A few warfarin pharmacogenetic dosing algorithms have been proposed, based on multiethnic or homogeneous populations, to estimate warfarin therapeutic doses. However, it remains to be proven which algorithm is accurate in predicting warfarin dose in Chinese people. We selected eight warfarin dose predictive pharmacogenetic algorithms and retrospectively assessed the predictive accuracy of each algorithm in a total of 368 eligible outpatients by comparing the actual stable therapeutic dose to the dose predicted by the algorithm. Our results showed that a high level of performance was demonstrated by three algorithms, Gage et al., Anderson et al. and Wu et al., having a similar performance in coefficient of determination (R²) and percentage of patients predicted dose within 20% of actual dose. The Gage et al. algorithm had the lowest mean absolute error (MAE). These results indicated that the algorithm by Gage et al. provided a more accurate prediction than did the others, which suggests that this pharmacogenetic algorithm may be used in clinical practice to guide rational administration of warfarin. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Patients with chemosensitive aggressive non-Hodgkin lymphoma (NHL) could benefit from high-dose chemotherapy (HDC) followed by autologous stem cell transplantation (auto-SCT). We report clinical outcomes of HDC using a novel regimen consisting of mitoxantrone, etoposide, cytarabine, and melphalan (NEAM) with auto-SCT. A total of 69 patients were consecutively enrolled. Median age was 42 years (range, 20-66 years). Median event-free survival (EFS) was 17.9 months. Median overall survival (OS) has not been reached yet and estimated 2-year OS was 64.2%. Among patients with measurable lesions, response rate was 79.5%. Median time to recovery of neutrophil (>500 /microliter) and platelet (>20,000 /microliter) was 12.5 days and 13.5 days, respectively. Febrile neutropenia developed in 61 patients (88.4%). Grade 3 or 4 hepatic toxicity developed in 7 patients (10.1%), grade 3 or 4 renal toxicity in 2 patients (2.9%), and grade 3 or 4 cardiac toxicity in 2 patients (2.9%). Transplant-related mortality (TRM) developed in 2 patients (2.9%). Multiple prior treatments before transplantation, auxiliary bone marrow harvest for stem cell collection, and high serum lactate dehydrogenase level were related to unfavorable treatment outcomes. In conclusion, NEAM conditioning with auto-SCT demonstrated considerable efficacy with modest toxicity in patients with chemosensitive aggressive NHL. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome is a rare multisystemic disease associated with plasma cell dyscrasia and increased serum or plasma Vascular Endothelial Growth Factor (VEGF) levels, the latter likely responsible for several POEMS syndrome manifestations. Whereas peripheral neuropathy is the main neurological feature and a mandatory diagnostic criterium, central nervous system involvement is less common except for papilledema and stroke. We recently reported the frequent occurrence at brain MRI of cranial pachymeningeal involvement in a series of POEMS syndrome patients. Meningeal histopathology revealed hyperplasia of meningothelial cells, neovascularisation and obstructive vessel remodelling without inflammatory signs pointing to a role of VEGF in the meningeal manifestations. Here we report the dramatic pachymeningeal improvement in patients undergoing lenalidomide therapy. These findings support the therapeutic role of lenalidomide and might shed further light on the pathophysiology of the disease. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Recombinant DNA technology and protein engineering are creating hope that we can address ongoing challenges in hemophilia care such as reducing the costs of therapy, increasing the availability to the developing world, and improving the functional properties of these proteins. Technological advances to improve the half-life of recombinant clotting factors have brought long acting clotting factors for hemophilia replacement therapy closer to reality. Preclinical and clinical trial results are reviewed as well as the potential benefits and risks of these novel therapies. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

The last decade has seen increased use of aggressive, catheter-based methods of treating venous thromboembolism (DVT). In this article, we outline the risks, benefits, and uncertainties surrounding endovascular DVT therapies, describe clinical situations in which endovascular treatment options should reasonably be considered, and update the reader on new outcomes data that pertains to catheter-based DVT interventions. Endovascular thrombolytic therapy is reasonable to perform for selected patients with DVT causing acute limb-threatening circulatory compromise, acute IVC occlusion, or acute iliofemoral DVT for the purposes of limb salvage and relief of presenting DVT symptoms, and appears likely to prevent post-thrombotic syndrome (PTS) in patients with proximal DVT. A multicenter randomized trial, the ATTRACT Study, is currently underway in the United States to determine if pharmacomechanical catheter-directed thrombolysis (PCDT) is sufficiently safe and effective to be recommended for routine use in proximal DVT patients. Selected patients with established moderate-to-severe PTS in association with an occluded iliac vein or a refluxing saphenous vein may also be amenable to endovascular intervention to reduce venous hypertension, alleviate symptoms, and improve limb function and quality of life. Pending the results of further studies, an individualized approach to patient selection for interventional DVT therapies is recommended. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Thromboembolism, including both venous and arterial events, occurs commonly amongst patients with cancer. The occurrence of thromboembolism has significant consequences for cancer patients, including direct and indirect associations with mortality, morbidity, requirement for long-term anticoagulant therapy and consumption of healthcare resources. Recent studies have resulted in a better understanding of clinical risk factors and biomarkers of cancer-associated thrombosis, and a risk assessment model incorporating both has now been validated in multiple settings. Thromboprophylaxis with either unfractionated heparin or low-molecular-weight heparins (LMWHs) has been shown to be safe and effective in high-risk settings such as hospitalization for medical illness and the post-surgical period. Emerging new data from randomized studies have focused on outpatient prophylaxis, suggesting potential benefits in this setting as well. Treatment of cancer-associated thrombosis requires long-term anticoagulation with LMWH. Results from ongoing and planned trials of novel anticoagulants in the cancer setting are awaited. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

During the past 25 years, our knowledge concerning the pathogenesis, diagnostic strategies and treatment of von Willebrand disease (VWD) has increased significantly. Following the immunological differentiation of factor VIII (FVIII) and von Willebrand factor (VWF) in the 1970s and the cloning of the FVIII and VWF genes in the mid-1980s, substantial progress has been made in our understanding of this, the most common inherited bleeding disorder. We now recognize that VWD represents a range of genetic diseases all with the clinical endpoint of increased mucocutaneous bleeding. The molecular pathology of Type 2 and Type 3 VWD is now comprehensively documented and involves rare sequence variants at the VWF locus. In contrast, the genetic causation of Type 1 disease remains incompletely defined and in many cases appears to involve genetic determinants in addition to or instead of VWF. The diagnostic triad of a personal history of excessive mucocutaneous bleeding, laboratory tests for VWF that are consistent with VWD, and a family history of the condition remain the keystone to VWD identification. In the laboratory, measurement of VWF antigen and function continue to be the most important diagnostic studies, and while our understanding of the molecular genetic pathology of VWD has advanced considerably in the past decade, genetic testing as a component of diagnosis is limited to certain distinct subtypes of the disorder. Treatment of VWD has been relatively unchanged for the past decade and continues to involve either stimulation of the release of intrinsic VWF with desmopressin or the infusion of VWF concentrates. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Primary myelofibrosis is a chronic myeloproliferative neoplasm characterized by cytopenias, leukoerythroblastosis, extramedullary hematopoiesis, hepatosplenomegaly and bone marrow fibrosis. Primary myelofibrosis is a rare disorder in adults; children are even less commonly affected by this entity, with the largest pediatric case series reporting on three patients. Most literature suggests spontaneous resolution of myelofibrosis without long term complications in the majority of affected children. We describe the clinical, pathological and molecular characteristics and outcomes of nineteen children with primary myelofibrosis treated in our center from 1984 to 2011. Most patients had cytopenia significant enough to require supportive therapy. No child developed malignant transformation and only 5 of the 19 children (26%) had spontaneous resolution of disease. Sequence analyses for JAK2V617F and MPLW515L mutations were performed on bone marrow samples from 17 and 6 patients, respectively, and the results were negative. In conclusion, analysis of this large series of pediatric patients with primary myelofibrosis demonstrates distinct clinical, hematologic, bone marrow, and molecular features from adult patients. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

In AL amyloidosis prognosis depends on the severity of heart dysfunction which is best assessed by natriuretic peptides (BNP and NT-proBNP). However, their clearance relies on glomerular filtration rate (GFR) and their concentration increases with renal failure. We evaluated the diagnostic and prognostic performance of NT-proBNP and BNP in 248 patients with AL amyloidosis with different degrees of renal failure.

Patients were grouped according to GFR. Group 1 comprised 109 patients with GFR ≥60 mL/min/1.73m², Group 2, 77 subjects with GFR <60 and ≥15 mL/min/1.73m², and Group 3, 62 patients with GFR <15 mL/min/1.73m². The ability of natriuretic peptide to detect heart involvement and to predict survival in the 3 groups was assessed.

Decreasing eGFR required higher cutoffs of both NT-proBNP and BNP for detecting heart involvement and predicting survival. Both natriuretic peptides were independent prognostic markers in Groups 1 and 2, whereas in Group 3 only BNP independently predicted survival. Natriuretic peptides are powerful and useful markers of cardiac dysfunction and prognosis, provided that eGFR is considered in interpreting their clinical meaning. BNP should be preferred in patients with end-stage renal failure. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

The new oral anticoagulants are rapidly replacing warfarin for several indications. In contrast to warfarin, which lowers the functional levels of all of the vitamin K-dependent clotting factors, the new agents target either factor Xa or thrombin. With targeted inhibition of coagulation, the new oral anticoagulants have pharmacological, biochemical and clinical features distinct from those of warfarin. Focusing on laboratory perspectives, this paper compares and contrasts the pharmacological and biochemical properties of the new oral anticoagulants with those of warfarin and uses this information to speculate on the underlying mechanisms responsible for the clinical features that differentiate the new agents from warfarin. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Essential Thrombocythaemia (ET) is a rare type of myeloproliferative neoplasm (MPN) characterized by clonal expansion of the megakaryocyte and platelet lineage. Here, we describe a novel mutation (Y252H) in the thrombopoietin (TPO) receptor, or MPL, in a JAK2 mutation-negative ET patient. The bone marrow examination revealed increased numbers of dysmorphic megakaryocytes with focal clustering. The x-inactivation pattern suggested clonal expansion of hematopoietic cells in the bone marrow. Furthermore we found that the patient's bone marrow cells were hypersensitive to TPO in generating megakaryocyte colonies in vitro. More importantly, we demonstrated that this MPL Y252H mutant confers increased TPO/MPL-mediated cell growth and increased cell survival upon cytokine withdrawal in BaF3 cells, indicating it is a disease-driving mutation and may contribute to the development of ET in vivo. In summary, this is the first report describing a mutation in the extracellular domain of MPL underlying ET. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Cardioembolic strokes account for one-sixth of all strokes and are an important potentially preventable cause of morbidity and mortality. Vitamin K antagonists (e.g., warfarin) are effective for the prevention of cardioembolic stroke in patients with atrial fibrillation (AF) and in those with mechanical heart valves but because of their inherent limitations are underutilized and often suboptimally managed. Antiplatelet therapies have been the only alternatives to warfarin for stroke prevention in AF but although they are safer and more convenient they are much less efficacious. The advent of new oral anticoagulant drugs offers the potential to reduce the burden of cardioembolic stroke by providing access to effective, safe and more convenient therapies. New oral anticoagulants have begun to replace warfarin for stroke prevention in some patients with AF, based on the favorable results of recently completed phase III randomized controlled trials, and provide for the first time an alternative to antiplatelet therapy for patients deemed unsuitable for warfarin. The promise of the new oral anticoagulants in patients with mechanical heart valves is currently being tested in a phase II trial. If efficacy and safety are demonstrated, the new oral anticoagulants will provide an alternative to warfarin for patients with mechanical heart valves and may also lead to increased use of mechanical valves for patients who would not have received them in the past because of the requirement for long term warfarin therapy. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Venous thromboembolism (VTE) is an increasingly common complication encountered in tertiary care pediatric settings. The purpose of this review is to summarize the epidemiology, current and emerging pharmacotherapeutic options, and management of this disease. Over 70% of VTE occur in children with chronic diseases. Although they are seen in children of all ages, adolescents are at greatest risk. Pediatric VTE is associated with an increased risk of in-hospital mortality; recurrent VTE and post-thrombotic syndrome are commonly seen in survivors. In recent years, anticoagulation with low molecular weight heparin has emerged as the mainstay of therapy, but compliance is limited by its onerous subcutaneous administration route. New anticoagulants either already approved for use in adults or in the pipeline offer the possibility of improved dose stability and oral routes of administration. Current recommended anticoagulation course durations are derived from very limited case series and cohort data, or extrapolations from adult literature. However, the pathophysiologic underpinnings of pediatric VTE are dissimilar from those seen in adults and are often variable within groups of pediatric patients. Clinical studies and trials in pediatric VTE are underway which will hopefully improve the quality of evidence from which therapeutic guidelines are derived. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Management of adults with primary immune thrombocytopenia (ITP) has changed dramatically in the past 10 years. New regimens of corticosteroids for 1^st-line treatment have been introduced and are currently being evaluated in a randomized clinical trial. Many patients may not have durable remissions with initial corticosteroid regimens and may require additional, 2^nd-line, treatment. For these patients, rituximab has been increasingly used, as it has for other autoimmune disorders, and new thrombopoietin (TPO)-receptor agonists have been developed. Although splenectomy was the first effective and remains the most effective treatment for ITP, inducing durable complete remissions in 66% of patients, rituximab and TPO-receptor agonists are now additional options for 2^nd-line treatment. For patients who continue to have severe and symptomatic thrombocytopenia following failure of multiple treatments, including splenectomy and rituximab, the TPO-receptor agonists are effective as 3^rd-line treatment for maintaining safe platelets counts to prevent bleeding symptoms in most patients. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Children with Down syndrome (DS) have a high risk of acute megakaryoblastic leukemia (AMKL). Unlike AMKL in children without DS, AMKL-DS shows a uniquely favorable response to chemotherapy, especially to cytarabine. Because increase in treatment intensity in AMKL-DS enhances treatment-related mortality and decreases survival rate, lowering intensity of chemotherapy has been attempted. However, the risk-adapted appropriate intensity is not known. We describe an AMKL-DS case who suffered from severe pulmonary fibrosis and required tracheostomy. We identified the unique GATA1 mutation site in the leukemic cells of the patient and succeeded in performing real time quantitative polymerase chain reaction (RQ-PCR) for measurement of minimal residual disease (MRD). To prevent fatal respiratory infection during myelosuppression with conventional aggressive chemotherapy, the patient basically received 12 courses consisting of 14 days of ultra low-dose cytarabine (10mg/m²/dose, twice per day) for 12 months using the GATA1 mutation to monitor MRD and has been in complete remission for more than 23 months after achieving her 1^st complete remission. Ultra low-dose cytarabine treatment using GATA1 mutation by RQ-PCR to monitor MRD is an attractive candidate treatment in AMKL-DS. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Venous thromboembolism (VTE) is a chronic disease with a 30% ten-year recurrence rate. The highest incidence of recurrence is in the first 6 months. Active cancer significantly increases the hazard of early recurrence, and the proportions of time on standard heparin with an APTT≥0.2 anti-X_a U/mL, and on warfarin with an INR≥2.0, significantly reduce the hazard. The acute treatment duration does not affect recurrence risk after treatment is stopped. Independent predictors of late recurrence include increasing patient age and body mass index, leg paresis, active cancer and other persistent VTE risk factors, idiopathic VTE, antiphospholipid antibody syndrome, antithrombin, protein C or protein S deficiency, hyperhomocysteinemia and a persistently increased plasma fibrin D-dimer. A recommendation for secondary prophylaxis should be individualized based on the risk for recurrent VTE (especially fatal pulmonary embolism) and bleeding. The appropriateness of secondary prophylaxis should be continuously re-evaluated, and the prophylaxis stopped if the benefit no longer exceeds the risk. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Two decades of research into heparin-induced thrombocytopenia (HIT) permit a personal historical perspective on this fascinating syndrome. Previously, the frequency of HIT was unknown, although complicating thrombosis was believed to be rare and primarily arterial. The opportunity to apply a remarkable test for “HIT antibodies”—the ¹⁴C-serotonin-release assay (SRA)—to serial plasma samples obtained during a clinical trial of heparin thromboprophylaxis, provided insights into the peculiar nature of HIT, such as: its prothrombotic nature—including its strong association with venous thrombosis (RR=11.6 [95%CI, 6.4-20.8; p<0.0001); its more frequent occurrence with unfractionated versus low-molecular-weight heparin; the “iceberg” model, which states that among the many patients who form anti-PF4/heparin antibodies during heparin therapy, only a minority whose antibodies evince strong platelet-activating properties develop HIT; and the characteristic HIT timeline, whereby serum/plasma antibodies are readily detectable at or prior to the HIT-associated platelet count fall. Applying the SRA also to patients encountered in clinical practice led to recognition of warfarin-induced venous limb gangrene (for which HIT is a major risk factor via its extreme hypercoagulability) and delayed-onset HIT (whereby thrombocytopenia begins or worsens following heparin discontinuation, due to the ability of HIT antibodies strongly to activate platelets even in the absence of heparin—so-called heparin-“independent” platelet activation). Recent concepts include the increasing recognition of HIT “overdiagnosis” (due to the low diagnostic specificity of the widely-applied PF4-dependent immunoassays), and the observation that HIT-associated consump-tive coagulopathy is a risk factor for treatment failure with PTT-adjusted direct thrombin inhibitor therapy (“PTT confounding” secondary to HIT-associated coagulopathy). Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Although treatment with plasma exchange increased the survival of patients with thrombotic thrombocytopenia purpura (TTP) to 80% in the 1980s, no further increase of survival occurred over the next 20 years. However more consistent use of adjuvant treatment with corticosteroids and rituximab in recent years has begun to further increase survival as well as decrease the frequency of relapse. With adjuvant treatment, durable remissions can be achieved more quickly, requiring fewer days of plasma exchange. Fewer days of plasma exchange have resulted in fewer complications, such as central venous catheter-related systemic infections. Future potential options for adjuvant treatment, recombinant ADAMTS13 to correct severe ADAMTS13 deficiency and agents to block von Willebrand factor-mediated platelet thrombosis, are being investigated. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Bleeding disorders commonly result from deficiencies or defects in von Willebrand factor (VWF), platelets, coagulation factors, or fibrinolytic proteins. The primary goal of our study was to assess current North American coagulation laboratory practices for diagnosing bleeding disorders, using an on-line patterns-of-practice survey of diagnostic laboratory members of the North American Specialized Coagulation Laboratory Association (NASCOLA). The survey examined laboratory approaches to evaluating bleeding disorders, with specific questions about the tests and test panels offered and compliance to recent guideline recommendations on diagnosing von Willebrand disease (VWD) and platelet function disorders.

All laboratories responding to the survey performed a prothrombin time/international normalized ratio, an activated partial thromboplastin time, and coagulation factor assays, and many tested for VWD and platelet disorders. However, few laboratories had test panels that evaluated the more common bleeding disorders and few performed some assays, including VWF multimer assessments and assays for fibrinolytic disorders. Additionally, the cutoffs used by laboratories to diagnose type 1 VWD varied considerably, with only a minority following the National Heart Lung Blood Institute recommendations. In contrast, laboratories that tested for platelet function disorders mostly complied with aggregation testing recommendations, as published in the recent North American guidelines.

Our results indicate that there are some gaps in the strategies used by laboratories to diagnose bleeding disorders that might be addressed by development of further guidelines and test algorithms that emphasize evaluations for common bleeding disorders. Laboratories may also benefit from guidelines on test interpretation, and external evaluation of their bleeding disorder testing strategies. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Congenital or familial erythrocytosis/polycythemia can have many causes, and an emerging cause is genetic disruption of the oxygen-sensing pathway that regulates the ERYTHROPOIETIN (EPO) gene. More specifically, recent studies have identified erythrocytosis-associated mutations in the HIF2A gene, which encodes for Hypoxia Inducible Factor-2α (HIF-2α), as well as in two genes that encode for proteins that regulate it, Prolyl Hydroxylase Domain protein 2 (PHD2) and the von Hippel Lindau tumor suppressor protein (VHL). We report here the identification of two new heterozygous HIF2A missense mutations, M535T and F540L, both associated with erythrocytosis. Met-535 has previously been identified as a residue mutated in other patients with erythrocytosis, although the mutation of this particular residue to Thr has not been reported. In contrast, Phe-540 has not been reported as a residue mutated in erythrocytosis, and we present evidence here that this mutation impairs interaction of HIF-2α with both VHL and PHD2. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

No data is present regarding the management of cancer patients requiring interruption of long-term vitamin-K antagonist (VKA) therapy. For this purpose, we tested the efficacy and safety of fixed doses of low-molecular weight heparin (LMWH) in substitution of VKA because of invasive procedures or chemotherapy-induced thrombocytopenia. In cancer patients on VKA, therapy was discontinued 5 + 1 days before surgery or chemotherapy. Heparin was given at prophylactic dosage in patients at low risk and at fixed sub-therapeutic doses (3,800 or 4,000 UI anti-FXa, b.i.d.) in those at high-risk for thrombosis. LMWH was reinitiated 12 hours after surgery and VKA the day after. In patients receiving chemotherapy, LMWH was reinitiated 12/24 h after obtaining a stable platelet count > 30,000 mmc³, and VKA after a stable platelet count > 50,000 mmc³. Thromboembolism and major bleeding events were recorded from the time of VKA suspension to 30 + 2 days post-procedure, or until the next chemotherapy. Overall,156 patients (56.4% at low risk and 43.5% at high risk for thrombosis) were enrolled; 34.6% underwent major surgery, 40.4% non-major surgery, and 25% chemotherapy. Thrombotic events occurred in 5 patients (3.2%, 95% CI 1.41-7.27), 4 belonging to the high-risk and 1 to the low-risk group. Major bleeding occurred in 5 patients (3.2%, 95 CI 1.41-7.27), all belonging to the high-risk group (3 during major surgery and 2 during chemotherapy). In conclusion, LMWH given at fixed sub-therapeutic is a feasible and relatively safe approach for bridging therapy in cancer patients on long-term VKA. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Mutations in GBA1 gene result in defective acid β-glucosidase and the complex phenotype of Gaucher disease (GD) related to the accumulation of glucosylceramide-laden macrophages. The phenotype is highly variable even among patients harboring identical GBA1 mutations. We hypothesized that modifier gene(s) underlie phenotypic diversity in GD and performed a GWAS study in Ashkenazi Jewish patients with type 1 GD (GD1), homozygous for N370S mutation. Patients were assigned to mild, moderate or severe disease category using composite disease severity scoring systems. Whole-genome genotyping for >500,000 SNPs was performed to search for associations using OQLS algorithm in 139 eligible patients. Several SNPs in linkage disequilibrium within the CLN8 gene locus were associated with the GD1 severity: SNP rs11986414 was associated with GD1 severity at p value 1.26 × 10⁻⁶. Compared to mild disease, risk allele A at rs11986414 conferred an odds ratio of 3.72 for moderate/severe disease. Loss of function mutations in CLN8 causes neuronal ceroid-lipofuscinosis but our results indicate that its increased expression may protect against severe GD1. In cultured skin fibroblasts, the relative expression of CLN8 was higher in mild GD compared to severely affected patients in whom CLN8 risk alleles were over-represented. In an in vitro cell model of GD, CLN8 expression was increased which was further enhanced in the presence of bioactive substrate, glucosylsphingosine. Taken together, CLN8 is a candidate modifier gene for GD1 that may function as a protective sphingolipid sensor and/or in glycosphingolipid trafficking. Future studies should explore the role of CLN8 in pathophysiology of GD. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Heat shock protein (HSP) 70 and HSP90 are released by primary human acute myeloid leukemia (AML) cells during stress-induced spontaneous in vitro apoptosis. The AML cells also show constitutive release of several cytokines, and the systemic serum levels of several soluble mediators are altered in patients with untreated AML. In the present study we investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving AML-stabilizing palliative treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Patients with untreated AML showed increased HSP90 levels and a distinct serum cytokine profile compared with healthy controls, and low pre-therapy HSP90 levels were associated with a prolonged survival during treatment with ATRA + valproic acid + theophyllin. Hierarchical cluster analysis showed a close association between HSP70, HSP90, IL-1 receptor antagonist (IL-1ra) and Hepatocyte growth factor (HGF) levels. Furthermore, diseasestabilizing therapy altered the serum cytokine profile, but the correlations between HSP70/ HSP90/ IL-1ra/ HGF were maintained only when ATRA + valproic acid were combined with theophyllin but not when combined with cytarabine. We conclude that both HSP levels and serum cytokine profiles are altered and may represent possible therapeutic targets or prognostic markers in human AML. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are mature CD5⁺ B-cell malignancies with different biological/clinical characteristics. We recently reported an association between different prognostic subgroups of CLL (i.e. IGHV mutated and unmutated) and genomic methylation pattern. However, the relationship between DNA methylation and prognostic markers, such as the proliferation gene expression signature, has not been investigated in MCL. We applied high-resolution methylation microarrays (27,578 CpG sites) to assess the global DNA methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 poor-prognostic IGHV unmutated subset #1 and 15 good-prognostic IGHV mutated subset #4) samples. Notably, MCL and each CLL subset displayed distinct genomic methylation profiles. After unsupervised hierarchical clustering 17/20 MCL cases formed a cluster separate from CLL, while CLL subsets #1 and #4 formed sub-clusters. Surprisingly, few differentially methylated genes (n=6) were identified between high vs. low proliferation MCL. In contrast, distinct methylation profiles were demonstrated for MCL and CLL. Importantly, certain functional classes of genes were preferentially methylated in either disease. For instance, developmental genes, in particular homeobox transcription factor genes (e.g. HLXB9, HOXA13), were more highly methylated in MCL, whereas apoptosis-related genes were enriched among targets methylated in CLL (e.g. CYFIP2, NR4A1). Results were validated using pyrosequencing, RQ-PCR and re-expression of specific genes. In summary, the methylation profile of MCL was homogeneous and no correlation with the proliferation signature was observed. Compared to CLL, however, marked differences were discovered such as the preferential methylation of homeobox genes in MCL. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Romidepsin is the second histone deacytelase inhibitor (HDACi) approved for the treatment of advanced stages of cutaneous T cell lymphoma (CTCL). Recent in vitro data suggests that HDACi suppress immune function although these findings have not been confirmed in patients. Thus, we serially examined the cellular immune function of eight CTCL patients undergoing treatment with three cycles of romidepsin. We measured the patient's natural killer (NK) and dendritic cell (DC) function, and performed an in vitro TUNEL assay to measure cellular apoptosis. Patients' NK cell cytolytic activity decreased from baseline to the third cycle of treatment (P=.018) but stimulation with a TLR agonist increased this activity (P=.018). At baseline a TLR agonist could both activate patients' DC (P=.043) and stimulate IL-12 protein production (P=.043) but both were suppressed after the first cycle of romidepsin. Finally, we observed increased specificity for romidepsin induced CD4+ tumor cell apoptosis and dose dependent increases in cellular apoptosis of healthy cells in multiple lineages (P<.05). These findings raise concern that HDACi suppress immune function in CTCL patients and support the concurrent use of multiple immune stimulatory agents to preserve the host immune response. Am. J. Hematol., 2012. © 2012 Wiley-Liss, Inc.

Neonatal alloimmune thrombocytopenia (NAIT) is the most common cause of severe thrombocytopenia and intracranial hemorrhage (ICH) in the perinatal period. While the gold standard for making a diagnosis of NAIT is detection of an HPA-specific antibody in maternal serum, together with identifying an incompatibility between the parents for the cognate HPA antigen, platelet genotyping is the gold standard method for HPA typing. Platelet genotyping is critical in screening at-risk fetuses for the presence of the HPA corresponding to the maternal antibody. In addition, platelet genotyping may play a role in population screening to identify women at risk for sensitization, and thus, fetuses at risk for NAIT. The most commonly used methods of platelet genotyping are sequence-specific primer-polymerase chain reaction (PCR-SSP), restriction fragment length polymorphism-PCR (PCR-RFLP), and TaqMan real-time PCR. PCR-SSP and PCR-RFLP are relatively inexpensive and technically simple methods, but are not easily automated and require expertise for reliable interpretation of results. Newer methods that allow for multiplexing, automation and easily interpretable results, such as bead arrays, are currently in development and are available for research purposes. Am. J. Hematol., 2011. © 2011 Wiley-Liss, Inc.

In patients with recurrent Hodgkin or non-Hodgkin's lymphoma, autologous stem cell transplantation (ASCT) can offer potential for cure or long term remission. Because of potential toxicity, elderly patients are usually not considered candidates, but data regarding tolerability and efficacy in this group are lacking. The transplant database at Weill Cornell Medical College was reviewed to identify patients with lymphoma undergoing ASCT at age 69 or greater. Clinical data and comorbidities were correlated with outcome. Twenty-one patients were identified. Sixteen of 19 evaluable patients (76%) achieved complete remission following ASCT, while 2 patients died before response assessment. Median progression-free survival following ASCT was 8 months and median overall survival was 18 months. Age was not predictive of overall survival, but patients 75 and older had inferior progression free survival compared to younger patients. High risk status by hematopoietic stem cell transplant comorbidity index (HCT-CI) was associated with short overall survival and high transplant-related mortality. ASCT is feasible and of potential benefit in selected elderly lymphoma patients. Consideration of comorbidities, rather than age alone, may allow selection of patients likely to tolerate and benefit from ASCT. Am. J. Hematol., 2011. © 2011 Wiley-Liss, Inc.

Despite advances in our understanding of the pathophysiology of thrombotic thrombocytopenic purpura (TTP), there remains significant room for improvement in the treatment of acute TTP. A novel approach to the treatment of TTP using ARC1779 to target the A1-domain of von Willebrand Factor (VWF) to prevent the formation of microthombi has been developed. Preliminary data suggests that blockade of the A1-domain of VWF by ARC1779 can inhibit VWF activity, resulting in clinically significant improvements in the platelet count and lactate dehydrogenase(1-5). ARC1779 is a nucleic acid macromolecule, or aptamer, that inhibits the prothrombotic function of VWF by binding to the A1-domain of VWF, blocking its interaction with the platelet GPIb receptor. It has been hypothesized that ARC1779 prevents the formation of new microthrombi in patients with acute TTP, and may result in shorter courses of plasma exchange(PEX) and fewer end organ complications via the more rapid inhibition of microthrombotic disease. Prior to the premature closure of the study, 9 patients were treated with either ARC1779 of placebo as an adjunct to PEX. Although limited, these data support the safety of this targeted approach to the treatment of TTP, providing the basis for continued study of this unique approach to therapy. Am. J. Hematol., 2011. © 2011 Wiley-Liss, Inc.

Anemia of chronic disease (ACD) or anemia of inflammation is prevalent in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease. ACD is associated with poor prognosis and lower quality of life. Management of ACD using intravenous iron and erythropoiesis stimulating agents are ineffective for some patients and are not without adverse effects, driving the need for new alternative therapies. Recent advances in our understanding of the molecular mechanisms of iron regulation reveal that increased hepcidin, the iron regulatory hormone, is a key factor in the development of ACD. In this review, we will summarize the role of hepcidin in iron homeostasis, its contribution to the pathophysiology of ACD, and novel strategies that modulate hepcidin and its target ferroportin for the treatment of ACD. Am. J. Hematol. 2012. © 2011 Wiley Periodicals, Inc.

Disease relapse still greatly interferes with the success of allogeneic hematopoietic stem cell transplantation (HSCT) for acute lymphoblastic leukemia (ALL). This study retrospectively evaluated the long-term safety and efficacy of a conditioning regimen consisting of total body irradiation (TBI; 12 Gy), cyclophosphamide (CY; 60 mg kg⁻¹, two doses), and high-dose cytarabine (Ara-C; 2 g m⁻²; four doses) for patients with ALL. Fifty-five patients (median age: 31-years old) were evaluated. Stem cells were from human leukocyte antigen-identical siblings in 22 patients and from alternative donors in 33. There were no cases of early death before engraftment, and 100-day transplant-related mortality was 7.3%. With a median follow-up period of 9.6 years, 5-year overall and disease-free survival were 63.2% (95% CI: 46.5–79.9%) and 63.6% (95% CI: 47.1–80.1%) in patients with complete remission, respectively, both of which were significantly higher than the values of 27.3% (95% CI: 8.7–46.0%) and 22.7% (95% CI: 5.3–40.1%) for patients in advanced stages (P < 0.01). These results suggest that TBI and CY (TBI-CY) plus Ara-C could be a feasible and effective conditioning regimen for adult patients with ALL both in remission and in advanced stages, and a future study to compare this combination therapy with TBI-CY is required. Am. J. Hematol. 2012. © 2011 Wiley Periodicals, Inc.

The prevention of Rhesus D alloimmunization through Rh immune globulin (RhIg) administration is the major indication for the accurate detection and quantification of fetomaternal hemorrhage (FMH). In the setting of D incompatibility, D-positive fetal cells can sensitize the D-negative mother, resulting in maternal anti-D alloantibody production. These anti-D alloantibodies may lead to undesirable sequelae such as hemolytic disease of the newborn (HDN). Since the widespread adoption of FMH screening and RhIg immunoprophylaxis, the overall risk of Rh alloimmunization and infant mortality from HDN has substantially decreased. The rosette screen, the initial test of choice, is highly sensitive in qualitatively detecting 10 mL of fetal whole blood in the maternal circulation. As the screen is reliant on the presence of the D antigen to distinguish fetal from maternal cells, it cannot be used to detect FMH in D-positive mothers or in D-negative mothers carrying a D-negative fetus. The Kleihauer-Betke acid-elution test, the most widely used confirmatory test for quantifying FMH, relies on the principle that fetal RBCs contain mostly fetal hemoglobin (HbF), which is resistant to acid-elution whereas adult hemoglobin is acid-sensitive. Although the Kleihauer-Betke test is inexpensive and requires no special equipment, it lacks standardization and precision, and may not be accurate in conditions with elevated F-cells. Anti-HbF flow cytometry is a promising alternative, although its use is limited by equipment and staffing costs. Hematology analyzers with flow cytometry capabilities may be adapted for fetal cell detection, thus giving clinical laboratories a potentially attractive automated alternative for quantifying FMH. Am. J. Hematol., 2011. © 2011 Wiley Periodicals, Inc.

Chronic myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) have an inherent tendency to progress to acute myeloid leukemia (AML). Using high-resolution SNP microarrays, we studied a total of 517 MPN and MDS patients in different disease stages, including 77 AML cases with previous history of MPN (N = 46) or MDS (N = 31). Frequent chromosomal deletions of variable sizes were detected, allowing the mapping of putative tumor suppressor genes involved in the leukemic transformation process. We detected frequent deletions on the short arm of chromosome 6 (del6p). The common deleted region on 6p mapped to a 1.1-Mb region and contained only the JARID2 gene—member of the polycomb repressive complex 2 (PRC2). When we compared the frequency of del6p between chronic and leukemic phase, we observed a strong association of del6p with leukemic transformation (P = 0.0033). Subsequently, analysis of deletion profiles of other PRC2 members revealed frequent losses of genes such as EZH2, AEBP2, and SUZ12; however, the deletions targeting these genes were large. We also identified two patients with homozygous losses of JARID2 and AEBP2. We observed frequent codeletion of AEBP2 and ETV6, and similarly, SUZ12 and NF1. Using next generation exome sequencing of 40 patients, we identified only one somatic mutation in the PRC2 complex member SUZ12. As the frequency of point mutations in PRC2 members was found to be low, deletions were the main type of lesions targeting PRC2 complex members. Our study suggests an essential role of the PRC2 complex in the leukemic transformation of chronic myeloid disorders. Am. J. Hematol. 2012. © 2011 Wiley Periodicals, Inc.

No studies exist documenting that chemotherapy alone eradicates tumors composed of leukemic cells in a large group of patients with tumors at any one site. Yet, its use has continued over 40 years in the absence of data. Consensus protocols exist only for testis and meningeal tumors, relying on local therapy. To constitute a body of knowledge about tumors at one site, the breast was chosen and all published cases were analyzed, with follow-up obtained, to document the behavior of acute leukemia tumors and survival after presentation. Among 235 cases (52% published since 2000), overall survival was poor, particularly for the 43% with concurrent morphologic marrow relapse, with 66-73% one-year mortality. Only 4 of 106 patients treated with chemotherapy alone survived 4 years. The majority of AML and ALL tumors were only transiently responsive to anti-leukemia treatments, including transplant, and next relapses were as, or more, common in further tumors than in marrow. A pattern of tumors similar to the metastases of invasive lobular breast cancer was revealed. When relapse occurred in marrow, durable remission was only rarely obtained. These data suggest a potential benefit of incorporating extent of disease workup at diagnosis and relapse into prospective trials. This could yield an accurate incidence of extramedullary tumors and a means to identify occult residual disease which could lead to marrow relapse. This approach could potentially result in greater success in curing acute leukemias. Am. J. Hematol. 2012. © 2011 Wiley Periodicals, Inc.

Recently, nontumor specific circulating DNA was shown to be elevated in a broad range of lymphomas, implicating a role as a potential biomarker. Epstein-Barr virus' (EBV) presence within a proportion of lymphomas implies EBV-DNA has potential as a lymphoma-specific disease response biomarker. However, application would be restricted to EBV-associated lymphomas. Neither detailed comparison has been performed of lymphoma-specific versus nonspecific DNA as disease response biomarkers nor have the kinetics of circulating DNA during treatment been established, and the optimal methodology remains unknown. We prospectively evaluated DNA levels and clinical response of 63 lymphoma patients. DNA was measured in paired serum, plasma, and cell samples at five predetermined time-points taken prior, during and following treatment. Both cell-free (c-f) circulating EBV-DNA (in EBV-associated lymphoma) and nonspecific c-f DNA levels (in all lymphomas) were elevated and discriminatory at presentation compared to healthy controls. Nonspecific c-f DNA was significantly associated with baseline serum lactate dehydrogenase. Within EBV-associated lymphomas at presentation, there was a strong correlation between specific and nonspecific circulating c-f DNA (r = 0.9, P < 0.0001). However, only c-f EBV-DNA correlated with clinical/radiological response. In addition, c-f EBV-DNA, and not nonspecific c-f DNA, provided an early marker of relapsed and refractory disease. Serum versus plasma, and single versus multiple-copy EBV-gene targets were equivalent. Lymphoma-specific DNA is a disease response biomarker; however, nonspecific DNA reflected neither lymphoma-specific DNA nor therapeutic response. Lymphoma disease response can be monitored by blood tests, but new lymphoma-specific biomarkers need to be identified to broaden applicability. Am. J. Hematol. 2012. © 2011 Wiley Periodicals, Inc.

D antigen is the most immunogenic and clinically relevant antigen within the complex Rh blood group system. Variability of D antigen expression was first described decades ago but has rarely been investigated quantitatively, particularly in the context of RHD zygosity along with RhCcEe serological phenotype. With IRB approval, 107 deidentified blood samples were analyzed. Rh phenotypes were determined serologically by saline technique using monoclonal antibodies against D, C, c, E, and e antigens. RHD zygosity was determined using both PCR-restriction fragment length polymorphisms and quantitative real-time PCR techniques. A novel and robust method was developed for quantitation of erythrocyte D antigen sites using calibrated microspheres and flow cytometry, allowing correlation of D antigen density with RHD zygosity and expression of Rh CcEe antigens. Subjects homozygous for RHD expressed nearly twice the number of D antigen sites compared with RHD hemizygotes (33,560 ± 8,222 for DD versus 17,720 ± 4,471 for Dd, P < 0.0001). Expression of c or E antigens was associated with significantly increased erythrocyte D antigen expression, whereas presence of C or e antigens reduced expression. These data and this novel quantitation method will be important for future studies investigating the clinical relevance of D antigen variability. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.

A total of 149 patients with multiple myeloma (MM) who received allogeneic hematopoietic stem cell transplantation (allo-HCT) with myeloablative (MAC; n = 38) or reduced-intensity conditioning (RIC; n = 110) regimens at MD Anderson Cancer Center were evaluated. Of the total, 120 (81%) patients had relapsed or had refractory disease. Median age of MM patients was 50 (28–70) years with a followup time of 28.5 (3–164) months. The 100-day and 5-year treatment related mortality (TRM) rates were 17% and 47%, respectively. TRM was significantly lower with RIC regimens (13%) vs. 29% for MAC at 100 days (P = 0.012). The cumulative incidence of Grade II–IV acute graft-versus-host disease (GVHD) was 35% and chronic GVHD was 46%. PFS and OS at 5 years were 15% and 21%, respectively. In multivariate analysis, allo-HCT for primary remission consolidation was associated with longer PFS (HR 0.35; 95% CI, 0.18–0.67) and OS (HR 0.29; 95% CI 0.15–0.55), while absence of high-risk cytogenetics was associated with longer PFS only (HR 0.59; 95% CI 0.37–0.95). We observe that TRM has decreased with the use of RIC regimens, and long-term disease control can be expected in a subset of MM patients undergoing allo-HCT. Further studies should be conducted in carefully designed clinical trials in this patient population. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.

Non-Hodgkin lymphoma (NHL) represents a heterogenous group of neoplasias originating from lymphoid cells. Increased angiogenesis and expression of Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFR) have been found to be associated with NHL disease progression. Increase in VEGF and other cytokines stimulate signaling cascades, including the Ras/Raf/Mek/Erk pathway, resulting in increased proliferation and decreased apoptosis. Here, we report the in vitro antilymphoma activity of sorafenib, an inhibitor of VEGFR and Raf kinase. Sorafenib induced potent cytotoxicity in NHL cell lines and patient samples. This induction of cytotoxicity was associated with a corresponding increase in apoptotic cell death. Mechanism of action of sorafenib was investigated in follicular (DoHH2) and Burkitt lymphoma (Raji) cell lines. pStat3, pAkt, Mcl1, and Xiap were downregulated in both cell lines, whereas pErk decreased in Raji but not in DoHH2 cells following sorafenib treatment. IL6 was unable to prevent sorafenib induced repression of pStat3, pAkt, Mcl1, and Bcl-Xl. Sorafenib in combination with an mTORC1 inhibitor rapamycin demonstrated synergy in inducing cytotoxicity in NHL cells. Sorafenib/rapamycin combination resulted in downregulation of pAkt, pmTOR, p-p70S6K, p4EBP1, pGSK3β, Mcl1, and Bcl-Xl. On the basis of our results, a clinical trial is underway using sorafenib with everolimus in NHL patients., 2012. © 2011 Wiley Periodicals, Inc.

Disease overview: Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms primarily characterized by erythrocytosis and thrombocytosis, respectively. Other disease features include leukocytosis, splenomegaly, thrombohemorrhagic complications, vasomotor disturbances, pruritus, and a small risk of disease progression into acute myeloid leukemia or myelofibrosis.

Diagnosis: Almost all patients with PV harbor a JAK2 mutation. When PV is suspected, the presence of a JAK2 mutation confirms the diagnosis and its absence, combined with normal or increased serum erythropoietin level, excludes the diagnosis. Differential diagnosis of ET had to include chronic myelogenous leukemia and prefibrotic myelofibrosis. A JAK2 mutation is found in approximately 60% of patients with ET.

Risk stratification: Current risk stratification in PV and ET is designed to estimate the likelihood of thrombotic complications: high-risk is defined by the presence of age >60 years or presence of thrombosis history; low-risk is defined by the absence of both of these two risk factors. Presence of extreme thrombocytosis (platelet count >1,000 × 10⁹/L) might be associated with acquired von Willebrand syndrome (AvWS) and, therefore, risk of bleeding. Risk factors for shortened survival in both PV and ET include advanced age, leukocytosis, and history of thrombosis.

Risk-adapted therapy: Survival is near-normal in ET and reasonably long in PV. The 10-year risk of leukemic/fibrotic transformation is <1%/1% in ET and <3%/10% in PV. In contrast, the risk of thrombosis exceeds 20%. The main goal of therapy is therefore to prevent thrombohemorrhagic complications and this is effectively and safely accomplished by the use of low-dose aspirin (PV and ET), phlebotomy (PV) and hydroxyurea (high risk PV and ET). Treatment with busulfan or interferon-α is usually effective in hydroxyurea failures. Screening for clinically significant AvWS is recommended before administrating aspirin in the presence of extreme thrombocytosis. Am. J. Hematol. 87:285–293, 2012. © 2012 Wiley Periodicals, Inc.

The introduction of tyrosine kinase inhibitors (TKIs), starting with imatinib and followed by second and third generation TKIs, has significantly changed the clinical management of patients with chronic myeloid leukemia (CML). Despite their unprecedented clinical success, a proportion of patients fail to achieve complete cytogenetic remission by 12 months of treatment (primary resistance) while others experience progressive resistance after an initial response (secondary resistance). BCR-ABL1 kinase domain (KD) mutations have been detected in a proportion of patients at the time of treatment failure, and therefore their identification and monitoring plays an important role in therapeutic decisions particularly when switching TKIs. When monitoring KD mutations in a clinical laboratory, the choice of method should take into account turnaround time, cost, sensitivity, specificity, and ability to accurately quantify the size of the mutant clone. In this article, we describe in a “manual” style the methods most widely used in our laboratory to monitor KD mutations in patients with CML including direct sequencing, D-HPLC, and pyrosequencing. Advantages, disadvantages, interpretation of results, and their clinical applications are reviewed for each method. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.