Mahmood Ayub

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA

The application of precision medicine requires in-depth characterisation of a patients tumours and the dynamics of their responses to treatment. We used next-generation sequencing of cfDNA to monitor therapy responses of a metastatic vaginal mucosal melanoma and show that cfDNA can be used to monitor tumour evolution and subclonal responses to therapy even when biopsies are not available.

Development of a circulating miRNA assay to monitor tumor burden: From mouse to man

Circulating miRNA stability suggests potential utility of miRNA based biomarkers to monitor tumor burden and/or progression, particularly in cancer types where serial biopsy is impractical. Assessment of miRNA specificity and sensitivity is challenging within the clinical setting. To address this, circulating miRNAs were examined in mice bearing human SCLC tumor xenografts and SCLC patient derived circulating tumor cell explant models (CDX). We identified 49 miRNAs using human TaqMan Low Density Arrays readily detectable in 10 μl tail vein plasma from mice carrying H526 SCLC xenografts that were low or undetectable in non‐tumor bearing controls. Circulating miR‐95 measured serially in mice bearing CDX was detected with tumor volumes as low as 10 mm3 and faithfully reported subsequent tumor growth.

twoddpcr: an R/Bioconductor package and Shiny app for Droplet Digital PCR analysis

Abstract Summary Droplet Digital PCR (ddPCR) is a sensitive platform used to quantify specific nucleic acid molecules amplified by polymerase chain reactions. Its sensitivity makes it particularly useful for the detection of rare mutant molecules, such as those present in a sample of circulating free tumour DNA obtained from cancer patients. ddPCR works by partitioning a sample into individual droplets for which the majority contain only zero or one target molecule. Each droplet then becomes a reaction chamber for PCR, which through the use of fluorochrome labelled probes allows the target molecules to be detected by measuring the fluorescence intensity of each droplet. The technology supports two channels, allowing, for example, mutant and wild type molecules to be detected simultaneously in the same sample. As yet, no open source software is available for the automatic gating of two channel ddPCR experiments in the case where the droplets can be grouped into four clusters. Here, we present an open source R package ‘twoddpcr’, which uses Poisson statistics to estimate the number of molecules in such two channel ddPCR data. Using the Shiny framework, an accompanying graphical user interface (GUI) is also included for the package, allowing users to adjust parameters and see the results in real-time. Availability and implementation twoddpcr is available from Bioconductor (3.5) at https://bioconductor.org/packages/twoddpcr/. A Shiny-based GUI suitable for non-R users is available as a standalone application from within the package and also as a web application at http://shiny.cruk.manchester.ac.uk/twoddpcr/. Package maintainer anthony.chiu@cruk.manchester.ac.uk

Abstract 3960: Combined circulating tumour cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer

Background The challenge to improve outcomes for patients diagnosed with advanced pancreatic cancer remains very real with only small improvements in median survival gained by the use of systemic chemotherapy and little improvement in 5-year survival over the past decades. The advent of next generation sequencing (NGS) of tumor nucleic acids has opened up the possibility of improving outcomes through personalized therapies selected on the basis of tumor genetics. Whilst NGS biomarkers can be measured in tumor biopsies sampled shortly before and after treatment this is often not practical for ethical, logistical and simple lack of availability. To circumvent problems associated with tumor sampling we have developed and evaluated blood-borne nucleic acids biomarkers for patients diagnosed with advanced pancreatic cancer. Approach We developed a NGS circulating free DNA (cfDNA) analysis pipeline based on the generation of whole genome NGS libraries followed by sequencing of over 600 cancer-associated genes using Agilent SureSelect. Using whole blood collected with Streck cell free DNA blood collection tubes (cfDNA BCT) we optimised combined circulating tumor cell (CTC) enrichment and cfDNA isolation. Quantitative measurements of KRAS mutations using both NGS and droplet digital PCR (ddPCR) were used to compare the tumor component present in both CTCs and cfDNA. We evaluated the overall approach using External Quality Assessment (EQA) controls and over 50 advanced pancreatic cancer patient samples. For CTC analysis we also compared epitope dependent (CellSearch) and independent (Parsortix) enrichment. Results After applying our NGS pipeline to 5 EQA genomic controls we identified all 14 known mutations correctly indicating high sensitivity and specificity. Both ddPCR and NGS identified KRAS mutations in patient cfDNA with a higher success rate seen for ddPCR consistent with the higher sensitivity of this methodology. From the NGS output additional mutations were detected in samples which either harboured or lacked detectable KRAS mutations. Consistent with previous observations CellSearch CTCs were detected at low levels in around 20% of patients with a similar frequency seen in initial analysis of CTCs obtained by epitope independent enrichment (Parsortix). In general KRAS mutations were detected at a higher level in patient cfDNA compared to enriched CTCs although some patients showed detectable CTC KRAS mutations with no KRAS mutations detected in their cfDNA by either ddPCR or NGS. Ongoing analysis is aimed at establishing if the molecular observations correlate with clinical outcome. Conclusion Combined CTC enrichment and cfDNA isolation is readily achievable using a single Streck cfDNA BCT. Results indicate that combined CTC and cfDNA analysis is more sensitive than either approach alone. Citation Format: Ged Brady, Dominic Rothwell, Mahmood Ayub, Nigel Smith, Sumitra Mohan, Jakub Chudziak, Kyaw Aung, Richard Hubner, Crispin Miller, Alison Backen, Hui Sun Leong, Sakshi Gulati, Chang Sik Kim, Angela Lamarca, Mairead McNamara, Juan Valle, Caroline Dive. Combined circulating tumour cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer. [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 3960.

Abstract A176: RET inhibition: Development of novel compounds and a personalized medicine strategy in lung adenocarcinoma

Background:RET is a receptor tyrosine kinase (RTK) and forms part of a macromolecular receptor complex containing dimerised RET receptor, two co-receptors and a bound ligand. Signalling networks downstream of RET play an important role in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activating mutations in RET (e.g. C634W and M918T) are known drivers in medullary thyroid carcinomas (MTC). More recently, oncogenic RET fusions (e.g. CCDC6-RET and KIF5B-RET) have been identified in 1-2% of lung adenocarcinoma patients. We are currently developing novel, selective inhibitors of RET, and at the same time, investigating a number of biomarker approaches for the stratification of RET fusion-positive lung cancer patients who might benefit from such therapy. Methods: We have undertaken collaborative studies using established techniques including immunohistochemistry (IHC) and FISH (DNA break apart and RNA). In addition, we have investigated hybrid capture DNA sequencing of both biopsy material and circulating tumour DNA in the blood. Here we, compare and contrast the benefits of each biomarker assay evaluated and consider how these approaches could be translated for use in Phase I clinical trials at The Christie. Conclusion: Our data supports the successful implementation of predictive biomarkers to identify patients who might benefit from treatment with selective RET inhibitors. Acknowledgements:This work was funded by Cancer Research UK (Grant numbers C480/A1141 and C5759/A17098). Citation Format: Mandy Watson, Helen Small, Phil Chapman, Gemma Hopkins, Habiba Begum, Ian D. Waddell, Garry Ashton, Caron Abbey, Jade Harris, Mahmood Ayub, Sumitra Mohan, Dominic Rothwell, Ged Brady, Caroline Dive, Allan Jordan, Donald Ogilvie. RET inhibition: Development of novel compounds and a personalized medicine strategy in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A176.

Abstract 3060: Circulating tumor cells from small cell lung cancer patients are tumorigenic

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small cell lung cancer (SCLC), is a highly aggressive and metastatic disease with a 5 year survival of 5%. SCLC represents 15-20% of all lung cancers and causes >160,000 deaths a year. The majority of patients present with metastatic disease and consequently resections are rare, while biopsies are small and only for diagnostic purposes thus hampering the study of SCLC. However, circulating tumour cells (CTCs) are highly prevalent in SCLC patients and may represent an avenue for the better understanding this disease. Our aim was to determine whether CTCs isolated from SCLC patients were able to form tumours in immunocompromised mice. This was accomplished by erythrocyte and leukocyte depletion and implantation of the remaining cells. Of the 6 initial patients whose CTCs were implanted, 4 gave rise to tumours in less than 5 months. Immuno-histochemical analysis of the tumours revealed them to be human in nature and express markers consistent with SCLC. Whole exome sequencing demonstrated that the tumours had mutations (e.g. TP53 and RB1) and CNV (e.g. loss of 3p and 13q) commonly observed in SCLC samples. Furthermore, single cell analysis of CTCs isolated from the corresponding patient revealed that genetic abnormalities detected in the tumour were also present in the patients CTCs. This confirmed that the tumours (termed CDX for CTC derived explant), were indeed derived from CTCs. In all 4 successful cases, analysis of parallel blood samples by CellSearch demonstrated that the patients had more than 400 CTCs/7.5 ml blood. Two of these patients were initially sensitive to platinum/etoposide therapy, while 2 were refractory. The doubling times of CDX derived from refractory and sensitive patients were 7.2 and 5.0 days compared to 14.2 and 13.4 days, consistent with refractory SCLC being more aggressive than sensitive SCLC. We have been able to successfully passage, freeze and resurrect all the CDX models and aim to report whether the patient response to therapy is mirrored in their CDX. These data demonstrate that SCLC CTCs are tumorigenic and we are investigating whether the CTC derived tumours represent a faithful model of the clinical disease. Citation Format: Christopher J. Morrow, Cassandra L. Hodgkinson, Yaoyong Li, Robert Metcalf, Dominic Rothwell, Francesca Trapani, Radoslaw Polanski, Debbie Burt, Kathryn Simpson, Karen Morris, Stuart Pepper, Daisuke Nonaka, Alastair Greystole, Paul Kelly, Matthew Krebs, Jenny Antonello, Mahmood Ayub, Suzanne Faulkner, Lynsey Priest, Louise Carter, Catriona Tate, Crispin J. Miller, Fiona Blackhall, Ged Brady, Caroline Dive. Circulating tumor cells from small cell lung cancer patients are tumorigenic. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3060. doi:10.1158/1538-7445.AM2014-3060