ALBERT

Description: The purpose of this study was to determine the facility and reliability of the World Health Organization (WHO) classification of myelodysplastic syndromes (MDSs) with several observers reviewing the same diagnostic specimens. We also wanted to determine if the WHO classification provided additional information about predictability of clinical response outcome. To accomplish these goals we reviewed 103 previously diagnosed cases of low-risk MDS. We found 92% interobserver agreement (P 〈 .001). Sixty-four of these patients had been entered into clinical trials using growth factors by the Nordic MDS Study Group. The WHO classification reliably predicted therapeutic response to the combination of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo). The response rate differed significantly between refractory anemia with ringed sideroblasts (RARS) and refractory anemia with multilineage dysplasia and ringed sideroblasts (RCMD/RS) with regard to therapeutic response (75% versus 9%; P = .003). Also, in the group of patients with less than 5% marrow blasts, there was a difference in median survival between patients with unilineage dysplasia (51% surviving at 67 months) and those with multilineage dysplasia (median survival, 28.5 months; P = .03). (Blood. 2004;103:3265-3270)

Description: 1) The growth from human leukemic bone marrow particles begins with the emigration of cells having the morphologic characteristics of erythrocytic, granulocytic, monocytic and lymphocytic elements. Proliferation of fibroblastic-like cells replaces these elements within 3 to 4 weeks. Various types of immature leukocytes occur in larger numbers, and persist longer, in tissue cultures of human leukemic bone marrow than in bone marrow cultures from patients with no hematological disease. 2) Lymphocytes from either leukemic or non-leukemic bone marrow cultures often appear to enter the cytoplasm of fibroblast-like cells, but those derived from cultures of chronic lymphocytic leukemic bone marrow appeared intracellularly in much larger numbers and persisted there longer. One of the mechanisms of this phenomenon may be the feeder layer principle. Extensive injury of certain fibroblast-like cells in relation to relatively few small lymphocytes suggests the possibility of an autoimmune attack. It is possible that both the lymphocytes and the "target cells" may disintegrate. 3) Two types of multinucleated giant cells have been observed. One type is syncytial in structure, suggesting a viral mechanism of initiation. Another type resembles the polyploid giant cells which are commonly seen in old tissue cultures of whatever origin, and in various malignant diseases in vivo as well as in vitro. 4) At times, poorly differentiated round cells may persist in old fibroblastic cultures. These cells may be derived from the original explant which continued to produce them, or they may originate from fibroblast-like cells reconverted into a morphologically undifferentiated mesenchymal cell type.

Description: Various theories can explain the expansion of glycosyl phosphatidyl inositol anchored protein (GPI-AP)-deficient clones in PNH. The differential growth advantage of PIG-A mutant stem cell scould be due to immune escape in the context of an autoimmune attack on hematopoietic targets explaining the close clinical relationship to aplastic anemia. The causes of evolution of the PNH clone may be related to a less efficient immune recognition of PNH cells, their relative resistance to apoptosis or slower senescense. These changes may be related to additional mutations or genetic damage present in either normal or GPI-AP deficient hematopoietic clone. Using traditional karyotyping, chromosomal defects are not common in PNH but the limited resolution of this method may preclude detection of smaller aberrations. Due to these limitations, novel technologies which improve resolution and sensitivity are under development. In array-based comparative genomic hybridization (A-CGH), differentially labeled test and reference DNA samples are hybridized to genomic microarrays. Differences in sequence copy number between the samples are reflected in a shift of the fluorescence spectrum. The principle of A-CGH allows for detection of unbalanced chromosomal changes of the whole genome and its resolution is limited solely by the number of clones. In preliminary studies we found significantly decreased telomerase activity in normal vs. CD59−CD55− CD34+ cells derived from patients with PNH suggesting: 1) more accelerated telomere shortening in phenotypically normal hematopoietic cells derived from PNH patients, or 2) the possibility of chromosomal instability and acquisition of karyotypic defects should a critical telomere length be reached. We hypothesized that higher resolution analysis of the karyotypic changes in PNH using CGH may result in detection of cryptic karyotypic abnormalities present in either the normal or GPI-AP deficient clone. We have analyzed a cohort of patients (N=6) with hemolytic PNH and major expansion of a GPI-AP deficient clone (50–95% of PNH granulocytes). All patients had normal cytogenetics by metaphase karyotyping. We have separated GPI-AP-deficient myeloid peripheral blood cells using CD55 and CD59 as well as CD2 and CD19 staining to exclude lymphocytes. Separated CD55−CD59− and CD55+CD59+ non lymphoid cells were sorted and, following DNA extraction, subjected to A-CGH analysis (Vysis microarrays containing 287 probes). In one patient, PNH cells appeared normal by A-CGH but the corresponding “normal” cells contained deletions of telomeric region of several chromosomes (2, 5, 16, 18, 19, 20qtel as well as 5, 11,19ptel). However, in the remaining patients telomeric deletions were present in both normal and PNH cells. There was no difference in the numbers of chromosomes affected and diverse chromosomal telomeric deletions were present. No other lesions were found. By comparison when A-CGH analysis was performed in 8 normal individuals telomeric deletions were only rarely found and only occasional gains of contiguous clones on chromosomes were detected. Deletion of the telomeric portions of multiple chromosomes is compatible with accelerated global telomere loss in PNH. However, it appears that this phenomenon was not restricted to either normal or PNH cells. Our results are in agreement with previously described telomere shortening measured by FISH, flow cytometry or Southern blot analysis in PNH, a change not consistently restricted to normal cells.

Description: Forty patients with advanced hematologic malignancies or severe aplastic anemia received marrow grafts from partially mismatched, unrelated marrow donors. All patients were administered conventional prophylaxis for acute graft-v-host disease (GVHD) consisting of methotrexate and low-dose glucocorticoids. All but two patients who survived at least 30 days showed durable engraftment. Six patients survive 17+ to 36+ months following transplantation. Severe acute GVHD was seen in 47% of the patients; however, no direct correlation between GVHD and the degree of mismatching could be determined. Fatal infections were seen in 29 patients, and in the majority the infection occurred after the granulocyte count had risen to greater than 500 cells/microL. We conclude that the problems encountered in this pilot study can potentially be solved, and that further studies with this type of marrow grafting are warranted.

Description: Background: As previously shown, the risk of cardiovascular disease is higher after a maternal placental syndrome, especially in the presence of fetal compromise. The HPA1-b allele of the ß3 subunit of the essential platelet integrin Alphallbbeta3 is a risk factor for increased platelet thrombogenicity, leading to arterial vascular occlusion also triggered by inflammatory endothelial alterations. We performed a case-control study to assess hereditary risk factor for arterial thrombosis in addition to the known risk determinants for venous thrombosis as risk determinants for fetal IGR. Materials and Methods: 121 women with severe fetal IGR (defined by birth weight below the 5th- percentile for gestational age and sex) and 300 normal women were evaluated. Women with other reasons of IGR (history of venous thrombosis, fetal loss, and preeclampsia, neonatale immunthrombocytopenia) were excluded. The fetuses were born alive after the 24th week of gestation. Results: A significant risk association could be shown for the HPA-1b/1b genotype OR 3.1 (95%CI 1.2–8.8), increased levels of lipoprotein (a) (〉0.2g/l) OR 1.7 (95%CI 1.0–2.9), increased levels of fibrinogen (〉median 284mg/dl) OR 2.8 (95%CI 1.8–4.4) increased levels of FVIII:C (〉median 147%) OR 1.7 (95%CI 1.1–2.7), and increased levels of von-Willebrand-factor antigen (vWF-Ag) (〉median 129%) OR 2.7 (95%CI 1.1–2.7). The combination of risk determinants led to a further increase in risk (e.g. HPA-1b allele, increased lipoprotein (a) and vWF-Ag OR 14.0) There was no significant risk association for factor V Leiden G1691A OR 1.3 (95% CI 0.7–2.81), and prothrombin G20210A mutation. OR 2.0 (95% CI 0.6–6.5) Conclusions: Increased platelet thrombogenicity via interaction with an inflammatory vascular process and/or via plasma levels of components of hemostasis/receptor ligands appear to be more important in women with IGR than the established risk determinants for venous thrombosis. Pharmacological control of the observed platelet thrombogenicity in patients at risk may be of clinical relevance. In consequence, it will be of importance to examine whether the critical subgroup of patients can benefit from prevention with specific antiplatelet agents.

Description: Large granular lymphocyte leukemia (LGL) is a clonal lymphoproliferation of CTL associated with immune cytopenias. Hematopoietic progenitor cells may be targets for the immune attack by clonal CTL. The rearranged unique portion of the T cell receptor (TCR), the complementarity determining region 3 (CDR3) of the variable beta chain can serve as molecular marker of the clonal process. Previously, we applied molecular analysis of TCR repertoire to the analysis of CDR3 regions of the variable beta-chain in LGL leukemia and obtained 70 individually specific CDR3 clonotypes. Moreover, using clonotype-specific PCR clonal size was determined. Shared or highly homologous clonotypic CDR3 sequences in some patients provide evidence for the non-random nature of the clonal transformation in LGL and the presence of a common antigenic target. Such an antigen may constitute a drive for the expansion of LGL and its distribution may explain lineage-specificity of the LGL CTL. Identification of the target antigen is essential for understanding of immune-mediated hematologic disorders, including autoimmune cytopenias such as those observed in LGL. T-LGL has inherent advantage of the availability of clonal CTL for experimentation. We hypothesized here that generation of a soluble TCR (sTCR) derived from T-LGL clone could facilitate identification of target cells and analysis of the cellular distribution of peptide presentation. For cloning of sTCR we selected 4 patients with extreme clonal CTL expansion and profound neutropenia. Flow cytometry with VB specific antibodies was applied to determine VB family utilization by the clonal TCR. B-chains were amplified with specific primers so that C region is truncated and the transmembrane and intracellular portions are deleted. Due to the lack of VA specific antibodies, a 5′RACE RT-PCR with a constant A primer was applied. Expanded LGL-specific A chain was determined and cloned in its truncated form as described for B-chain. For example, clonal TCR for patient #1 had a structure VB1/JB1.6/CB1 and VA9-2/JA41*01/CA. To construct a sTCR truncated VA and VB products were subcloned into a dual promoter baculovirus expression vector containing the k and H-chain cassettes (pAC-k-CH3 or alternatively pAC-Fc). This vector allows for expression of a bivalent TCR:Ig protein that can be produced in a baculovirus system. A flow cytometry on fluorochrome conjugated sTCR after co-culture with HLA matched normal bone marrow cells allows for the detection of cells expressing target peptides. Therapeutically, malignant T cell clone-specific sTCR may be used as a potential idiotypic vaccine for T cell lymphomas/leukemias expressing TCR. Figure Figure

Description: Acquired pure red cell aplasia (PRCA) is a rare disorder that may be associated with malignancy, infections, connective tissue disorders, pregnancy and drugs. Recently, we observed PRCA in pancreas transplant patients treated with a new immunosuppressive regimen. Since 1966, the University of Minnesota Medical Center (UMMC) has performed 1577 pancreas transplants. Since February 2003, 280 pancreas transplants were treated with a new immunosuppressive protocol involving induction and maintenance with (n=190) or conversion to (n=90) alemtuzumab (humanized anti-CD52) and mycophenolate mofetil (MMF). Daclizumab (humanized anti-CD25, IL-2 receptor) was added when MMF dosing was limited by neutropenia. Between September 2004 and July 2005, 12 patients receiving the triple drug combination were diagnosed with PRCA. All 12 had prolonged exposure to MMF and/or a calcineurin inhibitor (cyclosporine, tacrolimus) prior to induction with [n=8] or conversion to [n=4] the above protocol. Eight subjects had bone marrow biopsies performed and reviewed at UMMC, and 4 additional patients received the diagnosis of PRCA at other institutions. All cases showed similar morphologic features, including dysgranulopoiesis, erythroid aplasia or marked hypoplasia with evidence of maturation, megakaryocytic hyperplasia with normal or low peripheral platelet counts, and atypical lymphoid aggregates in bone marrow trephine sections. Cytogenetic studies showed normal karyotypes. Severe reticulocytopenia was present in all subjects. In one subject, an extramedullary post-transplant lymphoproliferative disorder was diagnosed simultaneously. Another patient developed atypical reactive lymphadenopathy. Acute parvovirus infection was excluded by serology or polymerase chain reaction in 11 cases. Erythropoietin, when used, was started after the diagnosis or maintained through recovery of PRCA. Two patients died, one (post conversion) of PRCA associated with autoimmune hemolytic anemia and one (post induction) of ischemic heart disease. MMF was either discontinued (n=8) or dose reduced (n=2) in the remaining 10 subjects, with improvement in hemoglobin or decreased transfusion requirements in 8 patients. The single patient in whom a follow-up bone marrow biopsy was obtained after clinical recovery showed normal erythropoiesis. Ten patients developed infections with cytomegalovirus (n=7), BK virus (n=3), Zygomyces (n=1), Aspergillus versicolor (n=1), and Mycobacteria Simiae/Interjectum (n= 1). Hypogammaglobulinemia was a common finding. We conclude that reversible PRCA is associated with the immunosuppressive protocol including alemtuzumab, MMF, and daclizumab.

Description: The integration of a retroviral vector as used in hematopoietic gene therapy trials produces a transition sequence from the vector DNA into the genomic DNA and may thus serve as a stable molecular marker unique for each cell clone. High sensitive linear amplification mediated PCR (LAM-PCR) allows the detection of specific retroviral integration sites. Thus it is possible to determine the clonal composition of the hematopoietic system in vivo (1). We could show that the hematopoietic repopulation in human SCID-X1 patients was derived from various, long-lived progenitor cell clones indicating retroviral transduction into pluripotent cells (manuscript submitted). In two cases of lymphoproliferative disorder after successful SCID-X1 gene therapy integration of the retroviral vector into the LMO-2 oncogene was probable the main reason for malignancy (2). The distribution of integration sites over the whole genome, the potential preference for integration at certain loci and which cells receive genetic correction and engraftment are therefore of considerable interest. Recently, another gene therapy trial has successfully corrected 4 infants suffering from SCID-X1. A GALV-pseudotyped MLV-based vector carrying the therapeutic common gamma chain gene was used for transduction of autologous CD34+ cells. The patients did not receive any conditioning therapy before transplantation. We analyzed lymphoid and myeloid DNA from all patients. The transduction efficiency of T lymphocytes and myeloid cells was up to 100 and 1 %, respectively. In vivo clonality analysis of CD3+ cells showed a polyclonal composition 1 to 2 years after transplantation. The myeloid repopulation also consisted of various different clones. These data may indicate transduction of pluripotent and long term active stem or progenitor cells. We here report on more than 300 sequenced integration sites of the patients, whereas 250 sequences could be assigned unequivocally to a unique locus. So far, no vector integration in the LMO-2 oncogene could be detected in this trial, and the patients do not reveal any other evidence for malignancy or clonal deformation of their stem cell compartment. We could show that integration of the mammalian gammaretroviral vector in this gene therapy trial is not random. Integration of the vector happens generally within or close to specific regions of genes. We found common integration sites (CIS) in RefSeq gene regions. The targeted RefSeq genes were classified according to the Gene Ontology database. Our data strongly support the presumption that curative gene therapeutic treatment requires a sustained polyclonal contribution of ex vivo manipulated stem and progenitor cells and provide an important insight into the integration manner of GALV-pseudotyped MLV-based vectors.

Description: Abstract 3104 Diffuse large B-cell lymphoma (DLBCL) is the most common sub-type of non-Hodgkin's lymphoma.1 DLBCL is a heterogeneous, clinically aggressive disease, which has recently been sub-categorized based upon gene expression profiling. The prognosis of patients with DLBCL is presently assessed by the International Prognostic Index (IPI), which predicts estimated five-year survival based on clinical criteria.1,2 Over the past decade, a number of reports have established that the intra-tumoral content of the phospholipid-related metabolites phosphoethanolamine and phosphocholine (Etn-P and Cho-P) is increased in clinically aggressive malignant disease, and decreases when the malignancy responds to anticancer treatment. These data suggest that phospholipid metabolism is an intrinsic part of the disease process.3,4 Based on these observations, we hypothesized that intra-tumor levels of Etn-P and Cho-P may be a reliable predictive biomarker for therapeutic outcome in aggressive malignant diseases. To test this hypothesis, we measured noninvasively the tumor content of the sum of Etn-P plus Cho-P normalized by the tumor content of nucleoside triphosphates ([Etn-P+Cho-P]/NTP) in patients with DLBCL prior to initiating therapy. These data show that the pretreatment metabolic ratio (PMR) of [Etn-P+Cho-P]/NTP, is a potentially valuable biomarker of outcome, which identifies a subset of DLBCL patients likely to experience early failure to standard therapy, and for whom alternatives to therapy should be considered. Methods: Under ethical review board approval, 27 previously untreated DLBCL patients prior to receiving standard doxorubicin-based therapy were studied noninvasively using 3D localized, 1H-decoupled, nuclear Overhauser-enhanced phosphorus MR spectroscopy at 1.5 T. Result: As only complete response (CR) is clinically meaningful in achieving durable remissions of DLBCL, we divided these patients by response at six months into CR and all the other responses (not complete response, NCR). In the 17 CR patients, the PMR was significantly lower than in the ten NCR patients (PMR mean ± 95% CI, 1.46 ± 0.21 vs. 2.47 ± 0.24, p 〈 3 × 10-6). The prediction of response using a Fisher test was significant for the PMR alone (p 〈 1 × 10-4; sensitivity of 1.00, specificity of 0.70) and improved further when combined with the IPI (p 〈 2 × 10-6; sensitivity of 1.0, specificity of 0.90). The progression-free survival (PFS) strongly correlated with the PMR alone (p 〈 4 × 10-8) and with the PMR and IPI as covariates (p 〈 1 × 10-7) by the Cox regression model. Using the Kaplan-Meier model, the PMR discriminated 64% of patients with PFS below 11 months (p 〈 1 × 10-7), while as covariate with the IPI it discriminated 82% of these patients (p 〈 2 × 10-7). Discussion: Our results show that the PMR contains information that predicts outcome to standard therapy in DLBCL patients. This prediction improves when the PMR is combined with the IPI. While the PME alone identifies 64% of those patients who will go on to show an extremely poor response with a progression-free survival below 11 months, the combination of the PME and IPI identify 82% of these patients. In conclusion, we have successfully demonstrated that the PMR can be an important determinant in predicting response to treatment in patients with DLBCL, especially when integrated with the IPI. References: 1) Cancer: principles and practice of oncology. 6th ed. Philadelphia, PA, Lippincott, Williams & Wilkins, 2001; 2) Smith MR, Non-Hodgkin's lymphoma. St. Louis, MO, Mosby, 1966; 3) Arias-Mendoza F, Brown TR, Dis. Markers, 2003;19:49-68; 4)Griffiths JR, et al, Lancet 1983;1:1435-6 Disclosures: No relevant conflicts of interest to declare.

Description: The need for safer gene therapy vectors was highlighted by the occurrence of three cases of retroviral vector-induced leukemia in children after the cure of severe combined immunodeficiency by gene therapy. These severe adverse events enhanced the development of new gene therapy vector systems, which aim to reduce influence of insertions on integrity and expression of genomic host DNA. Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) have no LTR enhancer activity and are not expected to produce insertional gene activation. In our study we analyzed the integrations sites of three different lentiviral SIN-HIV-based- vectors. These vectors differed in their internal elements (Promotor, Transgene, WPRE) which allowed us to investigate whether internal elements are influencing target site selection and clone survival. A total of 1422 integration sites were analyzed at three different time points (1 day, 30 days, 60 days) after transduction of identical HeLa cells by LAM- PCR. Our results showed similar gene involvement and chromosomal distribution of integration sites for all vector types analyzed, independent of vector composition. 62–63% of the integrations were detected in Refseq genes, 82–86% were found within Refseq genes and their surrounding 10kb. Surprisingly, 271 of 1422 integration sites were clustered as common integration sites (CIS). Computer simulations allowed us to show that this high number of CIS was significantly different from a modeled random distribution of integration sites. Gene ontology analysis showed no difference in significantly overrepresented gene categories between the distinct vector types. Interestingly, we detected a time dependent increase or decrease in significance. Specific gene categories like phosphorylation, protein kinase or ATP binding activity increased in significance from freshly transduced cells (1 day) to 30 days. This observation was even more pronounced at the latest time point analyzed (60 days). Other gene categories showed the exactly opposite effect. Gene ontology analysis of freshly transduced cells showed a significant overrepresentation of genes involved in cell cycle regulation, whereas the analysis of the later time points did not show overrepresentation of this gene category. These results suggest that lentiviral SIN-HIV-based vectors may induce clonal selection in vitro independently of internal vector elements. The character of such effects as well as any putative relevance of such genotoxicity for the in vivo situation will have to be investigated in detail for the role of individual vector/cell type configurations.