Aric Parnes

Resveratrol ameliorates TNFα-mediated suppression of erythropoiesis in human CD34(+) cells via modulation of NF-κB signalling.

Overexpression of pro‐inflammatory cytokines, including tumour necrosis factor alpha (TNFα), has been implicated in the pathogenesis of anaemia of inflammation. TNFα suppresses erythroid colony formation via both direct and indirect effects on haematopoietic progenitors, often involving activation of nuclear factor (NF)‐κB signalling resulting in downregulation of transcription factors critical for erythropoiesis. There is a dearth of effective and safe therapies for many patients with inflammatory anaemia. Resveratrol is a flavanol found in red wine grapes that possesses potent anti‐inflammatory properties, but studies of its impact on human erythropoiesis have proven contradictory. We investigated whether resveratrol ameliorates TNFα‐mediated suppression of erythropoiesis in human CD34+ haematopoietic progenitors. We found that resveratrol partially reverses the erythroid suppressive effects of TNFα, leading to significant recovery in burst forming unit‐erythroid colony formation in human CD34+ cells. CD34+ cells pre‐incubated with resveratrol for 72 h in the presence of TNFα inhibited NF‐κB activation via decreased NF‐κB nuclear localization without altering total NF‐κB protein levels and independent of IκB degradation. Resveratrol also significantly restored the baseline expression of erythroid transcription factors NFE2 and the GATA1/GATA2 ratio in CD34+ cells treated with TNFα. In conclusion, resveratrol may inhibit TNFα‐mediated NF‐κB activation and promote erythropoiesis in primary human CD34+ cells.

Single nucleotide polymorphisms in the human TNF gene are associated with anaemia and neutropenia in a cohort of patients with de novo myelodysplastic syndrome.

Anaemia is the most common haematological abnormality in the myelodysplastic syndromes (MDS), contributing to morbidity and mortality via reduced oxygen carrying capacity, transfusional iron overload, and a reduced quality of life (Delforge, 2003). Infection also remains a challenging complication and the most common cause of death in MDS patients (Kouides & Bennett, 1997). The expression of several proinflammatory cytokines, including tumour necrosis factoralpha (TNFa), is increased in MDS patients versus normal subjects (Gersuk et al, 1998). The TNF gene, which encodes TNFa, contains functional single nucleotide polymorphisms (SNPs) that have been linked to cytokine hypersecretion (Imahara & O’ Keefe, 2004). The high-expressing TNF -308A/ A genotype was found to be overrepresented 14-fold in a cohort of 21 MDS patients (Powers et al, 2007), but the extent to which TNF SNPs affect the phenotype or natural history of MDS remains to be elucidated. We postulated that underlying host factors including proinflammatory mediators are genetic modifiers of disease severity in MDS, influencing the degree of cytopenias, severity of infections, and disease progression. We therefore examined whether functional TNF polymorphisms altered the MDS phenotype. Our studies revealed that high-expressing SNPs in TNF are independently associated with National Cancer Institutes Grade II or greater anaemia and with neutropenia in our cohort of treatment naı̈ve, de novo MDS patients, supporting their role as disease modifiers in MDS. This study was approved by Dana-Farber Cancer Institute’s (DFCI) Investigational Review Board. DNA samples isolated from the bone marrows of 328 individuals with de novo MDS with accompanying clinical annotation and from the blood of 150 normal healthy subjects were generously provided by Dr. Benjamin Ebert and the DFCI tissue repository, respectively. MDS patients were defined according to FrenchAmerican-British (FAB) criteria. DNA was genotyped for the )308G/A and )238G/A polymorphisms of TNF by polymerase chain reaction (PCR) amplification with primer pairs: TNF 308G/A [5¢-AATAGGTTTTGAGGGCCATG-3¢ (forward) and 5¢-ATCTGGAGGAAGCGGTAGTG-3¢ (reverse)]; TNF -238G/ A [5¢-AGAAGACCCCCCTCGGAACC-3¢ (forward) and 5¢-ATCTGGAGGAAGCGGTAGTG-3¢ (reverse)]. PCR amplification conditions were as follows: 94 C for 5 min; 10 cycles of 94 C for 30 s, 65 C for 30 s (decreasing 1 C each cycle), and 72 C for 30 s (decreasing 1 C each cycle); 30 cycles of 94 C for 30 s, 55 C for 30 s, 72 C for 30 s; terminal extension at 72 C for 7 min. PCR products were digested with MspI (New England Biolabs Inc., Beverly, MA, USA) for 6 h at 37 C, and restriction fragment length polymorphism products analyzed by gel electrophoresis using 5-20% Tris-HCl polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA). Differences in allele frequencies were analyzed by chi-square testing. Logistic regression was used to assess whether TNF polymorphisms were associated with the following outcomes: age, FAB classification, International Prognostic Scoring System (IPSS) score, karyotype, blast percentage, survival, anaemia [haemoglobin (Hb) <130 g/l or <120 g/l for men and women, respectively], neutropenia [absolute neutrophil count (ANC) <1Æ5 · 10/l], thrombocytopenia (platelet count < 150 · 10/l), requirement for red blood cell (RBC) or platelet transfusions, and therapy with erythropoiesis stimulating agents (ESAs). Data was analyzed using the Statistical Package for the Social Sciences (spss) for Windows, version 15.0. MDS cases were classified by FAB criteria: Refractory Anaemia (n = 155), Refractory Anaemia with Ringed Sideroblasts (n = 30), Refractory Anaemia with Excess Blasts (n = 110), and Refractory Anaemia with Excess Blasts in Transformation (n = 33). Our MDS cohort was predominantly treatment naı̈ve, with only 20/328 patients having received ESA therapy as their only form of disease management at the time clinical data were collected. Within our MDS cohort, multivariate analysis of TNF -308 genotypes revealed that the AA genotype (high-expressing) was independently associated with neutropenia at ANC levels of less than 1Æ5 · 10/l (P = 0Æ04, Logistic regression) (Table I). Multivariate analysis of TNF 238 genotypes in our MDS cohort revealed that the presence of a high-expressing genotype, either GA or AA, was independently associated with the presence of NCI Grade II or greater anaemia (Hb < 100 g/l) (P = 0Æ035, Logistic regression) (Table I), which retained statistical significance after controlling for RBC transfusions and ESA therapy (P = 0Æ03). Furthermore, whereas the A allele in our MDS cohort was present in 14% of anaemic individuals (n = 41), no patients with a normal Hb level (n = 26) carried an A allele (P = 0Æ03, Chi-Square Test), which also retained statistical significance after controlling for RBC transfusions or ESA therapy (P = 0Æ02). TNF -308 or TNF -238 genotypes were not significantly associated with other outcomes analyzed. The frequencies of the GG, GA, and AA genotypes at both correspondence

A systematic review of religious beliefs about major end-of-life issues in the five major world religions.

OBJECTIVE The objective of this study was to examine the religious/spiritual beliefs of followers of the five major world religions about frequently encountered medical situations at the end of life (EoL). METHOD This was a systematic review of observational studies on the religious aspects of commonly encountered EoL situations. The databases used for retrieving studies were: Ovid MEDLINE In-Process & Other Non-Indexed Citations, Ovid MEDLINE, Ovid EMBASE, Ovid PsycINFO, Ovid Cochrane Central Register of Controlled Trials, Ovid Cochrane Database of Systematic Reviews, and Scopus. Observational studies, including surveys from healthcare providers or the general population, and case studies were included for review. Articles written from a purely theoretical or philosophical perspective were excluded. RESULTS Our search strategy generated 968 references, 40 of which were included for review, while 5 studies were added from reference lists. Whenever possible, we organized the results into five categories that would be clinically meaningful for palliative care practices at the EoL: advanced directives, euthanasia and physician-assisted suicide, physical requirements (artificial nutrition, hydration, and pain management), autopsy practices, and other EoL religious considerations. A wide degree of heterogeneity was observed within religions, depending on the country of origin, level of education, and degree of intrinsic religiosity. SIGNIFICANCE OF RESULTS Our review describes the religious practices pertaining to major EoL issues and explains the variations in EoL decision making by clinicians and patients based on their religious teachings and beliefs. Prospective studies with validated tools for religiosity should be performed in the future to assess the impact of religion on EoL care.

A novel regimen incorporating the concomitant administration of fludarabine and alemtuzumab for the treatment of refractory adult acute lymphoblastic leukaemia: a report of three cases.

Treatment of refractory acute lymphoblastic leukaemia (ALL) in adults remains a challenge, with allogeneic stem cell transplant (SCT) representing the only potential curative intervention. Alemtuzumab, a humanized IgG1j anti-CD52 antibody, has been approved for fludarabine-resistant B-cell chronic lymphocytic leukaemia and has been evaluated in other lymphoid malignancies, including ALL (Laporte et al, 2004; Piccaluga et al, 2004; Tibes et al, 2006). However, the data for single-agent alemtuzumab in adult relapsed/refractory ALL appears modest. In previous reports involving 10 patients treated with single-agent alemtuzumab, complete remission (CR) was achieved in only one (Laporte et al, 2004; Piccaluga et al, 2004). Interestingly, the combination of alemtuzumab and fludarabine has been shown to be highly cytotoxic to CD52+ ALL cells in vitro (Golay et al, 2006). We report three cases of refractory adult ALL whereby the concomitant administration of alemtuzumab, fludarabine and polyethylene-glycol conjugated asparaginase (PEG-asparaginase) resulted in clearance of leukaemic blasts and enabled the successful use of allogeneic SCT. Case 1 was a 39-year-old female presenting with precursor B-cell ALL. The patient initially achieved CR with the Larson protocol (Larson et al, 1998) but subsequently relapsed 4 years later. Her ALL proved refractory to two salvage chemotherapy regimens, including hyper-fractionated cyclophosphamide, vincristine, adriamycin and dexamethasone (hyper-CVAD; Kantarjian et al, 2000) and methotrexate, vincristine, PEGasparaginase and prednisone (MOAP; Aguayo et al, 1999), with >60% leukaemic blasts persisting on serial bone marrow examinations. The patient then received FCM (fludarabine 25 mg/m days 1–3, cyclophosphamide 600 mg/m day 1, and mitoxantrone 8 mg/m day 1) in combination with PEGasparaginase 2500 U/m once every 2 weeks for two doses and alemtuzumab 30 mg subcutaneously 3 d per week starting on day 5 (11 doses total). A day + 35 bone marrow examination revealed near total bone marrow aplasia without morphological evidence of leukaemic blasts. Her course was complicated by Staphylococcus aureus bacteraemia. The patient required no further chemotherapy and successfully underwent a one antigen-mismatched unrelated donor SCT. She remained in CR for 8 months post-SCT but then suffered disease relapse and expired shortly thereafter. Case 2 involved a 50-year-old male with primary refractory biphenotypic leukaemia who had failed to achieve CR after having received four standard ALL regimens. The patient’s bone marrow examination revealed >75% leukaemic blasts with a peripheral white blood cell count of 10 · 10/l, including 10% leukaemic blasts. He was given FLAG (fludarabine 30 mg/m days 1–5, Ara-C 2000 mg/m days 1–5, and G-CSF 300 lg/d) in combination with PEG-asparaginase 2500 U/m once every 2 weeks for two doses and alemtuzumab 30 mg subcutaneously 3 d per week starting on day 6 (14 doses total). The patient was noted to have progressive clearance of lymphoblasts from his peripheral blood and subsequently developed persistent pancytopenia over the next 28 d, with a day + 42 bone marrow examination revealing bone marrow aplasia with no morphological evidence of leukaemia (Fig 1). The patient’s course was complicated by neutropenic fevers and vancomycin-resistant enterococcal bacteraemia. He subsequently underwent a successful matched-sibling donor allogeneic SCT and remains in CR 2 years later. Case 3 was a 24-year-old male with precursor B-cell ALL who initially achieved CR with induction chemotherapy but subsequently relapsed within 8 months with ALL that proved resistant to hyper-CVAD, intravenous methotrexate and FLAG. Bone marrow examination revealed >40% leukaemic blasts without peripheral blood involvement. FCM in combination with PEG-asparaginase and alemtuzumab (14 doses total) was initiated as described in case 1. The patient’s course was complicated by Listeria bacteraemia. By day + 42 the patient’s pancytopenia began to resolve and subsequent bone marrow examination revealed trilineage hematopoiesis consistent with CR. The patient subsequently underwent a successful matched unrelated donor SCT and remains in a CR 6 months post-SCT. In all cases, patients presented with relapsed leukaemia which had proved refractory to numerous salvage regimens commonly used in ALL. Although bone marrow examinations revealed >30% involvement with leukaemia prior to the initiation of therapy, all cases exhibited a relatively low burden of disease, with only minimal peripheral blood involvement with leukaemia in one case and absence of hepatosplenomegaly or bulky lymphadenopathy. In addition to the three cases described here, we used this precise regimen in one other patient with refractory pre-B cell ALL. However, this patient withdrew from the regimen early in the course of therapy and opted for supportive care, making it impossible to assess the effectiveness of the regimen in this case. Correspondence

Pregnancy outcomes in inherited bone marrow failure syndromes

To the editor: Inherited bone marrow failure syndromes include entities such as Fanconi anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, severe congenital neutropenia, and telomere biology disorders/dyskeratosis congenita (TBDs/DKC). These are potentially devastating conditions that

Polycythemia and Thrombocytosis

Myeloproliferative neoplasms (MPNs) are diseases of excess cell proliferation from bone marrow precursors. Two classic MPNs, polycythemia vera (PV) and essential thrombocytosis (ET), are conditions of excess proliferation of red blood cells and platelets, respectively. Although PV and ET involve different cells in the myeloid lineage, their clinical presentations have shared features, consistent with overlapping mutations in growth factor signaling. The management of both diseases involves minimizing the risk of thrombotic and hemorrhagic complications. Both PV and ET can progress to myelofibrosis or acute myeloid leukemia, portending a poor prognosis. MPNs can also present as primary myelofibrosis.

Approach to pancytopenia: Diagnostic algorithm for clinical hematologists

Pancytopenia is a relatively common phenomenon encountered in clinical practice. The evaluation of a patient with pancytopenia requires a comprehensive approach and identifying the underlying cause can be challenging given the wide range of etiologies including drugs, autoimmune conditions, malignancies, infections, hemophagocytosis, and inheritable conditions. Recent advances in molecular hematology which include genomic profiling and next-generation sequencing have helped gain major insights into various hematological conditions and can guide diagnosing specific diseases in a shorter time at lower costs. However the approach to manage patients with pancytopenia in the current era of genomics is not well defined in the literature and is widely variable in practice. Herein, we conducted a systematic review to help devise an algorithm and management approach for pancytopenia, which serves as a general consultative approach.

A single center experience with romiplostim for the management of chemotherapy-induced thrombocytopenia

e Angers, UFR Sant e, Angers, France CHU d’Angers, Laboratoire d’H ematologie, Angers, France CRCINA, INSERM, Universit e d’Angers, Angers, France F ed eration Hospitalo-Universitaire ‘Grand Ouest Against Leukemia’ (FHU GOAL), Angers, France CHU de Brest, Service d’H ematologie Clinique, Brest, France CHU de Nantes, Laboratoire d’H ematologie, Nantes, France CHU de Brest, Laboratoire d’H ematologie, Brest, France INSERM U1078, CHRU Brest, Brest, France Universit e de Bretagne Occidentale, UFR M edecine, Brest, France CHU d’Angers, Service des Maladies du Sang, Angers, France AP-HP, CHU Henri Mondor, Laboratoire d’H ematologie, Cr eteil, France Polyclinique Bordeaux Nord Aquitaine, Service d’h emato-oncologie, Bordeaux, France INSERM 1066 MINT, Universit e d’Angers, Angers, France Service de M edecine-H ematologie, CH Robert Boulin, Libourne, France CHU de Limoges, Service d’H ematologie, Limoges, France CHU de Limoges, Laboratoire d’H ematologie, CRBS, Limoges, France UMR CNRS 7276, Limoges, France Service de M edecine Interne-Maladies Infectieuses, CHU de Bordeaux, Pessac, France

Therapy-related myeloid neoplasms - what have we learned so far?

Therapy-related myeloid neoplasms are neoplastic processes arising as a result of chemotherapy, radiation therapy, or a combination of these modalities given for a primary condition. The disease biology varies based on the etiology and treatment modalities patients receive for their primary condition. Topoisomerase II inhibitor therapy results in balanced translocations. Alkylating agents, characteristically, give rise to more complex karyotypes and mutations in p53. Other etiologies include radiation therapy, high-dose chemotherapy with autologous stem cell transplantation and telomere dysfunction. Poor-risk cytogenetic abnormalities are more prevalent than they are in de novo leukemias and the prognosis of these patients is uniformly dismal. Outcome varies according to cytogenetic risk group. Treatment recommendations should be based on performance status and karyotype. An in-depth understanding of risk factors that lead to the development of therapy-related myeloid neoplasms would help developing risk-adapted treatment protocols and monitoring patients after treatment for the primary condition, translating into reduced incidence, early detection and timely treatment.

Management of refractory autoimmune hemolytic anemia via allogeneic stem cell transplantation

Management of refractory autoimmune hemolytic anemia via allogeneic stem cell transplantation