Transformation of castration-resistant prostate cancer (CRPC) into an aggressive neuroendocrine disease (CRPC-NE) represents a major clinical challenge and experimental models are lacking. A ...CTC-derived eXplant (CDX) and a CDX-derived cell line are established using circulating tumor cells (CTCs) obtained by diagnostic leukapheresis from a CRPC patient resistant to enzalutamide. The CDX and the derived-cell line conserve 16% of primary tumor (PT) and 56% of CTC mutations, as well as 83% of PT copy-number aberrations including clonal TMPRSS2-ERG fusion and NKX3.1 loss. Both harbor an androgen receptor-null neuroendocrine phenotype, TP53, PTEN and RB1 loss. While PTEN and RB1 loss are acquired in CTCs, evolutionary analysis suggest that a PT subclone harboring TP53 loss is the driver of the metastatic event leading to the CDX. This CDX model provides insights on the sequential acquisition of key drivers of neuroendocrine transdifferentiation and offers a unique tool for effective drug screening in CRPC-NE management.
Manipulating single biological objects is a major unmet challenge of biomedicine. Herein, we describe a lab-on-a-chip platform based on dielectrophoresis (DEP). The DEParray is a prototypal version ...consisting of 320 × 320 arrayed electrodes generating >10 000 spherical DEP cages. It allows the capture and software-guided movement to predetermined spatial coordinates of single biological objects. With the DEParray we demonstrate (a) forced interaction between a single, preselected target cell and a programmable number of either microspheres or natural killer (NK) cells, (b) on-chip immunophenotypic discrimination of individual cells based on differential rosetting with microspheres functionalized with monoclonal antibodies to an inhibitory NK cell ligand (HLA-G), (c) on-chip, real-time (few minutes) assessment of immune lysis by either visual inspection or semiautomated, time-lapse reading of a fluorescent dye released from NK cell-sensitive targets, and (d) manipulation and immunophenotyping with limiting amounts (about 500) cells. To our knowledge, this is the first report describing a DEP-based lab-on-a-chip platform for the quick, arrayed, software-guided binding of individually moved biological objects, the targeting of single cells with microspheres, and the real-time characterization of immunophenotypes. The DEParray candidates as a discovery tool for novel cell:cell interactions with no prior (immuno)phenotypic knowledge.
Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs ...with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non‐random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC‐positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high‐quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre‐existing cells resistant to ERBB2‐targeted therapies suggesting ongoing microevolution at late‐stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.
Synopsis
A novel workflow enabling detection, isolation and characterization of single circulating tumors cells (CTCs) from blood suggests that CTCs may harbor genetic alterations undetectable in the primary tumor and associated with therapy resistance.
Single circulating tumor cells (CTCs) are analyzed by a semi‐automated workflow combining CellSearch® enrichment, DEPArrayTM isolation and Ampli1TM whole genome amplification (WGA).
The WGA quality of single CTCs is assessed by a genome integrity index (GII).
The GII predicts outcome of downstream sequence‐based molecular assays.
Single cell analysis reveals the existence of rare potential therapy escape variants.
The diagnostic precision of the workflow enables molecular monitoring of CTCs under iatrogenic selection.
A novel workflow enabling detection, isolation and characterization of single circulating tumors cells (CTCs) from blood suggests that CTCs may harbor genetic alterations undetectable in the primary tumor and associated with therapy resistance.
Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome ...analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.
Abstract
Gatekeeper mutations are identified in only 50% of the cases at resistance to
Anaplastic Lymphoma Kinase
(
ALK
)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are ...relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in
ALK
-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six
ALK
-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1
st
and 3
rd
ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK
+
/cytokeratins
-
, 46 (56%) ALK
-
/cytokeratins
+
and 4 (5%) ALK
+
/cytokeratins
+
. Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial
ALK
-negative CTCs may drive resistance through activation of bypass signaling pathways, while
ALK
-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies.
Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the ...presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.
Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical–pathological investigations. CUP patients ...are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH
®
and DEPArray
TM
NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with
Ampli
1
TM
kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in
APC
gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne.
FGFR2
and
CCNE1
gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in
ARID1A
gene (p.R1276
∗
) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in
ASPM
and
SEPT9
genes and loss of
FANCC
. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs
∗
19) and a somatic mutation (p. A1487V) in
ARID1A
gene and a point mutation in
FGFR2
gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.
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