International Journal of Gynecological Pathology 33:554–559, Lippincott Williams & Wilkins, Baltimore r 2014 International Society of Gynecological Pathologists
Original Article
The Immunohistochemistry Signature of Mismatch Repair (MMR) Proteins in a Multiethnic Asian Cohort With Endometrial Carcinoma Yin Ling Woo,
F.R.C.O.G., Ph.D.,
Phaik Leng Cheah, F.R.C.Path., Shazni I. Shahruddin, Siti Z. Omar, M.O.G., and Mark Arends, F.R.C.Path.
M.B.B.S.,
Summary: Endometrial cancer is the most common gynecologic cancer in developed countries and is rising in incidence globally. Although the 5-year survival rates are >80%, factors beyond conventional pathologic features that predict clinical outcomes are still being elucidated. The aims of this study were to define the prevalence and associations of deficient mismatch repair (dMMR) protein expression (MLH1, MSH2, MSH6, PMS2) by immunohistochemistry in a multiethnic Southeast Asian cohort with endometrioid endometrial cancer. A total of 77 patients with adequate formalin-fixed paraffin-embedded specimens were identified. The sections were stained in 2 centers for 4 MMR proteins and examined by 2 independent specialist histopathologists. The mean age for the cohort was 58.6 yr, with 19.4% (15/77) of patients’ cancers showing loss of 2 MMR proteins. All 13 cancers with absent MLH1 showed PMS2 loss (13/15), whereas absent MSH2 correlated with MHS6 loss (2/15). There were no significant differences for dMMR cases in age, body mass index, histopathologic characteristics, and clinical outcomes. In dMMR cases, an overrepresentation of patients of Indian ethnic origin was observed compared with Chinese and Malays. These findings suggest that dMMR protein expression in a Southeast Asian endometrial cancer cohort does not correlate with disease outcomes. Key Words: Mismatch repair proteins—Immunohistochemistry—Endometrial carcinoma—Hereditary gynecologic cancer syndromes.
Endometrial cancer is the most common gynecologic cancer in developed countries. Globally, 6 of every 100 women develop endometrial cancer by the age of 65 yr. The rate is twice as high in developed compared with developing countries (1). The burden of endometrial cancer is expected to rise with an increasing prevalence of obesity and an ageing population (2,3). Although the 5-year survival rates for endometrial cancer are >80%, factors beyond conventional pathologic characteristics that correlate with clinical outcomes are still being elucidated (4,5). With the recent classification of endometrial carcinomas into 4 groups by the Cancer Genome Atlas research network using an integrated genomic, transcriptomic, and proteomic approach, this should allow clinicians to further triage cases and tailor
From the Department of Obstetrics and Gynaecology (Y.L.W., S.I.S., S.Z.O.), UM Cancer Research Institute, Faculty of Medicine; Health and Translational Medicine Cluster (Y.L.W.), University Malaya Cancer Research Institute; Department of Pathology (P.L.C.), Faculty of Medicine, University of Malaya; Department of Pathology (P.L.C.), University of Malaya Medical Centre, Kuala Lumpur, Malaysia; Centre for Systems Pathology (M.A.), Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Division of Pathology, Edinburgh; and Department of Pathology (M.A.), Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK. Supported by the University of Malaya UMRG Grants RG272/ 10HTM and RG207/10HTM. The authors declare no conflict of interest. Address correspondence and reprint requests to Yin Ling Woo, FRCOG, PhD, Department of Obstetrics and Gynaecology, UM Cancer Research Institute, Faculty of Medicine, University Malaya, Kuala Lumpur 50603, Malaysia. E-mail: [email protected].
DOI: 10.1097/PGP.0000000000000099
554
MISMATCH REPAIR PROTEINS IHC IN ENDOMETRIAL CANCER management plans accordingly (6). In this collaborative study, around 40% of endometrioid carcinomas had defective DNA mismatch repair (MMR) from acquired silencing of expression of one of the MMR genes (MLH1), resulting from its promoter hypermethylation (6). Deficient mismatch repair (dMMR) is one of the more common mutational changes observed in endometrial cancer but its full clinical significance remains unclear despite intensive study. MMR is a wellconserved mechanism to maintain DNA integrity, predominantly by correcting DNA replication errors. Abnormalities of the MMR proteins due to gene mutation or deletion are associated with accelerated carcinogenesis and microsatellite instability (MSI), the hallmarks of Lynch Syndrome (LS) also known as hereditary nonpolyposis colorectal cancer (HNPCC). In fact, the cumulative lifetime risk of endometrial cancer in women with LS is reported to exceed the risk of colorectal cancer (7). Endometrial cancer is often the sentinel cancer of a patient with LS. All of the critical components of the MMR system (including the accessory proteins) are yet to be fully characterized, with the human MutS homolog 2 (hMSH2) and human MutL homolog 1 (hMLH1) genes being the first 2 mutated genes found to be causative for LS/HNPCC and also the most commonly mutated genes in LS. Subsequently, human postmeiotic 2 (hPMS2) and hMSH6 mutations were shown to contribute to the LS/HNPCC phenotype (8). MMR abnormalities detected in endometrial cancer can be due to germline mutations or epigenetic modifications (such as promoter hypermethylation) of MLH1, both leading to MSI (9). The clinical significance of defective MMR protein expression is yet to be fully determined, although dMMR/MSI Group (2) cancers have an intermediate prognosis between that of the Group (1) POLE mutant tumors with an excellent prognosis, and that of Group (4) cancers with a poor prognosis (6). This observational study aims to define the prevalence and associations of abnormal MMR expression by immunohistochemistry (IHC) in an unselected Southeast Asian cohort with endometrioid endometrial cancer. The pathologic characteristics and clinical outcomes will be correlated with the MMR expression patterns. MATERIALS AND METHODS Study Group A total of 77 patients, aged 30 to 77 yr (average 58.9 yr), diagnosed with endometrioid endometrial carcinoma between 2006 and 2012, identified from
555
the Department of Pathology, University of Malaya Medical Centre (UMMC) database formed the study cohort. All cases were reviewed and histologically reconfirmed. The patient demographics and clinical information were obtained from their clinical notes. Patients with subtypes other than endometrioid, insufficient tumor material in the paraffin blocks for analysis, or inadequate clinical documentation were excluded. The study was approved by the local research ethics committee, University of Malaya, Kuala Lumpur (Ref: 818.13). IHC Staining for MMR Proteins Sections of 4 mm were cut from formalin-fixed paraffin-embedded endometrial cancer blocks and mounted on glass slides. IHC staining was performed independently in 2 centers. In 1 center (UMMC), the immunostaining was carried out on the Ventana Benchmark XT automated stainer (Ventana Medical Systems Inc., Tucson, AZ). Briefly, antigen retrieval was performed using a tris-based alkaline buffer (CC1: Ventana Medical Systems Inc.) and this was followed by incubation with mouse monoclonal antibodies to respective Mismatch Repair Proteins viz. MLH1 (BD Pharmingen, clone G168-728 at 1:100 dilution), MSH2 (BD Pharmingen, clone G219-1129 at 1:800 dilution), MSH6 (BD Transduction Laboratories, clone 44/ MSH6 at 1:500 dilution), and PMS2 (BD Pharmingen, clone A16-4 at 1:100 dilution). In the other center (Cambridge), the Bond-max automated staining system (Leica Microsystems, Wetzlar, Germany) was used. Antigen retrieval was performed using Bond Epitope Retrieval Solution 1 (pH 6.0) at 1001C and endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 5 min. Sections were incubated with a 1:50 dilution of each of the following mouse monoclonal antibodies against the MLH1, MSH2, MSH6, and PMS2 proteins [clone numbers 25B12 (MLH1), 25D12 (MSH2), PU29 (MSH6), and MOR4G (PMS2), Novocastra, Leica Microsystems] for 30 min at room temperature as previously described (10–12). Interpretation of IHC Staining Each of the stained sections was examined by 2 specialist histopathologists (M.J.A. and P.L.C.) and scored for absence or presence of the MMR protein (MLH1, MSH2, MSH6, or PMS2) without knowledge of the patient data. Tumors were considered to show abnormal protein expression where there was evidence of clear absence of staining in the nuclei of the tumor epithelial cells, and positive staining of Int J Gynecol Pathol Vol. 33, No. 6, November 2014
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Y. L. WOO ET AL.
lymphocytes or stromal cells in the adjacent stroma that act as an internal positive control (13,14).
Statistics The 2-sided Fisher exact test was used to determine the statistical significance of the differences between those that were MMR deficient compared with those with normal MMR expression and the Student t test was used to compare the means. The values of Po0.05 were regarded as significant. Statistical analyses were performed using GraphPad InStat (San Diego, CA) software. RESULTS A total of 77 patients with the diagnosis of endometrioid endometrial adenocarcinoma were selected for analysis. The mean age of the study cohort was 58.6 yr (SD = 11.7, 95% confidence interval, 56–61.3). This cohort consisted of 39 (50.6%) Malays, 29 (37.7%) Chinese, and 9 (11.7%) Indians. The mean body mass index (BMI) was 29.7 (SD = 7.4, 95% confidence interval, 27.9–31.5). It was not possible to obtain an accurate family history from the clinical notes. The more detailed demographics and pathologic features of the patients are summarized in Table 1. In this study 15/77 patients’ cancers had abnormal MMR protein expression, with 13/15 (86.7%) showing concurrent absence of MLH1 and PMS2 protein expression, whereas 2/15 (13.3%) displayed abnormal staining for both MSH2 and MSH6. All cancers with absent MLH1 showed loss of PMS2, whereas absent MSH2 correlated with MHS6 loss. See Figure 1 for examples of endometrial cancers with either negative MLH1/PMS2 or MSH2. The characteristics of patients with normal MMR protein expression (n = 62), positive for MLH1, MSH2, PMS2, and MSH6 (MMR-positive) were compared with those with negative MLH1/PMS2 or negative MSH2/MSH6 (MMR-negative, n = 15) cancers (Table 1). There were no significant differences in age, BMI, age of menarche or menopause, histopathologic characteristics, and clinical outcomes among these 2 groups. Interestingly, there was an overrepresentation of MMRnegative cases among the Indians and this was statistically significant (P = 0.012). DISCUSSION In this multiethnic cohort with endometrioid endometrial cancer, 15/77 (19.4%) of patients were Int J Gynecol Pathol Vol. 33, No. 6, November 2014
found to have abnormal MMR protein expression by IHC using antibodies against MLH1, MSH2, MSH6, and PMS2. The absence of MLH1 expression was always accompanied by loss of PMS2 expression, whereas MSH2 loss of expression was always associated with absence of MSH6 expression. These observations are consistent with the fact that MLH1 protein dimerizes with PMS2 protein during the MMR process and that a functional defect in MLH1 would lead to instability and degradation of PMS2. Similarly, defects in MSH2 protein also result in instability and degradation of MSH6. The endometrial cancers were independently immunostained for MMR proteins in 2 centers and the sections examined separately by 2 specialist histopathologists (M.J.A. and P.L.C.) and full agreement was reached on the IHC diagnosis of defective MMR in the relevant cancers, thus considerably strengthening the IHC evidence base for this investigation with independent confirmation. Two of 15 (13.3%) of those who had MMR deficient staining were MSH2/MSH6 negative. These cases are most likely to represent germline mutations consistent with LS or HNPCC. This is because the positive predictive value of absent MSH2 and/or MSH6 is very high and that loss of these proteins by IHC may be sufficient for the diagnosis of LS/ HNPCC (15,16). In contrast, loss of expression of MLH1 and PMS2 proteins in an endometrial cancer is much more likely to be due to sporadically and somatically acquired ML1 promoter hypermethylation than LS due to germline mutation, as sporadic dMMR accounts for 20% to 30% of all endometrial cancers, whereas LS accounts for only 2% to 4% of all endometrial cancers, of which inherited MLH1 mutation is responsible for 1% to 2% (10,17). MMR protein expression by IHC has not been previously described in a multiethnic Asian population with endometrial cancer before. However, in a Malaysian cohort of unselected colorectal cancers, 18.9% (28/148) demonstrated absence of 1 to 2 MMR proteins. In this series, only 57% of these abnormal cases showed absence of MLH1/PMS2 and MSH2/MSH6 staining (18). When the patients with endometrial cancers that expressed MMR abnormally (n = 15) were compared with those with normal MMR expression (n = 62), there were no differences in age, BMI, age of menarche, menopause, grade, stage, and disease outcomes. LS/ HNPCC-associated endometrial cancers are reported to occur at a mean age of 50 yr, 10 yr younger than the general population (7). However, in this cohort (which
MISMATCH REPAIR PROTEINS IHC IN ENDOMETRIAL CANCER
557
TABLE 1. Demograhics and clinical features of patients who had endometrial cancers that either had normal expression of MMR proteins (MMR-positive) or abnormal MMR protein expression (MMR-negative) MMR-positive n = 62 (%) Age Average o40 41–50 51–60 61–70 470 Ethnicity Malay Chinese Indian Age of menarche (yr) 10–12 13–16 416 Unknown Age of menopause Premenopausal o45 46–50 51–55 455 Unknown BMI Average o20 21–25 26–30 31–35 36–40 440 Unknown FIGO Stage I II III IV Grade I II III Clinical outcomes Alive/without disease Alive with recurrence/relapse Dead Status unknown Mean follow-up (median, SD) (mo)
5 11 18 22 6
MMR-negative n = 15 (%)
58.7 (8.1) (17.7) (29) (35.5) (9.7)
1 4 2 6 2
58.5 (6.7) (26.7) (13.3) (40) (13.3)
P 0.95 1 0.47 0.32 0.77 0.64
33 (53) 25 (40) 4 (7)
6 (40) 4 (27) 5 (33)
0.4 0.38 0.012*
20 15 3 24
(32.3) (24.2) (4.8) (38.7)
4 4 0 7
(26.7) (26.7) (0) (46.7)
0.7 1.0 1.0 0.57
12 2 21 12 1 14
(19.4) (3.2) (33.9) (19.4) (1.6) (22.6)
0 1 2 3 1 8
(0) (6.7) (13.3) (20) (6.7) (53.3)
0.1 0.48 0.21 1.0 0.35 0.02*
2 18 15 11 3 8 5
30.2 (3.2) (29) (24.2) (17.7) (4.8) (12.9) (8.1)
0 6 4 5 0 0 0
27.9 (0) (40) (26.7) (33.3) (0) (0) (0)
0.30 1 0.53 1 0.28 1 0.34 0.57
43 6 8 5
(69.4) (9.7) (12.9) (8)
9 1 3 2
(60) (6.7) (20) (13.3)
0.3 1 0.43 0.61
4 (26.7) 8 (53.3) 3 (20)
1.0 0.77 0.39
18 (29) 37 (59.7) 7 (11.3) 43 9 2 8 18.6
(69.4) (14.5) (3.2) (12.9) (16,13.3)
8 2 1 4 24.7
(53.3) (13.3) (6.7) (26.7) (26, 19.1)
0.36 1 0.48 0.23 0.13
*The 2 groups were compared and a P-value of
Original Article
The Immunohistochemistry Signature of Mismatch Repair (MMR) Proteins in a Multiethnic Asian Cohort With Endometrial Carcinoma Yin Ling Woo,
F.R.C.O.G., Ph.D.,
Phaik Leng Cheah, F.R.C.Path., Shazni I. Shahruddin, Siti Z. Omar, M.O.G., and Mark Arends, F.R.C.Path.
M.B.B.S.,
Summary: Endometrial cancer is the most common gynecologic cancer in developed countries and is rising in incidence globally. Although the 5-year survival rates are >80%, factors beyond conventional pathologic features that predict clinical outcomes are still being elucidated. The aims of this study were to define the prevalence and associations of deficient mismatch repair (dMMR) protein expression (MLH1, MSH2, MSH6, PMS2) by immunohistochemistry in a multiethnic Southeast Asian cohort with endometrioid endometrial cancer. A total of 77 patients with adequate formalin-fixed paraffin-embedded specimens were identified. The sections were stained in 2 centers for 4 MMR proteins and examined by 2 independent specialist histopathologists. The mean age for the cohort was 58.6 yr, with 19.4% (15/77) of patients’ cancers showing loss of 2 MMR proteins. All 13 cancers with absent MLH1 showed PMS2 loss (13/15), whereas absent MSH2 correlated with MHS6 loss (2/15). There were no significant differences for dMMR cases in age, body mass index, histopathologic characteristics, and clinical outcomes. In dMMR cases, an overrepresentation of patients of Indian ethnic origin was observed compared with Chinese and Malays. These findings suggest that dMMR protein expression in a Southeast Asian endometrial cancer cohort does not correlate with disease outcomes. Key Words: Mismatch repair proteins—Immunohistochemistry—Endometrial carcinoma—Hereditary gynecologic cancer syndromes.
Endometrial cancer is the most common gynecologic cancer in developed countries. Globally, 6 of every 100 women develop endometrial cancer by the age of 65 yr. The rate is twice as high in developed compared with developing countries (1). The burden of endometrial cancer is expected to rise with an increasing prevalence of obesity and an ageing population (2,3). Although the 5-year survival rates for endometrial cancer are >80%, factors beyond conventional pathologic characteristics that correlate with clinical outcomes are still being elucidated (4,5). With the recent classification of endometrial carcinomas into 4 groups by the Cancer Genome Atlas research network using an integrated genomic, transcriptomic, and proteomic approach, this should allow clinicians to further triage cases and tailor
From the Department of Obstetrics and Gynaecology (Y.L.W., S.I.S., S.Z.O.), UM Cancer Research Institute, Faculty of Medicine; Health and Translational Medicine Cluster (Y.L.W.), University Malaya Cancer Research Institute; Department of Pathology (P.L.C.), Faculty of Medicine, University of Malaya; Department of Pathology (P.L.C.), University of Malaya Medical Centre, Kuala Lumpur, Malaysia; Centre for Systems Pathology (M.A.), Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Division of Pathology, Edinburgh; and Department of Pathology (M.A.), Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK. Supported by the University of Malaya UMRG Grants RG272/ 10HTM and RG207/10HTM. The authors declare no conflict of interest. Address correspondence and reprint requests to Yin Ling Woo, FRCOG, PhD, Department of Obstetrics and Gynaecology, UM Cancer Research Institute, Faculty of Medicine, University Malaya, Kuala Lumpur 50603, Malaysia. E-mail: [email protected].
DOI: 10.1097/PGP.0000000000000099
554
MISMATCH REPAIR PROTEINS IHC IN ENDOMETRIAL CANCER management plans accordingly (6). In this collaborative study, around 40% of endometrioid carcinomas had defective DNA mismatch repair (MMR) from acquired silencing of expression of one of the MMR genes (MLH1), resulting from its promoter hypermethylation (6). Deficient mismatch repair (dMMR) is one of the more common mutational changes observed in endometrial cancer but its full clinical significance remains unclear despite intensive study. MMR is a wellconserved mechanism to maintain DNA integrity, predominantly by correcting DNA replication errors. Abnormalities of the MMR proteins due to gene mutation or deletion are associated with accelerated carcinogenesis and microsatellite instability (MSI), the hallmarks of Lynch Syndrome (LS) also known as hereditary nonpolyposis colorectal cancer (HNPCC). In fact, the cumulative lifetime risk of endometrial cancer in women with LS is reported to exceed the risk of colorectal cancer (7). Endometrial cancer is often the sentinel cancer of a patient with LS. All of the critical components of the MMR system (including the accessory proteins) are yet to be fully characterized, with the human MutS homolog 2 (hMSH2) and human MutL homolog 1 (hMLH1) genes being the first 2 mutated genes found to be causative for LS/HNPCC and also the most commonly mutated genes in LS. Subsequently, human postmeiotic 2 (hPMS2) and hMSH6 mutations were shown to contribute to the LS/HNPCC phenotype (8). MMR abnormalities detected in endometrial cancer can be due to germline mutations or epigenetic modifications (such as promoter hypermethylation) of MLH1, both leading to MSI (9). The clinical significance of defective MMR protein expression is yet to be fully determined, although dMMR/MSI Group (2) cancers have an intermediate prognosis between that of the Group (1) POLE mutant tumors with an excellent prognosis, and that of Group (4) cancers with a poor prognosis (6). This observational study aims to define the prevalence and associations of abnormal MMR expression by immunohistochemistry (IHC) in an unselected Southeast Asian cohort with endometrioid endometrial cancer. The pathologic characteristics and clinical outcomes will be correlated with the MMR expression patterns. MATERIALS AND METHODS Study Group A total of 77 patients, aged 30 to 77 yr (average 58.9 yr), diagnosed with endometrioid endometrial carcinoma between 2006 and 2012, identified from
555
the Department of Pathology, University of Malaya Medical Centre (UMMC) database formed the study cohort. All cases were reviewed and histologically reconfirmed. The patient demographics and clinical information were obtained from their clinical notes. Patients with subtypes other than endometrioid, insufficient tumor material in the paraffin blocks for analysis, or inadequate clinical documentation were excluded. The study was approved by the local research ethics committee, University of Malaya, Kuala Lumpur (Ref: 818.13). IHC Staining for MMR Proteins Sections of 4 mm were cut from formalin-fixed paraffin-embedded endometrial cancer blocks and mounted on glass slides. IHC staining was performed independently in 2 centers. In 1 center (UMMC), the immunostaining was carried out on the Ventana Benchmark XT automated stainer (Ventana Medical Systems Inc., Tucson, AZ). Briefly, antigen retrieval was performed using a tris-based alkaline buffer (CC1: Ventana Medical Systems Inc.) and this was followed by incubation with mouse monoclonal antibodies to respective Mismatch Repair Proteins viz. MLH1 (BD Pharmingen, clone G168-728 at 1:100 dilution), MSH2 (BD Pharmingen, clone G219-1129 at 1:800 dilution), MSH6 (BD Transduction Laboratories, clone 44/ MSH6 at 1:500 dilution), and PMS2 (BD Pharmingen, clone A16-4 at 1:100 dilution). In the other center (Cambridge), the Bond-max automated staining system (Leica Microsystems, Wetzlar, Germany) was used. Antigen retrieval was performed using Bond Epitope Retrieval Solution 1 (pH 6.0) at 1001C and endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 5 min. Sections were incubated with a 1:50 dilution of each of the following mouse monoclonal antibodies against the MLH1, MSH2, MSH6, and PMS2 proteins [clone numbers 25B12 (MLH1), 25D12 (MSH2), PU29 (MSH6), and MOR4G (PMS2), Novocastra, Leica Microsystems] for 30 min at room temperature as previously described (10–12). Interpretation of IHC Staining Each of the stained sections was examined by 2 specialist histopathologists (M.J.A. and P.L.C.) and scored for absence or presence of the MMR protein (MLH1, MSH2, MSH6, or PMS2) without knowledge of the patient data. Tumors were considered to show abnormal protein expression where there was evidence of clear absence of staining in the nuclei of the tumor epithelial cells, and positive staining of Int J Gynecol Pathol Vol. 33, No. 6, November 2014
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Y. L. WOO ET AL.
lymphocytes or stromal cells in the adjacent stroma that act as an internal positive control (13,14).
Statistics The 2-sided Fisher exact test was used to determine the statistical significance of the differences between those that were MMR deficient compared with those with normal MMR expression and the Student t test was used to compare the means. The values of Po0.05 were regarded as significant. Statistical analyses were performed using GraphPad InStat (San Diego, CA) software. RESULTS A total of 77 patients with the diagnosis of endometrioid endometrial adenocarcinoma were selected for analysis. The mean age of the study cohort was 58.6 yr (SD = 11.7, 95% confidence interval, 56–61.3). This cohort consisted of 39 (50.6%) Malays, 29 (37.7%) Chinese, and 9 (11.7%) Indians. The mean body mass index (BMI) was 29.7 (SD = 7.4, 95% confidence interval, 27.9–31.5). It was not possible to obtain an accurate family history from the clinical notes. The more detailed demographics and pathologic features of the patients are summarized in Table 1. In this study 15/77 patients’ cancers had abnormal MMR protein expression, with 13/15 (86.7%) showing concurrent absence of MLH1 and PMS2 protein expression, whereas 2/15 (13.3%) displayed abnormal staining for both MSH2 and MSH6. All cancers with absent MLH1 showed loss of PMS2, whereas absent MSH2 correlated with MHS6 loss. See Figure 1 for examples of endometrial cancers with either negative MLH1/PMS2 or MSH2. The characteristics of patients with normal MMR protein expression (n = 62), positive for MLH1, MSH2, PMS2, and MSH6 (MMR-positive) were compared with those with negative MLH1/PMS2 or negative MSH2/MSH6 (MMR-negative, n = 15) cancers (Table 1). There were no significant differences in age, BMI, age of menarche or menopause, histopathologic characteristics, and clinical outcomes among these 2 groups. Interestingly, there was an overrepresentation of MMRnegative cases among the Indians and this was statistically significant (P = 0.012). DISCUSSION In this multiethnic cohort with endometrioid endometrial cancer, 15/77 (19.4%) of patients were Int J Gynecol Pathol Vol. 33, No. 6, November 2014
found to have abnormal MMR protein expression by IHC using antibodies against MLH1, MSH2, MSH6, and PMS2. The absence of MLH1 expression was always accompanied by loss of PMS2 expression, whereas MSH2 loss of expression was always associated with absence of MSH6 expression. These observations are consistent with the fact that MLH1 protein dimerizes with PMS2 protein during the MMR process and that a functional defect in MLH1 would lead to instability and degradation of PMS2. Similarly, defects in MSH2 protein also result in instability and degradation of MSH6. The endometrial cancers were independently immunostained for MMR proteins in 2 centers and the sections examined separately by 2 specialist histopathologists (M.J.A. and P.L.C.) and full agreement was reached on the IHC diagnosis of defective MMR in the relevant cancers, thus considerably strengthening the IHC evidence base for this investigation with independent confirmation. Two of 15 (13.3%) of those who had MMR deficient staining were MSH2/MSH6 negative. These cases are most likely to represent germline mutations consistent with LS or HNPCC. This is because the positive predictive value of absent MSH2 and/or MSH6 is very high and that loss of these proteins by IHC may be sufficient for the diagnosis of LS/ HNPCC (15,16). In contrast, loss of expression of MLH1 and PMS2 proteins in an endometrial cancer is much more likely to be due to sporadically and somatically acquired ML1 promoter hypermethylation than LS due to germline mutation, as sporadic dMMR accounts for 20% to 30% of all endometrial cancers, whereas LS accounts for only 2% to 4% of all endometrial cancers, of which inherited MLH1 mutation is responsible for 1% to 2% (10,17). MMR protein expression by IHC has not been previously described in a multiethnic Asian population with endometrial cancer before. However, in a Malaysian cohort of unselected colorectal cancers, 18.9% (28/148) demonstrated absence of 1 to 2 MMR proteins. In this series, only 57% of these abnormal cases showed absence of MLH1/PMS2 and MSH2/MSH6 staining (18). When the patients with endometrial cancers that expressed MMR abnormally (n = 15) were compared with those with normal MMR expression (n = 62), there were no differences in age, BMI, age of menarche, menopause, grade, stage, and disease outcomes. LS/ HNPCC-associated endometrial cancers are reported to occur at a mean age of 50 yr, 10 yr younger than the general population (7). However, in this cohort (which
MISMATCH REPAIR PROTEINS IHC IN ENDOMETRIAL CANCER
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TABLE 1. Demograhics and clinical features of patients who had endometrial cancers that either had normal expression of MMR proteins (MMR-positive) or abnormal MMR protein expression (MMR-negative) MMR-positive n = 62 (%) Age Average o40 41–50 51–60 61–70 470 Ethnicity Malay Chinese Indian Age of menarche (yr) 10–12 13–16 416 Unknown Age of menopause Premenopausal o45 46–50 51–55 455 Unknown BMI Average o20 21–25 26–30 31–35 36–40 440 Unknown FIGO Stage I II III IV Grade I II III Clinical outcomes Alive/without disease Alive with recurrence/relapse Dead Status unknown Mean follow-up (median, SD) (mo)
5 11 18 22 6
MMR-negative n = 15 (%)
58.7 (8.1) (17.7) (29) (35.5) (9.7)
1 4 2 6 2
58.5 (6.7) (26.7) (13.3) (40) (13.3)
P 0.95 1 0.47 0.32 0.77 0.64
33 (53) 25 (40) 4 (7)
6 (40) 4 (27) 5 (33)
0.4 0.38 0.012*
20 15 3 24
(32.3) (24.2) (4.8) (38.7)
4 4 0 7
(26.7) (26.7) (0) (46.7)
0.7 1.0 1.0 0.57
12 2 21 12 1 14
(19.4) (3.2) (33.9) (19.4) (1.6) (22.6)
0 1 2 3 1 8
(0) (6.7) (13.3) (20) (6.7) (53.3)
0.1 0.48 0.21 1.0 0.35 0.02*
2 18 15 11 3 8 5
30.2 (3.2) (29) (24.2) (17.7) (4.8) (12.9) (8.1)
0 6 4 5 0 0 0
27.9 (0) (40) (26.7) (33.3) (0) (0) (0)
0.30 1 0.53 1 0.28 1 0.34 0.57
43 6 8 5
(69.4) (9.7) (12.9) (8)
9 1 3 2
(60) (6.7) (20) (13.3)
0.3 1 0.43 0.61
4 (26.7) 8 (53.3) 3 (20)
1.0 0.77 0.39
18 (29) 37 (59.7) 7 (11.3) 43 9 2 8 18.6
(69.4) (14.5) (3.2) (12.9) (16,13.3)
8 2 1 4 24.7
(53.3) (13.3) (6.7) (26.7) (26, 19.1)
0.36 1 0.48 0.23 0.13
*The 2 groups were compared and a P-value of
