Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients.

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

Survival and cell cycle kinetics of human prostate cancer cell lines after single- and multifraction exposures to ionizing radiation.

PURPOSE: Fractionated radiation therapy is frequently used to treat prostate cancer with an underlying assumption that each daily dose of ionizing radiation (IR) results in equal cell killing. We used three human prostate cancer cell lines to evaluate how survival after a single 2-Gy dose may predict responses after daily repeated 2-Gy exposures. METHODS AND MATERIALS: LNCaP, CWR22R, and PC3 cells were used in these studies. Survival after IR exposures was assessed using clonogenic assays and cell cycle responses were determined by flow cytometry. RESULTS: The experimentally determined multifraction survival differed significantly from that predicted from their single-dose SF2. LNCaP and CWR22R cells showed lower than predicted survivals; PC3 cells exhibited greater than predicted survival. Daily IR exposures resulted in changes in the cell cycle distributions beyond those caused by a single exposure to IR. CONCLUSIONS: Our results show that in these prostate cancer cells: (1) survival after a clinically relevant dose of IR does not predict survival after multifraction IR, (2) cell cycle responses after a single 2 Gy dose can differ from those that occur when cells receive daily 2 Gy doses, and (3) some cell cycle changes that result from fractionated IR may predict their ultimate survival responses from such treatment.

Functional p53 increases prostate cancer cell survival after exposure to fractionated doses of ionizing radiation.

External beam radiation therapy is an effective therapy for localized prostate cancer, although failures occur at high rates. One variable that may affect the radiosensitivity of prostate tumor cells is their p53 status because this gene controls radiation-induced cell cycle arrest, apoptosis, and the repair of DNA damage. Using a system in which p53 function was conditionally restored to p53-null PC3 prostate cancer cells by stable transfection with a human temperature-sensitive p53 mutant allele, we tested the hypothesis that functional p53 increases cell cycle arrest and contributes to increased clonogenic survival after ionizing radiation (IR) of prostate carcinoma cells. Cell cycle arrest and clonogenic survival in response to single and multiple daily exposures to clinically relevant 2-Gy doses of IR were examined. Whereas the temperature-sensitive p53 protein was activated by phosphorylation after IR exposure at both the restrictive and permissive temperatures, Cdkn1/p21 was only induced by functional p53 (at the permissive temperature). In the presence of functional p53, the maintenance of G(2) arrest was significantly longer (P < 0.01), and a small increase in cell survival measured by clonogenic assay was seen after exposure to a single 2-Gy dose of IR. However, functional p53 significantly increased clonogenic survival (P < 0.01) after exposure to daily doses of 2 Gy of IR and contributed to a more sustained G(2) arrest and increased G(1) arrest in response to the multifraction regimen. These studies implicate the presence of wild-type p53 with increased survival of prostate carcinoma cells after fractionated exposure to radiation. Additionally, the data provide evidence that wild-type p53 in prostate tumor cells may reduce the effectiveness of radiation therapy.

BCL2 antisense reduces prostate cancer cell survival following irradiation.

Irradiation of the prostate, delivered as external beam radiation therapy (EBRT), is currently one of the few treatment options for localized prostate cancer. While it is relatively effective, the failure rate still remains unacceptably high with a 5-year biochemical failure rate of 10-40%. Utilizing genetically engineered LNCaP prostate cancer sublines that either overexpress Bcl2 (LNCaP/S22-d) or have down-regulated Bcl2 (LNCaP/AS17-f) we investigated the influence of this antiapoptotic protein on clonogenic survival following radiation. The radiation dose response curves (2-8 Gy) for the sublines differed significantly from the parental LNCaP (LNCaP/S22d: p < 0.001 and LNCaP/AS17-f: p = 0.008). The relative survival of the sublines revealed increased survival in the Bcl2 overexpressing cells, and decreased survival in the Bcl2 down-regulated cells. These data suggest a potentially important therapeutic approach for enhancing radiosensitivity in prostate tumors via antisense oligonucleotide or other drug therapies that down-regulate Bcl2. Strategies such as these likely hold the promise of enhancing the efficacy of EBRT by decreasing tumor cell survival, reducing the incidence of tumor recurrence and improving patient outcome.