Liquid biopsy-based precision medicine for extra-pulmonary neuroendocrine carcinomas

Abstract

Extra-Pulmonary NeuroEndocrine Carcinomas (EP-NECs) are aggressive tumours (Ki-67>20%, poorly differentiated morphology), with a median overall survival <12 months (ms). In most cases, EP-NECs are metastatic at diagnosis and palliative platinum/etoposide chemotherapy (chemo) is the only active management to offer. Improvements in clinical outcomes of EP-NECs have been hindered by the paucity of knowledge of their biology, and lack of validated biomarkers and preclinical models to inform patient management and therapeutic development. In the phenotypically similar pulmonary counterpart NEC, Small Cell Lung Cancer (SCLC), circulating tumour cells (CTCs) are abundant and prognostic1, and can be used for the generation of patient-relevant mouse models (CTC-Derived eXplants, CDX)2.Using SCLC research as a paradigm, a ‘proof-of-concept' prospective study has been designed to assess feasibility of CTC detection and CDX generation in patients with advanced stage EP-NEC undertaking palliative chemo. Serial CTC counts (pre-chemo, 6 weeks after start of chemo and at disease progression) are performed by using CellSearch (CS) which enumerates EpCAM expressing CTCs. CDX generation is attempted from matched blood samples after RosetteSep CTC-enrichment and engraftment in immunodeficient mice, following the same protocol optimised for SCLC CDX2. As of Nov 2020, pre-chemo CS-CTCs were detected in 11/17 patients (64.7%); mean 18.3, median 2, range 0-166 (per 7.5mL of blood). A CDX model was successfully developed from pre-chemo CTCs from a patient with a metastatic NEC of unknown origin. Histopathological analysis of the CDX tumour confirmed a NEC diagnosis (Ki-67=80%, strong expression of synaptophysin, chromogranin A, CD56) and closely matched the donor patient's biopsy. Notably, the CS-CTC count in a paired blood sample was low at 1/7.5mL of blood. As the CTC count predicts the likelihood of SCLC CDX generation2, this finding suggests that non-EpCAM expressing CTCs are present in the donor patient's bloodstream.In conclusion, the majority of advanced EP-NECs shed epithelial CTCs although at lower levels than SCLC, based on comparison with previous data1. Analysis of CTCs enriched by using a biomarker-independent platform may elucidate the phenotypic composition of CTC pools in EP-NECs. We report here on the feasibility of EP-NEC CDX generation. The model has been successfully passaged, and next steps will include its full phenotypic and molecular characterisation. In vivo drug studies are also underway using the CDX as an avatar to potentially guide future treatment for the donor patient. In addition, CDX-derived cell cultures have been established and hold the potential to serve as ex vivo platforms for functional validation of mechanisms of drug sensitivity/resistance and drug screening.