The use of blood-circulating cell-free DNA (cfDNA) as a “liquid biopsy” in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available.

Background. Mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells (PBMCs) has been suggested as a potential marker of mitochondrial toxicity associated with nucleoside analogue reverse-transcriptase inhibitor-containing therapy.Methods. We quantified mtDNA and mitochondrial RNA (mtRNA) in PBMCs over the course of 48 weeks in 78 patients infected with human immunodeficiency virus type 1 (HIV-1) who were randomly assigned to receive ritonavir-boosted indinavir and efavirenz with or without stavudine. Furthermore, we analyzed the association of mtDNA and mtRNA with clinical signs and symptoms and/or abnormalities in laboratory markers attributed to mitochondrial toxicity.Results. No statistically significant difference was found in mtDNA and mtRNA content over time between the 2 treatment arms. When arms were combined, both median mtDNA and mtRNA content showed statistically significant increases over the course of 48 weeks, from 206 to 278 copies/cell ( ) and from 154 to 288 P ! .001 copies/cell ( ), respectively. No statistically significant difference in mtDNA and mtRNA content was found P p .003 between patients with and those without adverse events attributed to mitochondrial toxicity.Conclusions. The observed increases in mtDNA and mtRNA content during the first year of treatment may represent a restorative trend resulting from suppression of HIV-1 infection, independent of the treatment used. Future studies should focus on well-defined mitochondrial toxicities and changes in these markers within the corresponding affected tissues simultaneously with those in PBMCs. Furthermore, with respect to studies of peripheral blood, mtDNA and mtRNA content in individual cell subtypes rather than in PBMCs may be better markers of toxicity and deserve further investigation.

Background:To study the clinical relevance of changes in mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells (PBMCs) attributable to HIV infection and/or combination antiretroviral therapy (cART), a high-throughput molecular assay to quantify mtDNA is required. Methods:We developed a quantitative real-time duplex nucleic acid sequence-based amplification assay in which both mtDNA and nuclear DNA are simultaneously amplified in 1 tube. The assay could accurately quantify mtDNA in a range of 15-1500 copies of mtDNA per 2 genomic copies with an intrarun variation of 11% and an interrun variation of 16%. We compared this real-time assay with the lactate/pyruvate ratios in fibroblasts incubated with glucose and exposed to zalcitabine. Additionally, we studied the effects of platelet contamination and the in vivo effects of cART on mtDNA in PBMCs from a small group of patients. Results: Decreases in mtDNA preceded the increase in lactate/pyruvate ratios and vice versa when zalcitabine was eliminated from the culture. Platelets affected the mtDNA in PBMCs if >5 platelets per PBMC were present. Within 12 weeks, mtDNA increased and remained increased in PBMCs from patients on continu-

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10 3 copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.Human immunodeficiency virus type 1 (HIV-1) can be differentiated into several genetic groups and subgroups, or subtypes. The viral isolates that belong to the genetic group M are responsible for more than 99% of all infections worldwide. This group can be divided into subtypes A through H, to which subtype K was recently added (32). As for subtype J, only two strains are known, whereas subtype I appears to be a mosaic of various subtypes. Besides the group M isolates, the groups N and O are also recognized (9, 34). Isolates that belong to these two groups are mainly found in patients in Cameroon, whereas group M isolates can be found worldwide, with distinct subtypes dominant in specific geographical areas. Epidemiological data is continuously being collected ...