Individual Circulating Tumor Cells Research Articles

Abstract P4-02-04: Serial monitoring of circulating tumor cells and circulating tumor DNA in metastatic lobular breast cancer identifies intra-tumor heterogeneity and precision and immuno-oncology biomarkers of therapeutic importance

Abstract Clinical decisions on precision and immuno-oncology therapies are based on predictive biomarkers commonly obtained from a single metastatic biopsy or archived primary tumor tissue. Circulating genomic biomarkers offer a minimally invasive approach to monitor intra-patient tumor heterogeneity and detect in real-time the clinically-relevant evolving clonal architecture. Although currently underutilized, we hypothesize that single-cell DNA next generation sequencing (scNGS) of circulating tumor cells (CTC) is a particularly well-suited method to complement biomarker information obtained from tissue and cell-free circulating tumor DNA (ctDNA). In this study we analyzed 113 individual CTC, 21 ctDNA, and 15 white blood cells (WBC) samples, from 15 CTC-positive lobular breast cancer patients, four of whom had CTC available at both metastatic baseline and after progression on a variety of therapies chosen at their physician’s discretion. Clinical NGS data from 15 tumor tissue biopsies obtained using a ~1700-gene DNA panel and whole transcriptome sequencing were available for comparison. CTC were enriched with the CellSearch® system and isolated as single cells with the DEPArray™ system. Whole genome amplified CTC and WBC, as well as ctDNA underwent scNGS with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space to detect mutations, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). 99.1% of single cells and 95.2% of ctDNA samples were informative, with a mean sequencing depth of 664x. Using our previously developed, CTC-based precision medicine reporting platform, MI-CTCSeq, CTC in 9 of 15 patients (60%) had mutations that were actionable by FDA-approved targeted therapies including in the oncogenes PIK3CA and FGFR2 and HER2. 3 of these 9 patients (33%) harbored actionable alterations not shared between all 3 analyte types (tissue, CTC and ctDNA). These included 3 actionable mutations found in CTC and ctDNA only, 1 in tissue and ctDNA only, and 1 in ctDNA only. However, 2 of those ctDNA mutations were identified near the limit of detection and with a priori knowledge of their presence from tissue or CTC. Further, 1 patient with plentiful CTC had no detectable ctDNA and one patient’s tissue biopsy was inadequate for sequencing while both liquid biopsy analytes were abundant. 13 patients (87%) displayed intra-patient, inter-CTC genomic heterogeneity of putative driver mutations. 1 of 4 (25%) patients with CTC available in >1 timepoint displayed fluctuations in their CTC subclonal makeup between timepoints. Data from this patient’s 2 tissue biopsies, 3 ctDNA samples, and 27 individual CTC over 4 timepoints combined to reveal in unprecedented detail inter-metastatic lesion and inter-CTC heterogeneity and tumor evolution in response to endocrine and immunotherapy selective pressures. ScNGS of CTC helped provide an additional level of detail not appreciated by sequencing of the other two analyte types. In another patient, CTC were composed of 2 subclones which were indistinguishable by ctDNA, 1 of which appears to have not been sampled by the tissue biopsy. Using a novel method, we enabled detection of single-cell CTC TMB and MSI. CTC TMB scores (dichotomized as above/below 10 mutations/Mb) were 100% concordant with those measured in the corresponding tissue biopsies. Further, in a novel observation, we detected intra patient, inter-CTC heterogeneity of TMB and MSI, which has potential implications for immunotherapy response and development of resistance. Taken together, these data support the non-invasive biomarker interrogation and monitoring by liquid biopsy that incorporates CTC scNGS and complements tissue in informing precision and immuno-oncology approaches. This may have important implications for appropriate treatment selection and identification of therapeutic resistance mechanisms. Citation Format: Andi Cani, Emily Dolce, Alissa Turnbull, Kevin Hu, Chia-Jen Liu, Elizabeth Darga, Dan Robinson, Yi-Mi Wu, Dafydd G. Thomas, Costanza Paoletti, Scott Tomlins, James Rae, Aaron Udager, Arul Chinnaiyan, Erin F. Cobain, Daniel F. Hayes. Serial monitoring of circulating tumor cells and circulating tumor DNA in metastatic lobular breast cancer identifies intra-tumor heterogeneity and precision and immuno-oncology biomarkers of therapeutic importance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-04.

Abstract P5-06-03: Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms

Abstract Background Liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is otherwise infeasible. In addition, the development of quantitative, reproducible, and more sensitive HER2 assays is expected to enable the identification of patients with HER2-low MBC that may benefit from novel HER2-targeted therapies. Here we report a comprehensive liquid biopsy platform including immunofluorescent HER2 and ER quantitative protein expression in CTCs (ctcIF) coupled with the determination of ERBB2 amplification and the number of Large-scale State Transitions (LST) by single-cell CTC genomics (ctcDNA), and ctDNA alterations in plasma. Methods Blood samples were collected for cell-based and cell-free analysis from 62 patients with documented MBC and from 24 blood donors (HD) with no known cancer history. After isolation, nucleated cells were plated, and slides and plasma were bio-banked. CTCs were identified using Epic Sciences digital imaging and machine learning algorithms, and ctcIF enables quantitation of HER2 and ER protein expression. ctcDNA was analyzed by low-pass whole-genome sequencing (WGS), allowing detection of ERBB2 amplification (ERBB2amp) and quantification of large-scale state transitions (LST+) in individual CTCs. cfDNA from plasma was analyzed using a validated NGS panel (56 genes of interest) to detect ctDNA alterations. Results Within this cohort of 62 MBC patients, the presence of CTCs, ctcDNA (LST+) and ctDNA alterations were detected in 87%, 70%, and 59%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2amp in MBC patients (11%, and 2%, respectively). A variant allele frequency (VAF) of > 40%, which is required for detecting a two-fold ERBB2 amplification by ctDNA, was not present among 86% and 0% of MBC detected by the ctcDNA and ctDNA platforms, respectively, suggesting that the ctcDNA platform can identify ERBB2amp among patients with a low ctDNA fraction. HER2+ or ER+ expression by ctcIF were detected in 37% and 58% of MBC patients, respectively. At the cellular level, across 62 patients, CTC with detectable ERBB2amp by ctcDNA had a higher median expression of HER2 protein by ctcIF compared to CTC with ERBB2nonamp (1772 MFI vs 122.5 MFI, respectively; p< 0.001). Similarly, at the patient level, among patients with circulating ERBB2amp, HER2 protein was detected by ctcIF in 100% of MBC patients (p< 0.001), suggesting a very high positive correlation between the presence of ctcDNA genomic ERBB2amp and ctcIF HER2 protein expression. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that among MBC, 11% were ERBB2amp, 26% were HER2 expressing (HER2+ and ERBB2nonamp), and 60% were HER2neg (HER2- and ERBB2nonamp). Combination models of the three individual platforms (ctcIF, ctcDNA, and ctDNA) were able to provide potentially clinically actionable biomarker data (LST+ CTC, ERBB2amp CTC, HER2+ CTC, ER+ CTC and 1A+ SNVs) to 79% of MBC patients while retaining 100% specificity. Conclusions Here we reported a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in late MBC patients. The comprehensive liquid biopsy platform’s combined utility can aid biomarker profiling of MBC among often biologically heterogeneous tumor sites of metastatic disease and those inaccessible by conventional tissue biopsy. Citation Format: Giuseppe Di Caro, Ernest T. Lam, Megan M. Slade, Anna Lundberg, Martin Blankfard, Nilesh Dharajiya, Alisa Tubbs, Rick Wenstrup, Lee Schwartzberg. Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-03.

A Rapid, Shallow Whole Genome Sequencing Workflow Applicable to Limiting Amounts of Cell-Free DNA.

Somatic copy number alterations (sCNAs) acquired during the evolution of breast cancer provide valuable prognostic and therapeutic information. Here we present a workflow for screening sCNAs using picogram amounts of cell-free DNA (cfDNA) and single circulating tumor cells (CTCs). We repurposed the Ion ReproSeq PGS™ preimplantation genetic testing kit to perform shallow whole genome sequencing on 178 cfDNA samples (300 pg) and individual CTCs from 10 MBC patients with metastatic breast cancer (MBC) recovered by CellSearch®/DEPArray™. Results were analyzed using a tailored ichorCNA workflow. sCNAs were detected in cfDNA of 41/105 (39%) patients with MBC and 3/23 (13%) primary breast cancers on follow-up (PBC FU), all of whom subsequently relapsed. In 8 of 10 MBCs, individual CTCs had a higher copy number count than matched cfDNA. The median tumor fraction detected by ichorCNA was 0.34 (range 0.17-0.58) for MBC and 0.36 (range 0.31-0.37) for PBC FU. Patients with detectable tumor fraction (≥ 0.1) and TFx and OncomineTM variants had significantly lower overall survival rates (P values P = 0.002 and P < 0.0001 for the log-rank test, respectively). The ReproSeq PGS assay is rapid, at approximately $120 per sample, providing both a sCNA profile and estimation of the tumor DNA fraction from limiting cfDNA template (300pg) and individual CTCs. The approach could be used to examine the copy number landscape over time to guide treatment decisions, support future trial designs, and be applied to low volume blood spot samples enabling remote monitoring.

Changes of protein expression during tumorosphere formation of small cell lung cancer circulating tumor cells.

Small cell lung cancer (SCLC) is frequently disseminated and has a dismal prognosis with survival times of approximately two years. This cancer responds well to initial chemotherapy but recurs within a short time as a globally chemoresistant tumor. Circulating tumor cells (CTCs) are held responsible for metastasis, the extremely high numbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines. These CTCs are distinguished by the spontaneous formation of large spheroids, termed tumorospheres, in regular tissue culture. These contain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to single cell cultures. Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as single cells or in the form of tumorospheres in Western blot arrays. With the exception of the UHGc5 line, all other CTC lines express EpCAM and lack a complete EpCAM-negative, vimentin-positive epithelial-mesenchymal transition (EMT) phenotype. Upon formation of tumorospheres the expression of EpCAM, that mediates cell-cell adhesion is markedly upregulated. Proteins such as E-Cadherin, p27 KIP1, Progranulin, BXclx, Galectin-3, and Survivin showed variable changes for the distinct CTC cell lines. In conclusion, EpCAM presents the most critical marker for individual SCLC CTCs and the assembly of highly chemoresistant tumorospheres.

Circulating Tumor Cell Cluster Sorting by Size and Asymmetry.

Circulating tumor cell (CTC) clusters are rare yet potent initiators of metastasis and may be useful as clinical biomarkers. Numerous techniques have been developed to isolate individual circulating tumor cells from the blood, but these techniques are often ineffective at capturing CTC clusters and may cause cluster damage or dissociation during processing or recovery. This chapter describes methods for fabricating and operating a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood or biological fluids using deterministic lateral displacement.

MAF Translocations Are Enriched in NDMM Patients with Elevated Levels of Circulating Tumor Cells Suggesting a Genetic Basis for Aggressive Disease Course

Introduction A subset of Multiple Myeloma (MM) patients responds poorly to treatment, highlighting the need to understand the pathobiology of high-risk MM. The correlation between aggressive disease and elevated levels of circulating tumor cells (CTCs) has been recognized. We recently identified a subset of high-risk newly diagnosed MM (NDMM) patients based on a transcriptomic classifier derived from patients with high levels of CTC, indicating that CTC levels are a relevant reflection of tumor cell biology. Here, we hypothesized that comparing bone marrow plasma cells (BM PCs) of MM patients with high and low CTC levels will provide an opportunity to identify mechanistic drivers of aggressive disease. Results We first set out to exclude the presence of a circulating aggressive subclone of MM cells in patients with high levels of CTCs. Paired single cell transcriptomes from BM and peripheral blood MM cells of 7 NDMM patients were generated. Analysis of 55,230 CTCs and BM PCs revealed patient specific clustering, but with large transcriptional overlap between individual paired CTCs and BM PCs. Since only 11 genes were significantly upregulated in the BM PCs (P < 0.05) compared to paired CTCs, we excluded the presence of circulating aggressive subclones in patients with high CTC levels. Next, we aimed to identify differences in MM cell pathobiology between patients with high and low levels of CTCs, we generated single cell transcriptomes from 89,092 BM MM cells from 8 NDMM patients with high (≥0.5%) and 14 patients with low (0.004-0.006%) percentages of CTCs. Single cell data integration across all patients led to the identification of 8 distinct PC clusters. Gene set enrichment analysis between both patient groups revealed upregulation of cell cycle pathways in MM cells of patients with high CTC levels. Based on cell cycle analyses an increase in cycling PCs (53% vs 32%, p=0.007) was associated with high CTC levels (Figure 1). Ki67 labeling of BM biopsies validated this finding. Interestingly, proliferation was increased across all transcriptomically-defined plasma cell clusters, suggesting a common cell-intrinsic driver of increased proliferation. To analyze whether the prevalence of genetic lesions previously associated with proliferation was increased in patients with high CTC levels, WGS was performed on the same 22 patients. This revealed an elevated mutation burden and a predilection for MAF translocations, as 50% (4/8) of the patients with high CTC levels carried MAF translocations versus none in the patients with low CTC levels (0/14). We also observed increased transcription of cyclin D2 (CCND2), CCR1 and IntegrinB7 (ITGB7) in patients with high CTC levels. These proliferation or tumor survival genes have been shown to be increased in patients with t(14;16), a translocation also resulting in overexpression of MAF. To validate these findings in a larger patient cohort, clinical, genomic and CTC data were retrieved from the MMRF-CoMMpass database (n=725). Strikingly, 21% of patients with CTC levels above 0.3% showed MAF translocations vs only 3% in patients with CTC levels below 0.3% (p<0.001), solidifying the correlation between MAF translocations and high levels of CTCs in NDMM patients with a high-risk disease profile. Conclusion Single cell RNA sequencing and WGS of MM cells from patients with high and low CTC levels identified a correlation between CTC levels and overrepresentation of MAF translocations in NDMM patients. This finding suggests that MAF target genes and downstream proliferation could be drivers of aggressive disease and might be used for future patient stratification. Figure 1. Based on cell cycle analyses in single cell transcriptomics an increase in cycling PCs was associated with high CTC levels. a) UMAP of cell cycle analysis of 89,902 BM MM cells from 22 patients with high (n=8) and low (n=14) CTC levels. Colors represent cell cycle phase. b) Bar graph representing percentage of cycling cells. Data points are individual patients (high CTC levels, n=8; low CTC levels, n=14). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Shallow WGS of individual CTCs identifies actionable targets for informing treatment decisions in metastatic breast cancer

BackgroundWe report copy-number profiling by low-pass WGS (LP-WGS) in individual circulating tumour cells (CTCs) for guiding treatment in patients with metastatic breast cancer (MBC), comparing CTC results with mutations detected in circulating tumour DNA (ctDNA) in the same blood samples.MethodsAcross 10 patients with MBC who were progressing at the time of blood sampling and that had >20 CTCs detected by CellSearch®, 63 single cells (50 CTCs and 13 WBCs) and 16 cell pools (8 CTC pools and 8 WBC pools) were recovered from peripheral blood by CellSearch®/DEPArray™ and sequenced with Ampli1 LowPass technology (Menarini Silicon Biosystems). Copy-number aberrations were identified using the MSBiosuite software platform, and results were compared with mutations detected in matched plasma cfDNA analysed by targeted next-generation sequencing using the Oncomine™ Breast cfDNA Assay (Thermo Fisher).ResultsLP-WGS data demonstrated copy-number gains/losses in individual CTCs in regions including FGFR1, JAK2 and CDK6 in five patients, ERBB2 amplification in two HER2-negative patients and BRCA loss in two patients. Seven of eight matched plasmas also had mutations in ctDNA in PIK3CA, TP53, ESR1 and KRAS genes with mutant allele frequencies (MAF) ranging from 0.05 to 33.11%. Combining results from paired CTCs and ctDNA, clinically actionable targets were identified in all ten patients.ConclusionThis combined analysis of CTCs and ctDNA may offer a new approach for monitoring of disease progression and to direct therapy in patients with advanced MBC, at a time when they are coming towards the end of other treatment options.

Chromosomal instability (CIN) in circulating tumor cells (CTC) predicts for taxane sensitivity in metastatic castration-resistant prostate cancer (mCRPC).

5026 Background: Few clinically validated biomarkers can inform therapy (tx) sequencing in mCRPC. Previously, we credentialed a biomarker of CIN in individual CTCs using phenotypic identification of cells with ≥9 large scale transitions (LSTs) and classification of CIN as high when detected in ≥3 CTC/mL (CIN3+). CIN3+ was associated with shorter radiographic progression survival (rPFS) and overall survival (OS) following taxane (T) or androgen receptor signaling inhibitor (ARSi) tx in mCRPC. Similarly, high CIN defined as ≥1 CTC/mL with ≥9 LSTs (CIN1+) predicts worse rPFS and OS following ARSi. Here, we explored if CIN1+ or CIN3+ predicted differential outcomes in pts on-tx with T or ARSi for mCRPC. Methods: We analyzed 208 banked CTC samples from 173 mCRPC pts collected within 30 days prior to starting a new tx with either T (n=88) or ARSi (n=120) in 2012-2017. CTCs were detected using Epic Sciences platform and CIN defined using the phenotypic classifier (PMID 32816908). Retrospective clinical annotation was completed for OS, defined as time from index tx start date to death, and rPFS, defined as index date to date of new or/and increasing lesion size in on-tx radiology report or death. Associations with rPFS and OS were studied according to samples’ CIN1+ and CIN3+ status. T vs. ARSi comparisons were adjusted using Cox models that incorporated pre-tx covariates, i.e. prior T or ARSi txs, total CTC count, presence of visceral disease, and prognostic lab values. Results: The median (range) follow-up from tx start to date of death or last follow-up was 7 (5-9) years. There were 53/120 (44%) pts starting ARSi that had prior ARSi txs and 41/88 (46%) pts starting taxane that had prior taxane txs. CTCs were detected in 184 (88%) samples, CIN1+ in 73 (35%; 64% pre-T, 36% pre-ARSi), and CIN3+ in 47 (22%; 60% pre-T,40% pre-ARSi). Compared to CIN1+, model results (Table) suggest that CIN3+ predicts a longer rPFS (p&lt;0.01), and possibly OS (p=0.05), with T therapy. Compared to CIN1-, CIN3- also predicts a longer OS with ARSi therapy (p=0.04). Conclusions: CIN3+ CTC may be a more discriminating predictive biomarker than CIN1+ to help guide selection of T or ARSi tx in pts with mCRPC. Adequately powered prospective studies are planned. [Table: see text]

Demonstration of the value of liquid biopsy to identify metastatic breast cancer: Comparison and combination of cell and cell-free platforms.

e13004 Background: Liquid biopsy analysis detecting circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) is a non-invasive approach that may provide clinically actionable information for patients’ (pts) treatment decisions when conventional biopsy is otherwise inaccessible or infeasible. Here we report a liquid biopsy analysis method including CTC characterization and ctDNA alterations in late-stage metastatic breast cancers (MBC). Methods: Blood samples were collected for cell and cell-free DNA analysis from 57 pts with MBC. 44 pts were progressing on treatment and 13 were responding to treatment. Blood was also collected from 25 blood donors (HD) with no known cancer history. After plasma isolation, nucleated cells were plated, and slides were bio-banked. Immunofluorescent staining and subsequent imaging were performed on replicate slides. CTCs were identified using Epic Sciences digital imaging and machine learning algorithms trained to identify breast cancer cells. Single-cell (sc) isolation for genomic quantification of large-scale transitions (scLST) was used to further characterize individual CTCs. cfDNA from bio-banked plasma was analyzed using a validated NGS panel to detect class 1A ctDNA alterations. Results: Within this cohort of 57 MBC pts, CTCs, scLST+CTCs and ctDNA alterations were detected in (77%, 49% and 33%, respectively). All MBC pts on active therapy (23%) had no CTC detected. No CTCs, scLST+CTCs or ctDNA were detected in the HD cohort, suggesting high specificity (100%, 100% and 100%, respectively). The presence of CTCs or of scLST+CTCs had low concordance with the presence of ctDNA+ alterations in MBC pts, suggesting that these platforms provide independent and complementary value when identifying MBC. To test this hypothesis, we designed combination models which added ctDNA to cell analysis methods ([CTC+][ctDNA+] and [scLST+CTC+][ctDNA+]). Adding ctDNA to either cell analysis method increased sensitivity by 5% and 12%, respectively, over the individual methods and retained 100% specificity. Conclusions: Here we demonstrate the value of comprehensive cell and cell-free liquid biopsy analysis for the detection of MBC pts. Our data suggest there may be clinical utility of adding cell-free to cell analysis for the identification of MBC. Analysis of the comparative and combined utility of these liquid biopsy methods in earlier settings is ongoing.

Improved sensitivity in identification of first recurrences of metastatic breast cancer with a combination of cell and cell-free liquid biopsy analysis.

e13003 Background: Liquid biopsy analysis detecting circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) is a non-invasive approach that may provide clinically actionable information for patients’ (pts) treatment decisions when conventional biopsy is otherwise inaccessible or infeasible. Here we report a liquid biopsy method including CTC characterization &amp; ctDNA alterations in pts symptomatic of first metastatic recurrence with a history of primary breast cancer (BC). Methods: Blood samples from 34 symptomatic BC pts were collected for cell &amp; cell-free DNA analysis before undergoing contemporaneous tissue biopsy &amp; initiating treatment for metastatic BC (MBC). Blood was also collected from 25 blood donors (HD) with no known cancer history. After plasma isolation, nucleated cells were plated, &amp; slides were bio-banked. Immunofluorescent staining &amp; subsequent imaging were performed on replicate slides. CTCs were identified using Epic Sciences digital imaging &amp; machine learning algorithms trained to identify breast cancer cells. Single-cell (sc) isolation for genomic quantification of large-scale transitions (scLST) was used to further characterize individual CTCs. cfDNA from bio-banked plasma was analyzed to detect Class IA alterations in ctDNA. Results: Within this cohort of 34 patients, CTCs &amp; ctDNA alterations were detected in 80% &amp; 58%, respectively while no CTCs &amp; ctDNA were detected in the HD cohort suggesting high specificity (100% &amp; 100%). In this study, 26 pts were confirmed MBC (M1-BC) by tissue biopsy, 3 pts were non-MBC by tissue biopsy but still diagnosed as metastatic (Mx-BC), &amp; 5 pts were confirmed non-MBC (M0-BC). To benchmark the cellular (CTCs &amp; scLST+CTCs) &amp; genomic (ctDNA) individual platforms, we designed thresholds of CTCs &gt; 4, scLST+CTC+, &amp; ctDNA+ that optimally identified M1-BC pts with high specificity (100%, 100%, &amp; 100%) but low sensitivity (46%, 46%, &amp; 50%). CTCs &gt; 4 &amp; scLST+CTC+ cutoffs had low concordance with the presence of ctDNA alterations, suggesting these platforms provide independent &amp; complementary value when identifying MBC. To test this hypothesis, we designed combination models [CTC &gt; 4][ctDNA+] &amp; [scLST+CTC+][ctDNA+] that significantly improved sensitivity (75% &amp; 67%) over individual platforms while maintaining high specificity (100% &amp; 100%) when identifying the first recurrence of metastasis in BC pts. Conclusions: To our knowledge, this is the first report showing the additive &amp; complementary value of models combining cellular &amp; genomic liquid biopsy platforms for the detection of the first MBC recurrence. Our data suggest the clinical utility of combined CTC &amp; ctDNA platforms for the identification of MBC when tissue biopsy is inaccessible or infeasible.

Single-Cell Digital Microfluidic Mass Spectrometry Platform for Efficient and Multiplex Genotyping of Circulating Tumor Cells.

Gene mutation profiling of heterogeneous circulating tumor cells (CTCs) offers comprehensive and real-time molecular information of tumors for targeted therapy guidance, but the lack of efficient and multiplex genotyping techniques for single-CTC analysis greatly hinders its development and clinical application. This paper reports a single-CTC mass spectrometry analysis method for efficient and multiplex mutation profiling based on digital microfluidics. Digital microfluidics affords integrated single-CTC manipulation, from single-CTC isolation to high-performance whole genome amplification, via nanoliter droplet-based wettability trapping and hydrodynamic adjustment of cell distribution. Coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, multiplex mutation information of individual CTCs can be efficiently and accurately identified by the inherent mass differences of different DNA sequences. This platform achieves Kirsten rat sarcoma viral oncogene mutation profiling of heterogeneous CTCs at the single-cell level from cancer patient samples, offering new avenues for genotype profiling of single CTCs and cancer therapy guidance.

Serial monitoring of genomic alterations in circulating tumor cells of ER-positive/HER2-negative advanced breast cancer: feasibility of precision oncology biomarker detection.

Nearly all estrogen receptor (ER)‐positive (POS) metastatic breast cancers become refractory to endocrine (ET) and other therapies, leading to lethal disease presumably due to evolving genomic alterations. Timely monitoring of the molecular events associated with response/progression by serial tissue biopsies is logistically difficult. Use of liquid biopsies, including circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), might provide highly informative, yet easily obtainable, evidence for better precision oncology care. Although ctDNA profiling has been well investigated, the CTC precision oncology genomic landscape and the advantages it may offer over ctDNA in ER‐POS breast cancer remain largely unexplored. Whole‐blood (WB) specimens were collected at serial time points from patients with advanced ER‐POS/HER2‐negative (NEG) advanced breast cancer in a phase I trial of AZD9496, an oral selective ER degrader (SERD) ET. Individual CTC were isolated from WB using tandem CellSearch®/DEPArray™ technologies and genomically profiled by targeted single‐cell DNA next‐generation sequencing (scNGS). High‐quality CTC (n = 123) from 12 patients profiled by scNGS showed 100% concordance with ctDNA detection of driver estrogen receptor α (ESR1) mutations. We developed a novel CTC‐based framework for precision medicine actionability reporting (MI‐CTCseq) that incorporates novel features, such as clonal predominance and zygosity of targetable alterations, both unambiguously identifiable in CTC compared to ctDNA. Thus, we nominated opportunities for targeted therapies in 73% of patients, directed at alterations in phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 2 (FGFR2), and KIT proto‐oncogene, receptor tyrosine kinase (KIT). Intrapatient, inter‐CTC genomic heterogeneity was observed, at times between time points, in subclonal alterations. Our analysis suggests that serial monitoring of the CTC genome is feasible and should enable real‐time tracking of tumor evolution during progression, permitting more combination precision medicine interventions.

CNA Profiling of Single CTCs in Locally Advanced Esophageal Cancer Patients during Therapy Highlights Unexplored Molecular Pathways.

Simple SummaryIn the present work, we describe the evolution of circulating tumor cells (CTCs) released into the bloodstreams of 11 patients affected by locally advanced esophageal cancer (EC) during clinical treatments. We aimed at characterizing identified CTCs in depth both phenotypically as well as through single cell copy number aberrations profiles and to investigate the features of CTCs from relapsed patients, if present. We found that locally advanced EC spreads circulating tumor cells with both epithelial and mesenchymal phenotypes during the course of therapy. CTCs of relapsed patients display higher levels of genome disruption to those of disease-free patients. Specific enriched terms emerged from copy number aberration analysis of CTCs of relapsed patients.Background: Here, we monitored the evolution of CTCs spread in 11 patients affected by locally advanced EC who were undergoing therapy. Methods: In this perspective study, we designed multiple blood biopsies from individual patients: before and after neoadjuvant chemo-radio therapy and after surgery. We developed a multi-target array, named Grab-all assay, to estimate CTCs for their epithelial (EpCAM/E-Cadherin/Cytokeratins) and mesenchymal/stem (N-Cadherin/CD44v6/ABCG2) phenotypes. Identified CTCs were isolated as single cells by DEPArray, subjected to whole genome amplification, and copy number aberration (CNA) profiles were determined. Through bioinformatic analysis, we assessed the genomic imbalance of single CTCs, investigated specific focal copy number changes previously reported in EC and aberrant pathways using enrichment analysis. Results: Longitudinal monitoring allowed the identification of CTCs in at least one time-point per patient. Through single cell CNA analysis, we revealed that CTCs showed significantly dynamic genomic imbalance during treatment. Individual CTCs from relapsed patients displayed a higher degree of genomic imbalance relative to disease-free patients’ groups. Genomic aberrations previously reported in EC occurred mostly in post-neoadjuvant therapy CTCs. In-depth analysis showed that networks enrichment in all time-point CTCs were inherent to innate immune system. Transcription/gene regulation, post-transcriptional and epigenetic modifications were uniquely affected in CTCs of relapsed patients. Conclusions: Our data add clues to the comprehension of the role of CTCs in EC aggressiveness: chromosomal aberrations on genes related to innate immune system behave as relevant to the onset of CTC-status, whilst pathways of transcription/gene regulation, post-transcriptional and epigenetic modifications seem linked to patients’ outcome.

Circulating Tumor Cell Clusters Are Cloaked with Platelets and Correlate with Poor Prognosis in Unresectable Pancreatic Cancer

Simple SummaryDespite recent advances, some patients with pancreatic cancer are refractory to treatment and the disease rapidly progresses, resulting in early death. The potential prognostic value of circulating tumor cells (CTCs) has been demonstrated in other cancer types, but the clinical validity in pancreatic cancer remains elusive. Here, we show that CTC clusters, which show mesenchymal characteristics and platelet marker expression, are highly correlated with poor prognosis in patients with unresectable pancreatic cancer.Circulating tumor cells (CTCs) are known to be heterogeneous and clustered with tumor-associated cells, such as macrophages, neutrophils, fibroblasts, and platelets. However, their molecular profile and clinical significance remain largely unknown. Thus, we aimed to perform a comprehensive gene expression analysis of single CTCs and CTC clusters in patients with pancreatic cancer and to identify their potential clinical relevance to provide personalized medicine. Epitope-independent, rapid (>3 mL of whole blood/min) isolation of single CTCs and CTC clusters was achieved from a prospective cohort of 16 patients with unresectable pancreatic cancer using a centrifugal microfluidic device. Forty-eight mRNA expressions of individual CTCs and CTC clusters were analyzed to identify pancreatic CTC phenotype. CTC clusters had a larger proportion of mesenchymal expression than single CTCs (p = 0.0004). The presence of CTC clusters positively correlated with poor prognosis (progression-free survival, p = 0.0159; overall survival, p = 0.0186). Furthermore, we found that most CTCs in these patients (90.7%) were cloaked with platelets and found the presence of a positive correlation between the increase in CTC clusters and rapid disease progression during follow-ups. Efficient CTC cluster isolation and analysis techniques will enhance the understanding of complex tumor metastasis processes and can facilitate personalized disease management.

The value of liquid biopsy in the diagnosis and staging of hepatocellular carcinoma: a systematic review.

Blood-borne tumour markers in the form of circulating tumour cells (CTCs) are of intense research interest in the diagnostic and prognostic work-up of hepatocellular carcinoma (HCC). This is a meta-analysis. Using a PICO strategy, adults with HCC was the population, with the individual CTCs as the intervention and comparators. The primary outcome was the sensitivity and specificity of HCC detection with tumour specific single gene methylation alteration. Secondary outcomes were the comparison using specific assay methods and the effect of early vs. late stages on CTC positivity. We included patients with HCC who had samples taken from peripheral blood and had sufficient data to assess the outcome data. ASSIA, Cochrane library, EMbase, Medline, PubMed and the knowledge network Scotland were systematically searched with appropriate Mesh terms employed. The quality assessment of diagnostic accuracy studies (QUADAS) was used to ensure quality of data. Statistical analysis was performed using the 'Rev Man' meta-analysis soft ward for Windows. The review included 36 studies, with a total of 5,853 patients. Here, we found that AFP has the highest overall diagnostic performance. The average Youden index amongst all CTC was 0.46 with a mode and median of 0.5 with highest of 0.87 and lowest of 0.01. The available literature provides weak evidence that there is potential in the use of CTC, however the lack of a standardised procedure in the study of CTC contribute to the lack of consensus of use. Future research should include large scaled, standardized studies for the diagnostic accuracy of CTCs.

Abstract 3143: Monitoring circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) genomic alterations in ER positive (POS)/HER2 negative (NEG) advanced breast cancer during endocrine therapy: correlative study of AZD9496 oral SERD phase I trial

Abstract Purpose: The vast majority of advanced ER POS breast cancers eventually cease responding to endocrine (ET) and other therapies leading to evolution of lethal disease. However, timely monitoring of the molecular events associated with response/progression in tissue biopsies is logistically difficult. The use of liquid biopsies, such as CTC and ctDNA, in this context has been of recent interest. Patients and Methods: Individual CTC and ctDNA were obtained at different time points from patients with advanced ER POS/HER2 NEG breast cancer enrolled in a Phase I trial of AZD9496, an oral selective estrogen receptor degrader (SERD) ET. The CTC, purified using tandem CellSearch®/DepArray™ technologies, were genomically profiled by DNA single cell next generation sequencing (scNGS). Plasma ctDNA was isolated from blood collected in Streck BCT tubes. Genomic profiling was performed by targeted gene panel scNGS for CTC and ddPCR for ERα gene (ESR1) mutations in ctDNA. Results: 123 high-quality CTCs from 12 patients profiled by scNGS showed 100% concordance with ctDNA in detection of driver ESR1 somatic mutations. CTC scNGS additionally revealed extensive intra-patient heterogeneity of driver alterations, that would have been unresolvable by bulk ctDNA profiling, including separate subclonal CTC populations emerging within the same patient. ScNGS revealed potential opportunities for targeted therapies in 73% of patients, directed at alterations in PIK3CA, FGFR2, KIT and BRAF, at times present as 2 or more targets in the same or different cell populations. In one patient, an emergent, distinct, BRAF p.V600E targetable alteration was detected in a subpopulation of CTCs collected at the progression time point but not at baseline. Conclusion: Serial monitoring of CTC and ctDNA genomic alterations is feasible and should enable real-time tracking of response/progression, tumor evolution and opportunities for precision medicine interventions. Citation Format: Andi K. Cani, Emily M. Dolce, Elizabeth P. Darga, Kevin Hu, Martha Brown, Chia-Jen Liu, Jackie Pierce, Kieran Bradbury, Kimberly Aung, Gaia Schiavon, Danielle Carroll, T. H. Carr, Teresa Klinowska, Justin Lindemann, Gayle Marshall, Vicky Rowlands, Elizabeth A. Harrington, J. Barrett, Anne Armstrong, Richard Baird, Erika Hamilton, Seock-Ah Im, Komal Jhaveri, Manish R. Patel, Caroline Dive, Scott A. Tomlins, Aaron M. Udager, Daniel F. Hayes, Costanza Paoletti. Monitoring circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) genomic alterations in ER positive (POS)/HER2 negative (NEG) advanced breast cancer during endocrine therapy: correlative study of AZD9496 oral SERD phase I trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3143.

Abstract 587: Genomic copy number profiling of single CTCs reveals clonal evolution in metastatic breast cancer and identifies actionable targets for informing treatment decisions

Abstract Introduction: Genomic profiling of circulating tumor cells (CTCs) is an approach to help to understand tumour heterogeneity and evolution in metastatic breast cancer (MBC) to inform prognosis and guide treatment options. We showed previously that single CTCs provide complementary information on hot spot mutations to those detected in circulating tumor DNA (ctDNA) and the primary tumour tissue, and now extend analyses to include genomic copy number changes. Herein, we report that identification of actionable targets through copy number profiling of single CTCs is achievable at any time point during metastatic progression and could be a valuable tool to guide treatment options. Methods: Fourteen patients (13 12 progressing and 1 2 responding) who were receiving treatment for MBC (12 13 HR pos, 1 HER2 pos, I1 TNBC) were recruited to the study with ethical consent and followed up with a single blood sample. A 7.5ml blood sample was collected into a CellSave Preservative tube and processed and counted within 96 hours of collection using the CELLSEARCH® system and the Circulating Tumor Cell Kit (Menarini Silicon Biosystems). The DEPArray™ System was used to identify and recover individual, intact CTCs of epithelial origin (Cytokeratin 8, 18, 19 +ve, CD45 -ve) and white blood cells (WBCs) as single cells or as a small pool of cells. Whole genome amplification was performed using the Ampli1 WGA kit. Libraries were generated using the Ampli1 LowPass Kit (Menarini Silicon Biosystems) and samples passing QC were analysed f by next generation sequencing on an S5 platform (Thermo fFisher). Results were analysed using the MSBiosuite software and data was compared with in- house informatics workfows. Results: From the 14 patients included in the study, we profiled a total of 97 samples using the Ampli1 LowPass sequencing workflow comprising 79 individual cells (across 14 patients) and 18 pools of approximately 10 cells. Successful genome- wide copy number profiles were generated in 12 of the 14 patients; samples from 2 patients had low QC scores and were inconclusive. In all, 12 patients individual CTCs from 12 patients showed similar copy number profiles, but with some heterogeneity, that revealed gene specific changes not detected by the cell pool. Moreover, 10 of the 12 patients had gene- specific changes (5 with CCND1 amplification, 2 with FGFR1 amplification, 1 with JAK2 amplification and 2 with STK11 deletion) that could have guided treatment decisions, for example offering an FGFR1 or JAK2 inhibitor. Conclusion: This study highlights the genomic heterogeneity in CTCs seen in MBC and the potential of monitoring gene specific changes to identify actionable targets in order to inform treatment decisions. The work requires further evaluation and validation but it may offer a new approach to managing treatment decisions in MBC for those patients with detectable CTCs. Citation Format: Daniel Fernandez Garcia, Georgios Nteliopoulos, Robert Hastings, Amelia Rushton, Karen Page, Luke J. Martinson, Molly Gray, David S. Guttery, Alberto Ferrarini, Nicolò Manaresi, Charles Coombes, Jacqui Shaw. Genomic copy number profiling of single CTCs reveals clonal evolution in metastatic breast cancer and identifies actionable targets for informing treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 587.

Schlafen 11 (SLFN11), a putative predictive biomarker of platinum/PARPi response, is frequently detected on circulating tumor cells (CTCs) in advanced prostate cancer.

e17039 Background: Schlafen 11 (SLFN11) is a DNA repair protein (DNA/RNA helicase homology) that is recruited to stressed replication forks and leads to cell death. Recent ph II trial data in Extensive Stage Small-Cell Lung Cancer (ES-SCLC) [Byers et al. JCO 2018 PMID: 29906251] and a retrospective analysis of Circulating Tumor Cells (CTCs) and tumor tissue in patients (Pts) with advanced prostate cancer [Conteduca et al. Mol Cancer Therapeutics 2020 PMID: 32127465] suggested that SLFN11 expression predicts sensitivity to DNA damage targeting agents. In both contexts, metastatic tumor biopsies may not provide adequate material for profiling. We assessed the frequency of SLFN11 expression in CTCs isolated from blood in men with progressing metastatic Castration Resistant Prostate Cancer (mCRPC) and related expression to Homologous Recombination Repair (HRR) alterations identified in metastatic tumor biopsies profiled by MSK-IMPACT. Methods: 95 patients with progressing mCRPC about to start a new systemic therapy who had undergone pre-treatment metastatic tumor profiling by MSK-IMPACT and a matched blood draw for CTC profiling were selected. Blood was sent overnight to Epic Sciences and processed onto glass pathology slides and bio-banked until analysis. Detected CTCs (cytokeratin (CK) positive and leukocyte (CD45) negative) were analyzed for SLFN11 protein expression by immunofluorescence and correlated to Homologous Recombination Repair (HRR) alterations in metastatic biopsy from the bone, lymph node, or visceral metastases (15 PROFOUND genes). A mean of 1.2 mL of blood was analyzed per patient. Results: CTCs with SLFN11 expression (DAPI+, CK+, CD45-, SLFN11+) were detected in 28.4% (27/95) of the patient sample analyzed. SLFN11 signal was found to overlap with the nuclear DAPI signal. Individual CTC expression of SLFN11 in a sample was heterogeneous and ranged from a minimum of 2.9% to 100%. Seven of 10 (70%) of patients with a BRCA1/2 or ATM alteration had a least one SLFN11 expressing CTC. In contrast, in patients with other HRR alterations, only 20% (1/5) had CTCs with SLFN11. Sequencing of single CTCs is ongoing. Conclusions: SLFN11 expression is detected with high frequency in CTCs in men with progressing mCRPC. In whom CTCs are detected, the majority of patients with BRCA1/2 or ATM altered tumors also had SLFN11 expressing CTCs. The results support the prospective evaluation of CTC SLFN11 expression as a predictive biomarker for PARPi or platinum agents.

Usefulness of monitoring circulating tumor cells as a therapeutic biomarker in melanoma with BRAF mutation

BackgroundWhile molecularly targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma, biomarkers are required to monitor drug responses. Circulating tumor cells (CTCs) are released from primary and/or metastatic tumors into the peripheral blood. We examined whether CTCs have potential as biomarkers by checking the number of CTCs, as well as the BRAF genotype of individual CTCs, in melanoma patients undergoing BRAF/MEK inhibitor treatment.MethodsCTCs were isolated from peripheral blood using a high-density dielectrophoretic microwell array, followed by labeling with melanoma-specific markers (MART-1 and/or gp100) and a leukocyte marker (CD45). The numbers of CTCs were analyzed in fifteen patients with stage 0–III melanoma. Furthermore, changes in CTC numbers were assessed in five patients with stage IV melanoma at four time points during BRAF/MEK inhibitor treatment, and the BRAF genotype was analyzed in CTCs isolated from one patient.ResultsWe examined CTCs in patients with stage 0–III (five samples per stage: stage 0–I, stage II, and stage III), and detected CTCs even in patients with early disease (stage 0 and I). Interestingly, recurrence occurred in the lymph nodes of one stage I patient 2 years after the detection of a high number of CTCs in the patient’s blood. The total number of CTCs in four of five patients with stage IV melanoma fluctuated in response to BRAF/MEK inhibitor treatment, suggesting that CTC number has potential for use as a drug response marker in advanced disease patients. Interestingly, one of those patients had CTCs harboring seven different BRAF genotypes, and the mutated CTCs disappeared upon BRAF/MEK inhibitor treatment, except for those harboring BRAFA598V.ConclusionsCTCs are present even in the early stage of melanoma, and the number of CTCs seems to reflect patients’ responses to BRAF/MEK inhibitor treatment. Furthermore, genetic heterogeneity of BRAF may contribute to resistance to BRAF/MEK inhibitors. Our findings demonstrate the usefulness of CTC analysis for monitoring responses to targeted therapies in melanoma patients, and for understanding the mechanism of drug resistance.

Abstract PS5-35: Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies

Abstract Background: Somatic genomic alterations that activate the phosphatidylinositol-3-kinase (PI3K) pathway signaling are found in 30-50% of breast cancers, and the p110a-specific inhibitor alpelisib is approved for use in combination with fulvestrant in PIK3CA mutated hormone receptor-positive (HR+) metastatic breast cancer. However, preclinical and clinical data has demonstrated significant variability in response to alpelisib and other PI3K inhibitors even in the presence of hotspot PIK3CA activating mutations, likely reflecting functional differences between somatic alterations, bypass signaling mechanisms, and intratumor clonal heterogeneity. Thus, there is an ongoing need for novel predictive biomarkers to help guide patient selection for these therapies. Circulating tumor cells (CTCs) represent an accessible source of tumor derived analytes that allow for the interrogation of protein level readouts of PI3K pathway activation, may better reflect the biologic heterogeneity of metastatic disease than single site solid tumor biopsy, and are amenable to longitudinal analysis. Here, we report the development of an assay to evaluate the expression of activated AKT (AKT pS473) in CTCs as a putative biomarker of sensitivity to PI3K inhibitor therapies in metastatic breast cancer. Methods: Peripheral blood for CTC isolation was collected serially from patients with metastatic breast cancer and known somatic PI3K pathway mutation status. Samples were processed using VERSA (Versatile Exclusion-based Rare Sample Analysis), a microfluidic technology that integrates CTC capture and downstream analysis. Following fixation, CTCs were captured immunomagnetically with antibodies for EpCAM and Trop2, permeabilized and stained on chip for pan-AKT plus AKT pS473. Quantitative fluorescent microscopy was used to enumerate CTCs, defined as cytokeratin positive cells with intact nuclei and negative for a group of normal blood cell markers (CD45/CD11b/CD34/CD66b), and to quantify activated AKT protein expression in CTCs and matched peripheral blood mononuclear cells (PBMCs). Results: 100% of patients with somatic PIK3CA mutations in our pilot cohort had CTCs with detectable phospho-AKT expression, and in 50% of patients, the median phospho-AKT/pan AKT ratio was higher in CTCs compared to matched peripheral blood cells, suggestive of increased PI3K pathway activity. All patients demonstrated heterogeneity in phospho-AKT expression and phospho-AKT/pan-AKT ratio among individual CTCs at a single timepoint and across timepoints in longitudinal analysis. Conclusion: We report here the feasibility of quantitative and longitudinal detection of activated AKT in EpCAM/Trop2 captured CTCs from metastatic breast cancer with somatic PIK3CA mutations. Future work will prospectively evaluate multiple clinical applications of this assay in patients receiving alpelisib plus endocrine therapy for PIK3CA mutated HR+ metastatic breast cancer, as well as expanding the assay to include the detection of additional phospho-protein readouts of PI3K pathway activity in CTCs. In addition to the potential to complement solid biopsy PIK3CA mutation status as a predictive biomarker of sensitivity to PI3K therapies, this assay has the unique potential to provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs while on treatment, which may serve as an early biomarker of clinical response or resistance. Citation Format: Marina N Sharifi, Jamie M Sperger, Cole Gilsdorf, Serena K Wolfe, Amanda Parkes, Kari B Wisinski, Ruth M O'Regan, Joshua M Lang. Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-35.