Global DNA hypomethylation in prostate cancer development and progression: a systematic review.

BACKGROUND: The role of global DNA methylation in prostate cancer (PCa) remains largely unknown. Our aim was to summarize evidence on the role of global DNA hypomethylation in PCa development and progression. METHODS: We searched PubMed through December 2013 for all studies containing information on global methylation levels in PCa tissue and at least one non-tumor comparison tissue and/or studies reporting association between global methylation levels in PCa tissue and survival, disease recurrence or at least one clinicopathological prognostic factor. We summarized results using non-parametric comparisons and P-value summary methods. RESULTS: We included 15 studies in the review: 6 studies with both diagnostic and prognostic information, 5 studies with only diagnostic information and 4 studies with only prognostic information. Quantitative meta-analysis was not possible because of the large heterogeneity in molecular techniques, types of tissues analyzed, aims and study designs. Summary statistical tests showed association of DNA hypomethylation with PCa diagnosis (P<0.006) and prognosis (P<0.001). Restriction to studies assessing 5-methylcytosine or long interspersed nucleotide element-1 revealed results in the same direction. Analyses restricted to specific clinicopathological features showed association with the presence of metastasis and tumor stage in all tests with P<0.03, and no association with Gleason score (all tests P>0.1 except for the weighted Z-test, P=0.05). CONCLUSION: DNA hypomethylation was associated with PCa development and progression. However, due to the heterogeneity and small sample sizes of the included studies, along with the possibility of publication bias, this association requires additional assessment.

High prevalence of oral human papillomavirus infection in Fanconi's anemia patients.

BACKGROUND: Fanconi's anemia (FA) is a rare recessive genetic disorder characterized by bone marrow failure, developmental and congenital abnormalities, which frequently evolves to aplastic anemia and neoplasias, primarily acute leukemia and head-neck carcinomas. Risk of malignancies increases after hematopoietic stem cell transplantation (HSCT), and the role of human papillomavirus (HPV) in FA carcinogenesis have been proposed. OBJECTIVE: To investigate prevalence of oral HPV in FA patients without oral malignant lesions. MATERIALS AND METHODS: After oral examination, 76 subjects without detectable oral malignant lesions were included and classified in four groups: 20 FA submitted to HSCT (I), 22 FA not submitted to HSCT (II), 18 severe aplastic anemia (SAA) submitted to HSCT (III) and 16 healthy subjects (IV). Liquid-based cytology sampling, HPV screening by polymerase chain reaction and genotyping by reverse hybridization were performed. RESULTS: The HPV detection rates were: group I 35%, group II 27.3%, group III 38% and group IV 6.25%. Prevalence of high risk HPV types, mainly HPV16, was detected. Compared with control group, suggestions for increased likelihood of being HPV infected in SAA (OR = 9.55, 95% CI: 1.01-125.41) and FA patients submitted to HSCT (OR = 8.08, 0.83-72.29) emerged. CONCLUSION: Patients without oral malignant lesions submitted to HSCT, have high prevalence of oral HPV. HPV screening and close follow up should be considered in these patients.

Interleukin-2 gene-transduced human leukemic cells induce major histocompatibility complex-restricted and -unrestricted anti-leukemic effectors in mixed lymphocyte-tumor cultures.

To explore the feasibility of designing vaccination protocols in acute leukemia patients with cytokine gene-transduced leukemic cells, we studied in vitro the growth potential of human leukemic cells transduced with the interleukin-2 (IL-2), IL-7, or IL-7 plus IL-2 genes, as well as the capacity of generating both autologous and allogeneic cytotoxic lymphocytes directed against the parental cells. A lymphoblastic T-cell line, ST4, obtained from a patient in long-lasting complete remission, was retrovirally engineered with the IL-2, IL-7, and IL-7 plus IL-2 genes; in addition, clones releasing different amounts of the cytokines were obtained by limiting dilution. Mixed lymphocyte-tumor cultures (MLTCs) were set up with parental or transduced leukemic cells as stimulators and with autologous or allogeneic lymphocytes as responders. When nonirradiated ST4 parental cells or clones producing <50 international units (IU)/mL/10(6) cells/72 hours of IL-2 were used as stimulators, leukemic overgrowth was observed in MLTCs within 16 days of culture. When clones producing >80 IU/mL/10(6) cells/72 hours of IL-2 were used as stimulators, the proliferation of leukemic cells was blocked and the transduced leukemic cells were completely cleared from the cultures by day 16; repeated restimulations with IL-2-producing leukemic cells were required to sustain long-term lymphocyte survival. On the contrary, when IL-7- or IL-7-IL-2-producing cells were used as stimulators, only a delay in leukemic cell overgrowth was observed, and lymphocytes were completely cleared from the cultures after day 60. IL-7 production by the different clones ranged between 11 and 36 ng/mL/10(6) cells/72 hours, whereas the highest IL-2-producing IL-7-IL-2 clone released 50 IU/mL/10(6) cells/72 hours of IL-2. When the stimulator efficacy of the highest IL-2-producing clone (ST4/IL-2#A7) was compared with that of exogenous IL-2 plus parental cells, a 7-fold higher amount of exogenous IL-2 was required to achieve the same results obtained with IL-2-producing leukemic cells. Autologous and allogeneic long-term MLTCs (up to 35 days) with ST4/IL-2#A7 as the stimulator were capable of generating cytotoxic effectors equally endowed with both major histocompatibility complex (MHC) class I-unrestricted and -restricted activity against parental ST4 cells. By day 18 of both autologous and allogeneic cultures, a substantial proportion of CD56+ cells was consistently recorded; this was coupled to a predominantly MHC-unrestricted cytotoxic activity directed against parental ST4 cells. CD56+ cells decreased considerably at the end of the different MLTCs, together with the unrestricted cytotoxic activity. At this time, >50% of the cells were CD8+, and 55% of the activity could be blocked by an anti-MHC class I monoclonal antibody. The results of this study demonstrate that IL-2 gene-transduced human acute leukemia cells cocultured with both autologous and allogeneic lymphocytes are capable of inducing a strong MHC-unrestricted anti-leukemic activity and subsequently "educating" MHC class I-restricted anti-leukemic effectors. The evidence that the immunogenic potential of human leukemic blasts can be boosted after transfer of the IL-2 gene suggests that the possibility of using leukemic cells engineered to release IL-2 as a therapeutic vaccine needs to be explored further.

Retroviral vector-mediated transfer of the tumor necrosis factor alpha gene into human cancer cells restores an apoptotic cell death program and induces a bystander-killing effect.

Tumor necrosis factor alpha (TNFalpha) may induce tumor cell death by apoptosis, the physiologic program of cell death usually lost during neoplastic progression. However, many tumor cells are resistant to its effect unless high doses are administered. By retroviral vector-mediated gene transfer, we have transduced the TNFalpha gene into the DNA of human tumor cells to investigate whether the indefinite neoplastic cell proliferation could be blocked and the lost physiologic program of cell death restored. Evidence is provided that high-TNFalpha-producing clones generated from a human lymphoma T-cell line (ST4) can undergo apoptosis following transduction of the TNFalpha gene. Internucleosomal DNA cleavage was documented by May-Grünwald-Giemsa and by propidium iodide staining, as well as by gel electrophoresis. The induced apoptotic phenomenon is TNFalpha-mediated, since it can be reverted following incubation with anti-TNFalpha monoclonal antibodies (MoAbs), and it occurs with cytokine levels released in the supernatant by the engineered cells much lower(>100 times) than those required to promote the same effect on parental ST4 cells following administration of exogenous recombinant TNFalpha. The process is associated with a downregulation of the apoptosis-preventing gene, bcl-2, while the expression of bax and p53, genes usually involved in promoting apoptosis, persists. Mixed-culture experiments performed coincubating TNFalpha-transduced and untransduced ST4 cells allowed documentation of a bystander-killing effect on the parental cells. This phenomenon still occurred at transduced to parental cell ratios as low as 1:20 and was blocked in the presence of an anti-TNFalpha MoAb. These findings indicated that TNFalpha may play a regulatory role in the proliferation of human tumor cells, and suggest potential new antitumor therapeutic strategies based on the direct delivery of the TNFalpha gene into cancer cells.

Cytokine gene therapy in oncology.

Over the last few years the possibility of introducing foreign material into the genome of host cells has become technically feasible. This has opened a new era in the treatment of congenital disorders, but has also offered potential innovative avenues in the management of cancer patients. Here, we shall discuss how cytokine genes may be successfully transduced into the DNA of different experimental tumours and how through this approach the tumorigenicity of the neoplastic cells may be abrogated. Emphasis will be given on the demonstration that following cytokine gene transfer, namely with the interleukin 2 (IL-2) gene, two of the primary goals of an optimal immunotherapeutic approach, i.e. anti-tumour specificity and memory, may be achieved. Attention will be focused, in particular, on the results so far obtained, as well as on ongoing studies, with human tumour cells engineered to release different cytokine genes. The design and activation of the first clinical protocols aimed at treating advanced cancer patients with cytokine gene-transduced neoplastic cells will also be discussed, together with other strategies of genetic engineering currently under investigation.

Constitutive production of tumor necrosis factor-alpha in hairy cell leukemia: possible role in the pathogenesis of the cytopenia(s) and effect of treatment with interferon-alpha.

PURPOSE: In view of the pleomorphic role cytokines play in human lymphoproliferative disorders, we investigated the possible involvement of tumor necrosis factor-alpha (TNF) in hairy cell leukemia (HCL). PATIENTS AND METHODS: The levels of TNF were measured in the serum of untreated patients, and in the culture supernatants of unstimulated and stimulated enriched hairy cells (HC). Furthermore, the presence of TNF mRNA transcripts in HC was analyzed. The possibility that HC could inhibit the in vitro growth of normal erythroid progenitors via the release of TNF was also investigated. Finally, in an attempt to correlate the circulating levels of TNF with the course of the disease, these were retested during and after treatment with interferon-alpha (IFN). RESULTS: Significantly increased levels of TNF were found in the sera of untreated HCL patients compared with normal control sera were seen from patients with other diseases (P less than .001), with values greater than 10 pg/mL in 21 of 42 samples tested. A significant decrease (P less than .01) of TNF levels was recorded following IFN-2a administration in 16 cases with detectable pretreatment serum levels of TNF. In two cases, an increase in TNF values was associated with persistence or progression of disease. The likelihood that the circulating levels of TNF were caused by the pathologic cells is supported by the evidence that purified HC may release TNF spontaneously. The values can be markedly increased following in vitro activation with the phorbol ester 12-0-tetradecanoylphorbol-13 acetate (PMA), with B-cell growth factor (BCGF), and, to a further extent, with the combination of PMA and BCGF. Furthermore, the constitutive mRNA for TNF was found in seven of eight HC samples analyzed. Although supernatants of enriched HC, were capable of reducing the growth of normal bone marrow erythroid progenitors by 50%, duplicate experiments using an anti-TNF antibody produced an almost complete disappearance of the inhibitory effect. CONCLUSION: The results of this study suggest that TNF plays an important role in the pathogenesis of the cytopenia(s) characteristically associated with HCL.

Thrombocytopenia in acute leukaemia patients treated with IL2: cytolytic effect of LAK cells on megakaryocytic progenitors.

In vivo administration of recombinant interleukin 2 (IL2) has been associated, in acute leukaemia as well as in other tumours, with a variable degree of thrombocytopenia. In two patients with acute myeloid leukaemia who showed a progressive and severe fall in platelet count during daily continuous i.v. infusion of IL2, we assessed whether peripheral blood IL2-generated lymphokine activated killer (LAK) lymphocytes could affect growth of the autologous bone marrow megakaryocytic progenitor cell compartment (CFU-MK) in vitro. Following overnight pre-incubation in liquid culture of the marrow cells with autologous LAK effectors, there was an almost complete abrogation of the CFU-MK colony growth (97% and 89% inhibition). Pre-incubation in the presence of a monoclonal antibody to tumour necrosis factor alpha (TNF) completely reversed the inhibitory effect. The role played by TNF was confirmed by the finding that recombinant TNF caused a dose-dependent inhibition of the growth of CFU-MK. IL2 alone was ineffective. These results suggest that the often severe thrombocytopenia observed in patients with acute leukaemia treated with IL2 is at least partly due to autologous LAK cells activated in vivo following the administration of IL2.

Peripheral blood and bone marrow immunophenotypic and functional modifications induced in acute leukemia patients treated with interleukin 2: evidence of in vivo lymphokine activated killer cell generation.

The effect of treatment with interleukin 2 (IL2) on the phenotypic and functional immune system of acute leukemia patients was investigated. Fifteen acute myeloid leukemia and acute lymphoid leukemia patients with evidence of persistent disease were further subdivided into two groups according to the percentage of bone marrow (BM) blasts: group a had 6-15% blasts and group b had 30-65%. Following two cycles of IL2 (Glaxo Imb, Geneva, Switzerland) given i.v. by continuous infusion at escalating doses, no major changes in the proportion of CD3-, CD4-, and CD8-positive cells were encountered in the blood or in the marrow of either group of patients. When these could be retested after four cycles of IL2, a significant increase of CD3+ and CD4+ cells was documented in the peripheral blood (PB), as well as a significant increase of CD3+ cells in the BM. Irrespective of the number of cycles administered, the proportion of CD16+ cells increased significantly in the blood in both groups of patients and in the marrow of group a patients only. The expression of CD25 was significantly enhanced in all samples tested. Following IL2 administration, an enhancement of the natural killer compartment was documented. This was consistently more evident in patients with more limited disease. A significant amplification of the in vitro-induced lymphokine-activated killer function was noted in the BM of the treated patients. Furthermore, we documented the presence both in the PB and in the BM of "spontaneous" lymphokine-activated killer cells generated in vivo following IL2 administration. These results demonstrate that in acute leukemia of both myeloid and lymphoid origin, treatment with IL2 is capable of inducing profound immunophenotypic and functional modifications in PB and in BM lymphocytes, particularly in patients with more limited disease. The evidence of the in vivo activation of cytotoxic cells, particularly in the BM, may help to explain the clinical responses preliminarily observed in individual acute leukemia patients.

Lymphokine activated killer (LAK) activity in lymphoproliferative disorders.

Lymphokine activated killer (LAK) cells are being considered as a new and promising form of immunotherapy in the management of patients with solid tumours. Few informations are instead available on these cytotoxic effectors in haematological neoplasias. Here we shall discuss the possible role of LAK cells in human leukaemias. Evidence will be provided for a rationale in the clinical exploitment of Interleukin 2 (IL2)/LAK cells in the treatment of acute leukaemia patients, whilst the implication of these cytotoxic populations appears more uncertain in chronic lymphoproliferative disorders.