Patients with Classic Hodgkin Lymphoma and Follicular Lymphoma Compared to Single Malignancy Controls.

Classic Hodgkin lymphoma (CHL) can arise in patients with low-grade B-cell lymphoma. The features of CHL arising in follicular lymphoma (FL) and its outcome are still unclear, mainly due to the very few cases reported. This study compares 17 patients with CHL and FL to 2 control groups: 1 of 26 patients with FL and a second of 60 patients older than 40 when diagnosed with CHL. Of the FL and CHL patients, 8 had simultaneous FL and CHL, while 9 had FL first, followed by CHL 4.7 years later on average. The age at the diagnosis of FL was 61 years for patients with synchronous FL and CHL and of 60 years for FL, followed by CHL at 65 years. Patients with FL only were, on average, 59 years old at presentation, while CHL patients were 61. FL was grade 1-2 in 75% of FL and CHL patients and 67% of FL first and CHL second patients, lower proportions than in the FL control group-92%. Epstein-Barr virus (EBV) was detected in a lower fraction (29%) of the FL and CHL group than in CHL-only controls (46%). BCL2 translocations were detected in 4 of the 7 cases with FL, but in positive cases, the rearrangement was also present in the CHL component, indicating a clonal relationship between FL and CHL. Patients with FL and CHL treated for CHL had an initial outcome more similar to FL than to CHL controls.

Formalin Fixation Followed by Paraffin Embedding Allows Long-Term Storage of Proteins for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

In an anatomical pathology laboratory, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to characterize amyloid deposits identified in formalin-fixed paraffin-embedded tissue (FFPET). However, the development of additional tests is partially limited by the lack of information the passage of time has on the proteins in FFPET. To investigate the reliability of LC-MS/MS in the analysis of old FFPET specimens, 1 bone marrow aspirate clot was analyzed by LC-MS/MS yearly from 2014 to 2018, in 3 consecutive months. Peptide-spectrum match, number of peptides identified, and percentage of the proteins covered were the parameters collected for the hemoglobin subunits alpha (HbA), beta (HbB), delta (HbD), and gamma (HbG). These proteins are constant components of the peripheral blood and are present in high and low abundance, allowing the monitorization of the performance of the test across varying protein concentrations. The hemoglobin subunits were stable over the years studied; 71% to 74% of HbA, 77% to 80% of HbB, 69% to 77% of HbD, and 57% to 63% of HbG were covered, with no statistical difference between 2014 and 2018. The number of peptides identified was also constant, 11 to 13 for HbA, 13 to 15 for HbB, 11 to 14 for HbD, and 7 to 9 for HbG. Peptide spectrum match was only slightly more variable: 209 to 327 for HbA, 569 to 1052 for HbB, 286 to 533 HbD, and 142 to 292 for HbG. In conclusion, high abundance hemoglobins, HbA and HbB, and relatively low abundance ones, HbD and HbG, are preserved in FFPET and confidently identified by LC-MS/MS for at least 5 years.

Myeloid Sarcoma Involving the Testis in Adults: Clonal Evolution of Acute Myeloid Leukemia.

To describe the clinical, histologic, immunophenotypic, and genetic characteristics of myeloid sarcoma (MS) diagnosed in the testes of adults, 3 cases were identified, and information on their presentation, clinical features, treatment, and outcome was retrieved from the medical records. In addition, histologic, immunophenotypic, and molecular characteristics were reviewed. This showed that all patients had a previous history of acute myeloid leukemia (AML), in 2 cases diagnosed >10 years before the testicular lesions. In 1 case, there was bilateral involvement, while in 2, involvement was unilateral. The neoplastic cells showed evidence of cytogenetic/molecular clonal evolution in all cases, 1 of which also had significant immunophenotypic changes. A mutational profile including NPM1 p.Trp288Cysfs*12, IDH1 p.Arg132His NRAS p.Gly12Asp was seen in 2 of the 3 cases. Concurrent bone marrow involvement by a myeloid neoplasm was diagnosed in 2 patients, in 1, there was AML in the second 8% blasts. These patients progressed rapidly after MS and had a dismal outcome. The patient with no concurrent bone marrow disease had a favorable outcome. In conclusion, MS involving the testes of adults is a rare event, and it may represent the clonal evolution of AML.

Pathology Residents as Testing Personnel in the Hematology Laboratory.

CONTEXT.­: Clinical laboratories and the training of pathology residents are tightly regulated environments. Compliance with regulatory requirements must be addressed when developing entrustable professional activities (EPAs) for pathology residents. OBJECTIVE.­: To describe the development of EPAs for peripheral blood and body fluid review in compliance with Clinical Laboratory Improvement Amendments and College of American Pathologists personnel and testing requirements. To examine the impact of EPA implementation on the workflow in a busy hematology laboratory. DESIGN.­: A training program was designed to prepare pathology residents to function as independent testing personnel in compliance with Clinical Laboratory Improvement Amendments. After a series of lectures, hands-on microscopy sessions, self-assessment quizzes, and achievement of a passing score on a training assessment exam, residents were deemed competent to release certain results independently. The volume and the turnaround time of hematology tests were compared before and after residents were integrated into the laboratory workflow. Faculty and residents were surveyed to assess satisfaction with the training. RESULTS.­: Empowering residents to independently release noncritical results from peripheral blood and body fluid reviews had no adverse impact on test turnaround time. The resident contribution to workflow resulted in a corresponding decrease in the number of cases that required attending pathologist review. Faculty and residents viewed the EPAs as beneficial to service and education. CONCLUSIONS.­: The implementation of the EPAs had a beneficial effect on the laboratory, the trainees, and faculty. Our experience may be helpful to other training programs as EPAs become more widely implemented in residency training.

A Phase II Trial of Imatinib Mesylate as Maintenance Therapy for Patients With Newly Diagnosed C-kit-positive Acute Myeloid Leukemia.

INTRODUCTION: Adults with acute myeloid leukemia (AML) have a high rate of remission; however, more than 50% relapse. C-kit is expressed in approximately 60% of patients with de novo AML and represents a potential therapeutic target. MATERIALS AND METHODS: Patients with newly diagnosed AML received 12 months of imatinib mesylate as maintenance therapy after the completion of post-remission therapy. The primary objective was to determine whether this approach improved progression-free survival (defined as no relapse and no death) compared with historical controls. RESULTS: The median progression-free survival of patients < 60 years of age was 52.1 months (historical control, 13 months) and for patients ≥ 60 years of age was 10.7 months (historical control, 8 months). The median level of AF1q expression was high (9.59), and 84% of patients had moderate or high levels of drug-resistance factors. CONCLUSIONS: Imatinib maintenance therapy may improve the outcome of newly diagnosed patients with AML who are < 60 years of age.

Amyloid in the breast: retrospective review with clinicopathological and radiological correlation of 32 cases from a single institution.

AIMS: Mammary amyloid is an uncommon and easily overlooked pathological diagnosis with ambivalent presentation. Herein, we delineate the clinicopathological and radiographic characteristics of mammary amyloid. METHODS AND RESULTS: The Department of Pathology database was searched from 1993 to 2019 for keywords 'breast' and 'amyloid', yielding 32 cases from 23 patients, including consultation cases. All patients were female, age range = 52-81 (mean = 67.4 years). The left breast was involved more than the right (43 versus 33%, respectively); bilateral amyloid involvement was also present (24%). Amyloid was most often associated with a benign histopathological diagnosis (57%), lymphoma in 39% [all B cell lymphomas; five of nine were mucosa-associated lymphoid tissue (MALT) lymphoma] and rarely with a concurrent epithelial malignancy (invasive lobular carcinoma, 4%). Of the 14 patients with available clinical history, amyloid presented as a mass clinically or radiographically (six patients, 43%), as microcalcifications (five patients, 36%), and only occasionally as an asymmetry (14%) or fibroglandular density (7%). Microscopic examination detected microcalcifications in an additional nine cases (total 14 patients; 44% of the cohort). Interestingly, one patient had concurrent epithelial and haematological malignancy and amyloid within an axillary lymph node. Co-morbidities included autoimmune diseases and multiple myeloma. CONCLUSION: The majority of mammary amyloid cases are associated with benign histopathological findings, while imaging most frequently noted microcalcifications or mass lesions. To avoid overlooking amyloid as simply fat necrosis or fibroelastotic stromal change, a low threshold for performing ancillary stains should be considered in elderly women with benign core needle findings performed for mass lesions or microcalcifications.

Extranodal Marginal Zone Lymphoma of the Central Nervous System Includes Parenchymal-Based Cases With Characteristic Features.

OBJECTIVES: To define the clinicopathologic features of extranodal marginal zone lymphoma (EMZL) of the central nervous system (CNS), including cases arising in CNS parenchyma, which have been reported only rarely. METHODS: Twelve cases of CNS EMZL were identified, including 5 based in CNS parenchyma and 7 nonparenchymal cases arising in dura or choroid plexus. RESULTS: Histologically, parenchymal cases were perivascular infiltrates without a dominant lymphoid mass, whereas nonparenchymal cases were masses of small lymphocytes. Plasma cells were a larger component of the infiltrate in parenchymal cases (median, 30%; range, 20%-50%) than nonparenchymal cases (median, 0%; range, 0%-5%; P < .001), and plasma cells were clonal by immunohistochemistry in 4 of 5 parenchymal vs 1 of 7 nonparenchymal cases (P = .07). Fluorescence in situ hybridization for MALT1 rearrangement was positive in 1 of 3 parenchymal and none of 3 nonparenchymal cases. Chromosomal microarray was abnormal in 5 of 7 cases (71%), with chromosome 6/6q alterations identified in 3 cases. No patients with parenchymal disease but all 6 (100%) with nonparenchymal disease achieved complete remission. CONCLUSIONS: This case series, the first to include multiple parenchymal cases, clarifies the spectrum of clinical, pathologic, and genetic findings in CNS EMZL and suggests that parenchymal-based lesions may show less favorable prognosis than dural-based disease.

IgA plasma cell neoplasms are characterized by poorer long-term survival and increased genomic complexity compared to IgG neoplasms.

The characteristics of the IgA plasma cell neoplasms are not clearly reported in the literature. The main goal of this study is to examine IgA plasma cell neoplasms (PCN) and to compare them to IgG lesions. After at least 5 years from the identification of an M protein, 98 cases were selected, the presentation and clinical evolution of 45 IgA neoplasms were compared to 43 cases of IgG gammopathies. The classification at presentation as monoclonal gammopathy of undermined significance (MGUS)-22 of 45 IgA and 20 of 43 IgG (49 vs 46%), plasma cell myeloma (PCM)-22 of 45 IgA and 22 of 43 IgG (49 vs 51%) and smoldering PCM (SPCM)-1 each (2% for both) was essentially identical. No solitary plasmacytomas were identified. At presentation, IgA patients were younger (66.5 ± 11.3 vs. 69.2 ± 10.7 years), less likely to have bone lesions (12/45 vs 18/43, p < 0.14) or immunoparesis (51% vs. 63%), differences statistically insignificant. Cases with normal fluorescence in-situ hybridization (FISH) results, 27% for IgA vs 61% for IgG (p < 0.037) were statistically different. The IgA patients had worse survival (80 vs 108 months median IgA vs IgG, p < 0.013), difference not detectable in the first 5 years, but substantial after 10. In conclusion, poorer long-term survival and increased genomic complexity by FISH are characteristics of IgA PCNs.

Quantification of the Effectiveness of a Residency Program Using the Resident In-Service Examination.

This study describes a quantitative tool in the assessment of residency programs, in which national ranking of residents after the resident in-service examination in postgraduate year 4 is compared to that in postgraduate year 1. The relationship between training and changes in ranking, resident in-service examination results before and after training in specific areas are also compared. To illustrate the use of this novel approach, data from a large residency program were analyzed. The 70 residents were ranked as a postgraduate year 1 group at the 50th national percentile. As postgraduate year 4 residents, they were ranked at the 59th percentile, a significant (P < .003) improvement. There was moderate correlation between performance in postgraduate year 1 and that in postgraduate year 4 (0.61); however, initial ranking was no indication of the final (R2 = .34), with the exception of high performers. Training in specific areas improved ranking, demonstrating association between training and performance. In conclusion, the effectiveness of training provided by a residency program can be quantified using the resident in-service examination. This should provide a quantitative tool in the assessment of postgraduate programs.

Leukemogenic nucleophosmin mutation disrupts the transcription factor hub that regulates granulomonocytic fates.

Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules.

Ocular Adnexal Amyloidosis: A Mass Spectrometric Analysis.

PURPOSE: Ocular adnexal amyloidosis (OAA) may represent localized manifestation of an underlying systemic process. Accurate identification of the amyloid fibrils can guide the systemic evaluation and treatment. The aim of this study was to characterize subtypes of OAA using immunohistochemistry and mass spectrometric analysis and to correlate with ocular involvement and systemic association. DESIGN: Retrospective case series. METHODS: Review of patients with OAA subtyped by immunohistochemistry and mass spectrometric analysis at the Cleveland Clinic from June 1995 to June 2017. RESULTS: While immunohistochemistry identified AL amyloid protein in 67% (4/6) of specimens tested, mass spectrometry identified AL amyloid protein in all specimens (10/10). AL lambda was identified in 5 (50%) samples, kappa in 3 (30%), and both kappa and lambda light chains in 2 (20%). The 5 cases of conjunctival amyloidosis were either AL lambda only (3 cases) or both lambda and kappa (2 cases). There were 3 cases that had associated systemic involvement. Two of these had eyelid skin involvement and AL kappa amyloidosis and the other patient had uveal involvement and AL lambda amyloidosis. CONCLUSIONS: Primary amyloidosis-AL is the most common form diagnosed by mass spectrometric analysis in patients with OAA. Immunohistochemistry is ineffective in the characterization of the amyloid deposits in a significant number of cases. Evaluation to exclude systemic involvement or associated underlying lymphoproliferative disorder is warranted.

Dendritic Cell Markers and PD-L1 are Expressed in Mediastinal Gray Zone Lymphoma.

AIMS: Mediastinal gray zone lymphoma (MGZL) is a rare entity with morphologic, immunophenotypic, and genetic features intermediate between classic Hodgkin lymphoma (CHL) and primary mediastinal large B-cell lymphoma (PMBL). It is challenging to differentiate from CHL and PMBL. A specific dendritic cell gene expression profile can distinguish CHL and MGZL from PMBL. We hypothesized that the dendritic markers fascin and CD123 may be helpful in distinguishing MGZL from CHL and PMBL. We also investigated programmed death-ligand 1 (PD-L1) expression in MGZL, which may have therapeutic significance in this difficulty to treat tumor. METHODS: Representative sections from 89 CHL, 20 PMBL, and 7 MGZL cases were stained for fascin, CD123, and PD-L1, and scored on a scale from 0 to 3+. Most (71%) MGZLs stained for CD123, as well as some (23%) CHLs, and few (11%) PMBLs. All MGZLs stained for fascin, as well as most (90%) CHLs, and approximately half (53%) of the PMBLs. PD-L1 was positive in all MGZLs, most (77%) CHLs and most (66%) PMBLs. CONCLUSIONS: Our study is the first to show CD123 is positive in a subset of formalin-fixed, paraffin-embedded MGZLs and CHLs, in contrast to PMBL which is largely negative. Staining for fascin was not significantly different between the lymphomas, but was less likely to be positive in PMBL. These findings suggest a role for CD123 and fascin in supporting diagnoses of MGZL and CHL, and in ruling out PMBL. By immunohistochemistry, PD-L1 is positive in MGZL, pointing to its therapeutic potential.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Intravascular T-cell lymphoma: A rare, poorly characterized entity with cytotoxic phenotype.

Intravascular T-cell lymphomas are rare, poorly characterized lesions. We discuss the clinical, radiologic and especially the laboratory characteristics of a lesion which presented in a 62-year-old woman with a history of progressive CNS abnormalities. Throughout the course of the disease, radiologic findings consisted mainly of multifocal mixed areas of ischemia and vasogenic edema involving cortical and subcortical regions. A brain biopsy identified an abnormal T-cell population confined to lumens of vessels. These T-cells were abnormal cytotoxic cells, positive for CD3, CD8, and negative for CD2, CD4, CD5, CD7 and CD30. While flow cytometry and immunohistochemistry failed to identify a similar population in the blood or bone marrow, molecular studies showed a clonal T-cell population in both the brain and the bone marrow. No other organs were involved. In spite of aggressive treatment, the patient's medical condition continued to progress and she passed away. In conclusion, this abnormal population of cytotoxic T-cells with intravascular localization probably represents a specific type of T-cell lymphoma with specific clinical, radiologic, molecular and immunophenotypic characteristics.

Thrombocytosis and STAT5 activation in chronic myelogenous leukaemia are not associated with JAK2 V617F or calreticulin mutations.

AIMS: Marked thrombocytosis is uncommon in chronic myelogenous leukaemia (CML) but may be associated with mutation of JAK2 V617F, calreticulin (CALR) and/or phospho-STAT5 (p-STAT5) activation in other myeloproliferative neoplasms (MPNs), particularly essential thrombocythaemia (ET). We investigated the JAK2 V617F, CALR and STAT5 activation status in patients with CML and thrombocytosis (CML-T) that mimicked ET, trying to identify a common mechanism for thrombocytosis in MPN. METHODS: Blood and bone marrow morphological findings were reviewed from seven CML-T, four otherwise typical CML and one CML in blast phase. All cases were analysed for BCR-ABL1, JAK2 V617F and CALR exon 9 mutation and p-STAT5 expression. RESULTS: Four of seven cases of CML-T had marked thrombocytosis (>1000×10(9)/L). Eleven of 12 cases had megakaryocyte morphology typical for CML. All cases were BCR-ABL1 positive. Eleven of 12 cases were negative for JAK2 V617F, while STAT5 was activated in six of seven CML-T and in four of five CML cases. No case had a detectable CALR exon 9 mutation. One case of CML developed ET-like morphology and had JAK2 V617F detected while in molecular remission for CML. CONCLUSIONS: Detection of BCR-ABL1 is critical in the distinction of ET from CML. Thrombocytosis and STAT5 activation in CML-T are not consistently associated with CALR exon 9 or JAK2 V617F mutation.

Transient abnormal myelopoiesis of a newborn not associated with chromosome 21 abnormalities or GATA1 mutations.

Transient abnormal myelopoiesis (TAM) is a disorder of Down syndrome newborns characterized by megakaryocytic blasts indistinguishable from acute myeloid leukemia (AML), which undergoes spontaneous remission. Acquired GATA1 mutations are present in blasts of both TAM and the subsequent AML which sometimes develops. We present a unique case of a newborn with leukemic megakaryoblasts indistinguishable from those of TAM who had neither extra material from chromosome 21 in the germline or blasts, nor evidence of GATA1 mutations. These findings suggest there are other genetic abnormalities that can lead to TAM besides GATA1 mutation in the setting of trisomy 21. Pediatr Blood Cancer 2015;62:353-355. © 2014 Wiley Periodicals, Inc.

Frequency, interobserver reproducibility and clinical significance of equivocal peaks in PCR clonality testing using Euroclonality/BIOMED-2 primers.

AIMS: PCR studies for lymphoid clonality are now widely employed, especially using Euroclonality/BIOMED-2 primers. Criteria for interpretation as a clonal result, however, have proven controversial. This study examines the frequency and clinical significance of equivocal amplification patterns and measures the interobserver reproducibility of clonality interpretations. METHODS: At our institution, results of each primer set are first classified as clonal, non-clonal or abnormal (equivocal peak on polyclonal background). Final results for all primer sets are then collectively reported as positive (≥1 clonal result), negative (non-clonal results) or indeterminate (≥1 abnormal result) for a clonal population. Results of 274 consecutive clonality cases were reviewed, and the interobserver reproducibility of individual primer set reactions and final results was determined in a subset of 30 cases. RESULTS: 44/161 (27%) B-cell and 50/163 (31%) T-cell cases contained at least one abnormal peak. Of these, 29 (64%) and 31 (62%), respectively, showed clonal results in another primer set. Interobserver reproducibility was excellent for most primer sets and for final interpretations, but only fair to good for IGK V-J and TCRB D-J1+2 primer sets. A definitive diagnosis of lymphoma was rendered in 93%, 20% and 6% of B-cell cases and 90%, 42%, and 14% of T-cell cases positive, indeterminate or negative for a clonal population, respectively. CONCLUSIONS: Using a subjective approach, abnormal (equivocal) peaks are frequently observed in routine practice. However, most cases with abnormal peaks contain clonal rearrangements in other primer sets, facilitating overall interpretation of final results with excellent interobserver reproducibility.