Author Correction: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia.

In the originally published version of this Article, the GAPDH loading control blot in Fig. 1a was inadvertently replaced with a duplicate of the DNMT2 blot in the same panel during assembly of the figure. This has now been corrected in both the PDF and HTML versions of the Article.

RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia.

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia.

Initial Diagnostic Workup of Acute Leukemia: Guideline From the College of American Pathologists and the American Society of Hematology.

CONTEXT: - A complete diagnosis of acute leukemia requires knowledge of clinical information combined with morphologic evaluation, immunophenotyping and karyotype analysis, and often, molecular genetic testing. Although many aspects of the workup for acute leukemia are well accepted, few guidelines have addressed the different aspects of the diagnostic evaluation of samples from patients suspected to have acute leukemia. OBJECTIVE: - To develop a guideline for treating physicians and pathologists involved in the diagnostic and prognostic evaluation of new acute leukemia samples, including acute lymphoblastic leukemia, acute myeloid leukemia, and acute leukemias of ambiguous lineage. DESIGN: - The College of American Pathologists and the American Society of Hematology convened a panel of experts in hematology and hematopathology to develop recommendations. A systematic evidence review was conducted to address 6 key questions. Recommendations were derived from strength of evidence, feedback received during the public comment period, and expert panel consensus. RESULTS: - Twenty-seven guideline statements were established, which ranged from recommendations on what clinical and laboratory information should be available as part of the diagnostic and prognostic evaluation of acute leukemia samples to what types of testing should be performed routinely, with recommendations on where such testing should be performed and how the results should be reported. CONCLUSIONS: - The guideline provides a framework for the multiple steps, including laboratory testing, in the evaluation of acute leukemia samples. Some aspects of the guideline, especially molecular genetic testing in acute leukemia, are rapidly changing with new supportive literature, which will require on-going updates for the guideline to remain relevant.

The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia.

The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues was last updated in 2008. Since then, there have been numerous advances in the identification of unique biomarkers associated with some myeloid neoplasms and acute leukemias, largely derived from gene expression analysis and next-generation sequencing that can significantly improve the diagnostic criteria as well as the prognostic relevance of entities currently included in the WHO classification and that also suggest new entities that should be added. Therefore, there is a clear need for a revision to the current classification. The revisions to the categories of myeloid neoplasms and acute leukemia will be published in a monograph in 2016 and reflect a consensus of opinion of hematopathologists, hematologists, oncologists, and geneticists. The 2016 edition represents a revision of the prior classification rather than an entirely new classification and attempts to incorporate new clinical, prognostic, morphologic, immunophenotypic, and genetic data that have emerged since the last edition. The major changes in the classification and their rationale are presented here.

Brca1 deficiency causes bone marrow failure and spontaneous hematologic malignancies in mice.

BRCA1 is critical for maintenance of genomic stability and interacts directly with several proteins that regulate hematopoietic stem cell function and are part of the Fanconi anemia (FA) double-strand break DNA repair pathway. The effects of complete BRCA1 deficiency on bone marrow (BM) function are unknown. To test the hypothesis that Brca1 is essential in hematopoiesis, we developed a conditional mouse model with Mx1-Cre-mediated Brca1 deletion. Mice lacking Brca1 in the BM have baseline cytopenias and develop spontaneous bone marrow failure or diverse hematologic malignancies by 6 months of age. Brca1(-/-) BM cells have a reduced capacity to form hematopoietic colonies in vitro and to reconstitute hematopoiesis in irradiated recipients, consistent with a hematopoietic progenitor functional defect. Brca1(-/-) BM cells also show FA-like hypersensitivity to the DNA crosslinking agent mitomycin C, and karyotypes feature genomic instability. Taken together, our results show that loss of Brca1 in murine BM causes hematopoietic defects similar to those seen in people with FA, which provides strong evidence that Brca1 is critical for normal hematopoiesis and that Brca1 is a bona fide FA-like gene.

Complex or monosomal karyotype and not blast percentage is associated with poor survival in acute myeloid leukemia and myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2): a Bone Marrow Pathology Group study.

Acute myeloid leukemia and myelodysplastic syndrome with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) have a poor prognosis. Indeed, the inv(3)(q21q26.2)/t(3;3)(q21;q26.2) has been recognized as a poor risk karyotype in the revised International Prognostic Scoring System. However, inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is not among the cytogenetic abnormalities pathognomonic for diagnosis of acute myeloid leukemia irrespective of blast percentage in the 2008 WHO classification. This multicenter study evaluated the clinico-pathological features of acute myeloid leukemia/myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and applied the revised International Prognostic Scoring System to myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2). A total of 103 inv(3)(q21q26.2)/t(3;3)(q21;q26.2) patients were reviewed and had a median bone marrow blast count of 4% in myelodysplastic syndrome (n=40) and 52% in acute myeloid leukemia (n=63) (P<0.001). Ninety-one percent of patients showed characteristic dysmegakaryopoiesis. There was no difference in overall survival between acute myeloid leukemia and myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) (12.9 vs. 7.9 months; P=0.16). Eighty-three percent of patients died (median follow up 7.9 months). Complex karyotype, monosomal karyotype and dysgranulopoiesis (but not blast percentage) were independent poor prognostic factors in the entire cohort on multivariable analysis. The revised International Prognostic Scoring System better reflected overall survival of inv(3)(q21q26.2)/t(3;3)(q21;q26.2) than the International Prognostic Scoring System but did not fully reflect the generally dismal prognosis. Our data support consideration of myelodysplastic syndrome with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) as an acute myeloid leukemia with recurrent genetic abnormalities, irrespective of blast percentage.

Dose intensification of daunorubicin and cytarabine during treatment of adult acute lymphoblastic leukemia: results of Cancer and Leukemia Group B Study 19802.

BACKGROUND: Cancer and Leukemia Group B (CALGB) Study 19802, a phase 2 study, evaluated whether dose intensification of daunorubicin and cytarabine could improve disease-free survival (DFS) in adults with acute lymphoblastic leukemia (ALL) and whether high-dose systemic and intrathecal methotrexate could replace cranial radiotherapy for central nervous system (CNS) prophylaxis. METHODS: One hundred sixty-one eligible, previously untreated patients ages 16 to 82 years (median age, 40 years) were enrolled, and 33 (20%) were aged ≥60 years. RESULTS: One hundred twenty-eight patients (80%) achieved complete remission (CR). Dose intensification of daunorubicin and cytarabine was feasible. At a median follow-up of 10.4 years for surviving patients, the 5-year DFS rate was 25% (95% confidence interval, 18%-33%), and the overall survival (OS) rate was 30% (95% confidence interval, 23%-37%). Patients aged <60 years who received the 80 mg/m(2) dose of daunorubicin had a DFS of 33% (95% confidence interval, 22%-44%) and an OS of 39% (95% confidence interval, 29%-49%) at 5 years. Eighty-four patients (52%) relapsed, including 9 patients (6%) who had isolated CNS relapses. The omission of cranial irradiation did not result in higher than historic CNS relapse rates. CONCLUSIONS: Intensive systemic, oral, and intrathecal methotrexate dosing permitted the omission of CNS irradiation in adult patients with ALL. This intensive approach using higher doses of daunorubicin and cytarabine failed to result in an overall improvement in DFS or OS compared with historic CALGB studies. Future therapeutic strategies for adults with ALL should be tailored to specific age and molecular genetic subsets.

Myelodysplastic/myeloproliferative neoplasms.

The myelodysplastic/myeloproliferative neoplasms (MDS/MPN) include clonal myeloid neoplasms that overlap the MDS and MPN categories and at the time of initial diagnosis exhibit some clinical, laboratory, or morphologic features supporting the diagnosis of myelodysplastic syndrome (MDS) and at the same time show proliferative features in keeping with the diagnosis of a myeloproliferative neoplasm (MPN). Although the clinical, morphologic, and laboratory findings vary along a continuum from MDS to MPN, distinctive features are usually present that allow assignment of most of the cases to 1 of 3 distinct subtypes recognized by the 2008 World Health Organization (WHO) classification: chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR-ABL(-)(aCML, BCR-ABL1(-)), and juvenile myelomonocytic leukemia (JMML). The WHO classification also recognizes a provisional category of the MDS/MPN, unclassifiable (MDS/MPN, U), including the provisional entity of refractory anemia with ring sideroblasts and thrombocytosis (RARS-T). In the past 2 to 3 years since the publication of the WHO classification in 2008, dynamic progress in array technologies and next-generation amplicon deep sequencing has provided new insights into the molecular pathogenesis of MDS/MPN, especially CMML and JMML. In this review we will give an overview of these neoplasms and focus on adult MDS/MPN, especially CMML. We will give only brief updates for aCML and RARS-T; JMML will be discussed in a separate article.

P-glycoprotein inhibition using valspodar (PSC-833) does not improve outcomes for patients younger than age 60 years with newly diagnosed acute myeloid leukemia: Cancer and Leukemia Group B study 19808.

Cancer and Leukemia Group B 19808 (CALGB 19808) is the only randomized trial of a second-generation P-glycoprotein (Pgp) modulator in untreated patients with acute myeloid leukemia (AML) younger than age 60 years. We randomly assigned 302 patients to receive induction chemotherapy regimens consisting of cytosine arabinoside (Ara-C; A), daunorubicin (D), and etoposide (E), without (ADE) or with (ADEP) PSC-833 (P). The incidence of complete remission was 75% with both regimens. Reversible grade 3 and 4 liver and mucosal toxicities were significantly more common with ADEP. Therapy-related mortality was 7% and did not differ by induction arm. Excess cardiotoxicity was not seen with high doses of D in ADE. The median disease-free survival was 1.34 years in the ADE arm and 1.09 years in the ADEP arm (P = .74, log-rank test); the median overall survival was 1.86 years in the ADE arm and 1.69 years in the ADEP arm (P = .82). There was no evidence of a treatment difference within any identifiable patient subgroup. Inhibition of Pgp-mediated drug efflux by PSC-833 did not improve clinical outcomes in younger patients with untreated AML. This trial was registered at www.clinicaltrials.gov as #NCT00006363.

The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues: an overview with emphasis on the myeloid neoplasms.

The World Health Organization (WHO) classification of myeloid and lymphoid neoplasms utilizes morphology, immunophenotype, genetics and clinical features to define disease entities of clinical significance. It is a consensus classification in which a number of experts have agreed on the classification and diagnostic criteria. In general, the classification stratifies neoplasms according to their lineage (myeloid, lymphoid, histiocytic/dendritic) and distinguishes neoplasms of precursor cells from those comprised of functionally mature cells. Lymphoid neoplasms are derived from cells that frequently have features that recapitulate stages of normal B-, T-, and NK-cell differentiation and function, so to some extent they can be classified according to the corresponding normal counterpart, although additional features, such as genotype, clinical features and even location of the tumor figure into the final classification listing as well. Five major subgroups of myeloid neoplasms are recognized based mainly on their degree of maturation and biologic properties: myeloproliferative neoplasms (MPNs) which are comprised primarily of mature cells with effective proliferation; myeloid (and lymphoid) neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB and FGFR1, defined largely by the finding of significant eosinophilia and specific genetic abnormalities; myelodysplastic/myeloproliferative neoplasms (MDS/MPN), comprised mainly of mature cells with both effective and ineffective proliferation of various lineages; myelodysplastic syndromes (MDS), in which immature and mature cells are found with abnormal, dysplastic and ineffective maturation, and acute myeloid leukemia (AML), comprised of precursor cells with impaired maturation. Genetic abnormalities play an important role as diagnostic criteria for further sub-classification of some myeloid neoplasms, particularly of AML. Although therapy-related MDS and AML (t-MDS/AML) often have genetic defects identical to those found in de novo AML and de novo MDS, they are classified separately from de novo AML and MDS in order to emphasize their unique clinical and biologic properties.

Myeloproliferative neoplasms: contemporary diagnosis using histology and genetics.

The 2008 WHO classification system for hematological malignancies is comprehensive and includes histology and genetic information. Myeloid neoplasms are now classified into five categories: acute myeloid leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN, and myeloid and/or lymphoid malignancies associated with eosinophilia and PDGFR or FGFR1 rearrangements. MPN are subclassified into eight separate entities: chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, systemic mastocytosis, chronic eosinophilic leukemia not otherwise specified, chronic neutrophilic leukemia, and unclassifiable MPN. The diagnosis of chronic myelogenous leukemia requires the presence of BCR-ABL1, while its absence is required for all other MPN. Additional MPN-associated molecular markers include mutations of JAK2, MPL, TET2 and KIT. JAK2 V617F is found in most patients with polycythemia vera, essential thrombocythemia, or primary myelofibrosis and is, therefore, useful as a clonal marker in those settings. The diagnostic utility of MPL and TET2 mutations is limited by low mutational frequency. In systemic mastocytosis, presence of KIT D816V is expected but not essential for diagnosis. Chronic eosinophilic leukemia not otherwise specified should be distinguished from both PDGFR-rearranged or FGFR1-rearranged neoplasms and hypereosinophilic syndrome. We discuss histologic, cytogenetic and molecular changes in MPN and illustrate their integration into practical diagnostic algorithms.

Pomalidomide is active in the treatment of anemia associated with myelofibrosis.

PURPOSE: Thalidomide and lenalidomide can alleviate anemia in myelofibrosis. However, their value is undermined by their respective potential to cause peripheral neuropathy and myelosuppression. We therefore evaluated the safety and therapeutic activity of another immunomodulatory drug, pomalidomide. METHODS: In a phase II randomized, multicenter, double-blind, adaptive design study, four treatment arms were evaluated: pomalidomide (2 mg/d) plus placebo, pomalidomide (2 mg/d) plus prednisone, pomalidomide (0.5 mg/d) plus prednisone, and prednisone plus placebo. Pomalidomide was administered for up to 12 28-day treatment cycles. Prednisone (30 mg/d) was given in a tapering dose schedule during the first three cycles. Response was assessed by International Working Group criteria. RESULTS: Eighty-four patients with myelofibrosis-associated anemia were randomly assigned to the aforementioned treatment arms: 22, 19, 22, and 21, respectively. Response in anemia was documented in 20 patients, including 15 who became transfusion independent. Response rates in the four treatment arms were 23% (95% CI, 5% to 41%), 16% (95% CI, 0% to 33%), 36% (95% CI, 16% to 56%), and 19% (95% CI, 2% to 36%). The corresponding figures for patients receiving > or = 3 cycles of treatment (n = 62) were 38%, 23%, 40%, and 25%. Response to pomalidomide with or without prednisone was durable (range, 3.2 to 16.9+ months) and significantly better in the absence of leukocytosis (37% v 8%; P = .01); JAK2V617F or cytogenetic status did not affect response. Grade > or = 3 toxicities were infrequent and included (in each treatment arm) neutropenia (9%; 16%; 5%; 5%), thrombocytopenia (14%; 16%; 9%; 5%), and thrombosis (9%; 5%; 0%; 0%). CONCLUSION: Pomalidomide therapy at 0.5 or 2 mg/d with or without an abbreviated course of prednisone is well tolerated in patients with myelofibrosis and active in the treatment of anemia.

Chronic myelogenous leukemia, BCR-ABL1+.

Session 1 of the 2007 Workshop of the Society for Hematopathology/European Association for Haematopathology focused on chronic myelogenous leukemia, BCR-ABL1+ (CML). CML is a myeloproliferative neoplasm arising at the level of a pluripotent stem cell and consistently associated with the BCR-ABL1 fusion gene. CML most commonly manifests in a chronic phase of the disease with neutrophilic leukocytosis, and the demonstration of the Philadelphia chromosome is the ultimate confirmation of the diagnosis. However, in select cases, the initial diagnosis remains challenging, and a number of issues pertaining to the manifestations and disease evolution remain unresolved. These issues have been illustrated by the cases submitted to our workshop and include unusual manifestations of CML, including manifestation in the accelerated and/or blast phase, and patterns of disease progression and therapy resistance in the era of protein tyrosine kinase inhibitor therapy.

The 2008 World Health Organization classification system for myeloproliferative neoplasms: order out of chaos.

The first formal classification of chronic myeloid neoplasms is credited to William Dameshek, who in 1951 described the concept of "myeloproliferative disorders (MPD)" by grouping together chronic myelogenous leukemia, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2001 World Health Organization (WHO) classification of myeloid malignancies included these MPDs under the broader category of chronic myeloproliferative diseases (CMPD), which also included chronic neutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome (CEL/HES), and "CMPD, unclassifiable." The revised 2008 WHO classification system featured the following changes: 1) the term "CMPD" was replaced by "myeloproliferative neoplasm (MPN)," 2) mast cell disease was formally included under the category of MPN, and 3) the subcategory of CEL/HES was reorganized into "CEL not otherwise specified (CEL-NOS)" and "myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, and FGFR1"; CEL-NOS remained a subcategory of "MPN," whereas the latter neoplasms were now assigned a new category of their own. Furthermore, diagnostic criteria for PV, ET, and PMF were revised by incorporating recently described molecular markers (eg, JAK2 and MPL mutations) as well as underscoring the role of histology in differentiating reactive from clonal myeloproliferations. As a result, red cell mass measurement is no longer necessary for the diagnosis of PV, and ET can now be diagnosed at a lower platelet count threshold. The revised WHO document continues to promote the recognition of histologic categories as a necessary first step toward the genetic characterization of myeloid malignancies.

The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes.

Recently the World Health Organization (WHO), in collaboration with the European Association for Haematopathology and the Society for Hematopathology, published a revised and updated edition of the WHO Classification of Tumors of the Hematopoietic and Lymphoid Tissues. The 4th edition of the WHO classification incorporates new information that has emerged from scientific and clinical studies in the interval since the publication of the 3rd edition in 2001, and includes new criteria for the recognition of some previously described neoplasms as well as clarification and refinement of the defining criteria for others. It also adds entities-some defined principally by genetic features-that have only recently been characterized. In this paper, the classification of myeloid neoplasms and acute leukemia is highlighted with the aim of familiarizing hematologists, clinical scientists, and hematopathologists not only with the major changes in the classification but also with the rationale for those changes.

Treatment of myelodysplastic syndrome with 2 schedules and doses of oral topotecan: a randomized phase 2 trial by the Cancer and Leukemia Group B (CALGB 19803).

BACKGROUND: The Cancer and Leukemia Group B evaluated oral topotecan administered at 2 schedules and doses for myelodysplastic syndrome (MDS). METHODS: Patients with previously untreated primary or therapy-related MDS were eligible. Patients with refractory anemia (RA), RA with ringed sideroblasts, or refractory cytopenia with multilineage dysplasia (RCMD) were eligible only if they were dependent on erythrocyte transfusion, had a platelet count<50,000/microL, or had an absolute neutrophil count<1000/microL with a recent infection that required antibiotics. Patients were randomized to receive oral topotecan either at a dose of 1.2 mg/m2 twice daily for 5 days (Arm A) or once daily for 10 days (Arm B) repeated every 21 days for at least 2 cycles. Responding patients continued until they developed disease progression or unacceptable toxicity or until they had received 2 cycles beyond a complete response. RESULTS: Ninety patients received treatment, including 46 patients on Arm A and 44 patients on Arm B. Partial responses with improvement in all 3 cell lines occurred in 6 patients (7%), and hematologic improvement (in 1 or 2 cell lines) was observed in 21 patients (23%), for an overall response rate of 30%. Response duration was longer on Arm A (23 months vs 14 months; P=.02). Seven of 14 patients with chronic myelomonocytic leukemia responded. There were 8 treatment-related deaths from infection (6 deaths) and bleeding (2 deaths). Diarrhea was the most frequent nonhematologic toxicity (grade 3, 11%; grade 4, 2%; grading determined according to the National Cancer Institute Comman Toxicity Criteria v.2.0). CONCLUSIONS: Oral topotecan in the dose and schedules evaluated in this trial demonstrated only a modest response rate with a troublesome toxicity profile in the treatment of MDS.