Melanie Triboulet

High efficiency vortex trapping of circulating tumor cells

Circulating tumor cells (CTCs) are important biomarkers for monitoring tumor dynamics and efficacy of cancer therapy. Several technologies have been demonstrated to isolate CTCs with high efficiency but achieve a low purity from a large background of blood cells. We have previously shown the ability to enrich CTCs with high purity from large volumes of blood through selective capture in microvortices using the Vortex Chip. The device consists of a narrow channel followed by a series of expansion regions called reservoirs. Fast flow in the narrow entry channel gives rise to inertial forces, which direct larger cells into trapping vortices in the reservoirs where they remain circulating in orbits. By studying the entry and stability of particles following entry into reservoirs, we discover that channel cross sectional area plays an important role in controlling the size of trapped particles, not just the orbital trajectories. Using these design modifications, we demonstrate a new device that is able to capture a wider size range of CTCs from clinical samples, uncovering further heterogeneity. This simple biophysical method opens doors for a range of downstream interventions, including genetic analysis, cell culture, and ultimately personalized cancer therapy.

Enumeration and targeted analysis of KRAS , BRAF and PIK3CA mutations in CTCs captured by a label-free platform: Comparison to ctDNA and tissue in metastatic colorectal cancer

Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles.

Label-free isolation of prostate circulating tumor cells using Vortex microfluidic technology

There has been increased interest in utilizing non-invasive “liquid biopsies” to identify biomarkers for cancer prognosis and monitoring, and to isolate genetic material that can predict response to targeted therapies. Circulating tumor cells (CTCs) have emerged as such a biomarker providing both genetic and phenotypic information about tumor evolution, potentially from both primary and metastatic sites. Currently, available CTC isolation approaches, including immunoaffinity and size-based filtration, have focused on high capture efficiency but with lower purity and often long and manual sample preparation, which limits the use of captured CTCs for downstream analyses. Here, we describe the use of the microfluidic Vortex Chip for size-based isolation of CTCs from 22 patients with advanced prostate cancer and, from an enumeration study on 18 of these patients, find that we can capture CTCs with high purity (from 1.74 to 37.59%) and efficiency (from 1.88 to 93.75 CTCs/7.5 mL) in less than 1 h. Interestingly, more atypical large circulating cells were identified in five age-matched healthy donors (46–77 years old; 1.25–2.50 CTCs/7.5 mL) than in five healthy donors <30 years old (21–27 years old; 0.00 CTC/7.5 mL). Using a threshold calculated from the five age-matched healthy donors (3.37 CTCs/mL), we identified CTCs in 80% of the prostate cancer patients. We also found that a fraction of the cells collected (11.5%) did not express epithelial prostate markers (cytokeratin and/or prostate-specific antigen) and that some instead expressed markers of epithelial–mesenchymal transition, i.e., vimentin and N-cadherin. We also show that the purity and DNA yield of isolated cells is amenable to targeted amplification and next-generation sequencing, without whole genome amplification, identifying unique mutations in 10 of 15 samples and 0 of 4 healthy samples.Prostate cancer: a “liquid biopsy” test for circulating tumor cellsA microfluidic device can rapidly and efficiently isolate circulating tumor cells from the blood of prostate cancer patients. Elodie Sollier-Christen of Vortex Biosciences, Rajan Kulkarni of David Geffen School of Medicine at UCLA, Dino Di Carlo of UCLA and colleagues tested the company’s microfluidic technology on blood samples taken from 21 men with advanced prostate cancer and 10 healthy controls. They showed that, within an hour, the Vortex Chip could isolate circulating tumor cells in 80% of the cancer patients and that many of these cells did not display the usual surface markers that other approaches require to capture prostate cancer cells. The purities and DNA yields of the isolated cells were high enough to enable targeted genome sequencing, which revealed mutations potentially involved in tumor formation. The Vortex technology could help diagnose prostate cancer and inform therapeutic decision-making for those with the disease.

Label-free enumeration, collection and downstream cytological and cytogenetic analysis of circulating tumor cells

Circulating tumor cells (CTCs) have a great potential as indicators of metastatic disease that may help physicians improve cancer prognostication, treatment and patient outcomes. Heterogeneous marker expression as well as the complexity of current antibody-based isolation and analysis systems highlights the need for alternative methods. In this work, we use a microfluidic Vortex device that can selectively isolate potential tumor cells from blood independent of cell surface expression. This system was adapted to interface with three protein-marker-free analysis techniques: (i) an in-flow automated image processing system to enumerate cells released, (ii) cytological analysis using Papanicolaou (Pap) staining and (iii) fluorescence in situ hybridization (FISH) targeting the ALK rearrangement. In-flow counting enables a rapid assessment of the cancer-associated large circulating cells in a sample within minutes to determine whether standard downstream assays such as cytological and cytogenetic analyses that are more time consuming and costly are warranted. Using our platform integrated with these workflows, we analyzed 32 non-small cell lung cancer (NSCLC) and 22 breast cancer patient samples, yielding 60 to 100% of the cancer patients with a cell count over the healthy threshold, depending on the detection method used: respectively 77.8% for automated, 60–100% for cytology, and 80% for immunostaining based enumeration.

Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

Genomic characterization of circulating tumor cells (CTCs) may prove useful as a surrogate for conventional tissue biopsies. This is particularly important as studies have shown different mutational profiles between CTCs and ctDNA in some tumor subtypes. However, isolating rare CTCs from whole blood has significant hurdles. Very limited DNA quantities often can’t meet NGS requirements without whole genome amplification (WGA). Moreover, white blood cells (WBC) germline contamination may confound CTC somatic mutation analyses. Thus, a good CTC enrichment platform with an efficient WGA and NGS workflow are needed. Here, Vortex label-free CTC enrichment platform was used to capture CTCs. DNA extraction was optimized, WGA evaluated and targeted NGS tested. We used metastatic colorectal cancer (CRC) as the clinical target, HCT116 as the corresponding cell line, GenomePlex® and REPLI-g as the WGA methods, GeneRead DNAseq Human CRC Panel as the 38 gene panel. The workflow was further validated on metastatic CRC patient samples, assaying both tumor and CTCs. WBCs from the same patients were included to eliminate germline contaminations. The described workflow performed well on samples with sufficient DNA, but showed bias for rare cells with limited DNA input. REPLI-g provided an unbiased amplification on fresh rare cells, enabling an accurate variant calling using the targeted NGS. Somatic variants were detected in patient CTCs and not found in age matched healthy donors. This demonstrates the feasibility of a simple workflow for clinically relevant monitoring of tumor genetics in real time and over the course of a patient’s therapy using CTCs.Liquid biopsy: Simple workflow allows DNA analysis of circulating tumor cellsA microfluidic device that isolates cancer cells circulating in a blood sample allows for real-time genetic monitoring. A team led by Elodie Sollier-Christen of Vortex Biosciences, a cancer diagnostics company in Menlo Park, California, USA, in collaboration with Professor Stefanie Jeffrey at Stanford University School of Medicine, developed a simple workflow for analyzing the genomes of rare circulating tumor cells (CTCs) found in the bloodstream after they’ve been collected through a proprietary microfluidic system. They optimized rare cell DNA extraction, compared different whole genome amplification methods, and then tested the workflow on blood samples from patients with metastatic colorectal cancer. The analysis also included white blood cells from the same patients to parse cancer-causing mutations from inherited ones. The method could aid in the translation of liquid biopsies for the clinical care of cancer patients.

Abstract 4967: Label-free collection of prostate circulating tumor cells using microfluidic Vortex technology

BACKGROUND Prostate cancer is among the most common cancers in men worldwide. Better markers than Prostate Specific Antigen (PSA) are still needed for the detection and monitoring of disease progression. Circulating Tumor Cells (CTCs) are shed into the blood stream from primary tumor(s) and may play key roles in the metastatic process. Liquid biopsies have emerged as a promising approach, with a correlation between the CTC numbers and patient prognosis for prostate cancer. CTCs have also been shown to enable early detection of recurrence, and could be potential candidates for guiding cancer therapy in real-time [1]. Current CTC enrichment technologies, including immuno-affinity and size-based filtration methods, have focused on high capture efficiency with sometimes tedious sample preparation and overall low purity. METHOD Here, we describe the use of the microfluidic Vortex Chip [2] for rapid and size-based isolation of CTCs from the blood of 23 patients with advanced prostate cancer, and 10 healthy donors; 5 being RESULTS Preliminary work with LNCaP prostate cancer cells spiked in blood showed a 29% capture efficiency and 50% purity. In vitro cell assays confirmed that cells enriched with Vortex chip were alive and proliferating for up to 7 days. For 23 patient samples, CTCs were captured (0.5 - 20 CTCs/mL) with high purity (3.6 - 72.3%), in less than 1H, without prior sample preparation. 11.5% of the cells collected were CK and PSA-negative, but some were identified as undergoing epithelial-mesenchymal transition (EMT) following staining for vimentin and N-cadherin. Few atypical cells were also isolated from age-matched healthy donors (0.7 - 2.8 CTCs/mL), while none was detected in younger healthy donors. Using a threshold calculated from the age-matched healthy donors (3.31 CTCs/mL = mean + 2CV), 70% of the patients were characterized as “positive for CTCs”. No correlation was found between CTC counts and elevated PSA level. CONCLUSION These results demonstrate the ability to rapidly collect pure populations of CTCs in metastatic prostate cancer, independent of surface marker expression, without prior sample preparation. Future studies will use chips with optimized capture performance, sample recycling, and will include CTC molecular analysis by targeted panel sequencing. A larger cohort of healthy donors is also being examined to determine a statistically-robust CTC baseline for this size-based capture approach. [1] Scher Hi, et al., J. Clin. Oncol. 2015 [2] Sollier E, et al., Lab Chip 2014 Citation Format: Edward Pao, Corinne Renier, Clementine Lemaire, James Che, Melissa Matsumoto Di Carlo, Melanie Triboulet, Sandy Srivinas, Stefanie S. Jeffrey, Rajan P. Kulkarni, Matthew Rettig, Elodie Sollier, Dino Di Carlo. Label-free collection of prostate circulating tumor cells using microfluidic Vortex technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4967.

Abstract 1724: Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing

Background: Genomic characterization of circulating tumor cells (CTCs) provides insights into cancer genetic changes, and might be utilized for cancer prognosis, diagnosis, as well as monitoring of therapeutic efficacy. Targeted Panel Next Generation Sequencing (NGS) enables analyzing CTC genetic variants of a focused gene panel at a relatively lower cost 1 . However, CTCs are rare, often resulting in very limited DNA quantities available that require whole genome amplification (WGA). In previous studies, we introduced the Vortex technology, a platform enabling label-free enrichment of CTCs from blood samples of colorectal cancer (CRC) patients and their use for genomic assays downstream 2 . In this study, we developed a simple and efficient NGS workflow for CTC samples collected by this technology. Method: An optimized workflow using the Qiagen GeneRead DNAseq targeted panel and Illumina MiSeq NGS was first verified on HCT116 CRC cell line before being applied on patient CTCs. For patient blood samples, CTCs were collected with the Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. Matched white blood cell (WBC) DNA was included to subtract germline background. Fresh frozen liver metastasis tissue was collected and analyzed using the same NGS workflow. DNA from CTCs was extracted and amplified using Qiagen REPLI-g single cell WGA kit. Mutation detection on the WGA amplified DNA was performed using the GeneRead DNAseq CRC targeted panel of 38 genes and MiSeq sequencing. The sequencing data were analyzed by QIAGEN NGS Data Analysis Web Portal and Ingenuity Variant Analysis software. Results: The Vortex technology was validated for the capture of CTCs from CRC patients. REPLI-g performed a uniform, unbiased amplification on fresh rare cells with a coverage of 97.7%, which enabled further targeted panel NGS. Blood from 3 CRC patients (P1, P2, P3) and 2 healthy donors (HD1, HD2) was processed with Vortex platform. Less than 1 CTCs/mL blood were found in HD1 and HD2. P1 and P2 had 66 and 20 CTCs/ mL of blood respectively, with many vimentin positive CTC clusters. P3 had 2 CTCs/mL of blood. No somatic mutation was found in healthy donors. Somatic variants were only detected in the CTCs from patient samples that were not present in matched germline WBCs. For P1, more mutations were found in the CTCs than in the liver metastasis while it was the opposite for P2 and P3. Conclusion: For each patient, variants in CTCs and germline WBCs were analyzed from one blood sample using an optimized targeted NGS workflow and compared to liver mets. Our optimized workflow, using the Qiagen REPLIg and GeneRead DNAseq Targeted Panel NGS enabled the detection of CTC mutations for 38 CRC-focused genes. The inclusion of a germline WBC control in the workflow allowed the detection of mutations from pooled CTC samples collected using the Vortex technology. Altmuller J, et al. (2014). Biol Chem. Kidess-Sigal E, et al. (2016). Oncotarget. Citation Format: Haiyan E. Liu, Melanie Triboulet, Amin Zia, Meghah Vuppalapaty, Evelyn Kidess-Sigal, John Coller, Vanita S. Natu, Vida Shokoohi, James Che, Corinne Renier, Natalie Chan, Violet Hanft, Elodie Sollier-Christen, Stefanie S. Jeffrey. Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing [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 1724. doi:10.1158/1538-7445.AM2017-1724

Abstract 3149: Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background Colorectal cancer (CRC) is the 3rd most common cancer diagnosed worldwide in both men and women. Only 39% of cancers are diagnosed at a localized stage, and 5-year survival rates decrease rapidly for patients with advanced and metastasized disease (stage III 61%, stage IV 8%). Better markers for detection of disease progression, therapeutic resistance and minimal residual disease are still needed. Liquid biopsies, such as CTCs and ctDNA, are emerging biomarkers shed by the tumor into the blood stream. Both markers currently are attracting growing interest for their use in disease prognosis, early detection of recurrence and are promising candidates for guiding cancer therapy in real-time. Method For rapid label-free isolation of CTCs from peripheral blood we used the Vortex technology, a microfluidic device using inertia and laminar microvortices. From 15 patients with metastatic CRC to the liver that underwent liver metastatectomy with curative intent, we collected CTCs preoperatively, at the 5th postoperative day and during follow-up visits. Cells collected were immunostained for EpCAM, CD45 and DAPI, enumerated using standardized classification criteria, and subjected to Sanger sequencing. CTC enumeration and mutational patterns were compared to the primary tumor, liver metastases and ctDNA (detected by a multiplexed PCR and enrichment technology; Kidess E et al., 2015) as well as CEA levels when available. Results 41 blood samples from 15 patients were collected at different time points prior to and after surgical resection of liver metastases. More CTCs were found in preoperatively collected CRC patient samples (2.4 CTCs/mL, 0.1 - 5.5/mL) than in age-matched healthy controls (0.1 CTCs/mL, 0 - 0.4/mL). 80% of all CRC samples were identified as positive for CTCs (based on a calculated threshold from healthy controls), with varying levels of EpCAM expression (81.4% of CTCs being EpCAM+). The number of CTCs for each patient, showed a close correlation to clinical parameters and ctDNA levels: detection of CTCs, CTC mutational profiles as well as ctDNA revealed minimal residual disease and anticipated tumor recurrence earlier than carcinoembryonic antigen (CEA) value or imaging. For example, for P006, postoperative imaging surveillance revealed progressive disease, which was accompanied by rising levels of CTCs (up to 29 CTCs/mL at the last time point) and PIK3CA mutant DNA in both plasma ctDNA and CTC DNA, while CEA remained in the normal range. Conclusion Our data illustrate that CTCs as well as ctDNA can efficiently reveal disease recurrence as well as disease progression earlier than imaging and far more reliable compared to CEA, the currently standard biomarker for CRC. Beyond enumeration, CTC molecular analysis gives additional information and will potentially help to promote the development of tailored therapies for every individual patient. Citation Format: Evelyn Kidess-Sigal, Haiyan E. Liu, Melanie Triboulet, James Che, Georges A. Poultsides, Brendan C. Visser, Andre Marziali, Marc Lee, Valentina Vysotskaia, Matthew Wiggin, Vishnu C. Ramani, Ulrich Keilholz, Ingeborg Tinhofer, Amin Zia, John Coller, Jeffrey A. Norton, Elodie Sollier, Stefanie S. Jeffrey. Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3149.

Abstract 1525: Vortex technology for label-free enrichment of CTC from mouse xenograft models

Background Non-invasive liquid biopsies, such as CTCs, have been of growing interest due to their potential use in cancer detection, prognosis, and monitoring of therapeutic resistance [1]. Beyond enumeration, characterization of CTCs could help guide treatment selection and the development of targeted cancer therapy. Here, we show that Vortex technology can be used successfully for the size-based capture of CTCs in a preclinical mouse model of breast cancer. Method To establish that human epithelial cancer cells can be reliably detected in small volumes of mice blood, 50-100 MDA-MB231 and MCF7 breast cancer cells were spiked in 500 μL mice blood, diluted 20 fold, and processed through Vortex chip [2]. Recovered cells were immunostained (CK, CD45, DAPI), and enumerated. For breast cancer xenograft model, 8×106 MDA-MB-231-fLuc/GFP cells were implanted orthotopically into the mammary fat pad of NOD-SCID Gamma mice (n = 35). Tumors were measured in 2 dimensions 3 times/week and tumor volume calculated. Blood from cardiac puncture (500 μl) and lateral saphenous vein (100 μl) was collected starting 1 week post implantation, diluted 40X and processed. Mice were euthanized, organs harvested, formalin fixed, paraffin embedded and HE frequency 1/3), with number and frequency increasing over time up to 147-485 clusters/100 μl by day 42. No CTC were recovered from lateral saphenous vein blood until day 28 post implantation, and their number remained low (mean 2.15±0.65). Microscopic metastases were evident in lung of all mice starting at day 28 and in liver of all mice starting at day 35. Conclusion CTCs were isolated in a label-free manner from mice blood with both high capture efficiency and purity. In a preclinical model of metastatic breast cancer, CTC counts correlated well with primary tumor volume and metastases occurrence. Thus the Vortex chip appears to be well suited for the enrichment of CTCs from murine xenograft models. Future works will focus on mice implanted with patient derived xenograft (PDX) and their therapeutic response. Information gathered from these studies should facilitate discovery of new therapeutic targets and the development of personalized medicine. [1] Ignatiadis et al. Clin Cancer Res 2015. [2] Sollier et al. Lab Chip 2014.[K.H. and M.T. contributed equally to this work.] Citation Format: Kyra Heirich, Melanie M. Triboulet, Corinne M. Renier, Vishnu C. Ramani, Elodie Sollier, Stefanie S. Jeffrey. Vortex technology for label-free enrichment of CTC from mouse xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1525.