Search

Roughly one-third of acute myeloid leukemia (AML) patients exhibit mutations in the nucleophosmin (NPM1) gene, and multiple studies have linked these mutations with a more favorable clinical outcome. We developed an assay for the detection of NPM1 mutations in peripheral blood plasma, and compared the results with clinical outcomes from a single institution. Analyzing plasma from previously untreated AML patients revealed NPM1 insertion mutations in 24 of 98 (24%) patients, with greater sensitivity than existing peripheral blood cell-based tests which showed positivity in only 22 of the 24 patients. Plasma testing allowed the detection of a novel 4 bp deletion in NPM1 in one patient. Analysis of clinical data corroborated previous data linking NPM1 mutations with better clinical outcome. These data underline the significance of NPM1 in the biology and clinical behavior of AML, and demonstrate the reliability and efficacy of plasma-based testing for NPM1 mutations.

Rare cases of chronic myelogenous leukemia (CML) express high levels of alternatively spliced BCR-ABL mRNA with a 35-bp insertion (35INS) between ABL kinase domain exons 8 and 9. This insertion results in a frameshift leading to the addition of 10 residues and truncation of 653 residues due to early termination. Sensitive PCR-based testing showed that 32 of 52 (62%) imatinib-resistant CML patients in chronic phase and 8 of 38 (21%) in accelerated or blast crisis expressed varying levels of the alternatively spliced BCR-ABL mRNA. A three-dimensional structural model of the 35INS ABL kinase domain complexed with imatinib was built using homology modeling, followed by molecular dynamics simulations. Simulation results showed that the new residues cause a significant global conformational change, altering imatinib binding in a way similar to that of the T315I mutation and, therefore, providing resistance to imatinib that depends on the level of expression. [Mol Cancer Ther 2008;7(12):3834–41]

Background: Current reference methods for evaluating gene amplification and expression of ERBB2 (also known as HER-2)—cell-based fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC)—are subjective and influenced by methods of tissue preparation and fixation. We developed and evaluated a novel, quantitative liquid-based FISH (L-FISH) assay that uses flow cytometry to detect ERBB2 gene amplification in breast cancer patients.Methods: DNA was extracted from serum or tissue, biotinylated, hybridized to differentially labeled probes for ERBB2 and a chromosome 17–specific single-copy sequence (17-SSC), and immobilized to streptavidin-coated microspheres. The ERBB2/17-SSC signal ratio measured by flow cytometry was used to evaluate ERBB2 amplification. We used L-FISH to test 122 stored formalin-fixed, paraffin-embedded (FFPE) tissue samples and 22 serum samples from randomly selected breast cancer patients; results were compared with those obtained with conventional FISH and IHC.Results: The inter- and intraassay imprecisions were 3.7%–18.9% for FFPE tissue and 2.8%–6.3% for serum. Overall, L-FISH analyses of FFPE tissues demonstrated 84.4% concordance with results obtained with conventional FISH (P < 0.001) and 78.8% concordance with IHC results (P < 0.001). L-FISH analyses of serum samples showed 91% concordance with tissue-based IHC/FISH results (P = 0.038).Conclusions: Our data indicate that this PCR-free L-FISH method can be used to evaluate ERBB2 amplification in both cell-containing (paraffin-embedded tissue) and cell-free (serum) samples. This approach provides more objective results and is amenable to automation and quantitative measurement.

BACKGROUND. Tumor necrosis factor (TNF)-α and other cytokines are involved in the pathogenesis of acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS), but their prognostic significance in these diseases is unknown. In the current study, the authors assessed the association between serum levels of various cytokines and clinical outcomes in patients with untreated AML or high-risk MDS. METHODS. Serum levels of TNF-α, interleukin (IL)-1 receptor antagonist, IL-6, IL-10, and endostatin were measured in patients with AML or high-risk MDS who presented for treatment at The University of Texas M. D. Anderson Cancer Center from September 1994 through January 2001. Univariate and multivariate analyses were performed to test for correlations with clinical outcomes. RESULTS. Higher TNF-α levels were found to correlate with poorer performance status; higher leukocyte counts; higher levels of β2-microglobulin, creatinine, uric acid, and alkaline phosphatase; lower levels of creatinine clearance and albumin; baseline infection; and M4-M5 AML subtypes. TNF-α levels

We have developed a simplified sandwich immunoassay to measure free circulating total and phosphorylated fusion BCR-ABL protein in patients with the t(9;22)(q34;q11) chromosomal translocation. The assay is based on immunoprecipitating BCR-ABL protein using beads coated with anti-BCR antibody and detecting the fusion protein with anti-ABL antibody and flow cytometry. We show that this method allows the quantification of this protein in the plasma and may allow the measurement of tumor load. This method also allows the measurement of the level of phosphorylation of the immunoprecipitated BCR-ABL using antibodies against phosphorylated ABL protein, which can be used for monitoring of therapy with kinase inhibitors. The sensitivity of this immunoassay was comparable to the sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) assay. This technique is useful in monitoring patients with chronic myeloid leukemia (CML) or acute lymphoblastic leukemia (ALL), but the same approach can be used in other translocations and has the potential of multiplexing.

Purpose: Deforolimus (AP23573), a novel non-prodrug rapamycin analogue, inhibits the mammalian target of rapamycin, a downstream effector of the phosphatidylinositol 3-kinase/Akt and nutrient-sensing pathways. A phase 2 trial was conducted to determine the efficacy and safety of single-agent deforolimus in patients with relapsed or refractory hematologic malignancies. Experimental Design: Eligible patients were assigned to one of five disease-specific, parallel cohorts and given 12.5 mg deforolimus as a 30-minute infusion once daily for 5 days every 2 weeks. A Simon two-stage design was used for each cohort. Safety, pharmacokinetics, pharmacodynamics, and antitumor response were assessed. Results: Fifty-five patients received deforolimus as follows: cohort 1 23 acute myelogenous leukemia, two myelodysplastic syndrome and one chronic myelogenous leukemia in nonlymphoid blast phase; cohort 2, one acute lymphocytic leukemia; cohort 3, nine agnogenic myeloid metaplasia; cohort 4, eight chronic lymphocytic leukemia; cohort 5, nine mantle cell lymphoma and two T-cell leukemia/lymphoma. Most patients were heavily pretreated. Of the 52 evaluable patients, partial responses were noted in five (10%), two of seven agnogenic myeloid metaplasia and three of nine mantle cell lymphoma. Hematologic improvement/stable disease was observed in 21 (40%). Common treatment-related adverse events, which were generally mild and reversible, were mouth sores, fatigue, nausea, and thrombocytopenia. Decreased levels of phosphorylated 4E-BP1 in 9 of 11 acute myelogenous leukemia/myelodysplastic syndrome patients after therapy showed mammalian target of rapamycin inhibition by deforolimus. Conclusions: Deforolimus was well-tolerated in patients with heavily pretreated hematologic malignancies, and antitumor activity was observed. Further investigation of deforolimus alone and in combination with other therapeutic agents is warranted in patients with selected hematologic malignancies.

BACKGROUND Computational simulations have become powerful tools for understanding detailed interactions in biologic systems. To the authors' knowledge to date, the mechanism of imatinib resistance in BCR-ABL has not been clarified at the atomic level, and computational studies are required. METHODS Molecular dynamics (MD) simulations on the complex of imatinib with the wild-type, T315I mutant, and 10 other P-loop mutants of the tyrosine kinase BCR-ABL were performed to study the mechanism of imatinib resistance. RESULTS Simulations suggested that imatinib resistance of T315I results mainly comes from the breakdown of interactions between imatinib and both E286 and M290, contradictory to what was believed previously, in that the missing hydrogen bonding is the main contribution. The current results also demonstrated that the unfavorable electrostatic interaction between P-loop and imatinib is the main reason for resistance for the P-loop mutations. Furthermore, in Y253H, protonation of the histidine at the e position is essential for rendering this mutation resistant to imatinib. CONCLUSIONS The current results indicated that large-scale simulations may offer insight and information that other simple modeling methods cannot provide regarding the problem of BCR-ABL imatinib resistance, especially in the case of conformational changes because of remote mutations. Imatinib resistance mechanisms that were not anticipated previously were revealed by analyzing the interactions between imatinib and individual residues based on simulation results. This results demonstrated that MD is a powerful way to verify and predict the clinical response or resistance to imatinib and to other potential drugs. Cancer 2008. © 2008 American Cancer Society.

Recent advances in molecular biology have paved the way for the detection of minute quantities of cellular components with robust assays that are amenable for use in clinical laboratories. This review discusses the recently developed series of plasma-based assays that are changing the testing paradigm for hematologic diseases. These tests are based on the concept that a high turnover of neoplastic hematologic cells, relative to normal cells, enriches plasma with tumor-specific DNA, RNA and protein. Plasma-based testing promises to reduce the need for bone marrow biopsy, allow for more frequent and accurate monitoring of changes in bone marrow, allow the detection of more aggressive subclones of the malignant cells and provide a more quantitative means to measure the load of the malignant clone. We present data demonstrating that plasma, in some situations, allows even more sensitive detection than bone marrow cells. Moreover, the lessened impact of dilution by normal cells in plasma permits a distinction between homozygous and hemizygous abnormalities. Unlike solid tumors, currently available data suggest that in hematologic diseases, plasma is superior to cells in detecting molecular abnormalities.

Angiogenesis is part of the pathophysiology of myelofibrosis with myeloid metaplasia (MMM). PTK787/ZK 222584 (PTK/ZK) is a novel inhibitor of vascular endothelial growth factor receptors. Twenty-nine patients with MMM received a continuous dosing schedule of PTK/ZK doses of 500 or 750 mg twice daily (BID). Transient potentially PTK/ZK related mild nausea, vomiting, dizziness, fatigue, thrombocytopenia, or anorexia occurred in 15% of patients. Dose limiting toxicities of dyspepsia, proteinurea, and/or mucositis were observed in patients treated with 750 mg BID. One (3%) and five (17%) patients achieved complete remission and clinical improvement, respectively. PTK/ZK has modest activity in patients with MMM.

Summary The haematopoietic cell transplantation comorbidity index (HCTCI) predicts nonrelapse mortality and overall survival (OS) post-stem cell transplantation. HCTCI scores were assessed in 177 patients over 60 years of age receiving acute myeloid leukaemia (AML) induction therapy. HCTCI score was 0 in 22% of patients, 1–2 in 30%, and ≥3 in 48%. In patients with scores of 0, 1–2, or ≥3, early death rates were 3%, 11% and 29% (P

The V617F mutation of the JAK2 tyrosine kinase is found in a majority of patients with myeloproliferative disorders. Flow cytometry assays for quantitation of phosphorylated and total protein for JAK2, STAT5, and heat shock proteins (HSPs) were developed to facilitate the study of the JAK/STAT pathway. A cell line homozygous for V617F (HEL) was treated with inhibitors of JAK2 tyrosine kinase activity and the HSP90 inhibitor 17-AAG. 17-AAG reduced HSP90 levels, but increased HSP70 levels. Phospho-STAT5, total STAT5, and total AKT levels were also reduced by 17-AAG treatment. Further, phospho-JAK2, total JAK2, and cell viability were reduced to a greater extent by 17-AAG than by the pan-JAK kinase family inhibitor JKII or the JAK2-specific inhibitor AG490, and these inhibitors failed to synergize with 17-AAG. Flow-cytometry-based assays for JAK/STAT signaling pathway and HSPs are likely to have broad clinical utility for monitoring patients with abnormalities in the JAK2 pathway.

Purpose: Everolimus (RAD001, Novartis), an oral derivative of rapamycin, inhibits the mammalian target of rapamycin (mTOR), which regulates many aspects of cell growth and division. A phase I/II study was done to determine safety and efficacy of everolimus in patients with relapsed or refractory hematologic malignancies. Experimental Design: Two dose levels (5 and 10 mg orally once daily continuously) were evaluated in the phase I portion of this study to determine the maximum tolerated dose of everolimus to be used in the phase II study. Results: Twenty-seven patients (9 acute myelogenous leukemia, 5 myelodysplastic syndrome, 6 B-chronic lymphocytic leukemia, 4 mantle cell lymphoma, 1 myelofibrosis, 1 natural killer cell/T-cell leukemia, and 1 T-cell prolymphocytic leukemia) received everolimus. No dose-limiting toxicities were observed. Grade 3 potentially drug-related toxicities included hyperglycemia (22%), hypophosphatemia (7%), fatigue (7%), anorexia (4%), and diarrhea (4%). One patient developed a cutaneous leukocytoclastic vasculitis requiring a skin graft. One patient with refractory anemia with excess blasts achieved a major platelet response of over 3-month duration. A second patient with refractory anemia with excess blasts showed a minor platelet response of 25-day duration. Phosphorylation of downstream targets of mTOR, eukaryotic initiation factor 4E-binding protein 1, and/or, p70 S6 kinase, was inhibited in six of nine patient samples, including those from the patient with a major platelet response. Conclusions: Everolimus is well tolerated at a daily dose of 10 mg daily and may have activity in patients with myelodysplastic syndrome. Studies of everolimus in combination with therapeutic agents directed against other components of the phosphatidylinositol 3-kinase/Akt/mTOR pathway are warranted.

Purpose: LBH589 is a novel histone deacetylase inhibitor that inhibits proliferation and induces apoptosis in tumor cell lines. In this phase I study, LBH589 was administered i.v. as a 30-minute infusion on days 1 to 7 of a 21-day cycle. Experimental Design: Fifteen patients (median age, 63 years; range, 42-87 years) with acute myeloid leukemia (13 patients), acute lymphocytic leukemia (1 patient), or myelodysplastic syndrome (1 patient) were treated with LBH589 at the following dose levels (mg/m2): 4.8 (3 patients), 7.2 (3 patients), 9.0 (1 patient), 11.5 (3 patient), and 14.0 (5 patients). The levels of histone acetylation were measured using quantitative flow cytometry and plasma LBH589 concentrations were assayed. Results: Four dose-limiting toxicities (grade 3 QTcF prolongation) were observed, four at 14.0 mg/m2 and one at 11.5 mg/m2. QTcF prolongation was asymptomatic and reversed on LBH589 discontinuation. Other potentially LBH589-related toxicities included nausea (40%), diarrhea (33%), vomiting (33%), hypokalemia (27%), loss of appetite (13%), and thrombocytopenia (13%). In 8 of 11 patients with peripheral blasts, transient reductions occurred with a rebound following the 7-day treatment period. H3 acetylation increase was significant in B-cells (CD19+; P = 0.02) and blasts (CD34+; P = 0.04). The increase in H2B acetylation was highest in CD19+ and CD34+ cells [3.8-fold (P = 0.01) and 4.4-fold (P = 0.03), respectively]. The median acetylation of histones H2B and H3 in CD34+ and CD19+ cells significantly increased on therapy as did apoptosis in CD14+ cells. Area under the curve increased proportionally with dose with a terminal half-life of ∼11 hours. Conclusion: Intravenous administration of LBH589 was well tolerated at doses

We investigated the association of plasma thrombopoietin (TPO) and overall survival in 127 patients with previously treated and previously untreated chronic lymphocytic leukemia (CLL). Higher levels of TPO were associated with advanced Rai stage (P < .001), higher levels of β2-microglobulin (β2-M) (P < .001), and the absence of mutation in the immunoglobulin heavy chain variable region (IgVH) (P < .001), and were inversely correlated with platelet count (P = .002). We found that TPO correlated strongly in a continuous manner with overall survival in both previously treated and untreated patients. The univariate Cox proportional hazard model demonstrated that high TPO levels were associated with shorter survival (P < .001), and multiple variable Cox proportional hazards regression analysis demonstrated that this was independent of the IgVH mutation status, β2-M, and Rai stage. Recursive partitioning showed that a cutoff point of 639 pg/mL separated the CLL patients into 2 major survival groups (P < .001). The effects of β2-M were masked by the effects of TPO in the patients with TPO levels higher than 639 pg/mL, but in the remainder, patients with β2-M level higher than 4.95 mg/L had significantly shorter survival than those with lower values. Plasma TPO and β2-M may be useful for the prediction of clinical behavior in CLL and may replace the need for the determination of IgVH mutation status.

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.

Angiogenesis plays an important role in solid tumors and hematologic malignancies. The prognostic significance of angiogenic factors in adult acute lymphoblastic leukemia (ALL) remains ambiguous. We therefore analyzed the impact of angiogenic factor levels on overall survival of newly diagnosed adult ALL patients. Plasma levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-1 receptor alpha (IL-1Rα), IL-6, IL-8, VEGF receptors VEGFR1 and VEGFR2, and thrombopoietin (TPO) were measured in plasma samples of 95 patients by enzyme-linked immunosorbent assay (ELISA). In a univariate Cox proportional hazards model, higher levels of IL-1Rα, IL-8, VEGFR1, and VEGFR2 were predictive of poor survival. In contrast, higher levels of VEGF were predictive of longer survival, and higher levels of bFGF suggested a similar trend (P = .09). The multivariate model simultaneously included VEGF (relative risk [RR] for death, 8.01; P = .001 for levels less than or equal to 19.5 pg/mL), IL-1Rα (RR, 5.12; P = .007 for levels greater than 373 pg/mL), and VEGFR2 (RR, 4.01; P = .04 for levels greater than 8222 pg/mL) as independent factors for survival. Of interest is the association of high levels of VEGF with good prognosis and higher levels of VEGF receptors with poor outcome. These data reflect the complexity by which angiogenic factors may affect the clinical behavior of patients with ALL, and this complexity should be considered in any therapeutic strategy incorporating antiangiogenic agents.

Purpose: Cloretazine (VNP40101M) is a novel sulfonylhydrazine alkylating agent with significant antileukemia activity. A phase I study of cloretazine combined with cytarabine (1-β-d-arabinofuranosylcytosine, ara-C) was conducted in patients with refractory disease. Design: Ara-C was given i.v. at a fixed dose of 1.5 gm/m2/d by continuous infusion for 4 days (patients ages Results: Forty patients, including 32 with acute myeloid leukemia, received 47 courses of treatment. Complete responses were seen at cloretazine dose levels of ≥400 mg/m2 in 10 of 37 (27%) evaluable patients, and in this patient subset, AGT activity was significantly lower in patients that responded to treatment than in patients who did not (P ≤ 0.027). Dose-limiting toxicities (gastrointestinal and myelosuppression) were seen with 500 and 600 mg/m2 of cloretazine combined with the 4-day ara-C schedule but not seen with the 3-day schedule. Conclusion: The recommended cloretazine dose schedule for future studies is 600 mg/m2 combined with 1.5 gm/m2/d continuous infusion of ara-C for 3 days. The cloretazine and ara-C regimen has significant antileukemic activity. AGT activity may be a predictor of response to cloretazine.

MoAb-based therapies are evolving into the first broad-spectrum class of targeted anti-leukemic therapy. Developments in many areas, including computer modeling of receptors and ligands, and increasing sophistication in recombinant technologies may result in a rapid increase in the number and complexity of MoAb's available. We can anticipate an increase in the number of safer conjugates being delivered to leukemia cells. Further understanding of the in vitro mechanisms involved in tumor cell killing by MoAb will be important in maximizing the efficacy of this approach.

Alemtuzumab (MabCampath) is a humanized rat monoclonal antibody that targets the CD52 antigen. It has been approved for the treatment of patients with resistant chronic lymphocytic leukaemia (CLL). Measuring plasma/serum levels of alemtuzumab is important for optimizing the dosing and scheduling of therapy; however, current assays in serum or plasma, based on the capture of alemtuzumab using CD52, are complicated and difficult to adapt for high throughput testing. We developed a simple sandwich enzyme-linked immunosorbent assay (ELISA) to measure alemtuzumab that takes advantage of the remaining rat sequence in alemtuzumab. Using specific anti-rat immunoglobulin (Ig) antibodies (absorbed against human Ig), alemtuzumab levels were measured in the serum and plasma of patients treated with alemtuzumab. Levels were similar between plasma and serum samples, in fresh samples and samples stored at 4 degrees C for 24 h, but were significantly lower in samples stored at room temperature for 24h. The assay was successfully used to determine serum alemtuzumab pre- and post-treatment. This assay is simple and adaptable for high throughput testing, with a limit of detection of 0.05 microg/ml and a coefficient of variation of +/-12.5%. No false positivity was observed in >200 samples tested. This validated assay should help optimize the dosing and scheduling of alemtuzumab therapy. The underlying principles are also applicable to the measurement of other humanized antibodies using an appropriate anti-Ig.

We hypothesize that c-kit expression may be associated with disease-specific features and have prognostic value in acute leukemias. In acute lymphocytic leukemia (ALL), higher levels of c-kit expression predicted lower complete response (CR) rates, suggesting that these patients may benefit from acute myelogenous leukemia (AML) therapy. Despite a negative association with the Philadelphia-chromosome, there was no correlation with disease-free survival (DFS) in CR. In AML, c-kit was associated with older age and cytogenetic abnormality t(-5, -7). Consequently higher levels of c-kit predicted lower CR rates. However, after accounting for these covariates, multivariate analysis indicates that higher c-kit expression predicts higher CR rates, although there was no effect on DFS in CR.

Alemtuzumab is effective in reducing the risk of acute graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (ASCT). Alemtuzumab may also delay immune reconstitution and reduce graft-versus-leukemia effects. The optimal dose has not been established. We investigated engraftment, acute GVHD incidence and severity, and pharmacokinetics of alemtuzumab associated with the use of low-dose alemtuzumab/cyclophosphamide/total body irradiation and ASCT for patients with aggressive CD52-positive hematologic malignancies. In all, 12 patients were treated. Alemtuzumab 10 mg daily on days -7 to -3 was given intravenously. Tacrolimus and methotrexate were used for GVHD prophylaxis. Alemtuzemab was not detected in any of the 36 sequential serum samples tested between days -1 and +21 of transplant. All patients engrafted rapidly; the median time to an absolute neutrophil count >0.5 x 10(9)/l was 14 days (range 11-17 days), and the median time to a platelet count >20 x 10(9)/l was 16 days (range 6-30 days). By 1 month after transplant, nine patients had 100% donor chimerism, while three had mixed donor chimerism. At 3 months, 11 had achieved 100% donor chimerism. No cases of grade III/IV acute GVHD occurred. At a median follow-up interval of 14.7 months (range 4-24), seven patients remained alive, and five remained free of disease.

BACKGROUND Patients with chronic lymphocytic leukemia (CLL) that transforms to Richter syndrome (RS) frequently show atypical lymphocytes in bone marrow; however, a diagnosis of RS requires confirmation of the presence of sheets of large cells in bone marrow or lymph nodes. METHODS In this study, the authors evaluated the clinical significance of scattered large cells in bone marrow. They assessed the possibility of predicting transformation to RS in bone marrow smears by counting the percentages of prolymphocytes and large cells in 78 patients with CLL and 29 patients with lymph node biopsy-confirmed RS. RESULTS The percentage of large cells was found to be correlated with decreasing survival in a continuous fashion (P = 0.006). It is interesting to note that patients who had > 7% large cells in the bone marrow and elevated β2-microglobulin (β2-M) levels (> 5 mg/L) had a survival duration identical to that of patients with RS, and these factors together were a strong predictor of RS (P < 0.001). CONCLUSIONS Patients with CLL who had bone marrow that contained > 7% large cells and who had β2-M levels > 5 mg/L had a disease that was similar to RS, and the combination of large cells and β2-M can be used as a surrogate marker for RS. Cancer 2004. © 2004 American Cancer Society.

Purpose: Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are of adverse prognostic significance in patients with multiple myeloma (MM). VEGF, a soluble circulating angiogenic molecule, acts via receptor tyrosine kinases, including VEGF receptor 2. SU5416 is a small molecule VEGF receptor 2 inhibitor. Experimental Design: Adult patients with advanced MM were entered on a multicenter phase II study. Results: Twenty-seven patients (median age 69, range 39–79), median 4 (0–10) lines of prior therapy, 14 with prior thalidomide therapy, received SU5416 at 145 mg/m2 twice weekly i.v. for a median of two 4-week cycles (range 0.2–9). Grade 3/4 toxicities were rarely observed; the most frequent was thrombocytopenia (12%). Mild-to-moderate toxicities included nausea (63%), headache (56%), diarrhea, vomiting (both 37%), and fatigue (33%). There were three thromboembolic episodes and five cases of new onset hypertension. Two (7%) patients did not complete the first 4-week cycle of therapy because of adverse events (pneumonia and headache). There were no objective responses. Four patients had disease stabilization for ≥4 months. A decrease in median VEGF plasma levels was observed in patients with stable disease (n = 7) compared with patients with progressive disease (n = 5). Overall median survival was 42 weeks (range 3–92+). Conclusions: Although SU5416 had minimal clinical activity, signs of biological activity (decrease in plasma VEGF levels) suggest that angiogenic modulation may be of value in patients with MM.

Summary. The cell surface proteins CD20 and CD52 differ significantly in their structures and are expressed on the majority of B cells. Both circulating CD20 (cCD20) and circulating CD52 (cCD52) have been recently documented in patients with chronic lymphocytic leukaemia. A retrospective study to establish whether cCD20 and/or cCD52 were detectable in patients with lymphoma, and the clinical associations of these soluble antigens if detected, was conducted. cCD20 and cCD52 levels were analysed in a cohort of 65 patients with non-Hodgkin's lymphoma (NHL) and 37 with Hodgkin's disease (HD). Patients with NHL had elevated pretherapy levels of cCD20 and cCD52 compared with normal individuals. Patients with HD had significantly lower than normal pretherapy levels of both cCD20 and cCD52. cCD20 levels were marginally elevated post-therapy in NHL patients while in patients with HD, cCD20 levels remained significantly lower than normal after therapy. Serum cCD52 levels became significantly lower than normal post-therapy in NHL patients, and remained significantly lower than normal in HD patients. No predictive effects were found for pretherapy or post-therapy levels of cCD52 on survival for either cohort of patients. Post-therapy cCD20 levels independently highly correlated with survival in patients with NHL. Prospective evaluation will be required to establish if cCD20 and cCD52 may be used as biomarkers in the diagnosis, prognostic categorization, and monitoring of the clinical course in patients with lymphoma. The clinical significance of circulating antigen in patients receiving monoclonal antibody therapy directed against CD20 and/or CD52 warrants study.

Thalidomide is a putative anti-angiogenesis agent that has significant anti-tumour activity in haematological malignancies with increased bone marrow angiogenesis, including multiple myeloma (MM) and myelodysplastic syndromes (MDS). Increased levels of the mitogen for angiogenesis, vascular endothelial growth factor (VEGF), correlate with worse survival in acute myeloid leukaemia (AML). A phase II trial of thalidomide was conducted in patients with relapsed- or refractory-AML previously treated with cytarabine-containing regimens. A total of 16 patients with refractory- or relapsed-AML were treated with thalidomide 200-800 mg orally daily (median dose 400 mg daily) for a median of 27 d (range, 3-94 d). Overall, one patient (6%) achieved complete remission (CR) lasting for 36 months, and two patients had a transient reduction in marrow blasts from 8% and 7% to less than 5% in both cases. There was no correlation between reduction in levels of angiogenesis markers and response. Toxicities related to thalidomide were significant, and precluded dose escalation beyond 400 mg orally daily in most patients. Although there appears to be some evidence of biological activity, single agent thalidomide is not an optimal choice of therapy for salvaging patients with relapsed- or refractory-AML. Thalidomide analogues with more potent immunomodulatory activities and more favourable toxicity profiles may offer more promise as anti-AML therapy.

BACKGROUND Levels of the soluble form of CD86 (sCD86) are elevated in a proportion of patients with leukemia. Although it is a potential modulator of antitumor responses, the significance of sCD86 in patients with hematologic malignancies is unknown. METHODS The authors evaluated sCD86 levels by enzyme-linked immunosorbent assay in patients with acute myeloid leukemia (AML) (n = 57 patients) and patients with myelodysplastic syndrome (MDS) (n = 40 patients) and analyzed the relation between sCD86 levels and various clinical parameters. RESULTS Levels of sCD86 were elevated (> 2.32 ng/mL) relative to normal donors (0.22–2.32 ng/mL; n = 51 patients) in 25% of patients with AML and in 27% of patients with MDS. Patients with AML who had elevated sCD86 levels had significantly lower complete remission (CR) rates compared with patients with AML who had normal sCD86 levels. In multivariate analysis using sCD86 as a continuous variable and including the interaction of age and sCD86 as a variable, sCD86 was a significant prognostic factor (P = 0.014) independent of cytogenetics. Further analysis demonstrated that, in patients with AML age 60 years and younger, but not in patients older than 60 years, elevated sCD86 levels were associated with significantly shorter survival (P = 0.04). There was no correlation between sCD86 levels and CR rates or survival in patients with MDS. CONCLUSIONS The presence in patients with AML of elevated levels of circulating sCD86 were associated with lower CR rates and poor survival. The prognostic significance of sCD86 was independent of cytogenetics but was modulated by age, in that it was independently significant only in younger patients. The results suggest that sCD86 may play a role in modulating immune responses associated with the progression of AML. Cancer 2003. © 2003 American Cancer Society. DOI 10.1002/cncr.11693

The genetic events involved in the transformation of chronic lymphocytic leukemia (CLL) to Richter's syndrome (RS) are poorly understood. Frequently large cells are seen in the bone marrows of patients with CLL and evidence of RS. Using a laser-capture microdissection we analyzed small and large leukemic bone marrow cells from 19 patients with RS for loss of heterozygosity (LOH) on chromosome 11 (D11S2179 at the ATM gene), 17 (D17S938 and D17S1852 at the TP53 site), and 20 (Plc1, D20S96, D20S110, and D20S119). Megakaryocytes were also isolated and used as a control for normal cells. Four of 15 (27.7%) informative cases showed LOH in small cells in the ATM gene while seven (46.7%) showed LOH in large cells. Six of 15 (40%) informative cases had LOH in chromosome 17 in small cells, and eight (53%) showed LOH in large cells. Eleven of 19 informative cases (61.1%) showed LOH in chromosome 20 in large cells, and eight (42.1%) showed LOH in small cells. RS cases with LOH at chromosome 20 were associated with marginally shorter survival rates (P = 0.08). Our data suggest that there are significant molecular differences between large and small cells in patients with CLL. Further analysis of the genes on these chromosomes may provide new insight into our understanding of the transformation of small CLL cells to large (Richter) cells.

Background: Deletions or structural abnormalities in chromosomes 11 and 13 have been shown to be important in predicting clinical behavior in patients with multiple myeloma (MM). However, cytogenetic analysis in MM is frequently difficult because of poor yield of informative metaphases and the disease is frequently patchy, which complicates fluorescent in situ hybridization studies. Objectives: The purpose of this study was to explore the potential of using peripheral plasma DNA for the detection of loss of heterozygosity (LOH) in chromosomes 11 and 13 in patients with MM. Methods: Peripheral blood (PB) plasma of 81 patients with MM, was used as a source of DNA for the detection of LOH at chromosomes 13q14 (D13S319 and D13AFMaw301wb5), and 11q21 (D11S2179) using polymerase chain reaction. Results: Only 62 of the studied patients were informative for the two 13q microsatellite markers and 16 (26%) of these patients showed LOH. Only seven (11%) of 61 patients with informative D11S2179 microsatellite maker showed LOH. Purified plasma cells (PCs) from six bone marrow (BM) samples using anti-CD138-coated magnetic beads showed identical results to those detected in DNA isolated from PB plasma. Three patients with LOH underwent autologous BM transplantation, and two of three reverted to a normal state (no LOH) after transplantation. Seven of the patients with 13q LOH in PB plasma had

Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are adverse prognostic features in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDSs). VEGF is a soluble circulating angiogenic molecule that stimulates signaling via receptor tyrosine kinases (RTKs), including VEGF receptor 2 (VEGFR-2). AML blasts may express VEGFR-2, c-kit, and FLT3. SU5416 is a small molecule RTK inhibitor (RTKI) of VEGFR-2, c-kit, and both wild-type and mutant FLT3. A multicenter phase 2 study of SU5416 was conducted in patients with refractory AML or MDS. For a median of 9 weeks (range, 1-55 weeks), 55 patients (33 AML: 10 [30%] primary refractory, 23 [70%] relapsed; 22 MDS: 15 [68%] relapsed) received 145 mg/m2 SU5416 twice weekly intravenously. Grade 3 or 4 drug-related toxicities included headaches (14%), infusion-related reactions (11%), dyspnea (14%), fatigue (7%), thrombotic episodes (7%), bone pain (5%), and gastrointestinal disturbance (4%). There were 11 patients (20%) who did not complete 4 weeks of therapy (10 progressive disease, 1 adverse event); 3 patients (5%) who achieved partial responses; and 1 (2%) who achieved hematologic improvement. Single agent SU5416 had biologic and modest clinical activity in refractory AML/MDS. Overall median survival was 12 weeks in AML patients (range, 4-41 weeks) and not reached in MDS patients. Most observed toxicities were attributable to drug formulation (polyoxyl 35 castor oil or hyperosmolarity of the SU5416 preparation). Studies of other RTKI and/or other antiangiogenic approaches, with correlative studies to examine biologic effects, may be warranted in patients with AML/MDS.

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.

Summary. As the abnormal expression of p53 protein is prognostically significant in some human cancers, its significance in patients with B-cell chronic lymphocytic leukaemia (CLL) was assessed. Two investigators evaluated the percentage of bone marrow mononuclear cells that stained for p53, using biopsies stained with anti-p53 monoclonal antibody (DO-7), and graded the degree of staining (0, +, ++, +++). Samples from a cohort of 90 patients with CLL were studied (median age 60 years, range 30–89 years; 57 patients were (63%) previously untreated, 22 patients (24%) had received one or two prior regimens, 11 patients had received (12%) three to seven regimens. The overall percentage of cells positive for p53 staining was a median of 43 (range 1–88). No investigator effect was detected either in overall percentage cells rated p53 positive or on the degree of staining (Pearson's correlation coefficient 0·980, P-value

BACKGROUND CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]) is the ligand for CD38, a transmembrane glycoprotein that is expressed on the surface of leukemic cells in many patients with B-cell chronic lymphocytic leukemia (B-CLL). In a previous study, the authors showed that CD38 expression was correlated with a poor prognosis in patients with B-CLL. In the current study, blood samples from patients with B-CLL were examined to identify CD31 surface marker expression, and CD31 expression was correlated with several other known prognostic variables, including CD38. METHODS Using flow cytometry, peripheral blood samples from 120 patients with B-CLL were analyzed for CD31 and CD38 expression on CD19 positive leukemic B cells. RESULTS Thirteen of 120 patients (11%) had CD31 expression on < 20% of their B cells, and the remaining patients had various levels of CD31 expression. The median expression of CD31 was 76% of leukemic, CD19 positive cells. Levels of CD31 expression were not correlated with survival outcomes or with any of the known prognostic parameters when all patients were considered. Patients who had high CD38 expression (≥ 20%), as expected, had significantly shorter survival (P = 0.001) compared with patients who had low CD38 expression (< 20%). However, in patients with low CD38 expression, a subgroup with low CD31 expression (< 76%) had significantly longer survival compared with the survival for the entire group (P = 0.0001). Moreover, the survival pattern of patients with low CD38 expression and high CD31 expression was not significantly different from the survival pattern seen in patients with high CD38 expression. CONCLUSIONS CD31 expression further defined a subgroup of patients with B-CLL who had a different survival outcome. Defining the interaction between CD31 expression and CD38 expression in patients with CLL will require further exploration. Cancer 2003;97:1914–9. © 2003 American Cancer Society. DOI 10.1002/cncr.11264

BACKGROUND Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are of adverse prognostic significance in patients with myeloproliferative disorders (MPD), including agnogenic myeloid metaplasia (AMM), chronic myeloid leukemia in blastic phase (CML-BP), and chronic myelomonocytic leukemia (CMML). VEGF is a soluble, circulating, angiogenic molecule that acts through receptor tyrosine kinases (RTK), including VEGF receptor 2 (VEGFR-2). SU5416 is a small-molecule RTK inhibitor (RTKI) that targets VEGFR-2, c-kit, and fms-related tyrosine kinase Flk2. METHODS Adult patients with advanced CMML, AMM, CML-BP, or other BCR-ABL negative MPD were entered on a multicenter, Phase II study. RESULTS Thirty-two patients (19 patients with BCR-ABL negative MPD, 6 patients with CMML, 4 patients with CML-BP, and 3 patients with AMM) with a median age of 66 years (range, 29–85 years) received SU5416 145 mg/m2 twice weekly intravenously for a median of three 4-week cycles (maximum, 12 cycles). Drug-related Grade 3–4 toxicities included acute abdominal pain (13%), bone pain (9%), infusion-related dyspnea (9%) or headache (6%), fatigue (6%), diarrhea (3%), and catheter site reactions (3%). Eleven patients (34%) did not receive a second cycle of therapy (6 patients had progressive disease, 3 because of adverse events; 2 patients withdrew due to lack of response). One patient with AMM achieved a partial response. Eight patients received more than 6 months of therapy. CONCLUSIONS SU5416 had minimal clinical activity in patients with MPD. Long-term administration of a twice-weekly, hyperosmolar, intravenous solution containing polyoxyl 35 castor oil was difficult. More tolerable RTKI may be worthy of further investigation in patients with MPD. Cancer 2003;97:1920–8. © 2003 American Cancer Society. DOI 10.1002/cncr.11315

CD20 is a 33- to 36-kDa transmembrane phosphoprotein involved in the activation, proliferation, and differentiation of B lymphocytes. The predicted amino acid sequence of the CD20 suggests 4 transmembrane-spanning regions with both N- and C-termini located in the cytoplasm. We demonstrate herein that significant levels of circulating CD20 (cCD20) can be detected in the plasma of patients with chronic lymphocytic leukemia (CLL) and that cCD20 interferes with the binding of rituximab, a humanized anti-CD20 monoclonal antibody, to CLL cells. An enzyme-linked immunosorbent assay (ELISA) was developed to measure circulating cCD20 levels in the plasma. We measured cCD20 levels in the plasma of 180 patients with CLL and correlated these levels with clinical characteristics and outcome. Circulating CD20 levels correlated positively with β2-microglobulin level (p = .006) and percentage of CD38+ cells (p = .03) and negatively with platelet count (p = .004) and hemoglobin level (p = .02). Patients with advanced Rai (III/IV) or Binet (C) stage disease had significantly higher levels of cCD20 than did patients with earlier-stage disease (P = .01 and P = .006, respectively). There was no correlation between cCD20 level and age, lymphocyte count, or white blood cell count. Using a recursive classification method, we found that patients with a cCD20 level more than 1875 nM/L had significantly shorter survival than those with cCD20 1875 nM/L or below (P = .01). The prognostic value of cCD20 was independent of Rai staging or hemoglobin level. Prospective evaluation is indicated to establish whether rituximab dosing should be adjusted according to cCD20 levels.

Arsenic trioxide (As(2)O(3)), an inorganic arsenic compound, has recently been approved for the treatment of relapsed or refractory acute promyelocytic leukemia. However, systemic toxicity associated with As(2)O(3) treatment remains a problem. Inorganic arsenic is detoxified in vivo by methylation reactions into organic arsenic compounds that are less toxic.We investigated the antiproliferative and cytotoxic activity of dimethylarsinic acid (DMAA), an organic arsenic derivative and major metabolic by-product of As(2)O(3), against a panel of eight leukemia and multiple myeloma cell lines. As(2)O(3) was tested in comparison. In clonogenic assay, the average concentration of DMAA that suppressed cell colony growth by 50% was 0.5-1 m M, while for As(2)O(3) it was on average 1-2 microM. At those concentrations DMAA and As(2)O(3) had significantly less effect on colony growth of normal progenitor cells. Cytotoxic doses of DMAA and As(2)O(3) in 3-day trypan blue dye exclusion assay experiments were similar to doses effective in clonogenic assay. Assessment of apoptosis by annexin V assay revealed a high rate of apoptosis in all cell lines treated with DMAA and As(2)O(3), but significantly less effect on normal progenitor cells. DMAA, unlike As(2)O(3), had no effect on the maturation of leukemic cells.DMAA exerts differential antiproliferative and cytotoxic activity against leukemia and multiple myeloma cells, with no significant effect on normal progenitor cells. However, concentrations of DMAA needed to achieve such efficacy are up to 1000 times those of As(2)O(3). Evaluation of novel organic arsenic that would combine the high efficacy of As(2)O(3) and the low toxicity of DMAA is warranted.

BACKGROUND Therapy for patients with Richter syndrome (RS) or fludarabine-refractory chronic lymphocytic leukemia (CLL) is unsatisfactory. A Phase II study was conducted to evaluate an alternating combination cytotoxic regimen given with rituximab and granulocyte-macrophage–colony stimulating factor (GM-CSF) in these patients. METHODS Fludarabine-refractory CLL was defined as failure to respond to most recent prior fludarabine-containing regimen. Patients received up to six cycles of fractionated cyclophosphamide, vincristine, liposomal daunorubicin, and dexamethasone (hyper-CVXD) plus rituximab and GM-CSF alternating with methotrexate and cytarabine plus rituximab and GM-CSF. Response, toxicity, and survival data were compared with data from prior therapy with hyper-CVXD alone in this patient group. RESULTS Forty-nine patients with RS (n = 30 patients) or refractory CLL (n = 19 patients) were treated on study. Nine patients (18%) achieved a complete remission, and 11 patients achieved a partial remission (22%), for an overall objective response (OR) rate of 41%. With a median follow-up of 7.5 months and a maximum follow-up of 15.2 months, the 12-month failure free survival (FFS) rate was 27%, and the overall survival (OS) rate was 39%. Nine patients (18%) died during the first cycle of therapy, and two patients (4%) died during the second cycle. There were no significant differences between the rates of OR, OS, and FFS in the current study and those obtained with hyper-CVXD alone on a prior study. CONCLUSIONS The study regimen had activity and significant toxicity in patients with RS or fludarabine-refractory CLL. It was not clearly better compared with hyper-CVXD alone in this patient population. Cancer 2003;97:1711–20. © 2003 American Cancer Society. DOI 10.1002/cncr.11238

BACKGROUND Gemtuzumab is used to treat patients with previously untreated or recurrent acute myelogenous leukemia (AML). The fludarabine and cytarabine (ara-C) regimen is active in these patients. Resistance to gemtuzumab is associated with blast multidrug resistance (MDR). The objectives of this study were to evaluate the efficacy and toxicity of a combination regimen of gemtuzumab, fludarabine, ara-C, and the MDR modifier (cyclosporine [CyA]) in patients with previously untreated AML, refractory anemia with excess blasts (RAEB), or RAEB in transformation (RAEBT). METHODS The MFAC regimen was comprised of gemtuzumab (Mylotarg™) (6 mg/m2 intravenously [i.v.] on Day 1); fludarabine and ara-C (15 mg/m2 and 0.5 g/m2, respectively, twice daily on Days 2–6); and CSA (6 mg/kg loading dose before gemtuzumab, followed by 16 mg/kg continuous i.v. infusion on Days 1 and 2). RESULTS Fifty-nine evaluable patients were treated: 39 patients (66%) had AML and 20 patients (34%) had RAEB/RAEBT. Their median age was 57 years (range, 27–76 years). The MFAC regimen induced complete remission (CR) in 27 patients (46%) and CR with incomplete platelet recovery (CRp) in 1 patient (2%). The median survival period is 8 months. At 12 months, the survival rate is 38% and the event-free survival rate in patients with CR/CRp is 27%. Infections complicated 38% of the courses of chemotherapy. Grade 3/4 toxicity included hyperbilirubinemia in 31% and transaminitis in 7% of the patients. Four patients (7%) developed hepatic venoocclusive disease (VOD). CONCLUSIONS The MFAC regimen may merit further study in patients with AML if measures to avoid and/or treat VOD can be incorporated into the regimen. Cancer 2003;97:1481–7. © 2003 American Cancer Society. DOI 10.1002/cncr.11239

Summary. The malignant B cells of patients with chronic lymphocytic leukaemia (CLL) constitutively express interleukin 8 (IL-8) and IL-8 receptors. Ex vivo culture with exogenous IL-8 enhances IL-8 expression and prolongs leukaemia cell survival, partly through increased bcl-2 expression. IL-8 may function as an autocrine growth and apoptosis resistance factor in CLL. Therefore, we evaluated the prognostic relevance of plasma IL-8 levels in 151 CLL patients [median age 61 years (range, 32–84 years), median plasma IL-8 level 18·9 pg/ml (9·1–89·1 pg/ml)]. All Rai stages were represented; advanced stage was associated with significantly higher plasma IL-8 levels (P

BACKGROUND Aberrant methylation of promoter-associated cystosine-guanine (CpG) islands is an epigenetic modification of DNA frequently observed in adult patients with acute lymphocytic leukemia (ALL). This epigenetic modification has been associated with gene silencing, malignant transformation, and aging. It is not known whether there are epigenetic differences between pediatric patients and adult patients with ALL. METHODS To investigate the methylation characteristics of pediatric patients with ALL and to determine whether DNA methylation can explain prognostic or biologic differences between pediatric and adult patients, the authors analyzed the methylation status of 7 promoter-associated CpG islands in 16 pediatric patients with ALL and compared them with the methylation characteristics of a cohort of adult patients with ALL. The genes analyzed included the estrogen receptor gene (ER), multidrug resistance gene 1 (MDR1), p15, C-ABL, CD10, p16, and p73. RESULTS The mean methylation densities of ER, MDR1, CD10, p15, and C-ABL were 25.4%, 16.4%, 5.23%, 4.24%, and 4%, respectively. P16 was methylated in 11.7% of patients, and p73 was methylated in 17.6% of patients. One patient (6.2%) had methylation of 0 genes, 15 patients (93.7%) had methylation of ≥ 1 gene, and 4 patients (25%) had methylation of 3–4 genes. Methylation of all these genes was < 2% (or methylation specific polymerase chain reaction negative) in nonneoplastic tissues. A significant inverse correlation was observed between methylation of CD10 and CD10 expression. No differences were observed between the methylation characteristics of pediatric patients and adult patients. CONCLUSIONS The results indicate that DNA methylation is common in pediatric patients with ALL and that methylation of the genes studied does not account for prognostic differences between pediatric patients and adult patients with ALL. Cancer 2003;97:695–702. © 2003 American Cancer Society. DOI 10.1002/cncr.11090

BACKGROUND It has been reported that point mutations of the ras gene occur frequently in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, the prognostic significance of ras gene mutations in patients with these disorders has been a controversial issue. Although abnormalities in the neurofibromatosis 1 (NF1) gene, which is a gene involved in the ras pathway, have been observed frequently in patients with juvenile chronic myelogenous leukemia, the role of these abnormalities in adult patients with AML or MDS is not clear. METHODS In this study, bone marrow specimens that were obtained from previously untreated patients with AML and MDS were examined for ras mutations, and the levels of NF1 protein expression were measured. RESULTS Ras point mutations were detected in 16 of 83 patients with AML (19%) and in 21 of 90 patients with MDS (23%). Fourteen of 28 patients with chronic myelomonocytic leukemia (50%) had ras gene mutations. Decreased bone marrow levels of NF1 protein (< 70% of the median level detected in control bone marrow specimens) were observed in 20 of 66 patients with AML (30.3%) and in 8 of 29 patients with MDS (27.6%); none of the patients with ras gene mutations had decreased bone marrow levels of NF1 protein. The presence of a mutant ras gene was associated with a shorter complete remission duration (CRD) in patients with MDS (P = 0.05) but had no effect on survival in patients with AML or MDS. Patients with AML who had decreased NF1 protein levels had a slightly longer CRD compared with patients who had normal NF1 levels (P = 0.07). However, the NF1 level had no significant impact on survival among patients with AML or MDS. When patients with low NF1 levels were grouped with patients who had ras mutations, the patients who had AML had a significantly longer CRD compared with the patients who had MDS (P = 0.02). CONCLUSIONS The current results suggest that a reduction in NF1 protein expression and the presence of ras point mutations may complement each other and that they both play a complex role in the biology of AML and MDS. Cancer 2003;97:441–9. © 2003 American Cancer Society. DOI 10.1002/cncr.11036

BACKGROUND Significantly elevated telomerase activity (TA) has been found in samples from patients with almost all malignant hematologic diseases. The impact of elevated TA on the course of pediatric patients with acute myeloid leukemia (P-AML) is unknown. METHODS Using a modified polymerase chain reaction-based, telomeric repeat-amplification protocol assay, the authors measured TA in bone marrow samples from 40 patients with P-AML and, for comparison, in 65 adult patients with AML (A-AML), excluding patients with French–American–British M3 disease. The results were correlated with patient characteristics and survival. RESULTS TA in patients with P-AML was significantly lower compared with TA in patients with A-AML (P = 0.005). Patients who had P-AML with low TA had a projected 5-year survival rate of 88%, whereas patients who had P-AML with high TA had a projected 5-year survival rate of 43% (P = 0.009). Conversely, patients who had A-AML with very high TA (upper quartile) had significantly longer survival compared with patients who had A-AML with lower TA (P = 0.03). There was no correlation between complete remission rate or disease free survival and TA in P-AML or A-AML. In the A-AML group, when patients were separated by cytogenetic findings (poor prognosis vs. others), it was found that TA was significantly lower in patients with a poor prognosis, but the prognostic value of TA was not independent of cytogenetic status. CONCLUSIONS The current results suggest, that for patients with P-AML, bone marrow TA is a highly significant prognostic factor. Cancer 2003;97:2212–7. © 2003 American Cancer Society. DOI 10.1002/cncr.11313

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 =

The chromosomal abnormality t(14;18) is most commonly associated with neoplasms of follicular center cell origin. However, t(14;18) also has been reported in rare cases of chronic lymphocytic leukemia (CLL). In 2215 cases of CLL studied by conventional cytogenetics in our institution, we identified 2 cases of CLL carrying t(14;18). Both patients were men, aged 52 and 71 years at the time of diagnosis. One patient presented with leukemia and the other primarily with nodal disease. In addition to t(14;18), trisomy 12 (+12) was identified in the same clone in both cases. Atypical morphologic features were identified: case 1 contained more than 15% lymphoid cells with cleaved nuclei, whereas case 2 contained more than 15% plasmacytoid lymphoid cells. The immunophenotype of case 2 was also unusual for CLL, showing weak CD23 expression and FMC7 positivity. We identified 6 other t(14;18)-carrying CLL cases in the literature; 2 had t(14;18) as the sole abnormality and 2 contained +12 as the additional abnormality. To conclude, cases of CLL carrying t(14;18) are exceedingly rare, and +12 appears to be the most common cytogenetic abnormality coexisting with t(14;18) in CLL.

BACKGROUND. Up to 5% of patients with chronic myelogenous leukemia (CML) do not have the Philadelphia (Ph) translocation t(9;22)(q34;q11) or a bcr/abl molecular rearrangement. Although the diagnostic criteria of this entity are still under debate, there is general agreement that patients with Ph negative, bcr/abl negative CML have a severe clinical course that is not affected significantly by current treatment options. METHODS. A population of 76 patients with bcr/abl negative CML who had received minimal or no previous therapy was characterized carefully with the intent of investigating clinical and hematologic variables and their association with survival by univariate, correlation, and multivariate analyses. A group of 73 patients with Ph negative CML who were not tested for the bcr/abl rearrangement (bcr/abl unknown) was analyzed separately and used for extension of the analysis. RESULTS. In the bcr/abl negative patient population, the median overall survival was 24 months. At the time of the analysis, 38 patients (50%) had died, and blastic transformation preceded death in 31%. Chromosomal abnormalities were found in 30% of the 76 patients, with trisomy 8 the most common abnormality. Complex chromosomal abnormalities were rare, and monosomy 7 was not observed. Survival was not affected significantly by treatment. Multivariate analysis identified older age ( 65 years), anemia (hemoglobin 10 g/dL), and severe leukocytosis (white blood cells 50 10 9 /L) as variables with independent prognostic significance for poor survival. A prognostic scoring system stratified patients into a low-risk group (53%) and a high-risk group (47%), with median survivals of 38 months and 9 months, respectively. CONCLUSIONS. Bcr/abl negative CML is a distinct clinical entity associated with very poor prognosis. Two risk categories are identifiable using a simple scoring system based on age, hemoglobin level, and leukocyte number. Cancer 2002;95:1673– 84. © 2002 American Cancer Society. DOI 10.1002/cncr.10832

Summary. Increased angiogenesis is important in the pathophysiology of haematological malignancies. Cyclooxygenase-2 (Cox-2) converts arachidonic acid to prostaglandins, which induce expression of angiogenic factors, including vascular endothelial growth factor (VEGF), basic-fibroblast growth factor, transforming growth factor-β and interleukin 6. Cox-2 may also reduce apoptosis and reduce cellular attachment to the extracellular matrix (ECM). Increased bone marrow (BM) vascularity, increased BM cellular and plasma VEGF levels, and decreased progenitor adherence to BM ECM have all been observed in chronic myeloid leukaemia (CML). We investigated the prognostic significance of levels of Cox-2 in BM cells from patients with CML. Western blot and solid-phase radioimmunoassay (RIA) were used to measure Cox-2 BM levels in 149 patients with chronic phase CML (CP CML). Results were compared with those of normal controls. Expression of Cox-2 was significantly higher in CML than in normal controls (P

We measured the concentration of CD33 antigen on the surface of cells in 315 bone marrow (BM) samples and 114 corresponding peripheral blood (PB) samples from patients with various leukemias (acute myeloid leukemia [AML], chronic myelogenous leukemia [CML], myeloproliferative disorder [MPD] other than CML, myelodysplastic syndrome [MDS]) and from control subjects. Overall CD33 intensity in total CD33+ cells was significantly higher in BM than in PB. CD33 intensity in total BM CD33+ cells differed significantly with the type of disease. The median number of CD33 molecules per cell was highest in AML, followed by MDS, CML, and control subjects and lowest in MPD. When only CD34+/CD33+ cells were examined, CD33 molecules per cell were highest in CD34+ cells in AML and lowest in MPD (P = .027). Patients with AML or MDS younger than 60 years had significantly higher intensity of CD33 expression on CD34+ cells than patients 60 years or older. Levels of CD33 intensity did not correlate with cytogenetics in patients with AML or MDS. There was no correlation between CD33 intensity and response to therapy or overall survival in 35 patients treated with protocols including Mylotarg. These data demonstrate variation in CD33 intensity between various leukemias.

Purpose. Tumor necrosis factor-α (TNF-α) is an important effector and regulatory cytokine involved in the pathophysiology of hematologic malignancies, including hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), agnogenic myeloid metaplasia (AMM) and Philadelphia-negative myeloproliferative disorders (MPD). We conducted a pilot study to assess the safety of the soluble TNF receptor, etanercept (p75 TNFR:Fc; Enbrel) in patients with refractory hematologic malignancies. Methods. Patients were eligible if they had refractory HCL, CLL, AMM, or Philadelphia-negative MPD. Enbrel was administered twice weekly at a dose of 25 mg subcutaneously for a minimum of eight doses, and was continued in patients without overt progression. Results. Among the 26 patients enrolled on study, 25 patients were evaluable. Nine patients had AMM, eight CLL, three HCL, and five Philadelphia-negative MPD. Their median age was 60 years (range 30–83 years). A total of 70 courses consisting of 486 doses of Enbrel were administered. Enbrel was well tolerated, without any overt increase in infectious episodes. Stable disease/no objective response was seen in 22 patients (88%) and progression in 3 patients (12%). Three patients with AMM improved (two showed hematologic improvement, and one showed a reduction in liver and spleen size), and two patients (one with CLL and one with Philadelphia-negative MPD) showed improvement in disease-related symptoms. Conclusions. Enbrel was well tolerated, but no responses were noted in these immunosuppressed patients with refractory hematologic malignancies.

We have previously reported that high levels of cellular vascular endothelial growth factor (VEGF) protein correlated with short survival of patients with acute myeloid leukaemia (AML). As VEGF exerts its effects via two receptors, VEGF receptor 1 (VEGFR-1) and VEGFR-2, we evaluated the significance of VEGFR-1 and VEGFR-2 protein levels in AML and myelodysplastic syndrome (MDS), and their relationship to VEGF protein levels. Western blot analysis and radioimmunoassay confirmed and quantified specific protein levels in bone marrow samples from 41 MDS and 66 AML previously untreated patients. VEGFR-1 levels were significantly higher in AML than in MDS (P = 0.0004), but no significant difference was found in the VEGFR-2 levels (P = 0.5). No significant correlation between VEGFRs levels and duration of survival was found. VEGF protein levels were significantly higher in MDS than in AML (P < 0.0001). A Cox proportional-hazard regression model showed increasing VEGF levels to significantly correlate with shorter survival of patients with MDS (P = 0.008), a finding similar to our previous report of the inverse relationship between VEGF levels and survival of AML patients. We found a significant correlation between VEGF and VEGFR-2 levels in both AML and MDS (P < 0.0000001 andP < 0.0002 respectively) but not between VEGF and VEGFR-1 levels. These data suggest that VEGF expression, rather than the expression of its receptors, is the determining factor in the biological behaviour of AML and MDS, and that VEGFRs are differentially expressed in AML and MDS.

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 (P0.05) 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 with70% 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).

We reported previously that high levels of vascular endothelial growth factor (VEGF) were associated with shorter survival in adult acute myeloid leukemia (AML) patients. In this study, cellular VEGF and its receptor, VEGF-R2 (kinase domain receptor (KDR)), were analyzed in 45 pediatric AML patients using Western blot and solid-phase radioimmunoassay (RIA). Cellular VEGF levels were significantly lower in pediatric AML compared to adult AML patients. In contrast, there was no significant difference in VEGF-R2 levels between adult and pediatric AML. Higher VEGF and VEGF-R2 levels in pediatric AML patients correlated with higher white blood cell (WBC). Unlike in adults, VEGF and VEGF-R2 levels in pediatric AML patients did not correlate with survival. This data suggest that the role of VEGF and its receptor VEGF-R2 in pediatrics AML may be different from that in adult AML.