Principles of Analytic Validation of Immunohistochemical Assays: Guideline Update.

CONTEXT.­: In 2014, the College of American Pathologists developed an evidence-based guideline to address analytic validation of immunohistochemical assays. Fourteen recommendations were offered. Per the National Academy of Medicine standards for developing trustworthy guidelines, guidelines should be updated when new evidence suggests modifications. OBJECTIVE.­: To assess evidence published since the release of the original guideline and develop updated evidence-based recommendations. DESIGN.­: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and update the original guideline recommendations using the Grading of Recommendations Assessment, Development and Evaluation approach. RESULTS.­: Two strong recommendations, 1 conditional recommendation, and 12 good practice statements are offered in this updated guideline. They address analytic validation or verification of predictive and nonpredictive assays, and recommended revalidation procedures following changes in assay conditions. CONCLUSIONS.­: While many of the original guideline statements remain similar, new recommendations address analytic validation of assays with distinct scoring systems, such as programmed death receptor-1 and analytic verification of US Food and Drug Administration approved/cleared assays; more specific guidance is offered for validating immunohistochemistry performed on cytology specimens.

Markers of metastatic carcinoma of breast origin.

This review summarizes the three major breast-associated markers that can be of assistance in evaluating metastatic carcinomas for which a breast primary diagnosis is entertained. These markers include gross cystic disease fluid protein-15 (GCDFP-15), mammaglobin, and GATA3. The first two are cytoplasmic markers that show comparable sensitivities for breast cancer, although relatively few of the published studies have employed the same antibodies against the target molecule, making direct comparisons challenging. GATA3 is a nuclear transcription factor that shows superior sensitivity to GCDFP-15 and mammaglobin. However, the specificity of GATA3 can pose challenges, inasmuch as carcinomas of the bladder and other sites can show significant levels of positivity. Determination of the optimal panel of antibodies employed in a given clinical setting will thus depend on the non-breast tumours included in the differential diagnosis.

Practical Applications in Immunohistochemistry: Carcinomas of Unknown Primary Site.

CONTEXT: -Identification of the site of origin of carcinoma of unknown primary using immunohistochemistry is a frequent requirement of anatomic pathologists. Diagnostic accuracy is crucial, particularly in the current era of targeted therapies and smaller sample sizes. OBJECTIVES: -To provide practical guidance and suggestions for classifying carcinoma of unknown primary using both proven and new antibodies, as well as targeting panels based on integration of morphologic and clinical features. DATA SOURCES: -Literature review, the authors' practice experience, and authors' research. CONCLUSIONS: -With well-performed and interpreted immunohistochemistry panels, anatomic pathologists can successfully identify the site of origin of carcinoma of unknown primary. It is crucial to understand not only the diagnostic uses of the many available antibodies but also the potential limits and pitfalls.

Comparative Sensitivities and Specificities of Antibodies to Breast Markers GCDFP-15, Mammaglobin A, and Different Clones of Antibodies to GATA-3: A Study of 338 Tumors Using Whole Sections.

GATA-3 is a transcription factor that has recently been identified by immunohistochemistry to be highly expressed in urothelial and breast carcinomas (CAs). We sought to determine the potential utility of GATA-3 in identifying metastatic breast CA, and to compare its utility with the standard breast markers, GCDFP-15, and mammaglobin A. We identified an archival series of 338 formalin-fixed paraffin-embedded whole-tissue sections of various CAs. Using standard immunohistochemical (IHC) techniques we used mouse monoclonal antibodies to GATA-3 (clones L50-823, HG3-31), GCDFP-15 (23A3), and mammaglobin A (31A5). Both clones of GATA-3 showed positivity in 96% of non-triple-negative breast carcinomas (TNBCs), L50-823 and HG3-31, demonstrating expression in 87% and 63% of TNBCs, respectively; GCDFP-15 and mammaglobin A were expressed in 69% and 61% of non-TNBCs, respectively, and 10% and 17%, of TNBCs, respectively. The L50-823 clone manifested a lower specificity in identifying breast CAs (84%) than did the HG3-31 clone (97%). Both monoclonal antibodies to GATA-3 are very sensitive reagents for the identification of breast CA, surpassing antibodies to GCDFP-15 and mammaglobin A, and offer a significant improvement in identifying TNBCs. However, the L50-823 clone showed a lower level of specificity, which may qualify its utility in the setting of CAs of unknown primary.

The lung-restricted marker napsin A is highly expressed in clear cell carcinomas of the ovary.

OBJECTIVES: We recently observed expression of the "lung" marker napsin A in ovarian clear cell carcinomas and therefore sought to determine the extent of napsin A expression in a subset of ovarian neoplasms. METHODS: We identified an archival series of ovarian clear cell carcinomas (n = 36), serous borderline tumors (n = 21), high-grade serous carcinomas (n = 37), and endometrioid adenocarcinomas (n = 29). Using standard immunohistochemical techniques on whole sections of formalin-fixed, paraffin-embedded specimens, we employed a panel of antibodies: napsin A (IP64), estrogen receptor (SP1), WT-1 (6F-H2), PAX-8 (BC12), and TTF-1 (SPT24). RESULTS: Thirty-six of 36 clear cell carcinomas showed napsin A expression, typically in a uniform pattern. None of the serous borderline tumors or high-grade serous carcinomas manifested napsin A expression. Napsin A was expressed in three (10%) of 29 endometrioid adenocarcinomas, generally in a focal pattern. CONCLUSIONS: Our study showed that napsin A is an extremely sensitive (100%) marker of ovarian clear cell carcinomas and exhibits very high specificity (100%) in distinguishing clear cell carcinomas from high-grade serous carcinomas and serous borderline tumors and 90% specificity in discriminating clear cell carcinomas from endometrioid carcinomas.

Determining true HER2 gene status in breast cancers with polysomy by using alternative chromosome 17 reference genes: implications for anti-HER2 targeted therapy.

PURPOSE: The ratio of human epidermal growth factor receptor 2 (HER2) to CEP17 by fluorescent in situ hybridization (FISH) with the centromeric probe CEP17 is used to determine HER2 gene status in breast cancer. Increases in CEP17 copy number have been interpreted as representing polysomy 17. However, pangenomic studies have demonstrated that polysomy 17 is rare. This study tests the hypothesis that the use of alternative chromosome 17 reference genes might more accurately assess true HER2 gene status. PATIENTS AND METHODS: In all, 171 patients with breast cancer who had HER2 FISH that had increased mean CEP17 copy numbers (> 2.6) were selected for additional chromosome 17 studies that used probes for Smith-Magenis syndrome (SMS), retinoic acid receptor alpha (RARA), and tumor protein p53 (TP53) genes. A eusomic copy number exhibited in one or more of these loci was used to calculate a revised HER2-to-chromosome-17 ratio by using the eusomic gene locus as the reference. RESULTS: Of 132 cases classified as nonamplified on the basis of their HER2:CEP17 ratios, 58 (43.9%) were scored as amplified by using alternative chromosome 17 reference gene probes, and 13 (92.9%) of 14 cases scored as equivocal were reclassified as amplified. Among the cases with mean HER2 copy number of 4 to 6, 41 (47.7%) of 86 had their HER2 gene status upgraded from nonamplified to amplified, and four (4.7%) of 86 were upgraded from equivocal to amplified. CONCLUSION: Our results support the findings of recent pangenomic studies that true polysomy 17 is uncommon. Additional FISH studies that use probes to the SMS, RARA, and TP53 genes are an effective way to determine the true HER2 amplification status in patients with polysomy 17 and they have important potential implications for guiding HER2-targeted therapy in breast cancer.

High concordance between immunohistochemistry and fluorescence in situ hybridization testing for HER2 status in breast cancer requires a normalized IHC scoring system.

The American Society of Clinical Oncologists and College of American Pathologists have recently released new guidelines for laboratory testing of HER2 status in breast cancer, which require high levels (95%) of concordance between immunohistochemistry positive (3+) and fluorescence in situ hybridization-amplified cases, and between immunohistochemistry negative (0/1+) and fluorescence in situ hybridization-nonamplified cases; these required levels of concordance are significantly higher than those found in most published studies. We tested the hypothesis that a modification of the HER2 immunohistochemistry scoring system could significantly improve immunohistochemistry and fluorescence in situ hybridization concordance. A total of 6604 breast cancer specimens were evaluated for HER2 status by both immunohistochemistry and fluorescence in situ hybridization using standard methodologies. Results were compared when the standard immunohistochemistry scoring system was replaced by a normalized scoring system in which the HER2 score was derived by subtracting the score on the non-neoplastic breast epithelium from that on the tumor cells. Among the 6604 tumors, using a non-normalized immunohistochemistry scoring system, 267/872 (30.6%) of the immunohistochemistry 3+ cases proved to be fluorescence in situ hybridization nonamplified, whereas using the normalized scoring system only 30/562 (5.3%) of immunohistochemistry 3+ cases proved to be 'false positive'. The concordance rate between immunohistochemistry 3+ and fluorescence in situ hybridization-amplified cases using the normalized scoring method was 94.7%, whereas the concordance using the non-normalized method was only 69.4%. Extremely high concordance between immunohistochemistry and fluorescence in situ hybridization assessment of HER2 status in breast cancer is achievable, but to attain this high level of concordance, modification of the FDA-approved immunohistochemistry scoring system is required.