Among the 43 CK+ informative cases, an overall concordance of 93% between CTCs and the primary tumor was observed with regards to HER2 status. Concordance was observed in 18 of 19 cases with positive HER2 status (positive predictive value of 90%) and 22 of 24 cases with negative HER2 status (negative predictive value of 96%). There was no difference between the numbers of CK+ CTCs detected in the 19 patients with HER2+ status compared to the 24 HER2-cases (median 5 and 9 CTCs, respectively, p = 0.37). Discordance was observed in three cases as shown in Table 2. An overall test sensitivity of 95% and specificity of 92% was observed. A substantial agreement approaching the range of perfect agreement (0.81-1.00) was found using the Cohen's k statistic (k= 0.75) for concordance between HER2 status in the primary tumor and CTCs.
Introduction: Somatic mutations, including those in TP53, PIK3CA, and estrogen receptor alpha (ESR1), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity. Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch© (CXC kit). CTC and WBC were then purified using DEPArrayTM. DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1TM WGA). Genetic analysis was performed on individual CTC, pooled CTC and pooled WBC DNA by multiplexed PCR based targeted next generation sequencing (NGS) using the Oncomine Comprehensive Panel (targeting ∼130 onco- and tumor suppressor genes) and the Ion Torrent Proton. All patients had exome sequencing performed on research biopsies of metastases using an Illumina HiSeq 2500 platform. Results: This pilot study was conducted using high quality DNA from two patients assessed to date. Both patients had lobular carcinoma and as expected harbored somatic, deleterious CDH1 (E-cadherin) mutations (frameshift and non-sense) in both research biopsy and CTC-DNA. These data supported our approach. Patient #1 was TP53 wild type in her research biopsy, but multiple CTC harbored somatic TP53 frame-shift mutations (Table). Patient #2 harbored an ESR1 Y537S mutation in her research biopsy. However, only 4 of 7 CTC also harbored this somatic, heterozygous mutation. Prioritized mutations in CTCPt#Cell Type (CTC vs WBC), numberGeneMutationVariant fraction (expected 1=homozygous; 0.5=heterozygous)Found in research biopsy?1CTC_A2CDH1p.I584fs1YES CTC_A4 1 CTC_A7 0.54 CTC_pool* 0.74 WBC_pool 0 CTC_A2TP53p.152_156del1NO CTC_A4 1 CTC_A7 0.51 CTC_pool* 0.88 WBC_pool 0 2CTC_A9ESR1p.Y537S0.52YES CTC_D1 0.34 CTC_D2 0.46 CTC_D6 0.65 CTC_pool* 0.35 WBC_pool 0 CTC_A12 0 CTC_D3 0 CTC_D7 0 CTC_A12CDH1p.Q641X1YES CTC_A9 1 CTC_D1 1 CTC_D3 1 CTC_D6 1 CTC_pool* 1 WBC_pool 0 * pool of all CTC Conclusions: We demonstrate the ability to purify CTC, isolate, and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Both known and novel alterations were identified in comparison to research biopsy specimens. This approach allows single cell analysis demonstrating heterogeneity of mutational status in different single cells. Studies of CTC-ESR1 and other genetic abnormalities in patients with known tissue mutations who participated in Mi CTC-ONCOSEQ are now underway. Citation Format: Paoletti C, Cani AK, Aung K, Darga EP, Cannell EM, Hovelson DH, Yazdani M, Blevins AR, Tokudome N, Larios JM, Thomas DG, Brown ME, Gersch C, Schott AF, Robinson DR, Chinnaiyan AM, Bischoff F, Hayes DF, Rae JM, Tomlins SA. Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-19.
INTRODUCTION: Most circulating tumor cell (CTC) platforms rely on EpCAM for capture and cytokeratin (CK) for detection. However, an important population of cells that are CK-negative (i.e. cells with epithelial-mesenchymal transition (EMT) phenotype) will be missed. We report a new strategy to efficiently isolate a more heterogeneous population of CTCs using an antibody cocktail. METHODS: In the first prospective study, blood (20 mL) was collected from 89 patients diagnosed with various late stage metastatic/recurrent cancers (breast, CRC, lung, prostate) following IRB approval. PBMCs were incubated with either EpCAM alone or a mixture of 10 capture antibodies to target both epithelial and mesenchymal cells. CTCs were subsequently captured in the OncoCEE™ channels and detected with cytokeratin (CK) and CD45. A second prospective IRB approved study involving 54 patients diagnosed with late stage metastatic/recurrent breast cancer was performed using similar detection strategies (CK cocktail mixture and anti-CD45) with the addition of HER2 FISH to determine amplification status among captured CK+/CD45- and CK-/CD45-cells. RESULTS: In the first study, overall detection of CK+ cells was 83% with EpCAM alone and 93% with antibody cocktail. In addition, a median of 0.4 CK+ cells/mL and 1.0 CK+ cells/mL was observed using EpCAM and antibody cocktail, respectively. In the second study, CK+/CD45- cells were detected in 43 of 54 cases (80%). Among the 43 cases in which CK+/CD45- cells were detected, high concordance (93%) in HER2 status between primary tumor and CTCs was observed with HER2 amplification noted in both CK+/CD45- (50%) and CK-/CD45- (50%) cells. CONCLUSIONS: We have developed a novel and robust method for CTC enumeration that utilizes a cocktail of antibodies for the detection of a heterogeneous (CK+ and CK-) population of CTCs. Our findings suggest an important population of CK- cells is being missed by current stain criteria in breast cancer patients. Data also demonstrate that recovery of CTCs from peripheral blood using the OncoCEE™ platform is efficient and suitable for FISH-based laboratory testing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-13.
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