Elisa Paviati

Epigenetic alteration: new insights moving from tissue to plasma – the example of PCDH10 promoter methylation in colorectal cancer

Background:Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical–pathological features.Methods:A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay.Results:The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3–97.8%) and 5.9% (0–80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234).Conclusion:Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.

Comparison of Genetic and Epigenetic Alterations of Primary Tumors and Matched Plasma Samples in Patients with Colorectal Cancer

Background Although recent advances in circulating DNA analysis allow the prediction of tumor genomes by noninvasive means, some challenges remain, which limit the widespread introduction of cfDNA in cancer diagnostics. We analyzed the status of the two best characterized colorectal cancer (CRC) genetic and epigenetic alterations in a cohort of CRC patients, and then compared the degree to which the two patterns move from tissue to plasma in order to improve our understanding of biology modulating the concordance between tissues and plasma methylation and mutation profiles. Methods Plasma and tumor tissues were collected from 85 patients (69±14 years, 56 males). KRAS and SEPT9 status was assessed by allele refractory mutation system quantitative PCR and quantitative methylation-specific PCR, respectively. Six of the most common point mutations at codon 12 and 13 were investigated for KRAS analysis. Results KRAS mutations and SEPT9 promoter methylation were present in 34% (29/85) and in 82% (70/85) of primary tumor tissue samples. Both genetic and epigenetic analyses of cfDNA revealed a high overall concordance and specificity compared with tumor-tissue analyses. Patients presenting with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses. The median methylation rates in tumour tissues and plasma samples were 64.5% (12.2–99.8%) and 14.5% (0–45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.8–86.3%) in tissues and 2.9% (0–17.3) in plasma samples. The plasma/tissue (p/t) ratio of SEPT9 methylation rate was significantly higher than the p/t ratio of KRAS mutation load, especially in early stage cancers (p=0.0108). Conclusion The results of this study show a discrepant rate of epigenetic vs. genetic alterations moving from tissue to plasma. Many factors could affect mutation cfDNA analysis, including both presence of tumor clonal heterogeneity and strict compartmentalization of KRAS mutation profile. The present study highlights the importance of considering the nature of the alteration when analyzing tumor-derived cfDNA.

The impact of sensitive KIT D816V detection on recognition of Indolent Systemic Mastocytosis

Patients with Systemic Mastocytosis (SM) need a highly sensitive diagnostic test for D816V detection of the KIT receptor gene. Along with histology/cytology and flow cytometry evaluation, bone marrow (BM) from 110 consecutive adult patients referred with a suspicion of SM to Multidisciplinary Outpatient Clinic for Mastocytosis in Verona were tested both by Amplification Refractory Mutation System Reverse Transcriptase quantitative real time Polymerase Chain Reaction (ARMS-RT-qPCR) and RT-PCR+Restriction Fragment Length Polymorphism (RFLP) followed by Denaturing-High Performance Liquid Chromatography (D-HPLC) and Sanger sequencing. ARMS-RT-qPCR identified D816V mutation in 77 patients, corresponding to 100% of cases showing CD25(+) mast cells (MCs) whereas RT-PCR+RFLP/D-HPLC+sequencing revealed D816V mutations in 47 patients. According to the 2008 WHO criteria 75 SM, 1 Cutaneous Mastocytosis (CM), 1 monoclonal MC activation syndrome (MMAS), and 1 SM Associated with Haematologic Non-Mast Cell Disorder (SM-AHNMD) were diagnosed. Seventeen out 75 SM patients (23%) would have not satisfied sufficient WHO criteria on the basis of the sole RT-PCR+RFLP: these patients had significantly lower serum tryptase levels and amount of CD25(+) MCs. Therefore, ARMS-RT-qPCR might result particularly useful, in patients that do not fulfil major BM histological criterion, for the recognition of indolent SM with a very low MC burden.

Aberrant MicroRNA Expression in Patients With Endometrial Cancer

Objective Current evidence suggests that no single serum biomarker displays satisfactory diagnostic performance in patients with endometrial carcinoma (EC), the most frequent gynecological cancer in developed countries. However, aberrant tissue microRNA (miRNA) expression has been recently described in EC. Therefore, this study aimed to investigate the differential expression of 4 serum miRNAs and their association with CA125 (cancer antigen 125) and HE4 (human epididymis protein 4) in EC patients and in a control population. Methods Forty-six consecutive women with EC and 28 matched control subjects without a history of cancer or other diseases were enrolled. Total serum RNA was extracted using mirVana PARIS Kit. TaqMan MicroRNA Assay was used for quantitative real-time reverse transcriptase–polymerase chain reaction on ABI 7500 Sequence Detection System to assess differential miRNAs expression. The relative expression levels of 4 miRNAs (miR-222, miR-223, miR-186, and miR-204) were normalized to miR-16 and calculated using the 2-△Ct approach. Results Serum levels of miR-186, miR-222, and miR-223 appeared to be significantly higher in patients compared with control subjects (P = 0.004, P = 0.002, and P < 0.0001). Contrarily, serum miR-204 was found to be significantly lower in EC patients (P < 0.0001). The diagnostic performance of miRNAs was found to be significantly better than that of CA125. Among the various biomarker tested, serum miR-204 and HE4 exhibited the best diagnostic performance for discriminating EC patients from control subjects. Conclusions These results underpin that the 4 miRNAs that we have investigated are implicated in development and progression of EC, thus opening new avenues in EC diagnostics.

Role of JAK2 V617F mutation and aberrant expression of microRNA-143 in myeloproliferative neoplasms.

Abstract Background: Myeloproliferative neoplasms (MPNs) are clonal myeloid disorders characterized by the overproduction of mature blood cells. The pathogenetic hallmark of MPNs is the dysregulation of JAK-STAT signaling, usually associated with the JAK2 V617F mutation. Multiple additional genetic and epigenetic alterations that constitutively activate the JAK-STAT signaling pathway have been described, including the modulation of the microRNAs (miRs) expression levels. The aims of our study were to investigate JAK2 V617F mutation allele burden and miR-143 expression levels in MPNs patients and to investigate the correlation between these genetic signatures and hematological parameters. Methods: In total 78 patients with a clinical diagnosis of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IM), made according to the WHO 2008 criteria, were included in the study. Twenty healthy subjects were checked as controls. Quantification of JAK2 V617F mutation and miR-143 expression levels were determined by real-time quantitative polymerase chain reaction. Results: The miR-143 expression in MPNs patients was 2.97-fold higher than in controls. JAK2 V617F mutation allele burden and miR-143 expression level resulted higher in PV and IM respect to ET patients. Patients who had V617F allele burden >50% displayed a higher miRNA-143 expression level than patients with allele burden <50%. In MPNs patients, a statistically significant positive correlation was observed between JAK2 V617F mutation allele burden and hemoglobin and hematocrit values and between miR-143 expression levels and platelet count. Conclusions: Our findings of aberrant miR-143 expression support the concept that factors other than JAK2 V617F mutation may contribute to the pathogenesis and some clinical signs of MPNs.

Reference miRNAs for colorectal cancer: analysis and verification of current data

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

Evaluation of mir-203 Expression Levels and DNA Promoter Methylation Status in Serum of Patients with Endometrial Cancer

BACKGROUND Endometrial cancer (EC) is currently considered the fourth most frequent female cancer in Europe. In an attempt to achieve an early diagnosis, many studies have identified some putative biomarkers for gynecologic cancers, including circulating microRNAs (miRs) and aberrant promoter methylation status. Previous studies which have investigated miR-203 expression profiles in EC tissues and normal endometrial tissues concluded that miR-203 is regulated by methylation promoter. The aim of this study was to investigate the expression of miR-203 and promoter methylation levels in serum of EC patients and healthy controls (HC). METHODS Forty-five EC patients (64 ± 12 years) and 30 HC (63 ± 13 years) were enrolled before undergoing therapeutic procedures. RNA extraction from serum was performed with mirVana PARIS Isolation Kit (Thermo Scientific). miR expression was assessed by quantitative RT-PCR (Applied Biosystems). The expression levels of miR were normalized to miR-16 and calculated using the 2-ΔCt method. A quantitative methylation-specific PCR (MSP) technique was used to analyze miR-203 promoter methylation status. Differences between groups were assessed by Mann-Whitney test (for continuous variables) and chi-squared test (for categorical variables), whereas the correlation was calculated using Spearmans test. The diagnostic performance of miR-203 was defined using receiver operator characteristic (ROC) curves. RESULTS Serum expression levels of miR-203 were higher in EC patients compared to HC (p = 0.002). Aberrant miR-203 methylation was detected in 11/45 (24.4%) EC patients and in 2/30 (6.6%) HC (p = 0.046). The expression levels of miR-203 were not significantly correlated with promoter methylation status. The area under the curve of miR-203 expression was 0.71 (p = 0.002). CONCLUSIONS The high circulating miR-203 expression levels in EC patients compared to HC confirm the role of this miR as a potential biomarker for diagnosis of EC. Aberrant miR-203 methylation assessed in the peripheral blood does not apparently reflect cancer biology.

Influence of middle-distance running on muscular micro RNAs

Abstract A specific subset of micro RNAs (miRs), including miR-133 and miR-206, is specifically expressed in muscle tissue, so that they are currently defined as muscular miRs (myomiRs). To further elucidate the role of myomiRs in muscle biology, we measured miR-133a and miR-206 in plasma of 28 middle-age recreational athletes. The study population consisted of 28 middle aged, recreation athletes (11 women and 17 men; mean age, 46 years) who completed a 21.1 km, half-marathon. The plasma concentration of miR-133a and miR-206, the serum concentration of creatine kinase (CK) and high-sensitivity (HS) cardiac troponin T (cTnT), as well as capillary lactate, were measured before and immediately after the run. The median serum concentration of total CK (257 versus 175 U/L; p < .001), cTnT (17.8 versus 5.6 ng/L; p < .001), and the plasma values of both miR-133a (4.22 versus 0.64 × 10−4; p < .001) and miR-206 (1.36 versus 0.63 × 10−4; p = .001) were considerably increased immediately after the half-marathon run. In multivariate analysis only post-exercise capillary lactate was found to be independently associated with running time. A significant and independent correlation was observed between plasma variations of the two miRs, but not with other physiological or laboratory parameters. The results of this study suggest that the biological significance of miR-133a and 206 variation after middle-distance running parallels but not overlaps the release of biomarkers of nonspecific tissue damage. Enhanced plasma values of these myomiRs may hence reflect a physiological response to high-intensity and/or prolonged exercise rather than tissue injury.

The clinical significance of DJ-1 and HE4 in patients with endometrial cancer

The non‐invasive diagnostic approach for early detection of endometrial cancer (EC) remains limited. To date, human epididymis protein 4 (HE4) has been intensively studied but its diagnostic is controversial in EC. DJ‐1 is an oncoprotein secreted by cancer cells, recently identified as a potential diagnostic biomarker for breast cancer, melanoma, and pancreatic cancer. The aim of this study was to compare the diagnostic performances of DJ‐1 and HE4 measured in EC patients and healthy controls (HC).

A novel large-sized BCR-ABL transcript in a case of chronic myeloid leukaemia characterised by a favourable clinical course

Dear Editor, The reciprocal translocation t(9;22)(q34;q11) is detected in 95 % of chronic myeloid leukaemia (CML) as well as in 20– 30 % of adult [1] and 2–5 % of childhood acute lymphoblastic leukaemia [2]. It involves the breakpoint cluster region (BCR) gene locus on chromosome 22. Apart from the two principal breakpoints (the major BCR, between exons e12 and e16, and the minor m-BCR, in the first BCR intron) and the rare μ-BCR in exon e19, other breakpoints are seldom observed [3–5]. The correlation between BCR gene breakpoints and the CML phenotype remains a debatable issue. A few authors believe, however, that small BCR-ABL transcripts might have a stronger kinase activity than the large-sized ones, thus inducing more aggressive CML [6]. We describe here a novel large-sized BCR-ABL transcript, which was associated with a CML characterised by a favourable outcome. An 80-year-old female was referred to our institution in January 2008 due to detection of leukocytosis. The haemogram showed the following: haemoglobin 11.6 g/dl, platelet count 442×10/l and leukocyte count 63.0×10/l, with 71 % neutrophils, 3 % myelocytes, 2 % metamyelocytes, 12 % monocytes, 6 % lymphocytes, 5 % basophils and 1 % eosinophils. Bone marrow aspirate showed a mild hypercellularity and an increased myeloid/erythroid ratio; no blasts were detectable. Chromosome analysis showed a 46,XX,t (9;22)(q34;q11.2) karyotype in 21 out of 23 metaphases. The physical examination did not show spleen enlargement. Following the diagnosis of intermediate risk [7] chronicphase CML, the patient underwent treatment with imatinib at 400 mg/day, subsequently reduced to 300 mg/day due to myelotoxicity. Before treatment, peripheral blood standard qualitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed [8]: single step analysis evidenced a major BCR-ABL transcript larger than the one deriving from e14a2 (Fig. 1a). The presence of a larger transcript was subsequently confirmed by multiplex RT-PCR (Fig. 1b) [4]. The sequencing analysis (Beckman Coulter CEQ 8800) of RT-PCR products detected the joining of BCR exon e14 and ABL exon a2, with a 60-bp insert in-between showing a complete homology to the entire BCR exon e17 (NCBI BLAST) (Fig. 1c). The transcript conserved the annealing sites for real time quantitative PCR (RQ-PCR) primers [9], thus allowing the monitoring of the molecular response to treatment. Accordingly, the BCR-ABL/ABL ratio was 47 % at diagnosis, 0.04 % at 5 months and 0.0313 % at 12 months. At present, after a 48-month follow-up, the patient presents a BCRABL/ABL ratio of 0.0007 %. Concerning the e13/a2 and e14/a2 CML rearrangements, few cases of in-frame insertion of a pseudo-exon between coding BCR and ABL sequences have been reported [10] and an e14/a2 rearrangement including exon e17 has not previously been described. Noteworthy, the insertion of exon e17 with the loss of BCR exons e15 and e16 allowed the restoration of the reading frame, thus generating a functional oncoprotein, which induced a disease with a favourable outcome. With regard to clinical relevance, we believe that only a thorough description of cases associated with atypical transcripts will help to establish the possible prognostic role of different, unusual BCR-ABL transcripts. G. De Matteis (*) : F. Aprili :M. Benati : E. Paviati :G. C. Guidi Department of Life and Reproduction Sciences, Clinical Biochemistry Laboratory, University of Verona, Policlinico G.B. Rossi, P.le L.A. Scuro, Verona, Italy e-mail: giovanna.dematteis@ospedaleuniverona.it