Hans A. Messner

Clinical Relevance of a Pharmacogenetic Approach Using Multiple Candidate Genes to Predict Response and Resistance to Imatinib Therapy in Chronic Myeloid Leukemia

Purpose: Imatinib resistance is major cause of imatinib mesylate (IM) treatment failure in chronic myeloid leukemia (CML) patients. Several cellular and genetic mechanisms of imatinib resistance have been proposed, including amplification and overexpression of the BCR/ABL gene, the tyrosine kinase domain point mutations, and MDR1 gene overexpression. Experimental Design: We investigated the impact of 16 single nucleotide polymorphisms (SNP) in five genes potentially associated with pharmacogenetics of IM, namely ABCB1, multidrug resistance 1; ABCG2, breast-cancer resistance protein; CYP3A5, cytochrome P450-3A5; SLC22A1, human organic cation transporter 1; and AGP, α1-acid glycoprotein. The DNAs from peripheral blood samples in 229 patients were genotyped. Results: The GG genotype in ABCG2 (rs2231137), AA genotype in CYP3A5 (rs776746), and advanced stage were significantly associated with poor response to IM especially for major or complete cytogenetic response, whereas the GG genotype at SLC22A1 (rs683369) and advanced stage correlated with high rate of loss of response or treatment failure to IM therapy. Conclusions: We showed that the treatment outcomes of imatinib therapy could be predicted using a novel, multiple candidate gene approach based on the pharmacogenetics of IM.

Long-term pulmonary function abnormalities and survival after allogeneic marrow transplantation

Summary:We studied long-term pulmonary function testing (PFT) in a retrospective cohort of 6-month survivors of allogeneic marrow transplant (BMT) between 1980 and 1997. Of 593 patients, 73, 71 and 65% had adequate data to assess for obstruction, restriction and diffusion impairments respectively. Over 5 years, mean declines in 1-s forced expiratory volume/forced vital capacity (FEV1/FVC), total lung capacity (TLC) and diffusion were 4, 7 and 17%, respectively. TLC and diffusion tended to subsequently increase. In all, 6, 12 and 35% of patients met criteria for obstruction, restriction and impaired diffusion, respectively. Obstruction was less common in recent transplants (5 vs 15%, P=0.004), while restriction and diffusion impairment rates remained stable. There was significantly greater mortality with obstruction (HR 2.0 (1.04–3.95)), and a nonstatistically significant higher mortality rate with restriction (HR 1.6 (0.95–2.75)), but not with impaired diffusion (HR=0.99 (0.65–1.50)). cGVHD (OR 16.7 (2.2–129.8)) and busulfan (OR 2.9 (1.01–8.24)) were associated with obstruction. Marrow from nonsibling or mismatched donors (OR 4.9 (2.2–10.7)) was associated with restriction. In summary, after BMT, decreased diffusion capacity is common and benign; obstruction has decreased in frequency, is rare without cGVHD, and is associated with mortality; nonsibling and mismatched donor are risk factors for restriction.

Anti-Erythropoietin Antibody-Mediated Pure Red Cell Aplasia after Treatment with Recombinant Erythropoietin Products: Recommendations for Minimization of Risk

Since 1998, there has been a marked increase in incidence of pure red cell aplasia secondary to development of anti-erythropoietin antibodies (Ab+ PRCA) in patients who have chronic kidney disease (CKD) and receive recombinant erythropoietin. The relationship between incidence of Ab+ PRCA and specific erythropoietin products has not been examined rigorously. Manufacturers provided data regarding exposure to erythropoietin products and incidence of Ab+ PRCA between January 1998 and March 2003 in patients with CKD. Assuming a Poisson distribution, a maximum likelihood estimate for the Poisson rate parameter was calculated for each product. A test for homogeneity of Poisson rates was conducted to compare likelihood estimates between products. Global incidence of Ab+ PRCA was relatively low. Likelihood estimates were not significantly different for Epogen, Procrit, and Aranesp, independent of their formulation or route of administration. Eprex lacking human serum albumin (HSA) and administered subcutaneously was associated with the greatest risk of Ab+ PRCA. HSA-containing Eprex administered subcutaneously was associated with a lower risk than HSA-free Eprex administered subcutaneously, but this risk exceeded that of intravenous Epogen and intravenous HSA-free Eprex. NeoRecormon administered subcutaneously was associated with less risk than subcutaneous HSA-free Eprex but more risk than intravenous Epogen. HSA-free Eprex should not be administered subcutaneously to patients with CKD due to increased risk of Ab+ PRCA. Although the subcutaneous administration of HSA-containing Eprex is riskier than intravenous Epogen and intravenous HSA-free Eprex, and the use of subcutaneous NeoRecormon is riskier than intravenous Epogen, there is currently no evidence that other products are safer.

Effect of Noncompetitive Proteasome Inhibition on Bortezomib Resistance

BACKGROUND Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance. METHODS 5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ-proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided. RESULTS 5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [IC(50)] of AMC release in intact cells ranged from 0.57 to 5.03 microM), induced cell death in intact cells from leukemia and myeloma cell lines (mean IC(50) values for cell growth ranged from 0.94 to 3.85 microM), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding beta5 proteasome subunit (mean IC(50) for cell death in the absence of bortezomib, wild-type THP1: 3.7 microM, 95% confidence interval = 3.4 to 4.0 microM; THP1/BTZ500: 6.6 microM, 95% confidence interval = 5.9 to 7.5 microM). 5AHQ interacted with the alpha subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002). CONCLUSIONS 5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro.

Pulmonary function abnormalities after allogeneic marrow transplantation: a systematic review and assessment of an existing predictive instrument.

Pulmonary function testing (PFT) is used to characterize non-infectious pulmonary complications after allogeneic BMT. Identifying high-risk patients could facilitate preventive or early therapeutic measures. The objectives of the study were first, to review available data on PFT changes after BMT and second, to validate a previously published predictive index for PFT obstruction in patients transplanted at one center. For the systematic review, frequency, severity and time course of PFT changes after BMT and for the validation study, retrospective cohort comparing predicted with observed PFT, and calculation of indices of predictive accuracy were summarized. The validation study involved 434 patients from Princess Margaret Hospital, Toronto, Canada, who received their first BMT between 1980 and 1997, survived for at least 6 months and had adequate PFT follow-up. The systematic review included 20 studies. After BMT, decreased diffusion and total lung capacity were common and partially reversible. Obstruction was less common. The validation study of a previously published index, performed in 434 patients, found a sensitivity and specificity of 48% and 68% for identifying patients who develop obstruction. We concluded that PFT changes after BMT are common. A published predictive index is not sufficiently accurate to identify high-risk patients for potential preventive or early therapeutic strategies.

Allogeneic or autologous bone marrow transplantation (BMT) for non-Hodgkin’s lymphoma (NHL): results of a provincial strategy

In 1986, the bone marrow transplant centers in Ontario agreed to a strategy for the treatment of patients with NHL. Suitable patients would undergo autotransplant but be referred for allotransplant if they had persistent marrow involvement or an inadequate marrow/stem cell harvest. Data of all patients were recorded in a database. We reviewed this database to compare these transplant modalities with respect to overall survival, rate of relapse and treatment-related mortality. Between January 1986 and August 1997, 429 patients underwent BMT for NHL – 385 autotransplants and 44 allotransplants. Sixty-eight percent of patients received their transplant for aggressive NHL, while the others had indolent lymphoma. Three-year actuarial survival did not differ between allogeneic and autologous BMT: 71% vs 62%, respectively (P = 0.5330 by log-rank testing). Three-year actuarial rate of relapse was lower after allotransplant than autotransplant: 6% vs 41%, respectively (P = 0.0006 by log-rank testing). Treatment-related mortality was higher after allotransplant than autotransplant: 23% vs6%, respectively (P = 0.001 by χ2 analysis). For further comparison, autotransplant patients were randomly matched 2:1 with the allotransplant patients for age ± 5 years, disease status at BMT, disease histology, and year of BMT. In the matched comparison, survival did not differ (relative risk of death after allotransplant: 0.711 (95% CI: 0.309–1.637)). Relapse rate was significantly lower in the allotransplant group (relative risk of relapse for allotransplant: 0.190 (95% CI: 0.043–0.834)) and treatment-related mortality was not significantly different (relative risk for allotransplant: 1.425 (95% CI: 0.527–3.851)). In conclusion, a review of a provincial strategy for treatment of NHL, shows that survival is not different after allogeneic or autologous BMT, but the rate of relapse is lower after allotransplant. These data support continuing the current provincial strategy. Bone Marrow Transplantation (2000) 26, 859–864.

Monitoring soluble interleukin-2 receptor levels in related and unrelated donor allogeneic bone marrow transplantation

Acute graft-versus-host disease (GVHD) is effected by donor T lymphocytes which have been stimulated by host antigens. Activated donor T lymphocytes express interleukin-2 receptor (IL-2R), which is comprised of three subunits (α, β, γ). During activation, the α IL-2R subunit (CD25) is shed from the receptor complex and can be measured in the circulation. Soluble IL-2Rα (sIL-2R) levels are increased in states of immune activation including GVHD, and could theoretically be used as a guide to therapy. Since IL-2Rα expression is an early marker of T cell activation, we investigated: (1) if an increase in sIL-2R is specific for acute GVHD; and (2) if serial sIL-2R levels can identify patients with early GVHD, prior to the onset of clinical tissue damage (effector function). Weekly sIL-2R levels were monitored in 36 patients undergoing matched related (n = 23) or matched unrelated (n = 13) allogeneic bone marrow transplantation (BMT). There was no significant difference in sIL-2R levels between matched related and matched unrelated recipients. Patients with acute GVHD (n = 19, 53%) demonstrated higher sIL-2R levels, than those without during weeks 2 and 3 post-BMT (P = 0.02 and 0.04, Mann–Whitney U test, two-tailed). In patients with acute GVHD, the rise in sIL-2R preceded the clinical signs of GVHD (16/19 patients). However, patients with sepsis demonstrated a trend towards higher sIL-2R levels at week 1 and significantly greater levels by week 4 (P = 0.02). Furthermore, patients with veno-occlusive disease (VOD) (25%) also had significantly higher sIL-2R levels at week 2 (P = 0.03). We conclude that although sIL-2R levels increase in patients with acute GVHD, similar increases are seen in patients with VOD and/or sepsis and therefore, as a single biochemical marker, we find that serial measurements of sIL-2R lacks sufficient specificity to guide GVHD therapy.

A systematic review of transfusion-associated graft-versus-host disease.

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare complication of blood transfusion. The clinicolaboratory features of TA-GVHD and the relative contributions of recipient and component factors remain poorly understood. We conducted a systematic review of TA-GVHD reports. The HLA relationship between donor and recipient was classified as D = 0 when no donor antigens were foreign to the recipient vs D ≥ 1 when ≥1 donor antigen disparity occurred. We identified 348 unique cases. Criteria for component irradiation were met in 48.9% of cases (34.5% immune-compromised, 14.4% related-donor), although nonirradiated components were transfused in the vast majority of these (97.6%). Components were typically whole blood and red cells. When reported, component storage duration was ≤10 days in 94%, and 23 (6.6%) were leukoreduced (10 bedside, 2 prestorage, and 11 unknown). Among 84 cases with HLA data available, the category of D = 0 was present in 60 patients (71%) at either HLA class I or II loci and was more common among recipients without traditional indications for component irradiation. These data challenge the historic emphasis on host immune defects in the pathogenesis of TA-GVHD. The dominant mechanism of TA-GVHD in both immunocompetent and compromised hosts is exposure to viable donor lymphocytes not recognized as foreign by, but able to respond against, the recipient.

Treatment outcomes following leukemic transformation in Philadelphia-negative myeloproliferative neoplasms

Leukemic transformation (LT) is a rare but fatal complication of Philadelphia-negative myeloproliferative neoplasms (MPNs) for which optimal treatment strategies are not known. At our center, we have adopted a treatment approach for LT where patients within the transplant age group who have a reasonable fitness level are treated with curative intent and offered induction chemotherapy. Subsequently, those who respond and have a suitable donor are considered for allogeneic hematopoietic cell transplantation (HCT). In this study, we evaluated the clinical outcomes of this treatment approach in 75 patients with LT. The 2-year overall survival (OS) from the time of LT was 15%. A total of 39 patients (52%) were treated with curative intent (induction ± HCT) and had a 2-y OS of 26% compared with 3% in those noncuratively treated (P < .0001). In the curative intent group, 18 individuals (46%) achieved complete remission (CR) or CR with incomplete recovery and 12 (31%) reverted to a chronic MPN phase, with 17 patients undergoing HCT. Survival of patients posttransplant was significantly improved compared with those who responded to induction but were not transplanted (2-y OS of 47% vs 15%; P = .03). Thus, induction chemotherapy followed by HCT has the potential for long-term disease control in select patients with LT preceded by a MPN.

RBC transfusion requirements after allogeneic marrow transplantation: impact of the before-transplant Hb level on transfusion and early survival

BACKGROUND : Most patients undergoing allogeneic marrow transplantation (alloBMT) require transfusions of RBCs. A retrospective analysis was performed to evaluate the utilization and risk factors for RBC transfusions including age and sex of recipient, HLA matching between donor and recipient, disease status at time of BMT, the occurrence of GVHD, ABO blood group compatibility, the source of progenitor cells and the Hb level before BMT (PT‐Hb).