Clinical Challenges Associated with Universal Screening for Lynch Syndrome–Associated Endometrial Cancer

Endometrial cancer is the most common gynecologic cancer in the United States (1). Risk factors for endometrial cancer include increasing age, obesity, hypertension, diabetes, menstrual irregularities, nulliparity, and unopposed estrogen (2). Another significant risk factor for endometrial cancer is Lynch syndrome, an inherited cancer syndrome due to a germline mutation in one of the four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2), which accounts for 2% to 6% of all endometrial cancers. For women with Lynch syndrome, the lifetime risk of endometrial cancer is 64%, and the lifetime risk of colorectal cancer is 54% (3). Prior studies have shown that use of clinical screening (patient age and family history of cancer) alone misses a substantial subset of endometrial cancer patients that may harbor a germline Lynch syndrome mutation (4, 5).

PCR-based microsatellite instability (MSI) testing, IHC evaluation of expression for the DNA MMR proteins, and PCR-based MLH1 methylation analysis have emerged as useful clinical laboratory tests to screen endometrial cancer patients for Lynch syndrome. Tumors with high levels of MSI (MSI-H) or immunohistochemical loss of expression of DNA MMR proteins in the absence of MLH1 gene methylation are suggestive of Lynch syndrome. Many recently published studies have advocated for universal screening of endometrial carcinomas with MSI and/or IHC (4, 6, 7). The American College of Obstetricians and Gynecologists and the Society of Gynecologic Oncology have recently issued a practice bulletin recommending that universal tissue testing as a rational approach for identifying women at risk for Lynch-associated endometrial cancer (8). With the adoption of universal testing, clinical and diagnostic conundrums may potentially emerge, including discordances between MSI and IHC results, tumor testing suggestive of Lynch syndrome with negative germline testing, and germline testing that results in a variant of unknown significance (9, 10). The incidences of these problems in endometrial cancer testing are not well documented. In addition, it is known that a subset of Lynch-associated endometrial cancers may have low levels of MSI (MSI-L). The detection of MSI-L in colorectal cancer does not typically warrant a genetic counseling referral unless there is IHC loss of an MMR protein or the patient has an informative family history (11).

The purpose of this study was to prospectively identify the incidence of clinically and diagnostically significant challenges when universal tissue testing is applied to patients with endometrial carcinoma.

Institutional Review Board approval was obtained for this prospective study, and a waiver of informed consent granted as universal testing is the standard clinical practice at our institution. Women ages 18 and older with newly diagnosed endometrial cancer undergoing surgery at The University of Texas MD Anderson Cancer Center (Houston, TX) from August 2012 to 2014 underwent tumor testing of their pathology specimens. Patient demographics were retrieved from the electronic medical record and included age at endometrial cancer diagnosis, body mass index (BMI), FIGO surgical stage, tumor histology, FIGO tumor grade (nonendometrioid tumors were considered grade 3), depth of myometrial invasion, and tumor location within the uterus (corpus vs. lower uterine segment). Data extraction was performed primarily by A.S. Bruegl and validated by K.L. Ring. Tumor testing consisted of IHC for the expression of DNA MMR proteins MLH1, MSH2, MSH6, and PMS2, with MLH1 promoter methylation analysis in cases of IHC loss of MLH1, and PCR-based MSI testing. Preanalytic, analytic, and postanalytic barriers to definitively classifying an endometrial carcinoma as sporadic or Lynch syndrome related were recorded.

IHC for the expression of the DNA MMR proteins was performed in a Clinical Laboratory Improvement Amendments (CLIA)-approved laboratory using previously described methods (9). The absence of nuclear staining in tumor cells with retained stromal staining was classified as loss of expression for the corresponding DNA MMR protein. Endometrial carcinomas exhibiting loss of MSH2, MSH6, or PMS2 were considered suggestive of a Lynch syndrome–associated tumor. For cases in which there was IHC loss of MLH1 protein expression, the PCR-based MLH1 promoter methylation assay was utilized to distinguish between sporadic epigenetic silencing of MLH1, methylated, and suspected MLH1 loss due to Lynch syndrome, unmethylated as described previously (12). Briefly, DNA isolated from formalin-fixed, paraffin-embedded endometrial carcinoma tissue sections was treated with bisulfite to convert unmethylated cytosines to uracil using the Zymo EZ DNA Methylation-Gold Kit according to the manufacturer's instructions (Zymo Research). Methylation of MLH1 was assessed by methylation-specific PCR followed by capillary electrophoresis using FAM-labeled reverse primer and unlabeled forward primers (Integrated DNA Technology). The following primer sequences were used: methylated forward, 5′-GAT AGC GAT TTTTAA CGC-3′; unmethylated forward, 5′-AGA GTG GAT AGT GAT TTT TAA TGT-3′; and labeled reverse primer, 5′- FAM-TCT ATA AAT TAC TAA ATC TCT TC-3′. The forward primers were designed to distinguish the methylated amplicon from the unmethylated by difference in size. The bisulfite-treated DNA was then subjected to PCR using primers specific for methylated and unmethylated DNA. The methylated PCR product of 85 bp was separated from unmethylated PCR product of 91 bp by capillary electrophoresis using an ABI Prism 3130 Genetic Analyzer. MSI was assessed using a panel of six NCI (Bethesda, MD) recommended microsatellites with the addition of TGFBR2. A tumor with allelic shift in three or more markers was designated as MSI-H, 1–2 markers as MSI-L, and no allelic shift as microsatellite stable (MSS). For cases with insufficient tissue for molecular analysis, a referral to genetic counselor was based on patient clinical characteristics and family history.

Patients with endometrial carcinomas with IHC loss of MLH1, MSH2, MSH6, or PMS2 with lack of MLH1 methylation (for patients with IHC loss of MLH1) were referred to a genetic counselor for consideration of germline testing, no matter the MSI testing results. Patients with MSI-H cancers lacking MLH1 methylation were also referred to genetic counselors, no matter the IHC results. There are no established guidelines for patients with MSI-L endometrial cancers. If MSI-L was associated with IHC loss of a MMR protein and lack of MLH1 methylation, patients were referred to genetic counseling and germline testing. Patients with MSI-L with intact IHC protein expression were also referred to genetic counselors, who might recommend germline testing if the family history of cancer was informative. Germline testing of MMR genes was performed by commercial clinical laboratories, usually Ambry Genetics or Myriad Genetics Briefly, next-generation sequencing was performed using the Illumina HiSeq2500 (Illumina Inc.). Sanger sequencing was performed for any regions with insufficient read depth coverage. Large rearrangements were identified using quantitative dosage analysis of the data obtained from next-generation sequencing. In addition, deletions and duplications were identified using a custom microarray comparative genomic hybridization chip (Agilent Technologies). Multiplex ligation-dependent probe amplification analysis for large rearrangements in PMS2 was performed to distinguish homologous pseudo-genes and actual gene regions. Variants were classified using American College of Medical Genetics and Genomics recommendations (13, 14).

Summary statistics were calculated to describe the clinical and demographic characteristics of the study population using Stata v14.1 software. To determine the concordance between IHC and MSI-H, we calculated the proportions that agree and disagree along with their 95% confidence intervals.

There were 213 surgeries performed for endometrial carcinoma during the study period. Clinical and pathologic characteristics of the study population are listed in Table 1. The median age at diagnosis was 61.3 years with a range of 23–86. Most women were diagnosed at age greater than 50, were obese with a BMI greater than 30, had cancers with endometrioid histology (74.2%), and had early-stage disease. Thus, this patient cohort is a good representation of the endometrial cancer patient population in general.

MSI analysis was successfully performed in 199 cases, and IHC was carried out in 203 cases. Reasons for not performing the tests included failure of insurance to authorize testing, patient declining tumor screening, and insufficient tissue to perform the evaluation. Results of MSI and IHC testing are summarized in Table 2. Of the evaluable MSI testing cases, 71.9% were MSS, 25.1% MSI-H, and 3% MSI-L. Of the cases with IHC testing, 22.8% of patients had tumors with loss of MLH1/PMS2, 1.0% loss of MSH2/MSH6, 1.5% loss of MSH6, and 1.5% loss of PMS2. A total of 72.6% of patients had tumors with intact staining for all MMR proteins.

Detailed discordance and concordance data for MSI and IHC are summarized in Table 3 and Fig. 1. Overall concordance and discordance between IHC and MSI was 96.9% and 3.1%, respectively. Cases exhibiting MSI-H results had a concordant loss of IHC expression in 98.0% of cases. For the one discordant case, IHC exhibited heterogeneous loss of MLH1/PMS2 and had methylation of the MLH1 promoter and was thus considered a sporadic tumor (Fig. 1). The lowest level of agreement occurred in tumors with IHC loss of at least one MMR protein, with only 89.1% of these tumors being MSI-H. MSS cases were concordant with intact IHC protein expression in 96.5% of cases. Of the 5 discordant cases, 4 exhibited heterogeneous loss of MLH1/PMS2 with 3 having MLH1 promoter methylation and therefore considered sporadic tumors. The one patient with a tumor with unmethylated MLH1 promoter underwent genetic testing for a germline mutation in the MLH1 gene, and this was found to be negative. The final discordant case exhibited IHC loss of MSH6, and genetic testing revealed a germline mutation in MSH6. For endometrial cancer tumors with intact IHC expression for all proteins, there was concordance with an MSS result in 96.5% of cases. Of the 5 discordant cases, one tumor was MSI-H with a methylated MLH1 promoter, and the remaining 4 were MSI-L. All MSI-L cases are discussed in more detail below. The table in Fig. 1 summarizes the tumor testing and germline testing results, when applicable, for cases with discordant IHC and MSI results.

Six patients had MSI-L tumors (Table 4). Five of these had intact immunohistochemical staining of all four DNA MMR proteins, and five were aged less than 60 at the time of diagnosis, thus meeting the Society of Gynecologic Oncology clinical screening criteria for referral to a genetic counselor. The insurance carrier declined genetic testing for one of these patients. For 2 patients, no MSH6 germline mutations were identified. One patient had a germline MSH6 variant of uncertain significance. The fifth patient with an MSI-L tumor and intact MMR IHC was not offered genetic testing, as she had a synchronous ovarian granulosa cell tumor, which can be associated with endometrial hyperplasia and cancer. One patient with an MSI-L endometrial cancer had a tumor with IHC loss of MLH1 and PMS2 and MLH1 promoter methylation, which is consistent with a sporadic tumor.

Table 5 summarizes the germline testing results, when available, of all endometrial cancer cases with tissue testing suggesting possible Lynch syndrome. Included is one endometrial cancer patient who had genetic testing prior to her hysterectomy because of the diagnosis of colorectal adenocarcinoma at a young age; a PMS2 germline mutation was detected, so no subsequent tissue testing was performed. Seven germline Lynch syndrome mutations were identified in this cohort, representing 3.4% of patients undergoing some type of tissue testing. Note that tissue testing identified 12 of 213 (5.6%) patients as being suspected of having a potential Lynch germline mutation. For 5/12 of these patients, either germline testing did not identify a mutation or was declined by insurance.

Various clinical and diagnostic challenges that can occur with the implementation of universal tissue testing for Lynch syndrome were identified in this study. These challenges are significant, as they prevented us from definitively classifying these cancers as hereditary or sporadic. These include pretesting factors (n = 10, 4.7%; patient does not want testing; insurance denies reimbursement for testing; not enough tumor for testing), IHC/MSI discordances (n = 6, 2.8%), MSI-L with intact MMR protein expression by IHC (n = 5, 2.3%), and post-tissue testing issues (n = 8, 3.8%; insurance denies reimbursement for germline testing; patient declines germline testing; VUS detected; no germline mutation identified). In summary, we encountered clinical or diagnostic challenges when utilizing a universal tissue testing approach to screen for Lynch syndrome in 13.6% (29/213) of endometrial cancer patients.

National organizations support screening patients with endometrial adenocarcinoma for Lynch syndrome either through family history or tissue-based screening modalities. There have been several studies evaluating the effectiveness of universal tumor testing and its ability to identify endometrial cancers secondary to Lynch syndrome; however, there have not been any published studies evaluating the clinical and diagnostic challenges that result from a universal tumor testing approach. To maximize capturing clinical and diagnostic challenges that can emerge through universal tumor testing, data were prospectively collected on a sequential, unselected cohort of endometrial carcinoma patients who underwent hysterectomy. The demographic and pathologic characteristics in this study population are similar to published national data; thus, findings from this study can presumably be applied to other endometrial cancer patients (15). Results from this cohort also show tumor testing results of MSI-H in 25.1% of cases, with 93.5% of these with MLH1 promoter methylation, a finding consistent with other published literature evaluating both colorectal and endometrial carcinomas (12, 16–21). Using the universal tumor screening approach, clinical and diagnostic challenges in definitively designating an endometrial cancer patient as sporadic or Lynch syndrome would be expected to occur in approximately 13.6% of patients.

Looking first at tumor testing strategies, the choice of best tumor-based screening method for Lynch syndrome is not clear. For endometrial cancer, the National Comprehensive Cancer Network guidelines recommend IHC or MSI screening of all women less than age 50 or those with a significant past medical history of family history concerning for Lynch syndrome (22). The American College of Obstetrics and Gynecology and the Society of Gynecologic Oncology practice guidelines recommend that all women should undergo comprehensive clinical screening or molecular tumor-based testing, deferring the choice of specific approaches to individual practices (8). These national guidelines do not favor one form of tissue testing over the other. Goodfellow and colleagues, in a recent large cooperative group study of more than 1,000 endometrial cancer patients, recommend a combination of MSI, IHC, and reflexive MLH1 promoter methylation for all patients with endometrial cancer with endometrioid histology, regardless of age, suggesting that cases of Lynch syndrome could be missed if one method was used in place of another (6).

MSI testing by itself has an overall sensitivity for detecting germline Lynch syndrome mutations of 83% with a range of 25% to 93% (23). These numbers are largely derived from colorectal cancer family registries and colorectal cancer literature. The greatest sensitivity is for detecting MSI-H associated with MLH1 and MSH2 germline mutations and to a lesser degree MSH6 and PMS2. If an institution were to solely use MSI testing as a tissue screen for Lynch syndrome, as is the case for many of the colorectal carcinoma screening protocols, the number of missed clinically significant Lynch syndrome cases in our cohort is 1/199 (0.5%). Drawbacks to using only MSI are that it is more expensive (2016 Medicare reimbursement associated with CPT81391 = $394.44), and it is technically more complex than IHC. In addition, it does not target the possible gene of interest, and subsequent evaluation would include either adding IHC to identify the source of MMR defect or germline testing of all four DNA MMR genes.

IHC for the four DNA MMR proteins has an overall sensitivity for detecting germline Lynch syndrome mutations of 94% with a range of 92% to 100% (23). These numbers are also extrapolated from the colorectal cancer literature and some studies that include endometrial cancers. IHC with reflexive MLH1 promoter methylation for cases with MLH1 protein loss has been proposed to be more cost effective than IHC alone (12). 2016 Medicare reimbursement for DNA MMR is $233.81 (CPT 88360), and PCR-based MLH1 promoter methylation is $159.64. If an institution chose to only perform IHC with reflexive MLH1 promoter methylation in indicated cases, the number of missed Lynch syndrome cases based on our prospective population is 2/203 (0.98%). Benefits of this approach include that it is a reliable indicator for identifying at-risk individuals, targets gene of interest for subsequent germline testing, is less expensive than MSI, is simpler to perform, and the presence of an internal positive control with each IHC sample. Drawbacks to IHC are ambiguous results such as heterogeneous staining, which occurred in 4 of 203 (2.0%) of our patient population as well as false-negative results in which a nonfunctional protein is translated and stains positive on IHC. Although the latter circumstance was not encountered in this study, it was reported by Goodfellow and colleagues and has been occasionally encountered during the clinical practice of one of the authors (R.R. Broaddus; ref. 6). Although the numbers of missed Lynch syndrome cases are relatively small when considering IHC or MSI as single screening tests, one does need to consider that each missed patient may have multiple siblings and children who would also be at risk for having a deleterious germline mutation. This results in a larger number of missed opportunities for early intervention and cancer screenings.

There have been two major studies examining the concordance between MSI and IHC. A retrospective analysis by Bartley and colleagues found discordance between MSI and IHC results in 13 of 591 (2.2%) cases; nearly all of the examined tumors were colorectal, with only seven endometrial cancers in the entire cohort. Two of the 13 identified discordances were endometrial adenocarcinomas (9). A prospective study by Leenen and colleagues found a 100% concordance between MSI and IHC in a series of 179 endometrial carcinomas (7). In a large cooperative group study of more than 900 endometrial cancer patients, 2.0% of endometrial cancers were MSI-high with intact IHC expression of MMR proteins (6). In our prospective endometrial cancer population, MSI and IHC were discordant in 3.9% of cases.

In addition to discordances between tumor testing methodologies, there are other sources of diagnostic difficulty. For example, MSI-L is not clearly associated with a risk for Lynch syndrome, but some tumors that are MSI-L have a corresponding germline mutation in one of the DNR MMR genes. In the study by Goodfellow and colleagues, their cohort of 1,043 had a 2.8% incidence of MSI-L tumors. Patients with tumors that were MSI-L with intact IHC did not undergo germline sequencing, and there were no tumors with MSI-L and a loss of expression on IHC (6). In another study, de Leeuw and colleagues found that 3 of 37 (8.1%) of the MSH6 mutation carriers with endometrial carcinoma had an MSI-L tumor and concomitant IHC loss of MSH6 (24). In the prospective study in the Netherlands, there were no cases of MSI-L tumors, but their MSI testing included five microsatellites rather than the seven microsatellites in the Bethesda Panel (7). Our study detected MSI-L detected in 3% of patients; one of these patients had a germline MSH6 variant of unknown significance but no definite MSH6 deleterious mutations. Of the small number of MSI-L endometrial carcinomas reported in the published literature, the number with a germline mutation is low but not zero (25). Kuismanen and colleagues examined endometrial cancers from families with known MLH1 or MSH2 germline mutations and found that, compared with colon cancers arising in these families, there was a lower proportion of unstable microsatellites, with 23% being MSS (26). For institutions using only MSI testing as a screening method, patients with MSI-L tumors may benefit from reflexive IHC or germline testing for MSH6 mutation.

Heterogeneous protein expression on IHC presents another tumor testing interpretive challenge (Fig. 1). Our cohort showed 2.0% (4/203) of tumors expressed a partial loss of protein expression for MLH1 and PMS2. These tumors either had associated MLH1 methylation or no germline mutation detected. Patient numbers are small for this finding, but they do suggest that a heterogeneous pattern on IHC is not associated with Lynch syndrome.

Tumor testing results associated with germline MSH6 mutations are less consistent than other Lynch syndrome germline mutations. Individuals with an MSH6 mutation often have a unique phenotype in that probands are often older at diagnosis and endometrial cancer is more commonly seen than colorectal cancer in the family history (27, 25). The study by Goodfellow and colleagues had 21 endometrial cancer cases with IHC loss of MSH6 (6). Of these, 15 were MSI-H, 1 was MSI-L, and 5 were MSS. Seven of nine confirmed MSH6 germline mutations were from patients who had tumors that were MSI-H cases with corresponding IHC loss of MSH6. Two other confirmed patients with MSH6 mutations were also MSI-H; one had intact IHC for DNA MMR proteins and one had inconclusive IHC results. MSI-L cases did not undergo genetic testing in their study. Our cohort had two germline MSH6 mutations and one variant of unknown significance. For these, one was MSI-H with concordant loss of MSH6, one was MSS with discordant loss of MSH6, and one was MSI-L with intact IHC expression of all four MMR proteins.

Other potential tissue testing problems have been reported, but they were not encountered in our study. The Mayo Clinic group recently identified 22/13,100 (0.2%) cases of MSH6 heterogeneous loss by IHC using a retrospective analysis (28). At least 23% of these were associated with germline mutations in MMR genes other than MSH6. Goodfellow and colleagues identified two cases of germline PMS2 mutations (one variant of undetermined significance and one deleterious) in which the PMS2 IHC showed intact positive protein expression, but there was IHC loss of MSH2 and MSH6 (6).

After receiving tumor testing results suggestive of Lynch syndrome (MSI-H and/or IHC loss of DNA MMR proteins), a referral to a genetic counselor with subsequent germline testing is typically performed. The recommendations are clear when a germline Lynch syndrome mutation is identified. Challenges that ensue for the genetic counselor are when there is no identifiable germline mutation or a variant of unknown significance occurs. Prior studies have shown that approximately 60% of tumors that have testing consistent with a diagnosis of Lynch syndrome will have a germline mutation subsequently identified (29, 30). In our series, 13/213 (6.1%) of endometrial carcinomas exhibited tumor testing suggestive of Lynch syndrome, and 6/13 (46.2%) of these had a germline mutation confirmed. For the patients without a germline mutation, guidelines for the patient's subsequent colon cancer screening and recommendations for family members are less clear. One study showed that the risk for subsequent cancers in these cases was lower than patients with a germline mutation but higher than the general population (31). There have also been some studies delving in to etiology of this phenomenon. Two studies have recently identified somatic mutations of MMR genes in endometrial and colorectal cancers, but the exact incidence of such mutations is not certain (32, 33).

In this study, 13.6% of endometrial cancer cases encountered clinical or diagnostic challenges, which limited our ability to definitively classify a patient as Lynch syndrome or sporadic. Note that the number of instances in each of the preanalytic (patient or insurance declining screening), analytic (insufficient tissue, ambiguous results, discordance between IHC, and MSI), and postanalytic (patient or insurance declines genetic counseling/testing, informative tumor testing with negative germline testing, or germline testing showing a variant of unknown significance) categories is relatively small. However, it is clear that problems can arise at numerous steps along the path to universal testing. It should also be noted here that only some of these problems can likely be resolved. For example, over time medical insurance companies will become more educated as to the relationship of endometrial cancer to Lynch syndrome. With more cumulative experience, we will likely be able to confidently classify MSI-low/intact MMR IHC cases as sporadic. Implementation of somatic sequencing of MMR genes will help to classify some of the cases with no germline mutation as sporadic. Other problems, however, such as IHC/MSI discordance and detection of a germline VUS, will continue to limit our ability to accurately classify a subset of patients.

No potential conflicts of interest were disclosed.

Conception and design: A.S. Bruegl, D.L. Urbauer, R.R. Broaddus

Development of methodology: A.S. Bruegl, D.L. Urbauer, R.R. Broaddus

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.S. Bruegl, K.L. Ring, R.R. Broaddus

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.S. Bruegl, K.L. Ring, B.M. Fellman, D.L. Urbauer, R.R. Broaddus

Writing, review, and/or revision of the manuscript: A.S. Bruegl, K.L. Ring, M. Daniels, B.M. Fellman, D.L. Urbauer, R.R. Broaddus

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.S. Bruegl

Study supervision: R.R. Broaddus

This work was supported by NIH 2P50 CA098258-06 SPORE in Uterine Cancer (RRB), NIH T32 Training Grant (ASB), and M.D. Anderson Cancer Center Support Grant NIHCA016672.

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