ALBERT

Description: The role of the HOX gene family in leukemia development has been extensively studied. However, these studies have focused almost exclusively on the potential oncogenic role of HOX gene family members. In contrast to the oncogenic function often attributed to HOX genes, our studies have identified several HOX gene family members as candidate tumor suppressor genes and shown that inactivation of HOX genes, particularly HOXA4, is associated with poor prognosis. We have used multiple quantitative methylation assays to search for epigenetic inactivation of HOX genes in adult and childhood leukemia. In both adult myeloid and lymphoid leukemia two members of the HOXA cluster (HOXA4 and A5) were found to be frequently inactivated by promoter hypermethylation (26–64% of cases were hypermethylated). In contrast, a further 12 HOXA, B and C cluster genes were found to be essentially devoid of hypermethylation (except HOXA6 in CLL, where 34% of samples exhibited hypermethylation). HOXA4 and HOXA5 were also frequently inactivated in childhood ALL and AML (39–79% of samples). However, in contrast to the adult leukemias, all but one of the additional HOX genes analyzed were also found to be targets for hypermethylation in both ALL and AML (4–26% of samples), suggesting that HOX genes are differentially regulated in childhood versus adult leukemia. Hypermethylation of HOX genes (HOXA4, HOXA5 and HOXA6) was associated with loss of expression of the corresponding gene. Expression analysis also suggests that interaction between different HOX genes may be crucial. In normal karyotype AML samples, those expressing of high levels of HOXA9, but not those with low HOXA9 expression, were associated with invariable HOXA4 hypermethylation (p=0.01). Interestingly HOXA4 hypermethylation also correlates with poor prognosis in all types of leukemia tested. Hypermethylation of HOXA4 correlates with progression to blast crisis (p=0.007) and poor response to imatinib in CML (p=0.04), with cytogenetic status in AML (33%, 72% and 100% in good, intermediate and poor prognostic groups respectively, p=0.0004) and with IgVh mutational status (p=0.003) and poor survival in CLL (median survival 159 versus 199 months in hypermethylated and non hypermethylated patients, respectively). Furthermore transfection of a HOXA4 expressing construct into a CML blast crisis cell line results in re-expression of markers of myeloid differentiation, suggesting that loss of HOXA4 is functionally relevant in leukemic cells. These results indicate that aberrant epigenetic regulation of HOXA4, and indeed other frequently inactivated HOX genes such as HOXA5 and HOXA6, may play a key role in the development of multiple types of leukemia. Thus co-ordinated up and down regulation of expression of HOX gene family members may be crucial in the leukemogenic process.

Description: The aim of this study was to determine the efficacy and toxicity of methotrexate (MTX) and/or rituximab treatment in a novel central nervous system (CNS) lymphoma model, using either intravenous (IV) or intraarterial (IA) drug administration in conjunction with osmotic blood-brain barrier disruption (BBBD). Female athymic nude rats (rnu/rnu) were inoculated with human MC116 B-cell lymphoma cells in the caudate putamen. On the day of treatment and first magnetic resonance imaging (MRI) (day 16–20), rats were randomized into 7 groups: control group with saline IV (n = 15) or IA/BBBD (n = 7); rituximab 375 mg/m2 IV (n = 6) or IA/BBBD (n = 3); MTX 1000 mg/m2 IV (n = 6) or IA/BBBD (n = 6); and rituximab 375 mg/m2 IV + MTX 1000 mg/m2 IV (n = 6). A second MRI scan was done 1 week after treatment. Study end points included tumor response on MRI (defined as ≤75% tumor volume on post-treatment MRI), toxicity, and tumor volumes on histology. In the IV groups, the percent change in tumor volume on T2/FLAIR images in the control versus rituximab group was statistically significant (p = 0.0051). In the IA/BBBD groups, there was a statistically significant difference between control and rituximab (p = 0.0167) for absolute and percent changes in tumor volumes on both T2/FLAIR and T1 with gadolinium sequences. For T1 with gadolinium, the difference between control and MTX was also significant for both absolute change (p = 0.0066) and percent change (p = 0.0043). Response rates in the IV groups differed significantly between controls and rituximab (p = 0.0017 on T2/FLAIR and p = 0.0049 on T1 with gadolinium). A good correlation between tumor volumes (in log mm3) on histology and fluid-attenuated inversion recovery (FLAIR)/T2 (R2 = 0.83) or contrast-enhanced T1 weighted images (R2 = 0.73) for tumors located in the caudate putamen was observed. Results of complete blood counts at sacrifice did not differ significantly between the groups. The MC116 CNS lymphoma model is valuable for preclinical testing of efficacy and toxicity of various treatment regimens. Rituximab and MTX were particularly effective in this model, regardless of the route of administration.