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Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms characterized by recurrent somatic mutations in JAK2, CALR, and MPL. This short review addresses (1) the spectrum of mutations seen in PV, ET, and PMF, (2) the emerging genotype-phenotype correlations, (3) the current role of molecular testing in disease classification and management, and (4) several important considerations for selecting an appropriate molecular test. In our view, sequential testing algorithms and simultaneous assessment of multiple mutations by next-generation sequencing are both valid approaches to testing. Am. J. Hematol. 91:1277-1280, 2016. © 2016 Wiley Periodicals, Inc.

Primary mediastinal large B-cell lymphoma (PMBL) is a distinct subtype of diffuse large B-cell lymphoma (DLBCL) that shows overlap with classical Hodgkin lymphoma (CHL) and a favorable prognosis compared to mediastinal gray-zone lymphoma (MGZL). We performed immunohistochemistry on initial diagnostic specimens of 49 cases of uniformly treated PMBL to determine the frequency and clinical significance of expression of antigens commonly seen in CHL and MGZL, along with markers previously shown to be prognostic in DLBCL, not otherwise specified. The median age was 37 years with a female:male ratio of 2.3. After a median follow-up of 78 months, 24% of patients had relapsed or refractory disease and 22% had died; the 5-year PFS was 70%. Variable CD15 expression was seen in 31% of cases, but was not associated with adverse outcome. Hans cell-of-origin, proliferation index, and MYC/BCL2 coexpression were not associated with outcome, while low PDL1 (P = 0.011) and high MUM1 (P = 0.065) staining were each associated with shorter PFS. A biologic risk score (one point each for low PDL1 and high MUM1) stratified patients into three prognostic risk groups for PFS (P = 0.001) and OS (P = 0.032). On separate multivariate models, low PDL1 was independent of R-IPI risk group for PFS (HR 6.0, P = 0.023), as was a biologic risk score of 2 (HR 5.6, P = 0.011). Incorporation of the biologic risk score sub-stratified patients within R-IPI groups for both PFS (P

Bone marrow (BM) evaluation is an important part of lymphoma staging, which guides patient management. Although positive staging marrow is defined as morphologically identifiable disease, such samples often also include flow cytometric analysis and conventional karyotyping. Cytogenetic analysis is a labor-intensive and costly procedure and its utility in this setting is uncertain. We retrospectively reviewed pathological reports of 526 staging marrow specimens in which conventional karyotyping had been performed. All samples originated from a single institution from patients with previously untreated Hodgkin and non-Hodgkin lymphomas presenting in an extramedullary site. Cytogenetic analysis revealed clonal abnormalities in only eight marrow samples (1.5%), all of which were positive for lymphoma by morphologic evaluation. Flow cytometry showed a small clonal lymphoid population in three of the 443 morphologically negative marrow samples (0.7%). Conventional karyotyping is rarely positive in lymphoma staging marrow samples and, in our cohort, the BM karyotype did not contribute clinically relevant information in the vast majority of cases. Our findings suggest that karyotyping should not be performed routinely on BM samples taken to stage previously diagnosed extramedullary lymphomas unless there is pathological evidence of BM involvement by lymphoma.

Recent work has identified distinct molecular subgroups of acute myeloid leukemia (AML) with implications for disease classification and prognosis. NPM1 is one of the most common recurrently mutated genes in AML. NPM1 mutations often co-occur with FLT3-ITDs and mutations in genes regulating DNA methylation, such as DNMT3A, TET2, and IDH1/2. It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis. We identified 133 cases of NPM1-mutated AML and correlated sequencing data with immunophenotypic and clinical findings. Of 84 cases (63%) that lacked monocytic differentiation ("myeloid AML"), 40 (48%) demonstrated an acute promyelocytic leukemia-like (APL-like) immunophenotype by flow cytometry, with absence of CD34 and HLA-DR and strong myeloperoxidase expression, in the absence of a PML-RARA translocation. Pathologic variants in TET2, IDH1, or IDH2 were identified in 39/40 APL-like cases. This subset of NPM1-mutated AML was associated with longer relapse-free and overall survival, when compared with cases that were positive for CD34 and/or HLA-DR. The combination of NPM1 and TET2 or IDH1/2 mutations along with an APL-like immunophenotype identifies a distinct subtype of AML. Further studies addressing its biology and clinical significance may be especially relevant in the era of IDH inhibitors and recent work showing efficacy of ATRA therapy in NPM1 and IDH1-mutated AML.

The purpose of the study was to assess consensus and interobserver agreement among an international panel of six hematopathologists regarding characterization and reproducibility of bone marrow (BM) histologic features used to diagnose early stage myeloproliferative neoplasms, in particular differentiation of so-called masked/prodromal polycythemia vera (mPV) from JAK2-mutated essential thrombocythemia (ET). The six members of the hematopathology panel evaluated 98 BM specimens independently and in a blinded fashion without knowledge of clinical data. The specimens included 48 cases of mPV according to the originally published hemoglobin threshold values for this entity (male: 16.0-18.4 g/dL, female: 15.0-16.4 g/dL), 31 cases with overt PV according to the updated 2016 WHO criteria, and 19 control cases. The latter group included cases of JAK2-mutated ET, primary myelofibrosis, myelodysplastic syndrome, and various reactive conditions. Inter-rater agreement between the panelists was very high (overall agreement 92.6%, kappa 0.812), particularly with respect to separating mPV from ET. Virtually all cases of mPV were correctly classified as PV according to their BM morphology. In conclusion, a central blinded review of histology slides by six hematopathologists demonstrated that highly reproducible specific histological pattern characterize PV and confirmed the notion that there are no significant differences between mPV and overt PV in relation to BM morphology.

Flow cytometry (FCM) is an adjunct study to routine analysis of cerebrospinal fluid (CSF) to investigate for involvement by a hematologic malignancy. However, in our experience, FCM only infrequently detects abnormalities in CSF. To help optimize resources without forfeiting clinically important data, we sought to determine evidence-based indications and criteria for performing FCM on CSF. FCM results of 316 consecutive CSF specimens were retrospectively reviewed and correlated with clinical history, total nucleated cell (TNC) counts, and results of concurrent cytologic review. Of 255 samples adequate for analysis, 54% were from patients with a prior history of hematologic malignancy, of which 12% (17 cases) were abnormal by FCM. Corresponding TNC counts among samples with abnormal FCM ranged from 0–1050 cells/µL, and only 44% showed abnormal morphology on concurrent cytology. Of the remaining 46% of samples from patients with no known history of hematologic malignancy who had CSF sampling for neurological indications, only one (1%) was abnormal by FCM. This specimen had an elevated TNC count (39 cells/µL) but lacked clearly abnormal findings on concurrent cytology. These results support the use of CSF FCM only in patients with a history of hematologic malignancy or, in the absence of such a history, in samples showing pleocytosis. If these criteria were applied to the current cohort using a TNC count cut-off of >5 cells/µL, 23% of samples would have been deferred from testing, resulting in decreased cost, improved efficiency, and reduction in the need for unnecessary testing without a negative impact on clinical care. Am. J. Hematol. 89:978–984, 2014. © 2014 Wiley Periodicals, Inc.

It is controversial whether acute myeloid leukemia (AML) patients with 20–29% bone marrow (BM) blasts, formerly referred to as refractory anemia with excess blasts in transformation (RAEBT), should be considered AML or myelodysplastic syndrome (MDS) for the purposes of treatment and prognostication. We retrospectively studied 571 de novo AML in patients aged >50 years, including 142 RAEBT and 429 with ≥30% blasts (AML30), as well as 151 patients with 10–19% BM blasts (RAEB2). RAEBT patients were older and had lower white blood count, but higher hemoglobin, platelet count, and karyotype risk scores compared to AML30, while these features were similar to RAEB2. FLT3 and NPM1 mutations and monocytic morphology occurred more commonly in AML30 than in RAEBT. RAEBT patients were treated less often with induction therapy than AML30, whereas allogeneic stem cell transplant frequency was similar. The median and 4-year OS of RAEBT patients were longer than those of AML30 patients (20.5 vs 12.0 months and 28.6% vs 20.4%, respectively, P = 0.003); this difference in OS was manifested in patients in the intermediate UKMRC karyotype risk group, whereas OS of RAEBT patients and AML30 patients in the adverse karyotype risk group were not significantly different. Multivariable analysis showed that RAEBT (P

Bone marrow (BM) evaluation is an important part of lymphoma staging, which guides patient management. Although positive staging marrow is defined as morphologically identifiable disease, such samples often also include flow cytometric analysis and conventional karyotyping. Cytogenetic analysis is a labor-intensive and costly procedure and its utility in this setting is uncertain. We retrospectively reviewed pathological reports of 526 staging marrow specimens in which conventional karyotyping had been performed. All samples originated from a single institution from patients with previously untreated Hodgkin and non-Hodgkin lymphomas presenting in an extramedullary site. Cytogenetic analysis revealed clonal abnormalities in only eight marrow samples (1.5%), all of which were positive for lymphoma by morphologic evaluation. Flow cytometry showed a small clonal lymphoid population in three of the 443 morphologically negative marrow samples (0.7%). Conventional karyotyping is rarely positive in lymphoma staging marrow samples and, in our cohort, the BM karyotype did not contribute clinically relevant information in the vast majority of cases. Our findings suggest that karyotyping should not be performed routinely on BM samples taken to stage previously diagnosed extramedullary lymphomas unless there is pathological evidence of BM involvement by lymphoma.

Flow cytometry (FCM) is an adjunct study to routine analysis of cerebrospinal fluid (CSF) to investigate for involvement by a hematologic malignancy. However, in our experience, FCM only infrequently detects abnormalities in CSF. To help optimize resources without forfeiting clinically important data, we sought to determine evidence-based indications and criteria for performing FCM on CSF. FCM results of 316 consecutive CSF specimens were retrospectively reviewed and correlated with clinical history, total nucleated cell (TNC) counts, and results of concurrent cytologic review. Of 255 samples adequate for analysis, 54% were from patients with a prior history of hematologic malignancy, of which 12% (17 cases) were abnormal by FCM. Corresponding TNC counts among samples with abnormal FCM ranged from 0-1050 cells/µL, and only 44% showed abnormal morphology on concurrent cytology. Of the remaining 46% of samples from patients with no known history of hematologic malignancy who had CSF sampling for neurological indications, only one (1%) was abnormal by FCM. This specimen had an elevated TNC count (39 cells/µL) but lacked clearly abnormal findings on concurrent cytology. These results support the use of CSF FCM only in patients with a history of hematologic malignancy or, in the absence of such a history, in samples showing pleocytosis. If these criteria were applied to the current cohort using a TNC count cut-off of5 cells/µL, 23% of samples would have been deferred from testing, resulting in decreased cost, improved efficiency, and reduction in the need for unnecessary testing without a negative impact on clinical care.