Among two populations of gynecologic oncology patients that are recommended to undergo genetic testing by the 1996 Bethesda guidelines, we found 12% (6/50) of the early-onset endometrial cancer group and 14% (3/22) of the synchronous endometrial and ovarian cancer group to have tumors with molecular characteristics suggestive of LS. Only three of these nine patients had a pedigree pattern that met the revised Amsterdam criteria for LS.
Screening by IHC for MMR proteins followed by selective MSI testing and evaluation for MLH1 promoter methylation may provide a useful algorithm for triage of patient samples toward genetic testing to identify a deleterious mutation in a MMR gene. We demonstrate a high concordance between absent MMR protein IHC and the MSI-H phenotype and conclude that further MSI testing is not necessary in these cases.
The prediction of MLH1 mutations by IHC has been problematic in the past, due to the occurrence of MLH1
missense mutations that result in a deficient, but antigenically-active protein [20
]. We approached this problem by using MSI testing as a triage tool for further work-up of tumors with weak MLH1 protein staining [22
]. An alternative approach would be to add the PMS2 antibody to the IHC panel. Addition of PMS2 increases the sensitivity of IHC in predicting MLH1
mutation to 92%; up from 85% with the three-antibody panel composed of MLH1, MSH2 and MSH6 [21
]. MLH1 dimerizes with PMS2 and mutations of MLH1
will often cause a concurrent loss of the two proteins [24
]. Our study potentially underestimates the prevalence of LS by the omission of this fourth antibody in our screening panel.
We chose to use IHC as the primary screening tool based on studies that suggest similar effectiveness of this method when compared to screening by MSI [25
]. Addition of an IHC panel of MMR proteins to the pathological evaluation of a tumor is relatively easy for the clinical pathologist and the pattern of MMR protein staining abnormalities can direct genetic testing towards the gene most likely to be affected [21
]. Furthermore, IHC is more likely than MSI testing to detect a MSH6 deficient tumor that may be characterized by low or absent MSI [23
]. However, IHC can miss cases resulting from a deleterious missense mutation that encodes a functionally-deficient but antigenically-intact protein [27
]. Furthermore, while most cases of LS are due to mutations in MLH1
, or PMS2
; cases arising from an as of yet undefined mutated gene would not be detected by the IHC antibody panel [28
]. Notably, the concordance rate between IHC and MSI testing is only 92%, with both tests missing some cases that would be detected by the other [29
The relatively high rate of MLH1
promoter methylation found in the synchronous primary cancer group suggests the benefit of adding MLH1
methylation analysis to the diagnostic algorithm. Those cases found to have MLH1
promoter methylation would not require additional genetic testing for LS. Studies in colorectal cancer have demonstrated the utility of adding BRAF
V600E mutation analysis to determine the sporadic nature of tumors with decreased MLH1 expression [31
]. Sparse data are available to this approach to the work-up of endometrial cancer. However, one recent report suggests the BRAF
V600E mutation is not found in sporadic endometrial carcinomas [34
In our diagnostic algorithm, we classified two patterns of abnormalities to be virtually diagnostic of LS: (1) absent MLH1 staining and non-methylated MLH1
gene promoter and (2) absent MSH2 and/or MSH6 staining [35
]. Three of the nine patients classified as LS based on these patterns of molecular abnormalities underwent commercial genetic testing and all three (100%) were confirmed to carry a deleterious mutation. The highly predictive nature of these molecular findings raises the issue that IHC for MMR proteins could be interpreted as a genetic test. As such, the clinician should consider whether appropriate informed consent protocols should be in place before immunohistochemical testing is performed.
We did not perform germline testing on all patients in this study, nor did we study a population-based sample. Both of these limitations could result in either overestimation or underestimation of LS among our two study populations. Nevertheless, using our diagnostic algorithm, we found 12% of patients with endometrial cancers before the age of 45 to have molecular findings consistent with LS, which aligns with findings from prior studies. In patients diagnosed with endometrial cancer before 50 years of age, three studies utilizing germline gene sequencing reported LS in 4.9%[37
], 8.6% [38
], and 9% [39
]. We found 14% of patients with synchronous endometrial and ovarian cancers to have molecular findings suggestive of LS. Our results are slightly higher than the those of two retrospective studies (also based on tumor molecular profiling) that suggested LS incidence rates of 3 – 7% [40
]. However, in a study utilizing germline mutation analysis in early-onset endometrial cancer patients less than 50 years of age, one of nine (11%) patients with a synchronous primary ovarian cancer had a LS mutation [39
]. Our study also demonstrates a substantial proportion of MMR and MSI abnormalities in synchronous cases to result from MLH1
Among patients with synchronous endometrial and ovarian cancers, the tumors at both sites often showed similar IHC staining patterns and/or similar patterns of MLH1 promoter methylation, possibly reflecting either a still undefined genetic or environmental field effect that impacts tumor development at both sites. We included only tumors where the clinical impression of synchronous malignancies was favored, but the possibility that the two tumor sites represent a metastasis from one site to the other must also be considered. Nevertheless, the concordance of molecular findings in tumor pairs raises the feasibility of restricting molecular testing to the endometrial cancer in these patients.
The optimal population of endometrial cancer patients for referral to genetic testing has yet to be defined. In this study, we evaluated patients diagnosed with endometrial cancer before the age of 45 as recommended by the 1996 Bethesda guidelines. However, evidence suggests that a cutoff age of 45 will miss a large proportion of LS patients. In a population-based study, the median age of diagnosis among ten LS mutation carriers was 54.6 years (range 39–69), with six of the ten probands more than 50 years of age at the time of endometrial cancer diagnosis [37
]. The use of family history as a triage tool may also miss LS cases. Berends et al [38
] reported that among early-onset endometrial cancer patients (before the age of 50), 23% were found to have a germline LS mutation if they had a first-degree relative with a LS-associated cancer. In a population-based study of unselected endometrial cancer patients, seven of ten LS mutation carriers did not fulfill either the Amsterdam criteria or the Bethesda guidelines for screening [37
Identification of LS individuals and families is important because it has been shown to decrease colorectal cancer mortality with the institution of heightened cancer screening protocols [4
]. The use of immunohistochemistry followed by selective MSI and MLH1
promoter methylation studies may represent a useful algorithm for the identification of patients who should undergo analysis for a germline MMR gene mutation. We did not find family history to be a useful triage tool. Based on our findings, we would recommend screening for both gynecologic cancer populations identified by the original Bethesda guidelines, irrespective of family history. However, the optimal age cut-off for LS screening has not yet been defined and remains to be determined with future study.