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Toward New Strategies to Select Young Endometrial Cancer Patients for Mismatch Repair Gene Mutation Analysis

Maran J.W. Berends, Ying Wu, Rolf H. Sijmons, Tineke van der Sluis, Wietske Boersmavan Ek, Marjolijn J.L. Ligtenberg, Neeltje J.W. Arts, Klaske A. ten Hoor, Jan H. Kleibeuker, Elisabeth G.E. de Vries, Marian J.E. Mourits, Harry Hollema, Charles H.C.M. Buys, Robert M.W. Hofstra, Ate G.J. van der Zee

From the Departments of Gastroenterology, Clinical Genetics, Pathology, Obstetrics and Gynecology, and Medical Oncology, University Hospital Groningen, Groningen; and Departments of Human Genetics and Pathology, University Medical Center Nijmegen, Nijmegen, the Netherlands.

Address reprint requests to A.G.J. van der Zee, MD, PhD, Department of Gynaecology, University Hospital Groningen, Hanzeplein 1, PO Box 30.001, 9700 RB Groningen; e-mail: A.G.J.van.der.Zee{at}og.azg.nl.

	ABSTRACT

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Purpose: To determine the frequency of mismatch repair (MMR) gene germline mutations in endometrial cancer patients who were diagnosed at less than 50 years of age; to relate the presence of mutations to family history, histopathologic data, presence of tumor microsatellite instability (MSI), and immunostaining; and to formulate criteria for genetic testing in these patients.

Patients and Methods: Endometrial cancer patients (N = 58), who were diagnosed at less than 50 years of age, were included and questioned about their family history. Mutation analysis of the MLH1, MSH2, and MSH6 genes was performed (denaturing gradient gel electrophoresis and sequence analysis to detect small mutations and multiplex ligation-dependent probe amplification to detect large deletions or duplications). For MSI analysis, five consensus markers were used, and immunostaining of the three MMR proteins was performed.

Results: In five of 22 patients with a positive first-degree family history for hereditary nonpolyposis colorectal cancer (HNPCC)-related cancers, pathogenic germline mutations were found (one MLH1, three MSH2, and one MSH6). Four mutation carriers belonged to families fulfilling the revised Amsterdam criteria. No mutations were found in the 35 patients without such family history (P = .006). MSI was detected in 20 of 57 cancers, among which four were from mutation carriers. In 23 of 51 cancers, one or more MMR protein was absent; in all five mutation carriers, immunostaining indicated the involved MMR gene.

Conclusion: In 23% of the young endometrial cancer patients with at least one first-degree relative with an HNPCC-related cancer, an MMR gene mutation was detected. Therefore, presence of an HNPCC-related cancer in a first-degree relative seems to be an important selection criterion for mutation analysis. Subsequent immunostaining of MMR proteins will point to the gene(s) that should be analyzed.

	INTRODUCTION

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HEREDITARY NONPOLYPOSIS colorectal cancer (HNPCC) is a dominantly inherited disorder characterized by a marked increase in cancer susceptibility. Endometrial cancer is, after colorectal cancer, the second most common tumor in HNPCC.¹ Most germline mutations have been detected in the following three mismatch repair (MMR) genes: MSH2, MLH1, and MSH6. In the Netherlands, in families with germline MMR mutations, the cumulative lifetime risk at age 70 for developing endometrial cancer is approximately 25% for MLH1 germline mutation carriers and 35% to 40% for MSH2 mutation carriers,² whereas this risk is 3% for the general population.³ The mean age for female carriers of an MLH1 or MSH2 mutation to develop endometrial cancer is approximately 47 years, whereas in sporadic endometrial cancer, the mean age is approximately 62 years.^3,4 In MSH6 mutation carriers, the mean age at diagnosis for endometrial cancer might be higher than the mean age at diagnosis in MLH1 or MSH2 mutation carriers, which is similar to what has been reported for colorectal cancer.^2,5,6

In 1991, the Amsterdam criteria I (AC I) were formulated to define families that are clinically affected by HNPCC. At that time, endometrial cancer was not included in the AC I.⁷ In the last decade, however, it became clear that these original AC I were too stringent because they did not take into account extracolonic cancers. Therefore, the criteria were revised, which resulted in the Amsterdam criteria II (AC II).⁸ The clinical diagnosis of HNPCC is now made when at least three relatives have histologically verified cancer of the colorectum, endometrium, small bowel, ureter, or renal pelvis, one of whom is a first-degree relative of the other two; at least two consecutive generations are affected; at least one of the patients with cancer is diagnosed before the age of 50 years; and familial adenomatous polyposis has been excluded.

In the AC II, a family history of cancer is considered an important indicator of HNPCC. However, reduced penetrance and occurrence of de novo mutations should be taken into account when performing genetic counseling.

In 1997, an international workshop on HNPCC formulated the Bethesda criteria, comprising clinicopathologic criteria that should lead to the identification of more MMR gene mutation carriers than by the AC I.⁹ According to these criteria, among others, individuals with endometrial cancer who are diagnosed at less than 45 years of age, irrespective of their positive family history, should be screened by microsatellite instability (MSI) analysis.¹⁰ MSI has been recognized as a hallmark of HNPCC¹¹ and is found in nearly all cancers from patients with an MLH1 or MSH2 germline mutation.¹⁰ MSI is also found in 9% to 25% of sporadic endometrial cancers, in particular because of hypermethylation of MLH1.^12,13

In this study, a relatively large number of endometrial cancer patients selected for young age was included, in whom MLH1, MSH2, and MSH6 mutation analysis was performed, irrespective of cancer MSI. The primary aim of this study was to determine the frequency of MLH1, MSH2, and MSH6 germline mutations in patients with endometrial cancer who were diagnosed at less than 50 years of age and to relate the results of mutation analysis to clinicopathologic data, such as age at diagnosis, family history, stage, and histology, the results of MSI analysis, and immunostaining of the three MMR proteins. The second aim of this study was to formulate appropriate recommendations for young endometrial cancer patients, their families, and their physicians with respect to genetic testing.

	PATIENTS AND METHODS

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All patients with endometrial cancer who were diagnosed at less than 50 years of age, irrespective of family history, were eligible for this cross-sectional study. Patients were identified by using the database of the regional cancer registry of the Comprehensive Cancer Center North-Netherlands (Groningen, the Netherlands) from 1989 onwards. Their doctors at participating hospitals or their general practitioners were asked to invite patients to participate in the study and to refer them to the study coordinator. Patients, who were newly diagnosed with endometrial cancer from September 1997 onwards, were also offered participation in the same way. Patients diagnosed with endometrial cancer before 1989 and who came to our attention for whatever medical reason were also included. Inclusion was ended by December 2000. When patients had given informed consent after written and verbal pretest counseling, 20 mL of blood was collected. Clinical data were reviewed. Cancer material was obtained, and the pathology was revised. A thorough family history for HNPCC-related cancers was taken. Colorectal cancer, endometrial cancer, cancer of the small bowel, the stomach, the pancreas, the biliary tract, and the ovaries, and transitional cell cancer of the pelvis, ureter, and bladder were considered to be HNPCC-related cancers. With the permission of relatives affected with HNPCC-related cancer, the relatives’ medical records were collected whenever possible to verify the nature of reported tumors. The participating patients were informed of the results of the genetic test, if they wished so. In that case, they received verbal posttest counseling and a written summary afterwards. The medical ethical committees of the University Hospital Groningen and other participating hospitals approved the study.

Histopathologic Review
Histologic classification and grading, based on the WHO criteria, were performed on the corresponding hematoxylin and eosin–stained endometrial cancer tissue sections.¹⁴ The tumors were grouped into endometrioid and nonendometrioid cancers. Only the endometrioid tumors were graded. Staging was based on histopathology after surgery, according to the International Federation of Gynecology and Obstetrics criteria.¹⁵

Mutation Analysis
For the purpose of this study, mutations were defined as pathogenic if changes in the gene sequences would cause inactivation of the allele, either because of the production of a truncated protein product or because of an exonic deletion or duplication. Mutation analysis of the MLH1, MSH2, and MSH6 genes in DNA obtained from peripheral-blood lymphocytes was carried out using denaturing gradient gel electrophoresis followed, in case of aberrant band patterns, by direct sequencing of independently amplified polymerase chain reaction products, as previously described.^16,17 For the detection of large deletions or duplications of whole exons or a complete gene, we used the multiplex ligation-dependent probe amplification (MLPA) test (MRC-Holland, Amsterdam, the Netherlands), as described earlier.¹⁸ The probe mix contains 16 exon probes for the MSH2 gene, 19 exon probes for the MLH1 gene, and seven control probes specific for DNA sequences outside the MSH2 and MLH1 genes (for details, see http://www.mrc-holland.com). Patients in whom no mutation was found using the denaturing gradient gel electrophoresis method were selected for the MLPA when absence of immunostaining of the MSH2 protein was noted or the tumor was MSI high (MSI-H) in absence of immunohistochemical results. Because MLH1 promoter hypermethylation has been frequently described in MSI-H endometrial cancer with absence of MLH1 protein staining¹³ and large deletions in the MLH1 gene seem to represent only a small proportion of the disease-causing mutations,¹⁹ patients with absence of MLH1 staining were not selected for the MLPA test. Confirmation of the MLPA data, for those patients who had deletions of one or more exons in the MSH2 gene, was performed by Southern blot analysis, according to Wijnen et al.²⁰ The restriction enzymes used for Southern blot analysis were XbaI, HindIII, and BglII (AP Biotech Inc, Piscataway, NJ). The following three amplicons of the MLH1 cDNA were used as probes for the hybridization: probe 1, exon 1 to 7, base pairs -12 to 1,265 (the A of the ATG is used as base pair No. 1); probe 2, exon 7 to 12, base pairs 1,070 to 2,006; and probe 3, exon 10 to 16, base pairs 1,511 to 2,875.

MSI
MSI primers were used as defined at an international workshop on HNPCC in Bethesda (National Cancer Institute, Bethesda, MD).¹⁰ These primers include two mononucleotide repeats (BAT25 and BAT26) and three dinucleotide repeats (D2S123, D5S346, and D17S250). DNA was extracted from formalin-fixed, paraffin-embedded tumor sections. Normal control DNA was obtained from normal tissue from paraffin-embedded sections or from peripheral blood of the same patients. MSI analysis was performed as described previously.⁶ Tumors were classified as MSI-H when two or more of the five consensus markers showed MSI and as MSI low (MSI-L) when no or one marker showed MSI. Because a limited number of markers were analyzed, the classification of microsatellite stable was not used.

Immunohistochemistry
MLH1, MSH2, and MSH6 immunohistochemistry was performed as described previously.⁶ Protein expression in normal tissue next to the cancer served as internal control. The sections were scored as either negative (ie, absence of detectable nuclear staining of cancer cells) or positive for MLH1, MSH2, and MSH6 staining. Scoring of tumor staining was performed without knowledge of the MSI or mutation status.

Statistical Analysis
For statistical analysis, the ² test, the Fisher’s exact test (in case of small numbers), and the Mann-Whitney test (to compare median ages) were performed. P values less than .05 were considered significant.

	RESULTS

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Clinical Data
A total of 58 apparently unrelated patients, who were diagnosed with endometrial cancer at less than 50 years of age, were included. The participating patients were treated between 1979 and 2001. The data of 34 of these patients were partly described in a previous report.²¹ The median age at diagnosis of endometrial cancer was 45 years (Table 1). Twenty-two patients (39%) had a first-degree relative with an HNPCC-related cancer (Table 1). One patient had no information about her family history. No difference was seen in first-degree family size (parents and siblings) between patients with and without a positive first-degree family history (5.4 v 5.2, respectively; P = .76). Thirteen patients had one or more cancers in addition to the endometrial cancer (Table 1). Five of these patients had one parent with an HNPCC-related cancer. Seven of the nine patients with a concurrent ovarian cancer did not have a first-degree relative with an HNPCC-related cancer. At the time of inclusion, families of eight (14%) of the 58 patients fulfilled the AC II; in five of these families, diagnoses of affected family members (predominantly affected with colorectal cancer) could not be completely histologically verified.

Three different missense variants and one intronic insertion were found in four patients (Table 3). Two hundred healthy Dutch individuals served as controls. The MSH6 variant Ser144Ile (previously reported⁶) was tested in a Saccharomyces cerevisiae–based functional assay.²² In this assay, the mutant showed loss of function, indicating a probable pathogenic mutation. However, in a more recent study applying an in vitro MMR assay the variant appeared as functional as the wild type.²³ Therefore, for this study, this mutation was not included as pathogenic.

MSI Analysis
Endometrial cancer material of 57 patients was available for MSI analysis. In 20 patients (35%), the endometrial cancer was MSI-H, and in 37 patients (65%), the cancer was MSI-L. In patients with a positive first-degree family history, the percentage of MSI-H endometrial cancers was 45% (10 of 22 patients), whereas this percentage was 29% otherwise (10 of 34 patients; not significant). In the 13 patients with multiple cancers, MSI data were available for nine patients; for three patients with an additional colorectal cancer (all mutation carriers), both cancers were MSI-H. Of the six patients with an additional ovarian cancer, the MSI results showed discrepancies in three patients.

Mutations were more often found in patients with an MSI-H endometrial cancer than in patients with an MSI-L cancer (four of 20 patients v one of 37 patients, P = .047). Three of the eight patients from AC II families had an MSI-L endometrial cancer, and one of these patients had a truncating MSH6 mutation (Table 2).

MSI was not related to age or to stage or grade of the tumor. The clear-cell and the papillary serous endometrial cancers were all MSI-H (n = 4); the undifferentiated endometrial cancer was MSI-L. The nonendometrioid endometrial cancers were more often MSI-H than the endometrioid cancers (four of five cancers v 16 of 52 cancers, P = .047).

Immunohistochemical Analysis
From 51 cancers, adequate specimens were available for MLH1 and MSH2 immunostaining, whereas adequate cancer specimens from 36 of these 51 tumors were available for MSH6 immunostaining. All endometrial cancers from the five mutation carriers showed absence of the concurrent MMR protein; the three MSH2 mutation carriers also showed absence of the MSH6 protein. Mutations were only detected in patients with absence of at least one of the MMR proteins in their tumors compared with patients with no absence of the MMR proteins in their tumors (five of 23 patients v zero of 28 patients, P = .018). The results of the immunohistochemical versus MSI analysis in 51 patients are listed in Table 4. Tumors with absence of one or more of the MMR proteins were more often MSI-H (14 of 24 tumors) than the tumors with positive staining (five of 27 tumors, P = .003). In seven of the eight patients from families fulfilling the AC II, a complete immunohistochemical analysis could be performed; apart from the four mutation carriers, two patients without a mutation had absence of staining of the MLH1 protein and of both the MSH2 and MSH6 protein, respectively. In patients without a first-degree family history, absence of the MLH1 protein was mainly seen (Table 4).

	DISCUSSION

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It is remarkable that three of the five mutations, as found in our study, were large MSH2 genomic deletions. To the best of our knowledge, no reports have yet been published specifically on the frequency of MSH2 deletions in young endometrial cancer patients.^{18,19,28–30} Because the number of mutations observed is small, our finding of only MSH2 deletions and no other MSH2 pathogenic mutations might well be coincidental. However, it is known that MSH2 mutation carriers have a higher probability of developing endometrial cancer than MLH1 mutation carriers.² A high incidence of endometrial cancer has also been noticed in families with MSH6 mutations but with a higher mean age of onset of 55 years.^5,6 This might explain the low frequency of MSH6 gene mutations in our study population.

Classic clinicopathologic characteristics of the endometrial tumor do not seem to be useful in predicting the presence of a germline mutation.³¹ In addition, we did not observe a relationship between MSI and stage or grade, which supports similar findings in endometrial cancer patients unselected for age.^32–34 Data, including ours, on MSI in nonendometrioid tumors are conflicting, most likely because of small sample size in all studies published so far or because of different panels of MSI markers.^34,35

Because endometrial cancer in MSH6 mutation carriers predominantly showed to be MSI-L in this study and others^5,6,36, the use of MSI analysis in endometrial cancer as a prescreening for mutation analysis may be insufficient. In clinically well-defined HNPCC families, immunohistochemistry in endometrial cancer proved to be a good prescreening method for mutation detection.²¹ In this study, six of the seven patients from families fulfilling the AC II indeed showed loss of at least one MMR protein. Furthermore, in five (22%) of 23 patients with absence of one of the MMR proteins, a germline mutation was found. Therefore, it seems that first-degree family history in combination with immunohistochemistry is a good selection criterion for mutation analysis. However, the numbers are small, and there might be a selection bias because of the exclusion of deceased endometrial cancer patients. Therefore, these data should be further evaluated in a prospective study of young endometrial cancer patients from different populations to show the validity of this policy. When no first-degree family history of HNPCC-related tumors is present but suspicion of HNPCC still exists (eg, based on an extended family history or because of the occurrence of another HNPCC-related cancer in the patient), one may decide to perform immunohistochemical analysis anyway because this is a relatively simple and cheap method.

Immunohistochemical analysis showed MLH1 protein absence in 13 cancers, of which one was from an MLH1 germline mutation carrier. This may be explained predominantly by the occurrence of MLH1 hypermethylation, as described by others^13,37, or by somatic MMR gene mutations or germline mutations in gene sequences not yet studied by us (eg, in promoter regions¹⁹). In seven MSI-L tumors, loss of MLH1 protein was observed. Although it has been shown that most tumors with MLH1 hypermethylation will be MSI-H, MLH1 hypermethylation in MSI-L tumors (two colorectal, one endometrial, and six gastric cancers) has previously been described,^13,38,39 but the underlying mechanism is not known yet. In tumors with MSH2 protein absence, concomitant MSH6 protein absence was observed, as reported before.^40,41 This may be a result of the fact that the heterodimeric complex of the MSH2 and the MSH6 protein cannot be formed, possibly leading to degradation of the MSH6 protein soon after synthesis.

In this study, little attention was given to the occurrence of missense variants. It is notable that the tumor of the carrier of the Val180Gly variant showed absence of MLH1 protein staining. Because the family history was negative, segregation analysis could not be performed. To further analyze the relevance of this variant and the cause of the absence of staining, the methylation status of the promoter of the MLH1 gene could be studied, and analysis of loss of heterozygosity might be performed. In general, pathogenicity of missense variants is hard to assess and, in fact, can only be determined by functional assays. However, these are currently not routinely performed. Once these functional assays become available for the clinic, their results will be included in counseling families.

Twenty-three percent of all young endometrial cancer patients with a positive first-degree family history in this study carried a germline MMR gene mutation. Three of the five mutations that were found were large MSH2 genomic deletions. As a prescreening method for mutation analysis in young endometrial cancer patients, immunohistochemistry in patients with a first-degree relative with an HNPCC-related cancer seems to be valuable. MSI analysis in our series of young endometrial patients did not add to immunohistochemical analysis with respect to identification of germline MMR gene mutations. A prospective analysis of more young endometrial cancer patients will be necessary to validate our proposed strategy for mutation analysis.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	ACKNOWLEDGMENTS

 
We thank, in addition to the referring specialists in our hospital, the following specialists for referring the patients: A.W.C. Wester (Assen), M.M. Henselmans (Drachten), Tj.D. Ypma (Emmen), J.F. Blacquiere (Heerenveen), A.B. Koster (Meppel), P.F.J. Donderwinkel, L. Emmen (Groningen, Martini Hospital), J.J. van Beek, H.H. de Haan, and H.B. Rethmeier (Zwolle). Furthermore, we thank J. Schakenraad and M. Schaapveld of the Comprehensive Cancer Centre North-Netherlands, Groningen, the Netherlands, for selection of patients fulfilling our inclusion criteria from the regional cancer registry.

	NOTES

 
Supported by grant no. RUG 97-1544 of the Dutch Cancer Society.

Presented at the Annual Meeting of the Society of Gynecological Oncology, New Orleans, LA, February 1–5, 2003.

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Submitted April 11, 2003; accepted September 16, 2003.

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