Small cell lung cancer (SCLC) continues to have a poor prognosis, with a 5-year survival rate of 7%. The standard treatment combines chemo-immunotherapy, but only 15% of patients benefit durably from this combination. Since 2019, a new classification based on the expression of transcription factors has been proposed: ASCL1, NEUROD ₁ , POU2F3 and the triple-negative subtype. Preliminary data suggest a therapeutic vulnerability according to subtype. The aim of our work is to identify the transcriptomic subtype at diagnosis using circulating tumour cells (CTCs) obtained from liquid biopsies [1 Nat Rev Cancer 2019; 19:289-97.

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Since 2016, the monocentric, non-interventional CTC-SCLC study has developed an immuno-magnetic sorting method for CTC collection. To detect transcriptomic subtypes, multiplex digital PCR–highly sensitive due to the partitioning of the sample into thousands of micro-droplets–was chosen. Probes and primers targeting the markers of interest were first developed and then validated on commercial SCLC cell lines of known subtypes: NCI-H ₆₉ (ASCL1), NCI-H ₄₄₆ (NEUROD ₁ ), NCI-H ₅₂₆ (POU2F3) and NCI-H ₁₉₆ (triple negative). To experimentally simulate the conditions of CTCs isolation from patient samples, these cell lines were introduced into blood collected from healthy donors [2 Sci Rep 2023; 13:3626.

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Transcriptomic profiles of four commercial cell lines–NCI-H ₆₉ (ASCL1), NCI-H ₄₄₆ (NEUROD ₁ ), NCI-H ₅₂₆ (POU2F3), and NCI-H ₁₉₆ (triple-negative)–were validated by qPCR and western blotting. ddPCR assays were developed for each subtype, detecting 2.425, 0.413 and 631 copies per μL of ASCL1, NEUROD ₁ , and POU2F3 in NCI-H ₆₉ , NCI-H ₄₄₆ , and NCI-H ₅₂₆ , respectively. Healthy donor blood spiked with NCI-H ₆₉ and NCI-H ₅₂₆ cells showed respectively 7.327 ASCL1 copies per well by ddPCR and POU2F3 expression with a mean Ct of 31.047 by qPCR (18S housekeeping gene Ct averaged 19.652).

Our results demonstrate the feasibility of detecting the ASCL1 and POU2F3 transcriptomic subtypes by experimentally reproducing CTC isolation using commercial lines. The next step will be to apply this methodology to patient samples and compare the subtype defined by the liquid biopsy with the primary tissue.