Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practice.

Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray.

Programmed death 1 ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index tissue microarray (TMA) to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index TMA containing a panel of ten isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142 FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other four assays. The assays for 22C3 FDA, 28-8-FDA, SP263 FDA, and E1L3N LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.

SATB2 protein expression by immunohistochemistry is a sensitive and specific marker of appendiceal and rectosigmoid well differentiated neuroendocrine tumours.

AIMS: Neuroendocrine neoplasms (NNs) range from well to poorly differentiated and indolent to highly aggressive. The site of origin in metastatic NNs has therapeutic and prognostic implications. SATB2 is a transcriptional regulator involved in osteoblastic and neuronal differentiation and is a sensitive and specific marker of colorectal epithelium. This study aimed to evaluate the expression of SATB2 in NNs from various primary sites and its utility as a marker in determining the site of origin of these neoplasms. METHODS AND RESULTS: SATB2 immunohistochemistry was performed on 266 NNs, including lung small cell carcinomas (n = 39) and carcinoids (n = 30), bladder (n = 21) and prostate (n = 31) small cell carcinomas, and gastrointestinal (GI)/pancreatic NNs of various primary sites (n = 145) consisting of well-differentiated neuroendocrine tumours (WDNET)s (n = 124) and poorly differentiated neuroendocrine carcinomas (PDNEC)s (n = 21). SATB2 was expressed in prostatic (10 of 31, 32%) and bladder (eight of 21, 38%) small cell carcinomas, lung carcinoid tumours (one of 30, 3%), and lung small cell carcinomas (eight of 39, 21%). Among primary GI NNs, SATB2 was expressed in 37 of 124 (30%) WDNETs and four of 21 (19%) PDNECs. Of the former, 15 of 15 (100%) rectal/rectosigmoid and 22 of 22 (100%) appendiceal neoplasms expressed SATB2. Using receiver operator characteristic analysis, SATB2 was a sensitive and specific marker for rectal (100.0%, 80.0%) and appendiceal (100.0%, 84.5%) WDNETs, respectively. CONCLUSIONS: In summary, SATB2 is a sensitive and specific marker for rectal/rectosigmoid and appendiceal WDNETs, and may represent a useful diagnostic tool when these sites of origin are considered in the differential diagnosis.

Analytical validation of a standardised scoring protocol for Ki67 immunohistochemistry on breast cancer excision whole sections: an international multicentre collaboration.

AIMS: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. METHODS AND RESULTS: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot-spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799-0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot-spot method (0.83; 95% CI = 0.74-0.90) did not. Visually, interobserver concordance in location of selected hot-spots varies between cases. The median times for scoring were 9 and 6 min for global and hot-spot methods, respectively. CONCLUSIONS: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further.

Concordance study of PD-L1 expression in primary and metastatic bladder carcinomas: comparison of four commonly used antibodies and RNA expression.

Therapy with anti-PD-L1 immune check-point inhibitors is approved for several cancers, including advanced urothelial carcinomas. PD-L1 prevalence estimates vary widely in bladder cancer, and lack of correlation between expression and clinical outcomes and immunotherapy response may be attributed to methodological differences of the immunohistochemical reagents and procedures. We characterized PD-L1 expression in 235 urothelial carcinomas including 79 matched pairs of primary and metastatic cancers using a panel of four PD-L1 immunoassays in comparison with RNAscope assay using PD-L1-specific probe (CD274). The antibody panel included three FDA-approved clones (22C3 for pembrolizumab, 28.8 for nivolumab, SP142 for atezolizumab), and a commonly used clone E1L3N. Manual scoring of tissue microarrays was performed in each of 235 tumors (624 tissue cores) and compared to an automated image analysis. Expression of PD-L1 in tumor cells by ≥1 marker was detected in 41/142 (28.9%) primary tumors, 13/77 (16.9%) lymph nodes, and 2/16 (12.5%) distant metastases. In positive cases, high PD-L1 expression (>50% cells) was detected in 34.1% primary and 46.7% metastases. Concordant PD-L1 expression status was present in 71/79 (89.9%) cases of matched primary and metastatic urothelial carcinomas. PD-L1 sensitivity ranked from highest to lowest as follows: RNAscope, clone 28.8, 22C3, E1L3N, and SP142. Pairwise concordance correlation coefficients between the four antibodies in 624 tissue cores ranged from 0.76 to 0.9 for tumor cells and from 0.30 to 0.85 for immune cells. RNA and protein expression levels showed moderate to high agreement (0.72-0.87). Intra-tumor expression heterogeneity was low for both protein and RNA assays (interclass correlation coefficients: 0.86-0.94). Manual scores were highly concordant with automated Aperio scores (0.94-0.97). A significant subset of 56/235 (23.8%) urothelial carcinomas stained positive for PD-L1 with high concordance between all four antibodies and RNA ISH assay. Despite some heterogeneity in staining, the overall results are highly concordant suggesting diagnostic equivalence of tested assays.

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.

An international study to increase concordance in Ki67 scoring.

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.

PAX8 expression in uterine adenocarcinomas and mesonephric proliferations.

PAX8 is a useful immunohistochemical marker for the diagnosis of gynecologic tract malignancies. Several studies have described PAX8 expression in a wide variety of epithelial neoplasms, including ovarian and endometrial carcinomas. The goal of this study was to evaluate PAX8 expression in various types of uterine adenocarcinomas and mesonephric proliferations. Ninety-four cases of uterine adenocarcinomas (52 endometrial endometrioid carcinomas, 21 endometrial serous carcinomas, and 21 human papillomavirus-related endocervical carcinomas), 11 cases of benign mesonephric proliferations (remnants/hyperplasia), and normal endometrial and endocervical glandular epithelium in 58 cases were studied. Immunohistochemical staining was performed with the rabbit polyclonal anti-PAX8 antibody. All adenocarcinoma groups demonstrated a high frequency of PAX8 expression but with relatively high variability in the extent of staining among different subtypes. Both serous carcinomas and endometrioid carcinomas were positive in most cases (95% and 96%, respectively), but serous carcinomas displayed a significantly higher level of expression (immunohistochemical composite scores based on combined extent and intensity of expression) compared with endometrioid carcinomas (mean immunohistochemical composite scores: 8.3 vs. 5.3, respectively; P<0.006). Endocervical adenocarcinomas also had a high frequency of PAX8 expression (86% of cases), but the level of expression was significantly less than that of endometrial adenocarcinomas (mean immunohistochemical composite scores: 2.9 vs. 5.3-8.3, respectively; P<0.004). Among benign glandular epithelia, normal endocervical glands exhibited a significantly lower level of expression compared with either normal endometrial glands or benign mesonephric proliferations (mean immunohistochemical composite scores: 2.6 vs. 6.6-11.2, respectively; P<0.0006). We conclude that PAX8 is expressed in the vast majority of uterine adenocarcinomas, including those of both endometrial and endocervical origin, and that the level of expression based on combined extent and intensity is highest in endometrial serous carcinoma and lowest in endocervical adenocarcinoma. However, the high prevalence of PAX8 expression in the various types of uterine adenocarcinomas precludes use of this marker for distinguishing these tumors. In extrauterine sites, PAX8 can serve as a useful marker for adenocarcinomas of uterine origin (also positive in the majority of ovarian carcinomas), being most sensitive for identification of endometrial adenocarcinomas (both serous and endometrioid). The sensitivity for identifying metastatic endocervical adenocarcinomas is likely less and dependent on the degree to which the significantly lower extent of expression in these tumors is maintained in metastatic sites.

PAX8 expression in uterine malignant mesodermal mixed tumor (carcinosarcoma).

PAX8 has emerged as a useful immunohistochemical marker for epithelial neoplasms of gynecologic origin. Expression of PAX8 in uterine malignant mesodermal mixed tumors (MMMT, carcinosarcoma) has not been characterized in detail. The goal of this study is to evaluate PAX8 expression in uterine MMMTs, with particular attention to its distribution in specific tumor components. Thirty-seven cases were studied. PAX8 expression was assessed by immunohistochemistry and scored separately in the epithelial and mesenchymal components of the tumors. The extent of staining was scored based on the estimated percentage of positive tumor cells as 1+: 1% to 25%; 2+: 26% to 50%; 3+: 51% to 75%; 4+: 76% to 100%. The epithelial component expressed PAX8 in all but 1 tumor, with 92% of tumors displaying 3+ and 4+ extent of staining. The mesenchymal component lacked PAX8 expression in 27 cases (73%) with variable expression in the remaining 10 cases. In addition, 12 tumors contained undifferentiated areas that were not readily classifiable as carcinoma or sarcoma based on morphologic features. Of these, 8 (67%) were negative for PAX8, whereas 4 (33%) demonstrated variable extent of expression. Thus, PAX8 is expressed in the carcinomatous components of nearly all uterine MMMTs (97%), with expression in sarcomatous and undifferentiated components being less common and less extensive. The uniform, extensive expression in the carcinomatous components makes PAX8 a useful marker for diagnosis of carcinomatous metastases of uterine MMMT at extrauterine sites. Its infrequent expression in the sarcomatous and undifferentiated components limits its utility in identifying sarcoma-predominant metastases as gynecologic in origin.

A Pilot Validation Study Comparing Fluorescence-Imitating Brightfield Imaging, A Slide-Free Imaging Method, With Standard Formalin-Fixed, Paraffin-Embedded Hematoxylin-Eosin-Stained Tissue Section Histology for Primary Surgical Pathology Diagnosis.

CONTEXT.­: Digital pathology using whole slide images has been recently approved to support primary diagnosis in clinical surgical pathology practices. Here we describe a novel imaging method, fluorescence-imitating brightfield imaging, that can capture the surface of fresh tissue without requiring prior fixation, paraffin embedding, tissue sectioning, or staining. OBJECTIVE.­: To compare the ability of pathologists to evaluate direct-to-digital images with standard pathology preparations. DESIGN.­: One hundred surgical pathology samples were obtained. Samples were first digitally imaged, then processed for standard histologic examination on 4-µm hematoxylin-eosin-stained sections and digitally scanned. The resulting digital images from both digital and standard scan sets were viewed by each of 4 reading pathologists. The data set consisted of 100 reference diagnoses and 800 study pathologist reads. Each study read was compared to the reference diagnosis, and also compared to that reader's diagnosis across both modalities. RESULTS.­: The overall agreement rate, across 800 reads, was 97.9%. This consisted of 400 digital reads at 97.0% versus reference and 400 standard reads versus reference at 98.8%. Minor discordances (defined as alternative diagnoses without clinical treatment or outcome implications) were 6.1% overall, 7.2% for digital, and 5.0% for standard. CONCLUSIONS.­: Pathologists can provide accurate diagnoses from fluorescence-imitating brightfield imaging slide-free images. Concordance and discordance rates are similar to published rates for comparisons of whole slide imaging to standard light microscopy of glass slides for primary diagnosis. It may be possible, therefore, to develop a slide-free, nondestructive approach for primary pathology diagnosis.

Immunohistochemical characterization of squamous differentiation and morular metaplasia in uterine endometrioid adenocarcinoma.

Squamous differentiation (SD) and morular metaplasia (MM) are frequently present in uterine endometrioid adenocarcinoma (EAC) and can mimic areas of solid tumor. We used immunohistochemical stains to further characterize these lesions, and to determine which markers would help to distinguish these metaplasias from areas of solid growth in EAC. The pathology database was searched for diagnoses of EAC from 1997 to 2007, the hematoxylin and eosin-stained slides were reviewed, and 143 cases with SD, MM, or both (SD+MM) were identified. A panel of immunohistochemical stains was performed. In particular, we were interested in PAX2 and PAX8, recently studied markers of Müllerian tissue as potential markers for differentiation of metaplasias and tumor. In addition, estrogen receptor and progesterone receptor, and Her-2/neu, were examined to determine whether there was a differential expression between the metaplasias and solid tumor that may be diagnostically useful. In addition, to further characterize MM and SD, bcl-2 as a marker of cell regulation and inhibition of apoptosis, p16 as a surrogate marker for human papillomavirus, and p63 as a marker of mature SD were studied. Adjacent normal endometrium (NEM), when present, and 20 EAC cases (FIGO Grades 1-3) without SD or MM served as controls. PAX2 was positive in NEM (58/61, 95%) and was lost in SD (15/136, 11%), MM (1/25, 4%), and EAC (57/163, 35%), whereas PAX8 was positive in all NEM (61/61, 100%) and in the majority of SD (125/136, 92%), MM (19/25, 73%), and EAC (162/163, 99%). The estrogen receptor and the progesterone receptor were expressed by the majority of EAC (148/163, 91% and 144/163, 88%, respectively), whereas both were markedly diminished in SD (56/136, 41% and 58/136, 43%) and MM (4/25, 16% and 2/25, 8%). Approximately half of the MM was positive for bcl-2 (12/25, 48%), making it an unreliable marker. Her-2/neu was negative in all cases (0%). p16 was patchy in SD (111/136, 82%), MM (22/25, 88%), and EAC (154/163, 94%), whereas p63 was predominantly positive only in SD (96/136, 71%). Estrogen receptor and progesterone receptor, PAX2, and PAX8 were helpful in differentiating MM from SD, EAC, or NEM (P<0.05). In addition, p63 distinguished between SD and MM, supporting the theory that morules do not show characteristic mature SD.

Coordinated expression of galectin-3 and caveolin-1 in thyroid cancer.

Galectin-3 (Gal3) is the single most accurate marker for the diagnosis of differentiated thyroid cancer (DTC). Gal3 overrides the tumour suppressor activity of caveolin-1 (Cav1) and functions in concert with Cav1 to promote focal adhesion turnover and tumour cell migration and invasion. To study their coordinated role in progression of a human cancer, we investigated the expression of Gal3 and Cav1 in specimens of human benign thyroid lesions, DTC and anaplastic thyroid cancer (ATC). Gal3 and Cav1 expression is significantly associated with DTC and ATC, but not benign nodules. Essentially all Cav1-positive DTC cancers express Gal3, supporting the synergistic activity of these two proteins in DTC progression. Similarly, coordinated elevated Gal3/Cav1 expression was observed in three DTC-derived cell lines (papillary TCP1 and KTC1 and follicular FTC133) but only one (ACT1) of five ATC-derived cell lines. Using siRNA knockdown, Gal3 and Cav1 were shown to be required for RhoA GTPase activation, stabilization of focal adhesion kinase (FAK; a measure of focal adhesion signalling and turnover) and increased migration of the DTC cell lines studied, but not the ATC cell lines, including ACT1, which expresses elevated levels of Gal3 and Cav1. Co-expression of Gal3 and Cav1 in the T238 anaplastic cell line stabilized FAK-GFP in focal adhesions. Gal3 and Cav1 therefore function synergistically to promote focal adhesion signalling, migration and progression of DTC.

The Biomarker Ki-67: Promise, Potential, and Problems in Breast Cancer.

Ki-67 is a nuclear protein serendipitously discovered by monoclonal antibody selection in the early 1980s. While it has been applied for decades in the context of breast cancer as a putative prognostic and, more recently, predictive, biomarker, even after all this time there is incomplete agreement as to the validity of the immunohistochemical assays employed for Ki-67 assessment, given possible effects of the disparate methodologies employed and possible confounding preanalytical, analytical, and interpretive variables. In this brief review, the history of Ki-67 and the problems, particularly with the analytical and interpretive variables, are highlighted through a selective review of the published literature. The contributions of the International Ki-67 Breast Cancer Working Group are highlighted, and in particular, the recommendations made by this group are reviewed. The potential of Ki-67 as a biomarker for breast cancer has not yet been fully realized, but an understanding of the power as well as the limitations of the methods of Ki-67 assessment are important if this biomarker can realize its potential.

Diagnostic utility of galectin-3 in thyroid cancer.

Galectin-3 (Gal-3), which has received significant recent attention for its utility as a diagnostic marker for thyroid cancer, represents the most well-studied molecular candidate for thyroid cancer diagnosis. Gal-3 is a protein that binds to beta-galactosidase residues on cell surface glycoproteins and has also been identified in the cytoplasmic and nuclear compartment. This marker has been implicated in regulation of normal cellular proliferation and apoptosis, as well as malignant transformation and the metastasis of cancer cells. We here present a mechanistic review of Gal-3 and its role in cancer development and progression. Gal-3 expression studies in thyroid tissue and cytologic tumor specimens and their methodological considerations are also discussed in this article. Despite great variance in their methodology, the majority of immunohistochemical studies found that Gal-3 was differentially expressed in thyroid carcinoma compared with benign and normal thyroid specimens, suggesting that Gal-3 is a good diagnostic marker for thyroid cancer. Recent studies have also demonstrated improved methodological reliability. On the other hand, Gal-3 genomic expression studies have shown inconsistent results for diagnostic utility and are not recommended. Overall, the development of Gal-3 as a diagnostic marker for thyroid cancer represents a promising avenue for future study, and its clinical application could significantly reduce the number of diagnostic thyroid operations performed for cases of indeterminant fine needle aspiration biopsy cytology, and thus positively impact the current management of thyroid nodular disease.

Phosphorylated transducer and activator of transcription-3 (pSTAT3) immunohistochemical expression in paired primary and metastatic colorectal cancer.

BACKGROUND: Signal Transducer and Activator of Transcription-3 (STAT3) mediates cellular functions. We assessed the IHC expression of phosphorylated STAT3 (pSTAT3) in paired primary tumors and liver metastases in patients with advanced stage colorectal cancer (CRC). METHODS: We included patients with tissue blocks available from both the primary CRC and a surgically resected liver metastasis. The IHC pSTAT3 expression agreement was measured using Cohen's kappa statistic. RESULTS: The study included 103 patients, 55% male, median age was 64. 43% tumors originated in rectum, and 63% of the primary tumors were synchronous. Expression of pSTAT3 was 76% in liver metastases and 71% in primary tumors. A difference in pSTAT3 staining between the primary tumor and liver metastases was noted in 64%. There was lost expression of pSTAT3 in the liver metastases in 28% and gained expression in 36% of cases compared to the primary. The kappa statistic comparing agreement between staining patterns of the primary tumors and liver metastases was a "less-than-chance", at -0.02. Median survival was 4.9 years, with no difference in survival outcomes by pSTAT3 expression in the primary tumor or liver metastases. DISCUSSION: STAT3 is not a prognostic marker in the selective setting of metastatic CRC to liver, but it may remain a potential therapeutic target given most liver metastases expressed pSTAT3. Discordant pSTAT3 expression in between primary tumors and paired liver metastases suggests that use of this class of drug to treat liver predominant metastatic colorectal cancer in a biomarker-driven approach may require confirmatory liver tumor biopsy.

Immunohistochemistry for TFE3 lacks specificity and sensitivity in the diagnosis of TFE3-rearranged neoplasms: a comparative, 2-laboratory study.

TFE3 rearrangements are characteristic of alveolar soft part sarcomas (ASPS), Xp11.2 translocation renal cell carcinomas (Xp11-RCC), and other rare tumors. Immunohistochemistry for TFE3 protein has been considered by some to be a reliable surrogate for TFE3 molecular studies, although others disagree. We compared 2 methods for TFE3 immunohistochemistry to determine if technical differences underlie these differences. Ninety-eight archival cases of mixed type, 19 ASPS, and 8 Xp11-RCC were stained for TFE3 at Laboratory A and Laboratory B using routine protocols. Positive controls were normal human testis (Laboratory A) and Xp11-RCC (Laboratory B). Nuclear staining was scored as "negative," "1+" (<10%), "2+" (10%-50%), and "3+" (>50%). Intensity was scored as "negative," "weak," "moderate," or "strong." Only moderate-strong, 2+ or 3+ staining was considered positive. Laboratory A results were as follows: archival cases (42 of 98, 43%), ASPS (16 of 19, 84%), and Xp11-RCC (7 of 8, 88%). Laboratory B results were as follows: archival cases (5 of 98, 5%), ASPS (14 of 19, 74%), and Xp11-RCC (5 of 8, 63%). TFE3 fluorescence in situ hybridization was positive in all tested ASPS and Xp11-RCC. The overall sensitivity and specificity of TFE3 immunohistochemistry for TFE3-rearranged neoplasms were 85% (23/27) and 57% (56/98) at Laboratory A and 70% (19/27) and 95% (93/98) at Laboratory B. Technical differences, in particular, the type of control tissue, likely account for these different results. The results of our study and prior studies suggest that TFE3 immunohistochemistry should play only a minor role (if any) in the diagnosis of TFE3-rearranged tumors, with fluorescence in situ hybridization representing the preferred method.

A Multi-Institutional Study to Evaluate Automated Whole Slide Scoring of Immunohistochemistry for Assessment of Programmed Death-Ligand 1 (PD-L1) Expression in Non-Small Cell Lung Cancer.

Assessment of programmed death-ligand 1 (PD-L1) expression is a critical part of patient management for immunotherapy. However, studies have shown that pathologist-based analysis lacks reproducibility, especially for immune cell expression. The purpose of this study was to validate reproducibility of the automated machine-based Optra image analysis for PD-L1 immunohistochemistry for both tumor cells (TCs) and immune cells. We compared conventional pathologists' scores for both tumor and immune cell positivity separately using 22c3 antibody on the Dako Link 48 platform for PD-L1 expression in non-small cell lung carcinoma. We assessed interpretation first by pathologists and second by PD-L1 image analysis scores. Lin's concordance correlation coefficients (LCCs) for each pathologist were measured to assess variability between pathologists and between pathologists and Optra automated quantitative scores in scoring both tumor and immune cells. Lin's LCCs to evaluate the correlation between pathologists for TC was 0.75 [95% confidence interval (CI), 0.64-0.81] and 0.40 (95% CI, 0.40-0.62) for immune cell scoring. Pathologists were highly concordant for tumor scoring, but not for immune cell scoring, which is similar to previously reported studies where agreement is higher in TCs than immune cells. The LCCs between conventional pathologists' read and the machine score were 0.80 (95% CI, 0.74-0.85) for TCs and 0.70 (95% CI, 0.60-0.76) for immune cell population. This is considered excellent agreement for TCs and good concordance for immune cells. The automated scoring methods showed concordance with the pathologists' average scores that were comparable to interpathologist scores. This suggests promise for Optra automated assessment of PD-L1 in non-small cell lung cancer.

HER2 in well differentiated breast cancer: is testing necessary?

BACKGROUND: In addition to providing a timely and accurate diagnosis, pathologists routinely provide prognostic and predictive information to assist in the treatment of patients with invasive breast cancer. As our understanding of breast cancer at the molecular and genetic level improves, sophisticated new treatment options have become available to patients. The demonstrated improvements in disease-free and overall survival with the use of trastuzumab (Herceptin) has made HER2 testing a standard of care in the evaluation of patients with breast cancer. Specialized breast centers have accumulated sufficient experience to recognize that HER2 positive tumors tend to be of higher grade and to be estrogen receptor negative, whereas well-differentiated breast cancers rarely are HER2 positive. METHODS: To determine whether HER2 testing is necessary in well-differentiated breast cancer, we analyzed the frequency of HER2 positivity among 1,162 cases from 7 major breast centers or commercial laboratories in the United States and Europe. RESULTS: Well-differentiated breast cancers, defined by either nuclear grading or the Scarff-Bloom-Richardson system, rarely are HER2 positive (mean 1.6%, range 0-2.8%). CONCLUSIONS: Given the low rate of well differentiated HER2 positive tumors, falling within the range reported for false negative IHC tests for HER2, and the absence of published data demonstrating a beneficial effect of trastuzumab therapy in this subset of patients, HER2 testing should not be considered a standard of care for all patients with well-differentiated breast cancer.