In vivo effects of decitabine in myelodysplasia and acute myeloid leukemia: review of cytogenetic and molecular studies.

Low-dose demethylating agents such as 5-aza-2'-deoxycytidine (decitabine, DAC) and 5-azacytidine (azacitidine, Vidaza) have been explored for the treatment of myelodysplasia, acute myeloid leukemia, and hemoglobinopathies since the early 1980s, aiming to revert a methylator phenotype. Originally, the treatment rationale in hemoglobinopathies was to achieve demethylation of the hypermethylated and hence silent gamma-globin gene locus, thus reactivating synthesis of hemoglobin F (HbF). In myelodysplastic syndrome (MDS), cytogenetic analyses are mandatory for risk stratification and for monitoring response to drug treatment. The current knowledge regarding cytogenetic subgroups as predictors of response to low-dose decitabine in MDS as well as cytogenetic responses caused by demethylating agents is summarized in this review. Decitabine treatment is associated with a response rate that is higher in patients with high-risk cytogenetics (i.e., complex karyotype and/or abnormalities of chromosome 7) than in patients with intermediate-risk cytogenetics (two abnormalities or single abnormalities excluding 5q-, 20q-, and -Y). Following decitabine treatment of patients with abnormal karyotype, approximately one-third achieve a major cytogenetic response that can be confirmed by FISH analyses, while in two-thirds of patients, the abnormal karyotype persists but hematologic improvement may be observed during continued treatment. The most frequently studied gene in myelodysplasia is the cell cycle regulator p15(INK4b). Hypermethylation of p15(INK4b) in MDS is reversed during treatment with decitabine, resulting in reactivation of this gene. In hemoglobinopathies, treatment with demethylating agents leads to reactivation of fetal HbF (the gamma-globin gene locus also possibly being another target for reactivation in MDS), and thus, HbF may potentially act as surrogate marker for activity of decitabine. Other, thus far unidentified hypermethylated genes may also be targets for demethylating agents.

Farnesyltransferase inhibitors inhibit T-cell cytokine production at the posttranscriptional level.

Several cytoplasmic proteins, such as GTPases of the Ras family, containing a C-terminal CAAX motif are prenylated by farnesyltransferase to facilitate localization to cellular membranes where activation occurs. Farnesyltransferase inhibitors (FTIs) interfere with this farnesylation process, thereby preventing proper membrane localization and rendering the proteins unavailable for activation. Currently, FTIs are being explored as antineoplastic agents for the treatment of several malignancies. However, since farnesylated proteins like Ras are also involved in intracellular signaling in lymphocytes, FTIs might interfere with T-cell activation. Based on this hypothesis we examined the effect of several FTIs on cytokine production in response to anti-CD3 + anti-CD28 monoclonal antibodies or PMA + ionomycin. Murine Th1 and Th2 clones, stimulated in the presence of FTIs, showed a dose-dependent reduction of lineage-specific cytokine secretion (IFN-gamma, IL-2, IL-4, IL-5). However, no inhibition of ERK or JNK MAP kinases was observed, nor was induction of cytokine mRNA affected. Rather, intracellular cytokine protein synthesis was blocked. Inhibition of human T-cell INF-gamma production also was observed, correlating with reduced phosphorylation of p70S6K. These results indicate that FTIs inhibit T-cell activation at the posttranscriptional level and also suggest that they may have potential as novel immunosuppressive agents.

Vaccination of advanced prostate cancer patients with PSCA and PSA peptide-loaded dendritic cells induces DTH responses that correlate with superior overall survival.

Prostate stem cell antigen (PSCA) and prostate-specific antigen (PSA) are overexpressed in most prostate cancers. PSCA- and PSA-derived, HLA-A2 binding peptides are specific targets for T-cell responses in vitro. A phase I/II trial was performed to demonstrate feasibility, safety and induction of antigen-specific immunity by vaccination with dendritic cells (DC) presenting PSCA and PSA peptides in patients with hormone- and chemotherapy-refractory prostate cancer. Patients received 4 vaccinations with a median of 2.7 x 10(7) peptide-loaded mature DC s.c. in biweekly intervals. Clinical responses were assessed 2 weeks after the 4th vaccination. Immune monitoring was performed by DTH and HLA multimer analysis. Twelve patients completed vaccination without relevant toxicities. Six patients had stable disease after 4 vaccinations. One patient had a complete disappearance of lymphadenopathy despite rising PSA. Four patients with SD and 1 progressor developed a positive DTH after the 4th vaccination. With a median survival of all patients of 13.4 months, DTH-positivity was associated with significantly superior survival (p = 0.003). HLA tetramer analysis detected high frequencies of peptide-specific T cells after 2 vaccinations in 1 patient who was also the sole responder to concomitant hepatitis B vaccination as an indicator of immune competence and survived 27 months after start of vaccination. Vaccination with PSA/PSCA peptide-loaded, autologous DCs may induce cellular responses primarily in immunocompetent patients, which appear to be associated with clinical benefit. Testing of DC-based vaccination is warranted for patients at earlier stages of prostate cancer.

Expression spectrum and methylation-dependent regulation of melanoma antigen-encoding gene family members in pancreatic cancer cells.

Human MAGE and GAGE genes encode tumor-specific antigens presented by HLA I molecules recognized on tumor cells by cytolytic T lymphocytes. To determine if pancreatic cancer patients would be suitable for MAGE- or GAGE-based immunotherapy, the expression frequency of MAGE-A1, -A2, -A3, -A4, -A6 and GAGE1-8 genes was assessed in 15 pancreatic tumor cell lines and 23 pancreatic tumor specimens using reverse transcription-polymerase chain reaction (RT-PCR). In 67% of the cell lines at least one of the MAGE-A genes was detected, 53% revealed concomitant expression of two or more genes. GAGE1-8 expression was detected in 47% of the cell lines. In the primary pancreatic tumors, MAGE-A analysis revealed exclusive MAGE-A1 and MAGE-A2 gene expression in 26 and 30% of the specimens, respectively, independent from clinicopathologic factors. Treatment of MAGE-A expression-negative pancreatic tumor cells with the demethylating agent 5-aza-2'-deoxycytidine could activate MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4 and GAGE transcription suggesting silencing due to promoter methylation. Interestingly, a metastatic lesion to the liver revealed concomittant expression of MAGE-A1, -A2, -A3 and -A6 consistent with a more pronounced genome-wide hypomethylation in metastases. Therefore, a subset of pancreatic cancer patients could be eligible for active, specific immunotherapy directed against MAGE-A antigens and demethylating agents could increase the number of candidate patients.