Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

A combination of the immunohistochemical markers CK7 and SATB2 is highly sensitive and specific for distinguishing primary ovarian mucinous tumors from colorectal and appendiceal metastases.

Primary ovarian mucinous tumors can be difficult to distinguish from metastatic gastrointestinal neoplasms by histology alone. The expected immunoprofile of a suspected metastatic lower gastrointestinal tumor is CK7-/CK20+/CDX2+/PAX8-. This study assesses the addition of a novel marker SATB2, to improve the diagnostic algorithm. A test cohort included 155 ovarian mucinous tumors (105 carcinomas and 50 borderline tumors) and 230 primary lower gastrointestinal neoplasms (123 colorectal adenocarcinomas and 107 appendiceal neoplasms). All cases were assessed for SATB2, PAX8 CK7, CK20, and CDX2 expression on tissue microarrays. Expression was scored in a 3-tier system as absent, focal (1-50% of tumor cells) and diffuse ( >50% of tumor cells) and then categorized into either absent/present or nondiffuse/diffuse. SATB2 and PAX8 expression was further evaluated in ovarian tumors from an international cohort of 2876 patients (expansion cohort, including 159 mucinous carcinomas and 46 borderline mucinous tumors). The highest accuracy of an individual marker in distinguishing lower gastrointestinal from ovarian mucinous tumors was CK7 (91.7%, nondiffuse/diffuse cut-off) followed by SATB2 (88.8%, present/absent cut-off). The most effective combination was CK7 and SATB2 with accuracy of 95.3% using the 3-tier interpretation, absent/focal/diffuse. This combination outperformed the standard clinical set of CK7, CK20 and CDX2 (87.5%). Re-evaluation of outlier cases confirmed ovarian origin for all but one case. The accuracy of SATB2 was confirmed in the expansion cohort (91.5%). SATB2 expression was also detected in 15% of ovarian endometrioid carcinoma but less than 5% of other ovarian histotypes. A simple two marker combination of CK7 and SATB2 can distinguish lower gastrointestinal from ovarian primary mucinous tumors with greater than 95% accuracy. PAX8 and CDX2 have value as second-line markers. The utility of CK20 in this setting is low and this warrants replacement of this marker with SATB2 in clinical practice.

Current usage and future trends in gross digital photography in Canada.

BACKGROUND: The purpose of this study was to assess the current usage, utilization and future direction of digital photography of gross surgical specimens in pathology laboratories across Canada. METHODS: An online survey consisting of 23 multiple choice and free-text questions regarding gross digital photography was sent out to via email to laboratory staff across Canada involved in gross dissection of surgical specimens. RESULTS: Sixty surveys were returned with representation from most of the provinces. Results showed that gross digital photography is utilized at most institutions (90.0%) and the primary users of the technology are Pathologists (88.0%), Pathologists' Assistants (54.0%) and Pathology residents (50.0%). Most respondents felt that there is a definite need for routine digital imaging of gross surgical specimens in their practice (80.0%). The top two applications for gross digital photography are for documentation of interesting/ complex cases (98.0%) and for teaching purposes (84.0%). The main limitations identified by the survey group are storage space (42.5%) and security issues (40.0%). Respondents indicated that future applications of gross digital photography mostly include teaching (96.6%), presentation at tumour boards/ clinical rounds (89.8%), medico-legal documentation (72.9%) and usage for consultation purposes (69.5%). CONCLUSIONS: The results of this survey indicate that pathology staff across Canada currently utilizes gross digital images for regular documentation and educational reasons. They also show that the technology will be needed for future applications in teaching, consultation and medico-legal purposes.

Differentiating neurotized melanocytic nevi from neurofibromas using Melan-A (MART-1) immunohistochemical stain.

CONTEXT: Neurotized melanocytic nevi and neurofibromas are common, benign cutaneous neoplasms. Usually they are histologically distinct from each other; however, neurotized melanocytic nevi and neurofibromas can be clinically and histologically similar. OBJECTIVE: To determine whether Melan-A (MART-1) immunohistochemical stain is sufficient to differentiate neurotized melanocytic nevi from neurofibromas. DESIGN: Forty-nine consecutive specimens of melanocytic nevi with neurotization and 49 specimens of neurofibromas were selected. We used antibodies against Melan-A, S100, and neurofilament protein. RESULTS: All of the melanocytic nevi showed Melan-A staining within the neurotized areas, with most of the areas staining strongly positive, whereas all the neurofibromas were completely absent of Melan-A stain. All of the nevi, including the neurotized areas, stained strongly and diffusely for S100, whereas all the neurofibromas showed a distinctive, sharp, wavy pattern of S100 staining. Neurofilament protein showed scattered staining of both melanocytic nevi and neurofibromas. CONCLUSIONS: Our data indicate that Melan-A immunohistochemical staining is helpful in differentiating neurotized melanocytic nevi from neurofibromas when distinction on histomorphology alone is difficult.

Oral cavity neuroendocrine carcinoma: a comparison study with cutaneous Merkel cell carcinoma and other mucosal head and neck neuroendocrine carcinomas.

OBJECTIVES: Published cases of oral high-grade neuroendocrine carcinoma (HGNEC) variably call the tumors Merkel cell carcinoma (MCC) or small-cell/high-grade neuroendocrine carcinoma. We studied cases of cutaneous MCC and mucosal HGNEC to better distinguish them and to better define oral cases. STUDY DESIGN: Twelve cutaneous MCC and 14 mucosal HGNEC cases were identified. We reviewed the hematoxylin-eosin (H&E) morphology and performed immunohistochemistry for cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), p63, neurofilament (NF), and achaete-scute homolog 1 (MASH-1). We also performed polymerase chain reaction (PCR) for the Merkel cell polyomavirus (MCPyV). RESULTS: By morphology and immunohistochemistry, MCC and HGNEC showed many differences. CK20, NF, and TTF-1 stains were the most discriminatory. MASH-1 was expressed by both MCC and HGNEC. MCPyV was present in MCC and absent in all HGNEC. The 2 oral cavity mucosal carcinomas segregated into MCC and HGNEC types, the former having the H&E nuclear morphology and classic dot-like perinuclear CK20 staining typically associated with MCC. CONCLUSIONS: Oral cavity neuroendocrine carcinoma can be segregated into MCC and small-cell/HGNEC types by morphology and CK20 immunohistochemistry. MCPyV was present by PCR in cutaneous MCC but was not found in mucosal HGNEC.

Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis.

Microarray analysis of multiple sclerosis (MS) lesions obtained at autopsy revealed increased transcripts of genes encoding inflammatory cytokines, particularly interleukin-6 and -17, interferon-gamma and associated downstream pathways. Comparison of two poles of MS pathology--acute lesions with inflammation versus 'silent' lesions without inflammation--revealed differentially transcribed genes. Some products of these genes were chosen as targets for therapy of experimental autoimmune encephalomyelitis (EAE) in mice. Granulocyte colony-stimulating factor is upregulated in acute, but not in chronic, MS lesions, and the effect on ameliorating EAE is more pronounced in the acute phase, in contrast to knocking out the immunoglobulin Fc receptor common gamma chain where the effect is greatest on chronic disease. These results in EAE corroborate the microarray studies on MS lesions. Large-scale analysis of transcripts in MS lesions elucidates new aspects of pathology and opens possibilities for therapy.