In silico predicted compound targeting the IQGAP1-GRD domain selectively inhibits growth of human acute myeloid leukemia.

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the 'fittest hit' to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

Gamma-delta T-cell acute lymphoblastic lymphoma/leukemia: a report of a rare entity.

Gamma delta (γδ) T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is a rare, aggressive subtype of T-lymphoid leukemia that accounts for only 9-12% of all T-ALL cases. Herein, we report the case of an 8-year-old boy who presented with facial swelling, shortness of breath, and progressive cervical and axillary lymphadenopathy. Pathological examination, flow cytometry (Navios, Beckman Coulter ClearLLab 10C 10-color T-cell panel [containing FITC-labeled TCR γδ antibody]), chromosomal analysis, interphase FISH, and targeted DNA-based NGS (34-gene Illumina TruSeq Myeloid Panel) were performed. Flow cytometry evaluation of a lymph node biopsy specimen revealed an immature T-cell population positive for CD4, CD3, CD2 (subset positive), CD5, CD7, CD38, CD1a, cytoplasmic terminal deoxynucleotidyl transferase (cyto-TdT), CD30 (subset positive), and T-cell receptor (TCR) gamma delta (γδ). Microscopic examination of an enlarged lymph node and bone marrow showed involvement by a dense, diffuse, neoplastic infiltrate. Interphase FISH revealed a copy number loss of PDGFRB (5q32) in 90.5% of interphase nuclei. Targeted DNA-based NGS detected a tier II oncogenic variant in NOTCH1 (c.7375C > T, p.Gln2459Ter) at a VAF of 21%. This case of γδ T-ALL highlights a rare entity and adds to the literature, albeit scant, which may aid in better recognition and classification.

Unicentric Castleman Disease: Illustration of Its Morphologic Spectrum and Review of the Differential Diagnosis.

CONTEXT.­: Unicentric Castleman disease (UCD) is a dynamic entity with a wide spectrum of morphologic findings. UCD can be further subdivided into hyaline-vascular and mixed/plasmacytic variants. Hyaline-vascular UCD has both follicular and interfollicular (stromal) changes, and occasionally these lesions show a skewed representation of either the follicular or stromal compartments. Plasmacytosis is usually minimal in the hyaline-vascular variant. The mixed/plasmacytic variant of UCD is composed of sheets of plasma cells often associated with a variable number of follicles with regressive changes. OBJECTIVE.­: To illustrate the differential diagnosis of UCD, as it is quite broad and includes lymphomas, plasma cell neoplasms, stromal neoplasms such as follicular dendritic cell sarcoma and vascular neoplasms, immunoglobulin G4-related disease, infections, and other rare lesions. An additional objective is to enhance awareness of the morphologic features of UCD in excisional and in small core-needle biopsy specimens, the latter of which may inadvertently target follicle- or stroma-rich areas, causing diagnostic challenges. DATA SOURCES.­: In this review, we provide readers a concise illustration of the morphologic spectrum of UCD that we have encountered in our practice and a brief discussion of entities in the differential diagnosis. CONCLUSIONS.­: UCD exhibits a broad spectrum of morphologic changes, and awareness of these morphologic variations is key to avoid misdiagnosis.

T-Cell-Rich Hodgkin Lymphoma With Features of Classic Hodgkin Lymphoma and Nodular Lymphocyte-Predominant Hodgkin Lymphoma: A Borderline Category With Overlapping Morphologic and Immunophenotypic Features.

CONTEXT.­: It is known that a subset of cases of classic Hodgkin lymphoma (CHL) with B-cell-rich nodules (lymphocyte-rich CHL) exhibits morphologic and immunophenotypic features that overlap with nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), raising diagnostic difficulties that can be resolved in most cases by performing an adequate battery of immunohistochemical studies. OBJECTIVE.­: To fully characterize cases of T-cell-rich Hodgkin lymphoma where a specific diagnosis of NLPHL (ie, pattern D) or CHL could not be made even after complete immunophenotypic investigation. DESIGN.­: The clinical, immunomorphologic, and molecular (when applicable) presentation of 3 cases of T-cell-rich Hodgkin lymphoma was thoroughly investigated. RESULTS.­: These 3 cases harbored lymphocyte-predominant-like and Hodgkin and Reed-Sternberg-like cells that partially expressed B-cell and CHL markers and were negative for Epstein-Barr virus-encoded small RNA, in a T-cell-rich background with residual follicular dendritic cell meshworks; 1 case had frequent and the other 2 cases scant/absent eosinophils and plasma cells. Two patients with advanced-stage (III or IV) disease presented with axillary and supraclavicular lymphadenopathy, respectively, and without B symptoms. These patients underwent NLPHL-like therapeutic management with 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride [hydroxydaunorubicin], vincristine sulfate [Oncovin], and prednisone) chemotherapy; both are in complete remission 7 years posttherapy. One patient presented with stage I disease involving an internal mammary lymph node without B-symptoms and was treated with surgical excision alone; this patient is also in complete remission 1 year later. CONCLUSIONS.­: These cases illustrate overlapping features of T-cell-rich NLPHL and CHL with neoplastic cells expressing both B-cell program and CHL markers. This underrecognized overlap has not been fully illustrated in the literature, although it portrays a therapeutic challenge. These neoplasms may deserve in-depth investigation in the future that may bring up diagnostic or theragnostic implications.

Clonal cytopenia of undetermined significance (CCUS)-associated reversion of donor-derived, transient αß T-cell large granular clonal lymphocytosis, emerging post-transplant in a patient with a history of γδ T-cell large granular lymphocytic leukemia.

Autologous and allogeneic hematopoietic stem cell transplantation (HSCT) has revolutionized the therapy of hematolymphoid malignancies. Yet, how to best detect or predict the emergence of HSCT-related complications remain unresolved. Here, we describe a case of donor-derived, transient Alpha Beta (αß) T-cell large granular clonal lymphocytosis and cytopenia that emerged post-HSCT in a patient with a history of gamma delta (γδ) T-cell large granular lymphocytic leukemia (T-LGLL). Clonal unrelatedness of post-transplant T-LGL lymphocytosis to the patient's pretransplant T-LGLL was first identified by T-cell receptor (TCR) PCR showing different sized fragments of rearranged gamma chains, in addition to shift from γδ to αß TCR expression by flow cytometry analyses. Donor-derivation of the patient's post-transplant clonal lymphocytosis was confirmed by serial chimerism analyses of recipient's blood specimens demonstrating 100% donor DNA. Moreover, oncogenic DNMT3A and RUNX1 mutations were detected by next-generation sequencing (NGS) only in post-transplant specimens. Intriguingly, despite continued increase in DNMT3A and RUNX1 mutation load, the patient's clonal lymphocytosis and anemia eventually largely resolved; yet, the observed mutation profile with persistent thrombocytopenia indicated secondary clonal cytopenia of undetermined significance (CCUS) in the absence of overt morphologic evidence of myeloid neoplasm in the marrow. This case illustrates the utility of longitudinal chimerism analysis and NGS testing combined with flow cytometric immunophenotyping to evaluate emerging donor-derived hematolymphoid processes and to properly interpret partial functional engraftment. It may also support the notion that driver mutation-induced microenvironmental changes may paradoxically contribute to reestablishing tissue homeostasis.

The role of splenectomy in management of splenic B-cell lymphomas.

INTRODUCTION: Splenic B-cell lymphomas are rare and understudied entities. Splenectomy is frequently required for specific pathological diagnosis in patients with splenic B-cell lymphomas other than classical hairy cell leukemia (cHCL), and can be effective and durable therapy. Our study investigated the diagnostic and therapeutic role of splenectomy for non-cHCL indolent splenic B-cell lymphomas. METHODS: Observational study of patients with non-cHCL splenic B-cell lymphoma undergoing splenectomy between 1 August 2011 and 1 August 2021 at the University of Rochester Medical Center. The comparison cohort was patients categorized as having non-cHCL splenic B-cell lymphoma who did not undergo splenectomy. RESULTS: Forty-nine patients (median age 68 years) had splenectomy (SMZL n = 33, HCLv n = 9, SDRPL n = 7) with median follow up of 3.9 years post splenectomy. One patient had fatal post-operative complications. Post-operative hospitalization was ≤ 4 days for 61% and ≤ 10 days for 94% of patients. Splenectomy was initial therapy for 30 patients. Of the 19 patients who had previous medical therapy, splenectomy changed their lymphoma diagnosis in 5 (26%). Twenty-one patients without splenectomy were clinically categorized as having non-cHCL splenic B-cell lymphoma. Nine required medical treatment for progressive lymphoma and of these 3 (33%) required re-treatment for lymphoma progression compared to 16% of patients following first line splenectomy. CONCLUSION: Splenectomy is useful for the diagnosis of non-cHCL splenic B-cell lymphomas with comparable risk/benefit profile and remission duration to medical therapy. Patients with suspected non-cHCL splenic lymphomas should be considered for referral to a high-volume center with experience in performing splenectomies for definitive diagnosis and treatment.

Common clonal origin of three distinct hematopoietic neoplasms in a single patient: B-cell lymphoma, T-cell lymphoma, and polycythemia vera.

The potential for more than one distinct hematolymphoid neoplasm to arise from a common mutated stem or precursor cell has been proposed based on findings in primary human malignancies. Particularly, angioimmunoblastic T-cell lymphoma (AITL), which shares a somatic mutation profile in common with other hematopoietic malignancies, has been reported to occur alongside myeloid neoplasms or clonal B-cell proliferations, with identical mutations occurring in more than one cell lineage. Here we report such a case of an elderly woman who was diagnosed over a period of 8 years with diffuse large B-cell lymphoma, polycythemia vera, and AITL, each harboring identical somatic mutations in multiple genes. Overall, at least five identical nucleotide mutations were shared across multiple specimens, with two identical mutations co-occurring at variable variant allele frequencies in all three specimen types. These findings lend credence to the theory that a common mutated stem cell could give rise to multiple neoplasms through parallel hematopoietic differentiation pathways.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) arising in the setting of polycythemia vera (PV): An illustration of the emerging role of flow cytometry analysis in monitoring progression of myeloproliferative neoplasms.

This report highlights the value of flow cytometry analysis, particularly in the setting of myeloproliferative neoplasms showing features of progression, as neoplastic plasmacytoid dendritic cell (PDC) proliferations may be present, representing either a clonal expansion of mature PDCs related to the underlying myeloproliferative neoplasm or transformation to blastic plasmacytoid dendritic cell neoplasm (BPDCN). BPDCN should always be considered in patients with myeloid neoplasms in progression and/or who develop new cutaneous findings, as it may prompt change of management.

Allogeneic Bone Marrow Transplant as a Cure for Refractory T-Cell Large Granular Lymphocytic Leukemia in an Adolescent.

T-cell large granular lymphocytic (T-LGL) leukemia is a rare, typically indolent neoplasm with a median age of onset above 60 years. Pathogenesis involves clonal T-cell expansion, and nearly all reported pediatric cases have been associated with concurrent autoimmune disease. Immunosuppressive therapy often mitigates sequelae, but definitive cure is not routinely achieved. Here we present an otherwise healthy 13-year-old with T-LGL leukemia refractory to all standard treatments. Our patient ultimately underwent allogeneic bone marrow transplant (BMT) and is now stable in remission 3 years post-BMT. BMT may offer a viable definitive cure for refractory T-LGL leukemia in very young patients.

Nodular Lymphocyte-predominant Hodgkin Lymphoma With Nodular Sclerosis: An Underrecognized Feature Associated With Pattern D.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) with unusual features, including some that can overlap morphologically with classic Hodgkin lymphoma (CHL), have been described. Herein, we describe 12 cases of NLPHL with fibrous bands and capsular fibrosis resembling, in part, nodular sclerosis (NS) CHL. Seven of 12 cases harbored Reed-Sternberg-like cells, further suggestive of CHL, but all cases lacked associated eosinophils and/or plasma cells in the background. In this cohort, all cases had areas of so-called pattern D (nodular T-cell rich) as a sole component in 7 (58%) cases or as a hybrid pattern along with pattern E (diffuse T-cell/histiocyte-rich) in 5 (42%) cases. The immunophenotype of the large neoplastic cells in these cases supported their being lymphocyte predominant cells of NLPHL, positive for CD20, CD79a, and OCT2, and negative for CD15 and CD30. However, PAX5 was weak in 9 of 11 cases similar to Hodgkin/Reed-Sternberg cells in CHL. We conclude that some cases of NLPHL are associated with fibrous bands and capsular fibrosis and resemble, in part, NS CHL. In our experience, NLPHL with NS-like features occurs in 10% to 15% of cases of NLPHL and is associated with a variant pattern (D and/or E). In addition, all patients in this cohort were not treated before biopsy, suggesting that the prominent sclerosis in these cases is inherent to disease biology. Recognition of NLPHL with NS-like features further expands the morphologic spectrum of NLPHL and helps avoid potential misdiagnosis as CHL.

Targeted therapy for treatment of patients with classical hairy cell leukemia.

Most patients with treatment naïve classical hairy cell leukemia (cHCL) have durable responses with purine nucleoside analogues. In contrast, options are limited for cHCL patients with co-morbidities, purine analogue intolerance, or resistant disease. We report the utility of targeted therapy for nine cHCL patients presenting with treatment naïve cHCL and severe neutropenia and infection (n = 3), purine analogue intolerance (n = 2), or purine analogue resistant disease (n = 4). BRAF inhibitor vemurafenib was started at 240-480 mg twice daily (planned 90-day treatment) and combined with rituximab in seven patients. Therapy was tolerable with no severe adverse events. All patients responded with rapid blood count recovery (median time 1.52 months, range 0.43-4.33). Median progression free and overall survival was not reached at a median follow up of 18.1 months (range 3.2-68.9). These data suggest targeted therapy could be an option for patients unable to be treated with purine analogues.

Hepatic involvement by T-cell neoplasms: a clinicopathologic study of 40 cases.

Hepatic involvement by a T-cell neoplasm is rare and often challenging to diagnose in liver biopsies. We collected 40 cases of T-cell neoplasms diagnosed in the liver from five large academic institutions to assess the clinicopathologic features. The patients included 11 women and 29 men, with a median age of 54 (range: 2-75) years and a high mortality rate (31/37, 83.8%). Fourteen (35%) patients were diagnosed with hepatosplenic T-cell lymphoma (HSTCL), 13 (32.5%) peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), and 13 (32.5%) other types of T-cell neoplasms. Patients with HSTCL were much younger and had worse survival than PTCL-NOS and other T-cell neoplasms (P < 0.05). On imaging studies, 20 cases (50%) showed abnormalities, including 10 with mass lesions that correlated with normal or cholestatic pattern enzyme elevation. Histomorphological analysis revealed four main patterns; with the exception of mass forming lesions (pattern 4; n = 8), cases with sinusoidal predominant (pattern 1; n = 12), portal predominant with sinusoidal infiltrates (pattern 2; n = 13) or lobular aggregates (pattern 3; n = 5) demonstrated small to medium lymphocytes resembling a reactive/inflammatory process. In addition, we described two cases of T-cell large granular lymphocytic leukemia that mimicked HSTCL, and a case of aggressive post-transplant lymphoproliferative disorder that developed after chronic Epstein-barr virus (EBV) infection, suggesting the importance of EBV testing in some lymphoma cases. As the largest cohort of T-cell neoplasms in liver, our study provides critical data on disease frequency, distribution, and clinicopathologic features that are essential for accurate diagnosis.

Multiple B cell malignancies in patients with chronic lymphocytic leukemia: epidemiology, pathology, and clinical implications.

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) is associated with increased risk for certain cancers, but relatively little is known about the risk for these patients to develop additional B cell malignancies. Here, we review the available epidemiological data on multiple B cell malignancies in CLL, discuss diagnostic methods and proper pathologic evaluation to distinguish CLL from other B cell malignancies, and address clinical challenges and unmet needs in caring for CLL patients with unrelated B cell malignancies and disease transformation. Considerations include CLL patients with unrelated monoclonal B cell lymphocytosis, biclonal CLL, secondary B cell non-Hodgkin lymphomas, and Richter syndrome - both clonally related transformation and de novo large B cell lymphoma. We address the challenges that remain in order to better understand the underlying risk factors and biology that may put CLL patients at increased risk of developing multiple B cell neoplasia.

Incidental splenic findings in pancreatosplenectomy specimens resected for primary pancreatic lesions.

AIMS: Little has been written on the frequency and nature of incidental splenic lesions diagnosed on histopathological examination of pancreatosplenectomy specimens. METHODS AND RESULTS: For 191 such specimens, incidental histological findings after haematopathologist re-review were tabulated. Cases suspicious for lymphoid malignancy underwent molecular analysis for immunoglobulin heavy and kappa light chain rearrangement. Follow-up was obtained on selected cases. In five cases (3%), the spleen was sampled but not mentioned in the original microscopic report; all were normal on re-review. Otherwise, most (171 of 186, 92%) were initially diagnosed as normal, with 160 (94%) remaining so on re-review. Findings on re-review not initially described (n = 11, 6%) included four cases with splenic morphology suspicious for possible leukaemia/lymphoma involvement. Additional findings included abscess formation, foamy macrophages, necrotising granulomas and simple cysts. Fifteen spleens were initially diagnosed as abnormal; the histopathological process was confirmed in all, including non-necrotising granulomas, cysts, Gamna-Gandy bodies, foamy macrophages, involvement by pancreatic neoplasm and involvement by known chronic lymphocytic leukaemia (CLL). Molecular analysis was performed on the five cases of known/suspected lymphoma and two were positive for monoclonal gene rearrangement, including the known CLL and a previously undiagnosed case with similar immunophenotype. CONCLUSIONS: Incidental splenic findings are not uncommon in pancreatosplenectomy specimens. While most are of limited clinical significance, low-grade lymphoproliferative disorders may go undetected if the spleen is overlooked. We recommend careful observation of splenic findings in these specimens, with a low threshold for haematopathological consultation when in doubt.

Novel 1.3 Mb germline duplication in chromosome 8q21.11 by microarray comparative genomic hybridization plus single nucleotide polymorphism analysis in an adult patient with pancytopenia and urinary bladder complications.

We present the case of a 30-year-old woman with a history of perinatal complications as well as bladder and urinary disease through her childhood and adult life. Microarray comparative genomic hybridization (aCGH) analysis revealed a 1.3 megabase duplication at chromosome 8q21.11 encompassing the CASC9 and HNF4G genes.

miR-150 downregulation contributes to the high-grade transformation of follicular lymphoma by upregulating FOXP1 levels.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

Loss of Full-Length GATA1 Expression in Megakaryocytes Is a Sensitive and Specific Immunohistochemical Marker for the Diagnosis of Myeloid Proliferative Disorder Related to Down Syndrome.

OBJECTIVES: Myeloid proliferative disorders associated with Down syndrome (MPD-DS), including transient abnormal myelopoiesis and myeloid leukemia associated with Down syndrome (DS), harbor mutations of GATA1, a transcription factor essential for erythroid and megakaryocytic development. These mutations result in a N-terminally truncated GATA1 (GATA1s) and prohibit the production of the full-length GATA1 (GATA1f). Here, we demonstrate the utility of immunohistochemical GATA1f reactivity in diagnosing MPD-DS. METHODS: Immunohistochemical studies for GATA1f expression were performed on bone marrow biopsy specimens. RESULTS: In all cases of MPD-DS, megakaryocytes lacked GATA1f expression. In contrast, GATA1f expression was detected in megakaryocytes in all specimen types from patients without DS (normal bone marrows, pediatric myelodysplastic syndrome, juvenile myelomonocytic leukemia, adult acute megakaryocytic leukemia [pediatric and adult; without trisomy 2]), as well as normal bone marrows from patients with DS. CONCLUSIONS: The lack of GATA1f expression is a sensitive and specific immunohistochemical marker for MPD-DS.