Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3 and MPL mutations.

Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to 2 patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients, rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.

A Kindred with a ß-Globin Base Substitution [ß89(F5)Ser→Arg (AGT>AGG); HBB: c.270T>G] Resulting in Hemoglobin Vanderbilt.

High oxygen affinity hemoglobins (Hbs), characterized by a decreased ability to release oxygen to the tissues and a left-shifted oxygen dissociation curve, are a rare cause of secondary erythrocytosis. Here, we report a base substitution in the ß-globin gene at codon 89 (AGT>AGG) in a kindred with familial erythrocytosis resulting in Hb Vanderbilt, a high oxygen affinity variant.

Genomic landscape of neutrophilic leukemias of ambiguous diagnosis.

Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.

MYC Regulates the HIF2α Stemness Pathway via Nanog and Sox2 to Maintain Self-Renewal in Cancer Stem Cells versus Non-Stem Cancer Cells.

Cancer stem cells (CSC) maintain both undifferentiated self-renewing CSCs and differentiated, non-self-renewing non-CSCs through cellular division. However, molecular mechanisms that maintain self-renewal in CSCs versus non-CSCs are not yet clear. Here, we report that in a transgenic mouse model of MYC-induced T-cell leukemia, MYC, maintains self-renewal in Sca1+ CSCs versus Sca-1- non-CSCs. MYC preferentially bound to the promoter and activated hypoxia-inducible factor-2α (HIF2α) in Sca-1+ cells only. Furthermore, the reprogramming factors, Nanog and Sox2, facilitated MYC regulation of HIF2α in Sca-1+ versus Sca-1- cells. Reduced expression of HIF2α inhibited the self-renewal of Sca-1+ cells; this effect was blocked through suppression of ROS by N-acetyl cysteine or the knockdown of p53, Nanog, or Sox2. Similar results were seen in ABCG2+ CSCs versus ABCG2- non-CSCs from primary human T-cell lymphoma. Thus, MYC maintains self-renewal exclusively in CSCs by selectively binding to the promoter and activating the HIF2α stemness pathway. Identification of this stemness pathway as a unique CSC determinant may have significant therapeutic implications. SIGNIFICANCE: These findings show that the HIF2α stemness pathway maintains leukemic stem cells downstream of MYC in human and mouse T-cell leukemias. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4015/F1.large.jpg.

Revisiting diagnostic criteria for myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis: Borderline cases without anemia exist.

INTRODUCTION: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a rare disease in the 2016 revised World Health Organization (WHO) classification. Diagnostic criteria include the following: persistent thrombocytosis (>450 × 109 /L) with clustering of atypical megakaryocytes, refractory anemia, dyserythropoiesis with ring sideroblasts, and the presence of the spliceosome factor 3b subunit (SF3B1) mutation. It is unclear if anemia should be a required criterion for this diagnosis as cases which show all other features of MDS/MPN-RS-T but without anemia exist. METHODS: We searched for borderline cases of MDS/MPN-RS-T in which refractory anemia was absent at diagnosis in two major academic institutes. RESULTS: Three cases without anemia were identified. These cases all showed other classic morphologic and clinical features of MDS/MPN-RS-T, including thrombocytosis, atypical megakaryocytes with clustering, and characteristic SF3B1 and JAK2 V617F mutations. CONCLUSION: Given these findings, the requirement of refractory anemia as a diagnostic criterion for MDS/MPN-RS-T should be re-evaluated. Removal of refractory anemia as a diagnostic criterion would incorporate current borderline cases and extend the spectrum of this disorder.

Predictive models for splenic response to JAK-inhibitor therapy in patients with myelofibrosis.

JAK inhibitors for myelofibrosis (MF) reduce spleen size, control constitutional symptoms, and may improve survival. We studied the clinical characteristics of 548 MF patients treated with JAK inhibitors from 2008 to 2016 to better understand predictors of splenic response. Response was defined as a 50% decrease in spleen size at early (3-4 months on therapy) and late (5-12 months) timepoints after therapy initiation. Early response positively correlated with higher doses of JAK inhibitor, baseline spleen size 5-10 cm, and hemoglobin. Early response negatively correlated with baseline spleen size >20 cm and high WBC. The strongest predictor of late response was whether a patient had a response at the earlier timepoint (OR 8.88). Our response models suggest that clinical factors can be used to predict which patients are more likely to respond to JAK inhibitors, and those who do not achieve an early response, i.e. within 3-4 months, should consider alternative treatments.

Impact of somatic and germline mutations on the outcome of systemic mastocytosis.

Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of ≥1 multilineal somatic mutation involving genes other than KIT D816V, ≥3 germline variants, and ≥1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and ß2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of ≥1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort.

Treatment With JAK Inhibitors in Myelofibrosis Patients Nullifies the Prognostic Impact of Unfavorable Cytogenetics.

INTRODUCTION: In the era before Janus kinase (JAK) inhibitors, cytogenetic information was used to predict survival in myelofibrosis patients. However, the prognostic value of cytogenetics in the setting of JAK inhibitor therapy remains unknown. PATIENTS AND METHODS: We performed a retrospective analysis of 180 patients with bone marrow biopsy-proven myelofibrosis from 3 US academic medical centers. We fit Cox proportional hazards models for overall survival and transformation-free survival on the bases of 3 factors: JAK inhibitor therapy as a time-dependent covariate, dichotomized cytogenetic status (favorable vs. unfavorable), and statistical interaction between the two. The median follow-up time was 37.1 months. RESULTS: Among patients treated with best available therapy, unfavorable cytogenetic status was associated with decreased survival (hazard ratio = 2.31; P = .025). At initiation of JAK inhibitor therapy, unfavorable cytogenetics was (nonsignificantly) associated with increased survival compared to favorable cytogenetics (hazard ratio = 0.292; P = .172). The ratio of hazard ratios was 0.126 (P = .034). These findings were similar after adjusting for standard clinical prognostic factors as well as when measured against transformation-free survival. CONCLUSION: The initiation of JAK inhibitor therapy appears to change the association between cytogenetics and overall survival. There was little difference in survival between treatment types in patients with favorable cytogenetics. However, the use of JAK inhibitor therapy among patients with unfavorable cytogenetics was not associated with worse survival compared to favorable cytogenetics. Our analyses suggest that initiation of JAK inhibitor therapy nullifies the negative prognostic implication of unfavorable cytogenetics established in the pre-JAK inhibitor therapy era.

Mast cells in systemic mastocytosis have distinctly brighter CD45 expression by flow cytometry.

OBJECTIVES: We sought to determine the significance of bright CD45 expression on mast cells in cases of systemic mastocytosis vs mast cells in bone marrows uninvolved by systemic mastocytosis and compare this CD45 expression with CD25 and CD2 expression on mast cells. METHODS: Multiparameter flow cytometry was performed on 31 cases of systemic mastocytosis and 70 bone marrow cases that were not involved by systemic mastocytosis. Bright expression of CD45 was defined as more than 20% of CD117+ mast cells showing brighter CD45 expression than the average expression level of lymphocytes. RESULTS: Mast cells with bright CD45 expression were seen in 26 systemic mastocytosis cases and three bone marrows uninvolved by systemic mastocytosis (sensitivity, 84%; specificity, 96%). CD25 alone had a greater sensitivity (100%) but lower specificity (93%) compared with bright CD45 for identifying abnormal mast cells, while CD2 alone had lower sensitivity but higher specificity. To reach a specificity of 100%, CD25 together with bright CD45 on mast cells was the optimal combination to detect cases of systemic mastocytosis. CONCLUSIONS: A combination of bright CD45 and CD25 appears to specifically identify abnormal mast cells in cases of systemic mastocytosis. Further studies will be necessary to confirm these results.

Phase II evaluation of IPI-926, an oral Hedgehog inhibitor, in patients with myelofibrosis.

The clinical safety and efficacy of IPI-926 was evaluated in 14 patients with myelofibrosis in a phase II study. Patients received 160 mg IPI-926 orally in continuous 28-day cycles. The median treatment duration was 5.1 months, and all patients had discontinued treatment by 7.5 months. Nine patients discontinued due to lack of response as determined by the treating physician, two after developing acute leukemia and one due to disease progression/loss of response. Twelve patients had slight reductions in spleen size (less than 50% from baseline), but symptoms did not improve consistently. One patient achieved transfusion independence lasting 5 months. Reductions in GLI1 mRNA and protein levels, JAK2V617F allele burden, degree of fibrosis or cytokine levels were observed in some patients, but were not significant when evaluated for the cohort. Low-grade gastrointestinal/liver abnormalities were the most common toxicities. The results did not support continued evaluation of IPI-926 as a monotherapy in myelofibrosis.

Next-generation sequencing of acute myeloid leukemia identifies the significance of TP53, U2AF1, ASXL1, and TET2 mutations.

We assessed the frequency and clinicopathologic significance of 19 genes currently identified as significantly mutated in myeloid neoplasms, RUNX1, ASXL1, TET2, CEBPA, IDH1, IDH2, DNMT3A, FLT3, NPM1, TP53, NRAS, EZH2, CBL, U2AF1, SF3B1, SRSF2, JAK2, CSF3R, and SETBP1, across 93 cases of acute myeloid leukemia (AML) using capture target enrichment and next-generation sequencing. Of these cases, 79% showed at least one nonsynonymous mutation, and cases of AML with recurrent genetic abnormalities showed a lower frequency of mutations versus AML with myelodysplasia-related changes (P<0.001). Mutational analysis further demonstrated that TP53 mutations are associated with complex karyotype AML, whereas ASXL1 and U2AF1 mutations are associated with AML with myelodysplasia-related changes. Furthermore, U2AF1 mutations were specifically associated with trilineage morphologic dysplasia. Univariate analysis demonstrated that U2AF1 and TP53 mutations are associated with absence of clinical remission, poor overall survival (OS), and poor disease-free survival (DFS; P<0.0001), whereas TET2 and ASXL1 mutations are associated with poor OS (P<0.03). In multivariate analysis, U2AF1 and TP53 mutations retained independent prognostic significance in OS and DFS, respectively. Our results demonstrate unique relationships between mutations in AML, clinicopathologic prognosis, subtype categorization, and morphologic dysplasia.

Acute myeloid leukemia with monosomal karyotype: morphologic, immunophenotypic, and molecular findings.

OBJECTIVES: Acute myeloid leukemia (AML) with monosomal karyotype (MK) recently has been reported to be associated with worse outcome than the traditional complex karyotype. METHODS: In this retrospective study of 111 patients with AML, we identified 14 patients with MK (13% of all patients with AML) using the definition proposed by Breems et al. RESULTS: Five (36%) of these 14 patients had a loss of a single chromosome in the presence of other structural abnormalities, and nine (64%) had a loss of two or more autosomal chromosomes. Patients with AML-MK presented at an older age, with lower bone marrow blasts, and their blasts less frequently expressed CD34. Most patients with AML-MK had morphologic multilineage dysplasia and were predominantly subclassified as having AML with myelodysplasia-related changes (AML-MRC). Molecular analysis showed a significant absence of NPM1 and FLT3 in patients with AML-MK. CONCLUSIONS: Outcome data showed that patients with AML-MK had significantly worse overall survival, disease-free survival, and complete response compared with the rest of the patients with AML as well as within the AML-MRC group.

Altered translation of GATA1 in Diamond-Blackfan anemia.

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA), congenital asplenia and T cell leukemia. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type- and tissue-specific defects remains unknown. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.

Hidden mastocytosis in acute myeloid leukemia with t(8;21)(q22;q22).

OBJECTIVES: To assess the frequency of systemic mastocytosis (SM) in a large series of acute myeloid leukemia (AML) with t(8;21)(q22;q22). METHODS: We retrospectively characterized 40 bone marrow aspirate smears and biopsy specimens from patients with AML with t(8;21) for the presence of SM. Cases were assessed for mast cell morphology and immunohistochemistry, as well as KIT exon 8 and 17 mutational assessment by reverse transcription polymerase chain reaction. RESULTS: Four patients met criteria for SM, 1 met criteria for myelomastocytic leukemia, and 8 demonstrated the benign finding of mast cell hyperplasia. CONCLUSIONS: We recommend examining all cases of AML with t(8;21) for the presence of SM via morphology, immunophenotyping, and KIT mutational analysis studies.

Kinase pathway dependence in primary human leukemias determined by rapid inhibitor screening.

Kinases are dysregulated in most cancers, but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here, we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of kinase targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options.

Sequential azacitidine plus lenalidomide combination for elderly patients with untreated acute myeloid leukemia.

There are limited treatment options for older patients with acute myeloid leukemia and prognosis of these patients remains poor, thereby warranting development of novel therapies. We evaluated the efficacy and safety of azacitidine in combination with lenalidomide as front-line therapy for older patients with acute myeloid leukemia. Patients ≥ 60 years of age with untreated acute myeloid leukemia received azacitidine 75 mg/m2 for 7 days followed by escalating doses of lenalidomide daily for 21 days starting on day 8 of each cycle every 6 weeks. Patients received continued therapy until disease progression, unacceptable toxicity, or completion of 12 cycles. Forty-two patients (median age, 74 years) were enrolled with equal distribution according to European LeukemiaNet risk. The overall response rate was 40% (rate of complete remission with or without complete recovery of blood counts = 28%). The median time to complete remission with or without complete recovery of blood counts was 12 weeks, and duration of this status was 28 weeks (range, 4 - >104 weeks). Therapy-related acute myeloid leukemia and a high score on the Hematopoietic Cell Transplantation Comorbidity Index were negative predictors of response. Early death was noted in 17% of patients. Grades ≥ 3 toxicities were uncommon and most adverse events were gastrointestinal, fatigue and myelosuppression. In conclusion, a sequential combination of azacitidine plus lenalidomide has clinical activity in older patients with acute myeloid leukemia, and further studies of this combination are underway.

DNA methylation analysis of ALOX12 and GSTM1 in acute myeloid leukaemia identifies prognostically significant groups.

To determine the role of DNA methylation in the progression of acute myeloid leukaemia (AML), we analysed the methylation status of ALOX12, GSTM1, HS3ST2 and FZD9 in 127 AML patients. Aberrant methylation of ALOX12 was associated with the subcategory AML with myelodysplasia-related changes (P = 0·0439) and specifically with megakaryocytic dysplasia (P = 0·0003). An association between HS3ST2 and AML patients with favourable cytogenetic risk was identified (P = 0·0469). In univariate and multivariate analysis, methylation of GSTM1 was associated with worse overall survival (OS) and disease-free survival (DFS), with hazard ratios of 2·57 and 1·86, respectively. Furthermore, the significance of methylation of GSTM1 in predicting poor prognosis was maintained within the subcategories of AML not otherwise specified (NOS), AML with intermediate cytogenetic risk and normal karyotype AML. Finally, patients with both GSTM1 and ALOX12 methylated, demonstrated worse outcomes when all AML patients were assessed (OS; P = 0·000411) as well as within AML NOS (DFS; P = 0·0023), AML with intermediate cytogenetic risk (OS; P = 0·0104) and normal karyotype AML (OS; P = 0·00636). This study implicates methylation of specific genes in the classification and prognostication of AML and suggests that the morphological feature of multilineage dysplasia may be a surrogate marker of gene methylation in at least a subset of AML cases.

Treatment advances have not improved the early death rate in acute promyelocytic leukemia.

Early mortality in acute promyelocytic leukemia has been reported to occur in less than 10% of patients treated in clinical trials. This study reports the incidence and clinical features of acute promyelocytic leukemia patients treated at Stanford Hospital, CA, USA since March 1997, focusing on early mortality. We show that the risk of early death in acute promyelocytic leukemia patients is higher than previously reported. In a cohort of 70 patients who received induction therapy at Stanford Hospital, 19% and 26% died within seven and 30 days of admission, respectively. High early mortality was not limited to our institution as evaluation of the Surveillance, Epidemiology and End Results Database demonstrated that 30-day mortality for acute promyelocytic leukemia averaged 20% from 1977-2007 and did not improve significantly over this interval. Our findings show that early death is now the greatest contributor to treatment failure in this otherwise highly curable form of leukemia.

Phase I trial of a novel human monoclonal antibody mAb216 in patients with relapsed or refractory B-cell acute lymphoblastic leukemia.

BACKGROUND: This phase I trial was conducted to determine the safety and pharmacokinetics of monoclonal antibody 216, a human monoclonal Immunoglobulin M antibody targeting a linear B-cell lactosamine antigen, administered alone and in combination with vincristine in patients with relapsed or refractory B-cell acute lymphoblastic leukemia, and to preliminarily assess tumor targeting and efficacy. DESIGN AND METHODS: Three cohorts of patients received escalating doses of monoclonal antibody 216 administered as an intravenous infusion. In the case of poor response to the first dose of monoclonal antibody 216 alone, defined as less than 75% reduction in peripheral blood blast count, a second dose of the antibody with vincristine was given between days 4 and 7. Responses were assessed weekly until day 35. Serum concentration of monoclonal antibody 216 was measured before and after infusion. Monoclonal antibody 216 targeting was determined with an anti-idiotypic antibody to monoclonal antibody 216 and preliminary efficacy was analyzed by changes in peripheral blood blasts. RESULTS: Thirteen patients were enrolled. One episode of grade 3 epistaxis was the only dose-limiting toxicity observed. All patients showed a poor response to the first monoclonal antibody 216 infusion with a decrease in peripheral blasts from 6-65% in 9 patients. In 8 patients, addition of vincristine to monoclonal antibody 216 resulted in an average reduction of the peripheral blasts of 81%. One patient without peripheral blasts achieved a hypoplastic marrow without evidence of leukemia after one infusion of monoclonal antibody 216 and monoclonal antibody 216/vincristine each. Monoclonal antibody 216 was detected on peripheral blasts in all patients. CONCLUSIONS: Treatment with monoclonal antibody 216 in combination with vincristine is feasible and well tolerated in patients with relapsed or refractory B-cell acute lymphoblastic leukemia. Binding of monoclonal antibody 216 to leukemic blasts was efficient, and favorable early responses were observed.