We present Beyondcell, a computational methodology for identifying tumour cell subpopulations with distinct drug responses in single-cell RNA-seq data and proposing cancer-specific treatments. Our method calculates an enrichment score in a collection of drug signatures, delineating therapeutic clusters (TCs) within cellular populations. Additionally, Beyondcell determines the therapeutic differences among cell populations and generates a prioritised sensitivity-based ranking in order to guide drug selection. We performed Beyondcell analysis in five single-cell datasets and demonstrated that TCs can be exploited to target malignant cells both in cancer cell lines and tumour patients. Beyondcell is available at: https://gitlab.com/bu_cnio/beyondcell.

Despite impressive and durable responses, non-small cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase inhibitors (ALK-Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next-generation sequencing (NGS) upon disease progression. We collected26 plasma and two cerebrospinal fluid samples from 24 advanced ALK-positive NSCLC patients at disease progression to an ALK-I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool).We identified at least one resistance mutation in the ALK locus in ten (38.5%) plasma samples; the G1269A and G1202R mutations were the most prevalent among patients progressing to first-and secondgeneration ALK-Is, respectively. Overall, 61 somatic mutations were detected in 14 genes: TP53, ALK,

Background: Despite impressive and durable responses, patients treated with ALK inhibitors (ALK-Is) ultimately progress. We investigated potential resistance mechanisms in a series of ALK-positive non-small cell lung cancer (NSCLC) patients progressing on different types of ALK-Is.Methods: 26 plasma and 2 cerebrospinal fluid samples collected upon disease progression to an ALK-I, from 24 advanced ALK-positive NSCLC patients, were analyzed by next-generation sequencing (NGS). A tool to retrieve variants at the ALK locus was developed. Results: 61 somatic mutations were detected in 14 genes: TP53, ALK, PIK3CA, SMAD4, MAP2K1 (MEK1) FGFR2, FGFR3, BRAF, EGFR, IDH2, MYC, MET, CCND3 and CCND1. Overall, We identified at least one mutation in ALK locus in 10 (38.5%) plasma samples, being the G1269A and G1202R mutations the most prevalent among patients progressing to first- and second-generation ALK-I treatment, respectively. An exon 19 deletion in EGFR was identified in a patient showing primary resistance to ALK-I. Likewise, the G466V mutation in BRAF and the F129L mutation in MAP2K1 (MEK1) were identified as the underlying mechanism of resistance in three patients who gained no or little benefit from second-line treatment with an ALK-I. Putative ALK-I resistance mutations were also found in PIK3CA and IDH2. Finally, a c-MYC gain, along with a loss of CCND1 and a FGFR3, were detected in a patient progressing on a first-line treatment with crizotinib. Conclusions: NGS analysis of liquid biopsies upon disease progression identified putative ALK-I resistance mutations in most cases, being a valuable approach to devise therapeutic strategies upon ALK-I failure.

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