Advances in Molecular Understanding of Polycythemia Vera, Essential Thrombocythemia, and Primary Myelofibrosis: Towards Precision Medicine.

Myeloproliferative neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF), are characterized by the clonal proliferation of hematopoietic stem cells leading to an overproduction of hematopoietic cells. The last two decades have seen significant advances in our understanding of the molecular pathogenesis of these diseases, with the discovery of key mutations in the JAK2, CALR, and MPL genes being pivotal. This review provides a comprehensive update on the molecular landscape of PV, ET, and PMF, highlighting the diagnostic, prognostic, and therapeutic implications of these genetic findings. We delve into the challenges of diagnosing and treating patients with prognostic mutations, clonal evolution, and the impact of emerging technologies like next-generation sequencing and single-cell genomics on the field. The future of MPN management lies in leveraging these molecular insights to develop personalized treatment strategies, aiming for precision medicine that optimizes outcomes for patients. This article synthesizes current knowledge on molecular diagnostics in MPNs, underscoring the critical role of genetic profiling in enhancing patient care and pointing towards future research directions that promise to further refine our approach to these complex disorders.

Clonotypic VDJ rearrangements in Mixed Phenotype Acute Leukemia can be Successfully Utilized to Track Minimal Residual Disease (MRD).

INTRODUCTION: Multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) can both be used to identify a neoplastic clonotype by targeting CDR3 and assessing rearrangements in IgH, IgK, IgL, TCR-ß, and TCR-gamma loci. The clonotypic sequence can be robustly used to track minimal residual disease (MRD). The ability to track MRD by NGS in mixed phenotype acute leukemia (MPAL) is unknown and warrants investigation. METHODS: Institutional Review Board (IRB) approval was obtained. Central Moffitt Cancer Center (MCC) database was searched to locate any patients with MPAL from over 600,000 entries. Patient charts were manually curated to identify those with clonoSEQ data, and clinical data was procured from the electronic medical record (EMR). RESULTS: Twenty-nine patients with MPAL were identified. Only 2 patients with clonoSEQ testing were found. Both demonstrated a B/myeloid phenotype, and both were bilineal. NGS (clonoSEQ) identified 4 dominant (IGH) (patient A; 8/2019) and 2 dominant sequences (patient B; 10/2019), respectively. In both patients, clonoSEQ testing successfully tracked minimal residual disease and mirrored clinical disease burden. CONCLUSIONS: This report is the first to confirm the utility of NGS-based MRD tracking in patients with MPAL and shows increased sensitivity of NGS over MRD flow cytometry.

Mutations highly specific for secondary AML are associated with poor outcomes in ELN favorable risk NPM1-mutated AML.

ABSTRACT: Acute myeloid leukemia (AML) is a heterogeneous malignancy with outcomes largely predicted by genetic abnormalities. Mutations of NPM1 are common in AML, occurring in ∼30% of cases, and generally considered a favorable risk factor. Mutations highly specific for secondary AML (sMut) have been shown to confer poor prognosis, but the overall impact of these mutations in the setting of favorable-risk AML defined by mutant NPM1 remains unclear. In this multicenter study of patients with AML (n = 233) with NPM1 mutation at diagnosis, we observed that patients with sMut had worse overall survival (OS) than those without sMut (15.3 vs 43.7 months; P = .002). Importantly, this finding persisted in the European LeukemiaNet (ELN) 2017-defined favorable risk subset (14.7 months vs not reached; P < .0001). Among patients who achieved NPM1 measurable residual disease (MRD) negativity, longer OS was observed in the entire cohort (P = .015) as well as in both the sMut subset (MRD negative: median OS (mOS) 73.9 months vs MRD positive: 12.3 months; P = .0170) and sMut ELN 2017-favorable subset (MRD negative: mOS 27.3 vs MRD positive: 10.5 months; P = .009). Co-occurrence of sMut and mutant NPM1 confers a poor prognosis in AML.

Sclerotic marginal zone lymphoma: A case report.

BACKGROUND: Marginal zone lymphoma (MZL) is an indolent non-Hodgkin B cell lymphoma with various architectural pattern including perifollicular, follicular colonization, nodular, micronodular, and diffuse patterns. A sclerotic variant has not been previously reported and represents a diagnostic pitfall. CASE SUMMARY: A 66-year-old male developed left upper extremity swelling. Chest computed tomography (CT) in September 2020 showed 14 cm mass in left axilla. Needle core biopsy of axillary lymph node showed sclerotic tissue with atypical B lymphoid infiltrate but was non-diagnostic. Excisional biopsy was performed for diagnosis and showed extensive fibrosis and minor component of infiltrating B cells. Flow cytometry showed a small population of CD5-, CD10-, kappa restricted B cells. Monoclonal immunoglobulin heavy chain and light chain gene rearrangement were identified. Upon being diagnosed with MZL, patient was treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone and achieved complete remission by positron emission tomography/CT. CONCLUSION: This is an important case report because by morphology this case could have easily been overlooked as non-specific fibrosis with chronic inflammation representing a significant diagnostic pitfall. Moreover, this constitutes a new architectural pattern. While sclerotic lymphomas have rarely been described (often misdiagnosed as retroperitoneal fibrosis), we do not know of any cases describing this architectural presentation of MZL.

Clinicopathologic characteristics, genetic features, and treatment options for acute lymphoblastic leukemia with JAK2 rearrangement-A 10-case study and literature review.

JAK2 rearrangement (JAK2-R) in acute lymphoblastic leukemia (ALL) is rare and often categorized as B-ALL with BCR::ABL1-like features based on the World Health Organization classification. We report 10 patients with JAK2-R ALL, 9 males and 1 female, with a median age 40.5 years. Eight patients presented with marked leukocytosis (median WBC, 63 × 10 9/L) and hypercellular (>95%) bone marrow with increased lymphoblasts (72%-95%). There was no evidence of bone marrow fibrosis or hypereosinophilia. Immunophenotypic analysis showed 9 B-cell and 1 T-cell neoplasms. Using fluorescence in situ hybridization (FISH) and RNA sequencing analysis, JAK2 partners were identified for 7 cases and included PCM1 (n = 4), ETV6 (n = 2) and BCR (n = 1). All patients received upfront polychemotherapy. Additionally, 2 patients received ruxolitinib, 2 received allogeneic stem cell transplant, and 1 received CAR-T therapy. The 1- and 3-year overall survival rates were 55.6% and 22.2%, respectively. A literature review identified 24 B-ALL and 4 T-ALL cases with JAK2-R reported, including 16 males, 6 females and 6 gender not stated. Many JAK2 partner-genes were reported with the most common being PAX5 (n = 7), ETV6 (n = 5), BCR (n = 4) and PCM1 (n = 2). Survival data on 13 reported cases showed 1- and 3-year overall survival rates of 41.7% and 41.7%, respectively. In summary, JAK2-R ALL occurs more often in adult males, are mostly of B-cell lineage, and associated with an aggressive clinical course. Absence of eosinophilia and bone marrow fibrosis and no evidence of preexisting/concurrent JAK2-R myeloid neoplasms distinguish JAK2-R ALL from other myeloid/lymphoid neoplasms with eosinophilia and JAK2-R.

Genomic Landscape of Mixed-Phenotype Acute Leukemia.

Mixed-phenotype leukemia (MPAL) is a type of acute leukemia in which the blast population shows mixed features of myeloid, T-lymphoid, and/or B-lymphoid differentiation. MPALs are rare and carry a poor prognosis, thus, often pose both a diagnostic and therapeutic challenge. Conventionally, the diagnosis of MPAL requires either a single blast population with a lineage-defining phenotypic expression of multiple lineages (myeloid, B-cell and/or T-cell) (biphenotypic) or two distinct blast populations that each independently satisfy criteria for designation as AML, B-ALL, and/or T-ALL (bilineage). Given the rarity of MPAL, minimal studies have been performed to describe the genomic landscape of these neoplasms. IRB approval was obtained. Central MCC database was searched for any patient with a diagnosis of acute undifferentiated leukemia (AUL), acute leukemia of ambiguous lineage (ALAL), and MPAL. All patient diagnoses were manually reviewed by a hematopathologist to confirm the diagnosis of MPAL. Genomic and molecular data were collated from the EMR and bioinformatically from MCC genomics repositories. Twenty-eight patients with MPAL were identified. Thirteen were female and 15 were male. Average age was 56 years old (range = 28-81). Ten cases were biclonal and 18 were biphenotypic. Diagnoses were as follows: B/myeloid (n = 18), T/myeloid (n = 9), and T/B (n = 1). Cytogenetic analysis (Karyotype +/- FISH) was available for 27 patients. The most frequent recurrent abnormalities were complex karyotype (n = 8), BCR/ABL1 translocation (n = 6), Del 5q/-5 (n = 4), Polysomy 21 (n = 4). Mutational analysis was available for 18 patients wherein mutations were detected in 45 unique genes. The most frequently mutated genes were TP53 (7), RUNX1 (6), WT1 (4), MLL2 (3), FLT3 (3), CBL (2), ASXL1 (2), TET2 (2), MAP3K6 (2), MLL (2), and MAP3K1 (2). Targetable or potentially targetable biomarkers were found in 56% of cases. Overall survival was 19.5 months (range = 0-70 m). Ten patients were treated with an allogeneic stem cell transplant and had superior outcome (p = 0.0013). In one the largest series of MPAL cases to date, we corroborate previous findings with enriched detection of RUNX1 and FLT3-ITD mutations along with discovery of unreported mutations (MAP3K) that may be amenable to therapeutic manipulation. We also report the frequent occurrence of AML with MDS-related changes (AML-MRC)-defining cytogenetic abnormalities (26%). Finally, we show that those patients that received stem cell transplant had a better overall survival. Our findings support the need to genomically profile MPAL cases to exploit opportunities for targeted therapies in this orphan disease with dismal prognosis.

Fatty acids secreted from head and neck cancer induce M2-like Macrophages.

Tumor-infiltrating monocytes can mature into Macrophages that support tumor survival or that display antitumor properties. To explore mechanisms steering Macrophage maturation, we assessed the effects of supernatants from squamous cell carcinoma cell lines (FaDu and SCC) on monocyte-derived Macrophage maturation. Purified monocytes were incubated in medium or medium supplemented with supernatants from FaDu and SCC9 or the leukemia monocytic cell line, THP-1. Macrophages were examined for markers of maturation (CD14, CD68), activation (HLA-DR, CD86, IL15R), scavenger receptor (CD36), toll-like receptor (TLR4), M2 marker (CD206), immune checkpoint (PD-L1), and intracellular chemokine expression (IP-10). Compared to other conditions, cells incubated with FaDu or SCC9 supernatants displayed enhanced survival, down-regulation of cell surface HLA-DR, CD86, IL-15R, CD36, and intracellular IP-10 expression, and increased cell surface PD-L1, CD14, and CD206 expression. Despite expressing TLR4 and CD14, Macrophages matured in tumor supernatants failed to respond to stimulation with the canonical TLR4 agonist, LPS. These changes were accompanied by a decrease in intracellular phospho-p38 expression in tumor supernatant conditioned Macrophages. Depletion of fatty acids from tumor supernatants or treatment of cell cultures with an inhibitor of fatty acid oxidation, Etomoxir, reversed a number of these phenotypic changes induced by tumor supernatants. Additionally, Macrophages incubated with either palmitic acid or oleic acid developed similar phenotypes as cells incubated in tumor supernatants. Together, these data suggest that fatty acids derived from tumor cells can mediate the maturation of Macrophages into a cell type with limited pro-inflammatory characteristics.

Enhancing Molecular and Cytogenetic Fellow Education With an Integrative Hematopathology/Molecular Genetic Pathology Joint Conference.

OBJECTIVES: To overcome the challenges associated with molecular and cytogenetic (MG) education in hematopathology (HP), a monthly joint HP/MG conference with specific curricular goals was established and evaluated by the participants. METHODS: All cases from the HP/MG conference over 56 months were reviewed. To assess the educational impact, a survey was distributed to current/former HP/molecular genetic pathology fellows and faculty. RESULTS: During the study period, a total of 252 cases covering MG testing considered important for HP fellowship training were presented. The 100 most recent cases since 2018 discussed findings of diagnostic (85%), prognostic (40%), or therapeutic (10%) importance. A broad range of technologies were discussed such as karyotyping, cytogenetic fluorescence in situ hybridization studies, microarrays, polymerase chain reaction-based tests, next-generation sequencing, and Sanger sequencing. Twenty-three (95.8%) of 24 survey respondents agreed that the conference achieved all of its goals, and all agreed it was worth implementing. CONCLUSIONS: This educationally based HP/MG conference supplements existing rotations, didactic presentations, and consensus case conferences and enhances MG education in HP without excessive time commitment or need for extensive in-house MG testing. It also contributes to enhancing HP knowledge among the MG faculty and fellows.

A Review of Advances in Hematopoietic Stem Cell Mobilization and the Potential Role of Notch2 Blockade.

Hematopoietic stem cell (HSC) transplantation can be a potential cure for hematological malignancies and some nonhematologic diseases. Hematopoietic stem and progenitor cells (HSPCs) collected from peripheral blood after mobilization are the primary source to provide HSC transplantation. In most of the cases, mobilization by the cytokine granulocyte colony-stimulating factor with chemotherapy, and in some settings, with the CXC chemokine receptor type 4 antagonist plerixafor, can achieve high yield of hematopoietic progenitor cells (HPCs). However, adequate mobilization is not always successful in a significant portion of donors. Research is going on to find new agents or strategies to increase HSC mobilization. Here, we briefly review the history of HSC transplantation, current mobilization regimens, some of the novel agents that are under investigation for clinical practice, and our recent findings from animal studies regarding Notch and ligand interaction as potential targets for HSPC mobilization.

Divergent clonal evolution of a common precursor to mantle cell lymphoma and classic Hodgkin lymphoma.

Clonal heterogeneity and evolution of mantle cell lymphoma (MCL) remain unclear despite the progress in our understanding of its biology. Here, we report a 71-yr-old male patient with an aggressive MCL and depict the clonal evolution from initial diagnosis of typical MCL to relapsed blastoid MCL. During the course of the disease, the patient was diagnosed with classic Hodgkin lymphoma (CHL) and received a CHL therapeutic regimen. Molecular analysis by next-generation sequencing of both MCL and CHL demonstrated clonally related CHL with characteristic immunophenotype and PDL1/2 gains. Moreover, our data illustrate the clonal heterogeneity and acquisition of additional genetic aberrations including a rare fusion of SEC22B-NOTCH2 in the process of clonal evolution. Evidence obtained from our comprehensive immunophenotypic and genetic studies indicates that MCL and CHL can originate from a common precursor by divergent clonal evolution, which may pose a therapeutic challenge.

Ultrastructural changes in peripheral blood leukocytes in α-synuclein knockout mice.

Effects of α-synuclein deficiency on cellular blood components have not been extensively investigated. This study evaluated ultrastructural changes of leukocytes in α-synuclein knockout (KO) mice using electron microscopy (EM). The following ultrastructural characteristics were quantified in leukocytes: mitochondria, primary granules, specific granules (SG), Golgi apparatus (GA), inclusions, rough-endoplasmic reticulum (RER), smooth-endoplasmic reticulum (SER), and cellular projections (CP). EM showed increased numbers or amounts of SG, inclusions, and SER in KO group (5.3 ±â€¯4.5 in WT vs. 14.1 ±â€¯10.3 in KO, p = 0.02; 0.4 ±â€¯0.9 in WT vs. 3.2 ±â€¯2.8 in KO, p = 0.007; and 7.7 ±â€¯6.7 in WT vs. 17.7 ±â€¯12.2 in KO, p = 0.03, respectively). Although CP number was not significantly different between the two groups (13.4 ±â€¯5.3 in WT vs. 16.3 ±â€¯7.5 in KO, p = 0.32), their size and shapes were altered in KO mice. Notably, findings occurred in the setting of significant lymphopenia. α-Synuclein deficiency leads to changes in size and shape of secretory particles and increases in SER, SG, and inclusions, indicating a potential role for α-synuclein in vesicular trafficking in leukocytes. Further studies are needed to elucidate functions mediated by α-synuclein.