Darryl Irwin

Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Methodology/Principal Findings Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. Conclusions/Significance We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.

Sensitive Detection of BCR-ABL1 Mutations in Patients With Chronic Myeloid Leukemia After Imatinib Resistance Is Predictive of Outcome During Subsequent Therapy

PURPOSE BCR-ABL1 mutation analysis is recommended to facilitate selection of appropriate therapy for patients with chronic myeloid leukemia after treatment with imatinib has failed, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. However, mutations could be present below the detection limit of conventional direct sequencing. We developed a sensitive, multiplexed mass spectrometry assay (detection limit, 0.05% to 0.5%) to determine the impact of low-level mutations after imatinib treatment has failed. PATIENTS AND METHODS Mutation status was assessed in 220 patients treated with nilotinib or dasatinib after they experienced resistance to imatinib. RESULTS Mutations were detected by sequencing in 128 patients before commencing nilotinib or dasatinib therapy (switchover). In 64 patients, 132 additional low-level mutations were detected by mass spectrometry alone (50 of 132 mutations were resistant to nilotinib and/or dasatinib). When patients received the inhibitor for which the mutation confers resistance, 84% of the low-level resistant mutations rapidly became dominant clones detectable by sequencing, including 11 of 12 T315I mutations. Subsequent complete cytogenetic response rates were lower for patients with resistant mutations at switchover detected by sequencing (0%) or mass spectrometry alone (16%) compared with patients with other mutations or no mutations (41% and 49%, respectively; P < .001). Failure-free survival among the 100 patients with chronic phase chronic myeloid leukemia when resistant mutations were detected at switchover by sequencing or mass spectrometry alone was 0% and 0% compared with 51% and 45% for patients with other mutations or no mutations (P = .003). CONCLUSION Detection of low-level mutations after imatinib resistance offers critical information to guide subsequent therapy selection. If an inappropriate kinase inhibitor is selected, there is a high risk of treatment failure with clonal expansion of the resistant mutant.

Gene‐wide association study between the aromatase gene (CYP19A1) and female pattern hair loss

Background  Female pattern hair loss (FPHL) is a common trait in which androgens and oestrogens may have a pathogenic role. The aromatase enzyme converts androgens to oestrogens in scalp hair follicles and is differentially expressed in balding and nonbalding scalps of women. Sequence variation in the gene encoding aromatase, CYP19A1, might influence the risk of developing FPHL.

Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor

A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR‐tyrosine kinase inhibitors (EGFR‐TKIs). A non‐invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non‐small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR‐TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression‐free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M‐positive patients (n = 11) was significantly shorter than that of the T790M‐negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR‐TKI therapy.

Association analysis of oestrogen receptor beta gene (ESR2) polymorphisms with female pattern hair loss

in the vicinity. Zinc treatment in patients with EPP may therefore have provided antioxidant protection of cellular membranes against the deleterious photodynamic effects of PpIX accumulation. In accordance with the above, measurement of ultraweak photon emission with a photomultiplier revealed reduced oxidative stress in the skin during zinc treatment. In conclusion, oral treatment with a high daily dosage of zinc sulphate during the spring and summer reduced light sensitivity and cutaneous pain in the majority of our patients with EPP. However, as the intervention was based on an open-label procedure, such a study design may be subject to major bias.

Assessment of common somatic mutations of EGFR, KRAS, BRAF, NRAS in pulmonary non-small cell carcinoma using iPLEX® HS, a new highly sensitive assay for the MassARRAY® System

Increased early detection and personalized therapy for lung cancer have coincided with greater use of minimally invasive sampling techniques such as endobronchial ultrasound-guided biopsy (EBUS), endoscopic ultrasound-guided biopsy (EUS), and navigational biopsy, as well as thin needle core biopsies. As many lung cancer patients have late stage disease and other comorbidities that make open surgical procedures hazardous, the least invasive biopsy technique with the highest potential specimen yield is now the preferred first diagnostic study. However, use of these less invasive procedures generates significant analytical challenges for the laboratory, such as a requirement for robust detection of low level somatic mutations, particularly when the starting sample is very small or demonstrates few intact tumor cells. In this study, we assessed 179 clinical cases of non-small cell lung carcinoma (NSCLC) that had been previously tested for EGFR, KRAS, NRAS, and BRAF mutations using a novel multiplexed analytic approach that reduces wild-type signal and allows for detection of low mutation load approaching 1%, iPLEX® HS panel for the MassARRAY® System (Agena Bioscience, San Diego, CA). This highly sensitive system identified approximately 10% more KRAS, NRAS, EGFR and BRAF mutations than were detected by the original test platform, which had a sensitivity range of 5–10% variant allele frequency (VAF).

Abstract 5596: Impact of blood collection tubes on CTC-, ctDNA- and miRNA recoveries in malignant melanoma patients

Liquid biopsy represents a powerful clinical tool that makes use of the detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes in peripheral blood. Each different analysis is complementary to each other and can give additional valuable clinical information. These biomarkers are susceptible to quick degradation, presenting a challenge in a clinical environment and may require different blood collection tubes. The aim of this CANCER-ID study was to analyze the influence of different blood tubes on quantity and quality of CTCs, ctDNA and total and exosomal miRNA isolated from the same blood tube. After confirming that plasma removal does not influence CTC recovery rates, peripheral blood from 20 cases of metastatic melanoma patients was collected in EDTA, Streck and Transfix tubes. CTC isolation from the PBMC fraction was performed by the ClearCell device (Clearbridge BioMedics). CTCs were identified by immunofluorescence staining. CtDNA was extracted from Streck and EDTA plasma samples, quantity (Qubit) and quality (Tapestation) measurements were performed before analyzing 86 hot-spot mutations in 13 genes by the UltraSEEK chemistry (Agena Bioscience). Ten of the 20 metastatic melanoma patients and 5 healthy donors were chosen for miRNA analysis. MiRNAs were extracted with miRNeasy Serum/Plasma Advanced Kit and miRNeasy Serum/Plasma Kit (QIAGEN) from total plasma and from extracellular vesicles (EVs), respectively. EVs were isolated by ultracentrifugation. QIAseq miRNA libraries were produced and sequenced (Illumina NextSeq 550). The reads were mapped to miRBase and normalized (geNorm). Volcano plots of fold change versus p-value were used to display the miRNAs that are significantly regulated. The CTC enrichment results showed that 3/20 of the EDTA blood samples were positive, whereas samples from Streck and Transfix tubes were negative. The quantity or quality of the ctDNA did not significantly differ between the EDTA and Streck tubes. Mutation analysis of ctDNA performed so far showed similar detection sensitivities between the two tubes. The morphology, particle concentration and size distribution of EVs did not either differ between the two tubes. MiRNA-NGS analyses from plasma revealed that seven (EDTA) and four (Streck) miRNAs are significantly differentially expressed in patients compared with healthy donors. No overlap of these miRNAs was found between the two tubes. In the EV fraction 24 and 31 miRNAs were found significantly differentially expressed in the EDTA and Streck tubes, respectively. Here, one EV miRNA is up-regulated and six are down-regulated in both tubes. In conclusion, the CTC recovery rates and minimal overlap of circulating miRNAs indicate that the different tubes affect CTC, ctDNA and miRNA results. Thus, the outcome of liquid biopsy analyses strongly depends on the choice of blood collection tubes as important pre-analytical variable. Citation Format: Svenja Schneegans, Lelia Luck, Leonie Bluhm, Janina Staub, Rudiger Greinert, Beate Volkmer, Alexander Sartori, Darryl Irwin, Taija af Hallstrom, Melanie Hussong, Jonathan Shaffer, Markus Sprenger-Haussels, Stefan W. Schneider, Peter Mohr, Klaus Pantel, Harriet Wikman. Impact of blood collection tubes on CTC-, ctDNA- and miRNA recoveries in malignant melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5596.

Abstract 750: Detection of mutations in single circulating tumor cells using MALDI-TOF mass spectrometry

Background: As cancer therapeutics are increasingly selected based upon molecular genetic information, having reliable, accurate, rapid and inexpensive methods for mutational analysis are extremely desirable. Circulating tumor cells (CTCs) allow non-invasive “liquid biopsy” access to intact cells for molecular analysis. Here we demonstrate the successful detection of mutations in model CTCs individually isolated from blood (AccuCyte® - CyteFinder® system, RareCyte) using MALDI-TOF Mass Spectrometry (MassARRAY, Agena Bioscience). Methods: Breast (MDA-MD-231) and lung (NCI-H1975) cancer cells with a set of known mutations were spiked into blood and processed by AccuCyte onto microscope slides and stained on an automated immunostainer. Slides were imaged using the CyteFinder digital fluorescence scanning microscope and mCTCs were identified by positive nuclear, EpCAM, and cytokeratin staining, and negative CD45 staining. mCTCs and white blood cell (WBC) negative controls were picked from the slides and put into PCR tubes using the CytePicker® module. DNA from individual or small pools of cells (3-5) was amplified using the PicoPLEX® (Rubicon) whole genome amplification (WGA) kit; alternatively cells were lysed and directly entered into the ensuing iPLEX® Pro workflow. Specific regions surrounding 5 different mutations in each of the mCTC lines were amplified from the WGA product or the lysed cells and the products were detected and scored for the mutations using a single PCR reaction iPLEX® Pro panel that includes a combination of 10 common lung and breast cancer mutations using the MassARRAY® platform. Results: Five point mutations in four different genes (CDKN2A, EGFR, PIK3CA, and TP3) were measured in the NCI-H1975 lines and four point mutations in four genes (BRAF, KRAS, NF2, and TP53) and a deletion in one gene (CDKN2A) were measured in the MDA-MB-231 cells by iPLEX® Pro chemistry on the MassARRAY® system. All mutations were accurately detected in the WGA single and pooled cell samples and most were also detected in cells that did not undergo WGA before PCR with with iPLEX Pro panel. Allelic frequency observed was consistent with known zygosity of the mutation. Conclusions: MassARRAY successfully detected mutations in single model CTCs that were individually picked from a blood sample processed by the AccuCyte - CyteFinder system. Integrating CTC isolation with MassARRAY may be a practical way to identify and monitor known cancer mutations non-invasively. Citation Format: Jackie L. Stilwell, Ryan T. Birse, Arturo Ramirez, Melinda Duplessis, Brennan Enright, Darryl Irwin, Eric Kaldjian. Detection of mutations in single circulating tumor cells using MALDI-TOF mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2017-750