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Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1–positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-transretinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1–positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1–positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1–positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients.

High-risk human papillomaviruses (HPVs) have been proposed to be associated with a subset of head and neck cancers (HNSCCs). However, clear biological evidence linking HPV-mediated oncogenesis to the development of HNSCC is hardly available. An important biological mechanism underlying HPV-mediated carcinogenesis is the inactivation of p53 by the HPV E6 oncoprotein. In the present study we investigated this biological relationship between HPV and HNSCC. In total 84 HNSCC tumors were analyzed for the presence of high-risk HPV nucleic acids by DNA polymerase chain reaction-enzyme immunoassay (PCR-EIA) and E6 reverse transcriptase (RT)-PCR as well as for the presence of mutations in the p53 gene. We found 20/84 HPV16 DNA-positive cases with one or more DNA assays, 10 of which were consistently positive with all assays. Only 9/20 cases showed E6 mRNA expression, indicative for viral activity. Only these nine E6 mRNA-positive cases all lacked a p53 mutation, whereas both the other HPV DNA-positive and HPV-DNA negative tumors showed p53 mutations in 36% and 63% of the cases, respectively. Moreover, only in lymph node metastases of HPV E6 mRNA-positive tumors both viral DNA and E6 mRNA were present. Our study provides strong biological evidence for a plausible etiological role of high-risk HPV in a subgroup of HNSCC. Analysis of E6 mRNA expression by RT-PCR or alternatively, semiquantitative analyses of the viral load, seem more reliable assays to assess HPV involvement in HNSCC than the very sensitive DNA PCR analyses used routinely. © 2001 Wiley-Liss, Inc.

Dendritic cell (DC)-based immunotherapy faces new challenges since efficacy of DC vaccines in clinical trials has been inconsistent. Strategies to improve immune responses induced by DC are currently being explored. We have recently shown the feasibility of generating fully functional DC from Acute Myeloid Leukemic (AML) blasts, but with varying expression levels of the important costimulatory molecule CD86. To overcome this variability, we developed a novel bispecific diabody (BsDb) simultaneously and agonistically targeting CD40 on AML-DC and CD28 on nai¨ve T cells. Beside optimization of CD28-mediated signaling, the resulting cellular cross-linking was also hypothesized to increase the strength and duration of T cell/AML-DC interactions, thus increasing T cell responsiveness to AML antigens. Indeed the aCD40/aCD28-bispecific diabody provokes increased T cell-DC cluster formation as assessed by light microscopy. Significant increased cluster formation was observed when T cells and AML-DC were cocultured in presence of the BsDb as compared to T cells incubated with a control protein (46%±2 versus 22%±1 respectively, p<0.05). Prior incubation of T cells and/or AML-DC with CD28 or CD40, respectively, completely prevented cluster formation in presence of the BsDb indicating specific binding of the BsDb to CD40 and CD28. The aCD40/aCD28 BsDb significantly increases T cell proliferation induced by AML-DC as compared to the unstimulated cocultures, in a dose dependent manner, as evaluated by mixed lymphocyte reactions (fold increased T cell proliferation of cocultures stimulated with BsDb as compared to unstimulated cocultures:170%±12, p<0.05). In addition, BsDb is capable of DC maturation induction as shown by significant increased mean fluorescence index (MFI) of the maturation markers CD80 (MFI of AML-DC cultured in presence of control protein vs AML-DC cultured in presence of BsDb: 22±5 vs 12±3, p<0.05) and CD83 (4±1 vs 1.5±0.5, p<0.05). In order to determine the effect of aCD40/aCD28-bispecific diabody-mediated cross-linking of AML-derived DC and CD8+ T cells on the induction efficiency of tumor-specific CTL, AML-DC derived from the HLA-A2+ AML cell line MUTZ-3 were pre-incubated with the aCD40/aCD28-bispecific diabody, loaded with the heteroclitic variant of the aa988 epitope of hTERT, and used as stimulator cells in an HLA-A2-matched allogeneic in vitro CTL induction protocol. In total nine parallel bulk cultures, were stimulated twice with peptide-loaded MUTZ-3 DC, either pulsed with control protein or the aCD40/aCD28-bispecific diabody. hTERT988Y-specific CD8+ T cells could be detected in 5/9 individual cultures when stimulated with DC pulsed with the aCD40/aCD28-bispecific diabody, whereas in only 1/9 individual cultures hTERT988Y-specific CD8+ T cells could be detected when stimulated with DC pulsed with the control protein. Thus, priming efficacy of tumor-specific cytotoxic T cells can also be improved by cross-linking AML-DC and T cells with the aCD40/aCD28 diabody. We propose that the aCD40/aCD28-bispecific diabody can serve as a potent therapeutic tool to effectively augment anti-tumor T cell responses elicited by AML-DC.

The proteins of the Fanconi anemia/BRCA pathway are thought to function in specific DNA repair processes. Disruption of these genes results in genomic instability and hypersensitivity to DNA cross-linking agents. As illustrated by patients with Fanconi anemia (FA) [OMIM#227650] this cellular phenotype confers an increased risk for the development of malignancies, in particular acute myeloid leukemia (AML). In the past years several studies have shown that inherited and somatic abnormalities in individual FA genes play a role in the development of sporadic cancers. To study the involvement of the FA genes in sporadic cases of leukemia we analyzed the presence of aberrant promoter methylation of the 11 known FA genes by Methylation Specific-Multiplex Ligation dependent Probe Amplification (MS-MLPA).

In acute myeloid leukemia (AML) promoter methylation has been observed for the estrogen receptor (ESR1) as well as for a number of Tumor Suppressor Genes (TSGs). These individual aberrancies were suggested to be part of a general methylation defect in subsets of AML patients, rather than random events. The objective of this study was to assess whether aberrant promoter methylation of multiple genes, as observed in AML samples, are associated and whether such associations render impact on clinical outcome. By Methylation-Specific Multiplex Ligation Probe Amplification (MS-MLPA) the methylation status of 26 TSGs was determined in bone marrow samples of 119 primary AML patients and 5 control individuals. No promoter methylation was detected in any of the controls, while at least one TSG was methylated in 59/119 patients. Methylation was observed in 12 out of 26 assessed sites, most frequently for ER, CDKN2B/p15,and IGSF4(28–36% of all patients). A substantial intra-class correlation of 0.38 existed between methylation of different TGSs. ESR1methylation (34/119) strongly predicted concurrent methylation of TSGs, OR 7.33 (95%CI 4.13–12.99). A regression model that included both the ESR1methylation status and the number of methylated TSGs (methylation index), showed both parameters to be independent oppositely directed predictors for overall survival (OS), HR 0.06 (95%CI 0.01–0.33; p=.001) and HR 1.92 (95%CI 1.19–3.10; p=.007), respectively. In line with this observation, a higher methylation index was found to yield a significant negative effect on patient OS in both the ESR1methylated (ESR1+) and ESR1unmethylated (ESR1−) subgroups.