Lung cancer is the leading cause of cancer-related death in the world. It is broadly divided into small cell (SCLC, approx. 15% cases) and non-small cell lung cancer (NSCLC, approx. 85% cases). The main histological subtypes of NSCLC are adenocarcinoma and squamous cell carcinoma, with the presence of specific DNA mutations allowing further molecular stratification. If identified at an early stage, surgical resection of NSCLC offers a favourable prognosis, with published case series reporting 5-year survival rates of up to 70% for small, localized tumours (stage I). However, most patients (approx. 75%) have advanced disease at the time of diagnosis (stage III/IV) and despite significant developments in the oncological management of late stage lung cancer over recent years, survival remains poor. In 2014, the UK Office for National Statistics reported that patients diagnosed with distant metastatic disease (stage IV) had a 1-year survival rate of just 15–19% compared with 81–85% for stage I.

Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.

Mycoplasma, Acholeplasma, and Ureaplasma sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the Mycoplasma, Acholeplasma, and Ureaplasma sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of M. arginini, M. orale, M. hyorhinis, M. fermentans, M. genitalium, M. hominis, M. pneumoniae, M. salivarium, M. pirum, A. laidlawii, and U. urealyticum. Limit of detection values varied between 125–300 and 50–100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from Mycoplasma, Acholeplasma, and Ureaplasma in tested cell cultures.

Background: Prostate cancer (CaP) is the second-leading cause of male cancer-related mortality in Western societies. Localised prostate cancer (Loc-CaP) can be classified into low-, intermediate-, or high-risk groups. Active surveillance is recommended for low-risk patients, whereas radiotherapy or surgery is often indicated in intermediate- and high-risk patients, with possible intensification using hormone therapy treatment. Despite advances in radiation delivery and surgery, ~20% of patients will not be cured with local treatment and require androgen deprivation therapy (ADT). The majority will develop resistance to ADT resulting in castration resistant prostate cancer (CRPC) with poor prognosis and increased risk of metastatic disease (mCRPC). Biomarkers are being developed based on tumour biopsies which may help stratify patients and allow more effective treatment. However tumour biopsies are invasive, demanding for patients and surgeons as well as challenging for longitudinal sampling. Here we evaluate a range of blood biomarkers suitable for longitudinal sampling including circulating tumour cells (CTCs), circulating tumour DNA (ctDNA) and Tumour educated platelet mRNA (TEP-RNA). Methods: A blood sampling, processing and banking pipeline has been established for CaP patients providing ctDNA, enriched and single CTCs as well as TEP-RNA. Following ctDNA isolation, next generation sequencing (NGS) libraries are prepared to generate copy number alteration (CNA) data and mutation profiles. CTCs are enriched using both the epitope independent Parsortix (P-CTC) (Chudziak J. et al, 2016) and the epitope dependent (EPCAM and KRT19) CellSearch (CS-CTC) platforms. Genomic analysis of CTCs is carried out by NGS of whole genome amplified (WGA) single and pooled CTCs. CTC and TEP-RNA mRNA profiles are established using both the Fluidigm RT qPCR (Fluidigm Biomark™ HD system) and RNA-Seq. Results: Over 160 Loc-CaP and 100 mCRPC blood samples have been collected, processed and banked. In keeping with published data 53% of the 100 mCRPC patients have &gt5 CS-CTCs (a poor prognostic indicator) compared to 100% of Loc-CaP having <2 CTCs. Control cell “spike in” RNA data showed =>1000 fold enrichment of epithelial mRNAs post Parsortix CTC enrichment indicate an equivalent enrichment of the “spiked in” cells. Similar epithelial mRNA enrichment was seen in 3/5 mCRPC clinical samples tested. Tumour associated CNA changes were clearly detected in at least 15 of the 1st 22 mCRPC ctDNA samples. Sample processing and statistical analysis is ongoing to evaluate if these blood biomarkers, either alone or in combination, can be linked to clinical status or outcome. Note: This abstract was not presented at the meeting. Citation Format: Jenny Antonello, Jakub Chudziak, Alan Redfern, Victoria Foy, Shambhavi Srivastava, Adnan Syed, Deborah Burt, Mahmood Ayub, Bedirhan Kilerci, Marina Parry, Richard Marais, Esther Baena, Crispin Miller, Dominic Rothwell, Noel Clarke, Caroline Dive, Ged Brady. An evaluation of DNA and RNA based blood biomarkers in prostate cancer [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 5685. doi:10.1158/1538-7445.AM2017-5685