Predictors of clinical outcome in myeloproliferative neoplasm, unclassifiable: A Bone Marrow Pathology Group study.

OBJECTIVES: Myeloproliferative neoplasm, unclassifiable (MPN-U, revised to MPN, not otherwise specified in the fifth edition of the World Health Organization classification) is a heterogeneous category of primary marrow disorders with clinical, morphologic, and/or molecular features that preclude classification as a more specific MPN subtype due to stage at diagnosis, overlapping features between MPN subtypes, or the presence of coexisting disorders. Compared with other MPN subtypes, the contribution of the mutational landscape in MPN-U in conjunction with other clinical and morphologic biomarkers to prognosis has been less well investigated. METHODS: We performed a multicenter, retrospective study of MPN-U (94 cases) to better define the clinicopathologic features, genetic landscape, and clinical outcomes, including subgroups of early-stage, advanced-stage, and coexisting disorders. The Dynamic International Prognostic Scoring System (DIPSS) plus scoring system was applied to assess its relevance to MPN-U prognosis. RESULTS: Multivariate analysis demonstrated bone marrow blast count and DIPSS plus score as statistically significant in predicting overall survival. Univariate analysis identified additional potential poor prognostic markers, including abnormal karyotype and absence of JAK2 mutation. Secondary mutations were frequent in the subset analyzed by next-generation sequencing (26/37 cases, 70.3%) with a borderline association between high molecular risk mutations and overall survival. CONCLUSIONS: This study, as one of the largest of MPN-U studies incorporating both clinicopathologic and molecular data, moves toward identification of biomarkers that better predict prognosis in this heterogeneous category.

The Spectrum of Non-neoplastic Changes Associated With Breast Implants: Histopathology, Imaging, and Clinical Significance.

Breast implant-associated anaplastic large cell lymphoma has been recognized as a distinct entity in the World Health Organization classification of hematolymphoid neoplasms. These neoplasms are causally related to textured implants that were used worldwide until recently. Consequently, there is an increased demand for processing periprosthetic capsules, adding new challenges for surgeons, clinicians, and pathologists. In the literature, the focus has been on breast implant-associated anaplastic large cell lymphoma; however, benign complications related to the placement of breast implants occur in up to 20% to 30% of patients. Imaging studies are helpful in assessing patients with breast implants for evidence of implant rupture, changes in tissues surrounding the implants, or regional lymphadenopathy related to breast implants, but pathologic examination is often required. In this review, we couple our experience with a review of the literature to describe a range of benign lesions associated with breast implants that can be associated with different clinical presentations or pathogenesis and that may require different diagnostic approaches. We illustrate the spectrum of the most common of these benign disorders, highlighting their clinical, imaging, gross, and microscopic features. Finally, we propose a systematic approach for the diagnosis and handling of breast implant specimens in general.

The Clinicopathologic Features and Molecular Signatures of Blastoid High-Grade B Cell Lymphoma, Not Otherwise Specified.

A small subset of high-grade B-cell lymphoma (HGBL) with blastoid morphology remains poorly understood. We assessed 55 cases of blastoid HGBL, not otherwise specified (NOS) and compared their clinicopathologic characteristics with those of 81 non-blastoid HGBL-NOS and 62 blastoid HGBL with MYC and BCL2, with or without BCL6 rearrangements (double/triple-hit lymphoma [D/THL]). Patients with blastoid HGBL-NOS showed similar clinicopathologic features to patients with blastoid D/THLs and non-blastoid HGBL-NOS, except more frequently with a history of low-grade B-cell lymphoma, bone marrow involvement, and BCL2 rearrangement (P < .05) compared to the latter. MYC rearrangement (MYC-R), detected in 40% of blastoid HGBL-NOS, was associated with aggressive clinicopathologic features and poorer overall survival, even worse than that of blastoid D/THL (P < .05). Transcriptome profiling revealed a distinct gene expression pattern with differentially expressed genes enriched in MYC and P53-targeted genes in MYC-R blastoid HGBL-NOS. Fifty-two percent of blastoid HGBL-NOS had a double hit-like signature, similar to non-blastoid HGBL-NOS (P = .73). The overall survival of the blastoid HGBL-NOS group was similar to that of the blastoid D/THL group but appeared poorer than that of its non-blastoid counterparts (P = .07). Taken together, blastoid HGBL-NOS is an aggressive B-cell lymphoma that shares overlapping clinicopathologic and genetic features with non-blastoid HGBL-NOS. MYC-R in patients with blastoid HGBL-NOS identifies a highly aggressive subgroup with distinct aggressive clinicopathologic features, unique molecular signatures, and a dismal clinical outcome.

Monocytic and blastic plasmacytoid dendritic cell differentiation in acute leukemia with KMT2A rearrangement.

Acute leukemia with KMT2A rearrangement shows a spectrum of immunophenotypic presentation, but blastic plasmacytoid dendritic cell differentiation is extremely rare. Here we present a case of KMT2A rearranged acute leukemia with a hybrid immunophenotype in which a single blast population showed both blastic plasmacytoid dendritic cell and monocytic differentiation. This unusual case contributes to the diversity of the immunophenotypic spectrum in KMT2A rearranged acute leukemia and also sheds light on the cell of origin of plasmacytoid dendritic cells.

Guide to the Diagnosis of Myeloid Neoplasms: A Bone Marrow Pathology Group Approach.

OBJECTIVES: The practicing pathologist is challenged by the ever-increasing diagnostic complexity of myeloid neoplasms. This guide is intended to provide a general roadmap from initial case detection, often triggered by complete blood count results with subsequent blood smear review, to final diagnosis. METHODS: The integration of hematologic, morphologic, immunophenotypic, and genetic features into routine practice is standard of care. The requirement for molecular genetic testing has increased along with the complexity of test types, the utility of different testing modalities in identifying key gene mutations, and the sensitivity and turnaround time for various assays. RESULTS: Classification systems for myeloid neoplasms have evolved to achieve the goal of providing a pathology diagnosis that enhances patient care, outcome prediction, and treatment options for individual patients and is formulated, endorsed, and adopted by hematologists/oncologists. CONCLUSIONS: This guide provides diagnostic strategies for all myeloid neoplasm subtypes. Special considerations are provided for each category of testing and neoplasm category, along with classification information, genetic testing requirements, interpretation information, and case reporting recommendations based on the experience of 11 Bone Marrow Pathology Group members.

Myeloid sarcoma with NPM1 mutation may be clinically and genetically distinct from AML with NPM1 mutation: a study from the Bone Marrow Pathology Group.

Myeloid sarcoma (MS) is currently considered equivalent to de novo acute myeloid leukemia (AML); however, the relationship between these entities is poorly understood. This retrospective multi-institutional cohort study compared 43 MS with NPM1 mutation to 106 AML with NPM1 mutation. Compared to AML, MS had more frequent cytogenetic abnormalities including complex karyotype (p = .009 and p = .007, respectively) and was enriched in mutations of genes involved in histone modification, including ASXL1 (p = .007 and p = .008, respectively). AML harbored a higher average number of gene mutations (p = .002) including more frequent PTPN11 mutations (p < .001) and mutations of DNA-methylating genes including DNMT3A and IDH1 (both p < .001). MS had significantly shorter overall survival (OS) than AML (median OS: 44.9 vs. 93.2 months, respectively, p = .037). MS with NPM1 mutation has a unique genetic landscape, and poorer OS, compared to AML with NPM1 mutation.

First study comparing genetic profiles of MS and AML with a common disease-defining lesion.NPM1^Mut MS may be genetically distinct from NPM1^Mut AML.NPM1^Mut MS may have inferior overall survival compared to NPM1^Mut AML.

Clinicopathologic and Molecular Analysis of Normal Karyotype Therapy-Related and De Novo Acute Myeloid Leukemia: A Multi-Institutional Study by the Bone Marrow Pathology Group.

PURPOSE: Therapy-related acute myeloid leukemias (t-AML) are a heterogenous group of aggressive neoplasms that arise following exposure to cytotoxic chemotherapy and/or ionizing radiation. Many therapy-related myeloid neoplasms (t-MN) are associated with distinct chromosomal aberrations and/or TP53 alterations, but little is known about the clinicopathologic and molecular features of normal karyotype t-AML (NK-t-AML) and whether this t-MN subtype is distinctly different from NK de novo AML (NK-dn-AML). METHODS: This multi-institutional study by the Bone Marrow Pathology Group retrospectively evaluated clinicopathologic and molecular characteristics of 335 patients with NK-AML, comprising 105 t-AML and 230 dn-AML cases. RESULTS: Patients with t-AML compared with dn-AML exhibit significantly shorter overall survival (OS; median months: 17.6 v 44.2; P < .0001) and relapse-free survival (RFS; median months: 9.1 v 19.2; P = .0018). Frequency of NPM1, FLT3, KRAS, and GATA2 mutations were significantly different in NK-t-AML compared with NK-dn-AML (NPM1 35% v 49%; P = .0493; FLT3 23% v 36%; P = 0494; KRAS 12% v 5%; P = .0465; GATA2 9% v 2% P = .0105), while TP53 mutations were rare. Patients with t-AML more often stratified into intermediate or adverse 2017 ELN genetic risk groups. Favorable ELN risk predicted favorable OS (hazard ratio [HR], 0.4056; 95% CI, 0 to 0.866; P = .020) and RFS (HR, 0.355; 95% CI, 0 to 0.746; P = .006). Among all patients with NK-AML, stem-cell transplant and favorable ELN risk both significantly affected RFS, while therapy-relatedness and age had a borderline significant impact on OS (HR, 1.355; 95% CI, 0.975 to 1.882; P = .070). CONCLUSION: To our knowledge, this is the largest study to date to comprehensively evaluate NK-t-AML and provides a framework that may inform our understanding of NK-t-AML disease biology and could potentially help guide therapeutic management and improved disease classification in t-MNs that lack cytogenetic aberrations.

Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferation in mice via altered Runx1 splicing.

Acute myeloid leukemia (AML) is driven by numerous molecular events that contribute to disease progression. Herein, we identify hnRNP K overexpression as a recurrent abnormality in AML that negatively correlates with patient survival. Overexpression of hnRNP K in murine fetal liver cells results in altered self-renewal and differentiation potential. Further, murine transplantation models reveal that hnRNP K overexpression results in myeloproliferation in vivo. Mechanistic studies expose a direct functional relationship between hnRNP K and RUNX1-a master transcriptional regulator of hematopoiesis often dysregulated in leukemia. Molecular analyses show that overexpression of hnRNP K results in an enrichment of an alternatively spliced isoform of RUNX1 lacking exon 4. Our work establishes hnRNP K's oncogenic potential in influencing myelogenesis through its regulation of RUNX1 splicing and subsequent transcriptional activity.

Clinicopathologic spectrum of myeloid neoplasms with concurrent myeloproliferative neoplasm driver mutations and SRSF2 mutations.

Myeloproliferative neoplasms (MPNs) are frequently associated with classic driver mutations involving JAK2, MPL or CALR. SRSF2 is among the most frequently mutated splicing genes in myeloid neoplasms and SRSF2 mutations are known to confer a poor prognosis in patients with MPNs. In this study, we sought to evaluate the clinicopathologic spectrum of myeloid neoplasms harboring concurrent MPN-driver mutations and SRSF2 mutations. The study cohort included 27 patients, 22 (82%) men and five (19%) women, with a median age of 71 years (range, 51-84). These patients presented commonly with organomegaly (n = 15; 56%), monocytosis (n = 13; 48%), morphologic dysplasia (n = 11; 41%), megakaryocytic hyperplasia and/or clustering (n = 10; 37%) and bone marrow fibrosis >MF-1 (17/22; 77%). About one third of patients either initially presented with acute myeloid leukemia (AML) or eventually progressed to AML. Eighteen (68%) patients had a dominant clone with SRSF2 mutation and nine (33%) patients had a dominant clone with a classic MPN-associated driver mutation. Our data suggest that the presence of an SRSF2 mutation preceding the acquisition of a MPN driver mutations is not a disease-defining alteration nor is it restricted to any specific disease entity within the spectrum of myeloid neoplasms. In summary, patients with myeloid neoplasms associated with concurrent SRSF2 and classic MPN driver mutations have clinical and morphologic features close to that of classic MPNs often with frequent dysplasia and monocytosis.

International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data.

The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

Immunohistochemical loss of enhancer of Zeste Homolog 2 (EZH2) protein expression correlates with EZH2 alterations and portends a worse outcome in myelodysplastic syndromes.

EZH2 coding mutation (EZH2MUT), resulting in loss-of-function, is an independent predictor of overall survival in MDS. EZH2 function can be altered by other mechanisms including copy number changes, and mutations in other genes and non-coding regions of EZH2. Assessment of EZH2 protein can identify alterations of EZH2 function missed by mutation assessment alone. Precise evaluation of EZH2 function and gene-protein correlation in clinical MDS cohorts is important in the context of upcoming targeted therapies aimed to restore EZH2 function. In this study, we evaluated the clinicopathologic characteristics of newly diagnosed MDS patients with EZH2MUT and correlated the findings with protein expression using immunohistochemistry. There were 40 (~6%) EZH2MUT MDS [33 men, seven women; median age 74 years (range, 55-90)]. EZH2 mutations spanned the entire coding region. Majority had dominant EZH2 clone [median VAF, 30% (1-92)], frequently co-occurring with co-dominant TET2 (38%) and sub-clonal ASXL1 (55%) and RUNX1 (43%) mutations. EZH2MUT MDS showed frequent loss-of-expression compared to EZH2WT (69% vs. 27%, p = 0.001). Interestingly, NINE (23%) EZH2WT MDS also showed loss-of-expression. EZH2MUT and loss-of-expression significantly associated with male predominance and chr(7) loss. Further, only EZH2 loss-of-expression patients showed significantly lower platelet counts, a trend for higher BM blast% and R-IPSS scores. Over a 14-month median follow-up, both EZH2MUT (p = 0.027) and loss-of-expression (p = 0.0063) correlated with poor survival, independent of R-IPSS, age and gender. When analyzed together, loss-of-expression showed a stronger correlation than mutation (p = 0.061 vs. p = 0.43). In conclusion, immunohistochemical assessment of EZH2 protein, alongside mutation, is important for prognostic workup of MDS.

Primary mediastinal germ cell tumor and clonally related and unique hematologic neoplasms with i(12p) and TP53 mutation: A report of two cases.

The development of clonally related hematologic neoplasms in the setting of primary mediastinal germ cell tumors (PMGCTs) has been recognized previously and is associated with a dismal prognosis. However, the presentation of hematologic neoplasms as chronic myelomonocytic leukemia (CMML) and hemophagocytic lymphohistiocytosis (HLH) has been rarely reported. Here we report two patients with PMGCTs and hematologic neoplasms. The PMGCT was composed mostly of yolk sac tumor whereas the hematologic neoplasms had morphologic features that resembled CMML and HLH. The hematologic neoplasms from both patients harbored isochromosome 12p [i(12p)] and TP53 mutations, supporting a clonal relationship between these tumors. This association represents a unique clinical syndrome that likely contributes to the poor clinical outcome of these patients.

Polarization Sensitive Photothermal Mid-Infrared Spectroscopic Imaging of Human Bone Marrow Tissue.

Collagen quantity and integrity play an important role in understanding diseases such as myelofibrosis (MF). Label-free mid-infrared spectroscopic imaging (MIRSI) has the potential to quantify collagen while minimizing the subjective variance observed with conventional histopathology. Infrared (IR) spectroscopy with polarization sensitivity provides chemical information while also estimating tissue dichroism. This can potentially aid MF grading by revealing the structure and orientation of collagen fibers. Simultaneous measurement of collagen structure and biochemical properties can translate clinically into improved diagnosis and enhance our understanding of disease progression. In this paper, we present the first report of polarization-dependent spectroscopic variations in collagen from human bone marrow samples. We build on prior work with animal models and extend it to human clinical biopsies with a practical method for high-resolution chemical and structural imaging of bone marrow on clinical glass slides. This is done using a new polarization-sensitive photothermal mid-infrared spectroscopic imaging scheme that enables sample and source independent polarization control. This technology provides 0.5 µm spatial resolution, enabling the identification of thin (≈1 µm) collagen fibers that were not separable using Fourier Transform Infrared (FT-IR) imaging in the fingerprint region at diffraction-limited resolution ( ≈ 5 µm). Finally, we propose quantitative metrics to identify fiber orientation from discrete band images (amide I and amide II) measured under three polarizations. Previous studies have used a pair of orthogonal polarization measurements, which is insufficient for clinical samples since human bone biopsies contain collagen fibers with multiple orientations. Here, we address this challenge and demonstrate that three polarization measurements are necessary to resolve orientation ambiguity in clinical bone marrow samples. This is also the first study to demonstrate the ability to spectroscopically identify thin collagen fibers (≈1 µm diameter) and their orientations, which is critical for accurate grading of human bone marrow fibrosis.

Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia.

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.