Venous thromboembolism prevention during asparaginase-based therapy for acute lymphoblastic leukemia.

BACKGROUND: Venous thromboembolism (vte) is a recognized complication in patients treated with asparaginase-containing chemotherapy regimens; the optimal preventive strategy is unclear. We assessed the safety and efficacy of prophylaxis using low-dose low molecular weight heparin in adult patients with acute lymphoblastic leukemia in complete remission treated with an asparaginase-based post-remission chemotherapy regimen. METHODS: As part of the intensification phase of the Dana-Farber Cancer Institute 91-01 regimen, asparaginase was administered weekly to 41 consecutive patients for 21-30 weeks; these patients also received prophylaxis with enoxaparin 40 mg daily (60 mg for patients ≥80 kg). Outcomes were assessed against outcomes in a comparable cohort of 99 patients who received the same chemotherapy regimen without anticoagulation prophylaxis. RESULTS: The overall rate of symptomatic venous thrombosis was not significantly different in the prophylaxis and non-prophylaxis cohorts (18.92% and 21.74% respectively). Among patients receiving prophylaxis, vte occurred in higher proportion in those who weighed at least 80 kg (42.86% vs. 4.35%, p = 0.0070). No major bleeding complications occurred in the prophylaxis group (minor bleeding: 8.1%). CONCLUSIONS: Prophylaxis with low-dose enoxaparin during the intensification phase was safe, but was not associated with a lower overall proportion of vte.

Do quality of life or physical function at diagnosis predict short-term outcomes during intensive chemotherapy in AML?

BACKGROUND: Intensive chemotherapy (IC) used to treat acute myeloid leukemia (AML) is associated with toxicity, particularly in older adults. Emerging data suggest that baseline quality of life (QOL) and physical function may predict outcomes in oncology, although data in AML are limited. We investigated the association between baseline QOL and physical function with short-term treatment outcomes in adults and elderly AML patients. MATERIALS AND METHODS: We conducted a prospective, longitudinal study of adults (age 18+) AML patients undergoing IC. Before starting IC, patients completed the European Organisation for the Research and Treatment of Cancer (EORTC) 30-item questionnaire (QLQ-C30) and Functional Assessment of Cancer Therapy Fatigue subscale (FACT-Fatigue) in addition to physical function tests (grip strength, timed chair stands, 2-min walk test). Outcomes included 60-day mortality, intensive care unit (ICU) admission and achievement of complete remission (CR). Logistic regression was carried out to evaluate each outcome. RESULTS: Of the 239 patients (median age 57.5 years), 56.7% were male and median Charlson comorbidity score was 0. Sixty-day mortality, ICU admission and CR occurred in 9 (3.7%), 15 (6.3%) and 167 (69.9%) patients, respectively. Using univariate regression, neither QOL nor physical function at presentation was predictive of 60-day mortality (all P > 0.05), whereas ICU admission (P < 0.001) and remission status at 30 days (P = 0.007) were. Fatigue (P = 0.004) and role functioning (P = 0.003) were predictors of ICU admission; QOL and physical function were not. A higher Charlson score predicted ICU admission (P = 0.01) and remission status (P = 0.002). The cytogenetic risk group was associated with achievement of CR (P = 0.02); QOL and physical function were not (all P > 0.05). Findings were similar when patients age 60+ were examined. Relationships between fatigue and role functioning with ICU admission deserve further exploration. CONCLUSIONS: Baseline QOL and physical function tests in this prospective study were not associated with short-term mortality, ICU admission or achievement of CR after the first cycle of chemotherapy.

T cell receptor and immunoglobulin gene rearrangements in acute myeloblastic leukemia.

The organization and expression of the beta chain of T cell antigen receptor gene (beta-TCR) and Ig H and L chain genes were analyzed by Southern blot technique in 24 patients with a diagnosis of acute myeloblastic leukemia (AML). Rearrangements of the beta-TCR genes were seen in DNA samples from 3 of the 24 patients. One of these three patients also showed rearrangement of the Ig H chain gene. RNA samples from all three patients expressed a beta-TCR gene transcript on dot blot analysis. However, on Northern blot analysis, one patient expressed an incomplete 1.0 kb transcript and no Ig H chain mRNA, despite a rearranged configuration. The karyotypes of two of these patients showed abnormalities involving chromosome 7. Rearrangements of T cell antigen receptor genes may occur in nonlymphoid malignancy, and is consistent with the concept of lineage infidelity in AML.

Sequences and diversity of human T cell receptor beta chain variable region genes.

The nucleotide sequences of 22 human T cell antigen receptor (TcR) beta chain variable region genes isolated from various T lymphocytes have been analyzed. Of the 19 variable gene segment (V beta)-containing sequences, 17 were unique. The V beta gene segments were grouped into 11 families. Comparisons were made with the data of Concannon et al. to unify the nomenclature. The data is consistent with a total V beta gene segment repertoire with a most probable value of 38 members and an upper bound of 104 members at the 95% confidence level. Southern blot data of germline DNA using selected TcR V beta cDNAs as probes support this estimate. The human repertoire is approximately three to four times greater than that reported for the mouse. Explanations for this discrepancy are proposed.

A tumor suppressor and oncogene: the WT1 story.

The Wilms' tumor 1 (WT1) gene encodes a transcription factor important for normal cellular development and cell survival. The initial discovery of WT1 as the causative gene in an autosomal-recessive condition identified it as a tumor suppressor gene whose mutations are associated with urogenital disease and the development of kidney tumors. However, this view is not in keeping with the frequent finding of wild-type, full-length WT1 in human leukemia, breast cancer and several other cancers including the majority of Wilms' tumors. Rather, these observations suggest that in those conditions, WT1 has an oncogenic role in tumor formation. In this review, we explore the literature supporting both views of WT1 in human cancer and in particular human leukemias. To understand the mechanism by which WT1 can do this, we will also examine its functional activity as a transcription factor and the influence of protein partners on its dual behavior.

Aspergillus galactomannan detection in exhaled breath condensate compared to bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in immunocompromised patients.

OBJECTIVES: Exhaled breath condensate (EBC) is a noninvasive means of sampling the airways that has shown significant promise in the diagnosis of many disorders. There have been no reports of its usefulness in the detection of galactomannan (GM), a component of the cell wall of Aspergillus. The suitability of EBC for the detection of GM for the diagnosis of invasive aspergillosis (IA) using the Platelia Aspergillus enzyme-linked immunosorbent assay was investigated. METHODS: Prospective, cross-sectional study of lung transplant recipient and haemotologic malignancy patients at a university centre. EBC samples were compared to concomitant bronchoalveolar lavage (BAL) samples among lung transplant recipients and healthy controls. EBC was collected over 10 minutes using a refrigerated condenser according to the European Respiratory Society/American Thoracic Society recommendations, with the BAL performed immediately thereafter. RESULTS: A total of 476 EBC specimens with 444 matched BAL specimens collected from lung transplant recipients (n = 197) or haemotologic malignancy patients (n = 133) were examined. Both diluted and untreated EBC optical density (OD) values (0.0830, interquartile range (IQR) 0.0680-0.1040; and 0.1130, IQR 0.0940-0.1383), respectively, from all patients regardless of clinical syndrome were significantly higher than OD values in healthy control EBCs (0.0508, IQR 0.0597-0.0652; p < 0.0001). However, the OD index values did not correlate with the diagnosis of IA (44 samples were associated with IA). Furthermore, no significant correlation was found between EBC GM and the matched BAL specimen. CONCLUSIONS: GM is detectable in EBC; however, no correlation between OD index values and IA was noted in lung transplant recipients.

Origin of leukemic relapse after bone marrow transplantation detected by restriction fragment length polymorphism.

Bone marrow transplantation has become an accepted modality in the treatment of acute leukemia. With this therapy, it is possible to obtain long-term disease-free survival. However, leukemia recurs occasionally. In most cases, leukemic relapse is of recipient origin. There have been several reports, though, of leukemia developing in donor cells. These cases have been limited to instances in which there is an easily identifiable chromosome difference or abnormality, usually a sex chromosome. In this paper we describe the use of restriction fragment-length polymorphism analysis to determine the origin of recurrent leukemia cells in which no identifying chromosome was present. We found that the leukemia had recurred in recipient cells. We also were able to demonstrate the presence of normal hemopoietic cells of donor origin.

c-KIT expression enhances the leukemogenic potential of 32D cells.

The growth of human leukemic cells in culture and in vivo is dependent upon the presence of hematopoietic growth factors. Most populations of human leukemic acute myeloblastic leukemia (AML) cells express c-Kit on their surface and respond to Kit ligand (KL) in culture. To determine if this interaction was of potential significance in vivo we used a mouse model system. 32D cells, a murine IL-3-dependent myeloid cell line, were rendered KL responsive by transfection of the murine c-Kit. After injection of 32D or 32D-Kit cells into syngeneic hosts, animals bearing 32D-Kit cells, but not 32D cells, became moribund and were killed. These animals had circulating leukemic blast cells, infiltration of bone marrow, spleen, brain, liver, lung, and kidney. Cells recovered from some of the animals continued to be dependent upon IL-3 or KL for growth while in other cases the cells were factor independent. This model illustrates that the constitutive expression of c-Kit enhances the leukemic potential of 32D cells. The model will be useful for studying the progression of leukemia in vivo and testing whether interruption of the interaction of Kit and KL can affect the growth of leukemic cells.

A human lymphoma cell line with multiple immunoglobulin rearrangements.

The development of a cell culture system efficient in the establishment of lymphoma cell lines has made it possible to dissect basic biological and molecular aspects of lymphoma cells. We have established a lymphoma cell line from a patient with B cell lymphoma. The cell line has a complex karyotype with translocations involving bands 8q24, 14q32, and 18q21. Molecular analysis revealed that the Myc gene was rearranged; we were unable to demonstrate rearrangement of the Bcl-2 gene. Evaluation of the structure of the heavy chain Ig genes revealed that the cell line carried the same rearrangements as the cells from which the cell line was derived. The pattern of rearrangement, however, was unusual in that there were at least four rearranged bands when DNA cut with HindIII was probed with a fragment of the heavy chain joining region. To further characterize the cell line, subclones were derived. Individual subclones had the same pattern of rearrangement as the parent cell line. The results of these studies provide evidence that multiple rearranged Ig genes may be present in a single clone of cells.

CD123 target validation and preclinical evaluation of ADCC activity of anti-CD123 antibody CSL362 in combination with NKs from AML patients in remission.

Despite the heterogeneity of acute myeloid leukemia (AML), overexpression of the interleukin-3 receptor-α (CD123) on both the more differentiated leukemic blast and leukemic stem cells (LSCs) provides a therapeutic target for antibody treatment. Here we present data on the potential clinical activity of the monoclonal antibody CSL362, which binds to CD123 with high affinity. We first validated the expression of CD123 by 100% (52/52) of patient samples and the correlation of NPM1 and FLT3-ITD mutations with the high frequency of CD123 in AML. In vitro studies demonstrated that CSL362 potently induced antibody-dependent cell cytotoxicity (ADCC) of AML blasts including CD34+CD38-CD123+ LSCs by natural killer cells (NKs). Importantly, compared with healthy donor (HD) NKs, NKs drawn from AML patients in remission had a comparable ADCC activity against leukemic cells; of note, during remission, immature NKs were five times higher in AML patients than that in HDs. Significantly, we report a case where leukemic cells were resistant to autologous ADCC; however, the blasts were effectively lysed by CSL362 together with donor-derived NKs after allogeneic hematopoietic stem cell transplantation. These studies highlight CSL362 as a promising therapeutic option following chemotherapy and transplant so as to improve the outcome of AML patients.

Biological and clinical consequences of NPM1 mutations in AML.

Acute myeloid leukemia (AML) is characterized by accumulation of myeloid cells in the bone marrow because of impaired differentiation and proliferation, resulting in hematopoietic insufficiency. NPM1 is one of the most commonly mutated genes in AML, present in 20-30% of cases. Mutations in NPM1 represent a distinct entity in the World Health Organization (WHO) classification and commonly indicate a better risk prognosis. In this review, we discuss the many functions of NPM1, the consequence of mutations in NPM1 and possible mechanisms through which mutations lead to leukemogenesis. We also discuss clinical consequences of mutations, associated gene expression patterns and the role of NPM1 mutations in informing prognosis and therapeutic decisions and predicting relapse in AML.

Expression of CD4 is correlated with an unfavorable prognosis in wild-type NPM1, FLT3-ITD-negative cytogenetically normal adult acute myeloid leukemia.

INTRODUCTION: In the cytogenetically normal population of AML (CN-AML), FLT3-ITD-positive and wild-type NPM1 is correlated with a worse outcome, and FLT3-ITD-negative with NPM1-mut is correlated with a better outcome. This leaves a large subpopulation of CN-AML patients without NPM1 or FLT3-ITD mutations with heterogeneous outcomes with overall survivals (OS) ranging from several weeks to years. Therefore, new prognostic markers are needed to better risk stratify this subset of patients. METHODS: The retrospective study included 60 de novo adult AML patients diagnosed at our institution with normal karyotype, no FLT3-ITD or NPM1 mutations, and who did not receive allogeneic hematopoietic stem cell transplantations. We investigated the prognostic significance of immunophenotypic markers and clinical laboratory features in this double-negative population. RESULTS: Older age (>60) and CD4 expression (14%) were significantly correlated with shorter event-free survival (EFS) (P < 0.001, P = 0.016, respectively). Expression of CD56 (12%), as well as lack of CD34 expression (19% of the cases), was also associated with a worse EFS (P = 0.048, P = 0.028, respectively). On multivariable analysis, CD4 expression and old age (>60) were identified as independent predictors for worse EFS (P = 0.016; P = 0.001, respectively) and OS (P = 0.048; P = 0.028, respectively). CONCLUSIONS: Our results indicate that CD4 expression and older age are adverse prognostic factors in wild-type NPM1, FLT3-ITD-negative CN-AML.

Oncogenic potential of the transcription factor LYL1 in acute myeloblastic leukemia.

The LYL1 gene encodes a basic helix-loop-helix transcription factor involved in T-cell acute lymphoblastic leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of LYL1 was at higher levels in the majority cases of acute myeloblastic leukemia (AML) or myelodysplastic syndrome when compared to normal bone marrow. Our study also showed that LYL1 was highly expressed in most AML cell lines and in CD34+ AML cells. To determine whether LYL1 had an affect on the phenotype and behavior of myeloid cells, we introduced full-length LYL1 cDNA into K562 cells using electroporation and U937 cells with retroviral infection. Both of the derivative cell lines with overexpression of LYL1 had an increased growth rate and clonogenecity. Forced expression of LYL1 in K562 cells enhanced spontaneous and hemin-induced erythroid differentiation but blocked spontaneous as well as PMA-induced megakaryocytic differentiation. Overexpression of LYL1 in U937 cells blocked all-trans retinoic acid-induced monocytic differentiation. The LYL1-transfected U937 cells were also more resistant to the cytotoxic drug cytarabine. These results demonstrate that LYL1 may play a role in early hematopoiesis and may be a potential oncogenic factor in AML.

The histone deacetylase inhibitor valproic acid alters sensitivity towards all trans retinoic acid in acute myeloblastic leukemia cells.

Acute myeloblastic leukemia (AML) may be classified in a number of ways. Using the French American British classification, the M3 form of the disease or acute promyelocytic leukemia (APL) has been found to be sensitive in vitro and in vivo to the retinoid all trans retinoic acid (ATRA). The mechanism for this is by restoration of normal gene expression through the release of histone deacetylase complexes (HDACs). In contrast to APL, other forms of AML are either nonresponsive or show blunted responses to ATRA. We evaluated if the inhibitor of HDAC activity, valproic acid (VPA), could mimic or enhance retinoid sensitivity in the AML cell line, OCI/AML-2, and clinical samples derived from patients with AML. An Affymetrix GeneChip experiment demonstrated that VPA modulated the expression of numerous genes in OCI/AML-2 cells that were not affected by ATRA including p21, a retinoid responsive gene in APL. VPA induced p21 expression in OCI/AML-2 cells and the majority of the AML samples tested; this was associated with cell cycle arrest and apoptosis not seen with ATRA alone. The addition of ATRA to VPA accentuated many of these responses, supporting the potential beneficial combination of these drugs in the treatment of AML.

Varying degrees of amplification of the N-ras oncogene in the human breast cancer cell line MCF-7.

The oncogene N-ras has been found to be amplified (congruent to 20 copies) in the human breast carcinoma cell line MCF-7. The amplified sequences have been localized to a marker chromosome by in situ hybridization. Sublines of MCF-7, serially passaged in different laboratories, have marked variation in the degree of N-ras amplification. The differing degrees of amplification of N-ras are further evidence of heterogeneity within MCF-7 subclones. The phenomenon may not have general relevance for breast cancer, since other breast cancer cell lines and DNA from patient biopsies failed to show evidence of N-ras amplification.

Identification of a human LIM-Hox gene, hLH-2, aberrantly expressed in chronic myelogenous leukaemia and located on 9q33-34.1.

We describe the isolation of human LH-2, a putative transcription factor containing two cysteine-rich regions (LIM domains) and a homeobox (Hox) DNA-binding domain. High levels of hLH-2 expression were observed in all cases of chronic myelogenous leukaemia (CML) tested, regardless of disease status. hLH-2 was mapped to chromosome 9Q33-34.1, in the same region as the reciprocal translocation that creates the BCR-ABL chimera of the Philadelphia chromosome (Ph'), the hallmark of CML; hLH-2 was retained on the derivative 9 chromosome and is therefore centromeric of c-ABL. The proximity of hLH-2 to the breakpoint on chromosome 9 raises the possibility of cis-activation by the t(9;22)(q34;q11) translocation. In addition to finding hLH-2 expression in all cases of CML, expression was observed in lymphoid malignancies and myeloid cell lines, but not in primary cases of acute myelogenous leukaemia. The role of hLH-2 in the development or progression of leukaemia is not known. However, hLH-2 may prove useful as a marker of CML for monitoring residual disease.

The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its alpha helical structure and independent of Bcl-2.

Since the over-expression of Bcl-2 is a common cause of multi-drug resistance, cytotoxic peptides that overcome the effects of Bcl-2 may be clinically useful. We harnessed the death-promoting alpha helical properties of the BH3 domain of BAD by fusing it to the Antennapedia (ANT) domain, which allows for cell entry (ANTBH3BAD). Treatment of 32D cells with the ANTBH3BAD peptide results in a 99% inhibition of colony formation. No significant toxicity is observed after treatment with ANT or BH3BAD alone. A mutant fusion peptide unable to bind Bcl-2 induces cell death as effectively as the wild-type ANTBH3BAD. Furthermore, 32D cells over-expressing Bcl-2 show no resistance to the ANTBH3BAD peptide. Therefore, the toxicity of the peptide was independent of the Bcl-2 pathway. We demonstrate that the toxicity of the peptide is due to its alpha helicity that disrupts mitochondrial function. Since this peptide overcomes major forms of drug resistance, it may be therapeutically useful if appropriately targeted to malignant cells.

Long-term medical outcomes and quality-of-life assessment of patients with chronic myeloid leukemia followed at least 10 years after allogeneic bone marrow transplantation.

PURPOSE: Benchmark analysis of patients with chronic myeloid leukemia (CML) alive for more than 10 years after allogeneic bone marrow transplantation (BMT) including data on disease status, bone marrow reserve, long-term complications, and quality of life (QOL). PATIENTS AND METHODS: Eighty-nine patients (46 in first chronic phase, 43 in advanced phase) received an allogeneic BMT for CML during the study period. Medical outcomes and QOL of patients were analyzed retrospectively. RESULTS: Twenty-eight (31.5%) of 89 patients were alive at 10 years and included in this analysis. Thirteen (46.4%) of 28 long-term survivors never relapsed. Fifteen patients relapsed between 0.5 and 16 years after transplantation. Ten patients showed a hematologic relapse and received salvage treatment. Five patients showed transient low levels of BCR-ABL-positive cells by Southern blot with no subsequent hematologic relapse. One of the 28 patients died in blast crisis at 12 years. The most frequent long-term complications were chronic graft-versus-host disease, osteoporosis, and cataracts. Frequency of clonogenic progenitors remained persistently decreased. QOL assessment yielded lower scores in physical performance as compared with an age-matched normative population, whereas social functioning was equivalent. A high degree of satisfaction was noted with interpersonal relationships. CONCLUSION: Patients with CML surviving their BMT long term do well in terms of medical outcomes. A constant rate of relapse was noted, with a high salvage rate of affected patients, suggesting the need for lifelong monitoring. QOL is perceived as good, particularly as related to social functioning; however, it is inferior to a normative population with regard to physical performance.

High-dose cytosine arabinoside in the treatment of acute myelogenous leukemia: contributions to outcome of clinical and laboratory attributes.

High-dose cytosine arabinoside (HDAra-C) has been used for remission induction, and in conventional doses for maintenance in a trial of single-agent therapy in 43 previously untreated patients with acute myelogenous leukemia (AML). Rationale for the trial was provided by the observed decrease in leukemic blast cell self-renewal in culture following exposure to Ara-C. Compared with a previous trial of 57 patients treated with multidrug therapy, single-drug Ara-C was associated with a significantly improved complete remission rate (P = .010), although the survival time was not increased. All patients with low self-renewal responded to HDAra-C in contrast to the previous trial where some patients with this phenotype failed remission induction. The clinical observations are consistent with the view that the antileukemic effect of Ara-C has some specificity for cellular events required for self-renewal of blast cells. Exposure in vivo to Ara-C was associated with an increase in blast stem cell renewal at relapse, indicating that maintenance with other drugs should be tested. The study demonstrates the importance of biological attributes in design and analysis of clinical trials.

Treatment of elderly patients with acute lymphoblastic leukemia--evidence for a benefit of imatinib in BCR-ABL positive patients.

All patients with acute lymphoblastic leukemia (ALL) over age 60 years seen at Princess Margaret Hospital over an 11-year period were analysed retrospectively. Of 53 patients, 45 received multiagent induction chemotherapy using a variety of regimens. There were 13 BCR-ABL positive patients, 9 of who received imatinib mesylate, either during induction or post-remission therapy. The overall complete remission (CR) rate of all 45-induction patients was 56%, with a 27% induction-related mortality rate. The CR rate was not influenced by induction regimen, age, initial WBC, LDH or BCR-ABL status. The median overall survival of the induction patients was 9 months, while the median progression-free survival (PFS) of the patients achieving CR was 10 months. The estimated overall survival (OS) at 3 years was 18.4% (95% CI: 9.8-34.3%). Age and initial WBC did not significantly predict for OS when evaluating the entire group of induction patients. However, there was a strong trend for BCR-ABL status to favorably predict for PFS, and for OS when only patients treated after July 2000 (when imatinib became available) were evaluated. The results indicate that ALL remains a poor prognosis disease in elderly patients, and that aggressive induction regimens designed for younger patients are very toxic for these patients. These data suggest that BCR-ABL+ ALL is becoming a relatively more favorable prognosis disease in the elderly, likely due to the influence of imatinib therapy. Further regimens should explore the use of less aggressive regimens in elderly patients and should evaluate the optimal way of combining imatinib with conventional agents in BCR-ABL+ patients.