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.

A Standardization Protocol for the In Situ Detection of SARS-CoV2 RNA and Proteins.

This manuscript details a stringent protocol for the in situ detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) RNA and 4 different viral proteins: envelope, spike, membrane, and nucleocapsid. Key aspects of the protocol are: (1) analysis of adjacent (serial) sections for viral RNA and at least 2 viral proteins; (2) cytologic alterations in the cells scored as virus positive based on an hematoxylin and eosin stain; (3) in situ demonstration of a host response in the cells scored as virus positive; (4) co-labeling experiments that show that the viral RNA and/or proteins co-localize with each other and the angiotensin converting enzyme 2 (ACE2) receptor; and (5) lack of signal in equivalent tissues obtained before the pandemic. Optimization conditions for the four viral proteins as well as the ACE2 receptor were each antigen retrieval in an EDTA solution which facilitates co-expression analyses. It is recommended not to use either electron microscopy or qRTPCR as methods to corroborate in situ SARS-CoV2 detection. This stringent protocol, that relies on sequentially labeled serial sections and can be completed in one working day, demonstrated the following: (1) infectious SARS-CoV2 is abundant in the lung in fatal coronavirus disease-2019 and is seen primarily in macrophages and endothelial cells; (2) circulating viral capsid proteins (spike, envelope, membrane without RNA) are evident in multiple organs including the skin and brain where it is endocytosed by ACE2+ cells and induce an endothelialitis; (3) both the infectious virus and circulating spike protein induce complement activation and cytologic changes in the viral positive cells.

Correlation of manual semi-quantitative and automated quantitative Ki-67 proliferative index with OncotypeDXTM recurrence score in invasive breast carcinoma.

BACKGROUND: Ki-67 immunohistochemistry (IHC) staining is a widely used cancer proliferation assay; however, its limitations could be improved with automated scoring. The OncotypeDXTM Recurrence Score (ORS), which primarily evaluates cancer proliferation genes, is a prognostic indicator for breast cancer chemotherapy response; however, it is more expensive and slower than Ki-67. OBJECTIVE: To compare manual Ki-67 (mKi-67) with automated Ki-67 (aKi-67) algorithm results based on manually selected Ki-67 "hot spots" in breast cancer, and correlate both with ORS. METHODS: 105 invasive breast carcinoma cases from 100 patients at our institution (2011-2013) with available ORS were evaluated. Concordance was assessed via Cohen's Kappa (κ). RESULTS: 57/105 cases showed agreement between mKi-67 and aKi-67 (κ 0.31, 95% CI 0.18-0.45), with 41 cases overestimated by aKi-67. Concordance was higher when estimated on the same image (κ 0.53, 95% CI 0.37-0.69). Concordance between mKi-67 score and ORS was fair (κ 0.27, 95% CI 0.11-0.42), and concordance between aKi-67 and ORS was poor (κ 0.10, 95% CI -0.03-0.23). CONCLUSIONS: These results highlight the limits of Ki-67 algorithms that use manual "hot spot" selection. Due to suboptimal concordance, Ki-67 is likely most useful as a complement to, rather than a surrogate for ORS, regardless of scoring method.

Discordance Between Immunohistochemistry and In Situ Hybridization to Detect HER2 Overexpression/Gene Amplification in Breast Cancer in the Modern Age: A Single Institution Experience and Pooled Literature Review Study.

BACKGROUND: Human epidermal growth factor 2 (HER2) amplification and/or overexpression occurs in 12% to 25% of breast cancers. Accurate detection of HER2 is critical in predicting response to HER2-targeted therapy. Both immunohistochemistry (IHC) and in situ hybridization (ISH) are FDA-approved methods for detecting HER2 status because its protein overexpression is largely attributable to gene amplification. However, variable discordant results between IHC and ISH have been reported. METHODS: We determined the frequency of HER2 IHC/ISH discordance in these patients and also performed a pooled literature review analysis. RESULTS: Of the 1125 consecutive primary or metastatic breast cancers with HER2 IHC and ISH performed simultaneously between 2015 and 2020, 84.6% had an unequivocal HER2 status. Discordance was found in 30 cases from 26 patients, including 13 IHC-/ISH+ and 17 IHC+/ISH-, representing 1.6% and 11.9% of IHC- and IHC+ cases, respectively. Review of the literature between 2001 and 2020 identified 46 relevant studies, with a total of 43,468 cases with IHC and ISH performed. The IHC-/ISH+ and IHC+/ISH- discordances were seen in all antibody clones and ISH methods used. The IHC+/ISH- discordance was significantly higher than IHC-/ISH+ (13.8% vs. 3%, P < .0001). The overall discordance constituted 4% of all cases and 5.4% of those with an unequivocal IHC status. Significantly lower incongruities for both IHC-/ISH+ and IHC+/ISH- were found in those published after 2018. The discordances probably reflect altered biology of HER2 oncogene/oncoprotein. Routinely performing both IHC and ISH may uncover such cases to prevent denial of potentially beneficial targeted therapy.

Diagnostic Accuracy of Immunohistochemistry in Detecting MGMT Methylation Status in Patients with Glioma.

BACKGROUND: The O6-methylguanine-DNA methyltransferase (MGMT) gene prevents mismatch in DNA replication and transcription by repairing mutagenic DNA lesions. MGMT is a predictor biomarker of chemotherapy in high-grade and low-grade gliomas based on high-risk clinical conditions. It also can be used for therapeutic decisions to predict hypermutation in recurrence in newly diagnosed low-grade gliomas. The gold standard  examination for the methylation is Polymerase Chain Reaction (PCR). However, this technique is not widely available in Indonesia for daily practice. Thus, an uncomplicated and simpler method such as immunohistochemistry (IHC) is needed as an alternative examination. This study aimed to predict the diagnostic accuracy of immunohistochemistry (IHC) in detecting the methylation status of O6-methylguanine-DNA methyltransferase (MGMT) in glioma. METHODS: This research was a cross-sectional study using formalin-fixed paraffin embedded (FFPE) tissue samples of glioma patients, dating between October 2017 until March 2021. Diagnosis of glioma was established based on clinical, radiological, and histopathological findings. MGMT methylation status was investigated using the IHC and PCR techniques. Diagnostic value of IHC was analyzed, with PCR as a gold standard method. Optimum threshold to determine positivity of IHC was determined by the Area Under the Curve (AUC) on Receiver Operating Characteristics (ROC) curve and Youden index. RESULTS: Among 75 samples examined, 29 (38.7%) patients were methylated. IHC detected MGMT methylation with sensitivity of 86.2%, specificity of 63.0%, positive predictive value of 59.5%, negative predictive value of 87.9% and accuracy of 72.0%. The AUC was 0.746, indicating moderate diagnostic value. Optimum positivity threshold of the IHC examination based on Youden Index was 10%. CONCLUSION: IHC examination can be used to detect MGMT methylation status of glioma patients in limited resources setting, where PCR technique is not available.

Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies.

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.

Evaluation of E-Cadherin and ß-Catenin Immunoreactivity for Determining Undifferentiated Cells in Anaplastic Thyroid Carcinoma.

INTRODUCTION: An immunohistochemical study has occasionally been performed to diagnose anaplastic thyroid carcinoma (ATC). However, antibodies to confirm the undifferentiated nature of ATC have not yet been evaluated. The aim of this study was to evaluate E-cadherin and ß-catenin expressions in immunoreactivity to determine undifferentiated carcinoma cells in the diagnosis of ATC. METHODS: We immunohistochemically examined 29 ATCs, 30 poorly differentiated thyroid carcinomas (PDTCs), 22 well-differentiated thyroid carcinomas (WDTCs), and 3 squamous cell carcinomas. Antibodies for thyroid transcription factor-1 (TTF-1), paired-box gene 8 (PAX8), ß-catenin, and E-cadherin were used. RESULTS: All WDTCs tested positive for TTF-1, PAX8, and E-cadherin. The positive rates of TTF-1, PAX8, and E-cadherin were 93.3, 93.3, and 100%, respectively, in PDTCs and 17.2, 51.7, and 10.3%, respectively, in ATCs. WDTC expressed the lateral cell membrane staining for ß-catenin and E-cadherin, whereas PDTC showed circumferential cell membranous expression (fishnet pattern). ß-catenin cell membrane expression in ATCs is lost or discontinuous. Carcinoma cells with ß-catenin nuclear expression without cell membranous expression were scattered in 72.4% of ATCs but were not observed in the other carcinomas. CONCLUSION: We propose 3 immunohistochemical findings to determine undifferentiated carcinoma cells in the diagnosis of ATC: (1) ß-catenin nuclear expression with no or reduced cell membranous expression, (2) the loss or discontinuous pattern of E-cadherin expression, and (3) the loss of PAX8 nuclear expression.

Applicability and interpretation of HER2/Neu & PD-L1 stains in gastrointestinal tract tumours.

The luminal gastrointestinal tract carcinomas are one of the major causes of cancer-related deaths. To improve overall survival, the current trend is to combine targeted therapeutic agents with conventional chemotherapies. Major trials have shown survival benefits with this approach and many more trials are being undertaken. However, pathologists often get perplexed by different methods of interpretation and reporting of these stains, vital for deciding therapeutic approaches.

Potential antigenic targets used in immunological tests for diagnosis of tegumentary leishmaniasis: A systematic review.

Immunological tests may represent valuable tools for the diagnosis of human tegumentary leishmaniasis (TL) due to their simple execution, less invasive nature and potential use as a point-of-care test. Indeed, several antigenic targets have been used with the aim of improving the restricted scenario for TL-diagnosis. We performed a worldwide systematic review to identify antigenic targets that have been evaluated for the main clinical forms of TL, such as cutaneous (CL) and mucosal (ML) leishmaniasis. Included were original studies evaluating the sensitivity and specificity of immunological tests for human-TL, CL and/or ML diagnosis using purified or recombinant proteins, synthetic peptides or polyclonal or monoclonal antibodies to detect Leishmania-specific antibodies or antigens. The review methodology followed PRISMA guidelines and all selected studies were evaluated in accordance with QUADAS-2. Thirty-eight original studies from four databases fulfilled the selection criteria. A total of 79 antigens were evaluated for the detection of antibodies as a diagnostic for TL, CL and/or ML by ELISA. Furthermore, three antibodies were evaluated for the detection of antigen by immunochromatographic test (ICT) and immunohistochemistry (IHC) for CL-diagnosis. Several antigenic targets showed 100% of sensitivity and specificity, suggesting potential use for TL-diagnosis in its different clinical manifestations. However, a high number of proof-of-concept studies reinforce the need for further analysis aimed at verifying true diagnostic accuracy in clinical practice.

Ambiguity about Splicing Factor 3b Subunit 3 (SF3B3) and Sin3A Associated Protein 130 (SAP130).

In 2020, three articles were published on a protein that can activate the immune system by binding to macrophage-inducible C-type lectin receptor (Mincle). In the articles, the protein was referred to as 'SAP130, a subunit of the histone deacetylase complex.' However, the Mincle ligand the authors aimed to investigate is splicing factor 3b subunit 3 (SF3B3). This splicing factor is unrelated to SAP130 (Sin3A associated protein 130, a subunit of the histone deacetylase-dependent Sin3A corepressor complex). The conclusions in the three articles were formulated for SF3B3, while the researchers used qPCR primers and antibodies against SAP130. We retraced the origins of the ambiguity about the two proteins and found that Online Mendelian Inheritance in Man (OMIM) added a Nature publication on SF3B3 as a reference for Sin3A associated protein 130 in 2016. Subsequently, companies such as Abcam referred to OMIM and the Nature article in their products for both SF3B3 and SAP130. In turn, the mistake by OMIM followed in the persistent and confusing use of 'SAP130' (spliceosome-associated protein 130) as an alternative symbol for SF3B3. With this report, we aim to eliminate the persistent confusion and separate the literature regarding the two proteins.

Comparison of breast cancer surrogate subtyping using a closed-system RT-qPCR breast cancer assay and immunohistochemistry on 100 core needle biopsies with matching surgical specimens.

BACKGROUND: Routine clinical management of breast cancer (BC) currently depends on surrogate subtypes according to estrogen- (ER) and progesterone (PR) receptor, Ki-67, and HER2-status. However, there has been growing demand for reduced immunohistochemistry (IHC) turnaround times. The Xpert® Breast Cancer STRAT4* Assay (STRAT4)*, a standardized test for ESR1/PGR/MKi67/ERBB2 mRNA biomarker assessment, takes less than 2 hours. Here, we compared the concordance between the STRAT4 and IHC/SISH, thereby evaluating the effect of method choice on surrogate subtype assessment and adjuvant treatment decisions. METHODS: In total, 100 formalin-fixed paraffin-embedded core needle biopsy (CNB) samples and matching surgical specimens for 98 patients with primary invasive BC were evaluated using the STRAT4 assay. The concordance between STRAT4 and IHC was calculated for individual markers for the CNB and surgical specimens. In addition, we investigated whether changes in surrogate BC subtyping based on the STRAT4 results would change adjuvant treatment recommendations. RESULTS: The overall percent agreement (OPA) between STRAT4 and IHC/SISH ranged between 76 and 99% for the different biomarkers. Concordance for all four biomarkers in the surgical specimens and CNBs was only 66 and 57%, respectively. In total, 74% of surgical specimens were concordant for subtype, regardless of the method used. IHC- and STRAT4-based subtyping for the surgical specimen were shown to be discordant for 25/98 patients and 18/25 patients would theoretically have been recommended a different adjuvant treatment, primarily receiving more chemotherapy and trastuzumab. CONCLUSIONS: A comparison of data from IHC/in situ hybridization and STRAT4 demonstrated that subsequent changes in surrogate subtyping for the surgical specimen may theoretically result in more adjuvant treatment given, primarily with chemotherapy and trastuzumab.

A new tool for technical standardization of the Ki67 immunohistochemical assay.

Ki67, a nuclear proliferation-related protein, is heavily used in anatomic pathology but has not become a companion diagnostic or a standard-of-care biomarker due to analytic variability in both assay protocols and interpretation. The International Ki67 Working Group in breast cancer has published and has ongoing efforts in the standardization of the interpretation of Ki67, but they have not yet assessed technical issues of assay production representing multiple sources of variation, including antibody clones, antibody formats, staining platforms, and operators. The goal of this work is to address these issues with a new standardization tool. We have developed a cell line microarray system in which mixes of human Karpas 299 or Jurkat cells (Ki67+) with Sf9 (Spodoptera frugiperda) (Ki67-) cells are present in incremental standardized ratios. To validate the tool, six different antibodies, including both ready-to-use and concentrate formats from six vendors, were used to measure Ki67 proliferation indices using IHC protocols for manual (bench-top) and automated platforms. The assays were performed by three different laboratories at Yale and analyzed using two image analysis software packages, including QuPath and Visiopharm. Results showed statistically significant differences in Ki67 reactivity between each antibody clone. However, subsets of Ki67 assays using three clones performed in three different labs show no significant differences. This work shows the need for analytic standardization of the Ki67 assay and provides a new tool to do so. We show here how a cell line standardization system can be used to normalize the staining variability in proliferation indices between different antibody clones in a triple negative breast cancer cohort. We believe that this cell line standardization array has the potential to improve reproducibility among Ki67 assays and laboratories, which is critical for establishing Ki67 as a standard-of-care assay.

Faecal immunochemical testing (FIT): sources of result variation based on three years of routine testing of symptomatic patients in English primary care.

Introduction: We aimed to determine the analytical capabilities of a commonly used faecal immunochemical test (FIT) to detect faecal haemoglobin (Hb) in symptomatic people attending primary care in the context of the English NICE DG30 guidance.Materials and Methods: Data obtained from independent verification studies and clinical testing of the HM-JACKarc FIT method in routine primary care practice were analysed to derive performance characteristics.Results: Detection capabilities for the FIT method were 0.5 µg/g (limit of blank), 1.3 µg/g (limit of detection) and 3.0 µg/g (limit of quantitation). Of 33 non-homogenized specimens, 31 (93.9%) analysed in triplicate were consistently categorized relative to 10 µg/g, compared to all 33 (100%) homogenized specimens. Imprecision was higher (median 27.8%, (range 20.5% to 48.6%)) in non-homogenized specimens than in homogenized specimens (10.2%, (7.0 to 13.5%)). Considerable variation was observed in sequential clinical specimens from individual patients but no positive or negative trend in specimen degradation was observed over time (p = 0.26).Discussion: The FIT immunoassay evaluated is capable of detecting faecal Hb at concentrations well below the DG30 threshold of 10 µg/g and is suitable for application in this context. The greatest practical challenge to FIT performance is reproducible sampling, the pre-analytical step associated with most variability. Further research should focus on reducing sampling variability, particularly as post-COVID-19 guidance recommends greater FIT utilization.

Exploring the role CD163-labeled TAMs and FOXP3-labeled Tregs in different types of breast cancer: Reflections and putative benefits.

CONTEXT: Tumor immune microenvironment (TIME) is heterogeneous and dynamic. It exerts bimodal pro and antitumor effects. Among the TIME contributors, TAMs and Tregs are condemned as cancer cells allies rather than enemies; however, such contribution is not universally equal in all tumors. AIMS: We aimed to explore and compare TAMs and Tregs in various breast cancers and link such findings to pathologic prognostic indices. SETTINGS AND DESIGN: This was a retrospective study. METHODS AND MATERIALS: Archival blocks of 108 breast cancers were immunohistochemically studied for CD163 and FOXP3 in tumor stroma (TS) and specialized DCIS periductal stroma. FOXP3 was additionally evaluated in tumor cells. CD163 and FOXP3 expressions were compared with different histopathological prognostic categories for statistical analysis. STATISTICAL ANALYSIS USED: Analysis of data was done using the Chi-Square test. RESULTS: Both CD163+ TAM and FOXP3+ Tregs. showed statistically significant association with high tumor grade, T stage, multifocality and hormone negativity. Synchronous expression was consistent for both markers in almost all compared parameters, dual high expression of both CD163 and FOXP3 yielded additional statistically significant association with lymphovascular invasion (LVI). Periductal stromal CD163 and FOXP3 high expression showed statistically significant association with DCIS. FOXP3 tumor cells expression was similar to TS FOXP3 but additionally showed significant association with LVI and N stage; moreover, Her-2 over-expressing breast cancer was significantly associated with low FOXP3+ tumor cells. CONCLUSIONS: Breast cancer TS TAMs and Tregs. abundance reflects unfavorable prognosis in various breast cancers particularly hormone negative cancers.

Diagnostic utility of p16 immunocytochemistry in metastatic cervical lymph nodes in head and neck cancers.

BACKGROUND: Human papilloma virus (HPV) testing can be useful in work-up of patients presenting with cervical node metastasis, suspected to be of head and neck origin as HPV positive tumors show better response to therapy. The current study was planned to detect HPV in aspirates from metastatic cervical nodes using p16 immunocytochemistry in head and neck squamous cell carcinoma (HNSCC). Further correlation of HPV status between node metastasis and primary tumor was done. METHODS: The prospective study included 50 patients diagnosed as metastatic SCC in cervical nodes on fine needle aspiration with either known head and neck primary or primary detected post cytodiagnosis. Immunostaining for p16 was carried out on both smears and tissue sections. RESULTS: Forty-three patients were male and seven were female. Age of the patients ranged from 35 to 80 years. Primary sites of HNSCC were oropharynx (25), oral cavity (14), and larynx (11). Immunocytochemistry for p16 on smears showed positivity in 28 cases. Immunohistochemistry for p16 in primary tumors was positive in 34. There was substantial agreement between p16 immunocytochemistry and immunohistochemistry (Kappa value: 0.823). The sensitivity of p16 immunocytochemistry for the detection of HPV in metastatic HNSCC was 82.4% while the specificity was 100%. The positive and negative predictive values were 100% and 72.7%, respectively. CONCLUSIONS: P16 immunocytochemistry in HNSCC metastatic to cervical node mirrors the HPV status of the corresponding primary tumor. Hence in tumors of unknown origin presenting as cervical node metastasis, p16 immunocytochemistry can be employed for localization of the primary.

Role of Cytology in the Current Guidelines for Malignant Mesothelioma: Largest Study from India.

INTRODUCTION: Cytology provides crucial window for early diagnosis of malignant mesothelioma (MM) since it is often the first and easily available material for evaluation, resulting in early treatment. Still, its role is overlooked in the current treatment guidelines. The aim of this study is to determine the sensitivity of cytomorphology and role of subsequent ancillary techniques in diagnosing MM. METHODS: This is a 5-year retrospective analysis of MM in the tertiary oncology center to determine sensitivity of cytomorphology and subsequent role of immunohistochemistry (IHC) in final diagnosis of MM according to the guidelines for cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma (GCDMM) laid by International Mesothelioma Interest Group. Cytomorphology and immunocytochemistry from effusions and fine needle aspirations were analyzed. RESULTS: Sixty-two of 128 cases of MM had cytology and cytomorphological criteria described in GCDMM were fulfilled in 61.3% cases. Architectural atypia was useful in identifying cases with low cytological atypia. Overall sensitivity of cytomorphology was 73.01%. Sensitivity of effusion cytology was 77.8%. Subsequent IHC on cell blocks revealed the sensitivity as 100% for mesothelin, calretinin, and cytokeratin 5/6; 87.5% for thrombomodulin; and 50% for WT1, while CEA and TTF1 showed 100% specificity. Treatment was given based on final diagnosis of MM given after IHC on cytology material in only 25.8% cases. However, it was possible in additional 35.5% cases. Mean survival was 10 months when diagnosed by cytology, compared to 7 months by histology. CONCLUSIONS: Rather than ignoring the role of cytology in the diagnosis and treatment guidelines for MM, it is important to understand its strengths and limitations. Standardized guidelines in future can play an important role in more streamlined communication between cytopathologist and clinician.

Malignancy is in the eye of the beholder: Pathologic diagnosis of challenging follicular neoplasms in the era of noninvasive follicular thyroid neoplasms with papillary-like nuclear features and immunohistochemical and molecular adjuncts.

BACKGROUND: Classification of thyroid follicular neoplasms can be challenging for pathologists. Introduction of noninvasive follicular thyroid neoplasms with papillary-like nuclear features, the utilization of immunohistochemistry, and molecular analysis are all thought to be valuable diagnostic adjuncts. Our aim was to determine whether interobserver variability for follicular neoplasms has improved since the application of these adjuncts. METHODS: One representative section from a cohort of follicular neoplasms previously proven difficult for pathologists were examined independently by 7 pathologists and assigned to 1 of 3 diagnostic categories (benign, neoplasms with papillary-like nuclear features, or malignant). This process was carried out separately 3 times: (1) after viewing hematoxylin and eosin stain slides, (2) hematoxylin and eosin stain in conjunction with immunohistochemistry, and (3) hematoxylin and eosin stain/immunohistochemistry in conjunction with molecular analysis. The interobserver variability and overall agreement were then calculated using the free-marginal kappa coefficient. RESULTS: Agreement on hematoxylin and eosin stain was 57%, with a kappa coefficient of 0.36 (minimal agreement). The agreement improved slightly with the application of immunohistochemistry (kappa coefficient = 0.49 [weak agreement] and a percentage agreement 67%). The level of agreement decreased slightly after the addition of molecular analysis (kappa coefficient = 0.43 [weak agreement] and percentage agreement 62%). CONCLUSION: Despite attempts to standardize the diagnostic criteria for neoplasms with papillary-like nuclear features and the utilization immunohistochemistry and molecular analysis, attaining pathologic consensus for difficult follicular neoplasms of the thyroid remains a challenge.

Biomarker testing in oncology - Requirements for organizing external quality assessment programs to improve the performance of laboratory testing: revision of an expert opinion paper on behalf of IQNPath ABSL.

In personalized medicine, predictive biomarker testing is the basis for an appropriate choice of therapy for patients with cancer. An important tool for laboratories to ensure accurate results is participation in external quality assurance (EQA) programs. Several providers offer predictive EQA programs for different cancer types, test methods, and sample types. In 2013, a guideline was published on the requirements for organizing high-quality EQA programs in molecular pathology. Now, after six years, steps were taken to further harmonize these EQA programs as an initiative by IQNPath ABSL, an umbrella organization founded by various EQA providers. This revision is based on current knowledge, adds recommendations for programs developed for predictive biomarkers by in situ methodologies (immunohistochemistry and in situ hybridization), and emphasized transparency and an evidence-based approach. In addition, this updated version also has the aim to give an overview of current practices from various EQA providers.

Immunohistochemical Validation of Rare Tissues and Antigens With Low Frequency of Occurrence: Recommendations From The Anatomic Pathology Patient Interest Association (APPIA).

Laboratories worldwide find it challenging to identify enough tissues and cases for verification and validation studies of low-incidence, rare antigens. These antigens have a low frequency of occurrence in the population, or have little or no expression in normal tissues. Validation studies are essential to assure testing standardization before introducing a new instrument, product, or test into the clinical laboratory. The College of American Pathologists has published comprehensive guidelines for the verification and validation of new immunohistochemical tests introduced into the laboratory menu. Within the guidelines, varied numbers of cases are required for nonpredictive versus predictive markers. However, regarding low-incidence antigens, the laboratory medical director determines the extent of validation required. Recommended practical solutions available to clinical laboratories for low-incidence validation include developing internal resources using the laboratory information system with retrospective and prospective search(s) of archival material and purchase of tissue microarray blocks, slides, or cell lines from external resources. Utilization of homemade multitissue blocks has proved to be extremely valuable in biomarker research and demonstrated great utility in clinical immunohistochemistry laboratories. Participation in External Quality Assessment program(s) may provide insufficient numbers or the ability to calculate concordance rates. However, supplementation with in-house tissues can allow a laboratory to reach the optimal number of cases needed for verification and/or validation schemes. An alternative approach is conducting a thorough literature search and correlating staining patterns of the new test to the expected results. These solutions may be used uniquely or together to assure consistent standardized testing.

Comparison of three FDA-approved diagnostic immunohistochemistry assays of PD-L1 in triple-negative breast carcinoma.

The Dako 28-8, Dako 22C3, and Ventana SP142 assays are among the approved programmed death ligand 1 (PD-L1) immunohistochemical companion/complementary diagnostics associated with cancer treatment. To address the concordance of these assays in triple-negative breast cancer (TNBC), we examined PD-L1 expression in 98 TNBC tumors and compared the positive rates using the three assays and three scoring methods: immune cell (IC), tumor cell (TC), and combined tumor cell and immune cell (TCIC) (an equivalent to combined positive score, or CPS). The positive rate for PD-L1 expression with a 1% cutoff was highest with 28-8, followed by the 22C3. These two assays demonstrated almost perfect or substantial agreement in all three scores. There was less agreement between SP142 and the other assays. Using the IC score or the TCIC score at a 1% cutoff (CPS 1), 4% of tumors were positive for PD-L1 with SP142 but negative with the other assays. Using SP142 with a 1% cutoff as a reference, the optimal cutoff for best agreement was at 1% for IC, 30% for TC, and 2% for TCIC (CPS 2) with the other two assays. A 2% cutoff for the 22C3 TCIC (CPS 2) yielded the best agreement with SP142 1% IC cutoff (kappa 0.65). Our study showed the lowest positive rate with SP142 among the three assays. However, the other two assays were not able to identify all tumors that would test positive with SP142 using IC or TCIC/CPS. It is unlikely to achieve high agreement between SP142 and the other two assays by changing the analytical cutoffs.