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Slide 1: Greeting

Hello, my name is Shabnam Zarei. I am a molecular genetic pathology fellow at the department of Laboratory Genomics and Genetics at Mayo Clinic in Rochester, MN. Welcome to this Pearl of Laboratory Medicine on “Hereditary Endometrial Carcinoma, with focus on Lynch syndrome.”

Slide 2: Overview

First, we will start with a definition of Lynch syndrome, review current screening guidelines and testing, mention available diagnostic tools, overview the molecular basis and then end with a few words on the role of genetic counseling in management of patients with Lynch syndrome.

Slide 3: Introduction

Lynch syndrome or Hereditary Non-Polyposis Colorectal Carcinoma (HNPCC) is a cancer predisposition syndrome associated with an increased risk of carcinoma in different organs, most commonly colorectal and endometrial carcinomas. It is associated with younger age at the time of cancer diagnosis, more than one organ involved by cancer, involvement of multiple family members and an overall better survival. Lynch syndrome is by far the most common cause of hereditary endometrial carcinoma.

Slide 4: Other Forms of Hereditary Endometrial Carcinoma

The second most common cause for hereditary endometrial carcinoma is PTEN germline mutation responsible for Cowden syndrome which is associated with increased risk for thyroid,

breast and endometrial carcinoma. The lifetime risk for developing endometrial carcinoma in patients harboring a germline PTEN mutation is about 28%. Other less common hereditary causes of endometrial carcinoma include EPCAM gene deletions (which inactivate MSH2), POLE and POLD1 germline mutations. However, today we will be focusing on Lynch syndrome.

Slide 5: Screening guidelines

In the past two decades several screening guidelines including Bethesda, Amsterdam and their revisions were introduced to aid with identifying patients at risk for Lynch syndrome. For example, the revised Bethesda criteria recommends testing for Lynch syndrome in cases of colon cancer diagnosed in patients younger than 50 year old, presence of synchronous or metachronous Lynch associated carcinomas (including colorectal, endometrium, small bowel, ovary, stomach, and pancreas), specific histology of the tumor in a patient younger than 60 year old, colorectal cancer diagnosed in >1 first-degree relatives with at least one cancer diagnosed before age 50, or colorectal cancer diagnosed in >=2 first or second degree relatives with Lynch syndrome, regardless of age. Due to a low sensitivity and specificity of these criteria for screening patients at risk for Lynch syndrome, the newest screening guideline by National Comprehensive Cancer Network (NCCN) and American Cancer Society (ACS) recommends screening testing in ANY patient, especially those younger than 50 years old with newly diagnosed endometrial or colorectal carcinomas.

Slide 6: Genes and pattern of inheritance

Four major genes located on three different chromosomes (3, 7 and 2) are involved in the pathogenesis of Lynch syndrome: MLH1, PMS2, MSH2 and MSH6. Lynch syndrome has an autosomal dominant pattern of inheritance with variable degree of mutation penetrance. About 3-5% of endometrial carcinomas diagnosed each year are associated with Lynch syndrome. On the other hand, patients with Lynch syndrome have 40-60% lifetime risk of developing endometrial and similar risk for colorectal carcinoma and lesser risk for developing ovarian, stomach, small bowel, ureter and renal pelvis carcinomas. The risk for cancer predisposition is higher with MLH1 and MSH2 mutations and it has the least association with PMS2 mutations. Therefore, the chance of cancer predisposition is different based on different genes.

Slide 7: Function of MMR proteins

Mistakes can happen during DNA replication specifically in the repetitive regions of the genome. Bubble and loop formation and slippage are the most common errors that are naturally removed by the normal product of these four genes, called mismatch repair enzyme proteins that function as protein dimers: MLH1 with PMS2 and MSH2 with MSH6. In the absence of normal gene or normal protein function, variation in the size of repetitive regions throughout the genome will happen. This phenomenon is called microsatellite instability and can be detected by microsatellite instability or MSI testing. The majority of Lynch syndrome cases (up to 90%) are due to germline mutations in the MLH1 or MSH2 genes.

Slide 8: Screening testing for Lynch syndrome

Different tests are available for screening of Lynch syndrome in endometrial carcinoma including immunohistochemistry (IHC) to look for mismatch repair protein expression, molecular testing for microsatellite instability to look into the repetitive regions of the genome, and MLH1 hypermethylation testing which is predominantly a somatic alteration and it is absent in Lynch syndrome. BRAF V600E mutation is another somatic alteration that if present with MLH1 promoter hypermethylation in colorectal carcinoma, makes the diagnosis of Lynch syndrome much less likely. The frequency of BRAF V600E mutation in sporadic endometrial carcinoma is very low therefore only MLH1 promoter methylation status is typically used in combination with MMR by IHC and MSI by molecular testing. After identifying patients with positive screening tests by either MMR IHC or MSI, the next step is to do germline mutation analysis targeted for the genes that showed loss of protein expression by IHC. These include: Sanger sequencing, MLPA for gene dosage and more.

Slide 9: Algorithmic testing for Lynch syndrome

Here we see an algorithmic approach for diagnosis of Lynch syndrome which begins by IHC and MSI screening testing and ends with gene sequencing. For example if the IHC shows loss of expression of MLH1/PMS2 and MSI testing shows microsatellite instability, it is recommended to perform MLH1 hypermethylation testing in patients with endometrial carcinoma to rule out MLH1 promoter hypermethylation which is seen mostly in patients with sporadic endometrial carcinoma. The absence of MLH1 hypermethylation is supportive of diagnosis of Lynch syndrome. In cases of colorectal carcinoma, BRAF V600E testing is also performed with MLH1 hypermethylation. Germline mutations in MMR genes do not typically co-exist with MLH1 hypermetheylation and/or BRAF V600E mutations. A positive MSI and negative MLH1 hypermethylation result in a patient with endometrial carcinoma should prompt germline testing. This test is done on patient’s blood sample and through the use of molecular methods, the exact molecular alteration may be identified and the diagnosis of Lynch syndrome confirmed.

Slide 10: MSI testing

Next, let’s take a closer look at how microsatellite instability testing is performed. Microsatellites are short tandem repeats of DNA sequences, scattered through the entire genome, each 1-3 or more nucleotides in size. The germline mutations will result in abnormal or absent mismatch repair protein function which can be detected by microsatellite instability (MSI) testing which is the consequence of mismatch repair enzyme deficiency. It is recommended to use 5 markers including both mono and di-nucleotide markers for detection of microsatellite instability. DNA is extracted from formalin fixed paraffin embedded (FFPE) tissue from tumor and normal areas. A PCR based assay using capillary electrophoresis for fragment analysis is used and based on the number of unstable markers, a final call will be made: If 30% or more (meaning 2 out of 5) markers show instability, it’s called MSI-high, if only 1 out of 5 markers shows instability, this is an MSI-low and if none of 5 markers shows instability that would be a case of microsatellite stable (or MSS).

Slide 11: Example case, H&E

Here we see a case of endometrial adenocarcinoma mostly present in the top part of this photomicrograph with few residual benign endometrial glands in the lower part of the photomicrograph. You can also appreciate tumor infiltrating lymphocytes present in this photo.

Slide 12: Example case, IHC

Here we see that in this case, by immunohistochemistry, the MLH1/PMS2 protein expression is lost whereas the MSH2 and MSH6 nuclear expression is retained. Please note the retained protein expression in the normal glands and stroma highlighted by black circles and in the tumor infiltrating lymphocytes marked by black arrow head. This pattern of staining is suggestive of mismatch repair enzyme deficiency in the MLH1/PMS2 complex. Since the immunohistochemistry is not 100% sensitive or specific for detection of Lynch syndrome, microsatellite instability (MSI) testing should also be performed. There are cases that show intact protein expression by IHC and MSI-H by MSI testing and vice versa. Such discordant results can be explained by the fact that the immunohistochemical staining only shows the presence or absence of mismatch repair protein expression, and cannot distinguish between functional and non-functional proteins. In cases of discordant results, it is recommended to proceed with germline testing.

Slide 13: Example case, MSI

Here we see that all 4 of 4 markers show instability in the form of increase in the size of the tandem repeat region seen in the tumor compared to normal. This is compatible with microsatellite unstable or MSI-H status. Since, microsatellite instability can also be due to MLH1 hypermethylation which is predominantly a somatic alteration, MLH1 hypermethylation testing is performed which showed no MLH1 hypermethylation (not shown in here). As mentioned earlier, BRAF V600E somatic mutation testing can also be done in conjunction with MLH1 hypermethylation in colorectal carcinomas, and the absence of BRAF V600E mutation is similarly supportive of diagnosis of Lynch syndrome. Per the algorithm that we discussed earlier, this case was sent for further genetic testing. The MLH1 gene sequencing showed a c.940delG deleterious alteration, confirming the diagnosis of Lynch syndrome. Identifying the exact genetic alteration in this case will be a guide for possible testing of the patients’ family members to identify those at risk for Lynch syndrome.

Slide 14: Histomorphology and prognosis

Some histologic findings are found to be more associated with Lynch syndrome, especially in colorectal carcinomas. These findings include: mucinous or signet cell morphology, increased intra-epithelial infiltrating lymphocytes, a Crohn’s like response at the edge of the tumor and medullary carcinoma like morphology. In endometrial carcinomas, Lynch syndrome is associated with tumors arising in the lower uterine segment, more peritumoral and intratumoral lymphocytes and tumor heterogeneity (mixed morphology including endometrioid and serous carcinoma). Patients with MSI-H colorectal carcinomas have a better prognosis. The data on prognosis in MSI-H endometrial carcinomas remains controversial. Despite being described and incorporated in some screening guidelines, these morphologic findings are not 100% specific for Lynch associated endometrial or colorectal carcinomas.

Slide 15: Genetic counseling

Patients with Lynch syndrome should be identified clinically as they are at higher risk for developing multiple primary malignancies. These patients will benefit from closer clinical follow- ups and/or prophylactic surgeries if applicable. Usually the genetic counselor will meet with the patient after screening testing showed abnormal IHC and/or MSI results. The genetic counselor will explain the findings and go over risk associated with developing other malignancies. He or she will also talk about the risk for the family members and will facilitate further specific germline genetic testing of the patient and in case of positive germline testing result in the patient, will recommend testing of the family members for the same known genetic alteration.

Slide 16: Summary

In summary, Lynch syndrome is a cancer predisposition syndrome associated with an increased risk of developing multiple cancers most importantly endometrial and colorectal carcinomas. It has an autosomal dominant pattern of inheritance with variable degree of penetrance. Most patients with Lynch syndrome have a detectable germline mutation in one of the four major genes responsible for DNA mismatch repair. MLH1 promoter hypermethylation results in gene silencing, and is predominantly a somatic alteration which also causes microsatellite instability. MLH1 promoter hypermethylation should be ruled out by molecular testing to support diagnosis of Lynch syndrome. Screening guidelines will help identify the patients and their relatives who are at risk for developing cancer in different organs. Earlier detection of cancer will improve patients’ prognosis and bring better quality of care for patients and their families.

Slide 17: References

  1. W Kohlmann, SB Gruber. Lynch Syndrome. 2014. Available from:
  2. Organisation mondiale de la santé, and Centre international de recherche sur le cancer. WHO classification of tumours of female reproductive organs. IARC press, 2014.
  3. Lancaster JM, Powell CB, Chen LM, Richardson DL, SGO Clinical Practice Committee. Society of Gynecologic Oncology statement on risk assessment for inherited gynecologic cancer predispositions. Gynecologic oncology. 2015 Jan 31;136(1):3-7.
  4. Stoffel EM et al. Hereditary colorectal cancer syndromes: American society of clinical oncology clinical practice guideline endorsement of the familial risk– colorectal cancer: European society for medical oncology clinical practice guidelines. Journal of clinical oncology. 2014 Dec 1;33(2):209-17.
  5. Umar A et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. Journal of the National Cancer Institute. 2004 Feb 18;96(4):261-8.
  6. American College of Obstetricians and Gynecologists Practice Bulletin. Lynch Syndrome. Available from: ACOG-bulletin.pdf
  7. Society of Gynecologic Oncology. Lynch Syndrome. November 2014. Available from:
  8. Society of Gynecologic Oncology. Screening for Lynch syndrome in Endometrial Cancer. March 2014. Available from: practice/guidelines/screening-for-lynch-syndrome-in-endometrial-cancer/
  9. NCCN Network for Resource Stratification of NCCN Guidelines. Uterine Neoplasms. August 2017. Available from:
    Online Mendelian Inheritance in Man. Lynch syndrome. Available from:

Slide 17: Disclosures


Slide 18: Thank You from

Thank you for joining me on this Pearl of Laboratory Medicine on “Hereditary Endometrial Cancer”