Ebru E. Akisik

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

Abstract 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. Clin Chem Lab Med 2008;46:311–7.

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.

Genotyping of the MTHFR Gene Polymorphism, C677T in Patients with Leukemia by Melting Curve Analysis

AbstractBackground: Methylenetetrahydrofolate reductase (MTHFR) plays a critical role in folate metabolism and displays common genetic polymorphisms affecting the enzyme activity. The MTHFR genetic polymorphisms have been associated with a decrease in the risk of developing the lymphoid but not myeloid form of pédiatric and adult leukemias. Aim: In this study we describe the genotyping of the MTHFR C677T polymorphism by melting curve analysis with the LightCycler® in a case-controlled study of patients with acute lymphocytic leukemia (ALL), myelogenous leukemia (AML), and chronic myelogenous leukemia (CML), and assess the effect of this common polymorphism on the leukemia risk in adult patients in Turkey. Methods: DNA from peripheral blood lymphocytes was used for genotyping in the LightCycler® PCR by melting curve analysis. The risk of leukemia associated with the MTHFR polymorphism was evaluated by comparing the genotype frequencies between the control and patient groups. Results: The frequency of the homozygote variant genotype (677TT) was lower than that in healthy individuals in all three leukemia groups. The 677TT genotype did not appear to have a protective effect in patients with ALL (Odds ratio [OR] = 0.78 with a 95% confidence interval [CI] = 0.24–2.59), compared with healthy controls. The difference was higher (4.3-fold) in patients with AML, but still non-significant (OR = 0.23 with a 95% CI = 0.03–1.83). In patients with CML, the frequencies of both heterozygous (677CT) and homozygote variant genotypes were lower (OR = 0.72 and 0.66, respectively). Conclusions: Our results suggest that the MTHFR C677T polymorphism displays a similar distribution pattern in lymphoid and myeloid leukemias and that the frequency of the homozygote variant genotype (677TT) is lower in all leukemia types.

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.

Investigation of themiR16-1 (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.

H3K9me3/H4K20me3 Ratio in Circulating Nucleosomes as Potential Biomarker for Colorectal Cancer

Detection of cancer-related histone modifications of nucleosomes in the circulating blood may be useful in early cancer diagnosis. We have analyzed the trimethylation of histone H3 lysine 9 (H3K9me3) and histone H4 lysine 9 (H4K20me3), two modifications involved in heterochromatin formation, at pericentric heterochromatin of circulating nucleosomes in the blood plasma of healthy individuals, patients with either colorectal cancer or multiple myeloma. H3K9me3 was found to be decreased in colorectal cancer and increased in multiple myeloma while H4K20me3 levels were similar in all study groups. Therefore, we used the H3K9me3/H4K20me3 ratio for normalizing H3K9me3 concentrations; it significantly distinguished patients with colorectal cancer (median 0.8) from the healthy group (median 3) and multiple myeloma (median 4.7) (both p < 0.001). We conclude that if validated in a larger series of cases the ratio H3K9me3/H4K20me3 might be a potential diagnostic biomarker for colorectal cancer.

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.

Abstract A08: Analysis of MRGBP and PPIL1 genes in colon cancer

Colorectal cancer is one of the most common causes of cancer death throughout the world. Colorectal carcinogenesis is a multi-step process and in addition to genetic changes significant changes in DNA methylation and epigenetic events such as histone modifications can also lead to deregulation of gene expression. The histone proteins play a dynamic role in the chromatin structure and transcription. Aberrant protein acetylation, particularly on histones, has been related to cancer while abnormal expression of histone deacetlytransferases (HDACs) are observed in a broad range of cancer types. Therefore, HDACs have emerged as promising targets in cancer therapeutics. The MRG-binding protein (MRGBP / C20orf20) is a component of the NuA4 histone acetyltransferase (HAT) complex and is involved in the transcriptional activation of specific genes. The MRGBP gene is frequently upregulated in tumors and is thought to play a role in cancer development. On the other hand removal of introns from pre-mRNA is carried out by a large macromolecular spliceosome complex. PPIL1 (Peptidyl-prolylisomerase-like 1) is a component of the human spliceosome and plays a primary role in pre-mRNA splicing for catalyzing isomerization of the peptide bonds. Overexpression of the PPIL1 protein in cancer suggest a potential role of PPIL1 in the in the development and promotion of colon cancer. In the present study we analyzed MRGBP and PPIL1 gene expression levels in matched tumor and normal tissue samples from patients with colorectal cancer. Tissue samples were collected from 51 patients with colorectal cancer who underwent surgery for tumor resection. The normal tissue specimens used in the study were histologically confirmed to be free of cancer. The HPRT (hypoxanthine-Guanine phosphoribosyltransferase) gene was used as reference. The target and reference genes were amplified in the same multiplex PCR using the LightCycler 480 System. 8-9 nucleotide long UPL probes were labeled with fluorescein (FAM) and TAMRA at the 5′ and 3′ ends, respectively. Expression levels were determined using the Basic Relative Quantification analysis software and the results were evaluated by the Mann-Whitney U test. Although the median MRGBP expression levels in the tumor tissue were higher than normal tissue (1,72 vs.1,41) the difference was not significant. The median expression levels of PPIL1 in the tumors and normal tissue samples (1,97 vs 1,86) were also similar. Our results indicate that neither MRGBP nor PPIL1 gene expression levels are upregulated in colon cancer cells indicating that these are not directly associated with colorectal cancer. Citation Format: Ebru Akisik, Nejat Dalay, Sumer Yamaner, Dursun Bugra. Analysis of MRGBP and PPIL1 genes in colon cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A08.