In this paper, we are working toward a problem of great importance to global health: determination of viral HIV and Hepatitis C (HCV) loads under point-of-care and resource limited settings. While antiretroviral treatments are becoming widely available, viral load must be evaluated at regular intervals to prevent the spread of drug resistance, and requires a quantitative measurement of RNA concentration over a wide dynamic range (from 50 up to 106 molecules/mL for HIV and up to 108 molecules/mL for HCV). “Digital” single molecule measurements are attractive for quantification, but the dynamic range of such systems is typically limited or requires excessive numbers of wells. Here we designed and tested two microfluidic rotational SlipChips to perform multivolume digital RT-PCR (MV digital RT-PCR) experiments with large and tunable dynamic range. These designs were characterized using synthetic control RNA and validated with HIV viral RNA and HCV control viral RNA. The first design contained 160 wells of each of four volumes (125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range of 5.2×102 – 4.0×106 molecules/mL at 3-fold resolution. The second design tested the flexibility of this approach, and further expanded it to allow for multiplexing while maintaining a large dynamic range by adding additional wells with volumes of 0.2 nL and 625 nL, and dividing the SlipChip into five regions to analyze five samples each at a dynamic range of 1.8×103 – 1.2×107 molecules/mL at 3-fold resolution. No evidence of cross-contamination was observed. The multiplexed SlipChip can be used to analyze a single sample at dynamic range of 1.7×102 – 2.0×107 molecules/mL at 3-fold resolution with limit of detection of 40 molecules/mL. HIV viral RNA purified from clinical samples were tested on the SlipChip, and viral load results were self-consistent and in good agreement with results determined using the Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test. With further validation, this SlipChip should become useful to precisely quantify viral HIV and HCV RNA for high-performance diagnostics in resource-limited settings. These microfluidic designs should also be valuable for other diagnostic and research applications, including detecting rare cells and rare mutations, prenatal diagnostics, monitoring residual disease, and quantifying copy number variation and gene expression patterns. The theory for the design and analysis of multivolume digital PCR experiments is presented in an accompanying paper by Kreutz et al (Anal. Chem. 2011).
Purpose The clinical relevance of targeting RAS/RAF/MEK/ERK pathway, activated in 70-80% of acute myeloid leukemia (AML) patients, is unknown. Experimental Design Selumetinib is an oral small molecule inhibitor of MEK1/2 kinase. Forty-seven patients with relapsed/refractory AML or ≥60 years old with untreated AML were enrolled on a phase II study. Patients were stratified by FLT3 ITD mutation status. The primary endpoint was response rate (complete, partial and minor). Leukemia cells were analyzed for ERK and mTOR phosphorylation. Results Common drug-related toxicities were grade I-II diarrhea, fatigue, nausea, vomiting and skin rash. In the FLT3 wild type cohort, 6/36 (17%) patients had a response [1 partial response, 3 minor responses, 2 unconfirmed minor responses (uMR)]. No patient with FLT3 ITD responded. NRAS and KRAS mutations were detected in 7% and 2% patients, respectively. The sole patient with KRAS mutation had uMR with hematologic improvement in platelets. Baseline p-ERK activation was observed in 85% of patients analyzed but did not correlate with a response. A single nucleotide polymorphism (SNP) rs3733542 in exon 18 of KIT gene was detected in significantly higher number of patients with response/stable disease compared with non-responders (60% vs 23%; p=0.027). Conclusion Selumetinib is associated with modest single agent antileukemic activity in advanced AML. However, given its favorable toxicity profile, combination with drugs that target other signaling pathways in AML should be considered. The potential association of SNP rs3733542 in exon 18 of KIT gene with antileukemic activity of selumetinib is intriguing, but will require validation in larger trials.
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