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October 2012

Editor:
Fredrick L. Kiechle, MD, PhD

Question Q. Our clinical colleagues have requested that we begin immunohistochemistry testing for mismatch repair proteins (Lynch syndrome). Are there any special considerations for these assays?

A. The hallmark of Lynch syndrome is a genetic mutation in one of the family of DNA mismatch repair (MMR) protein genes (MLH1, MLH3, MSH2, MSH6, PMS2). These proteins function to repair errors in replication of DNA at short repetitive sequences (microsatellites). Lynch syndrome is associated with a high risk of colon and endometrial cancers, as well as increased risk of urothelial, small bowel, hepatobiliary, and pancreatic cancer (see references 1 and 2 for comprehensive review). Further, 10 to 15 percent of sporadic colon, gastric, and endometrial tumors may harbor a somatic, non-germline MMR mutation or loss of expression. Colon cancers with MMR loss tend to have a somewhat more favorable prognosis and differ in chemotherapy sensitivity as compared with cancers from other molecular backgrounds.2

Complementary methods are available to test for defective mismatch repair function, including: 1) immunohistochemical analysis of mismatch repair protein expression in tumor tissue (MMR-IHC), and 2) demonstration of defective mismatch repair function via PCR analysis of a panel of microsatellite DNA sequences (MSI-PCR). Each of these methods has advantages and disadvantages.3-5 In short, MMR-IHC can reveal the identity of the defective protein for targeted genomic sequencing yet will “miss” a small percentage of cases with expressed but functionally defective MMR proteins. MSI-PCR, if abnormal, does not elucidate which gene is defective and will “miss” a small percentage of cases with a weak phenotype, as may be seen in MSH6 loss. It should also be noted that loss of MLH1 expression may be associated with non-heritable, epigenetic events, especially in older patients, as discussed later.

In developing MMR-IHC assays at your institution, a few issues should be considered at the outset, such as patient consent, antibody panels, antibody reagents, and validation.

Patient consent. Some institutions consider MMR-IHC akin to a genetic test, and thus recommend consent for genetic testing before performing the testing. However, the majority (84 percent per a 2011 CAP survey) do not obtain consent. Testing strategies vary among laboratories, with some testing all colon and/or endometrial cancer patients,6 some testing only those below a certain age, some testing only those with histologic features of Lynch tumors, and others testing as clinicians request.

Antibody panels. Most laboratories have an MMR-IHC panel consisting of antibodies to four MMR proteins: MLH1, MSH2, MSH6, PMS2. For efficiency and cost savings, recent publications have advocated primary screening with only MSH6 and PMS2.7,8 Intact expression of MSH6 and PMS2 generally translates to intact expression of MLH1 and MSH2; aberrant expression of MSH6/PMS2 then requires further IHC characterization. This strategy is based on the endogenous pairing of mismatch repair proteins, and an understanding of pairing is also necessary to interpret staining results. MLH1 and PMS2 form a heterodimer; if MLH1 is lost, PMS2 is also destabilized and expression is undetectable. However, since PMS2 has other binding partners, mutation or loss of PMS2 does not affect expression of MLH1. The MSH2/MSH6 heterodimer functions similarly; an MSH2 defect leads to loss of both MSH2 and MSH6, while an MSH6 mutation shows loss of MSH6 expression and intact MSH2 expression.

Antibody reagents. Several different antibody clones are available for each of the MMR proteins, with some new reagents under development. MMR staining of formalin-fixed tissue requires antigen retrieval, and several publications include detailed protocols.7-9

Validation. Because MMR-IHC testing has considerable implications for further molecular and genetic testing, as well as enhanced screening of patients and relatives, careful validation of these IHC assays is imperative, similar to that for predictive IHC tests. Thus, the validation cohort should include numbers of cancers with known protein/gene status, including both normal and abnormal cases, or cross comparison with an external expert laboratory. Further, CAP Surveys are available for both MMR-IHC and MSI-PCR testing.

Successful MMR-IHC assays require attention to tissue fixation, as well as careful evaluation of internal controls. MMR-IHC assays are particularly susceptible to under-fixation, especially MLH1 and PMS2.5,8-9 Since MMR proteins function in DNA repair, nuclear staining of replicating cells is expected normally. Epithelial cells at the base of normal colonic crypts are convenient internal controls, as are lymphocytes; in the uterus endometrial stroma and myometrium may serve as internal control.5,8-9 If internal controls are negative, repeat staining on another tissue block should be attempted; submission of additional tissue sections after longer fixation might also be helpful in difficult cases. MMR proteins may show patchy expression, especially MSH6 in post-chemotherapy specimens.9 Thus, it should be emphasized that even a low percentage of cells with nuclear staining should be scored as intact protein expression. For this same reason I prefer to test resection specimens rather than biopsies, although successful biopsy screening by MMR-IHC has been reported using a four-stain panel.9

In the case of MMR-IHC, positive staining is a normal result, while negative (lack of staining) is abnormal and suggests the need for further testing or genetic counseling. Thus, the conventional “positive” and “negative” descriptors in MMR-IHC reports can be confusing. We prefer to use the terminology “intact” or “normal” expression, as opposed to “loss of” or “aberrant” expression, with explanatory comments in pathology reports. Other considerations in interpretation of IHC results include the fact that loss of expression of MLH1 may be due to a germline mutation in the MLH1 gene (Lynch syndrome), but is perhaps more commonly due to hypermethylation of the MLH1 promoter in older individuals. BRAF point mutations (V600E) are closely associated with this hypermethylation, and many testing algorithms advocate BRAF mutational analysis before embarking on genetic testing in patients whose colon cancers show loss of MLH1 expression.1

In summary, MMR-IHC is an efficient means by which to screen cancers for aberrant mismatch repair protein expression. There are a few special considerations in assay development and interpretation; however, these are readily addressed with an understanding of the background biology and attention to internal controls, as is standard for all IHC assays.

References

1. Geiersbach KB, Samowitz WS. Microsatellite instability and colorectal cancer. Arch Pathol Lab Med. 2011;135:1269–1277.

2. Poulogiannis G, Frayling IM, Arends MJ. DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome. Histopathology. 2010;56:167–179.

3. Shia J. Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn. 2008;10:293–300.

4. Zhang L. Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part II. The utility of microsatellite instability testing. J Mol Diagn. 2008;10:301–307.

5. Bartley AN, Luthra R, Sariaya DS, et al. Identification of cancer patients with Lynch syndrome: clinically significant discordances and problems in tissue-based mismatch repair testing. Cancer Prev Res. 2012;5:320–327.

6. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group. Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genet Med. 2009;11:35–41.

7. Shia J, Tang LH, Vakiani E, et al. Immunohistochemistry as first-line screening for detecting colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome: a 2-antibody panel may be as predictive as a 4-antibody panel. Am J Surg Pathol. 2009;33:1639–1645.

8. Mojtahed A, Schrijver I, Ford JM, et al. A two-antibody mismatch repair protein immunohistochemistry screening approach for colorectal carcinomas, skin sebaceous tumors, and gynecologic tract carcinomas. Mod Pathol. 2011;24:1004–1014.

9. Shia J, Stadler Z, Weiser MR, et al. Immunohistochemical staining for DNA mismatch repair proteins in intestinal tract carcinoma: how reliable are biopsy samples? Am J Surg Pathol. 2011;35:447–454.

Megan L. Troxell, MD, PhD
Associate Professor
Department of Pathology
Oregon Health & Science University
Portland

Member, CAP Immunohistochemistry Committee

Question Q. I read in the August 2012 issue your question and answer about the best im-mu-no-histo-chemical markers to dis-tinguish squamous cell carcinoma from adenocarcinoma in the lung. In a presentation at the CAP ’11 annual meeting, Sanja Dacic, MD, PhD, previewed p40 as an even more specific marker for squamous cell carcinoma than p63. It is now commercially available from a large company. Do you have any comments or recommendations regarding the use of p40 in the panel of antibodies to distinguish adenocarcinoma from squamous cell carcinoma?

A. There have been two recently published papers (Bishop JA, et al. Mod Pathol. 2012;25:405–415; Nonaka D. Am J Surg Pathol. 2012;36:895–899) suggesting the superiority of antibodies to p40 over p63 in identifying squamous cell carcinomas. These papers notwithstanding, in my experience the currently available p40 reagent, a rabbit polyclonal antibody, is not robust enough to permit its routine use in diagnostic surgical pathology. It is possible that with the availability of new monoclonal antibodies this situation might change, but at the present time I would still recommend using the 4A4 mouse monoclonal antibody to p63 in this context.

Allen M. Gown, MD
Medical Director and Chief Pathologist
PhenoPath Laboratories
Seattle

Member, CAP Immunohistochemistry Committee

Question Q. What clinical laboratory thyroid tests in addition to TSH would be necessary to monitor subclinical hyperthyroidism in a patient on methimazole therapy? Iodine-123 thyroid scanning re-vealed a hyperfunctioning nodule in the right and left lobes of the thyroid with suppressed residual gland.

A. Methimazole is the most frequently used anti-thyroid medication. It is a thioamide, a class of drugs that inhibits the addition of iodine to tyrosine and the coupling of iodotyrosine residues in the thyroid gland, markedly suppressing thyroid hormone formation. Another thioamide, propylthiouracil, also inhibits the conversion of thyroxine (T4) to triiodothyronine (T3) outside of the thyroid.

The patient described probably has toxic multinodular goiter, not Graves disease. Therefore, it is likely that anti-thyroid medication is meant to allow the patient to become euthyroid in preparation for more definitive ablative therapy. Thyrotropin (TSH) may not be the most effective way of monitoring methimazole therapy because TSH may remain suppressed for some time after the euthyroid state is restored. Normalization of total T4 is probably the best indication that this has occurred, and total T3 should also be checked to ensure that thyroid production of T3 is also diminished. Methimazole may be less likely than propylthiouracil to produce serious side effects, such as hepatotoxicity and drug-induced anti-neutrophil cytoplasmic antibody vasculitis, but agranulocytosis occurs with both drugs and may develop suddenly. So it may also be a good idea to monitor white blood cell counts (as well as liver function tests).

References

Cooper DS. Antithyroid drugs. N Engl J Med. 2005;352:905–917.

James D. Faix, MD
Stanford Clinical Labs at Hillview
Stanford University School of Medicine
Palo Alto, Calif.

Member, CAP Chemistry Resource
Committee, Standards Committee,
Council on Scientific Affairs


Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla. submit your inquiries, or address them to Sherrie Rice, CAP TODAY, 325 Waukegan Road, Northfield, IL 60093; srice@cap.org.