A Consortium for Analytic Standardization in Immunohistochemistry.

CONTEXT.­: The authors announce the launch of the Consortium for Analytic Standardization in Immunohistochemistry, funded with a grant from the National Cancer Institute. As with other laboratory testing, analytic standards are important for many different stakeholders: commercial vendors of instruments and reagents, biopharmaceutical firms, pathologists, scientists, clinical laboratories, external quality assurance organizations, and regulatory bodies. Analytic standards are customarily central to assay development, validation, and method transfer into routine assays and are critical quality assurance tools. OBJECTIVE.­: To improve immunohistochemistry (IHC) test accuracy and reproducibility by integrating analytic standards into routine practice. To accomplish this mission, the consortium has 2 mandates: (1) to experimentally determine analytic sensitivity thresholds (lower and upper limits of detection) for selected IHC assays, and (2) to inform IHC stakeholders of what analytic standards are, why they are important, and how and for what purpose they are used. The consortium will then publish the data and offer analytic sensitivity recommendations where appropriate. These mandates will be conducted in collaboration and coordination with clinical laboratories, external quality assurance programs, and pathology organizations. DATA SOURCES.­: Literature review and published external quality assurance data. CONCLUSIONS.­: Integration of analytic standards is expected to (1) harmonize and standardize IHC assays; (2) improve IHC test accuracy and reproducibility, both within and between laboratories; and (3) dramatically simplify and improve methodology transfer for new IHC protocols from published literature or clinical trials to clinical IHC laboratories.

A deep learning image-based intrinsic molecular subtype classifier of breast tumors reveals tumor heterogeneity that may affect survival.

BACKGROUND: Breast cancer intrinsic molecular subtype (IMS) as classified by the expression-based PAM50 assay is considered a strong prognostic feature, even when controlled for by standard clinicopathological features such as age, grade, and nodal status, yet the molecular testing required to elucidate these subtypes is not routinely performed. Furthermore, when such bulk assays as RNA sequencing are performed, intratumoral heterogeneity that may affect prognosis and therapeutic decision-making can be missed. METHODS: As a more facile and readily available method for determining IMS in breast cancer, we developed a deep learning approach for approximating PAM50 intrinsic subtyping using only whole-slide images of H&E-stained breast biopsy tissue sections. This algorithm was trained on images from 443 tumors that had previously undergone PAM50 subtyping to classify small patches of the images into four major molecular subtypes-Basal-like, HER2-enriched, Luminal A, and Luminal B-as well as Basal vs. non-Basal. The algorithm was subsequently used for subtype classification of a held-out set of 222 tumors. RESULTS: This deep learning image-based classifier correctly subtyped the majority of samples in the held-out set of tumors. However, in many cases, significant heterogeneity was observed in assigned subtypes across patches from within a single whole-slide image. We performed further analysis of heterogeneity, focusing on contrasting Luminal A and Basal-like subtypes because classifications from our deep learning algorithm-similar to PAM50-are associated with significant differences in survival between these two subtypes. Patients with tumors classified as heterogeneous were found to have survival intermediate between Luminal A and Basal patients, as well as more varied levels of hormone receptor expression patterns. CONCLUSIONS: Here, we present a method for minimizing manual work required to identify cancer-rich patches among all multiscale patches in H&E-stained WSIs that can be generalized to any indication. These results suggest that advanced deep machine learning methods that use only routinely collected whole-slide images can approximate RNA-seq-based molecular tests such as PAM50 and, importantly, may increase detection of heterogeneous tumors that may require more detailed subtype analysis.

New Dimensions of Antigen Retrieval Technique: 28 Years of Development, Practice, and Expansion.

This review article summarized recent advances in the heat-induced antigen retrieval technique with numerous scientific fields in addition to immunohistochemistry. Particularly, proteomics including imaging mass spectrometry, extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues. Some novel approaches such as FFPE tissue-based renal immunopathology based on modified double heating protocols are also introduced in this review for further development. In general, the FFPE tissue housed in pathology worldwide is an invaluable treasure, and the simple method of heat-induced antigen retrieval is the gold key to open the door of this treasure.

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.

Whole Slide Imaging Versus Microscopy for Primary Diagnosis in Surgical Pathology: A Multicenter Blinded Randomized Noninferiority Study of 1992 Cases (Pivotal Study).

Most prior studies of primary diagnosis in surgical pathology using whole slide imaging (WSI) versus microscopy have focused on specific organ systems or included relatively few cases. The objective of this study was to demonstrate that WSI is noninferior to microscopy for primary diagnosis in surgical pathology. A blinded randomized noninferiority study was conducted across the entire range of surgical pathology cases (biopsies and resections, including hematoxylin and eosin, immunohistochemistry, and special stains) from 4 institutions using the original sign-out diagnosis (baseline diagnosis) as the reference standard. Cases were scanned, converted to WSI and randomized. Sixteen pathologists interpreted cases by microscopy or WSI, followed by a wash-out period of ≥4 weeks, after which cases were read by the same observers using the other modality. Major discordances were identified by an adjudication panel, and the differences between major discordance rates for both microscopy (against the reference standard) and WSI (against the reference standard) were calculated. A total of 1992 cases were included, resulting in 15,925 reads. The major discordance rate with the reference standard diagnosis was 4.9% for WSI and 4.6% for microscopy. The difference between major discordance rates for microscopy and WSI was 0.4% (95% confidence interval, -0.30% to 1.01%). The difference in major discordance rates for WSI and microscopy was highest in endocrine pathology (1.8%), neoplastic kidney pathology (1.5%), urinary bladder pathology (1.3%), and gynecologic pathology (1.2%). Detailed analysis of these cases revealed no instances where interpretation by WSI was consistently inaccurate compared with microscopy for multiple observers. We conclude that WSI is noninferior to microscopy for primary diagnosis in surgical pathology, including biopsies and resections stained with hematoxylin and eosin, immunohistochemistry and special stains. This conclusion is valid across a wide variety of organ systems and specimen types.

Development of a Diagnostic Programmed Cell Death 1-Ligand 1 Immunohistochemistry Assay for Nivolumab Therapy in Melanoma.

Nivolumab is a monoclonal antibody that blocks the interaction between programmed cell death 1 (PD1) and programmed cell death 1-ligand 1 (PD-L1), resulting in enhanced antitumor activity by the immune system. Nivolumab is currently approved by the US Food and Drug Administration (FDA) for melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, and urothelial carcinoma. PD-L1 IHC 28-8 pharmDx is FDA-approved as a complementary diagnostic for immunohistochemical (IHC) detection of PD-L1 in non-squamous NSCLC and melanoma. We report validation of PD-L1 IHC 28-8 pharmDx for PD-L1 detection on formalin-fixed, paraffin-embedded human melanoma specimens using Autostainer Link 48. A prevalence assessment of 104 melanoma specimens indicated that PD-L1 was detected across the full expression level range (0% to 100% of tumor cells). Assay robustness and precision studies were conducted at Agilent Technologies, with additional reproducibility studies performed at 3 external laboratories. Precision studies evaluated at ≥1% and ≥5% expression levels revealed a range of average negative agreement from 89.5%, 95% CI (83.2, 93.6) to 100%, 95% CI (97.3, 100), and average positive agreement from 85.5%, 95% CI (77.6, 90.9) to 100%, 95% CI (97.9, 100). For external reproducibility, precise results were obtained. These results demonstrate PD-L1 IHC 28-8 pharmDx is a precise, robust, and reproducible assay for determining PD-L1 expression in melanoma. This is the first PD-L1 IHC test to receive FDA approval as a complementary diagnostic in melanoma patients whereby positive PD-L1 expression is correlated with the magnitude of nivolumab treatment effect.

Distribution of RANK and RANK Ligand in Normal Human Tissues as Determined by an Optimized Immunohistochemical Method.

The expression and tissue distribution of RANK (Receptor Activator of Nuclear Factor κ B) and RANK Ligand (RANKL) are of critical interest in relation to efficacy and safety of antibodies against RANK or RANKL that are approved or under consideration as potential therapeutic agents. Data from the literature using protein or mRNA analyses of rodent and human tissues or immunohistochemical (IHC) studies with a variety of antibodies and methods have provided some background of the distribution of RANK and RANKL but have yielded inconsistent findings. The present study reports the generation of carefully validated antibodies to RANK and RANKL and the development of an optimized IHC method, with confirmatory data from 2 well-validated alternative protocols that were developed and performed in separate laboratories at USC and at Amgen. Tissue expression of RANK and RANKL is reported for the optimized IHC assay.

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine - Part 2: Immunohistochemistry Test Performance Characteristics.

All laboratory tests have test performance characteristics (TPCs), whether or not they are explicitly known to the laboratorian or the pathologist. TPCs are thus also an integral characteristic of immunohistochemistry (IHC) tests and other in situ, cell-based molecular assays such as DNA or RNA in situ hybridization or aptamer-based testing. Because of their descriptive, in situ, cell-based nature, IHC tests have a limited repertoire of appropriate TPCs. Although only a few TPCs are relevant to IHC, proper selection of informative TPCs is nonetheless essential for the development of and adherence to appropriate quality assurance measures in the IHC laboratory. This paper describes the TPCs that are relevant to IHC testing and emphasizes the role of TPCs in the validation of IHC tests. This is part 2 of the 4-part series "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine. Part 3: Technical Validation of Immunohistochemistry (IHC) Assays in Clinical IHC Laboratories.

Validation of immunohistochemistry (IHC) assays is a subject that is of great importance to clinical practice as well as basic research and clinical trials. When applied to clinical practice and focused on patient safety, validation of IHC assays creates objective evidence that IHC assays used for patient care are "fit-for-purpose." Validation of IHC assays needs to be properly informed by and modeled to assess the purpose of the IHC assay, which will further determine what sphere of validation is required, as well as the scope, type, and tier of technical validation. These concepts will be defined in this review, part 3 of the 4-part series "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine: Part 4: Tissue Tools for Quality Assurance in Immunohistochemistry.

The numbers of diagnostic, prognostic, and predictive immunohistochemistry (IHC) tests are increasing; the implementation and validation of new IHC tests, revalidation of existing tests, as well as the on-going need for daily quality assurance monitoring present significant challenges to clinical laboratories. There is a need for proper quality tools, specifically tissue tools that will enable laboratories to successfully carry out these processes. This paper clarifies, through the lens of laboratory tissue tools, how validation, verification, and revalidation of IHC tests can be performed in order to develop and maintain high quality "fit-for-purpose" IHC testing in the era of precision medicine. This is the final part of the 4-part series "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine: Part 1: Fit-for-Purpose Approach to Classification of Clinical Immunohistochemistry Biomarkers.

Technical progress in immunohistochemistry (IHC) as well as the increased utility of IHC for biomarker testing in precision medicine avails us of the opportunity to reassess clinical IHC as a laboratory test and its proper characterization as a special type of immunoassay. IHC, as used in current clinical applications, is a descriptive, qualitative, cell-based, usually nonlinear, in situ protein immunoassay, for which the readout of the results is principally performed by pathologists rather than by the instruments on which the immunoassay is performed. This modus operandi is in contrast to other assays where the instrument also performs the readout of the test result (eg, nephelometry readers, mass spectrometry readers, etc.). The readouts (results) of IHC tests are used either by pathologists for diagnostic purposes or by treating physicians (eg, oncologists) for patient management decisions, the need for further testing, or follow-up. This paper highlights the distinction between the original purpose for which an IHC test is developed and its subsequent clinical uses, as well as the role of pathologists in the analytical and postanalytical phases of IHC testing. This paper is the first of a 4-part series, under the general title of "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

An Audit of Failed Immunohistochemical Slides in a Clinical Laboratory: The Role of On-Slide Controls.

Appropriate controls are critical for the correct interpretation of immunohistochemistry (IHC) assays and help to detect unsuccessful/suboptimal slides. We performed an audit of slides that were designated as being "failed" by the IHC laboratory (ie, laboratory-failed slides) of a large North American oncology and transplant center. All slides were run with on-slide controls. The study included analysis of only those failed slides where staining of both internal and external controls were unsuccessful/suboptimal in a period of 65 days. Failed slides were categorized based on the reason why the laboratory failed the slides. The study compared frequencies of failed slides across 9 automated stainers from 2 manufacturers and between class 1 and class 2 biomarkers. Distinction between "failed slides" and "false-negative/false-positive tests" is emphasized. The study included 22,234 IHC slides in the study period. Of those, 452 (2%) were designated as "failed" by the laboratory. Class 1 and class 2 tests showed failure rates of 0.8% and 9%, respectively. The most frequent reason for failed slides on one platform related to "no or weak staining," whereas the other had more failed slides due to "high signal-to-noise ratio" (P<0.0001, χ test). Although the slides were run in groups of the same as well as different IHC protocols, unsuccessful/suboptimal testing typically manifested as individual slides (92%) and not as groups of slides; this indicates that so-called "batch controls" are not suitable as controls for automated platforms. We conclude that in the era of automated IHC staining platforms, on-slide controls allow for the proper identification of IHC slides that should be failed by the IHC laboratory and represent a powerful tool for preventing the reporting of false-negative/false-positive tests.

FOXC1 identifies basal-like breast cancer in a hereditary breast cancer cohort.

Breast cancers arising in the setting of the hereditary breast cancer genes BRCA1 and BRCA2 are most commonly classified as basal-like breast cancer (BLBC) or luminal breast cancer, respectively. BLBC is an aggressive subtype of breast cancer associated with liver and lung metastases and poorer prognosis than other subtypes and for which chemotherapy is the only systemic therapy. Multiple immunohistochemical markers are used to identify the basal-like subtype, including the absence of estrogen receptor alpha, progesterone receptor, and human epidermal growth factor receptor 2. Forkhead box C1 (FOXC1) has been identified as a specific marker expressed in BLBC in general breast cancer cohorts. We examined an institutional cohort of breast cancer patients with germline BRCA1 (n=46) and BRCA2 (n=35) mutations and found that FOXC1 expression on immunohistochemical staining is associated with BRCA1 vs BRCA2 mutations [30/46 vs. 6/35]. In BRCA1 mutant tumors, FOXC1 was expressed in 28/31 BLBC tumors and 2/13 non-BLBC tumors, In BRCA2 mutant tumors, FOXC1 was expressed in 5/5 BLBC tumors and 1/30 non-BLBC tumors. In cell culture models of BRCA1-mutant breast cancer, FOXC1 is associated with increased proliferation and may serve as a marker for sensitivity to PARP-inhibitor therapy with olaparib.