Yasuyuki Nakano

Molecular evolution of acute myeloid leukaemia in relapse : unstable N-ras and FLT3 genes compared with p53 gene

Relapse is a major cause of treatment failure in acute myeloid leukaemia (AML), and is usually accompanied by resistance to chemotherapy. To study whether relapse is accompanied by genetic alterations, we compared N‐ras, p53 and FLT3 gene mutations in paired samples obtained at initial diagnosis and first relapse. 28 patients with relapsed AML were studied, and their duration of complete remission ranged from 133 to 989 d (mean 318 d). Karyotype changes were observed at relapse in 11 patients. Point mutations of the N‐ras gene were positive at both stages (+/+) in three patients, positive at initial diagnosis and negative at relapse (+/−) in three patients, and negative at initial diagnosis and positive at relapse (−/+) in two patients. Internal tandem duplications of the FLT3 gene (FLT3/ITD) were +/+ in five patients, +/− in one patient, and −/+ in six patients. The p53 gene mutations were +/+ in two patients, +/− in one patient, and −/− in 25 patients. FLT3/ITD and mutant p53 at relapse were associated with short survival after relapse. These results indicate that relapse is frequently accompanied by molecular alterations that include the loss and/or acquisition of mutations. Thus relapse can be understood as clonal shift or collateral succession rather than clonal progression.

Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia

Abstract: In acute myeloid leukemia (AML), p53 mutations are reportedly infrequent but associated with a poor prognosis. The majority of mutations are missense mutations, which generally lead to accumulation of nuclear p53 protein. However, the prognostic significance of the accumulation remains unknown in AML. In this study, we compared the prognostic value of p53 mutations versus accumulation of the product. p53 mutations were found in 9 (4.5%) of 200 patients with de novo AML. The p53 mutation detectable (mutation+) group had a worse prognosis (p=0.0009) than the mutation not detectable (mutation−) group. Multivariate analysis showed that the p53 mutation was an independent factor (p=0.005) for short overall survival as well as 60 yr or older (p=0.001) and unfavorable karyotypes (p=0.001). In 79 of the 200 patients, the expression of p53 was studied by immunocytochemistry (ICC) using anti‐p53 monoclonal antibody (DO‐7). All samples carrying missense mutations (N=6) were positive for ICC in over 15% of nuclei of each sample, chosen as the optimized cutoff value of p53 accumulation. Accumulation was thus found in 14 of the 79 patients. However, there was no prognostic difference according to the accumulation, because the mutation−/accumulation+ group (N=8) tended to have a good prognosis. These findings indicate that molecular detection of p53 mutations yields better prognostic information than ICC. In a subset of AML, p53 protein might be accumulated without mutation presumably due to upstream signals of p53.

Lack of constitutive activation of MAP kinase pathway in human acute myeloid leukemia cells with N-Ras mutation

Mitogen-activated protein (MAP) kinases act as transducers of extracellular signaling via tyrosine kinase-growth factor receptors and G-protein-linked receptors to transcription factors. Constitutive activation of MAP kinase has been observed in a variety of solid tumors including renal cancer and breast cancer. Recently, we have reported that constitutively activated MAP kinase was observed in 50% of human primary acute myeloid leukemia (AML) cells. Ras is one of the components of G-proteins and transduces the signal from cytokine receptors to raf-1 theoretically resulting in the activation of MAP kinase pathway. In the present study, we have examined the correlation of Ras mutations and the activation of MAP kinase pathway in patients with AML. Twenty out of 22 AML cases with activating N-Ras mutations showed no phosphorylated forms of ERK2. ERK2 phosphorylation was tightly correlated with ERK1 phosphorylation and MAP kinase activity detected by in vitro kinase assay. Three samples with N-Ras mutations were stimulated with IL-3, GM-CSF and G-CSF separately but ERK2 activation was induced in none of these samples stimulated with these cytokines. In contrast, ERK2 was constitutively activated in all of four pancreatic carcinoma cases with K-Ras mutation at codon 12. These results suggest that function of the Ras mutations may be different between solid tumors, such as pancreatic carcinoma and colorectal carcinoma, and AML. Mutated Ras does not always stimulate MAP kinase pathway constitutively and may rather inhibit classical MAP kinase cascade in AML blasts from leukemia patients.

Trough plasma concentration of imatinib reflects BCR-ABL kinase inhibitory activity and clinical response in chronic-phase chronic myeloid leukemia : A report from the BINGO study

Pharmacokinetic (PK) factors have been suggested to be involved in the unfavorable clinical responses of chronic myeloid leukemia (CML) patients treated with imatinib. The purpose of this study was to clarify prognostic implications of PK factors in CML patients treated with imatinib. The plasma trough (Cmin) level of imatinib and serum α1‐acid glycoprotein (AGP) level were measured on two different days in 65 CML patients treated with imatinib for more than 12 months. We further examined whether the Cmin level of imatinib actually reflects inhibitory activity against BCR–ABL kinase using the plasma inhibitory activity (PIA) assay. Since the differences of five patients were statistically rejected by the Smirnov–Grubbs’ test, we excluded them for further analysis. The Cmin level was strongly associated with the achievement of MMR at the 12th month, and ROC analysis demonstrated Cmin levels and their discrimination potential for major molecular response (MMR) with the best sensitivity (63.2%) and specificity (68.2%) at a Cmin threshold of 974 ng/mL. The α1‐Acid glycoprotein (AGP) level was within the normal range in 57 of 60 patients, indicating little impact of AGP on our study. There was a weak correlation between PIA against phospho (P)‐BCR–ABL and the Cmin level of imatinib (r2 = 0.2501, P = 0.0007), and patient plasma containing >974 ng/mL imatinib sufficiently inhibited P‐BCR‐ABL. These results collectively indicated that maintaining ∼1000 ng/mL of Cmin was clinically and biologically important for the optimal response in CML patients treated with imatinib. A prospective intervention study is required to establish PK‐based management in CML patients treated with imatinib. (Cancer Sci 2010)

Poor clinical significance of p53 gene polymorphism in acute myeloid leukemia.

The cancer susceptibility according to the p53 polymorphism at codon 72 has been in controversy. In this study, the clinical significance of p53 polymorphism in de novo acute myeloid leukemia (AML) was examined. Although the allelic frequency of Arg in 200 patients with AML (64.3%) tended to be greater than that in normal controls (56. 6%), these frequencies were within the normal range according to the previous data in Japan (from 59.9 to 65.3%). p53 mutations, found in nine (4.5%) of the 200 patients, were not related to the polymorphism. Six of 93 patients showing heterozygosity at codon 72 had allelic imbalance according to the polymerase chain reaction assay, which occurred in either allele and was associated with p53 mutation and poor prognosis (P=0.01). However, the p53 polymorphism was not associated with clinical features, complete remission rates or prognosis of AML. These results indicate that the p53 genotype at codon 72 is useful to detect loss of heterozygosity but not associated with risk, pathophysiology or therapeutic response of AML.

Bevacizumab Exacerbates Paclitaxel-Induced Neuropathy: A Retrospective Cohort Study

Background Bevacizumab (BEV), a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody, enhances the antitumor effectiveness of paclitaxel (PTX)-based chemotherapy in many metastatic cancers. A recent study in mice showed that VEGF receptor inhibitors can interfere with the neuroprotective effects of endogenous VEGF, potentially triggering the exacerbation of PTX-induced neuropathy. In clinical trials, exacerbation of neuropathy in patients who received PTX combined with BEV (PTX+BEV) has generally been explained by increased exposure to PTX owing to the extended duration of chemotherapy. We investigated whether the concurrent use of BEV is associated with the exacerbation of PTX-induced neuropathy. Methods Female patients with breast cancer who had received weekly PTX or PTX+BEV from September 2011 through May 2016 were studied retrospectively. PTX-induced neuropathy was evaluated at the same time points (at the 6th and 12th courses of chemotherapy) in both cohorts. A multivariate Cox proportional-hazards model was used to assess the independent effect of BEV on the time to the onset of neuropathy. Results A total of 107 patients (median age, 55 years; range, 32–83) were studied. Sixty-one patients received PTX as adjuvant chemotherapy, 23 received PTX for metastatic disease, and 23 received PTX+BEV for metastatic disease. Peripheral sensory neuropathy was worse in patients who received PTX+BEV than in those who received PTX alone: at the 6th course, Grade 0/1/2/3 = 4/13/4/0 vs. 25/42/6/0 (P = 0.095); at the 12th course, 2/3/11/3 vs. 7/30/23/2 (P = 0.016). At the 12th course, the incidence of Grade 2 or higher neuropathy was significantly higher in patients treated with PTX+BEV than in those treated with PTX alone (74% vs. 40%; P = 0.017). In multivariate analysis, BEV was significantly associated with an increased risk of neuropathy (HR 2.32, 95% CI 1.21–4.44, P = 0.012). Conclusions The concurrent use of BEV could worsen PTX-induced neuropathy in patients with breast cancer.