The ALK inhibitor ASP3026 eradicates NPM-ALK+ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model

NPM-ALK⁺ T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK⁺ ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK⁺ solid tumors. However, NPM-ALK⁺ ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK⁺ ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK⁺ ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK⁺ ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK⁺ ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.     

p16INK4A and p15INK4B gene deletions in primary leukemias

The 9p21 locus has been deleted at a high frequency in a wide variety of tumors. Recently, two genes, p16INK4A and p15INK4B (also called MTS1 and MTS2), have been localized in close proximity at the 9p21 locus, encoding cyclin-dependent kinases 4/6 inhibitors of relative molecular mass 16 kD and 15 kD, respectively and also found to be deleted at a high frequency in tumor cell lines. We analyzed p16INK4A and p15INK4B genes in 178 cases of primary leukemias including 81 cases of chronic lymphocytic leukemia (CLL), seven of hairy cell leukemia (HCL), seven of chronic myelogenous leukemia (CML), 43 of acute myelogenous leukemia (AML), 27 of acute lymphoblastic leukemia (ALL), and 13 of myelodysplastic syndrome (MDS) by Southern blot analyses. The ALL cases showed a relatively high frequency of homozygous deletions (22%, 6 of 27) at the p16INK4A gene locus. Interestingly, of the six cases with p16INK4A homozygous deletions, only three showed homozygous deletions at the p15INK4B gene. In 81 CLL patients, we detected one homozygous and five heterozygous deletions at both the p16INK4A and p15INK4B genes and two heterozygous deletions at the p16INK4A gene alone. Deletion of these two genes in AML cases is relatively low (9%). We did not detect deletions in any of the MDS, HCL, and CML cases examined. Sequence analyses of p16INK4A gene of six CLL cases with heterozygous deletion at this locus showed a 27-bp deletion at the splice acceptor site of intron 1 in one case and changes in the coding sequence in three other cases. The data presented in this report showed that (1) p16INK4A and p15INK4B genes are preferentially deleted homozygously in ALL and heterozygously in CLL cases with frequent mutation in the second allele, and (2) p16INK4A gene appears to be more frequently deleted than p15INK4B gene.     

The novel tyrosine kinase inhibitor EXEL-0862 induces apoptosis in human FIP1L1-PDGFR-alpha-expressing cells through caspase-3-mediated cleavage of Mcl-1.

The FIP1-like-1 (FIP1L1)-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFR-alpha) fusion kinase causes hypereosinophilic syndrome (HES) in a defined subset of patients. Imatinib mesylate is a potent inhibitor of ABL but also of PDGFR-alpha, and has been associated with durable hematologic responses in patients with HES. However, development of mutations in the tyrosine kinase domain may hamper the activity of tyrosine kinase inhibitors (TKIs), which suggests that novel agents are warranted to prevent or overcome resistance. We evaluated the efficacy of the novel TKI EXEL-0862 in FIP1L1-PDGFR-alpha-expressing cell lines and in cells from a patient with HES harboring the FIP1L1-PDGFR-alpha gene. EXEL-0862 inhibited the proliferation of EOL-1 and imatinib-resistant T674I FIP1L1-PDGFR-alpha-expressing cells and resulted in potent inhibition of the phosphorylation of PDGFR-alpha and downstream proteins STAT3 and Erk1/2, both in vitro and ex vivo. Moreover, EXEL-0862 induced apoptotic death in EOL-1 cells and imatinib-resistant T674I FIP1L1-PDGFR-alpha-expressing cells, and resulted in significant downregulation of the antiapoptotic protein Mcl-1 through a caspase-dependent mechanism. Our data establish EXEL-0862 as a solid candidate for the targeted treatment of patients with FIP1L1-PDGFR-alpha-positive HES.     

ATM gene deletion in patients with adult acute lymphoblastic leukemia

BACKGROUND: Loss of heterozygosity (LOH) at the ATM gene (mutated in ataxia telangiectasia [AT] patients) and ATM protein deficiency occur in 14% and 34%, respectively, of patients with chronic lymphocytic leukemia (CLL). ATM protein deficiency also is associated with aggressive disease and worse patient survival. Considering the aberrations in the ATM gene in CLL and the high rate of incidence of lymphoid neoplasias in AT patients, the authors investigated its incidence rate and significance in patients with adult acute lymphoblastic leukemia (ALL). METHODS: Samples from 36 adults with ALL were analyzed for LOH and homozygous deletion (HD) using a panel of three microsatellite markers located at the ATM gene (D11S2179), the MLL gene (D11S1356), and the BCL1 gene (D11S987) loci. These markers are located within the 11q13-q23 locus. RESULTS: Of the 36 informative cases, 10 (28%) showed deletions (7 LOH and 3 HDs) at the D11S2179 marker. In two patients, the deletions were extended to the MLL gene locus. These deletions were submicroscopic because only 3% (1 of 36) of patients showed abnormalities involving 11q23 using cytogenetic studies. The authors also estimated the levels of ATM protein in 15 ALL patients and 12 healthy volunteers by radioimmunoassay. The ATM protein levels in cases with LOH at the ATM gene were between 15-50% of those from normal bone marrow. In contrast to CLL patients, patients with LOH or HD at the ATM gene locus showed better survival compared with patients without ATM gene deletions (P = 0.003). CONCLUSIONS: LOH of the ATM gene and protein deficiency are common in adult ALL, are not demonstrated at the cytogenetic level, and are associated with a favorable prognosis. The authors speculate that ATM deficiency may increase the sensitivity of leukemic blasts to the chemotherapy used during induction and after disease remission in patients with adult ALL. The relatively high frequency of deletion of the D11S2179 marker compared with the D11S1356 marker suggests that ATM is the target gene of the deletion at the 11q23 locus, and that such deletions may play a role in the pathogenesis of ALL.     

Variations in the low levels of cyclin D1/BCL1 have prognostic value in chronic lymphocytic leukemia

Cyclin D1 (CyD1)/BCL1 (PRAD1) is expressed at high levels in almost all cases of mantle cell leukemia/lymphoma (MCL) and in rare cases of chronic lymphocytic leukemia (CLL). The CyD1/BCL1 protein plays an important role in the progression of cells through the G1 phase of cell cycle. Most of the CyD1/BCL1 protein expression studies are performed using immunohistochemistry. We used a sensitive solid-phase radioimmunoassay (RIA) to quantify CyD1 protein expression in 199 patients with CLL. Of these 137 patients were previously untreated with the rest having had standard chemotherapeutic regimens including alkylating agents and fludarabine before being referred to our center. Median white cell count in these patients was 49x10(3) /microl (range 3.0-438.5x10(3)/microl), hemoglobin level 13.1 g/dl (range 5.2-17.3 g/dl), platelet count 157x10(3) /microl (range 10-377x10(3) /microl), age 58 (range 26-89), and beta2-microglobulin 2.75 mg/dl (range 1.1-14.3). The median radioactivity (CPM) of mononuclear cells obtained from 56 normal individuals was assigned a value of 1. There was no significant variation in CyD1 levels among normal individuals (SD=0. 12). While most CyD1 levels in MCL varied from 6.5 to 15.6, the median CyD1/BCL1 in CLL was 1.4 with 75th percentile under 2.12. Rare CLL cases (3.5%) showed levels between 4 and 8.83. When divided into two groups at the median level, patients with higher CyD1/BCL1 expression had shorter survival (P = 0.03). This remained true when applied only to the previously untreated patients (P=0.05). Despite the relatively low expression, the CyD1/BCL1 levels in univariate analysis were as good or better predictors of survival than Binet (P = 0.03) or Rai (P = 0.05) staging. Furthermore, CyD1/BCL1 levels correlated with serum beta2-microglobulin (P = 0.001), white blood cell count (P = 0.004) and hemoglobin levels at the time of collection (P = 0.0003) but not with lymphocyte count, platelet count or age. The data demonstrate that CyD1/BCL1 is likely to play a significant role in the biology of CLL and can be used as a prognostic indicator. Further studies to clarify the role of CyD1 in the biology of CLL and its value as a prognostic indicator at the time of diagnosis are encouraged.     

Downregulation of the p53 tumor suppressor gene and upregulation of the bcl-2 gene in retinoic acid receptor alpha-deficient transgenic mice

We recently demonstrated lymphoma development in transgenic mice deficient in retinoic acid receptor alpha (RARalpha). High incidence of lymphoma development in this transgenic mouse model system was similar to lymphoma development in p53 knockout mice. In an effort to understand the molecular basis of lymphomagenesis in RARalpha-deficient transgenic mice, we compared the levels of RARalpha to the levels of p53 mRNA, and Bcl-2, and Bax proteins in lymphoid and non-lymphoid tissues and in lymphomas derived from the RARalpha-deficient transgenic mice. The p53 mRNA levels were depleted in various tissues including spleen ( approximately 96%), thymus ( approximately 29%) and bone marrow ( approximately 62%) of RARalpha-deficient transgenic mice when compared with the normal littermates, and the reduction in p53 mRNA expression in the various tissues examined was proportional to the reduction in RARalpha expression. Bcl-2 to Bax ratios were highly increased in the lymphoid compartments (spleen >bone marrow >thymus) because of selective overexpression of Bcl-2 protein. In summary, RARalpha downmodulation in this transgenic mouse model system was accompanied by p53 downmodulation and deregulation of Bcl-2 to Bax ratios in the lymphoid compartments.     

Clinical relevance of circulating angiogenic factors in patients with non-Hodgkin's lymphoma or Hodgkin's lymphoma

Vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and angiogenin are important angiogenic factors. In 65 patients with non-Hodgkin's lymphoma (NHL), pre-treatment VEGF, bFGF, and HGF levels were significantly elevated compared to normal individuals, while angiogenin levels were significantly subnormal. In 37 patients with Hodgkin's disease, pre-treatment levels of VEGF and HGF were significantly elevated, bFGF levels were normal, and angiogenin levels were significantly subnormal. In patients with NHL, post-therapy levels of angiogenin were independently predictive of survival. Both pre-therapy and post-therapy VEGF levels were independently predictive of survival in patients with HD.     

Prognostic significance of cellular vascular endothelial growth factor expression in chronic phase chronic myeloid leukemia

The impact of elevated vascular endothelial growth factor (VEGF) expression on the course of chronic myeloid leukemia (CML) is unknown. By radioimmunoassay, we measured pretreatment cellular VEGF protein in bone marrow samples from 184 (148 chronic and 36 accelerated/blastic phases) CML patients and found the levels to be 1.6-fold higher than in 31 normal control bone marrow samples (P =.000 01). No significant differences were found in VEGF levels by different phases of CML (P =.1). VEGF levels correlated with older age (P =.01) and higher platelet count (P =.0003), but also with smaller spleen size (P =.004), lower white blood cell count (P =.0006), and lower percentage of peripheral blasts (P =.04). With the use of Cox proportional hazard model and VEGF levels as a continuous variable, high VEGF levels correlated with shorter survival of patients in chronic CML (P =.008). Multivariate analysis showed that VEGF was not independent of the synthesis stage (P =.09). These data suggest that VEGF plays a role in the biology of CML and that VEGF inhibitors should be investigated in CML.     

Clinical relevance of VEGF receptors 1 and 2 in patients with chronic myelogenous leukemia

Vascularity is increased in the bone marrow of patients with chronic myeloid leukemia (CML) and high vascular endothelial growth factor (VEGF) levels correlate with worse survival. We analyzed the significance of VEGF-receptor 1 (VEGF-R1) and VEGF-R2 levels in bone marrow samples from 170 CML patients (137 chronic, 24 accelerated, and 9 blastic phase). Median VEGF-R1 and VEGF-R2 levels were 4.66 and 2-fold, respectively, that in normal control samples. Receptor levels did not correlate with disease phase or other host and disease features examined. Chronic phase CML patients with increased VEGF-R2 levels had significantly inferior survival than patients without receptor up-regulation (P=0.009). Patients in accelerated/blastic phase CML with elevated VEGF-R2 expression had marginally worse survival (P=0.05). In contrast, high VEGF-R1 levels did not correlate with a specific CML phase, characteristic, or outcome. Our findings support VEGF-R2 over-expression as an independent prognostic indicator for shortened survival in patients with CML.     

Telomerase activity is prognostic in pediatric patients with acute myeloid leukemia: comparison with adult acute myeloid leukemia

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.