Immunohistochemical validation studies in effusion cytology: A cautionary tale

Our plain-English summary of Immunohistochemical validation studies in effusion cytology: A cautionary tale, by R. Pillappa, A.O. Kraft (Cancer Cytopathology, 2019).

The study

• Authors: R. Pillappa, A.O. Kraft
• Journal: Cancer Cytopathology
• Published: 2019;127(11):680-683
• Cited by: 6 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Summary of the article

In this commentary, the authors discussed the need to validate immunohistochemistry (IHC) assays used on cytological specimens from body-cavity effusions. Such testing is increasingly used for diagnosis, prognosis and treatment decisions. They noted that IHC assays are typically validated on formalin-fixed, paraffin-embedded tissue. Effusion cell blocks, by contrast, are prepared by more than 10 methods in the United States, and some of these use alcohol fixation, which has been reported to reduce staining or cause false-negative results. They summarized College of American Pathologists guidance: a minimum of 10 positive and 10 negative specimens for initial validation of nonpredictive assays, at least 20 of each for most predictive assays, and at least 90% overall concordance. They then described several examples. First, TTF1 antibody clones differ in sensitivity and specificity. Second, loss of BAP1 staining, CDKN2A deletion testing by fluorescence in situ hybridization, and MTAP staining can help diagnose malignant mesothelioma in effusions; they cautioned that good internal controls are needed and that negative BAP1 staining in their absence should be regarded as noncontributory. Third, progesterone receptor and HER2 results in breast cancer are unreliable after ethanol fixation. Fourth, PD-L1 scoring has pitfalls, including the need to evaluate at least 100 tumor cells. The authors concluded that analytical validation is needed, particularly for non-formalin-fixed material, and that awareness of preanalytic, analytic and postanalytic variables will lead to better patient care.

PubMed has no abstract for this study, and the article doesn't print one. We wrote this summary from the full article with the help of AI. It is not the authors' wording.

In plain English

This commentary by two pathologists reported no new data and did not discuss asbestos exposure. It addressed how laboratories should validate immunohistochemistry stains on cells from body-cavity fluids (effusions). Citing other published work, the authors noted that alcohol-fixed cells may stain more weakly or give false-negative results. They described College of American Pathologists guidance for tissue specimens: at least 10 positive and 10 negative specimens for initial validation of nonpredictive tests, and at least 90% agreement with expected results. For differently processed cytology specimens, the laboratory medical director decides how many cases to test. With 5 positive and 5 negative specimens, one wrong result still meets 90%, with a 95% confidence interval of 57% to 100%. They described loss of BAP1 nuclear staining in atypical mesothelial cells, with positive staining in internal control cells, as supportive of malignant mesothelioma in the appropriate clinical scenario, adding that correlation with cell appearance is essential. Without good internal controls, they cautioned, negative BAP1 staining should be read as noncontributory rather than negative. The authors concluded that effusion stains need analytical validation as recommended by the guidelines.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Commentary (opinion and discussion of published literature and guidelines); no original study
• Main results:
  • Citing other reports, the authors noted decreased signal or false-negative staining on alcohol-fixed cell blocks
  • Citing a recent study by others of Cellient cell blocks, the authors reported that half of the antibodies tested failed initial validation
  • The authors described College of American Pathologists guidance that, for tissue specimens, at least 10 positive and 10 negative specimens be tested for initial validation of nonpredictive tests
  • The guidance described requires at least 90% overall agreement with expected results
  • For cytology specimens not processed like the tissue used for initial validation, the laboratory medical director decides the number of positive and negative cases and the number of markers to test
  • With only 5 positive and 5 negative specimens, the 90% benchmark is met despite 1 wrong result, with a 95% confidence interval of 57% to 100%
  • Loss of BAP1 nuclear staining in atypical mesothelial cells, with positive staining in internal control cells, is considered supportive of malignant mesothelioma in the appropriate clinical scenario
  • The authors stated that correlation with cell appearance (cytomorphology) is essential, because BAP1 loss is more commonly found in epithelioid mesothelioma than in sarcomatoid/desmoplastic malignant mesothelioma
  • The chances of misinterpretation are high without good internal controls; in that situation, negative BAP1 staining of atypical mesothelial cells should be read as noncontributory, not negative
  • Citing a study by others, combining MTAP staining with BAP1 staining or CDKN2A FISH improved sensitivity for detecting malignant mesothelioma, compared with each test alone
  • For PD-L1 staining on cell blocks, at least 100 tumor cells must be evaluated
• The authors' conclusion: The authors concluded that stains on effusion samples need analytical validation as recommended by the guidelines. They added that in cytology, errors linked to fixation can contribute to risk to patient safety.

Topics

Medical terms in this study, explained in our Lexicon: Cytology, Immunohistochemistry, Prognosis, Cell Block, BAP1, CDKN2A, Fluorescence In Situ Hybridization (FISH), MTAP.

Related studies

• Usefulness of methylthioadenosine phosphorylase and BRCA-associated protein 1 immunohistochemistry in the diagnosis of malignant mesothelioma in effusion cytology specimens — K.B. Berg, A.M. Churg, et al. (2020)
• Utility of BAP1 Immunohistochemistry and p16 (CDKN2A) FISH in the Diagnosis of Malignant Mesothelioma in Effusion Cytology Specimens — H.C. Hwang, B.S. Sheffield, et al. (2016)
• Evaluation of androgen receptor and GATA binding protein 3 as immunohistochemical markers in the diagnosis of metastatic breast carcinoma to the lung — Y. Hattori, A. Yoshida, et al. (2015)
• The diagnostic utility of reduced immunohistochemical expression of SMARCB1 in synovial sarcomas: a validation study — J. Ito, N. Asano, et al. (2016)
• EZH2, a unique marker of malignancy in effusion cytology — H. Jiang, R. Gupta, et al. (2014)
• Sarcomatoid Mesothelioma: A CDKN2A molecular analysis of 53 cases with immunohistochemical correlation with BAP1 — M. Zaleski, N. Kalhor, et al. (2020)

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