Levels of soluble HLA-I and beta2M in patients with acute myeloid leukemia and advanced myelodysplastic syndrome: association with clinical behavior and outcome of induction therapy.

beta-2 Microglobulin (beta2M), a subunit of human leukocyte antigen-class I (HLA-I), is well established as a marker of prognosis in various solid tumors and hematologic malignancies. The prognostic role of intact free-circulating HLA-I (sHLA-I) is less well understood. We compared the clinical relevance of plasma levels of sHLA-I and beta2M in patients with acute myeloid leukemia (AML; n=209) or advanced myelodysplastic syndrome (MDS; n=98). sHLA-1 and beta2M levels were significantly higher in AML and MDS patients than in control subjects, but did not differ significantly between the two disease groups. In AML patients, multivariate analysis showed both sHLA-1 and beta2-M to be highly predictive of complete remission (CR), survival and duration of complete response (CRD). In MDS, the predictive value of the two markers differed substantially from one another: beta2M was associated with survival, CR and CRD, whereas sHLA-I was not. These findings not only establish the role of sHLA-I as a tumor marker in AML but also support that MDS is clinically and biologically distinct from AML. sHLA-I has been reported to be an immunomodulator inhibiting the cytotoxic effects of T-lymphocytes, which may offset its predictive value for disease aggressiveness in patients with MDS.

Soluble phosphorylated fms-like tyrosine kinase III. FLT3 protein in patients with acute myeloid leukemia (AML).

FLT3 ligand (FL) has a significant role in the proliferation and differentiation of hematopoietic cells. Mutations in the FLT3 receptor gene have been reported in 30% of patients with AML. We investigated whether abnormal phosphorylation of FLT3 may be more common in AML. We evaluated FLT3 protein and its phosphorylation in the plasma from 85 patients with AML, 16 patients with myelodysplastic syndrome (MDS) and 5 patients with acute lymphoblastic leukemia (ALL). There were no significant differences in the level of plasma FLT3 protein level in the different diseases (p=0.57). AML patients had a significantly higher level of phospho-FLT3:FLT3 ratio (p=0.02). FLT3-ITD and FLT3 point mutations were present in 27 (32%) of the AML patients. Phosphorylated FLT3 was significantly higher in the plasma from patients with FLT3 mutation (p=0.002). Overall, there was no correlation between survival and the plasma level of FLT3 protein or its phosphorylated form. However, amongst the patients without FLT3 mutations, those with a higher level of phosphorylated FLT3 had a significantly shorter duration of remission (p=0.04). Other mechanisms may be responsible for abnormal phosphorylation of FLT3 and inhibitors of FLT3 should also be investigated in patients without mutations.

Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Heterogeneity in detecting Abl kinase mutations and better sensitivity using circulating plasma RNA.

Most studies test for mutations in the kinase domain of the abl gene in chronic myeloid leukemia (CML) using peripheral blood (PB) cells. Frequently, progression of the disease manifests with increased blasts in bone marrow (BM) and not in PB. Simultaneous analysis of plasma, PB cells and BM cells from 41 imatinib-resistant CML patients showed mutations in 63% of PB cells and 68% of plasma or BM cells (P = 0.04). In discordant patients, 13 mutations were detected in plasma, 11 in BM cells and 9 in PB cells. The T315I mutation was detected in plasma and BM but not PB cells in one patient. We detected no mutations in the plasma of 45 previously untreated CML patients, but two of these patients showed mutations in plasma and not cells by 9 months on therapy. Circulating plasma mRNA is a reliable alternative to BM mRNA for detecting ABL mutations.

Variations in the detection of ZAP-70 in chronic lymphocytic leukemia: Comparison with IgV(H) mutation analysis.

Lack of immunoglobulin heavy chain genes (IgV(H)) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta-chain (T-cell receptor) associated protein kinase 70 kDa (ZAP-70) has been reported to be a surrogate marker for IgV(H) mutation status, and its expression in leukemic cells correlates with unmutated IgV(H). However, ZAP-70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP-70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP-70 by comparing results with clinical outcome and the mutation status of the IgV(H). Using stored samples, we show significant correlation between ZAP-70 expression and clinical outcome as well as IgV(H) mutation at a cut-off point of 15%. While positive samples (>15% positivity) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgV(H) mutation status when 15% cut-off was used. In these samples, cut-point of 10% correlated with the IgV(H) mutation (P = 0.0001). This data suggests that although ZAP-70 positivity correlates with IgV(H) mutation status and survival, variations in sample handling and preparation may influence results. We show that IgV(H) mutation results, unlike ZAP-70 remain correlated with CD38 expression and beta-2 microglobulin in shipped samples, and ZAP-70 testing should not be used as the sole criterion for stratifying patients for therapy.

Having a higher blast percentage in circulation than bone marrow: clinical implications in myelodysplastic syndrome and acute lymphoid and myeloid leukemias.

Determining the percentage of peripheral blood (PB) and bone marrow (BM) blasts is important for diagnosing and classifying acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Although most patients with acute leukemia or MDS have a higher percentage of BM blasts than PB blasts, the relative proportion is reversed in some patients. We explored the clinical relevance of this phenomenon in MDS (n = 446), AML (n = 1314), and acute lymphoblastic leukemia (ALL) (n = 385). Among patients with MDS or ALL, but not AML, having a higher blast percentage in PB than in BM was associated with significantly shorter survival. In multivariate analyses, these associations were independent of other relevant predictors, including cytogenetic status. Our findings suggest that MDS and ALL patients who have a higher percentage of PB blasts than BM blasts have more aggressive disease. These data also suggest that MDS classification schemes should take into account the percentage of blasts in PB differently from the percentage of blasts in BM.

ATM mutations in B-cell chronic lymphocytic leukemia.

Mutations in the ATM gene located on the long arm of chromosome 11 at 11q22-23 cause ataxia-telangiectasia, an autosomal recessive disorder that is associated with increased incidence of malignancy and, particularly, lymphoid tumors. A role for ATM in the development of sporadic T-cell chronic leukemias is supported by the finding of loss of heterozygosity at 11q22-23 and ATM mutations in leukemias carrying TCL-1 rearrangements. Approximately 14% of B-cell chronic lymphocytic leukemia (B-CLL), the most common adult leukemia, carry deletions of the long arm of chromosome 11 at 11q22-23. Loss of heterozygosity at 11q22-23 and, more recently, absence of ATM protein, have been associated with poor prognosis in B-CLL. To determine whether the ATM gene is altered in B-CLL, we have sequenced individual ATM exons in six B-CLL cases. We show that the ATM gene is mutated in a fraction of B-CLLs and that mutations can be present in the germ line of patients, suggesting that ATM heterozygotes may be predisposed to B-CLL.

Deficiency of the ATM protein expression defines an aggressive subgroup of B-cell chronic lymphocytic leukemia.

The gene mutated in ataxia telangiectasia, ATM, on human chromosome 11q22-q23 is implicated in cell cycle control and DNA repair. Ataxia telangiectasia patients as well as ATM-deficient mice are immune deficient and develop lymphoproliferative disease. Abnormalities in 11q22.3-q23.1 have also been described in B-cell chronic lymphocytic leukemia (B-CLL). We analyzed B-CLL samples for loss of heterozygosity (LOH) using microsatellite markers located at the ATM (D11S2179), mixed-lineage leukemia (MLL; D11S1356), and BCL1 (D11S987) loci, all of which are located around 11q23. Five (14%) of 36 informative cases showed LOH at the ATM gene, and two of these five cases had LOH at the MLL gene. No LOH was detected at the BCL1 locus, and none of the cases showed LOH at the MLL gene without LOH at the ATM gene. Four of these five cases with LOH at the ATM gene were studied for ATM protein expression by Western blot analysis. All four cases lacked ATM protein. An additional 111 cases of B-CLL were studied for expression of ATM protein by Western blot analysis and RIA. Thirty-eight (34%) of these cases showed ATM levels <50% of that seen in normal lymphoid cells. No morphological or immunophenotypic difference was observed between ATM-deficient B-CLL cases and cases with normal ATM expression. However, patients with ATM deficiency had significantly shorter survival times (35.66 versus 97.3 months; P = 0.003) and more aggressive disease, suggesting that ATM is involved in the leukemogenesis of B-CLL. These data also suggest that the ATM gene may play a role in the reported 11q23 abnormality in B-CLL, which also characterizes an aggressive disease.

Autocrine interleukin-1beta production in leukemia: evidence for the involvement of mutated RAS.

Interleukin (IL)-1beta is constitutively expressed in many leukemias and operates as an autocrine growth factor. To study the cellular basis for this aberrant production, we analyzed two cell lines, B1 (acute lymphoblastic leukemia) and W1 (juvenile chronic myelogenous leukemia), which express high levels of IL-1beta and have mutations in the K-RAS and N-RAS genes, respectively. Electromobility shift assays demonstrated transcription factor binding at multiple IL-1beta promoter elements [nuclear factor (NF)-IL6/CREB, NFB1, NFkappaB, and NF-IL6], consistent with the activation of an upstream signaling pathway. To determine whether activated Ras was involved, two structurally distinct classes of farnesyltransferase (FTase) inhibitors (the monoterpenes and a peptidomimetic) and an adenoviral vector expressing antisense targeted to K-RAS were used to specifically interfere with Ras function and/or expression. Treatment with the FTase inhibitors resulted in a concentration-dependent decrease in both NF-IL6/CREB binding to the IL-1beta promoter and IL-1beta protein levels, without a significant change in total cellular protein levels. Furthermore, exposure of the B1 cells to antisense against K-RAS resulted in an approximately 50% reduction in both p21Ras and IL-1beta protein levels. Growth suppression was observed after FTase inhibitor or antisense exposure, an effect that was partially reversible by the addition of recombinant IL-1beta to the cultures. Our observations suggest that mutated RAS genes may mediate autocrine IL-1beta production in some leukemias by stimulating signal transduction pathways that activate the IL-1beta promoter.

Positive selection and high sensitivity test for MYD88 mutations using locked nucleic acid.

INTRODUCTION: Detection of mutations in the myeloid differentiation primary response gene 88 (MYD88) has clinical implications on diagnosis and therapy, especially in patients with Waldenström's macroglobulinemia (WM) and IgM monoclonal gammopathy of unknown significance (IgM-MGUS). We describe a method that provides greatly increased sensitivity for detecting minority mutations in MYD88. METHODS: We used a locked nucleic acid oligonucleotide to block amplification of wild-type DNA during polymerase chain reaction (PCR). Sanger sequencing of amplified DNA was used for detecting mutations in MYD88 gene. This approach was used to test samples from patients with WM and IgM-MGUS. RESULTS: When compared to traditional PCR followed by Sanger sequencing, our methodology was significantly more sensitive (one mutant allele in a background of 200 wild-type alleles). Using sequencing allowed us to visualize the PCR product, giving advantages over other methodologies such as allele-specific PCR. Based on analyzing 36 randomly selected, MYD88 mutated, clinically tested samples, we demonstrate that traditional PCR failed to detect MYD88 mutations in 64% of the samples that were clearly positive by wild-type blocking PCR. CONCLUSION: The new methodology is essential for attaining accurate results in clinical testing.

Evaluation of tyrosinase mRNA as a tumor marker in the blood of melanoma patients.

PURPOSE: The value of tyrosinase messenger RNA (mRNA) detection in the peripheral blood by reverse-transcription polymerase chain reaction (RT-PCR) as a melanoma marker remains controversial. The purpose of this study was to compare the sensitivities of two different blood processing techniques for tyrosinase mRNA detection and evaluate its potential clinical value. METHODS: A total of 50 patients with progressive stage IV melanoma was studied. Two blood processing methods were used: RNA extraction from the whole blood and RNA extraction from density gradient-isolated peripheral-blood mononuclear cells (PBMC). The RNA samples were tested with a sensitive nested-primer RT-PCR assay. RT-PCR results were also correlated with serum lactate dehydrogenase (LDH), treatment status, and presence of visceral versus nonvisceral metastases. RESULTS: Thirteen (26%) of the density gradient and five (10%) of the whole blood processed samples were PCR positive (P = .011). Serum LDH levels were found to be significantly higher in PCR-positive PBMC-processed patients (P = .015). There was no significant difference in the detection rates between visceral versus nonvisceral metastases or between prior treatment versus no prior treatment. CONCLUSION: Using a density gradient method to process the blood samples resulted in a higher detection rate of tyrosinase mRNA than extracting the RNA from the whole blood. However, the relatively low sensitivity in patients with disseminated and progressive disease compared with other reports suggests that tyrosinase mRNA may be of limited value in the management of malignant melanoma.

Hyper-CVAD program in Burkitt's-type adult acute lymphoblastic leukemia.

PURPOSE: To evaluate response and outcome with a front-line intensive multiagent chemotherapy regimen in adults with Burkitt's-type acute lymphoblastic leukemia (B-ALL). PATIENTS AND METHODS: From September 1992 to June 1997, 26 consecutive adults with newly diagnosed untreated B-ALL received hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (Hyper-CVAD). Their median age was 58 years (range, 17 to 79 years), and 46% were > or = 60 years. Patients received Hyper-CVAD alternated with courses of high-dose methotrexate and cytarabine. Granulocyte colony-stimulating factor and prophylactic antibiotics were administered for all eight planned courses. CNS prophylaxis alternated intrathecal methotrexate and cytarabine on days 2 and 7 of each course. RESULTS: Complete remission (CR) was obtained in 21 patients (81%). There were five induction deaths (19%). The median time to CR was 22 days (range, 15 to 89 days); 70% achieved CR within 4 weeks. The 3-year survival rate was 49% (+/- 11%); the 3-year continuous CR rate was 61% (+/- 11%). Twelve CR patients (57%) were in continuous CR at a median follow-up of 3+ years (range, 13+ months to 6.5+ years). Characteristics predicting for worse survival were age > or = 60 years, poor performance status, anemia, thrombocytopenia, peripheral blasts, and increased lactate dehydrogenase level. The 3-year survival rate was 77% for 14 patients younger than 60 years and 17% for 12 patients > or = 60 years (P <.01). Regression analysis identified older age, anemia, and presence of peripheral blasts as independent factors associated with shorter survival. Patients could be stratified according to (1) no or one adverse feature, (2) two adverse features, and (3) all adverse features. The 3-year survival rates were 89%, 47%, and 0%, respectively (P <.01). CONCLUSION: Hyper-CVAD is effective in adult B-ALL. Identification of patients with high risk for relapse and improved methods to detect residual disease may result in risk-oriented approaches.

Significance of the levels of bone marrow lymphoid infiltrate in chronic lymphocytic leukemia patients with nodular partial remission.

Patients with chronic lymphocytic leukemia (CLL) are considered in nodular partial remission (nPR) when they are in remission but bone marrow biopsies show rare nodules. The significance of the level of residual disease in nPR is not known. We studied 91 previously untreated CLL patients who were treated with fludarabine alone, fludarabine with prednisone, or fludarabine with cyclophosphamide and achieved nPR at the end of six courses. We compared bone marrow lymphoid infiltration before therapy and at the end of three and six courses of therapy as evaluated by a pathologist in retrospective fashion with that of the routine evaluation at the time of performing bone marrow biopsy. We then compared these results with those obtained by computer-aided histomorphometry in 28 patients in nPR. There was significant correlation (P < 0.05) between pathologists as well as between pathologists and histomorphometry. Upon correlation with clinical characteristics, there was significant correlation (P 0.01) between marrow involvement before therapy and white blood cell counts (wbc), hemoglobin (hgb), absolute lymphocyte counts, and beta2-microglobulin (beta2-m) but none of these parameters correlated with the lymphoid infiltrate at the end of three or six courses of therapy. more importantly, lymphoid infiltration after three and six courses did not correlate with time to progression (ttp) or overall survival (os). however, patients with >70% marrow involvement before therapy had a significantly shorter TTP (P = 0.02). All 91 patients showed similar results. However, we found reverse correlation between marrow lymphoid infiltrate at the end of three courses and OS (P = 0.01).

Emergence of new clonal abnormalities following interferon-alpha induced complete cytogenetic response in patients with chronic myeloid leukemia: report of three cases.

New treatments which may change the course of a disease, or which have potential carcinogenicity, may result in the development of new cytogenetic or clinical disorders. Three patients with Philadelphia chromosome-positive (Ph-positive) chronic myeloid leukemia (CML) who developed new cytogenetic abnormalities after achieving a cytogenetic complete remission (CR) of their Ph-positive disease with interferon alpha (IFN-alpha) based therapy are described. Patient 1 developed chromosomal abnormalities involving chromosomes 5 (5q13-34) and later 7 (monosomy 7) 60 months after the start of therapy and 20 months after IFN-alpha was discontinued. A myelodysplastic syndrome was noted 83 months from the start of therapy. Patient 2 developed a myeloproliferative syndrome with 18p11 chromosomal abnormalities 90 months after the start of the therapy and 60 months after IFN-alpha was discontinued. Patient 3 developed a chromosome 11 abnormality (11q21-23) 23 months after the start of therapy, without hematological manifestations. All three patients remain in cytogenetic CR of Ph-positive disease with the hypermetaphase fluorescent in situ hybridization and polymerase chain reaction studies for BCR/ABL showing minimal residual disease. The emergence of new cytogenetic or clinical disorders in patients with CML on IFN-alpha therapy needs to be monitored. These findings may be related to changing the natural course of CML, to therapy, or to the emergence of suppressed clones in a stem cell disorder.

Acute lymphoblastic leukemia following preleukemic syndromes in adults.

Preleukemic syndromes (PLS) evolve to acute myeloid leukemia (AML) in 15-50% of cases, but rarely transform to acute lymphoblastic leukemia (ALL). AML following preleukemic syndromes has a poor prognosis, but little is reported about the outcome of secondary ALL. From the adult leukemia database at MD Anderson Cancer Center, nine patients with ALL following myelodysplastic syndrome (MDS) (n=6), smoldering leukemia (n=1), or cytopenias with dysplastic features (n=2) were identified. Clinical and laboratory features were abstracted from the database, patient charts, review of the bone marrows and special laboratory studies. The median interval from diagnosis of PLS to ALL was 6 months. Blasts with lymphoid morphology and terminal deoxynucleotidyltransferase (TdT) and periodic acid Schiff (PAS) staining were present in eight of nine cases and four of six cases respectively. T cell and myeloid markers were expressed in the majority of cases, but T cell gene rearrangements were not detected. Only one patient had electron microscopic myeloperoxidase-positive staining. Two patients later transformed to AML. Patients were predominantly male (89%) with a median age of 69 years. The complete remission (CR) rate with ALL-directed chemotherapy (78%) was comparable to that of other adult ALL patients (74%) (n=327) without excess myelosuppression. Marrow dysplasia persisted in CR in three of seven cases. The median remission duration of 16 months (range 4.5 to 51+ months) and the median overall survival of 21 months (range 1 to 55+ months) were comparable to that of ALL patients overall. ALL following preleukemic syndromes is a distinct syndrome with T cell and myeloid markers and responds well to ALL-directed therapy. The presence of myeloid and lymphoid markers suggests the transformation of an early stem cell.

Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical significance and therapeutic implications.

Mitochondrial DNA (mtDNA) codes for 13 respiratory chain subunits and is more vulnerable to damage than nuclear DNA due, in part, to a lack of histone protection and a weak repair capacity. While mtDNA alterations have been observed in human cancer, their roles in oncogenesis and chemosensitivity remain unclear. We investigated the relationship between mtDNA mutations, reactive oxygen species (ROS) generation, and clinical outcomes in chronic lymphocytic leukemia (CLL) patients. An analysis of mtDNA from 20 CLL patients revealed that primary CLL cells from patients with prior chemotherapy had a significantly higher frequency of heteroplasmic mutations than did those from untreated patients. Overall, mtDNA mutations appeared to be associated with increased ROS generation. Patients refractory to conventional therapeutic agents tended to have higher mutation rates than patients who responded to treatment. Analysis of paired blood samples from the same patient led to the identification of a heteroplasmic mutation in the cytochrome c oxidase II gene several months after chemotherapy. The mutation was associated with increased ROS generation. Our results suggest for the first time that chemotherapy with DNA-damaging agents may cause mtDNA mutations in primary leukemia cells, which often exist in heteroplasmy, and are associated with increased ROS generation.

More cell death in refractory anemia with excess blasts in transformation than in acute myeloid leukemia.

Refractory anemia with excess blasts in transformation (RAEB-T) is a subgroup of myelodysplastic syndrome (MDS) in which the bone marrow blast count ranges from 20% to 30%. The recently proposed World Health Organization Classification of Hematologic Malignancies eliminated this category from MDS by lowering the blast count cutoff for acute myeloid leukemia (AML) from 30% to 20%. However, MDS is distinguished from AML by a significant increase in apoptosis. To investigate the difference in apoptosis between RAEB-T, AML, and other categories of MDS, we prospectively analyzed fresh bone marrow samples using the Annexin V and mitochondrial potential assays. There was a significantly higher level of apoptosis in RAEB-T than in AML according to both assays, while no significant differences between RAEB-T and other categories of MDS were noted. The data suggest that RAEB-T is more likely to be an advanced stage of MDS and biologically different from AML.

Expression profile of 11 proteins and their prognostic significance in patients with chronic lymphocytic leukemia (CLL).

It has been suggested that the expansion of the leukemic cells in chronic lymphocytic leukemia (CLL) is due to dysregulation of pathways of programmed cell death (apoptosis) rather than cell proliferation, although differences may exist in early vs late and treated vs untreated patients. In the present study, we analyzed the expression of 11 proteins in CLL cells that are implicated in the control of apoptosis, proliferation, and differentiation, and correlated this expression profile with survival. Using a quantitative solid-phase radioimmunoassay (RIA), we measured the cellular protein levels of Bcl-2, cyclin D1, PCNA, ATM, Fas, Bax, retinoic acid receptor alpha (RARalpha), retinoic acid receptor beta (RXRbeta), Flt1, VEGF, and cellular beta2-microglobulin in 230 samples of CLL. Univariate analysis using the Cox proportional hazard model showed a correlation with survival of only the following proteins: Bcl-2 (P < 0.001), cyclin D1 (P = 0.027), Fas (P = 0.055), PCNA (P < 0.001), and ATM (P = 0.028). In a multivariate analysis using classification and regression tree analysis (CART), five groups of patients (nodes) could be generated with significant differences of survival expectation (P < 0.0001) based on levels of expression of the above proteins. Based on CART analysis, Bcl-2 levels emerge as the most important protein in predicting survival between all 11 proteins studied. Patients with marked elevation in Bcl-2 levels had the worst outcome while patients with intermediate levels, but with high levels of PCNA and cyclin D1 or abnormal ATM expression had intermediate survival. These data indicate that intracellular levels of proteins such as Bcl-2, ATM, cyclin D1, and PCNA can be used as markers to predict clinical behavior and survival in patients with CLL. The pathways in which these proteins are involved may also represent possible targets for future therapeutic trials in CLL.

Better detection of FLT3 internal tandem duplication using peripheral blood plasma DNA.

Somatic mutation of the FLT3 gene as an internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence causes constitutive tyrosine phosphorylation and activation. Tumor-specific DNA has been documented in the sera of patients with solid tumors even when it is in an early stage. We compared the detection of FLT3 ITD in DNA extracted from cells of bone marrow (BM) aspirations with DNA extracted from peripheral blood (PB) plasma in patients newly diagnosed with acute myeloid leukemia (AML; 85 patients), myelodysplastic syndrome (MDS; 16 patients), and acute lymphocytic leukemia (ALL; 16 patients). FLT3 ITD was detected in 18 (21%) AML samples and in one (6%) MDS sample in both cellular and plasma DNA but in none of the ALL samples. Hemizygous/homozygous FLT3 ITD was detected in five (28%) of the FLT3 ITD-positive AML using plasma DNA, whereas only four of these cases showed hemizygous/homozygous FLT3 ITD using cellular DNA. The presence of FLT3 ITD was associated with significantly shorter survival (P = 0.02) when only patients younger than 50 years of age (48 AML+MDS patients) were considered. This finding was independent of cytogenetics in this age group. However, patients with the FLT3 ITD hemizygous/homozygous phenotype had even shorter survival (P = <0.001). As expected, the presence of FLT3 ITD correlated with higher white blood cell (WBC) counts. These data demonstrate that plasma DNA is a reliable alternative resource for detecting FLT3ITD, especially the hemizygous/homozygous genotype. Furthermore, the data derived from this study support the notion that the presence of FLT3 ITD in conjunction with the absence of the wild-type FLT3 allele predicts an especially poor prognosis for patients with AML.

Analysis of the BCR-ABL protein in Philadelphia chromosome-positive adult acute lymphocytic leukemia.

Philadelphia chromosome (Ph)-positive acute lymphocytic leukemia (ALL) constitutes 15-35% of all ALL in adults. Its detection is prognostically significant. The Ph abnormality is usually detected through standard cytogenetic analysis but 20-30% of patients have insufficient metaphases (IM) with such analysis. To detect the BCR-ABL oncoprotein in peripheral blood specimen of patients with ALL at the time of diagnosis and at follow-up, a new sensitive technique of enhanced chemiluminescence Western blot (ECL-WB) analysis was investigated. Among 41 patients with newly diagnosed ALL, nine were Ph positive by cytogenetic studies; they were also BCR-ABL positive according to ECL-WB. Eight had p190 disease, and one had p210 disease. Among the 16 patients with IM, none demonstrated the oncoprotein through ECL-WB or through simultaneous Southern blot (SB) for p210 rearrangement. Follow-up studies were available for seven patients: four had detectable protein and three of them relapsed 4-20 weeks later; three had undetectable protein and one of them (who had low level protein at the time of diagnosis) relapsed 11 weeks later. Although none of the patients with IM at diagnosis had detectable protein according to ECL-WB, this was probably due to the small number of patients studied. One patient with IM studied at follow-up demonstrated the protein by ECL-WB. In summary, we describe a technique that is useful in the detection of p190/p210 ALL at diagnosis. It is less time consuming, and more cost effective than standard chromosome banding techniques. It may also detect the oncoprotein in cases with IM. Although a larger number of patients should be studied to prove its clinical usefulness, this technique may also be of value for monitoring residual disease at follow-up.