Brief report on the clinical characteristics of patients whose samples generate small cell lung cancer circulating tumour cell derived explants.

INTRODUCTION: Small cell lung cancer (SCLC) has a dismal prognosis. Circulating tumour cells (CTCs) can be used to generate CTC derived explants (CDX) for the study of SCLC biology and the development of novel therapeutics. We investigated whether there are demographic or clinical predictors of the success of CDX generation, and whether CDX models are representative of the SCLC patient population. METHODS: This was a single centre, retrospective analysis of SCLC patients who had participated in the CHEMORES Study. Paired blood samples were donated for CTC enumeration and CDX generation attempt at pre-treatment baseline, disease progression and intervening timepoints. Clinical and demographic data was collected from electronic records, and analysed for differences between patients whose samples did and did not generate a CDX. RESULTS: 231 paired blood samples were taken from 147 patients. 45 CDX were generated from 34 patients. CTC number was significantly higher in blood samples which successfully generated a CDX than those which didn't, at both baseline (p=<0.0001) and progression (p = 0.0001). The group with successful blood samples had a poorer performance status (p = 0.0067), and a higher proportion of patients with chemorefractory disease (p = 0.0077). Both progression free survival (PFS) (p = 0.0132) and overall survival (p=< 0.0001) were significantly shorter for patients with successful samples. CONCLUSIONS: Patients whose samples generate CDX models may have a higher disease burden and more aggressive disease. Thus, insights gained by study of SCLC CDX may have a significant impact, particularly in the SCLC subpopulation with the greatest clinical need.

Molecular analysis of single circulating tumour cells following long-term storage of clinical samples.

The CellSearch® semiautomated CTC enrichment and staining system has been established as the 'gold standard' for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time-consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch® -enriched cells in glycerol at -20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long-term glycerol storage for single-cell molecular analysis, we isolated individual CellSearch® -enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at -20 °C. All isolated cells were subjected to whole-genome amplification (WGA), and the efficacy of single-cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both 'spike-in' controls and clinical samples showed no statistical difference between values obtained pre- and postglycerol storage and that there is no further loss in integrity when DEPArray™-isolated cells are then stored at -80 °C for up to 2 years. In summary, we have established simple yet effective 'stop-off' points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.

Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi-site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours. In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype-directed therapies in multi-site clinical trials and represent a significant methodological improvement for clinical benefit.