Investigation of the miR16-1 (C > T) + 7 Substitution in Seven Different Types of Cancer from Three Ethnic Groups.

Background. MicroRNAs are a type of small noncoding RNA molecules that have been shown to control gene expression in eukaryotes. Aberrant expression and alteration of miRNAs may be responsible for human diseases including cancer. An miR16-1 (C > T) + 7 gene mutation has been previously found in familial chronic lymphocytic leukemia patients, one of which reported a family history of breast cancer. miR16-1 regulates the expression of bcl-2, which is important in retinoblastoma, and is located in a genomic region that is frequently lost in nasopharyngeal and hepatocellular carcinomas (HCCs). Therefore, miR16-1 may be potentially important in the etiology of several solid tumors. To understand the power of the miR16-1 (C > T) + 7 mutation as a prognostic and diagnostic risk factor, we investigated the mutation in patients with seven different types of cancer including 188 with breast, 102 with ovarian, and 22 nasopharyngeal carcinomas, 96 HCC, 872 chronic myeloid leukemia (CML), 39 chronic lymphocytic leukemia (CLL), and 46 retinoblastoma cases from three different ethnic groups and of hereditary and sporadic etiology. Methods. 5'Nuclease TaqMan SNP genotyping assay was used to detect the miR16-1 gene C > T substitution. Results. The miR16-1 (C > T) + 7 substitution was not detected in any of the groups studied. Conclusions. Considering the large scale of our study, the representation of different ethnicities and levels of hereditary risk, we conclude that the miR-16-1 (C > T) + 7 mutation is not a good diagnostic or prognostic indicator of risk for the cancers tested.

Effect of adjuvant chemotherapy on integrity of free serum DNA in patients with breast cancer.

It is not known how chemotherapy-induced cell death influences the size distribution of circulating free DNA (cf-DNA) in serum or plasma of cancer patients. In the present study, we investigated the integrity of cf-DNA during adjuvant systemic therapy in patients (n= 41) with invasive breast cancer. Sera taken at the beginning and the end of the adjuvant chemotherapy were comparatively analyzed for the integrity of cf-DNA. The assay was based on quantification of shorter and longer fragments representing apoptotic or non-apoptotic DNA from abundant genomic ALU repeats by quantitative real-time PCR. The ratio of longer to shorter fragments showed the integrity of free serum DNA. During chemotherapy, in half of the patients (51.2%), total DNA levels increased, but decreased in the other half. The distribution of the DNA integrity in the whole patient group after the systemic therapy (median 0.31) did not significantly differ from that at the beginning (median 0.29, P= 0.39). However, in the subgroups, the variation of the DNA integrity was related to the course of the total DNA level. In the subgroup with an increasing DNA level, the median DNA integrity was elevated from 0.27 to 0.39 (P= 0.005), whereas in the group with a decrease it declined from 0.34 to 0.28 (P= 0.044). Our results show that longer fragments released from non-apoptotic cells are the main contributors to increasing DNA levels during adjuvant systemic therapy. This information might be helpful in evaluating the response of patients to adjuvant systemic therapy.

Sequence-specific histone methylation is detectable on circulating nucleosomes in plasma.

BACKGROUND: alterations in DNA methylation and histone modifications have been implicated in carcinogenesis. Although tumor-specific alterations in DNA methylation can be detected in the serum and plasma of cancer patients, no data are available on the presence of histone modifications in circulating blood. We investigated whether histone methylation, as a model of histone modifications, is detectable in plasma. Because methylation at histone 3 lysine 9 (H3K9) has been demonstrated to be enriched at sites of repetitive ALU elements, we addressed the specificity of histone-methylation detection and hypothesized that if monomethylated H3K9 (H3K9me1) is detectable in plasma, the concentrations in mononucleosomes and oligonucleosomes would be different. We also analyzed a single-copy gene, CDKN2A. METHODS: we enrolled 21 multiple myeloma patients in the study. We used ELISA and real-time PCR analysis to evaluate nucleosomes and cell-free DNA, respectively, as evidence of the presence of histones and associated DNA in circulating blood. H3K9me1 was analyzed by chromatin immunoprecipitation. RESULTS: ELISA and real-time PCR assays indicated the presence of free nucleosomes and DNA in plasma, and the results were quantitatively correlated (P < 0.001). The detection of histone methylation on free nucleosomes was sequence dependent. Fragments representing mono- and oligonucleosomes differed with respect to H3K9me1 concentrations (P = 0.004), in accordance with our hypothesis. In addition, the detection rate and concentrations of H3K9me1 were significantly higher on the fragment covering both mononucleosomes and oligonucleosomes than on the CDKN2A promoter (P < 0.001). CONCLUSIONS: if validated in further studies, our findings may be a basis for investigations of cancer-specific alterations in histone modifications in the circulation.

Size distribution of circulating cell-free DNA in sera of breast cancer patients in the course of adjuvant chemotherapy.

BACKGROUND: The integrity of circulating cell-free DNA (cf-DNA) in serum or plasma appears to be of diagnostic and prognostic value in cancer. Here, we investigated the dynamics of serum DNA levels and the size distribution of cf-DNA during adjuvant chemotherapy of patients with breast cancer (n=73). METHODS: By evaluating sera taken at the beginning and the end of the adjuvant chemotherapy, variations of serum DNA levels and the size distribution were analyzed, based on quantification of shorter apoptotic and longer non-apoptotic fragments from abundant genomic ALU fragments amplified by quantitative real-time PCR. RESULTS: The mean DNA level did not change significantly during chemotherapy. However, individual cases revealed considerable variation in the amount of serum DNA. It increased in 43.8% of the patients, whereas it decreased in the remaining majority (56.2%). By calculating a "coefficient of variation" (both decrease and increase) in the level of total DNA and non-apoptotic DNA fragments, we compared the values at the beginning and the end of the therapy. For total DNA, the range was between 1.02- and 26-fold (mean 3.76-fold), whereas for non-apoptotic fragments it ranged from 1.01- to 73-fold (mean 6.9-fold) (p=0.033). In accordance with these findings, the integrity of serum DNA was higher in patients with increasing DNA levels and vice versa. CONCLUSIONS: Our findings suggest that non-apoptotic fragments contribute to a higher degree to the change of the DNA level during adjuvant chemotherapy.

Lack of association of XRCC1 codon 399Gln polymorphism with chronic myelogenous leukemia.

BACKGROUND: Recent studies suggest that single nucleotide polymorphisms in different genes may modulate the susceptibility to chronic myelogenous leukaemia (CML). Here, the association of the common XRCC1 gene polymorphism Arg399Gln at codon 399 in CML was investigated. PATIENTS AND METHODS: Genotyping was performed by melting curve analysis in samples from peripheral blood or bone marrow. RESULTS: The frequency of the variant allele 399Gln was similar between the control group and the patients (35.2% and 34.9%, respectively; p = 0.21). Similarly, the heterozygote and homozygote variant genotypes displayed a homogenous distribution in both groups (p > 0.05 for all comparisons). Moreover, distribution of the variant allele and subgenotypes did not significantly differ between the patient subgroups with a diagnosis age below or above 50 years. CONCLUSION: To our knowledge, this is the first study to investigate the role of any XRCC1 polymorphism in CML and our findings do not support a role of codon 399Gln polymorphism in CML.

MTHFR C677 T gene polymorphism in lymphoproliferative diseases.

Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, has been implicated in cancer risk. In the present study we used a melting curve analysis to investigate the association of the common MTHFR C677 T polymorphism with lymphoproliferative diseases. Patients (n=117) were compared with age- and sex-matched control subjects (n=154). Our results indicate that the 677 T variant occurred less frequently in patients (26%) than in the control group (33.7%; P=0.05). Investigation of the variant allele (677 T) frequency in the subgroups with Hodgkin's lymphoma (HL) and B-cell neoplasms (BCNs) revealed that this difference was a result of the significantly lower distribution of the variant allele in patients with HL (20.5%; P=0.01). This was accompanied by a significantly higher frequency of the homozygote normal genotype (677CC) among the patients with HL. In patients with BCNs the distribution of the variant allele (30.3%) was comparable to that in the control group (P=0.47). However, the difference between HL (20.5%) and BCNs (30.3%) did not reach statistical significance (P=0.09). Our results suggest that the distribution of the C677 T polymorphism may vary among lymphoproliferative diseases.

Circulating fragmented nucleosomal DNA and caspase-3 mRNA in patients with lymphoma and myeloma.

Elevated amounts of cell-free nucleic acids are detected in the circulation of cancer patients. The type and pattern of these may vary depending on the origin. Recently, we described the presence of circulating fragmented nucleosomal DNA. In the present study, our aim was to investigate the association between nucleosomal DNA, caspase-3 expression and circulating caspase-3 mRNA, the primary activator of nucleosomal DNA fragmentation. DNA fragmentation was analyzed by gel electrophoresis, and caspase-3 was analyzed by RT-PCR. Plasma samples from 65% of patients were positive for nucleosomal DNA fragmentation, whereas none of the healthy controls showed DNA fragmentation (P = 0.0004). Expression of caspase-3 differed significantly between the cases and controls (P < 0.0001). However, we observed no direct correlation between nucleosomal DNA fragmentation and caspase-3 expression in lymphocytes (P = 0.145). Circulating plasma caspase-3 mRNA was detected by nested RT-PCR, and no significant difference was observed between the patients and the control group (P = 0.5). Our results indicate that caspase-3 expression is increased in lymphocytes from patients. When compared to healthy individuals, no differences were observed in the amount of circulating mRNA. These findings suggest that nucleosomal DNA fragmentation is not correlated with elevated levels of apoptosis and circulating caspase-3 mRNA in circulating tumor cells.

Methylation of the INK4A/ARF locus in blood mononuclear cells.

In the detection of DNA hypermethylation as a tumor-specific epigenetic change in blood mononuclear cell fraction in patients with lymphoid and hematopoetic disorders, circulating tumor cells originating from the lymph nodes or bone marrow can be identified. However, it is still not clear whether methylation in mononuclear cells is disease specific. In the present study, we investigated whether methylation of the inhibitor of cyclin-dependent kinase (INK) 4A/alternative reading frame (ARF) locus is present in a disease-specific manner in the blood mononuclear cell fraction of patients with lymphoma, multiple myeloma, or leukemia. To increase the sensitivity of detection, a two-step methylation-specific PCR approach was used to analyze the methylation status of the promoter/exon 1 regions of both p14ARF and p16INK4A genes. Our findings indicate that although INK4A/ARF locus methylation is present in mononuclear cells, this event is not disease-specific since normal subjects also display methylated DNA in their mononuclear cells. In 85.1% of the patients and in 89% of the controls, p16INK4A gene was methylated, while the methylation rates for the p14ARF gene was 32.6 and 36.5%, respectively. The presence of methylated CpG sites in DNA in samples from normal subjects was confirmed by bisulfite genomic sequencing. The difference in the methylation rate between p16INK4A and p14ARF genes among the patients was highly significant (p<0.001). Our results demonstrate that methylation of the INK4A/ARF locus is not a disease-specific molecular change in mononuclear cell fraction and that the p14ARF and p16INK4A genes are differentially methylated.

Gender- and age-related distinctions for the in vivo prooxidant state in Fanconi anaemia patients.

Some selected oxidative stress parameters were measured in 56 Fanconi anaemia (FA) patients (42 untransplanted and 14 transplanted), 54 FA heterozygotes (parents) and 173 controls. Untransplanted FA patients showed a highly significant increase in leukocyte 8-hydroxy-2'-deoxyguanosine (8-OHdG) (P = 0.00003) and a borderline increase (P = 0.076) in urinary levels of 8-OHdG versus child controls. These increases were more pronounced in female FA patients (P = 0.00005 for leukocyte 8-OHdG and P = 0.021 for urinary 8-OHdG). Female FA patients also displayed a highly significant excess of spontaneous chromosomal breaks versus male patients (P = 0.00026), in the same female:male ratio ( approximately 1.4) as detected for both leukocyte and urine 8-OHdG levels. Plasma methylglyoxal (MGlx) levels were increased in untransplanted FA patients versus child controls (P = 0.032). The increases in leukocyte and urinary 8-OHdG and in MGlx levels were detected in young FA patients (< or =15 years), whereas patients aged 16-29 years failed to display any differences versus controls in the same age group. A significant increase in oxidized:reduced glutathione (GSSG:GSH) ratio was observed (P = 0.046) in the FA patients aged < or =15 years, whereas those aged 16-29 years, both untransplanted and transplanted, displayed a decrease (P = 0.06) in the GSSG:GSH ratio versus the controls of the respective age groups. No significant changes were detected in plasma levels of vitamin C, vitamin E or uric acid. Transplanted FA patients showed lesser alterations in leukocyte 8-OHdG and in GSSG:GSH ratio versus untransplanted patients. The parents of FA patients displayed a significant increase in plasma MGlx levels (P = 0.0014) versus adult controls. The results suggest a gender- and age-related modulation of oxidative stress in FA patients. The observed increase in urinary 8-OHdG in untransplanted FA patients suggests a proficient removal of oxidized DNA bases.