A Pilot Study of the Predictive Potential of Chemosensitivity and Gene Expression Assays Using Circulating Tumour Cells from Patients with Recurrent Ovarian Cancer.

Stefano Guadagni,Donatella Sarti,Giuseppe Zavattieri,Francesco Masedu,Karl Reinhard Aigner,Giammaria Fiorentini,Marco Clementi,Marcello Deraco,Panagiotis Apostolou,Antonietta Rossella Farina,Giovanni Scambia,Shigeki Kusamura,Andrew Reay Mackay,Giuseppe Vizzielli,Ioannis Papasotiriou

doi:10.3390/ijms21134813

Abstract

Circulating tumour cells (CTCs) from liquid biopsies are under current investigation in several cancers, including epithelial ovarian cancer (EOC) but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic EOC CTCs, from 10 patients, nine with stage IIIC and one with stage IV disease, in progression after systemic chemotherapy, submitted for hypoxic isolated abdominal perfusion (HAP), are both feasible and useful in predicting response to therapy. Viable metastatic EOC CTCs (>5 cells/mL for all 10 blood samples), enriched by transient culture and identified by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence (IF), were subjected to flow cytometry-based Annexin V-PE assays for chemosensitivity to several chemotherapeutic agents and by RT-PCR for tumour gene expression profiling. Using a cut-off value of >80% cell death, CTC chemosensitivity tests were predictive of patient RECIST 1.1 responses to HAP therapy associated with 100% sensitivity, 50% specificity, 33% positive predictive, 100% negative predictive and 60% accuracy values. We propose that the methodology employed in this study is feasible and has the potential to predict response to therapy, setting the stage for a larger study.

Highlights

Epithelial ovarian cancer (EOC) is the 7th most common cancer and 8th leading cause of cancer mortality in women, with a cumulative 5-year survival rate below 45% [1], and in cases with regional and distant invasion, 5-year survival rates are approximately 70% and 30%, respectively
We report that the methods employed for blood sampling, storage, transport and subsequent Circulating tumour cells (CTCs) purification and enrichment are feasible and reproducible, and that the subsequent CTC chemosensitivity and gene expression assays employed to select chemotherapeutic strategies are predictive of therapeutic response
The interval between blood sampling and quantitative reverse transcription polymerase chain reaction analysis did not exceed 80 h, previously reported to minimise alterations in gene and protein expression [41] and metastatic EOC CTC numbers in 10 liquid biopsies were greater than the 5 CTCs/mL cut-off value required for chemosensitivity and tumour gene expression analysis [41]

Summary

Introduction

Epithelial ovarian cancer (EOC) is the 7th most common cancer and 8th leading cause of cancer mortality in women, with a cumulative 5-year survival rate below 45% [1], and in cases with regional and distant invasion, 5-year survival rates are approximately 70% and 30%, respectively. The current standard of care for patients with advanced EOC is debulking surgery combined with systemic paclitaxel and carboplatin chemotherapy [2]. Patients have a good response to initial treatment, most experience relapse related to factors, including incomplete tumour debulking and presumed acquisition of resistance to platinum-based chemotherapy [3]. EOC patients considered to be platinum-resistant are commonly those who have relapse following a systemic platinum-based chemotherapeutic regimen. This assumption, is normally based upon disease-relapse and not molecular evidence, and is used to characterise four subsets based upon platinum free interval (PFI) duration (12 months), which correspond to platinum-refractory, platinum-resistant, partially platinum-sensitive and fully platinum-sensitive categories. (ADP-ribose) polymerases (PARPs) are important nucleoproteins involved in DNA damage-repair and PARP inhibitors have demonstrated efficacy, as maintenance therapy, in adult patients with recurrent EOC, who exhibit complete or partial responses to systemic platinum-based chemotherapy [4]

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