Microsatellite alterations and TP53 mutations in plasma DNA of small-cell lung cancer patients: Follow-up study and prognostic significance

Background: Small-cell lung cancer (SCLC), one of the major types of lung cancer, is associated with many different somatic molecular genetic changes. These alterations, observed in tumor DNA, have also been identified in the plasma DNA of patients. We undertook the present study to make a prospective investigation into the correlation between abnormal plasma DNA and patient survival. Patients and methods: Thirty-five patients with SCLC were selected after histological diagnosis. Polymorphic markers (ACTBP2, UT762 and AR) were chosen for their reported high rate of alterations in SCLC and analyzed in tumor tissue, normal blood cells and plasma DNA. Furthermore, we looked for mutations of the TP53 gene in tumor and plasma DNA. Results: In 25 patients (71%) at least one molecular change precisely matching that of the primary tumor was detected in the plasma DNA. No difference in survival was observed between patients with aberrant plasma DNA and patients without plasma DNA alterations. However, patients with microsatellite modifications and TP53 mutations concomitantly, showed a significant difference (P = 0.02) in survival compared with patients bearing only one of these molecular changes. In 15 cases it was possible to find a correlation either between tumor response and disappearance of abnormal plasma DNA, or tumor progression and persistence of plasma DNA alterations. Conclusions: Free plasma DNA with molecular alterations is present to a high degree in plasma DNA of SCLC patients and may have a role as a prognostic facto

Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index

Circulating cell-free DNA (cfDNA) has been considered an interesting diagnostic/prognostic plasma biomarker in tumor-bearing subjects. In cancer patients, cfDNA can hypothetically derive from tumor necrosis/apoptosis, lysed circulating cells, and some yet unrevealed mechanisms of active release. This study aimed to preliminarily analyze cfDNA in dogs with canine mammary tumors (CMTs). Forty-four neoplastic, 17 non-neoplastic disease-bearing, and 15 healthy dogs were recruited. Necrosis and apoptosis were also assessed as potential source of cfDNA on 78 CMTs diagnosed from the 44 dogs. The cfDNA fragments and integrity index significantly differentiated neoplastic versus non-neoplastic dogs (P<0.05), and allowed the distinction between benign and malignant lesions (P<0.05). Even if without statistical significance, the amount of cfDNA was also affected by tumor necrosis and correlated with tumor size and apoptotic markers expression. A significant (P<0.01) increase of Bcl-2 in malignant tumors was observed, and in metastatic CMTs the evasion of apoptosis was also suggested. This study, therefore, provides evidence that cfDNA could be a diagnostic marker in dogs carrying mammary nodules suggesting that its potential application in early diagnostic procedures should be further investigated

Human papillomavirus DNA in plasma of patients with cervical cancer

BACKGROUND: Human papillomavirus (HPV) is a crucial etiological factor for cervical cancer (CC) development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients. RESULTS: Whereas 6 out of 50 (12%) HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67). Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA. CONCLUSIONS: The plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy.

BACKGROUND: The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors. METHODS: Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA). RESULTS: The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047). CONCLUSION: Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA

Decreased serum cell-free DNA levels in rheumatoid arthritis

Purpose: Recent studies have demonstrated that serum/plasma DNA and RNA molecules in addition to proteins can serve as biomarkers. Elevated levels of these nucleic acids have been found not only in acute, but also in chronic conditions, including autoimmune diseases. The aim of this study was to assess cell-free DNA (cfDNA) levels in sera of rheumatoid arthritis (RA) patients compared to controls. Methods: cfDNA was extracted from sera of patients with early and established RA, relapsing-remitting multiple sclerosis patients (RRMS) and healthy subjects, and its concentration was determined by quantitative PCR using two amplicons, Alu115 and β-actin205, corresponding to Alu repetitive elements and the β-actin single-copy gene, respectively. Serum DNase activity was measured by a single radial enzyme diffusion method. Results: Reduced levels of cfDNA were observed in patients with establi

Identification of Mouse Serum miRNA Endogenous References by Global Gene Expression Profiles

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments

Detection of IL28B SNP DNA from Buccal Epithelial Cells, Small Amounts of Serum, and Dried Blood Spots

Background &amp; Aims: Point mutations in the coding region of the interleukin 28 gene (rs12979860) have recently been identified for predicting the outcome of treatment of hepatitis C virus infection. This polymorphism detection was based on whole blood DNA extraction. Alternatively, DNA for genetic diagnosis has been derived from buccal epithelial cells (BEC), dried blood spots (DBS), and genomic DNA from serum. The aim of the study was to investigate the reliability and accuracy of alternative routes of testing for single nucleotide polymorphism allele rs12979860CC. Methods: Blood, plasma, and sera samples from 200 patients were extracted (400 mL). Buccal smears were tested using an FTA card. To simulate postal delay, we tested the influence of storage at ambient temperature on the different sources of DNA at five time points (baseline, 48 h, 6 days, 9 days, and 12 days) Results: There was 100 % concordance between blood, plasma, sera, and BEC, validating the use of DNA extracted from BEC collected on cytology brushes for genetic testing. Genetic variations in HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10. A minimum of 0.04 mL, 4 mL, and 40 mL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma. No significant variation between each time point was observed for the different sources of DNA. IL28B SNPs analysis at these different time points showed the same results using the four sources of DNA

Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer

Citrullinated histone H3 (H3Cit) is a central player in the neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). NETs have been shown to elicit harmful effects on the host, and were recently proposed to promote tumor progression and spread. Here we report significant elevations of plasma H3Cit in patients with advanced cancer compared with age-matched healthy individuals. These elevations were specific to cancer patients as no increase was observed in severely ill and hospitalized patients with a higher non-malignant comorbidity. The analysis of neutrophils from cancer patients showed a higher proportion of neutrophils positive for intracellular H3Cit compared to severely ill patients. Moreover, the presence of plasma H3Cit in cancer patients strongly correlated with neutrophil activation markers neutrophil elastase (NE) and myeloperoxidase (MPO), and the inflammatory cytokines interleukin-6 and -8, known to induce NETosis. In addition, we show that high levels of circulating H3Cit strongly predicted poor clinical outcome in our cohort of cancer patients with a 2-fold increased risk for short-term mortality. Our results also corroborate the association of NE, interleukin-6 and -8 with poor clinical outcome. Taken together, our results are the first to unveil H3Cit as a potential diagnostic and prognostic blood marker associated with an exacerbated inflammatory response in patients with advanced cancer