ALBERT

Description: A mouse anti-interleukin-2 receptor A-chain-specific PC61-immunotoxin (PC61-IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61-IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodepletion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61-IT-treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allo-depletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 x 10(6) T-cell-depleted BM cells and 0.5 x 10(6) PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

Description: Fusion genes derived from the platelet-derived growth factor receptor beta (PDGFRB) or alpha (PDGFRA) play an important role in the pathogenesis of BCR-ABL–negative chronic myeloproliferative disorders (CMPDs). These fusion genes encode constitutively activated receptor tyrosine kinases that can be inhibited by imatinib. Twelve patients with BCR-ABL–negative CMPDs and reciprocal translocations involving PDGFRB received imatinib for a median of 47 months (range, 0.1-60 months). Eleven had prompt responses with normalization of peripheral-blood cell counts and disappearance of eosinophilia; 10 had complete resolution of cytogenetic abnormalities and decrease or disappearance of fusion transcripts as measured by reverse transcriptase–polymerase chain reaction (RT-PCR). Updates were sought from 8 further patients previously described in the literature; prompt responses were described in 7 and persist in 6. Our data show that durable hematologic and cytogenetic responses are achieved with imatinib in patients with PDGFRB fusion–positive, BCR-ABL–negative CMPDs.

Description: Group I Burkitt lymphoma (BL) cell lines, which retain the original biopsy phenotype, have been shown to enter apoptosis in response to a number of external stimuli including serum deprivation, thermal shock, addition of calcium ionophore, and ligation of surface immunoglobulin (Ig) by antibody. Transforming growth factor-beta 1 (TGF beta 1) is known to cause growth arrest in BL lines. Here we show that while it is by itself capable of promoting some degree of apoptosis in group IBL cells, TGF beta 1 cooperates with anti-immunoglobulin to this end. Trimeric soluble recombinant human CD40 ligand (sCD40L) was able to inhibit apoptosis induced by the combination of agonists to some degree, but such rescue proved to be short-lived. Both TGF beta 1 and anti-Ig individually caused BL cells to undergo growth arrest at the G1 phase of cell cycle before their entry into apoptosis: the consequence of sCD40L addition was to maintain the cells in cycle for longer. No induction of the apoptosis-protecting gene, bcl-2, occurred in the presence of sCD40L. These findings are discussed, particularly highlighting the relationship existing between survival and the cell cycle. The strong cooperative effects observed between anti-Ig and TGF beta 1 in promoting apoptosis and the inability of CD40 to signal for long-term rescue raise the potential for a novel therapeutic attack on B-cell lymphoma.

Description: A mouse anti-interleukin-2 receptor A-chain-specific PC61-immunotoxin (PC61-IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61-IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodepletion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61-IT-treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allo-depletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 x 10(6) T-cell-depleted BM cells and 0.5 x 10(6) PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

Description: Epidemiological data and family studies have indicated that inherited factors may predispose to the development of myeloproliferative neoplasms (MPN). It has also been suggested that single nucleotide polymorphisms (SNPs) within JAK2 are associated with specific MPN subtypes. To explore the role of inherited factors in more detail, we initially performed quantitative analysis of a series of JAK2 SNPs in homozygous PV cases (%V617F 〉50%; n=73). Most mutant haplotypes could be read directly from the distorted allele ratios brought about by expansion of the homozygous clone. In many cases with 50–90% V617F, the residual wild type haplotype could also be read. Strikingly, of the 144 V617F alleles that could be determined, 111 (77%) had an identical core haplotype (subsequently designated 46/1) whereas only 9/76 (12%) residual wild type alleles were 46/1 (P = 1.9e-21, Fisher’s exact test). To explore this observation in more detail we first determined the haplotype structure of JAK2 using 14 SNPs genotyped by the Wellcome Trust Case Control Consortium (WTCCC) in 1500 UK blood donors. Nine haplotypes were inferred using the program PHASE that accounted for 94% of alleles, with a frequency of 0.24 for haplotype 46/1. Haplotype inference and tag SNP analysis revealed that 46/1 was also more frequent in heterozygous V617F positive MPD cases (135/354 alleles) compared to 188 locally sourced healthy controls (92/376 alleles; P = 0.0001) as well as the WTCCC cohort (P = 3.3e-8). Haplotype 46/1 was more frequent in all V617F positive disease entities compared to controls: PV (n=203; P=1.2e-16), ET (n=81; P=1.2e-9) and MF (n=41; P=8.0e-5) however there was no difference in the frequency of 46/1 between controls and V617F negative MPD / idiopathic erythrocytosis (n=123). To determine if heterozygous V617F also preferentially arose on a 46/1 allele as seen for homozygous cases, we developed an allele specific PCR between V617F and a SNP that tags this haplotype. In an analysis of 67 informative heterozygous V617F cases, 50 V617F alleles were 46/1 compared to only 17 residual wild type alleles (P=9.4e-9). We conclude that the 46/1 JAK2 haplotype is a strong predisposition factor for development of V617F associated MPDs (RR=2.6; 95% CI 2.3–2.9). The reason for this predisposition is currently unknown but it is likely that 46/1 is in linkage disequilibrium with an unknown constitutional functional variant that interacts with V617F JAK2.

Description: Aberrant activation of tyrosine kinases (TKs), caused either by mutation or gene fusion, is of major importance for the development of many hematological malignancies, particularly myeloproliferative neoplasms (MPNs). In general, TKs activated by mutations are associated with less aggressive MPNs, whereas TK fusions are seen in more aggressive diseases. We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in atypical MPNs. To detect genomic copy number changes associated with such fusions, we performed a systematic search using custom designed, targeted high-resolution array comparative genomic hybridization (array CGH). Arrays contained 44,000 oligonucleotide probes that targeted all TKs (n=90) plus a further 450 genes encoding downstream TK signaling components, other translocation targets plus receptors and other factors known to be important for myelopoiesis. For each target, 50–100 probes were selected that spanned the gene plus flanking sequences of up to 200 kb, providing a resolution of approximately 5–10 kb. Pretreatment genomic DNA from 68 patients (44 males, 24 females; median age 62 years, range 16–86) with atypical MPNs was studied: atypical MPN associated with eosinophilia, n=17; chronic eosinophilic leukemia or hypereosinophilic syndrome (HES), n=17; chronic myelomonocytic leukemia (CMML), n=10; unclassified atypical MPN, n=9; atypical chronic myeloid leukemia (aCML), n=6; unclassifiable MDS/MPN, n=5; chronic neutrophilic leukemia, n=3; acute basophilic leukemia, (n=1). All patients were negative for BCR-ABL, FIP1L1-PDGFRA, JAK2 V617F and none had karyotypic abnormalities suggestive of other known TK fusions. Nine HES patients showed a significant response to imatinib treatment in the absence of any known imatinib-sensitive abnormality. Control experiments indicated that the arrays were readily able to identify FIP1L1-PDGFRA in a background of 50% normal cells. Seven cytogenetically cryptic abnormalities were detected in five (7%) patients: Pt 1 with aCML: 0.5 Mb del(21q22.12) (including RUNX1) plus 1 kb del(19p13.11) (JUND); Pt 2 with HES: 1 kb del(19p13.11) as seen in Pt 1; Pt 3 with CMML: 53 kb dup(19p13.3) (in 5’ proximity to MATK); Pt 4 with unclassified atypical MPN: 44 kb del(5p12) (FGF10) together with a 25 kb del(15q21.1) (FGF7); Pt 5 with atypical MPN and eosinophilia: 44 kb dup(22q13.2) (L3MBTL2). No abnormalities involving TKs were detected. Amplification of whole chromosomes or chromosome arms was observed in 8 patients (chr 8, n=3; 21q, n=2; 6p, 16, 17q, n=1 each) and loss of 17p in one patient. Forty different regions of copy number variation (CNV) were identified in genes on the array of which 17 (e.g. within BCL6, TLX3, CDKN1A, FUS) were novel. We conclude that cytogenetically cryptic TK fusion genes are rare in patients with atypical MPNs, even in cases who responded to imatinib. Other abnormalities were identified in a minority of cases which warrant further investigation.

Description: Introduction: Venous thromboembolism (VTE) is an important problem for medical patients. In spite of the existence of evidence-based guidelines for prevention of VTE, prophylaxis for venous thromboembolism is underutilized. Objective: To assess the global and individual adherences (adjust to risk groups) to venous thromboembolism prophylaxis guidelines (VTEPG) in medical ill patients. Desing: Prospective observational study. Setting: The 50-bed medical floors of a university hospital. Material and Methods: Patients: All consecutively medically ill patients (pt) admitted during a 6-month period (n: 452). VTEPG: the guide divided medical patients in risk groups (G); G1: No need of VTEP (35 pt); G2: myocardial infarction (9 pt); G3: Stroke (21 pt); G4: high risk with thrombophilia, previous VTE, cancer (113pt); G5: respiratory and congestive heart failure, pulmonary infection (137 pt); G6: trauma (5 pt); G7: not included in previous groups, required an individual evaluation to asses VTE risk (112pt). Prophylaxis methods included: low dose unfractionated heparin (UFH) or low molecular weight heparin (enoxaparin 40 mg/d) and mechanical methods. Main outcome measurements: 1- Global: correct application of the guide (defined by a coincidence between the prophylaxis received and the recommendation); 2-Adjust to risk group. Results: Global adherence: Correct application of the guide was noted in 252 pts (56%); Adjust to risk group: GR1:71%; GR2:44%; GR3:62%; GR4:76%; GR5:55%; GR6:60% and GR7:26%. Conclusion: In our experience in medical patients global adherence to guides was 56%, the best adherence was observed in high risk groups 76% and the lowest 26% in those patients that need an individual risk assessment. This study and follow up permitted us to detect inaccurate prophylaxis uses and take corrective measures.

Description: Hereditary hemorrhagic telangiectasia (HHT) is a relatively uncommon, autosomal dominant disorder characterized by telangiectases that develop in the skin, mucous membranes, and visceral organs. Mucous localization may seldom bleed profusely, especially epistaxis and upper gastrointestinal (GI) tract. Effective drug treatment is not well established, and multiple blood transfusions and endoscopic or surgical procedures may be the ultimate solution to the frequently bleeding HHT patient. Danazol (DZ) is a mild androgen that has been used in small series of HHT pts with ambiguous results. Its toxicity profile in long standing administration is now well known. Eighteen patients with HHT with transfusion requirements (TR) were treated with DZ at 400–600 mg/daily for the initial three months and 200–400 mg/daily thereafter as a maintenance treatment. At the time of initiation of DZ therapy, median age was 54 yr-old (32–73), nine were female and 9 male, and the median previous TR was 16 RBC units/yr. (2–46). All patients had epistaxis and oral cavity bleeding, with 2 additional upper gastrointestinal tract active bleeding that were detected in ten patients in which an upper GI endoscopy was performed. One patient had a cerebral angioma, surgically treated. None had pulmonary fistula. All patients had some kind of iron treatment. DZ was the first drug treatment intended to reduce the HHT bleeding in 10/18 pts. Median follow-up was 7 years (1.2–14) and two pts were lost to follow-up at 4 and 11 years respectively. At three months of DZ therapy, 12/18 pts (66.6%) showed a remarkable reduction of bleeding, and in 6 patients that showed no response, DZ treatment was stopped. In 7 pts (39%) TR dropped to none and in 5 pts median RT dropped from 22 RBC units/yr. to 10 units/yr. Two of responders had a relapse with upper GI tract bleeding and 1 pts with epistaxis within the first 2 years of DZ treatment. Attempts to reduce the maintenance dose below 200 mg/daily were related to new bleeding or worsening of the active sites. None of the long standing DZ therapy had any significant toxicity. DZ treatment have shown efficacy and safety in this cohort of HHT patients. Mecanism of action may involve the increase of synthesis or expression of ALK-1 dependent proteins and less likely of endoglin. We propose DZ as a first line treatment for the transfusion dependent HHT patients.

Description: Intravenous alemtuzumab is an effective and well-tolerated treatment for T-cell prolymphocytic leukemia (T-PLL). Alemtuzumab given intravenously as first-line treatment in 32 patients resulted in an overall response rate of 91% with 81% complete responses. Studies in B-cell chronic lymphocytic leukemia have shown subcutaneous alemtuzumab to be equally as effective as intravenous alemtuzumab. The UKCLL05 pilot study examined the efficacy and toxicity of this more convenient method of administration in 9 previously untreated patients with T-PLL. Only 3 of 9 patients (33%) responded to treatment. Furthermore, 2 of 9 patients (22%) died while on treatment. Recruitment was terminated because of these poor results. After rescue therapy with intravenous alemtuzumab and/or pentostatin, median progression-free survival and overall survival were similar to the intravenous group. Alemtuzumab delivered intravenously, but not subcutaneously, remains the treatment of choice for previously untreated T-PLL. This study is registered at www.eudract.ema.europa.eu as #2004-004636-31.

Description: Abstract 3185 Introduction: Lupus anticoagulant (LA) is one of the laboratory criteria for the diagnosis of antiphospholipid syndrome. To detect the presence of LA, ISTH recommends performing two assays based on different principles. Our LA profile includes dilute Russell viper venom time (dRVVT) and two different activated partial thromboplastin time (aPTT) reagents. The aim of the study was to evaluate sensitivity and specificity of aPTT and dRVVT in women with a history of pre-eclampsia, intra-uterine growth restriction, early recurrent abortion, late fetal loss and placental abruption collectively termed “placenta mediated complications”. Materials and Methods: We studied a total of 247 patients and 287 samples in a 10 months period. Based on consensus criteria, samples were retested within a period of three months. 62/247 plasmas samples were collected during pregnancy. Laboratory studies: Screening tests: aPTT was performed using PTT-LA (Stago) and an aPTT home made reagent with diluted cephalin. dRVVT was performed using Russel viper venom RVV (Stago) and diluted cephalin. Negative control plasma was prepared according to ISTH LA recommendations for the mixing tests. Confirmatory tests: We performed a home made reagent for platelet neutralization procedure (PNP)-aPTT and dRVVT; Rosner Index and % of Correction as criteria for mixing and confirmatory tests interpretation. Statistical evaluation was performed by ROC method using EP Evaluator 9.9 software. Results: 172/287 samples (59.9%) were negative, while 115/287 samples (40.1%) were positive for LA. 44/115 samples (38.26%) were positive for both tests (aPTT and dRVVT), 60/115 samples (52.17 %) were positive only for dRVVT and 11/115 samples (9.57%) were positive only for aPTT. LA was confirmed in 9/62 pregnant women samples (14.5 %). 7 (11.3%) had abnormal dRVVT, the remaining 2 samples were positive for both tests. Conclusions: Our results demonstrated: 1- High prevalence of dRVVT positive plasmas 104/115 (90.43%); 2- Despite showing a low performance regarding sensitivity when compared to dRVVT, both aPTT tests were able to detect LA in 47.83% of the positive studies. 3- According to our experience, considering this initial cohort of selected patients, the use of dRVVT as the first and aPTT as the second screening test may help improve the performance for the diagnosis of LA. Disclosures: No relevant conflicts of interest to declare.