Genetic profiling of cancer with circulating tumor DNA analysis

Circulating cell-free tumor DNA(ctDNA) in the blood is DNA released from apoptotic, circulating, and living tumor cells. ctDNA is about 140 nt in length and has a half-life of about 1.5 h. ctDNA analysis provides a noninvasive means to assess the genetic profile of cancer in real time. With the advent of molecular technologies, including digital PCR and massively parallel sequencing(MPS), ctDNA analysis has shown promise as a highly sensitive and specific alternative to conventional tissue biopsy in cancer detection, longitudinal monitoring, and precision therapy. This review provides an overview of the latest development in our understanding of the biologic characteristics, detection methodologies, and potential clinical implications of ctDNA, as well as the challenges in translating ctDNA analysis from the research arena to patient care.     

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer

The genomic profiling of circulating tumor cells (CTCs) in the bloodstream should provide clinically relevant information on therapeutic efficacy and help predict cancer survival. Here, we contrasted the genomic profiles of CTC pools recovered from metastatic colorectal cancer (mCRC) patients using different enrichment strategies (CellSearch, Parsortix, and FACS). Mutations inferred in the CTC pools differed depending on the enrichment strategy and, in all cases, represented a subset of the mutations detected in the matched primary tumor samples. However, the CTC pools from Parsortix, and in part, CellSearch, showed diversity estimates, mutational signatures, and drug-suitability scores remarkably close to those found in matching primary tumor samples. In addition, FACS CTC pools were enriched in apparent sequencing artifacts, leading to much higher genomic diversity estimates. Our results highlight the utility of CTCs to assess the genomic heterogeneity of individual tumors and help clinicians prioritize drugs in mCRC.     

Detection of oncogenic mutations in paired circulating tumor DNA and circulating tumor cells in patients with hepatocellular carcinoma

Background and aimsCirculating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients.MethodsTwenty-six mostly advanced HCC patients were enrolled. 30 mL peripheral blood from each patient was obtained. CellSearch system was used for CTC detection. A sequencing panel covering 14 cancer-relevant genes was used to identify oncogenic mutations. TERT promoter C228T and C250T mutations were determined by droplet digital PCR.ResultsCTCs were detected in 27% (7/26) of subjects but at low numbers (median: 2 cells, range: 1–15 cells) and ctDNA in 77% (20/26) of patients. Mutations in ctDNA were identified in several genes: TERT promoter C228T (77%, 20/26), TP53 (23%, 6/26), CTNNB1 (12%, 3/26), PIK3CA (12%, 3/26) and NRAS (4%, 1/26). The TERT C228T mutation was present in all patients with one or more ctDNA mutations, or detectable CTCs. The TERT C228T and TP53 mutations detected in ctDNA were present at higher levels in matched primary HCC tumor tissue. The maximal variant allele frequency (VAF) of ctDNA was linearly correlated with largest tumor size and AFP level (Log10). CtDNA (or TERT C228T) positivity was associated with macrovascular invasion, and positivity of ctDNA (or TERT C228T) or CTCs (≥ 2) correlated with poor patient survival.ConclusionsOncogenic mutations could be detected in ctDNA from advanced HCC patients. CtDNA analysis may serve as a promising liquid biopsy to identify druggable mutations.     

Mitochondrial DNA profiling via genomic analysis in mesial temporal lobe epilepsy patients with hippocampal sclerosis

Introduction

Mitochondria have an essential role in neuronal excitability and neuronal survival. In addition to energy production, mitochondria also play a crucial role in the maintenance of intracellular calcium homeostasis, generation of reactive oxygen species and mechanisms of cell death. There is a relative paucity of data about the role of mitochondria in epilepsy. Mitochondrial genome analysis is rarely carried out in the investigation of some diseases. In mesial temporal lobe epilepsies (MTLE) cases, genome analysis has never been used previously. The aim of this study is to show mitochondrial dysfunctions using genome analysis in patients with MTLE-hippocampal sclerosis (HS).

Methods

44 patients with MTLE-HS and 86 matched healthy unrelated controls were included in this study. The patients were divided into four groups according to their clinical presentation as the following: Group 1 consists of patients with intractable epilepsy who refused operation; Group 2 of operated seizure free patients; Group 3 of operated patients with seizures; and Group 4 unoperated seizure free patients with or without antiepileptic drugs. Blood samples were used to isolate DNA. Parallel tagged sequencing was employed to allow pyrosequencing of 130 samples. Complete mtDNA is amplified in two overlapping fragments (11 and 9 kb). The PCR amplicons were pooled in equimolar ratios. Titanium kits were used to produce shotgun libraries according to the manufacturer's protocol.

Results

The average coverage in total was 130 ± 30 and an average of 2365127 bases and 337 bp fragment length was received from all samples. The mean mtDNA heteroplasmy in patients was 26.35 ± 12.3 and in controls 25.03 ± 9.34. Three mutations had prominently high significance in patient samples. The most significantly associated variation was located in the MT-ATP-8 gene (8502 A > T, Asn46Ile) whereas the other two were in the MT-ND4 (11994 C > T, Thr412Ile) and MT-ND5 (13231 A > C, Lys299Gln) genes.

Conclusions

We have observed that three mutations were significantly related to the presence of epilepsy. These mutations were found at the 8502, 11994, and 13231 bp of mtDNA, which resulted in amino acid changes at the MT-ATP-8, MT-ND4 and MT-ND5 genes. Finding mutations can lead us to knowing more about the pathophysiology of the MTLE disease.     

儿童白血病患者血浆循环DNA定量分析

目的:通过对儿童白血病患者血浆循环DNA定量检测,分析血浆循环DNA含量用于儿童白血病诊断的价值.方法:用血液微量DNA抽提试剂盒提取血浆循环DNA,以SYBR GreenI荧光染色法分别检测46例儿童急性白血病患者、20例健康对照标本血浆循环DNA含量;利用受试者工作特征(ROC)曲线评价血浆循环DNA对儿童白血痛的诊断价值.结果:儿童白血病患者血浆循环DNA含量为(30.04±15.13)ng/mL,高于对照组(12.22±7.10)ng/mL,二者差异有统计学意义(P＜0.05);循环DNA含量16.31 ng/mL为诊断儿童白血病最佳临界值,敏感性和特异度分别为86.96%和75.00%,ROC曲线下面积(AUC)为0.879.结论:血浆循环DNA定量检测有可能成为一种新的用于儿童白血病诊断方法.     

Catalase levels in patients with aniridia and/or Wilms' tumor: utility and limitations

The gene for red blood cell (RBC) catalase has recently been mapped to 11p13, and a gene dosage effect has been demonstrated for individuals with triplication or deletion of that region. Deletion of the 11p13 band has also been associated with aniridia, with and without Wilm's tumor. We studied the RBC catalase levels in individuals without detectable chromosomal abnormalities but with aniridia, Wilm's tumor, and the combination of aniridia and Wilms' tumor, to determine whether catalase levels might provide evidence for a submicroscopic chromosomal deletion in the 11p13 region. All karyotypically normal patients were found to have normal catalase levels.     

Genome-scale profiling of circulating cell-free DNA signatures for early detection of hepatocellular carcinoma in cirrhotic patients

Dear Editor, Hepatocellular carcinoma (HCC) is the second most deadly cancer worldwide.1 Cirrhosis of different causes predisposes patients to HCC,increasing th...     

The prognostic value of circulating tumor DNA in patients with melanoma: A systematic review and meta-analysis

BackgroundCirculating tumor DNA (ctDNA) has been investigated as a potential prognostic biomarker to evaluate the therapeutic efficacy and disease progression in melanoma patients, yet results remain inconclusive. The purpose of this study was to illustrate the prognostic value of ctDNA in melanoma.ObjectivesTo describe the clinical prognostic value of ctDNA for melanoma patients.MethodsSearched for eligible articles from Pubmed, Web of Science and Embase. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to evaluate the association between ctDNA at baseline or during treatment and overall survival (OS) and progression-free survival (PFS).ResultsA total of 9 articles were obtained, involving 617 melanoma patients. The pooled HRs revealed that compared with baseline undetectable ctDNA patients, detectable ctDNA was highly correlated with poor OS (HR 2.91, 95% CI: 2.22–3.82; p < 0.001) and PFS (HR 2.75, 95% CI: 1.98–3.83; p < 0.001). A meta-analysis of these adjusted HRs was performed and confirmed that ctDNA collected at baseline was associated with poorer OS/PFS (OS: HR 3.00, 95% CI 2.19–4.11, p < 0.001/PFS: HR 2.68, 95% CI 1.77–4.06, p < 0.001). During treatment, a significant association was shown between ctDNA and poorer OS/PFS (OS: HR 6.26, 95% CI 2.48–15.80, p < 0.001; PFS: HR 4.93, 95% CI 2.36–10.33, p < 0.001).ConclusionInvestigation and application of ctDNA will improve "liquid biopsy" and play a role in early prediction, monitoring disease progression and precise adjusting treatment strategies in melanoma patients.     

Quantitative assessment of whole-body tumor burden in adult patients with neurofibromatosis

Purpose

Patients with neurofibromatosis 1 (NF1), NF2, and schwannomatosis are at risk for multiple nerve sheath tumors and premature mortality. Traditional magnetic resonance imaging (MRI) has limited ability to assess disease burden accurately. The aim of this study was to establish an international cohort of patients with quantified whole-body internal tumor burden and to correlate tumor burden with clinical features of disease.

Methods

We determined the number, volume, and distribution of internal nerve sheath tumors in patients using whole-body MRI (WBMRI) and three-dimensional computerized volumetry. We quantified the distribution of tumor volume across body regions and used unsupervised cluster analysis to group patients based on tumor distribution. We correlated the presence and volume of internal tumors with disease-related and demographic factors.

Results

WBMRI identified 1286 tumors in 145/247 patients (59%). Schwannomatosis patients had the highest prevalence of tumors (P = 0.03), but NF1 patients had the highest median tumor volume (P = 0.02). Tumor volume was unevenly distributed across body regions with overrepresentation of the head/neck and pelvis. Risk factors for internal nerve sheath tumors included decreasing numbers of café-au-lait macules in NF1 patients (P = 0.003) and history of skeletal abnormalities in NF2 patients (P = 0.09). Risk factors for higher tumor volume included female gender (P = 0.05) and increasing subcutaneous neurofibromas (P = 0.03) in NF1 patients, absence of cutaneous schwannomas in NF2 patients (P = 0.06), and increasing age in schwannomatosis patients (p = 0.10).

Conclusion

WBMRI provides a comprehensive phenotype of neurofibromatosis patients, identifies distinct anatomic subgroups, and provides the basis for investigating molecular biomarkers that correlate with unique disease manifestations.     

Clinical utility of plasma miR‐371a‐3p in germ cell tumors

Germ cell tumours predominantly of the testis ((T)GCTs) are remarkably chemotherapy sensitive. However, a small proportion of patients fail to be cured with cisplatin‐based combination chemotherapy. miR‐371a‐3p is a new liquid biopsy biomarker for (T)GCTs. The aim of this study was to evaluate clinical utility of plasma miR‐371a‐3p level in patients starting systemic chemotherapy. Patients were included before the first cycle (N = 180) and second cycle (N = 101) of systemic first line chemotherapy, treated between July 2010 and May 2017. Plasma miR‐371a‐3p levels were measured with the ampTSmiR test and compared to disease characteristics and outcome. Pretreatment plasma miR‐371a‐3p levels were increased in 51.7% of cases and associated with number of metastatic sites, presence of lung, retroperitoneal, and mediastinal lymph node metastases, S – stage, IGCCCG risk group, and response to therapy. Patients with a negative pretreatment plasma level had better progression‐free survival (PFS) and overall survival (OS) compared to patients being positive for miR‐371a‐3p (hazard ratio [HR] = 0.26, 95% confidence interval [CI] 0.09‐0.71, P = 0.02 for PFS and HR = 0.21, 95% CI 0.07‐0.67, P = 0.03 for OS, respectively). Patients negative for miR‐371a‐3p in both samples had a superior PFS (HR = 0.10, 95% CI 0.01‐21.49, P = 0.02) and OS (HR = 0.08, 95% CI 0.01‐27.81, P = 0.008) compared to patients with miR‐371a‐3p positive in both samples (multivariate analyses were non‐significant). In total 68% of the patients were S0. This study demonstrates clinical value of plasma miR‐371a‐3p level in chemotherapy naïve (T)GCT patients starting first line of chemotherapy to predict prognosis.     

LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors

Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.     

Human papillomavirus mutational insertion: specific marker of circulating tumor DNA in cervical cancer patients

Introduction

In most cases of cervical cancers, HPV DNA is integrated into the genome of carcinoma cells. This mutational insertion constitutes a highly specific molecular marker of tumor DNA for every patient. Circulating tumor DNA (ctDNA) is an emerging marker of tumor dynamics which detection requires specific molecular motif. To determine whether the sequence of the cell-viral junction could be used in clinical practice as a specific marker of ctDNA, we analyzed a series of cervical cancer patient serums.

Methods and Findings

Serum specimens of 16 patients diagnosed with HPV16/18-associated cervical cancer, and for which the viral integration locus had been previously localized, were analyzed. Sequential serum specimens, taken at different times during the course of the disease, were also available for two of these cases. ctDNA was found in 11 out of 13 patients with tumor size greater than 20 mm at diagnosis, and analysis of sequential serum specimens showed that ctDNA concentration in patients serum was related to tumor dynamics.

Conclusions

We report that HPV mutational insertion constitutes a highly specific molecular marker of ctDNA in HPV-associated tumor patients. Using this original approach, ctDNA was detected in most cervical cancer patients over stage I and ctDNA concentration was found to reflect tumor burden. In addition to its potential prognostic and predictive value, HPV mutation insertion is likely to constitute a new molecular surrogate of minimal residual disease and of subclinical relapse in HPV-associated tumor. This is of major importance in the perspective of specific anti-HPV therapy.     

Characteristics of microspheres formed in PCR with bacterial genomic DNA or plasmid DNA as templates

Earlier, we discovered that, along with linear DNA fragments, nano- and microparticles of DNA and their aggregates are formed in the PCR with yeast genomic DNA used as a template and gene-specific or partially complementary primers. The size of the microparticles (microspheres) varied in the range of 0.5 to 3–4 μm. Only thermostable KlenTaq polymerase but not Taq polymerase could effectively generate microspheres. In this work, we demonstrate that KlenTaq polymerase can produce microspheres of variable size (1 to 7 μm in diameter) if genomic DNA of the bacterium Acidithiobacillus ferrooxidans and partially complementary primers are present in the PCR mixture. Conditions for generation of DNA microparticles in PCR with Taq-polymerase and bacterial genomic DNA as template were also elaborated. It was also found that mainly large microspheres of up to 7 μm accumulated in PCR with plasmid DNAs used as templates and gene-specific primers in the presence of KlenTaq polymerase or mixtures of KlenTaq and Pfu polymerases. Besides, small aggregates, as well as linear branched structures and three-dimensional conglomerates of fused microspheres, were also revealed in the PCR mixtures. UV absorption spectra of native DNA microspheres and microspheres that had undergone heating at 93°C were registered. The key role of Mg²⁺ cations in the formation and stabilization of the microsphere structure was established.     

Prognostic impact of circulating tumor cells in patients with ampullary cancer

Circulating tumor cells (CTCs) are an important topic of investigation for both basic and clinical cancer research. In this prospective study, we evaluated the clinical role of CTCs in ampullary cancer. We analyzed blood samples from 62 consecutively diagnosed patients with ampullary adenocarcinoma and 24 healthy controls for their CTC content. Combined data from immunostaining of CD45, 4′,6‐diamidino‐2‐phenylindole (DAPI), and fluorescence in situ hybridization with a chromosome 8 centromere (CEP8) probe were used to identify CTCs; cells that were CD45‐/DAPI+/CEP8>2 were considered CTCs. The Cox proportional hazards model was used to assess the relationship between CTCs, clinical characteristics, and patient outcomes. We detected ≥2 CTCs/3.2 ml whole blood in 43 of 62 patients (69.4%), as well as ≥5 CTCs/3.2 ml in 16 of these patients (25.8%). A CTC cutoff value of 2 cells/3.2 ml achieved 69.4% sensitivity and 95.8% specificity as a diagnostic tool; CTCs were associated with tumor burden. CTC levels ≥3/3.2 ml (hazard ratio [HR]: 2.5, 95% confidence interval [CI]: (1.2–5.2), p = 0.014) and ≥5/3.2 ml (HR: 3.5, 95% CI: 1.7–7.3, p < 0.001) were both associated with shorter disease‐free survival. Moreover, ≥3 CTCs/3.2 ml (HR: 2.7, 95% CI: 1.2–6.3, p = 0.019) and ≥5 CTCs/3.2 ml (HR: 3.8, 95% CI: 1.8–8.5, p < 0.001) were predictive of shorter overall survival. CTC assessment may help identify patients with ampullary cancer who are at high risk of an unfavorable outcome.     

Transformation of rice with long DNA-segments consisting of random genomic DNA or centromere-specific DNA

Rice was transformed with either long DNA-segments of random genomic DNA from rice, or centromere-specific DNA sequences from either maize or rice. Despite the repetitive nature of the transgenic DNA sequences, the centromere-specific sequences were inserted largely intact and behave as simple Mendelian units. Between 4 and 5% of bombarded callus clusters were transformed when bombarded with just pCAMBIA 1305.2. Frequency of recovery dropped to 2–3% when BACs with random genomic inserts were co-bombarded with pCAMBIA, and fell to less than 1% when BACs with centromeric DNA inserts and pCAMBIA were co-bombarded. A similar effect was noted on regeneration frequency. Differences in transformation ability, regeneration and behavior of plants transgenic for BACs with random genomic DNA inserts, as compared to those with centromeric DNA inserts, suggests functional differences between these two types of DNA.     

Metabolite profiling of blood plasma of patients with prostate cancer

Prostate cancer is one of the most common types of cancer in men. It is though extremely important to search for specific markers including metabolites, which concentration in blood could be a diagnostic measure. In this regard, the metabolite profiling of blood plasma was performed with two groups of people: healthy volunteers (n = 30) and patients with prostate cancer, second stage (n = 40). The profiling protocol included proteins removal from blood plasma with methanol and direct analysis of metabolite fractions by mass spectrometry. Identification of the most abundant metabolites in samples was performed using an accurate mass tag and an isotope pattern methods. Cancer-specific metabolites were revealed by statistical analysis of metabolite intensities in the mass spectra. Six different metabolites were found to be cancer-specific. Two metabolites, acylcarnitine and arachidonoyl amine, have the AUC 0.97 and 0.86, respectively, which are higher than those from PSA test, 0.59.