Stains & CD markers
MLH1

Editorial Board Member: Catherine E. Hagen, M.D.
Deputy Editor-in-Chief: Raul S. Gonzalez, M.D.
Mieke R. Van Bockstal, M.D., Ph.D.

Last author update: 28 April 2021
Last staff update: 18 June 2021

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PubMed Search: MLH1[TI] stain

Mieke R. Van Bockstal, M.D., Ph.D.
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Cite this page: Van Bockstal MR. MLH1. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/stainsmlh1.html. Accessed March 19th, 2024.
Definition / general
    • Mismatch repair (MMR) protein
    • Deficiency results in microsatellite instability (MSI)
    • Germline mutations result in autosomal dominant hereditary Lynch syndrome
Essential features
  • Mismatch repair protein whose deficiency results in high microsatellite instability (MSI high)
  • Causes of deficiency include germline and somatic MLH1 mutations and somatic and constitutional epigenetic silencing through MLH1 promoter hypermethylation
  • Germline MLH1 mutations result in the autosomal dominant hereditary Lynch syndrome, which predisposes to colorectal cancer and several other noncolorectal neoplasms, such as endometrial cancer (Mod Pathol 2019;32:1)
  • Immunohistochemistry for the MMR proteins MLH1, PMS2, MSH2 and MSH6 is used for:
    • Identification of MMR deficient, MSI high neoplasms
    • Screening for Lynch syndrome
Terminology
Pathophysiology
Diagrams / tables

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MMR and Lynch syndrome screening algorithm

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Colorectal carcinoma: MMR status

Clinical features
  • MLH1 deficiency results in microsatellite instability (MSI), characterized by accumulation of numerous replication errors within short nucleotide repeat sequences (i.e. microsatellites)
  • MLH1 deficiency in cancer has 4 possible causes:
    • Somatic MLH1 promoter hypermethylation
    • Constitutional MLH1 promoter hypermethylation
    • Germline MLH1 mutation
    • Somatic MLH1 mutation
  • Somatic MLH1 promoter hypermethylation is the cause of MLH1 deficiency in 10 - 15% of all colorectal and endometrial carcinomas; colorectal carcinomas fitting this description often also have BRAF V600E mutations (J Med Genet 2021 Jan 15 [Epub ahead of print])
  • Constitutional MLH1 promoter hypermethylation is a rare cause (< 1%) of Lynch syndrome (Genes Chromosomes Cancer 2009;48:737)
  • In case of a heterozygous germline MLH1 mutation, a second hit can inactivate the remaining intact allele in a cell, resulting in deficient DNA mismatch repair and high microsatellite instability (J Mol Diagn 2004;6:308)
  • Biallelic germline mutations in 1 of the 4 mismatch repair genes give rise to the constitutional mismatch repair deficiency syndrome (CMMRD), a rare but aggressive cancer predisposition syndrome with cancers in multiple organs, often presenting in childhood; phenotypic overlap with neurofibromatosis 1 is common (J Med Genet 2021 Feb 23 [Epub ahead of print], Genet Med 2020;22:2081, Clin Genet 2020;97:296)
  • Somatic MLH1 mutation can occur either clonally or subclonally in a carcinoma (Am J Surg Pathol 2016;40:909)
Interpretation
  • Retained staining (i.e. MLH1 proficiency) is defined as diffuse nuclear immunoreactivity within neoplastic cells
  • Retained staining in neoplastic cells requires a similar or higher intensity than the nuclear immunoreactivity in normal epithelium, lymphocytes, fibroblasts and endothelium, which serve as internal control (Appl Immunohistochem Mol Morphol 2015;23:1)
  • Aberrant loss of staining (i.e. MLH1 deficiency) is defined as either:
    • Complete absence of nuclear immunoreactivity within neoplastic cells, in the presence of a positive internal control
    • PMS2 expression is almost always lost as well
    • Punctate, speckled, dot-like, nucleolar or granular heterogeneous nuclear MLH1 immunoreactivity within neoplastic cells, in the presence of a positive internal control (Histopathology 2019;74:795, Histopathology 2018;73:703)
    • Punctate or dot-like staining pattern is most often observed with the MLH1 M1 clone
  • MMR panel should generally use MLH1, MSH2, MSH6 and PMS2
    • 2 stain immunohistochemical screening (i.e. just PMS2 and MSH6) for Lynch syndrome may fail to detect MMR deficiency in rare cases and is not recommended (Mod Pathol 2018;31:1891)
  • Rarely, germline MLH1 missense mutation results in loss of function with retained MLH1 expression
    • If no germline PMS2 mutation is found in a carcinoma with isolated PMS2 loss, a germline MLH1 mutation should be excluded (Am J Surg Pathol 2016;40:e35)
  • MLH1 immunohistochemistry is fixation dependent; poor fixation results in false negative staining
    • MLH1 immunohistochemistry is preferentially performed on biopsies instead of resection specimens
Uses by pathologists
Prognostic factors
  • Patients with a colorectal carcinoma with combined deficiency of MLH1 and PMS2, in the absence of somatic BRAF mutations and MLH1 promoter hypermethylation, should be referred for germline mutation testing (Mod Pathol 2019;32:1)
  • MSI high neoplasms likely respond well to immune checkpoint inhibitors (Br J Cancer 2019;121:809, J Clin Oncol 2020;38:214, J Clin Oncol 2019;37:2786)
  • MMR deficient endometrial cancers have:
    • Better prognosis than copy number high / p53 abnormal endometrial cancers
    • Similar prognosis to copy number low / p53 wild type endometrial cancers
    • Worse prognosis than POLE mutant endometrial cancers (Nature 2013;497:67)
Microscopic (histologic) description
  • Normal finding: nuclear stain in at least some but generally most / all tumor cells, in addition to nonneoplastic background cells
  • Any other pattern could represent abnormality and should be further assessed (see Interpretation)
Microscopic (histologic) images

Contributed by Mieke R. Van Bockstal, M.D., Ph.D. and Shatavisha Dasgupta, M.D., Ph.D.
MLH1 deficient endometrioid carcinoma

MLH1 deficient endometrioid carcinoma

MLH1 deficient gastric carcinoma

MLH1 deficient gastric carcinoma

Moderately differentiated gastric adenocarcinoma

Moderately differentiated gastric adenocarcinoma

MLH1 proficient gastric adenocarcinoma

MLH1 proficient gastric adenocarcinoma

PMS2 deficient gastric carcinoma PMS2 deficient gastric carcinoma (paired interpretation)

PMS2 deficient gastric carcinoma (paired interpretation)


MLH1 proficient colon carcinoma MLH1 proficient colon carcinoma

MLH1 proficient colon carcinoma

Carcinosarcoma of the endometrium

Carcinosarcoma of the endometrium

MLH1 proficient endometrial carcinomsarcoma

MLH1 proficient endometrial carcinomsarcoma

Fixation artifact

Fixation artifact


MLH1 deficient endometrioid carcinoma MLH1 deficient endometrioid carcinoma MLH1 deficient endometrioid carcinoma

MLH1 deficient endometrioid carcinoma

Virtual slides

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MLH1 deficient endometrioid carcinoma

MLH1 deficient endometrioid carcinoma

Positive staining - normal
Positive staining - disease
  • "Positive" result generally indicates aberrant loss of staining (see Interpretation), so report should be carefully worded to avoid miscommunication
Negative staining
  • "Negative" result generally indicates retention of normal staining (see Interpretation), so report should be carefully worded to avoid miscommunication
Sample pathology report

MMR immunohistochemistry result Recommended sample report
Normal expression of all 4 MMR proteins MLH1, PMS2, MSH2 and MSH6 Background nonneoplastic tissue / internal control shows intact nuclear expression of MLH1, PMS2, MSH2 and MSH6.
The tumor shows normal nuclear expression of MLH1, PMS2, MSH2 and MSH6. There is no immunohistochemical evidence of a mismatch repair deficiency or Lynch syndrome. However, referral for genetic counseling should be considered if there is a strong family / clinical history suggestive of Lynch and related syndromes, despite this immunohistochemical result.
Abnormal result; combined MLH1 and PMS2 loss with expression of MSH2 and MSH6 Background nonneoplastic tissue / internal control shows intact nuclear expression of MLH1, PMS2, MSH2 and MSH6.
The tumor cells show loss of expression of MLH1 and PMS2, with normal nuclear expression of MSH2 and MSH6. This immunohistochemical profile suggests a mismatch repair deficiency, which requires MLH1 promoter hypermethylation testing.
  • If MLH1 promoter hypermethylation is not detected, the patient should be referred for germline genetic testing for Lynch syndrome.
  • If MLH1 promoter hypermethylation is detected, germline genetic testing is not required.
Abnormal result; isolated PMS2 loss with expression of MLH1, MSH2 and MSH6 Background nonneoplastic tissue / internal control shows intact nuclear expression of MLH1, PMS2, MSH2 and MSH6.
The tumor cells show loss of expression of PMS2, with normal nuclear expression of MLH1, MSH2 and MSH6. This immunohistochemical profile suggests a mismatch repair deficiency. The patient should be referred for germline genetic testing for Lynch syndrome.
Board review style question #1

Which of the following is true about this MLH1 immunohistochemical stain, performed on a well differentiated endometrioid carcinoma of the ovary?

  1. Immunohistochemical stain for PMS2 should be performed to exclude PMS2 deficiency
  2. Mismatch repair protein MLH1 is deficient
  3. This immunoreactivity pattern is likely due to MLH1 promoter hypermethylation
  4. This patient likely has Lynch syndrome and should be referred for germline mutation testing
Board review style answer #1
A. Immunohistochemical stain for PMS2 should be performed to exclude PMS2 deficiency

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Reference: MLH1
Board review style question #2
Which of the following is true about mismatch repair immunohistochemistry?

  1. A patient with an MLH1 deficient, PMS2 deficient colon carcinoma harboring a BRAF mutation should be referred to genetics to exclude an underlying germline MLH1 mutation
  2. Colorectal carcinomas with isolated PMS2 deficiency likely present with MLH1 promoter hypermethylation
  3. Immunohistochemistry for mismatch repair proteins should be performed on each carcinosarcoma of the endometrium to allow molecular classification
  4. Microsatellite instable endometrioid carcinomas of the endometrium generally have a worse prognosis than MMR proficient, p53 mutant endometrioid carcinomas of the endometrium
Board review style answer #2
C. Immunohistochemistry for mismatch repair proteins should be performed on each carcinosarcoma of the endometrium to allow molecular classification

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Reference: MLH1
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