Deep-penetrating nevi (DPNs) are characterized by activating mutations in the MAP kinase and Wnt/b-catenin pathways that result in large melanocytes with increased nuclear atypia, cytoplasmic pigmentation, and often mitotic activity. Together with a lack of maturation, this constellation of findings creates challenges for pathologists to distinguish DPN from DPN-like melanoma. To assess the utility of next-generation sequencing (NGS) in resolving this diagnostic dilemma, we performed NGS studies on 35 lesions including 24 DPNs and 11 DPN-like melanomas to characterize the specific genomic differences between the 2 groups and elucidate the genetic events involved in malignant transformation of DPNs. Compared with DPNs, DPN-like melanomas were clinically larger in size (1.1 vs 0.6 cm, P = .02) and on histopathologic examination more frequently showed high-grade nuclear atypia (11/11 vs 9/24; P = .00052), increased mitotic activity (mean 3.9 vs 1.3 per mm2; P = .0004), sheet-like growth pattern (5/11 vs 2/24; P = .01), and involvement of the subcutis (4/5 vs 3/13; P = .026). From a genomic standpoint, DPN-like melanomas had a higher tumor mutation burden (mean 37.1 vs 7.8 mutations/megabase; P = .002) than DPNs and more frequently harbored NRAS mutation (3/11 vs 1/24; P = .046), whereas MAP2K1 in-frame deletions were only identified in DPNs (0/11 vs 5/24). There was no statistically significant difference in the frequency or type of CTNNB1 or APC mutations between the 2 groups. Within progression genes, DPN-like melanomas were more frequently found to have pathogenic variants in TERT promoter (7/11 vs 0/24; P < .00001), CDKN2A (4/11 vs 0/24; P = .0008), and protein subunits of the SWI/SNF complex (7/11 vs 3/24; P =.02) compared with DPNs. Our findings provide a framework for employing NGS in the evaluation of deep-penetrating melanocytic tumors. (c) 2025 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

ABSTRACT:Characterizing the typical clinical, morphologic, and molecular findings of distinct subtypes of Spitz neoplasms may help facilitate diagnosis and management of these cases. Approximately 1%-2% of Spitz neoplasms have a MET fusion driver. However, there is minimal data in the current literature regarding this specific subtype of Spitz neoplasm. Most cases in our series (9/10) were diagnosed as atypical Spitz tumors, whereas 1 case was diagnosed as a Spitz nevus. We found 3 characteristic morphologic patterns, the first consisted of either expansile nests of spindle shaped spitzoid melanocytes with plexiform arrangement in the dermis forming a large nodular lesion or thin chords and fascicles of spindle shaped spitzoid melanocytes with a tightly interwoven plexiform pattern in the dermis forming a plaque-like architecture. The second pattern consisted of sheets of epithelioid melanocytes with spitzoid cytology, and the third pattern consisted of a both epithelioid and spindle shaped cytology with plexiform arrangement in the dermis as seen in various subtypes of Spitz neoplasms. Compared to a cohort of 81 control Spitz neoplasms, MET fusions were more likely to have spindle cytology and a plexiform growth pattern in the dermis. The most common fusion pattern was ZKSCAN1 (4/10). Copy number gains of the fusion gene were frequent, seen in 61% of cases. None of our cases had a TERT promoter mutation or homozygous deletions of CDKN2A . All of our patients had an uneventful clinical course with no evidence of recurrence after reexcision with average follow-up time of 35 months.

INTRODUCTION:There is an increasing demand to optimize the workflow and maximize tissue available for next-generation sequencing (NGS) for non-small cell carcinoma. We looked at transbronchial needle endobronchial ultrasound-guided bronchoscopy with transbronchial needle aspiration samples and evaluated the performance of supernatant (SN) fluid processed from a dedicated aspirate collected for NGS testing. MATERIALS AND METHODS:Nineteen samples were collected and processed using a new workflow. Five aspirates were collected in formalin. One additional dedicated pass was collected fresh and centrifuged. The resulting cell pellet was added to formalin for cell block (CB) processing. DNA and RNA were extracted from concentrated SN for targeted testing using the Oncomine Precision Assay (Thermo Scientific, Waltham, MA). NGS results from the corresponding CB samples were used as "controls" for comparison. RESULTS:Thirty-one mutations were detected in SN (Table 1). The most frequently mutated genes were TP53 (35%), EGFR (23%), KRAS (13%), CTNNB1 (6%), and ERBB2 (6%). There was 100% concordance between the mutations detected in SN and corresponding CBs with comparable variant allele frequencies. Turnaround time of NGS results was 1 day for SN compared to 4-10 days for CB. CONCLUSIONS:We were able to demonstrate the usefulness of SN for reliable rapid molecular results. We successfully incorporated the workflow for tissue handling and processing among our clinical, cytopathology, and molecular teams. Molecular results were available at the same time as the cytologic diagnosis, allowing for timely reporting of a comprehensive diagnosis. This approach is particularly useful in patients with advanced disease requiring urgent management.

Chronic myeloid leukemia (CML) is associated with several breakpoint regions that result in different BCR::ABL1 fusion transcripts. These include the major breakpoint region (M-BCR), minor breakpoint region (m-BCR), and mu breakpoint region (u-BCR) corresponding to p210, p190, and p230 fusion transcripts, respectively. This patient is a 38-year-old female with a new diagnosis of CML in chronic phase. A novel p210 fusion transcript splice variant was detected with qualitative reverse transcription PCR and capillary electrophoresis. Subsequent FISH study was performed, which revealed 86.5% positive for the BCR::ABL1 fusion. Quantitative real-time polymerase chain reaction (PCR) showed a negative result for the p210 fusion transcript. The variant was further characterized by Sanger sequencing. This variant is in-frame and predicted to be functional. This case illustrates the need for a combination of different testing techniques to fully characterize the rare BCR::ABL1 fusion transcripts.

ABSTRACT:Telomerase reverse transcriptase promoter mutations (TPMs) have been shown to be common in melanoma and uncommon in benign nevi. To assess the use of TPMs as an ancillary diagnostic tool, we report the concordance of the TPM status with the final diagnosis in clinical cases with distinct differential diagnostic scenarios: dysplastic nevus versus melanoma, atypical Spitz nevus versus melanoma, atypical deep penetrating nevus (DPN) versus melanoma, and atypical blue nevus versus malignant blue nevus. In a control cohort, we found a positive TPM in 51/70 (73%) of the total melanomas with the highest frequency in vertical growth phase melanoma cases. Conversely, only 2/35 (6%) dysplastic nevi in our control cases were TPM-positive and b were severely atypical dysplastic nevi. Our clinical cohort of 257 cases had a positive TPM in 24% of cases diagnosed as melanoma and in 1% of cases with a benign diagnosis. The overall concordance of the TPM status with the final diagnosis was 86%. The TPM status had the greatest concordance (95%) with the final diagnosis in the atypical DPN versus melanoma group, with the rest of the groups ranging between 50% and 88%. Overall, our results suggest that TPMs are most useful in the differential diagnosis of atypical DPN versus melanoma. It also has some value in the differential diagnosis of atypical Spitz tumor versus melanoma and dysplastic nevus versus melanoma, whereas in our cohort, it did not contribute meaningfully to differentiating malignant blue nevus and atypical blue nevus.

ABSTRACT:Proliferative nodules (PNs) are benign melanocytic proliferations that typically develop within congenital melanocytic nevi. These tumors have overlapping histological features with melanoma. Ancillary immunohistochemistry and genomic sequencing are often used in diagnostically challenging cases. To assess the utility of preferentially expressed antigen in melanoma (PRAME) immunoreactivity and telomerase reverse transcriptase (TERT) promoter mutation analysis in distinguishing PNs from melanoma arising in congenital nevi cases. Twenty-one PNs and 2 melanomas arising in congenital nevi were immunohistochemically stained with PRAME. Cases with adequate tissue were also assessed for TERT promoter mutations through sequencing studies. The positivity rates in the PN cases were compared with those of the melanomas. Two of 21 PN cases were diffusely positive for PRAME (≥75% of the tumor cells positive). Two of 2 melanomas arising in congenital nevus cases were also diffusely PRAME positive. The difference was statistically significant using a Fisher exact test. None of the tumors harbored TERT promoter mutations. PRAME immunohistochemical marker may have diagnostic value in distinguishing diagnostically challenging PNs from melanoma, but diffuse expression is not specific for melanoma.

medulloblastoma, meningioma and schwannoma show low misdiagnosis rate by DNA methylation profiling

The 2021 WHO Classification of Tumors of the Central Nervous System includes several tumor types and subtypes for which the diagnosis is at least partially reliant on utilization of whole genome methylation profiling. The current approach to array DNA methylation profiling utilizes a reference library of tumor DNA methylation data, and a machine learning-based tumor classifier. This approach was pioneered and popularized by the German Cancer Research Network (DKFZ) and University Hospital Heidelberg. This research group has kindly made their classifier for central nervous system tumors freely available as a research tool via a web-based portal. However, their classifier is not maintained in a clinical testing environment. Therefore, the Northwestern Medicine (NM) classifier was developed and validated. The NM classifier was validated using the same training and validation data sets as the DKFZ group. Using the DKFZ validation data set, the NM classifier's performance showed high concordance (92%) and compa-rable accuracy (specificity 94.0% versus 84.9% for DKFZ, sensitivity 88.6% versus 94.7% for DKFZ). Receiver-operator characteristic curves showed areas under the curve of 0.964 versus 0.966 for NM and DKFZ classifiers, respectively. In addition, in-house validation was performed and performance was compared using both classifiers. The NM classifier performed comparably well and is currently offered for clinical testing. (J Mol Diagn 2022, 24: 924-934; https://doi.org/10.1016/j.jmoldx.2022.04.009)

Monkeypox has recently been described as a public health emergency of international concern by the World Health Organization and a public health emergency by the United States. If the outbreak continues to grow, rapid scalability of laboratory testing will be imperative. During the early days of the coronavirus disease 2019 (COVID-19) pandemic, laboratories improved the scalability of testing by using a direct-to-PCR approach. To improve the scalability of monkeypox testing, a direct real-time PCR protocol for the detection of monkeypox virus was validated. The assay retains the sensitivity and accuracy of the indirect assay while eliminating the need for nucleic acid extraction kits, reducing laboratory technologist time per sample and decreasing exposure to an infectious agent. The direct method will make it easier for laboratories across the world to rapidly develop, validate, and scale testing for monkeypox virus.(J Mol Diagn 2022, 24: 1155-1159; https://doi.org/10.1016/j.jmoldx.2022.09.001)

Abstract Objectives The novel coronavirus, severe acute respiratory syndrome coronavirus 2, causing coronavirus disease 2019 (COVID-19) remains a global health threat and a significant source of human morbidity and mortality. While the virus primarily induces lung injury, it also has been reported to cause hepatic sequelae. Methods We aimed to detect the virus in formalin-fixed tissue blocks and document the liver injury patterns in patients with COVID-19 compared with a control group. Results We were able to detect viral RNA in the bronchioalveolar cell blocks (12/12, 100%) and formalin-fixed, paraffin-embedded tissue of the lung (8/8, 100%) and liver (4/9, 44%) of patients with COVID-19. Although the peak values of the main liver enzymes and bilirubin were higher in the patients with COVID-19 compared with the control group, the differences were not significant. The main histologic findings were minimal to focal mild portal tract chronic inflammation (7/8, 88%, P < .05) and mild focal lobular activity (6/8, 75%, P = .06). Conclusions We found that most patients who died of COVID-19 had evidence of mild focal hepatitis clinically and histologically; however, the virus was detected in less than half of the cases.

BACKGROUND:Transformation of follicular lymphoma most typically occurs as diffuse large B-cell lymphoma, however other forms of transformation such as classic Hodgkin lymphoma and lymphoblastic transformation can occur. Secondary malignant histiocytosis also represents a rare form of transformation, which is thought to occur due to a process of transdifferentiation whereby the lymphoma cells exhibit lineage plasticity and lose all evidence of B-cell phenotype and instead acquire the phenotype of a histiocytic neoplasm. Little is known about the underlying genetic alterations that occur during this unusual process. Comparative genetic analysis of pre- and post-transformation/transdifferentiation would be one tool by which we could better understand how this phenomenon occurs.CASE PRESENTATION:Here we report the clinical, immunophenotypic and genetic features of a rare case of secondary malignant histiocytosis, Langerhans cell-type (Langerhans cell sarcoma) arising from a previous low grade follicular lymphoma. FISH analysis confirmed the presence of IgH/BCL2 rearrangement in both the low grade follicular lymphoma (FL) and transformed Langerhans cells sarcoma (LCS) samples, demonstrating a clonal relationship. Comparative whole exome sequencing was then performed, which identified a KRAS p.G13D mutation in the LCS that was not present in the FL.CONCLUSIONS:This report highlights genetic alterations, in particular an acquired somatic KRAS mutation, that may occur during transdifferentiation, with additional significance of KRAS mutation as a possible therapeutic target in cases which otherwise would have limited treatment options.

Aims: Enhancer of Zeste Homolog 2 (EZH2) is a critical enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which provides inhibitory regulation of gene transcription through trimethylation of histone H3 on lysine 27 (H3K27me3). The somatic mutation EZH2 Y641 in its highly conserved catalytic SET domain has been reported in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) by methods of Sanger sequencing and whole exome sequencing. To develop a clinically applicable assay uses pyrosequencing method for accurate detection of EZH2Y641 mutations in formalin- fixed-paraffin-embedded (FFPE) tissue. Methods: We validated the pyrosequencing method for the detection of EZH2 Y641 mutation in DLBCL cell lines. We also screened a cohort of 43 DLBCL and 12 FL cases by pyrosequencing, and correlated with the results of Sanger sequencing of EZH2 exon 15. Results: We detected EZH2 Y641 mutations in 3/22 (13.6%) germinal center B cell-like DLBCL, 2/12 (16.7%) FL and none of the 21 activated B cell-like DLBCL. The mutations detected by pyrosequencing were in complete agreement with Sanger sequencing. We have identified the most common 4 types of mutations of EZH2 Y641. Conclusion: The rapid and accurate detection of the EZH2 mutation fromFFPE tissue sections in the clinical laboratory will facilitate screening patients with DLBCL and FL for enrollment of clinical trials using EZH2 inhibitors.

Background : The BCR-ABL negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF), share an increased risk of thrombotic and hemorrhagic complications. The risk of bleeding and thrombosis is less defined in MPN-Unclassifiable (MPN-U). Risk factors for hemorrhage are less defined compared to thrombosis, but acquired von Willebrand disease (aVWD) and thrombocytosis have been implicated. Because patients (pts) with CALR mutations have higher platelet counts compared to JAK2 V617F mutated pts, bleeding rates may be increased in the former group . Our aim was to define the prevalence and bleeding type and to evaluate whether aVWD, thrombocytosis, mutational status or treatment history associated with bleeding in a large cohort of MPN pts. Methods: The Northwestern University Electronic Data Warehouse identified MPN pts ≥18 years, last seen between 2005 and 2013. MPN was diagnosed based on clinical features, histopathology, and mutational status. A significant bleeding event was defined by a need for medical evaluation. Mutations in exon 9 of CALR were evaluated by PCR and fragment analysis. aVWD was defined by vW antigen (ag) and/or ristocetin cofactor activity below the laboratory reference range for blood type (<40% for blood group O or <53% for non blood group O; type defined by vW ag: activity ratio). Associations were tested using Fisher’s exact test; p<0.05 was considered statistically significant. Results I: Baseline characteristics 351 MPN pts were identified: 142 with ET (40.4%), 118 with PV (33.6%), 62 with MF (17.7%), 4 with MDS/MPN (1.1%), and 25 with MPN-U (7%). Median follow-up was 5 years, median age at diagnosis was 54.7 years, and 58.1% were female. 73.3% were treated with aspirin, and 51.6% were treated with hydroxyurea. JAK2 V617F was identified in 178/288 patients (61.8%). Among 110 JAK2 V617F -negative samples, residual DNA was available in 46 and 15/46 (32.6%) were CALR mutated, including primarily type 1 (52-bp deletion; c.1092\_1143del, N=11), type 2 (5-bp insertion; c.1154\_1155insTTGTC, N=3) mutations, and 1 with a 69-bp deletion. CALR mutated patients included 10/29 ET (34.5%), 2/7 MF (28.6%), and 3/9 MPN-U (33%). To date, 34 patients in the cohort (9.8%) progressed to MF and 18 (5.2%) had leukemic transformation. Results II: Bleeding complications 55 pts (15.6%) experienced 62 bleeding events, while thrombotic events were reported in 84 cases (23.9%). 19% of cases had both bleeding and thrombosis. In bleeding pts, the mean white blood count (WBC) at the time of diagnosis was 15.0 x 109/L, mean platelet count 803 x 109/L, and mean hemoglobin 12.6 x 109/L. Bleeding occurred at a median of 2 years after diagnosis. Gastrointestinal bleeding (GIB) was the most common type in 28/55 pts (50.9%), including 10 upper GIBs, 9 lower GIB, and 9 cases involving an unspecified location. Bleeding episodes from mucocutaneous sites were noted in 17/55 pts (30.9%; 9 with epistaxis, 1 with gum bleeding, and 7 with vaginal/uterine bleeding). There were 6 pts with intracranial hemorrhage. The mean platelet count at the time of bleeding was 473 x 109/L. Bleeding events were significantly more common in MPN-U (32%) than MF (19%), ET (10.6%), or PV (15.3%), (p=0.0163). Bleeding was associated with older age at diagnosis (59.4 yrs in bleeding pts vs 53.9 yrs in non-bleeders, p=0.03). There was no association between bleeding and mutational status ( JAK2 V617F, CALR ), gender, or aspirin use. The median ristocetin activity was 94% (N=38), vW ag 110% (N=37) and factor VIII activity 94% (N=28). Of 38 pts, there were 7 (18.4%) cases of VWD; 6 were acquired (5 with type 2, 1 with type 1) and 1 was congenital, respectively. Two of 7 pts with VWD (28.6%) had bleeding events. Conclusions Consistent with prior reports, bleeding was less prevalent than thrombosis, associated with older age and, uniquely MPN-U, an entity with a less defined natural history compared to ET, PV, or MF. VWD was infrequently tested for, even less commonly identified, and less represented in bleeding cases. The role of routine testing for aVWD requires further definition. We did not find any association between bleeding and aspirin use or mutational status. However, a limited number of CALR mutated patients may have precluded recognition of any association, and future studies with a larger sample of CALR mutated patients are needed to identify its impact on bleeding. Disclosures No relevant conflicts of interest to declare.

We compared 1 subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgVH) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgVH gene mutational status, but none of them demonstrated high concordance rates. Of the objective methods, ZAP-70 staining determined as a ratio of molecules of equivalent soluble fluorochrome intensity in CLL cells to that in normal B cells (ZAP-70+ staining in IgVH germline cases, 59%; ZAP-70- in IgVH mutated cases, 75%) or T cells (ZAP-70+ in IgVH germline cases, 66%; ZAP-70- in IgVH mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgVH germline and ZAP-70- status to IgVH mutated cases. The subjective method based on ZAP-70 expression in natural killer/T cells gave a similar result, but reproducibility between laboratories may be difficult. Further studies on ZAP-70 expression in relation to clinical parameters may address whether ZAP-70 is an independent prognostic marker for CLL.