Molecular Analysis of Familial Endometrial Carcinoma: A Manifestation of Hereditary Nonpolyposis Colorectal Cancer or a Separate Syndrome
http://www.100md.com
《临床肿瘤学》
the Departments of Medical Genetics, and Biological and Environmental Sciences, University of Helsinki
Laboratory of Molecular Genetics, and Department of Obstetrics and Gynecology, Helsinki University Central Hospital
Family Federation of Finland and Finnish Cancer Society, Helsinki, Finland
ABSTRACT
PURPOSE: Familial clustering of endometrial carcinoma (EC) may occur as part of hereditary nonpolyposis colorectal cancer (HNPCC), a multiorgan cancer syndrome with mismatch repair (MMR) deficiency. Clustering of EC alone, termed as familial site-specific EC, may constitute a separate entity. Because its genetic basis is unknown, our purpose was to characterize such families molecularly.
MATERIALS AND METHODS: Twenty-three families with site-specific EC were identified among 519 consecutive patients diagnosed with EC during 1986 to 1997. Tumor tissues were examined for MMR protein expression by immunohistochemical (IHC) analysis, and MMR genes pinpointed by IHC changes were screened for germline mutations by exon-by-exon sequencing, multiplex ligation-dependent probe amplification, and direct tests for mutations common in the population.
RESULTS: Among 33 ECs from 23 families, MLH1 protein was lost in seven tumors (21%), MSH2 together with MSH6 was lost in four tumors (12%), and MSH6 alone was lost in five tumors (15%). A truncating germline mutation in MSH6 (3261insC) was identified in one family and a likely pathogenic missense mutation in MSH2 (D603N) was identified in another family. Among the original 519 patients, nine (all with colon cancer in the family) were diagnosed with HNPCC at the outset—six with MLH1 and three with MSH2 mutations.
CONCLUSION: Our study gives a minimum overall frequency of 2.1% (11 of 519) for germline MMR defects ascertained through EC in the index patients. The fact that only two of 23 families with site-specific EC (8.7%) had germline mutations in MMR genes suggests another as yet unknown etiology in most families with site-specific EC.
INTRODUCTION
Endometrial cancer (EC) is among the three most common cancers in females in many industrialized countries. Known risk factors for this cancer include obesity, hypertension, diabetes mellitus, late menopause, and exogenous estrogen use.1,2 It has been estimated that 5% of patients with ECs diagnosed at age younger than 55 years have a family history of this cancer.3 Furthermore, epidemiologic studies have shown an association between EC and colorectal cancer.3,4 This may be explained in part by the fact that EC is the most common extracolonic malignancy in hereditary nonpolyposis colorectal cancer (HNPCC), a well-defined syndrome associated with inherited DNA mismatch repair (MMR) deficiency.5
Available information of the role of MMR genes in human endometrial tumorigenesis is derived from three main settings. First, on the basis of microsatellite instability (MSI) and immunohistochemical (IHC) findings, somatic inactivation of MMR genes occurs in 20% to 35% of sporadic endometrial tumors.6,7 Second, a number of studies have been conducted to determine the frequency of germline mutation carriers of MMR genes among unselected or consecutive patients with EC. These studies, which have typically used MSI for preselection, have arrived at carrier frequencies ranging from 0%8,9 to approximately 10%10,11 for MLH1 and MSH2, and from 0%11 to 23%12 for MSH6 (frequencies calculated among MSI-positive patients). Third, investigations of EC suggestive of having a hereditary basis by clinical presentation have typically focused on patients with selected HNPCC features, such as coexistence of EC and colon cancer or early age at onset. As expected, such studies report somewhat higher frequencies for germline mutation carriers compared with unselected series (up to 23% irrespective of MSI).13-16
Occasional families show clustering of EC alone, without colon or other cancers, termed as familial site-specific EC.3,17 The molecular basis of this proposed syndrome is entirely unknown, which prompted us to conduct a study on 23 patients with familial site-specific EC. We provide evidence of frequent MMR protein loss in tumors from such families, accompanied by a high degree of MSI (MSI-H) in only a minority of patients. We also demonstrate that whereas MMR gene involvement in the germline is rare, EC may be the only manifestation of an inherited MMR gene mutation even in extensive families.
MATERIALS AND METHODS
Patients and Specimens
This study was based on a consecutive series of patients treated for EC at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital (Helsinki, Finland) in 1986 to 1997 (Fig 1). Patients (N = 519) were interviewed for family history, which resulted in the identification of nine families with HNPCC based on a family history of colon cancer and the fulfillment of the Amsterdam criteria I18 or II.19 Another 23 families fulfilled our criteria for site-specific EC based on the presence of EC in at least one first-degree relative of the index patient (in family 9, the closest affected relative was of second degree) in the absence of any associated clustering of colon or other cancers. In addition to the information obtained during the interview, family histories were documented by identifying all first-degree relatives (parents, siblings, and children) in the official population registries (church registries) and verifying the cancer diagnoses through the nationwide Finnish Cancer Registry (the great majority of cancers reported to this registry are histologically or cytologically documented).
Paraffin-derived specimens of tumor and matching normal tissues of the index patients and their relatives were collected from the pathology departments of different hospitals and used for IHC analysis and DNA extraction. For family 15, blood specimens were also available. This study was approved by the appropriate institutional review boards of the Helsinki University Central Hospital.
IHC Analysis
Formalin-fixed, paraffin-embedded tissue sections were immunohistochemically stained with anti-MLH1 (clone G168-15; Pharmingen, San Diego, CA), anti-MSH2 (clone FE-11; Calbiochem/Oncogene Research, Darmstadt, Germany), and anti-MSH6 (clone 44; Transduction Laboratories, San Diego, CA). The EnVision+ System (Dako Cytomation, Glostrup, Denmark) was applied according to manufacturer's instructions with the antigen-retrieval step performed by boiling the sample in a microwave for 10 minutes in EDTA buffer (pH 8.0). The percentage of positively stained tumor cells varied between 10% and 95% (mean, 68%) for cases considered positive. Nuclear staining of normal endometrium and stromal cells included in each tumor section were used as a reference for the evaluation of the staining results.
MSI and Loss of Heterozygosity Analysis
DNA was prepared from archival paraffin-embedded tumor and normal tissue samples according to the method of Isola et al.20 Areas with pure normal or high tumor percentages were selected and verified histologically and subsequently dissected out. Tumor percentages ranged between 50 and 90.
MSI status was determined using the Bethesda panel of five markers (BAT25, BAT26, D5S346, D2S123, and D17S250).21 Tumors with two or more unstable markers were considered to have MSI-H and those with one unstable marker were considered to have low-degree microsatellite instability (MSI-L), whereas those with no unstable markers were microsatellite stable (MSS).
The results obtained with dinucleotide repeat markers from the Bethesda panel were also analyzed for loss of heterozygosity (LOH) in informative tumors (ie, those heterozygous and not showing MSI). In addition, LOH at 2p and 3p was evaluated using markers flanking MSH2/MSH6 and MLH1, respectively. The MSH2/MSH6-linked markers were ptel-D2S2378-MSH2/MSH6-CA7-D2S123-cen, spanning a 3.3-centimorgan (cM) region.22 The MLH1-linked markers were ptel-BAT21-D3S1611-D3S1298-cen, including two intragenic markers (BAT21 and D3S1611) and spanning a region of 0.6 cM. The primer sequences for the markers are available at http://www.gdb.org. The forward primers were fluorescently labeled, and the polymerase chain reaction (PCR) products were run on an automated sequencer and analyzed with the Genotyper 2.0 program (Applied Biosystems, Foster City, CA). For LOH analysis, the ratio of allelic peak areas was calculated as follows.23
where L is the LOH ratio, A1T is the area of allele 1 in the tumor, A2T is the area of allele 2 in the tumor; A1N is the area of allele 1 in a normal sample, and A2N is the area of allele 2 in a normal sample.
A sample was scored as showing LOH if L 0.6 or L 1.67 (indicating that one of the alleles had decreased 40% or more), and was scored as showing putative LOH or allelic imbalance if 0.6 < L < 0.8 or 1.25 < L < 1.67 (indicating the decrease of 21% to 39% for one allele).
MLH1 Promoter Methylation Analysis
The methylation status of three HpaII sites located at nucleotide positions –567, –347, and –341 relative to the A of the initiating codon of MLH1 (GenBank accession number U83845) was assessed by a method that relies on the methylation sensitivity of the HpaII restriction enzyme.24 The methylation status of tumor DNA was compared with that of normal endometrial DNA from the same individuals.
Mutation Analysis
The individual exons of MLH1, MSH2, and MSH6 were screened for mutations by direct sequencing of genomic PCR products using primers described in Chadwick et al.11 In addition, the Finnish founding mutation 1 (a large genomic deletion of MLH1 exon 16 and flanking introns)25 was tested in a PCR assay using two forward primers, one preceding (5'-GAGCCTCCAATACAATGTTGAATAGAAG-3') and the other one located within the deleted fragment (5'-ACATATGTGACATCCTCTCCACTCC-3'), and a common reverse primer (5'-CTCTCCATGTCTGGGTCCTTC-3'). Screening for two MSH6 mutations previously found in the Finnish population was performed with primers for fragment 4k26 for 3052delCT,27 and with 5'-GAAACAGCGCAACAGAATTG-3' and 5'-ATCCCTCCGTTCTTCAGCAT-3' for E995X.28 Furthermore, the possible presence of the frameshift mutation affecting the MSH6-C8, found in family 15 in this study, was evaluated in other families using primers described in Malkhosyan et al29
Multiplex ligation-dependent probe amplification (MLPA)30 was used to screen the samples for large genomic deletions or amplifications in MLH1, MSH2, and MSH6 using kits (SALSA P003, SALSA P008) and protocols (MRC-Holland, Amsterdam, the Netherlands). HNPCC samples from our collection known to carry large genomic deletions were used as positive controls.
RESULTS
Characteristics of Families and Tumors
Of 519 consecutive patients with EC, 62 indicated a family history of cancer (Fig 1). These included nine patients immediately diagnosed with HNPCC based on colorectal cancers in the family and fulfillment of the Amsterdam criteria. Of these, eight turned out to represent families with predisposing mutations identified previously (MLH1 in six families and MSH2 in two families; kindreds 3, 11, 28, 38, 39, 83, 93, and 108, reported in Nystrm-Lahti et al31 and Holmberg et al32), whereas the remaining family revealed a predisposing mutation (D603N in MSH2) in our investigation. An additional 26 patients reported nonspecific clustering of various cancers (other than EC) in the family. Excluding these patients and another four with unavailable samples, 23 patients with familial site-specific EC remained and formed our primary study series.
Clinicopathologic characteristics of the tumors (according to the International Federation of Gynecology and Obstetrics staging system)33 as well as family histories of the index patients are listed in Table 1. Twelve families showed evidence of vertical and 10 families showed evidence of horizontal transmission (the latter group included families 24 and 29 that were ascertained independently, but were subsequently found to unite into a single family). The mean age at EC diagnosis was 62 years.
Strategy for Molecular Characterization of Familial Site-Specific EC
In the first phase, archival tissue specimens were collected from the index patients and their relatives and screened for MLH1, MSH2, and MSH6 protein expression by IHC analysis, as well as for MSI, LOH, and MLH1 promoter hypermethylation (Fig 1). A total of 37 tumors were obtained for molecular studies (Table 2), including 34 ECs together with two breast cancers and one ovarian cancer occurring as additional primary cancers in the index patients. In the second phase, MMR genes implicated by IHC alterations were screened for germline mutations by exon-by-exon sequencing (for point mutations), MLPA (for large genomic deletions and amplifications), and direct tests for mutations previously detected in this population25,27,28,32,34 (see Materials and Methods). According to a recent report,16 alterations detectable by MLPA might constitute a majority of germline mutations occurring in EC patients.
IHC Alterations
Thirty-three EC tumors were available for IHC analysis of MLH1, MSH2, and MSH6, and abnormal protein expression was seen in 16 (48%; Table 2). Tumors with expression changes originated from 12 of 23 families (52%). MLH1 was lost in seven tumors of 33 (21%) and MSH2 together with MSH6 were lost in four tumors (12%), whereas five tumors (15%) showed abnormal MSH6 staining alone (loss of expression or cytoplasmic staining).
Germline Mutations
All patients with IHC changes were investigated for germline mutations of the respective genes. IHC alterations were mostly discordant in EC tumors from different members of the same family (eg, 14:1 displayed absent MLH1 and 14:2 absent MSH2 protein; Table 2), and it was therefore not surprising that no MMR gene germline mutations were found in such families. However, two families (15 and 13) showed intrafamilial concordance for IHC changes, and both revealed germline mutations in subsequent analysis. Three individuals tested from family 15 all showed MSH6 protein loss in EC (Table 2). Sequencing revealed a truncating germline mutation in MSH6 (insertion of C at nucleotides 3261 to 3262, codon 1088 in exon 5, designated as 3261insC), which cosegregated with EC in this family (Fig 2). Although MSH6 germline mutations are known to be associated with high risk for EC,35 EC typically coexists with colon and other cancers; family 15 was exceptional by displaying late-onset (mean, 60.8 years) EC in six members in the absence of colon cancer. An identical germline mutation was previously reported to occur in an HNPCC family from Australia (Bennett et al, www.insight-group.org); the clinical features of this family were not specified.
Family 13 was small, with two affected individuals (Table 1), and samples were available from the index patient (13:1) only (EC and ovarian cancer representing two different primary tumors; Table 2). These tumors showed a concordant MSH2/MSH6 change by IHC, and a germline mutation of the missense type (G>A at nucleotide 1807, codon 603, in exon 12 of MSH2) was detected by sequencing. This change, designated as D603N, is likely to be pathogenic for the following reasons. First, it substitutes asparagine for aspartic acid, which involves a change in the polarity group at a position that is evolutionarily conserved. Second, even though no segregation studies were possible in family 13, we detected the same germline alteration in a family diagnosed with HNPCC in the beginning (see Characteristics of Tumors and Families), and this family did show cosegregation of the mutation with HNPCC-type cancers. Furthermore, there is an earlier report of the D603N mutation occurring in a colon cancer patient with positive family history of cancer.36 Third, this change was absent in 129 healthy control individuals from the same population (this study and Salovaara et al36).
MSI, LOH, and MLH1 Promoter Methylation
Among 33 ECs analyzed by IHC, five (15%) demonstrated MSI-H and the remaining 28 had MSI-L (18%) or MSS (67%) by the Bethesda panel of markers (Table 2). Loss of MSH2 protein expression in EC (accompanied by abnormal MSH6 staining in all tumors) was regularly associated with MSI-H (three of four; 75%), whereas MSI-H was less frequent among tumors with MLH1 protein loss (two of seven; 29%) or solitary MSH6 expression change (one of five; 20%).
As possible somatic events that might underlie aberrant protein expression, LOH at 2p and 3p as well as MLH1 promoter methylation were addressed. Eight EC tumors with MSH2 and/or MSH6 protein loss were informative for the nearby markers D2S123, CA7, and D2S2378, and seven tumors (88%) showed LOH or putative LOH in at least one locus (Table 2). For comparison, the same tumors showed LOH frequencies of only one of seven (14%) for D5S346 and D17S250 each. Although these results could support the role of physical deletions at 2p as contributors to aberrant MSH2/MSH6 expression, this region might generally be prone to deletions and rearrangements, given that tumors with normal MSH2/MSH6 protein expression also showed frequent LOH or putative LOH at 2p (12 of 21; 57%). Six EC tumors with loss of MLH1 protein were informative for the intragenic markers BAT21 and D3S1611, and two (33%) showed LOH or putative LOH. Because this frequency did not significantly differ from LOH frequencies observed for D5S346 (three of six; 50%) or D17S250 (two of six; 33%) in the same tumors, or relative to 3p loss among tumors with normal MLH1 protein expression (three of 14; 21%; Table 2), the possible association of the loss of 3p genetic material with decreased MLH1 protein expression remains unclear. Finally, among seven tumors lacking MLH1 protein, MLH1 promoter hypermethylation could provide an explanation for one (14%; patient 14:1 in Table 2).
DISCUSSION
The existence of familial site-specific EC as a separate entity3,17 remains equivocal until the identification of predisposing gene defects specific for this disorder. HNPCC is the most important hereditary syndrome to be considered for differential diagnosis; for example, according to the Amsterdam criteria II,19 HNPCC diagnosis may be based on certain extracolonic cancers (including EC) alone, provided that at least one tumor is diagnosed in the patient younger than 50 years. To determine the extent of overlap between pure EC families and HNPCC, we analyzed the role of MMR genes in 23 families with site-specific EC. Our families had from two to six EC patients per family and an average age at EC diagnosis of 62 years, which is older than that for EC occurring in HNPCC (50 years37), but younger compared with the general population (66 years38). Our selection criterion (family history of EC alone in a newly diagnosed EC patient) led to the identification of two families with MMR gene germline mutations (two of 23; 8.7%). The observed frequency of germline mutation carriers was comparable to that reported for ECs selected for young age (younger than age 50 years, five of 57; 9%16). The simultaneous presence of colon or other GI cancer in an EC patient herself or in her family seems to be a somewhat stronger predictor of hereditary MMR defects, with reported frequencies of 16% to 23% for germline carriers in such series.13,14,16
On the basis of available data on known MMR gene mutation carriers showing that the gene mutated in the germline was always inactivated in their EC tumors,15,16,39,40 we used IHC as a guide for germline mutation analysis. In our study, expression changes were selective because only one MMR protein was altered in each tumor, with the exception of the previously described instability of MSH6 in the absence of its dimerization partner MSH2.39 Among 16 EC tumors with IHC alterations, four (which originated from two families) were associated with germline mutations, and these tumors, in contrast with the remaining ones, were distinguished by intrafamilial concordance for IHC changes, which therefore seems to be a useful indicator of a germline defect.
The correlation between IHC alterations and MSI was incomplete in our series, which questions the applicability of MSI alone, without IHC, for possible preselection of EC patients for germline mutation analysis. Although all tumors with MSI-H did show inactivation of at least one MMR protein, indicating that MLH1, MSH2, and MSH6 accounted for all patients with MSI-H, only five of 16 (31%) tumors with demonstrable MMR protein inactivation by IHC showed MSI-H. The lack of MSI-H was a special characteristic of inactivation of MLH1 or MSH6, but not MSH2. Accordingly, among families with identifiable germline mutations, two of two tumors tested from family 13 with MSH2 mutation showed MSI-H as opposed to zero of three ECs from family 15 with MSH6 mutation. We have reported previously that a significant fraction of ECs from known MLH1 mutation carriers fails to display MSI,41 and only 68% of sporadic EC tumors with IHC alterations investigated by Stefansson et al7 had MSI. On the basis of these observations, the predictive value of abnormal IHC for an MSI-H phenotype in EC is lower than in colorectal cancer41,42; the reasons for this are not known, but could be explained theoretically by normal tissue contamination or clonal heterogeneity, for example.41
Altogether, 11 families with inherited MMR defects were detected among 519 consecutive patients with EC on the basis of family history of EC (two families) or colorectal cancer (nine families). The present estimate for the incidence of MMR gene germline mutations ascertained through EC (11 of 519; 2.1%) is similar to previous estimates based on consecutive ECs (0.8% to 1.4%10,11) or colorectal cancers (0.3% to 3%36,43-47) using MSI for preselection. It is obvious that our figure represents a minimum estimate. With regard to families with colon cancers, only those fulfilling the Amsterdam criteria I or II were considered, and with regard to families with site-specific EC, no mutation detection strategy, including ours, is expected to be 100% accurate. Even with these limitations, it seems safe to conclude that only a small fraction of families with site-specific EC is explained by germline mutations in MMR genes. Additional studies should establish if the status of the remaining families is due to heritable defects in other genes, nongenetic factors, or chance.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank Gynel Arifdshan and Saila Saarinen for expert technical assistance, and Heli Surma-Aho and Katja Kuosa for assistance with sample collection.
NOTES
Supported by the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Cancer Foundation, the Finnish Cultural Foundation, the Paulo Foundation, and the Oskar flund Foundation.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Laboratory of Molecular Genetics, and Department of Obstetrics and Gynecology, Helsinki University Central Hospital
Family Federation of Finland and Finnish Cancer Society, Helsinki, Finland
ABSTRACT
PURPOSE: Familial clustering of endometrial carcinoma (EC) may occur as part of hereditary nonpolyposis colorectal cancer (HNPCC), a multiorgan cancer syndrome with mismatch repair (MMR) deficiency. Clustering of EC alone, termed as familial site-specific EC, may constitute a separate entity. Because its genetic basis is unknown, our purpose was to characterize such families molecularly.
MATERIALS AND METHODS: Twenty-three families with site-specific EC were identified among 519 consecutive patients diagnosed with EC during 1986 to 1997. Tumor tissues were examined for MMR protein expression by immunohistochemical (IHC) analysis, and MMR genes pinpointed by IHC changes were screened for germline mutations by exon-by-exon sequencing, multiplex ligation-dependent probe amplification, and direct tests for mutations common in the population.
RESULTS: Among 33 ECs from 23 families, MLH1 protein was lost in seven tumors (21%), MSH2 together with MSH6 was lost in four tumors (12%), and MSH6 alone was lost in five tumors (15%). A truncating germline mutation in MSH6 (3261insC) was identified in one family and a likely pathogenic missense mutation in MSH2 (D603N) was identified in another family. Among the original 519 patients, nine (all with colon cancer in the family) were diagnosed with HNPCC at the outset—six with MLH1 and three with MSH2 mutations.
CONCLUSION: Our study gives a minimum overall frequency of 2.1% (11 of 519) for germline MMR defects ascertained through EC in the index patients. The fact that only two of 23 families with site-specific EC (8.7%) had germline mutations in MMR genes suggests another as yet unknown etiology in most families with site-specific EC.
INTRODUCTION
Endometrial cancer (EC) is among the three most common cancers in females in many industrialized countries. Known risk factors for this cancer include obesity, hypertension, diabetes mellitus, late menopause, and exogenous estrogen use.1,2 It has been estimated that 5% of patients with ECs diagnosed at age younger than 55 years have a family history of this cancer.3 Furthermore, epidemiologic studies have shown an association between EC and colorectal cancer.3,4 This may be explained in part by the fact that EC is the most common extracolonic malignancy in hereditary nonpolyposis colorectal cancer (HNPCC), a well-defined syndrome associated with inherited DNA mismatch repair (MMR) deficiency.5
Available information of the role of MMR genes in human endometrial tumorigenesis is derived from three main settings. First, on the basis of microsatellite instability (MSI) and immunohistochemical (IHC) findings, somatic inactivation of MMR genes occurs in 20% to 35% of sporadic endometrial tumors.6,7 Second, a number of studies have been conducted to determine the frequency of germline mutation carriers of MMR genes among unselected or consecutive patients with EC. These studies, which have typically used MSI for preselection, have arrived at carrier frequencies ranging from 0%8,9 to approximately 10%10,11 for MLH1 and MSH2, and from 0%11 to 23%12 for MSH6 (frequencies calculated among MSI-positive patients). Third, investigations of EC suggestive of having a hereditary basis by clinical presentation have typically focused on patients with selected HNPCC features, such as coexistence of EC and colon cancer or early age at onset. As expected, such studies report somewhat higher frequencies for germline mutation carriers compared with unselected series (up to 23% irrespective of MSI).13-16
Occasional families show clustering of EC alone, without colon or other cancers, termed as familial site-specific EC.3,17 The molecular basis of this proposed syndrome is entirely unknown, which prompted us to conduct a study on 23 patients with familial site-specific EC. We provide evidence of frequent MMR protein loss in tumors from such families, accompanied by a high degree of MSI (MSI-H) in only a minority of patients. We also demonstrate that whereas MMR gene involvement in the germline is rare, EC may be the only manifestation of an inherited MMR gene mutation even in extensive families.
MATERIALS AND METHODS
Patients and Specimens
This study was based on a consecutive series of patients treated for EC at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital (Helsinki, Finland) in 1986 to 1997 (Fig 1). Patients (N = 519) were interviewed for family history, which resulted in the identification of nine families with HNPCC based on a family history of colon cancer and the fulfillment of the Amsterdam criteria I18 or II.19 Another 23 families fulfilled our criteria for site-specific EC based on the presence of EC in at least one first-degree relative of the index patient (in family 9, the closest affected relative was of second degree) in the absence of any associated clustering of colon or other cancers. In addition to the information obtained during the interview, family histories were documented by identifying all first-degree relatives (parents, siblings, and children) in the official population registries (church registries) and verifying the cancer diagnoses through the nationwide Finnish Cancer Registry (the great majority of cancers reported to this registry are histologically or cytologically documented).
Paraffin-derived specimens of tumor and matching normal tissues of the index patients and their relatives were collected from the pathology departments of different hospitals and used for IHC analysis and DNA extraction. For family 15, blood specimens were also available. This study was approved by the appropriate institutional review boards of the Helsinki University Central Hospital.
IHC Analysis
Formalin-fixed, paraffin-embedded tissue sections were immunohistochemically stained with anti-MLH1 (clone G168-15; Pharmingen, San Diego, CA), anti-MSH2 (clone FE-11; Calbiochem/Oncogene Research, Darmstadt, Germany), and anti-MSH6 (clone 44; Transduction Laboratories, San Diego, CA). The EnVision+ System (Dako Cytomation, Glostrup, Denmark) was applied according to manufacturer's instructions with the antigen-retrieval step performed by boiling the sample in a microwave for 10 minutes in EDTA buffer (pH 8.0). The percentage of positively stained tumor cells varied between 10% and 95% (mean, 68%) for cases considered positive. Nuclear staining of normal endometrium and stromal cells included in each tumor section were used as a reference for the evaluation of the staining results.
MSI and Loss of Heterozygosity Analysis
DNA was prepared from archival paraffin-embedded tumor and normal tissue samples according to the method of Isola et al.20 Areas with pure normal or high tumor percentages were selected and verified histologically and subsequently dissected out. Tumor percentages ranged between 50 and 90.
MSI status was determined using the Bethesda panel of five markers (BAT25, BAT26, D5S346, D2S123, and D17S250).21 Tumors with two or more unstable markers were considered to have MSI-H and those with one unstable marker were considered to have low-degree microsatellite instability (MSI-L), whereas those with no unstable markers were microsatellite stable (MSS).
The results obtained with dinucleotide repeat markers from the Bethesda panel were also analyzed for loss of heterozygosity (LOH) in informative tumors (ie, those heterozygous and not showing MSI). In addition, LOH at 2p and 3p was evaluated using markers flanking MSH2/MSH6 and MLH1, respectively. The MSH2/MSH6-linked markers were ptel-D2S2378-MSH2/MSH6-CA7-D2S123-cen, spanning a 3.3-centimorgan (cM) region.22 The MLH1-linked markers were ptel-BAT21-D3S1611-D3S1298-cen, including two intragenic markers (BAT21 and D3S1611) and spanning a region of 0.6 cM. The primer sequences for the markers are available at http://www.gdb.org. The forward primers were fluorescently labeled, and the polymerase chain reaction (PCR) products were run on an automated sequencer and analyzed with the Genotyper 2.0 program (Applied Biosystems, Foster City, CA). For LOH analysis, the ratio of allelic peak areas was calculated as follows.23
where L is the LOH ratio, A1T is the area of allele 1 in the tumor, A2T is the area of allele 2 in the tumor; A1N is the area of allele 1 in a normal sample, and A2N is the area of allele 2 in a normal sample.
A sample was scored as showing LOH if L 0.6 or L 1.67 (indicating that one of the alleles had decreased 40% or more), and was scored as showing putative LOH or allelic imbalance if 0.6 < L < 0.8 or 1.25 < L < 1.67 (indicating the decrease of 21% to 39% for one allele).
MLH1 Promoter Methylation Analysis
The methylation status of three HpaII sites located at nucleotide positions –567, –347, and –341 relative to the A of the initiating codon of MLH1 (GenBank accession number U83845) was assessed by a method that relies on the methylation sensitivity of the HpaII restriction enzyme.24 The methylation status of tumor DNA was compared with that of normal endometrial DNA from the same individuals.
Mutation Analysis
The individual exons of MLH1, MSH2, and MSH6 were screened for mutations by direct sequencing of genomic PCR products using primers described in Chadwick et al.11 In addition, the Finnish founding mutation 1 (a large genomic deletion of MLH1 exon 16 and flanking introns)25 was tested in a PCR assay using two forward primers, one preceding (5'-GAGCCTCCAATACAATGTTGAATAGAAG-3') and the other one located within the deleted fragment (5'-ACATATGTGACATCCTCTCCACTCC-3'), and a common reverse primer (5'-CTCTCCATGTCTGGGTCCTTC-3'). Screening for two MSH6 mutations previously found in the Finnish population was performed with primers for fragment 4k26 for 3052delCT,27 and with 5'-GAAACAGCGCAACAGAATTG-3' and 5'-ATCCCTCCGTTCTTCAGCAT-3' for E995X.28 Furthermore, the possible presence of the frameshift mutation affecting the MSH6-C8, found in family 15 in this study, was evaluated in other families using primers described in Malkhosyan et al29
Multiplex ligation-dependent probe amplification (MLPA)30 was used to screen the samples for large genomic deletions or amplifications in MLH1, MSH2, and MSH6 using kits (SALSA P003, SALSA P008) and protocols (MRC-Holland, Amsterdam, the Netherlands). HNPCC samples from our collection known to carry large genomic deletions were used as positive controls.
RESULTS
Characteristics of Families and Tumors
Of 519 consecutive patients with EC, 62 indicated a family history of cancer (Fig 1). These included nine patients immediately diagnosed with HNPCC based on colorectal cancers in the family and fulfillment of the Amsterdam criteria. Of these, eight turned out to represent families with predisposing mutations identified previously (MLH1 in six families and MSH2 in two families; kindreds 3, 11, 28, 38, 39, 83, 93, and 108, reported in Nystrm-Lahti et al31 and Holmberg et al32), whereas the remaining family revealed a predisposing mutation (D603N in MSH2) in our investigation. An additional 26 patients reported nonspecific clustering of various cancers (other than EC) in the family. Excluding these patients and another four with unavailable samples, 23 patients with familial site-specific EC remained and formed our primary study series.
Clinicopathologic characteristics of the tumors (according to the International Federation of Gynecology and Obstetrics staging system)33 as well as family histories of the index patients are listed in Table 1. Twelve families showed evidence of vertical and 10 families showed evidence of horizontal transmission (the latter group included families 24 and 29 that were ascertained independently, but were subsequently found to unite into a single family). The mean age at EC diagnosis was 62 years.
Strategy for Molecular Characterization of Familial Site-Specific EC
In the first phase, archival tissue specimens were collected from the index patients and their relatives and screened for MLH1, MSH2, and MSH6 protein expression by IHC analysis, as well as for MSI, LOH, and MLH1 promoter hypermethylation (Fig 1). A total of 37 tumors were obtained for molecular studies (Table 2), including 34 ECs together with two breast cancers and one ovarian cancer occurring as additional primary cancers in the index patients. In the second phase, MMR genes implicated by IHC alterations were screened for germline mutations by exon-by-exon sequencing (for point mutations), MLPA (for large genomic deletions and amplifications), and direct tests for mutations previously detected in this population25,27,28,32,34 (see Materials and Methods). According to a recent report,16 alterations detectable by MLPA might constitute a majority of germline mutations occurring in EC patients.
IHC Alterations
Thirty-three EC tumors were available for IHC analysis of MLH1, MSH2, and MSH6, and abnormal protein expression was seen in 16 (48%; Table 2). Tumors with expression changes originated from 12 of 23 families (52%). MLH1 was lost in seven tumors of 33 (21%) and MSH2 together with MSH6 were lost in four tumors (12%), whereas five tumors (15%) showed abnormal MSH6 staining alone (loss of expression or cytoplasmic staining).
Germline Mutations
All patients with IHC changes were investigated for germline mutations of the respective genes. IHC alterations were mostly discordant in EC tumors from different members of the same family (eg, 14:1 displayed absent MLH1 and 14:2 absent MSH2 protein; Table 2), and it was therefore not surprising that no MMR gene germline mutations were found in such families. However, two families (15 and 13) showed intrafamilial concordance for IHC changes, and both revealed germline mutations in subsequent analysis. Three individuals tested from family 15 all showed MSH6 protein loss in EC (Table 2). Sequencing revealed a truncating germline mutation in MSH6 (insertion of C at nucleotides 3261 to 3262, codon 1088 in exon 5, designated as 3261insC), which cosegregated with EC in this family (Fig 2). Although MSH6 germline mutations are known to be associated with high risk for EC,35 EC typically coexists with colon and other cancers; family 15 was exceptional by displaying late-onset (mean, 60.8 years) EC in six members in the absence of colon cancer. An identical germline mutation was previously reported to occur in an HNPCC family from Australia (Bennett et al, www.insight-group.org); the clinical features of this family were not specified.
Family 13 was small, with two affected individuals (Table 1), and samples were available from the index patient (13:1) only (EC and ovarian cancer representing two different primary tumors; Table 2). These tumors showed a concordant MSH2/MSH6 change by IHC, and a germline mutation of the missense type (G>A at nucleotide 1807, codon 603, in exon 12 of MSH2) was detected by sequencing. This change, designated as D603N, is likely to be pathogenic for the following reasons. First, it substitutes asparagine for aspartic acid, which involves a change in the polarity group at a position that is evolutionarily conserved. Second, even though no segregation studies were possible in family 13, we detected the same germline alteration in a family diagnosed with HNPCC in the beginning (see Characteristics of Tumors and Families), and this family did show cosegregation of the mutation with HNPCC-type cancers. Furthermore, there is an earlier report of the D603N mutation occurring in a colon cancer patient with positive family history of cancer.36 Third, this change was absent in 129 healthy control individuals from the same population (this study and Salovaara et al36).
MSI, LOH, and MLH1 Promoter Methylation
Among 33 ECs analyzed by IHC, five (15%) demonstrated MSI-H and the remaining 28 had MSI-L (18%) or MSS (67%) by the Bethesda panel of markers (Table 2). Loss of MSH2 protein expression in EC (accompanied by abnormal MSH6 staining in all tumors) was regularly associated with MSI-H (three of four; 75%), whereas MSI-H was less frequent among tumors with MLH1 protein loss (two of seven; 29%) or solitary MSH6 expression change (one of five; 20%).
As possible somatic events that might underlie aberrant protein expression, LOH at 2p and 3p as well as MLH1 promoter methylation were addressed. Eight EC tumors with MSH2 and/or MSH6 protein loss were informative for the nearby markers D2S123, CA7, and D2S2378, and seven tumors (88%) showed LOH or putative LOH in at least one locus (Table 2). For comparison, the same tumors showed LOH frequencies of only one of seven (14%) for D5S346 and D17S250 each. Although these results could support the role of physical deletions at 2p as contributors to aberrant MSH2/MSH6 expression, this region might generally be prone to deletions and rearrangements, given that tumors with normal MSH2/MSH6 protein expression also showed frequent LOH or putative LOH at 2p (12 of 21; 57%). Six EC tumors with loss of MLH1 protein were informative for the intragenic markers BAT21 and D3S1611, and two (33%) showed LOH or putative LOH. Because this frequency did not significantly differ from LOH frequencies observed for D5S346 (three of six; 50%) or D17S250 (two of six; 33%) in the same tumors, or relative to 3p loss among tumors with normal MLH1 protein expression (three of 14; 21%; Table 2), the possible association of the loss of 3p genetic material with decreased MLH1 protein expression remains unclear. Finally, among seven tumors lacking MLH1 protein, MLH1 promoter hypermethylation could provide an explanation for one (14%; patient 14:1 in Table 2).
DISCUSSION
The existence of familial site-specific EC as a separate entity3,17 remains equivocal until the identification of predisposing gene defects specific for this disorder. HNPCC is the most important hereditary syndrome to be considered for differential diagnosis; for example, according to the Amsterdam criteria II,19 HNPCC diagnosis may be based on certain extracolonic cancers (including EC) alone, provided that at least one tumor is diagnosed in the patient younger than 50 years. To determine the extent of overlap between pure EC families and HNPCC, we analyzed the role of MMR genes in 23 families with site-specific EC. Our families had from two to six EC patients per family and an average age at EC diagnosis of 62 years, which is older than that for EC occurring in HNPCC (50 years37), but younger compared with the general population (66 years38). Our selection criterion (family history of EC alone in a newly diagnosed EC patient) led to the identification of two families with MMR gene germline mutations (two of 23; 8.7%). The observed frequency of germline mutation carriers was comparable to that reported for ECs selected for young age (younger than age 50 years, five of 57; 9%16). The simultaneous presence of colon or other GI cancer in an EC patient herself or in her family seems to be a somewhat stronger predictor of hereditary MMR defects, with reported frequencies of 16% to 23% for germline carriers in such series.13,14,16
On the basis of available data on known MMR gene mutation carriers showing that the gene mutated in the germline was always inactivated in their EC tumors,15,16,39,40 we used IHC as a guide for germline mutation analysis. In our study, expression changes were selective because only one MMR protein was altered in each tumor, with the exception of the previously described instability of MSH6 in the absence of its dimerization partner MSH2.39 Among 16 EC tumors with IHC alterations, four (which originated from two families) were associated with germline mutations, and these tumors, in contrast with the remaining ones, were distinguished by intrafamilial concordance for IHC changes, which therefore seems to be a useful indicator of a germline defect.
The correlation between IHC alterations and MSI was incomplete in our series, which questions the applicability of MSI alone, without IHC, for possible preselection of EC patients for germline mutation analysis. Although all tumors with MSI-H did show inactivation of at least one MMR protein, indicating that MLH1, MSH2, and MSH6 accounted for all patients with MSI-H, only five of 16 (31%) tumors with demonstrable MMR protein inactivation by IHC showed MSI-H. The lack of MSI-H was a special characteristic of inactivation of MLH1 or MSH6, but not MSH2. Accordingly, among families with identifiable germline mutations, two of two tumors tested from family 13 with MSH2 mutation showed MSI-H as opposed to zero of three ECs from family 15 with MSH6 mutation. We have reported previously that a significant fraction of ECs from known MLH1 mutation carriers fails to display MSI,41 and only 68% of sporadic EC tumors with IHC alterations investigated by Stefansson et al7 had MSI. On the basis of these observations, the predictive value of abnormal IHC for an MSI-H phenotype in EC is lower than in colorectal cancer41,42; the reasons for this are not known, but could be explained theoretically by normal tissue contamination or clonal heterogeneity, for example.41
Altogether, 11 families with inherited MMR defects were detected among 519 consecutive patients with EC on the basis of family history of EC (two families) or colorectal cancer (nine families). The present estimate for the incidence of MMR gene germline mutations ascertained through EC (11 of 519; 2.1%) is similar to previous estimates based on consecutive ECs (0.8% to 1.4%10,11) or colorectal cancers (0.3% to 3%36,43-47) using MSI for preselection. It is obvious that our figure represents a minimum estimate. With regard to families with colon cancers, only those fulfilling the Amsterdam criteria I or II were considered, and with regard to families with site-specific EC, no mutation detection strategy, including ours, is expected to be 100% accurate. Even with these limitations, it seems safe to conclude that only a small fraction of families with site-specific EC is explained by germline mutations in MMR genes. Additional studies should establish if the status of the remaining families is due to heritable defects in other genes, nongenetic factors, or chance.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank Gynel Arifdshan and Saila Saarinen for expert technical assistance, and Heli Surma-Aho and Katja Kuosa for assistance with sample collection.
NOTES
Supported by the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Cancer Foundation, the Finnish Cultural Foundation, the Paulo Foundation, and the Oskar flund Foundation.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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