Current state of Grand Rounds in U.S. pathology training programs.

Grand Rounds are held with variable frequency in many academic pathology departments, but their exact goal is uncertain, and the type of subjects covered, and presenters have not been studied. We aimed to gather information about the current state of pathology grand rounds (PGR). We identified all US pathology residency programs accredited by the Accreditation Council for Graduate Medical Education (ACGME) and searched their websites for information regarding PGR, extracting data on their existence, frequency and timing. For a representative subgroup of institutions from all US regions and program sizes, we tabulated the 2017-2018 PGR titles and presenters (gender, degree(s), resident/fellow, faculty academic rank). We found that 71 of 142 (50%) ACGME-accredited programs had PGR, more often in programs with >12 residents (53/88, 60%). PGR were scheduled most commonly weekly, on Thursdays, and at noon. We analyzed 1019 PGR presentations from 41 institutions located in 26 US states. Among the 1105 presenters, 183 (16.56%) were trainees, 74 (6.7%) were non-academic, and 848 (76.7%) were faculty, 559 male and 289 female (M/F = 1.93). M/F ratio increased with academic rank, from 1.0 (117/115) for assistant, to 2.0 (135/68) for associate, and 2.9 (307/106) for full professors. Topics covered by PGR belonged to anatomic pathology (357), clinical pathology (209), research (184) or other medical or surgical specialties (149). Our study suggests that trainees are a major intended audience of pathology grand round. Unfortunately, there is a gender gap among pathology grand round presenters that widens with increasing academic rank of presenters.

Clonal highlights: Clonal seborrheic keratoses often demonstrates p16 expression.

BACKGROUND: At times, distinguishing Bowen disease (BD) and benign seborrheic keratosis (SK) is histologically challenging, especially when SK shows clonal features (clonal seborrheic keratosis [CSK]). While p16 is often reported as positive in BD and negative in SK, p16 expression in CSK is rarely studied. Here we investigate p16 immunohistochemistry in CSK, SK, and BD. METHODS: p16 immunohistochemistry with pattern of expression was noted for 14 CSK, 12 SK, and 18 BD. The degree of inflammation among lesions with respect to p16 expression was also noted. RESULTS: When examining p16 staining in clonal nests of CSK, 57% showed diffuse or patchy/diffuse positivity, 21% showed patchy positivity, and 21% showed clusters of single positive cells. 67% of BD showed diffuse positivity, 11% showed patchy/diffuse positivity, 17% showed patchy positivity, and 6% were negative. 25% of SK showed focal areas of patchy to full thickness positivity, 25% showed moderate number of positive single cells with or without patchy staining, and 50% showed negative/scattered single cell positivity. CONCLUSION: Our findings support that p16 positivity limited to clonal nests in CSK is normal. p16 positivity in clonal nests of CSK in isolation without concurrent atypical histologic features should not be used to support a diagnosis of BD.

Pure Erythroid Leukemia Mimicking Ewing Sarcoma/Primitive Neuroectodermal Tumor in an Infant.

Pure erythroid leukemia (PEL) is a rare type of acute myeloid leukemia (AML) with a very aggressive clinical course. Presentation as a myeloid/erythroid sarcoma is exceedingly rare. We describe an infantile PEL presenting as a multifocal myeloid sarcoma, clinically and pathologically mimicking Ewing sarcoma/PNET family of tumors. The patient died 8 weeks after the initial presentation due to widespread disease. Our case shows that PEL needs to be considered in the differential diagnosis of small round blue cell tumors in infancy. A meticulous workup including immunohistochemistry, flow cytometry, molecular, and cytogenetic studies was required to reach the diagnosis.

Central nervous system involvement by myeloid sarcoma: a report of 12 cases and review of the literature.

Myeloid sarcoma (MS) is an extramedullary malignancy of myeloid origin. It can occur in any organ. Common sites are skin, bone, lymph nodes, and soft tissue. Central nervous system (CNS) involvement is very uncommon. We report 12 new pathology-confirmed cases of CNS MS with literature review. Median age was 42.5 years (range: 0 - 84 years). Bone marrow involvement by hematologic neoplasia was co-incidental (n = 8) or occurred 8 - 51 months prior to CNS MS (n = 3). Abnormal radiological findings detected in all patients, included hemorrhagic (n = 5) or enhancing (n = 2) lesions, with multiple ring-enhancing dura-based masses in 1 patient. Seven tumors had abnormal cytogenetics including: t(11; 19) (q23; p13.3), +8, inv (16), t(9; 22), t(8; 21), del(5q), and +21. One had a complex karyotype and 2 were cytogenetically normal. One MS had the JAK2V617F mutation. Treatment modalities included surgery for decompression (n = 2), radiotherapy (n = 2), chemotherapy (n = 6), and stem cell transplant (n = 2). Nine patients died days to 12 months post CNS MS diagnosis (median = 4 months). Two patients were alive without evidence of disease at 16 and 50 months following MS diagnosis and one was lost to follow-up. The clinical and imaging features for CMS MS overlap with those of intracranial hemorrhage and primary CNS tumors. It is therefore important to maintain a high index of suspicion and perform a biopsy whenever clinically appropriate. A meticulous workup is necessary to avoid misdiagnosis of other hematopoietic or nonhematopoietic neoplasms. Since CNS MS is potentially curable, timely recognition is paramount.

Telecytology validation: is there a recipe for everybody?

INTRODUCTION: Telecytology offers a suitable solution to the cost and time efficiency questions on rapid onsite evaluation (ROSE). An increasing number of institutions are adopting new telecytology systems to meet the increasing ROSE requests, although there is no agreement on the details of how a telecytology validation study needs to be conducted. We propose a standardized approach for telecytology validation studies that could be done in a variety of practices. MATERIALS AND METHODS: Consecutive cases from 6 months prior were chosen to reflect a case mix comparable to real life. A fellow assessed the slides at the ROSE site while 6 cytopathology faculty convened in a conference room with a television screen, and noted the adequacy, diagnostic category, and specific diagnoses. All participants were blinded to the original adequacy assessment and final diagnoses. For each case, evaluation time and the slides counts were noted. RESULTS: Fine-needle aspiration specimens from 52 patients were included in the study. Of these, 13 cases were used in the first "test" session. The adequacy concordance rates ranged between 92.3% and 100%, with an overall concordance rate of 94.8%. The diagnostic category concordance rates ranged between 90.3% and 95.5%, with an overall concordance rate of 91.9%. The specific diagnosis concordance rates ranged between 84.6% and 92.9%, with an overall concordance rate of 88.1%. CONCLUSIONS: Validation of telecytology requires a standardized approach just like any other new technology. In this study, we propose an efficient and accurate method for cytopathology departments of various case volumes to conduct telecytology validation studies.

Accuracy of Subclassification and Grading of Renal Tumors on Fine Needle Aspiration Cytology Alone.

BACKGROUND: Fine needle aspiration (FNA) of renal masses can distinguish between benign and malignant neoplasms in 73-94% of cases. Previous studies suggested the correct subclassification of renal cell carcinomas (RCCs) by cytomorphology can be achieved in up to 80% of cases. However, as RCCs become increasingly subclassified by molecular signatures, correct subclassification based on cytology alone is increasingly difficult. DESIGN: Two FNA passes (2 stained with Diff-Quik® and 2 with the Papanicolaou method) were performed on all fresh nephrectomy specimens for a 1-year period. There were 30 cases in this study, with 29 primary renal tumors and 1 case of metastatic lung adenocarcinoma. Each case was assigned a random number and came with 2 slides (1 from each staining method). Eight cytopathologists were asked to provide a diagnosis and the World Health Organization/International Society of Urological Pathology (WHO/ISUP) grading if applicable. Fleiss' Kappa and Cohen's Kappa equations were used to look at inter-rater variability. RESULTS: When compared to the surgical pathology diagnosis, the average percent correct diagnosis for all cytopathologist was 35%. Chromophobe RCCs had the best average percent accuracy at 72% followed by clearcell RCC at 48%. Average accuracy for grading RCCs was 40%. Inter-rater variability among the cytopathologists for all RCC diagnoses was fair with a Fleiss' Kappa coefficient of 0.28. For the WHO/ISUP grade, the weighted coefficient for each pathologist ranged from 0.11 to 0.45, ranging from fair to moderate, respectively. CONCLUSIONS: Renal tumors are difficult to classify on cytopathology alone. Core needle biopsy and ancillary studies are necessary if diagnosis will change management.

The utilization and utility of immunostains in body fluid cytology.

BACKGROUND: Body fluid cytology (BFC) is an important tool in the diagnosis and staging of malignancy and is aided by the judicious use of immunohistochemistry (IHC). The aim of this study was to determine the usage rates of IHC stains in BFC, their type and indications, and their diagnostic impact. We also attempted to estimate the optimal rate of IHC use in BFC by comparing the entire laboratory's and each individual cytopathologist's IHC use rates with their respective indeterminate and malignant diagnosis rates. METHODS: We conducted a retrospective study of IHC stain use in BFC during a 5.5-year interval (2013-2018) and determined the laboratory's and each individual cytopathologist's IHC usage patterns according to the final diagnosis, site, and indications for their use. RESULTS: A total of 477 out of 4144 (11.5%) BFC cases had 2128 individual immunostains performed, with an average of 4.5 immunostains per case. Individual cytopathologists used IHC stains on 6.7% to 22% of their BFC cases. Pathologists with higher rates of IHC stain use than the laboratory's mean were less experienced and had higher rates of indeterminate but not of malignant diagnoses. The most common indication for the use of IHC stains was differentiating mesothelial from malignant cells. MOC31, calretinin, Ber-EP4, CD68, and D2-40 were the most commonly used of the 67 different IHC stains used in BFC. CONCLUSIONS: The laboratory's mean may represent the optimal IHC use rate, as higher IHC use rates did not lead to more diagnostic certainty or higher pickup rates of malignant cells.

Expression of renal cell markers and detection of 3p loss links endolymphatic sac tumor to renal cell carcinoma and warrants careful evaluation to avoid diagnostic pitfalls.

Endolymphatic sac tumor (ELST) is a rare neoplasm arising in the temporal petrous region thought to originate from endolymphatic sac epithelium. It may arise sporadically or in association with Von-Hippel-Lindau syndrome (VHL). The ELST prevalence in VHL ranges from 3 to 16% and may be the initial presentation of the disease. Onset is usually in the 3rd to 5th decade with hearing loss and an indolent course. ELSTs present as locally destructive lesions with characteristic computed tomography imaging features. Histologically, they show papillary, cystic or glandular architectures. Immunohistochemically, they express keratin, EMA, and variably S100 and GFAP. Currently it is recommended that, given its rarity, ELST needs to be differentiated from other entities with similar morphologic patterns, particularly other VHL-associated neoplasms such as metastatic clear cell renal cell carcinoma (ccRCC). Nineteen ELST cases were studied. Immunohistochemistry (18/19) and single nucleotide polymorphism microarray testing was performed (12/19). Comparison with the immunophenotype and copy number profile in RCC is discussed. Patients presented with characteristic bone destructive lesions in the petrous temporal bones. Pathology of tumors showed characteristic ELST morphology with immunoexpression of CK7, GFAP, S100, PAX-8, PAX-2, CA-9 in the tumor cells. Immunostaines for RCC, CD10, CK20, chromogranin A, synaptophysin, TTF-1, thyroglobulin, and transthyretin were negative in the tumor cells. Molecular testing showed loss of 3p and 9q in 66% (8/12) and 58% (7/12) cases, respectively. Immunoreactivity for renal markers in ELST is an important diagnostic caveat and has not been previously reported. In fact, renal markers are currently recommended in order to rule out metastatic RCC although PAX gene complex and CA-9 have been implicated in the development of the inner ear. Importantly copy number assessment of ELST has not been previously reported. Loss of 3p (including the VHL locus) in ELST suggests similar mechanistic origins as ccRCC.

Baseline gene expression predicts sensitivity to gefitinib in non-small cell lung cancer cell lines.

Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.

The PhenoGen informatics website: tools for analyses of complex traits.

BACKGROUND: With the advent of "omics" (e.g. genomics, transcriptomics, proteomics and phenomics), studies can produce enormous amounts of data. Managing this diverse data and integrating with other biological data are major challenges for the bioinformatics community. Comprehensive new tools are needed to store, integrate and analyze the data efficiently. DESCRIPTION: The PhenoGen Informatics website http://phenogen.uchsc.edu is a comprehensive toolbox for storing, analyzing and integrating microarray data and related genotype and phenotype data. The site is particularly suited for combining QTL and microarray data to search for "candidate" genes contributing to complex traits. In addition, the site allows, if desired by the investigators, sharing of the data. Investigators can conduct "in-silico" microarray experiments using their own and/or "shared" data. CONCLUSION: The PhenoGen website provides access to tools that can be used for high-throughput data storage, analyses and interpretation of the results. Some of the advantages of the architecture of the website are that, in the future, the present set of tools can be adapted for the analyses of any type of high-throughput "omics" data, and that access to new tools, available in the public domain or developed at PhenoGen, can be easily provided.

Mulberry cells in the thyroid: warthin-finkeldey-like cells in hashimoto thyroiditis-associated lymphoma.

Warthin-Finkeldey type giant cells were first described in autopsies performed on young children who died during the highly lethal measles epidemic in Palermo during the winter of 1908. The cells had 8-15 nuclei without identifiable cytoplasm within the germinal centers of lymphoid organs resembling megakaryocytes. We describe a case of Hashimoto thyroiditis with an enlarging substernal throid mass. The resection specimen contained many Warthin-Finkeldey-Like Cells (WFLC) in an extranodal marginal zone lymphoma (MALT type) with focal transformation to diffuse large B-cell lymphoma. The WFLC showed nuclear features similar to those of neighboring follicular dendritic cells (FDCs), favoring the hypothesis that these cells might be the product of fusion of FDCs. This is supported by immunostaining results and the occurrence of similar cells in follicular dendritic cell sarcomas and in "dysplastic" FDCs in hyaline vascular type Castleman disease, a possible precursor of follicular dendritic cell tumors. Diagn. Cytopathol. 2017;45:212-216. © 2016 Wiley Periodicals, Inc.

Red cell genotyping precision medicine: a conference summary.

This review summarizes the salient points of the symposium 'Red Cell Genotyping 2015: Precision Medicine' held on 10 September 2015 in the Masur Auditorium of the National Institutes of Health. The specific aims of this 6th annual symposium were to: (1) discuss how advances in molecular immunohematology are changing patient care; (2) exemplify patient care strategies by case reports (clinical vignettes); (3) review the basic molecular studies and their current implications in clinical practice; (4) identify red cell genotyping strategies to prevent alloimmunization; and (5) compare and contrast future options of red cell genotyping in precision transfusion medicine. This symposium summary captured the state of the art of red cell genotyping and its contribution to the practice of precision medicine.

Combined use of oligonucleotide and tissue microarrays identifies cancer/testis antigens as biomarkers in lung carcinoma.

High density oligonucleotide microarrays (OMAs) have been used recently to profile gene expression in lung carcinoma tissue homogenates. The length of the lists of potentially interesting genes generated by these studies is daunting, and biological and clinical relevance of these lists remains to be validated. Moreover, specific identification of individual biomarkers that might be used for early detection and surveillance has not been the objective of these early studies. We have developed a schema for combining the data derived from the OMA analysis of a few lung cancer cell lines with immunohistochemical testing of tissue microarrays to rapidly identify biomarkers of potential clinical relevance. Initially, we profiled gene expression in lung tumor cell lines using the Affymetrix HG-U95Av2 OMA. RNA from 2 non-small cell lung cancer (NSCLC) cell lines (A549 and H647) and 2 small cell lung cancer (SCLC) cell lines (SHP-77 and UMC-19) were tested. Cells from 1 histologically and cytogenetically normal bronchial epithelial primary culture from a volunteer who had never smoked and 10 samples of histologically unremarkable lung tissue from resection specimens served as normalization controls. Array results were analyzed with Gene Spring software. Results were confirmed by reverse transcription-PCR in an expanded number of cell lines. We then validated the cell line data by immunohistochemical testing for protein using a tissue microarray containing 187 NSCLC clinical samples. Of the 20 most highly expressed genes in the tumor lines, 6 were members of the cancer/testis antigen (CTAG) gene group including 5 MAGE-A subfamily members and NY-ESO-1. SCLC lines strongly expressed all of the MAGE-A genes as well as NY-ESO-1, whereas NSCLC lines expressed a subset of MAGE-A genes at a lower level of intensity and failed to express NY-ESO-1. Reverse transcription-PCR of an extended series of 25 lung cancer cell lines including 13 SCLC, 9 NSCLC, and 3 mesothelioma lines indicated that MAGE-A10 and NY-ESO-1 were expressed only by SCLC, and that MAGE-A1, 3, 6, 12, and 4b were expressed by both SCLC and NSCLC. By immunohistochemistry using the monoclonal antibody 6C1 that recognizes several MAGE-A gene subfamily members, 44% of NSCLC clearly expressed MAGE-A proteins in cytoplasm and/or nucleus. Expression of MAGE-A genes did not correlate with survival but did correlate with histological classification with squamous carcinomas more frequently MAGE-A positive than other NSCLC types (P < 0.00002). We conclude that expression of CTAG gene products, whereas apparently not of prognostic importance, may be useful for early detection and surveillance because of a high level of specificity for central airway squamous and small cell carcinomas.

Candidate genes and their regulatory elements: alcohol preference and tolerance.

QTL analysis of behavioral traits and mouse brain gene expression studies were combined to identify candidate genes involved in the traits of alcohol preference and acute functional alcohol tolerance. The systematic application of normalization and statistical analysis of differential gene expression, behavioral and expression QTL location, and informatics methodologies resulted in identification of 8 candidate genes for the trait of alcohol preference and 22 candidate genes for acute functional tolerance. Pathway analysis, combined with clustering by ontology, indicated the importance of transcriptional regulation and DNA and protein binding elements in the acute functional tolerance trait, and protein kinases and intracellular signal transduction elements in the alcohol preference trait. A rudimentary search for transcription control elements that could indicate coregulation of the panels of candidate genes produced modest results, implicating SMAD-3 in the regulation of four of the eight candidate genes for alcohol preference. However, the realization of the many caveats related to transcription factor binding site analysis, and attempts to correlate between transcription factor binding and function, forestalled any definitive global analysis of transcriptional control of differentially expressed candidate genes.

Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases.

Multicellular organisms have three well-characterized subfamilies of mitogen-activated protein kinases (MAPKs) that control a vast array of physiological processes. These enzymes are regulated by a characteristic phosphorelay system in which a series of three protein kinases phosphorylate and activate one another. The extracellular signal-regulated kinases (ERKs) function in the control of cell division, and inhibitors of these enzymes are being explored as anticancer agents. The c-Jun amino-terminal kinases (JNKs) are critical regulators of transcription, and JNK inhibitors may be effective in control of rheumatoid arthritis. The p38 MAPKs are activated by inflammatory cytokines and environmental stresses and may contribute to diseases like asthma and autoimmunity.

MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF-2 in fibroblasts.

Mitogen-activated protein kinases (MAPKs) are regulated by MAPK kinases (MKKs), which are in turn regulated by MKK kinases (MKKKs). While a single MKKK can regulate several different MAPK family members, and several MKKKs can often activate the same MAPK, emerging evidence indicates a unique role for individual MKKKs in acting as signaling nodes to coordinately activate different subsets of MAPKs in response to specific cellular stimuli. Thus, while there is much apparent overlap in MAPK regulation by different MKKKs, each MKKK serves a specific purpose in regulation of unique cellular functions. The purpose of this study was to define the specific role of MEKK2, an MKKK, in MAPK regulation and cell function. MEKK2 coordinately activates the ERK5 and JNK pathways. Targeted disruption of MEKK2 expression causes loss of ERK5 and JNK activation in response to FGF-2 in mouse embryonic fibroblasts (MEFs). FGF-2 receptor signaling requires MEKK2 for induction of mRNA for c-Jun, Fra-1, and Fra-2, components of the AP-1 transcription complex. In FGF-2-stimulated MEKK2-/- fibroblasts, c-Jun phosphorylation is inhibited, consistent with a loss of JNK activation. Thus, MEKK2 regulates AP-1 activity at two levels, by regulating both expression of AP-1 components and c-Jun N-terminal phosphorylation. One function of the AP-1 transcription complex is to regulate cytokine gene expression. Expression of IL-1alpha, IL-1beta, IL-6, and TNFalpha is inhibited in MEKK2-/- fibroblasts. Bacterial lipopolysaccharide (LPS) and TNFalpha neither activate ERK5 nor require MEKK2 for JNK activation, demonstrating specificity of MEKK2 in FGF-2 receptor signaling and control of cytokine gene expression.