The presence of estrogen receptors (ERs), as detected by immunohistochemistry (IHC), is a weak prognostic marker of clinical outcome in breast cancer, but a strong predictive marker for response, for ...example, to tamoxifen-based therapy. As with all IHC markers, factors such as tissue fixation (both type and duration), the choice of antibody, and the threshold for interpretation of positive immunostaining can dramatically affect test accuracy and reproducibility. For example, optimal fixation for detection of ER requires at least 6-8 h in formalin, and the use of newer antibodies such as SP1 may identify additional patients who might benefit from hormonal therapy. Although the threshold for positivity may be as few as 1% of tumor cells showing nuclear signal, recent studies appear to demonstrate a dichotomization of ER IHC, with the vast majority of cases showing all positive or all negative results. This may be helpful in dictating the appropriateness of hormonal therapy, but quantification of ER by IHC, or other methods, may play a more important role in the future. Breast cancers with human epidermal receptor protein-2 (c-erbB-2; HER2) alterations are critical to identify because such tumors require unique treatment, including the use of targeted therapies such as trastuzumab. HER2 alterations at the DNA (amplification) and protein (overexpression) level usually occur in concert, and both fluorescence in situ hybridization (FISH) or IHC can be accurate methods to assess these alterations. However, recent studies have suggested that serious reproducibility issues exist in both FISH and IHC HER2 studies. To address this, a joint committee of both the American Society for Clinical Oncologists and the College of American Pathologists has promulgated new guidelines for HER2 testing. These include the following: (a) recommendations for tissue fixation for more than 6 and less than 48 h; (b) new scoring criteria, including a new threshold of 30% strong immunostaining for classification of 3+; (c) introduction of the term 'equivocal' to characterize HER2 studies that are 2+ by IHC and/or show HER2/chromosome 17 ratios of between 1.8 and 2.2 by FISH; (d) requirements for laboratories to validate HER2 assays, generally through the cross-testing of cases with another HER2 methodology, with laboratories required to attain 95% concordance for both positive and negative tests; (e) participation in HER2 proficiency testing.
- Malignant mesothelioma (MM) is an uncommon tumor that can be difficult to diagnose.
- To provide updated, practical guidelines for the pathologic diagnosis of MM.
- Pathologists involved in the ...International Mesothelioma Interest Group and others with an interest and expertise in the field contributed to this update. Reference material included up-to-date, peer-reviewed publications and textbooks.
- There was discussion and consensus opinion regarding guidelines for (1) distinguishing benign from malignant mesothelial proliferations (both epithelioid and spindle cell lesions), (2) cytologic diagnosis of MM, (3) recognition of the key histologic features of pleural and peritoneal MM, (4) use of histochemical and immunohistochemical stains in the diagnosis and differential diagnosis of MM, (5) differentiating epithelioid MM from various carcinomas (lung, breast, ovarian, and colonic adenocarcinomas, and squamous cell and renal cell carcinomas), (6) diagnosis of sarcomatoid MM, (7) use of molecular markers in the diagnosis of MM, (8) electron microscopy in the diagnosis of MM, and (9) some caveats and pitfalls in the diagnosis of MM. Immunohistochemical panels are integral to the diagnosis of MM, but the exact makeup of panels employed is dependent on the differential diagnosis and on the antibodies available in a given laboratory. Depending on the morphology, immunohistochemical panels should contain both positive and negative markers for mesothelial differentiation and for lesions considered in the differential diagnosis. Immunohistochemical markers should have either sensitivity or specificity greater than 80% for the lesions in question. Interpretation of positivity generally should take into account the localization of the stain (eg, nuclear versus cytoplasmic) and the percentage of cells staining (>10% is suggested for cytoplasmic and membranous markers). Selected molecular markers are now being used to distinguish benign from malignant mesothelial proliferations. These guidelines are meant to be a practical diagnostic reference for the pathologist; however, some new pathologic predictors of prognosis and response to therapy are also included.
-There are a number of critical factors that can lead to incorrect results if the diagnostic pathologist performing immunohistochemistry is unaware of, or not vigilant about, their influence.
-To ...highlight 3 arenas in which errors may be introduced.
-For choosing the correct primary antibody, selection of the most appropriate antibodies for a given clinical application can be aided by obtaining information from the vendor; however, this can yield incomplete information. There are a number of online databases that have comparisons of antibodies from different vendors, particularly with respect to their use and properties. Reading the published literature can assist in this process, particularly with respect to determining antibody sensitivity and specificity, but it is a daunting task to keep up with all of the immunohistochemistry-related papers published. Finally, Web sites of a number of quality assurance organizations are accessible and can provide a wealth of information comparing the "real world" performance characteristics of different antibodies to the same target protein. False-positive signals can result from a number of factors, including the use of inappropriately high antibody concentration, and "pseudospecific" signal that is in the wrong compartment of the cell. False-negative signal can result from factors such as use of a nonoptimized epitope retrieval method. It is critical that epitope retrieval methods be optimized for each antibody employed in the laboratory.
-By paying attention to these potential problems, the "black box" of diagnostic immunohistochemistry can be made more transparent.
A variety of immunohistochemical (IHC) stains have been proposed to mark either benign or malignant mesothelial proliferations. Loss of the p16 tumor suppressor (CDKN2A), through homozygous deletions ...of 9p21, is a good marker of mesotheliomas but lacks sensitivity. Recent reports indicate that some mesotheliomas are associated with loss of BRCA-associated protein 1 (BAP1) expression. Here we investigate BAP1 and p16 as potential markers of malignancy and compare test characteristics with previously proposed markers using a well-characterized tissue microarray. BAP1 protein expression was interrogated by IHC. The p16 locus was examined by fluorescence in situ hybridization (FISH) directed toward chromosome 9p21. Loss of BAP1 was identified in 7/26 mesotheliomas and 0/49 benign proliferations. Loss of p16 was identified in 14/27 mesotheliomas and 0/40 benign proliferations, yielding 100% specificity and positive predictive value for each marker. Together, BAP1 IHC and p16 FISH were 58% sensitive for detecting malignancy. Various combinations of p53, EMA, IMP3, and GLUT1 showed reasonably high specificity (96% to 98%) but poor to extremely poor sensitivity. Combined BAP1 IHC/p16 FISH testing is a highly specific method of diagnosing malignant mesotheliomas when the question is whether a mesothelial proliferation is benign or malignant and is particularly useful when tissue invasion by mesothelial cells cannot be demonstrated. However, combined BAP1/p16 FISH testing is not highly sensitive, and negative results do not rule out a mesothelioma. The test characteristics of previously proposed markers EMA, p53, GLUT1, IMP3 suggest that, even in combination, these markers are not useful tools in this clinical setting.
The diagnosis of malignant mesothelioma in effusion cytology specimens is controversial. BAP1 immunohistochemistry and p16 fluorescence in situ hybridization (FISH) have recently been reported as ...reliable markers of malignancy in biopsies of mesothelioma. To determine whether these markers, singly or in combination, might also be useful in effusion cytology specimens, we examined 15 biopsies of epithelial mesotheliomas and 3 benign mesothelial reactions and corresponding effusion cytology paraffin-embedded cell blocks. Four cytology specimens were too scanty for p16 FISH analysis but were interpretable for BAP1 immunohistochemistry. Overall, loss of BAP1 and/or deletion of p16 was seen in 11/11 (100%) of matched cytology and tissue biopsy specimens. BAP1 loss alone was seen in 10/15 (67%) biopsies and 10/15 (67%) cytology specimens. Homozygous deletion of p16 by FISH was found in 12/15 (80%) biopsy specimens and 8/11 (73%) evaluable cytology specimens. Seven of 15 (47%) biopsies and 5/11 (42%) cytology specimens showed loss of both markers. All mesothelioma biopsy/cytology pairs showed exactly the same pattern of BAP1 or p16 retention or loss in the biopsy and cytology specimens. The 2 peritoneal mesothelioma cases demonstrated loss of BAP1 but not p16. None of the benign mesothelial reactions or corresponding cytology specimens showed loss of either marker. We conclude that both BAP1 immunohistochemistry and p16 FISH analysis provide reliable markers of mesothelial malignancy in effusion cytology specimens, especially where the atypical mesothelial proliferation is well sampled. BAP1 is easier to interpret with scanty specimens. On the basis of small numbers of cases, use of both markers appears to increase sensitivity.
Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring ...methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.
The separation of sarcomatous and desmoplastic mesotheliomas from benign organizing pleuritis can be morphologically very difficult. Deletion of p16 (CDKN2A) by fluorescence in situ hybridization ...(FISH) testing appears to be a reliable marker of malignancy in mesothelial proliferations, and more recently it has been reported that, in this setting, loss of BAP1 by immunohistochemistry is only seen in malignant mesotheliomas. To determine how useful these tests are with sarcomatous and desmoplastic mesotheliomas, we examined 20 such tumors. Loss of BAP1 was seen in 3/20 (15%) and deletion of p16 by FISH was seen in 16/20 (80%) cases. Loss of one or the other marker was observed in 17/20 (85%). We also examined 13 sarcomatoid carcinomas, an important differential diagnosis of sarcomatoid mesotheliomas, and found that BAP1 was never lost, but p16 was deleted in 3/11 (27%). We conclude that: (1) BAP1 immunohistochemistry is relatively insensitive in the context of sarcomatous and desmoplastic mesotheliomas, but as a matter of time and cost efficiency may nonetheless be a useful first approach to the problem; (2) deletion of p16 by FISH is considerably more sensitive, but there remain a proportion of cases in which p16 is not deleted; (3) a small improvement in sensitivity can be achieved by using both markers; (4) in the context of a spindle cell malignant tumor in the pleura or peritoneum, which morphologically might be a metastatic sarcomatoid carcinoma or a mesothelioma, the finding of BAP1 loss favors mesothelioma, but p16 FISH cannot be used to separate sarcomatous mesotheliomas from sarcomatoid carcinomas.
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.
The histologic subtype of non-small cell lung carcinoma is important in selecting appropriate chemotherapy for patients with advanced disease. As many of these patients are not operative candidates, ...they are treated medically after biopsy for diagnosis. Inherent limitations of small biopsy samples can make distinguishing poorly differentiated lung adenocarcinoma (ADC) from squamous cell carcinoma (SCC) difficult. The value of histochemical and immunohistochemical markers to help separate poorly differentiated ADC from SCC in resection specimens is well established; however, the optimal use of markers in small tissue samples has only recently been examined and the correlation of marker expression in small tissue samples with histologic subtype determined on resection specimens has not been well documented. We address this issue by examining the expression of 9 markers (p63, TTF1, CK5/6, CK7, 34βE12, Napsin A, mucicarmine, NTRK1, and NTRK2) on 200 cases of ADC and 225 cases of SCC in tissue microarray format to mimic small tissue specimens. The single best marker to separate ADC from SCC is p63 (for SCC: sensitivity 84%, specificity 85%). Logistic regression analysis identifies p63, TTF1, CK5/6, CK7, Napsin A, and mucicarmine as the optimal panel to separate ADC from SCC. Reduction of the panel to p63, TTF1, CK5/6, and CK7 is marginally less effective but may be the best compromise when tissue is limited. We present an algorithm for the stepwise application of p63, TTF1, CK5/6, CK7, Napsin A, and mucicarmine in situations in which separation of ADC from SCC in small specimens cannot be accomplished by morphology alone.
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