Purpose The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO™), an approved third generation EGFR inhibitor, in EGFR-mutant non-small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study. Experimental design AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting. Apoptosis was measured with annexin V/flow cytomentry. Growth-inhibitory effects of tested drugs were evaluated in vitro with cell number estimation and colony formation assay and in vivo with mouse xenogtaft models. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome. Gene knockdown were achieved with siRNA or shRNA. Results AZD9291 potently induced apoptosis in EGFR-mutant NSCLC cell lines, in which ERK phosphorylation was suppressed accompanied with Bim elevation and Mcl-1 reduction likely due to enhanced Mcl-1 degradation and increased Bim stability. Blocking Bim elevation by gene knockdown or enforcing Mcl-1 expression attenuated or abolished AZD9291-induced apoptosis. Moreover, AZD9291 lost its ability to modulate Bim and Mcl-1 levels in AZD9291-resistant cell lines. The combination of a MEK inhibitor with AZD9291 restores the sensitivity of AZD9291-resistant cells including those with C797S mutation to undergo apoptosis and growth regression in vitro and in vivo. Conclusions Modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and hence is an effective strategy to overcome acquired resistance to AZD9291.

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Summary. b-thalassaemia is a common inherited disorder of haemoglobin synthesis worldwide, with an estimated 3-10% frequency in certain regions. Rapid, accurate genotyping methodologies for specific, causative mutations of the b-globin gene are needed for pre-and postnatal screening and diagnosis of this disease in different ethnic populations. In this study, we performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) for simultaneously detecting and genotyping the five most common molecular lesions in the b-globin gene [codons (CDs) 41-42 (-TCTT), IVS-2-654 (C fi T), ) 28 (A fi G), CD17 (A fi T) and CD71-72 (+ A)] in Chinese populations. This method involved the amplification of b-globin target sequence followed by a purification step, a multiplex PE reaction that did not require labelled oligonucleotides, and a fully-denaturing DHPLC analysis on the Transgenomic Wave DNA fragment analysis system. In a blinded study, this technique accurately genotyped 100% (120/120) of samples previously characterized by reverse-dot blot and direct sequencing, and was used successfully for prenatal diagnosis of b-globin mutations in six Chinese families. This study validated the combined PE/DHPLC approach as simple, rapid, highly accurate and cost-effective for use in genotyping common disease-causing mutations, including substitutions, insertions and deletions in b-thalassaemia, and strongly suggests that this technique can be used successfully in other genetic diseases.

BACKGROUND Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-re-sequencing. Analysis of mutations below ~2–5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids, is problematic due to increased ‘noise’ from sequencing errors. Technologies that reduce noise via deep-sequencing unavoidably reduce throughput and increase cost. Here we provide proof-of-principle that COLD-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. METHODS We have developed a multiplex temperature-tolerant-COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. Following a multiplex pre-amplification from genomic DNA we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for Tm-increasing as well as Tm-decreasing mutations over all amplicons, in a single tube. RESULTS Using custom-made and commercial gene panels containing 8, 50, 190 or 16,000 amplicons we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of dITP/dDTP in place of dGTP/dATP enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit-of-detection of ~ 0.01–0.1% mutation abundance using Ion-Torrent and 0.1–0.3% using Sanger sequencing. CONCLUSIONS Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.

11044 Background: A variety of methods for isolation of CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM). Using classic phenotypic definition, a CTC is nucleated, cytokeratin CK(+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells which have undergone epithelial-mesenchymal transition (EMT). We report here the use of ApoStream, a novel dielectrophoresis field-flow-assisted, antibody-free method to isolate CTCs from blood. Methods: Blood was collected from consented NSCLC patients and processed using ApoStrea. For CTC enumeration comparison, CellSearch FDA-approved kit was used. Isolated cells were evaluated with multiplexed immunofluorescent assay and laser scanning cytometry analysis were applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI and EpCam. To determine specific EGFR mutations from captured CTCs, samples were analyzed using Improved and Complete Enrichment with CO-amplification at Lower Denaturation temperature (ICE COLD-PCR). Results: Blood samples from 32 NSCLC patients and 3 healthy volunteers were processed. ApoStream isolated 0 to 65 CK(+)/CD45(-) CTCs(n=32) and CellSearch isolated 0 to 13 EpCAM(+)/CK(+)/CD45(-) CTCs(n=7). Additionally, ApoStream™ recovered 37-3536 CK(-)/CD45(-) and 4-10702 CK(+)/CD45(+) cells. EpCAM expression was detected in 7-100% of CK(+)/CD45(-) and 0-5% of CK(-)/CD45(-) cells, and 18-100% of CK(+)/CD45(+) cells. EGFR mutations [exon 19 deletion and exon 21 L858R] were determined and found to be concordant when compared to tumor tissue analysis by Sanger sequencing. Conclusions: The ApoStream platform enriched EpCAM(+) and EpCAM(-) CTCs from the blood of NSCLC patients demonstrating utility in recovering cancer cells with multiple phenotypes. From recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and potential utility of CTCs as an alternative to tissue biopsy. Complete mutation analysis will be presented.