosin-stained tissue. These discrepancies have been attributed to differences in tissue fixation, staining, DNA extraction, and purification procedures. In the presented cases, MSI analysis of different markers, including those of the current international reference panel, (3) was consistently successful by direct fluorescence PCR amplification using tissue that was stained with hematoxylin-eosin according to a standard procedure. However, we noticed differences in signal intensities of PCR products. In general, shorter PCR fragments, for example, those of the markers BAT-25, BAT-26, BAT-40, and D5S346, which comprise 110 to 130 bp, gave stronger signals than the larger fragments of markers D2S123, D3S1266, and D17S250. This phenomenon may be related to DNA degradation. Nevertheless, we were able to amplify DNA fragments up to 300 bp in size using the described method.

In summary, MSI analysis is possible by direct fluorescence PCR amplification using hematoxylin-eosin-stained colorectal cancer specimen slides. Because hematoxylin-eosin-stained slides generally are archived for longer periods of time than paraffin-embedded tissue blocks, a time- and cost-effective molecular evaluation of suspected HNPCC is possible, even in cases in which access to paraffin-embedded tissue blocks is not possible.


(1.) Lynch HT, de la Chapelle A. Genetic susceptibility to non-polyposis colorectal cancer. J Med Genet. 1999;36:801-818.

(2.) Vasen HF, Watson P, Mecklin JP, Lynch HT. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology. 1999;116:1453-1456.

(3.) Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998;58:5248-5257.

(4.) Burns WC, Liu YS, Dow C, Thomas RJS, Phillips WA. Direct PCR from paraffin-embedded tissue. Biotechniques. 1997;22:638-640.

(5.) Raedle J, Brieger A, Trojan J, Herrmann G, Zeuzem S. Rapid microsatellite analysis of paraffin embedded tumour specimens from patients with hereditary non-polyposis colorectal cancer. J Clin Pathol. 1998;51:621-622.

(6.) Jass JR, Do KA, Simms LA, et al. Morphology of sporadic colorectal cancer with DNA replication errors. Gut. 1998;42:673-679.

(7.) Liu T, Wahlberg S, Burek E, Lindblom P, Rubio C, Lindblom A. Microsatellite instability as a predictor of a mutation in a DNA mismatch repair gene in familial colorectal cancer. Genes Chromosom Cancer. 2000;27:17-25.

(8.) Burton MP, Schneider BG, Brown R, Escamilla-Ponce N, Gulley ML. Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues. Biotechniques. 1998;24:86-92.

(9.) Banaschak S, Rolf B, Brinkmann B. Influence of different staining techniques on the DNA analysis of histological sections. Int J Legal Med. 2000;113:114-116.

Accepted for publication July 23, 2001.

From the Second Department of Internal Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany (Drs Trojan, Raedle, Brieger, and Zeuzem); and the Department of Pathology, Klinikum Ludwigsburg, Ludwigsburg, Germany (Dr Herrmann).

Reprints: Stefan Zeuzem, MD, Second Department of Internal Medicine, Klinikum der Johann Wolfgang Goethe-Universitat, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany (e-mail:
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Author:Trojan, Joerg; Raedle, Jochen; Herrmann, Guenter; Brieger, Angela; Zeuzem, Stefan
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:4EUGE
Date:Feb 1, 2002
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