PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

High-dose hypofractionated proton therapy demonstrated high LC rates for HCC and ICC safely, supporting ongoing phase III trials of radiation in HCC and ICC.

No abstract

Background. Autophagy is a catabolic pathway that permits cells to recycle intracellular macromolecules, and its inhibition reduces pancreatic cancer growth in model systems. We evaluated hydoxychloroquine (HCQ), an inhibitor of autophagy, in patients with pancreatic cancer and analyzed pharmacodynamic markers in treated patients and mice. Methods. Patients with previously treated metastatic pancreatic cancer were administered HCQ at 400 mg (n 5 10) or 600 mg (n 5 10) twice daily. The primary endpoint was 2-month progression-free survival (PFS).We analyzed peripheral lymphocytes from treated mice to identify pharmacodynamic markers of autophagy inhibition that were then assessed in peripheral lymphocytes from patients. Results. Among 20 patients enrolled, 2 (10%) were without progressive disease at 2 months. Median PFS and overall survival were 46.5 and 69.0 days, respectively. Treatmentrelated grade 3/4 adverse events were lymphopenia (n 5 1) and elevated alanine aminotransferase (n 5 1). Tolerability and efficacy were similar at the two dose levels. Analysis of treated murine lymphocytes suggested that LC3-II expression by Western blot is a reliable marker for autophagy inhibition. Analysis of LC3-II in patient lymphocytes demonstrated inconsistent autophagy inhibition. Conclusion. Mouse studies identified LC3-II levels in peripheral lymphocytes as a potential pharmacodynamic marker of autophagy inhibition. In patients with previously treated metastatic pancreatic cancer, HCQ monotherapy achieved inconsistent autophagy inhibition and demonstrated negligible therapeutic efficacy. The Oncologist 2014; 19:637-638