Int. J. Cancer: 122, 1077–1081 (2008) ' 2007 Wiley-Liss, Inc.
Hereditary nonpolyposis colorectal cancer in endometrial cancer patients Sang Nam Yoon1,2, Ja-Lok Ku3, Young-Kyoung Shin1, Kyung-Hee Kim1, Jin-Sung Choi1, Eun-Ja Jang1, Hyoung-Chul Park1,2, Duck-Woo Kim1,2, Min A Kim4, Woo Ho Kim4, Taek Sang Lee5, Jae Weon Kim5, Noh-Hyun Park5, Yong-Sang Song5, Soon-Beom Kang5, Hyo-Pyo Lee5, Seung-Yong Jeong6 and Jae-Gahb Park1,2,3* 1 Korean Hereditary Tumor Registry, Cancer Research Institute, Seoul National University, Seoul, Korea 2 Department of Surgery, Seoul National University College of Medicine, Seoul, Korea 3 Laboratory of Cell Biology, Cancer Research Institute, Seoul National University, Seoul, Korea 4 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea 5 Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea 6 Center for Colorectal Cancer, Research Institute and Hospital, National Cancer Center, Goyang, Korea Endometrial cancer is the second most common cancer in hereditary nonpolyposis colorectal cancer (HNPCC). It has often been overlooked to explore the possibility of HNPCC in endometrial cancer patients. Our study was to investigate how many HNPCC patients existed among endometrial cancer patients. Among patients who underwent hysterectomy for endometrial cancer at Seoul National University Hospital from 1996 to 2004, 113 patients were included, whose family history and clinical data could be obtained and tumor specimens were available for microsatellite instability (MSI) testing and immunohistochemical (IHC) staining of MLH1, MSH2 and MSH6 proteins. There were 4 (3.5%) clinical HNPCC patients fulfilling the Amsterdam criteria II, and 2 (2/ 4, 50%) of them carried MSH2 germline mutations. There were also 8 (7.1%) suspected HNPCC (s-HNPCC) patients fulfilling the revised criteria for s-HNPCC, and one (1/8, 12.5%) of them revealed MLH1 germline mutation. In 101 patients, who were not clinical HNPCC or s-HNPCC, 11 patients showed both MSI-high and loss of expression of MLH1, MSH2 or MSH6 proteins, and 2 (2/11, 18.2%) of them showed MSH6 germline mutations. In 113 patients with endometrial cancer, we could find 5 (4.4%) HNPCC patients with MMR germline mutation and 2 (1.8%) clinical HNPCC patients without identified MMR gene mutation. Family history was critical in detecting 3 HNPCC patients with MMR germline mutation, and MSI testing with IHC staining for MLH1, MSH2 and MSH6 proteins was needed in the diagnosis of 2 HNPCC patients who were not clinical HNPCC or s-HNPCC, especially for MSH6 germline mutation. ' 2007 Wiley-Liss, Inc. Key words: endometrial cancer; hereditary nonpolyposis colorectal cancer (HNPCC); microsatellite instability (MSI); mismatch repair gene (MMR); MLH1; MSH2; MSH6
The incidence of endometrial cancer has been increasing significantly among general population in Korea over the past decades.1 According to data on cancer incidence between 1999 and 2001 from the Korea Central Cancer Registry, the crude incidence rate per year is 3.11 cases per 100,000 Korean females.2 In a total of 43,627 new cases of female cancer registered by the Korea Central Cancer Registry in 2002, 825 cases were endometrial cancers, accounting for 1.9% of all malignancies in female.3 Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease characterized by high incidence of colorectal cancer (CRC), endometrial cancer and/or a variety of other cancers.4 Defective DNA mismatch repair (MMR) process is caused by germline mutations in the MMR genes, leading to the development of HNPCC. Germline mutations have been identified in five of these genes, MSH2, MLH1, PMS1, PMS2 and MSH6, and mutations in MSH2, MLH1 and MSH6 appear to account for the MMR defects seen in the majority of HNPCC families.5 Endometrial cancer is the second most common cancer found in HNPCC families.6 The lifetime risk of endometrial cancer for women with HNPCC is reported 40–60%, which is the same or greater than that of colorectal cancer.7 However, it has often been overlooked to explore the possibility of HNPCC in patients diagnosed as endometrial cancer. Publication of the International Union Against Cancer
Our study was to investigate how many HNPCC patients existed among the patients who underwent hysterectomy for endometrial cancer. Material and methods Patients The study population was drawn from a consecutive series of endometrial cancer patients who underwent hysterectomies at Seoul National University Hospital between January 1996 and December 2004. We enrolled 113 patients who answered the questions about their personal and family cancer histories, and whose paraffin-embedded tumor and normal tissues were available for microsatellite instability (MSI) analysis and immunohistochemical (IHC) staining. Patients were classified into clinical HNPCC when they fulfilled the Amsterdam criteria II,4 or into suspected HNPCC (s-HNPCC) when they fulfilled the revised criteria for s-HNPCC according to their personal and family cancer histories.8 Clinical data on pathologic result and patient demographics were retrospectively reviewed. Approval for taking personal and family cancer histories, as well as performing MSI analysis, IHC staining and mutational analysis was granted by the Institutional Review Board of the Seoul National University Hospital and the Seoul National University College of Medicine. MSI analysis For PCR-based MSI analysis, we utilized 2 mononucleotide repeats (BAT25 and BAT26) and 3 dinucleotide repeats (D2S123, D5S346 and D17S250); the MSI criteria and primers were as previously defined.9 The specific primer sequences of PCR amplification were obtained from http://www.gdb.org. Serial sections of paraffin-embedded matched normal and neoplastic primary tissues from each endometrial cancer patient were stained with H&E. Representative normal and tumor regions were identified by microscopic examination and separately microdissected. The dissected tissues were incubated overnight at 56°C in a lysis buffer containing proteinase K, the proteinase K was inactivated by heating for 15 min at 70°C, and the samples were used as template DNA for PCR analysis. The desired fragments were amplified in the presence of [a-P32] dCTP, using a programmable thermal cycler (PCR System 9700, Applied Biosystems; Foster City, CA). The PCR conditions consisted of a denaturation step at 94°C for The first three authors contributed equally to this work. Grant sponsors: Research Grant for National Cancer Center; BK21 Project for Medicine, Dentistry, and Pharmacy. *Correspondence to: Korean Hereditary Tumor Registry, Cancer Research Institute, Seoul National University, 28 Yongon-dong, Chongnogu, 110-744, Seoul, Korea. Fax: 182-2-742-4727. E-mail:
[email protected] Received 23 January 2007; Accepted after revision 19 April 2007 DOI 10.1002/ijc.22986 Published online 31 October 2007 in Wiley InterScience (www.interscience. wiley.com).
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10 min followed by 35 cycles of 95°C for 30 sec, 50–55°C for 1 min and 72°C for 1 min, and a final elongation at 72°C for 7 min. The PCR products were denatured and separated on 6 M urea/7% polyacrylamide gels run at 60 W. After electrophoresis, each gel was transferred to Whatmann 3 M paper, dried, and subjected to autoradiography. Tumors were classified as MSI-H (microsatellite instabilityhigh) when 2 or more of the 5 markers showed instability, as MSI-L (microsatellite instability-low) when only a marker showed instability, and as MSS (microsatellite stable) when none of the markers showed instability.9 Promoter hypermethylation in the MLH1 gene Promoter hypermethylation of the MLH1 gene was determined by the methylation-specific PCR (MS-PCR). The primers for MSPCR could amplify the methylated and unmethylated DNA in the promoter region of MLH1.10 Sodium bisulfite modification was done with a CpGenome DNA modification kit (Intergen, Oxford, UK). Amplified DNA fragments were fractionated in 2% agarose gel that was stained with ethidium bromide and visualized under UV. PCR reactions that demonstrated methylation were repeated for confirmation. Tissue array analysis Core tissue biopsies (2 mm in diameter) were taken from individual paraffin-embedded endometrial cancers (donor blocks) and arranged in a new recipient paraffin block (tissue array block) using a trephine apparatus (Superbiochips Laboratories, Seoul, Korea). Each tissue array block contained up to 50 cases, allowing 3 array blocks to contain the total of 113 cases. Samples were defined as adequate when the tumor occupied >10% of the core area. Each block contained an internal control consisting of nonneoplastic endometrium. Sections (4 lm) were cut from each tissue array block, deparaffinized and dehydrated for use in IHC staining. IHC staining for MLH1, MSH2 and MSH6 protein expression IHC staining for MLH1, MSH2 and MSH6 protein expression was performed as previously described,11 utilizing the following antibodies: MLH1, clone G168-15 (1 mg/mL; Pharmingen, San Diego, CA), a mouse MAb prepared with the full-length human recombinant MLH1 protein; MSH2, clone FE11 (210 mg/L; Zymed, San Francisco, CA), a mouse monoclonal antibody (MAb) generated with a carboxy-terminal fragment of the human MSH2 protein; MSH6, clone 44 (250 lg/mL; BD Transduction Laboratories, San Jose, CA), a mouse monoclonal antibody generated with an NH2terminal fragment (codons 225–333) of the MSH6 protein. Normal mouse sera were used as negative controls. Infiltrating lymphocytes and normal epithelial cells adjacent to the tumor cells served as internal positive controls. Tumors that demonstrated any evidence of MLH1, MSH2 or MSH6 expression, even in small foci, were considered positive for expression of that protein. Mutational analysis of the MLH1, MSH2 and MSH6 genes Mutational analysis was performed for patients who were identified as clinical HNPCC or s-HNPCC based on personal and family cancer history, and also for patients whose tumors showed both MSI-H and loss of MMR expression. Blood samples were obtained from patients and peripheral blood lymphocytes were isolated using Ficoll-Paque according to the manufacturer’s instructions (Amersham Biosciences, Uppsala, Sweden). Total genomic DNA was extracted using the TRI reagent following the manufacturer’s instructions (Molecular Research Center, Cincinnati, OH). When patient blood samples were unavailable, DNAs extracted from metastasis-free lymph nodes or microdissected normal tissues were used for mutational analysis. For identification of germline mutations in the MLH1, MSH2 and MSH6 genes, polymerase chain reaction—direct sequencing analysis was used to screen all gene coding regions, exon/intron
boundaries and core promoter sequences, as previously described.12 PCR products were bi-directionally sequenced using an ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Potentially mutated sequences were cloned into pCR2.1 (Invitrogen, San Diego, CA) for separate analysis of each allele, and to guard against sequencing errors. Statistical analysis Comparison of the variables was performed using Student’s t-test, Pearson’s v2 test, or Fisher’s exact test, depending on the nature of the data. Significance was assigned at the p < 0.05 level. Results are reported as mean 6 standard deviation. Results In 113 study patients, there were 4 (3.5%) clinical HNPCC patients fulfilling the Amsterdam criteria II. Two (1.8%) patients (SNU-H24 and SNU-H33) had already been registered as clinical HNPCC fulfilling the Amsterdam criteria II in Korean Hereditary Tumor Registry before hysterectomy.13 SNU-H33 carried MSH2 germline mutation, but not only SNU-H24 but also her younger sister with both endometrial cancer and colon cancer had no mutation of MMR genes.13 Another 2 (1.8%) (SNU-HE1 and SNUHE2) of 113 study patients were newly found to be clinical HNPCC during our study. Mutational screening revealed germline mutation of the MMR genes in 2 (SNU-H33 and SNU-HE1) of these 4 clinical HNPCC patients. Both had germline mutations in the MSH2 gene, and showed MSI-H and loss of MMR expression (Table I, Fig. 1). Eight (7.1%) patients met the revised criteria for s-HNPCC according to family history and mutational screening for mismatch repair (MMR) genes revealed germline mutation of the MLH1 gene in one (1/8, 12.5%) patient (SNU-HE1006). Another sHNPCC patient had a single nucleotide polymorphism in which cytosine was replaced by thymine (Leu to Phe) at codon 390 of exon7 in the MSH2 gene. In MSI testing, MSI-H phenotype was seen in 29 (25.7%) of 113 patients, MSI-L in 21 (18.6%), and MSS in 63 (55.8%). There were no difference in mean age, distribution of tumor stage, histologic grade and cellular type of cancer (data not shown). In IHC staining for MMR proteins, tumors from 26 (23.0%) patients showed loss of MMR protein expression. Loss of MLH1 expression was detected in 23 patients, loss of both MSH2 and MSH6 expression in 2, and loss of MSH6 expression in 1. No germline mutation was found in cases where the endometrial tumor sample exhibited MSI-L or MSS in MSI testing and retained MMR protein expression in IHC staining. Representative examples of MSI testing and IHC staining results are shown in Figures 2 and 3, respectively. Sixteen patients had MSI-H phenotype and concurrently showed loss of MMR protein expression. In these 16 patients, 3 patients were clinical HNPCC, 2 were s-HNPCC and the remaining 11 patients were not clinical HNPCC or s-HNPCC. Among these 11 patients, 2 (18.2%) carried germline mutations in MSH6 gene; one patient showed loss of both MSH2 and MSH6 expression, and the other exhibited loss of MSH6 expression. The other 9 patients, all of which showed loss of MLH1 expression, had no detectable germline mutations. Analysis for MLH1 promoter hypermethylation revealed that 7 of these samples showed MLH1 promoter hypermethylation, while the remaining 2 samples could not be analyzed due to the failure of the PCR amplification. Flow diagram for overall study results is illustrated at Figure 1. Discussion Recent studies reported that at least 1.8–2.1% of newly diagnosed endometrial cancer patients had germline mutations in the MMR genes.7,14 Five (4.4%) of 113 patients with endometrial cancer had germline mutation of the MMR genes in our series. In our
MSH6 MSH6 MSI-H None 72 SNU-HE5002
MSI, microsatellite instability; IHC staining, immunohistochemical staining; MMR, mismatch repair; HNPCC, hereditary nonpolyposis colorectal cancer; MSI–H, microsatellite-high; MSS, microsatellite stable. 1 SNU-H33 had already been registered as clinical HNPCC in the Korean Hereditary Tumor Registry before hysterectomy.–2Clinical HNPCC means that the patient fulfilled the Amsterdam criteria II.–3SNU-HE1 was newly diagnosed as clinical HNPCC in the present study.–4Suspected HNPCC means that the patient fulfilled the revised criteria for suspected HNPCC.
p.Gly1069Glu
Splice defect p.Cys697Arg p.Met587HisfsX6 p.Glu1274Lys, p.Cys1275X
c.2634_263411delGg c.2089T > C c.1757_1758insC c.3823G > A, c.3821_3824dupAATG c.3206G > A MSH2 MSH2 MLH1 MSH6 MSI-H MSI-H MSS MSI-H 43 44 56 48 SNU-H331 SNU-HE13 SNU-HE1006 SNU-HE5001
Clinical HNPCC2 Clinical HNPCC2 Suspected HNPCC4 None
MLH1, MSH2 (weak positive) MSH2, MSH6 MLH1 MSH2, MSH6
Mutational profiles Mutated genes Loss of MMR protein in IHC staining MSI testing Family history Age at diagnosis (years) Patient ID
TABLE I – CHARACTERISTICS OF 5 HNPCC PATIENTS WITH MMR GERMLINE MUTATION IN THIS STUDY
Consequences
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FIGURE 1 – Flow diagram for overall study results. In 113 patients, information of personal and family cancer histories was available and microsatellite instability and immunohistochemistry were assessed. Two known clinical HNPCC patients, and 2 newly found clinical HNPCC and 8 suspected HNPCC patients, and 11 patients showing MSI-H and loss of MMR protein expression without definite personal or family cancer history for clinical or suspected HNPCC were identified, and germline mutations of them are as illustrated. 1MMR protein is the abbreviation of mismatch repair protein; MLH1, MSH2 and MSH6 proteins.
FIGURE 2 – Representative examples of microsatellite instability. (a) BAT26; (b) D2S123. Arrows indicate microsatellite instability.
study, we used the revised criteria for s-HNPCC8 for mutation screening of MMR genes additional to the Amsterdam criteria II, and one mutation carrier could be found from 8 patients who met the revised criteria for s-HNPCC. MSI-H phenotype was seen in 29 (25.7%) of 113 patients in our study and this is consistent with the reported rate of 25–27%.15,16 Two previous studies reported that MSI-H phenotype were found in 18 and 45% of their endometrial cancer patients respectively, but these studies used different markers and criteria for MSI analysis, complicating the direct comparison of our results.11,12
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FIGURE 3 – Representative examples of immunohistochemical staining. (a) MLH1 negative; (b) MLH1 positive; (c) MSH2 negative; (d) MSH2 positive; (e) MSH6 negative; (f) MSH6 positive. Negative and positive mean the loss of expression and the retained expression, respectively. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
However, the rate of MSI-H in Korean endometrial cancer patients is higher than the 9.3% reported in primary sporadic CRC in Korea,17 and similarly, our finding that 23% of endometrial tumor samples showed loss of MMR protein expression indicates that this rate is higher than the 8.6% seen in primary sporadic CRC in Korean patients.18 We performed mutational analyses of patient samples from the identified clinical HNPCC, s-HNPCC and selected patients who showed both MSI-H and loss of MMR protein expression. Overall, the mutation rate in clinical HNPCC patients was 50% (2/4), and that in the s-HNPCC patients was 12.5% (1/8). Although our sample size was relatively small, these mutation rates are comparable to the 41.5% (22/53) and 19.8% (22/111) respectively found in clinical HNPCC and s-HNPCC patients identified through Korean
CRC probands and registered in the Korean Hereditary Tumor Registry.13 We did not test the two mutation-negative clinical HNPCC patients (SNU-H24, SNU-HE2) for possible large genomic rearrangements of DNA mismatch repair genes. Further test seems to be required to identify the role of large genomic rearrangements in the pathogenesis of our study patients. A previous report suggested that a combination of MSI and IHC analysis could be used to predict MMR gene germline mutations in patients with HNPCC.19 Consistent with this hypothesis, we found that the loss of MSH2 or MSH6 expression combined with MSI-H accurately predicted germline mutation in our patients, regardless of personal and family cancer history. Germline MSH6 mutations were detected in 2 of 11 patients who did not meet the Amsterdam criteria II or the revised criteria
HNPCC IN KOREAN ENDOMETRIAL CANCER PATIENTS
for s-HNPCC and showed both MSI-H and loss of MMR expression; one with loss of both MSH2 and MSH6 expression, and the other with loss of MSH6 expression. A previous study reported that germline MSH6 mutations have a relatively low penetrance with regard to neoplasm.20 However, endometrial cancer was also reported to be the most common cancer type among female carriers of MSH6 germline mutations.21 Among 9 patients who did not meet the Amsterdam criteria II or the revised criteria for s-HNPCC and showed both MSI-H and loss of MLH1 expression, we did not identify any germline MMR gene mutation. This prompted us to speculate the involvement of MLH1 promoter hypermethylation, since previous studies of randomly selected CRC samples revealed that MLH1 inactivation is most frequently due to MLH1 promoter hypermethylation.22 Fur-
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thermore, up to 70% of MSI-positive endometrial cancers have been associated with MLH1 promoter hypermethylation.15 Consistent with this, 7 of these 9 patients with both MSI-H and loss of MLH1 expression showed MLH1 promoter hypermethylation. In conclusion, we found 3 mutation carriers in MLH1 or MSH2 genes among 2 clinical HNPCC and 8 s-HNPCC patients identified by family history. Additional 2 mutation carriers in MSH6 gene were found by using MSI analysis and IHC staining. Thus, personal and family cancer histories should be obtained from all newly diagnosed endometrial cancer patients to identify HNPCC patients. In addition, MSI analysis and IHC staining for MMR proteins, especially for MSH6, might be helpful to find a small fraction of mutation carriers of MMR gene among patients with endometrial cancer.
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