Circulating Tumor Cells Expression Research Articles

Biomarker Associations with Efficacy of Abiraterone Acetate and Exemestane in Postmenopausal Patients with Estrogen Receptor–Positive Metastatic Breast Cancer

Abiraterone may suppress androgens that stimulate breast cancer growth. We conducted a biomarker analysis of circulating tumor cells (CTCs), formalin-fixed paraffin-embedded tissues (FFPETs), and serum samples from postmenopausal estrogen receptor (ER)+ breast cancer patients to identify subgroups with differential abiraterone sensitivity. Patients (randomized 1:1:1) were treated with 1,000 mg/d abiraterone acetate + 5 mg/d prednisone (AA), AA + 25 mg/d exemestane (AAE), or exemestane. The biomarker population included treated patients (n = 293). The CTC population included patients with ≥3 baseline CTCs (n = 104). Biomarker [e.g., androgen receptor (AR), ER, Ki-67, CYP17] expression was evaluated. Cox regression stratified by prior therapies in the metastatic setting (0/1 vs. 2) and setting of letrozole/anastrozole (adjuvant vs. metastatic) was used to assess biomarker associations with progression-free survival (PFS). Serum testosterone and estrogen levels were lowered and progesterone increased with AA. Baseline AR or ER expression was not associated with PFS in CTCs or FFPETs for AAE versus exemestane, but dual positivity of AR and ER expression was associated with improved PFS [HR, 0.41; 95% confidence interval (CI), 0.16-1.07; P = 0.070]. For AR expression in FFPETs obtained <1 year prior to first dose (n = 67), a trend for improved PFS was noted for AAE versus exemestane (HR, 0.56; 95% CI, 0.24-1.33; P = 0.19). An AA pharmacodynamic effect was shown by decreased serum androgen and estrogen levels and increased progesterone. AR and ER dual expression in CTCs and newly obtained FFPETs may predict AA sensitivity. Clin Cancer Res; 22(24); 6002-9. ©2016 AACR.

Non-Invasive Genetic Profiling Is Highly Applicable in Multiple Myeloma (MM) through Characterization of Circulating Tumor Cells (CTCs)

Chromosomal abnormalities (CA) play a pivotal role in predicting MM patients risk. Accordingly, well-defined CA have been associated with higher risk of transformation in smoldering MM (SMM), have been recently incorporated to the revised ISS, and may even guide treatment decisions; furthermore, because high-risk CA can be present in minor subclones at diagnosis it is recommended to reassess them at the time of relapse. Altogether, this implies that throughout their clinical course MM patients go through several bone marrow (BM) aspirates for cytogenetic risk assessment, and less invasive methods to screen for CA [ie. peripheral blood (PB) liquid biopsies] would be mostly welcomed providing they are fully comparable to its assessment in BM clonal plasma cells (PCs).Here, using the recently developed and sensitive next generation flow (NGF) method to monitor MRD in MM (median limit of detection, 3x10-6), we assessed the applicability of screening for CA in the PB, by determining the frequency of MGUS and MM patients with detectable CTCs and their respective absolute numbers. Using NGF, we detected CTCs in 22/37 (60%) MGUS cases, 9/12 (75%) SMM, 24/25 (96%) newly diagnosed MM, and 22/26 (85%) relapsed MM. Median numbers of CTCs/µL in the PB of MGUS, SMM, newly diagnosed and relapsed MM were 0.04, 0.1, 0.5 and 0.6, respectively. Thus, we noted highly significant differences (P<.001) between benign and malignant disease stages, with a 12.5-fold increment in CTCs/µL between MGUS and MM, and 5-fold between SMM and MM.By using whole exome sequencing, we reported recently that the concordance in CA between CTCs and BM clonal PCs was typically <50% due to the limited amount of DNA extracted from low numbers of CTCs, which required whole genome amplification. Accordingly, we investigated in 18 patient paired BM and PB (~5mL) samples (2 MGUS, 2 SMM and 14 MM) whether after NGF sorting and using the Affymetrix CytoScan HD (which requires significantly less DNA to provide molecular karyotypes), we could determine the pattern of CA in BM clonal PCs by screening that of CTCs. In 10 patients in which CTCs were successfully characterized, we obtained 100% concordance between BM clonal PCs and CTCs at the chromosome level (including trisomies of chromosomes 3, 5 and 21 which have been described to modulate the prognosis of patients with high-risk MM), for chromosomal arms, and for interstitial gains or losses (Panel A). Importantly, there was also 100% concordance with FISH performed on CD138+ BM clonal PCs for del(1p), +1q, del(13q) and del(17p) CA. In the remaining cases, ultra-low numbers of sorted CTCs (≤5.000) precluded their successful characterization, which could be readily overcome by using larger volumes of PB (ie. >5mL). Because the CytoScan HD does not assess the t(4;14), we included FGFR3 into the NGF panel of mAb in order to detect its surface expression in patients harboring the t(4;14). Similarly to that described above, we observed 100% concordance between FGFR3 expression in CTCs and the presence of t(4;14) in CD138+ BM clonal PCs, as confirmed by FISH (Panel B).Since gene expression profiling (GEP) has also been used to predict risk in MM, we investigated in 12 patient paired BM and PB samples whether after NGF sorting and using the Affymetrix Human Gene 2.0 ST Array, we could determine patients risk by screening the GEP of CTCs. Unsupervised analysis showed correct clustering between GEP of BM clonal PCs and CTCs in 9/12 cases, demonstrating that individual patient signatures typically outweighed GEP of MM clones located in different tissues (Panel C). Thus, CTCs could be used to predict risk according to well-established GEP signatures.In conclusion, by using sensitive NGF we showed that CTCs are present in 60% of MGUS and 87% of MM, which unravels high feasibility for liquid biopsying these patients. By also using NGF to sort CTCs, as well as commercially available, cost-effective and standardized arrays to determine CA and gene expression profiles, we demonstrated for the first time that the genetic features of BM clonal PCs can be determined with great accuracy by studying CTCs. Altogether, this combined approach could offer to many MM patients a less invasive cytogenetic characterization at certain stages of their clinical course. [Display omitted] DisclosuresPaiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

553P How can we bring the analysis of circulating tumor cells (CTCs) in the point-of-care testing (POCT)? A Tokyo experience

Background In the treatment of castration-resistant prostate cancer (CRPC), characterizing its molecular features is the key for the practice of precision medicine. CTC is the promising biomarker for CRPC since it directly reflects the profile of cancer cells with metastatic potential. However, isolation and analysis of CTC has been a challenge in clinical practice. Here, we report our experience of bringing CTC analysis to a POCT. Methods Detection and quantification of CTCs were performed by immunofluorescence using CytoQuest (Abnova, Taiwan). Cytoquest employs positive selection of CTCs using anti-EpCAM antibody, which enabled us multiple labeling with arbitrary antibodies such as AR-V7. Furthermore, in order to investigate gene expressions of CTCs, we utilized AdnaTest ProstateCancerSelect and AdnaTest ProstateCancerDetect (Qiagen, Germany); it enriches CTCs from peripheral blood by magnetic particles conjugated with an antibody mixture, followed by reverse transcription and PCR amplification. Subsequently, data obtained from the study were compared with the clinical course and prognosis. Results We streamlined the experimental procedures, such that the whole procedures of both methods can be completed within 24 hours from sample collection. CTCs were detected in 16 of 28 prostate cancer patients (57%) using AdnaTest. Among them, 5 patients were positive for AR-V7 expression (18%). Although the number of AR-V7- positive patients is limited so far, they appear to present more frequent resistance to hormonal therapy and poor prognosis. Furthermore, we observed a change in gene expression of CTCs before and after treatments. Specifically, a prostate cancer patient with treatment-naive advanced disease showed positive AR-V7 expression before hormonal therapy, which disappeared after the treatment. In addition, immunofluorescence by Cytoquest showed consistent results with AdnaTest. Conclusions: It is imperative to perform the analysis immediately and feasibly for on- the-spotclinicaldecision-makingofCRPCcases.CTCanalysiscouldbeperformedasa point-of-caretestmoreubiquitouslywithfurthertechnicaladvancessuchas automation ofCTC enumeration. Legal entity responsible for the study Naoya Nagaya Funding Grant-in-Aid for Scientific Research (C) Disclosure All authors have declared no conflicts of interest.

Correlation between epidermal growth factor receptor tyrosine kinase inhibitor efficacy and circulating tumor cell levels in patients with advanced non-small cell lung cancer.

ObjectiveThe aim of this study was to investigate the correlation between the efficacy of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and circulating tumor cell (CTC) levels in patients with advanced non-small cell lung cancer (NSCLC). The efficacy of EGFR-TKIs in reducing CTC counts in patients with advanced NSCLC was studied.Patients and methodsA total of 66 patients with advanced NSCLC were enrolled and divided into two groups (those with high CTC counts and those with low CTC counts) based on the patients’ median CTC counts. All the patients were treated with an EGFR-TKI, and the treatment efficacy and prognoses were compared.ResultsThe treatment efficacies were 53.3% (16/30) and 27.8% (10/36) for the low CTC group and high CTC group, respectively, and this difference was statistically significant (P<0.05). The median overall survival was 22.8 months (95% confidence interval [CI]: 18.9–26.8 months) for the low CTC group and 18.3 months (95% CI: 2.9–8.2 months) for the high CTC group. The median progression-free survival was 11.5 months (95% CI: 8.1–15 months) and 5.6 months (95% CI: 2.9–8.2 months) for the low and high CTC groups, respectively, and the difference was statistically significant (P<0.05).ConclusionThe CTC count can be used as an index for predicting the EGFR-TKI effect on patients with advanced NSCLC. Efficacy and prognosis of EGFR-TKI treatment and CTC count were considered important, and the CTC count could be used to predict the efficacy of EGFR-TKI treatment and prognosis of advanced NSCLC. The change in CTC expression levels can be used as an index for evaluating the prognosis of patients with advanced NSCLC.

Epidermal Growth Factor Receptor Status in Circulating Tumor Cells as a Predictive Biomarker of Sensitivity in Castration-Resistant Prostate Cancer Patients Treated with Docetaxel Chemotherapy.

Objective: We examined whether epidermal growth factor receptor (EGFR) expression in circulating tumor cells (CTCs) can be used to predict survival in a population of bone-metastatic castration-resistant prostate cancer (mCRPC) patients treated with docetaxel chemotherapy. Methods: All patients with mCRPC who had experienced treatment failure with androgen-deprivation therapy and had received docetaxel chemotherapy were eligible. CTCs and EGFR expression in CTCs were enumerated with the CellSearch System in whole blood. This system is a semi-automated system that detects and enriches epithelial cells from whole blood using an EpCAM antibody-based immunomagnetic capture. In addition, the EGFR-positive CTCs were assessed using CellSearch® Tumor Phenotyping Reagent EGFR. Results: The median CTC count at baseline before starting trial treatment was eight CTCs per 7.5 mL of blood (range 0–184). There were 37 patients (61.7%) who had ≥5 CTCs, with median overall survival of 11.5 months compared with 20.0 months for 23 patients (38.3%) with <5 CTCs (p < 0.001). A total of 15 patients (40.5%, 15/37) with five or more CTCs were subjected to automated immunofluorescence staining and cell sorting for EGFR protein. Patients with EGFR-positive CTCs had a shorter overall survival (OS) (5.5 months) than patients with EGFR-negative CTCs (20.0 months). CTCs, EGFR-positive CTCs, and alkaline phosphatase (ALP) were independent predictors of overall survival time (p = 0.002, p < 0.001, and p = 0.017, respectively). Conclusion: CTCs may be an independent predictor of OS in CRPC treated with docetaxel chemotherapy. The EGFR expression detected in CTCs was important for assessing the response to chemotherapy and predicting disease outcome.

Accession of Tumor Heterogeneity by Multiplex Transcriptome Profiling of Single Circulating Tumor Cells.

Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Vitamin D receptor status of circulating tumor cells (CTC) in metastatic breast cancer patients

Objective: In recent years, several trials suggested that optimal vitamin D levels, by binding to the vitamin D receptor (VDR), have a protective effect against development of breast cancer. VDR expressed in breast tumors may have the potential to be a prognostic biomarker for breast cancer. The detection of circulating tumor cells (CTCs) predicts outcomes of early and metastatic breast cancers. The evaluation of VDR expression in CTCs of breast cancers patients may contribute to better characterize CTCs.

411TiP - First-in-human trial of 4'-thio-2'-deoxycytidine (TdCyd) in patients with advanced solid tumors

Methylation-mediated silencing of genes is an epigenetic mechanism implicated in tumorigenesis. Hypermethylation of cytosines at CpG sites in the regulatory sequences of tumor suppressor genes silences them in a manner akin to inactivating mutations. Agents that inhibit methylation are of clinical interest because of their potential to re-activate silenced tumor suppressor genes, and two DNA hypomethylating nucleosides have been approved by the FDA for the treatment of patients (pts) with hematologic malignancies. The nucleoside analog 4'-thio-2'-deoxycytidine (TdCyd, NSC764276) is a cytidine analog in which the 4'-O moiety in deoxyribose has been replaced with sulfur where it engages the active site of DNA methyltransferase I (DNMT1), a maintenance methyltransferase that contributes to the hypermethylation and silencing of tumor suppressor genes. TdCyd and its active metabolites are phosphorylated and incorporated into the DNA of human cells in culture and in xenografts at rates exceeding those of the natural nucleoside or ara-analogs. Growth inhibition and tumor shrinkage were found during TdCyd dosing in several human tumour xenografts. Depletion of DNMT1 was observed also in certain xenograft models. Trial design: We are conducting an open label phase 1 first-in-human trial of TdCyd, following an accelerated titration design. TdCyd is administered PO qd x 5 days of each week for 2 weeks in 21-day cycles; the primary objective of the study is to establish its safety, tolerability, maximum tolerated dose, and recommended phase 2 dose. The secondary objectives of the study of oral TdCyd include preliminary efficacy assessment, pharmacokinetic (PK) characterization and exploratory pharmacodynamic (PD) evaluation of re-expression of select genes silenced by methylation in circulating tumor cells (CTCs). Eligibility criteria: solid tumor pts whose disease has progressed on standard therapy, ECOG ≤ 2, and normal organ function. Blood samples for PK/PD analyses and gene expression in CTCs are being evaluated. Currently, we are enrolling at DL5 in the escalation portion of the trial. Clinical trial identification: NCT02423057 Legal entity responsible for the study: Division of Cancer Treatment and Diagnosis, National Cancer Institute

Circulating tumour cells as biomarkers for evaluating cryosurgery on unresectable hepatocellular carcinoma.

We evaluated the efficacy of pre-cryosurgery and post-cryosurgery circulating tumour cells (CTCs) as biomarkers for unresectable hepatocellular carcinoma (HCC). Real‑time qPCR was used to detect potential biomarker genes in CTCs, and magnetic-activated cell sorting (MACS) and fluorescence‑activated cell sorting (FACS) was performed on 47patients with hepatocellular cancer who underwent cryosurgery. CTCs in the 47patients were assessed 1day before cryosurgery, and 7and30days after cryosurgery. The number of CTCs was 17.70±5.725, 14.64±6.761 and 10.28±5.598, respectively, and this decreased significantly over time(P<0.01). ΔCt values for MAGE-3, survivin and carcinoembryonic antigen (CEA) were elevated significantly compared with those obtained before cryosurgery; 2-ΔΔCt values were <1 before cryosurgery, and were 0.63±1.56, 0.21±0.22 and 0.22±0.34 for MAGE-3, survivin and CEA, respectively, at 7days after treatment. At 30days after treatment, 2-ΔΔCt values for MAGE-3, survivin and CEA were 0.24±0.82, 0.03±0.07 and 0.02±0.08, indicating that gene expression in CTCs significantly decreased over time (P<0.01). CTCs were useful biomarkers for evaluating the efficacy of cryosurgery on unresectable HCC.

Adhesion-based tumor cell capture using nanotopography

Circulating tumor cells (CTCs) shed from primary tumors, transport through the blood stream to distant sites, and cause 90% of cancer deaths. Although different techniques have been developed to isolate CTCs for cancer detection, diagnosis and treatment, the heterogeneity of expression of the target antigen and the significant size variance in CTCs limit clinical applications of antibody- and size-based isolation techniques. Cell adhesion using nanotopography has been suggested as a promising approach to isolate CTCs independent of surface marker expression or size of CTCs. However, the nanotopographies studied are mainly nanopillars; the influence of other nanotopography such as nanogratings and their dimensions on tumor cell capture remains to be investigated. This study examined capture performance of several cancer cell lines of different types, surface marker expression and metastatic status on nanotopographies of various geometries and dimensions without antibody conjugation. The cancer cells exhibited differential capture performance on the nanotopographies with an efficiency up to 52%. Compared with flat surfaces and isotropic, discrete nanopillars, nanogratings favored cancer cell adhesion, thus improving the capture efficiency. The influence of nanotopography height studied, on the other hand, was less significant. This study provides useful information to optimize nanotopography for further improvement of CTC capture efficiency.

Combined cell surface carbonic anhydrase 9 and CD147 antigens enable high-efficiency capture of circulating tumor cells in clear cell renal cell carcinoma patients.

Circulating tumor cells (CTCs) have emerged as promising tools for noninvasive cancer detection and prognosis. Most conventional approaches for capturing CTCs use an EpCAM-based enrichment strategy, which does not work well in cancers that show low or no expression of EpCAM, such as renal cell carcinoma (RCC). In this study, we developed a new set of cell surface markers including CA9 and CD147 as alternative CTC-capture antigens specifically designed for RCC patients. We showed that the expression of both CA9 and CD147 was prevalent in a RCC patient cohort (n=70) by immunohistochemical analysis, with both molecules in combination covering 97.1% of cases. The NanoVelcro platform combined with CA9-/CD147-capture antibodies demonstrated significantly higher efficiency for capturing both CTC-mimicking renal cancer cells and RCC CTCs in peripheral blood, compared to the conventional EpCAM-based method. Using immunofluorescence cytological validation at the single-cell level, we were able to identify bona fide CTCs in RCC patient blood following the well-accepted criteria in our CTC-capture system. We further demonstrated a significant association of CTC numbers as well as the CTC expression status of Vimentin, a mesenchymal marker, with disease progression, including pathologic features and clinical staging. These results provide new insights into developing novel, effective targets/approaches for capturing CTCs, making CTCs a valuable tool for improved cancer detection, prognosis and treatment in RCC.

Frequent detection of PIK3CA mutations in single circulating tumor cells of patients suffering from HER2-negative metastatic breast cancer

Modern technologies enable detection and characterization of circulating tumor cells (CTC) in peripheral blood samples. Thus, CTC have attracted interest as markers for therapeutic response in breast cancer. First studies have incorporated CTC analyses to guide therapeutic interventions and stratification of breast cancer patients. Aim of this study was to analyze characteristic features of CTC as biomarker for predicting resistance to HER2-targeted therapies. Therefore, CTC from metastatic breast cancer patients with HER2-negative primary tumors screened for the prospective randomized phase III trial DETECT III were explored for their HER2 status and the presence of PIK3CA mutations. Detection and characterization of HER2 expression of CTC were conducted with the CellSearch® system. Fifteen of 179 CTC-positive patients (8.4%) contained ≥1 CTC with strong HER2 expression. Genomic DNA from individual CTC isolated by micromanipulation was propagated by whole genome amplification and analyzed for PIK3CA mutations in exons 9 and 20 by Sanger sequencing. One or more CTC/7.5 mL were detected in 179/290 patients (61.7%). In 109 patients (34.8%), ≥5 CTC/7.5 mL were found. We detected at least one CTC with the mutation p.E542K, p.E545K, p.H1047R, p.H1047L or p.M1043V in 12/33 patients (36.4%). Thirty six of 114 CTC (31.6%) harbored one of these mutations. CTC in individual patients exhibited heterogeneity concerning PIK3CA mutations and HER2 expression. In conclusion, clinically relevant genomic aberrations such as mutations in the hotspot regions of exon 9 and 20 of the PIK3CA gene can be detected in single CTC and might provide insights into mechanisms of resistance to HER2-targeted therapies.

Abstract 496: Androgen receptor expression on circulating tumor cells in metastatic breast cancer

Abstract Background: Androgen receptor (AR) is a nuclear transcription factor and a member of the steroid hormone receptor superfamily that can be detected by immunohistochemical staining in all three subtypes of invasive breast cancer with frequency of &amp;gt;70% of estrogen receptor-positive tumors (ER+), &amp;gt;60% of HER2-positive tumors (HER2+), and 40-50% of triple-negative breast cancers (TNBC) respectively. AR-target therapies are showing early activity in AR+ breast cancer. Given early evidence of activity with agents targeting AR, it is desirable to improve our understanding of the AR biology in breast cancer. Although circulating tumor cells (CTCs) have been proposed as a liquid biopsy for biomarker detection in multiple cancer types, the rate and heterogeneity of AR expression in CTCs in breast cancer is still unclear. In this study, we determine the rate and heterogeneity of AR expression in CTCs of patients with metastatic breast cancer. Methods: Blood samples from patients with metastatic breast cancer were smeared onto glass slides and subjected to nuclear staining with DAPI and reacted with fluorescent-labeled antibodies to detect CD45 on leukocytes and cytokeratin and AR on epithelial cells; the slides were scanned on the Epic Sciences CTC platform and the data analyzed using established algorithms. Results: Among the 59 patients analyzed, 25 (42.4%) had ER+/HER2-, 12 (20.3%) had ER+/HER2+, 6 (10.2%) had ER-/HER2+, and 16 (27.1%) had TNBC based on the most recent staining. CTCs were detected in 50 of 59 (84.7%) patients. Characterization of CTCs identified CK+ CTCs (45 of 59, 76.3%), CK-negative CTCs (16 of 59, 27.1%), CK+ CTC clusters (16 of 59, 27.1%), and apoptotic CTCs (45 of 59, 76.3%), respectively. Number of CTCs per patient ranged from 0 to 282.1 per mL (median = 4.4). Six of the 50 patients (12%) with CTCs had AR+ CTCs. The number of AR+ CTCs per patient ranged from 0.3 to 52.6 per mL (median = 1.15). Among the patients with AR+ CTCs, the percentage of CTCs that were AR+ ranged from 5% to 50% (median = 21%). Among the six patients with AR+ CTCs, five patients had ER+ breast cancer and one had TNBC. Conclusions: We demonstrated that the Epic Sciences non-enriching or selecting platform can detect AR expression in CTCs in breast cancer. These preliminary results suggest the need for clinical validation of CTC AR expression as a potential test to stratify and identify patients who might benefit from AR therapy. The heterogeneity of intra-patient CTC AR expression leads us to another hypothesis that patients with AR+ CTCs might have heterogeneous disease with multiple drivers. Further studies are warranted to allow serial monitoring of changes in AR and to investigate the clinical applicability of AR+ CTCs and their heterogeneity. Citation Format: Takeo Fujii, Yipeng Wang, James M. Reuben, Rachel Krupa, Ryon Graf, Lyndsey Dugan, Jessica Kouw, Dena Marrinucci, Bora Lim, Carlos H. Barcenas, Angela N. Marx, Debu Tripathy, Ryan Dittamore, Naoto T. Ueno. Androgen receptor expression on circulating tumor cells in metastatic breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 496.

Abstract 2239: Isolation and characterization of circulating tumor cells (CTCs) in non small cell lung carcinoma

Abstract Purpose: Lung cancer is the most common cause of cancer-related deaths that frequently metastasizes prior to disease diagnosis. Circulating tumor cells (CTCs) are of great clinical interest in terms of prognosis and therapy intervention and may provide important prognostic and predictive information in non-small cell lung cancer (NSCLC). The purpose of this study was to evaluate diagnostic performance of a non-invasive method based on filtration-based technology (ISET) using cytomorphologic criteria in patients undergoing surgery comparing with histologic features of corresponding primary lesions. Materials and Methods: The CTCs analysis was performed through the following steps: isolation by ISET filtration method (ScreenCell®) from 5 ml of peripheral blood of 36 patients with NSCLC and 23 with benign lung lesions; classification of CTCs with haematoxylin and eosin (H&amp;E) staining; identification of cell marker with co-immunostaining for epithelial and mesenchymal markers (Vimentin and Cytocheratin). CTCs were classified as tumor cells if: (i) they have a large (≥12 μm), hyperchromatic, irregular-shaped nuclei; (ii) high nuclear-cytoplasmic ratio (&amp;gt;0.5). Diagnostic performance was evaluated against pathologist reported diagnoses of cancer from histological or cytological analysis. Results: CTCs were detected in 27 of 36 (75%) NSCLC patients and in 2 of 23 (9%) negative control patients (p = 0.001). No significant differences were found for the percentage of CTCs for primary and metastatic cancer as well as for cancer stages. A test sensitivity and specificity for diagnosing lung cancer was 75% and 91% respectively. After co-immunostaining (Cytocheratin, Vimentin, CD45) CTCs showed positivity (14/15, 93%) only to vimentin (mesenchymal marker) and completely absence of cytocheratin signal (epithelial marker). Conclusions: The performance of the tested filter-based antibody-independent technology to capture CTCs using standard cytomorphologic criteria provides the potential of a diagnostic blood test for lung cancer showed by high test sensitivity and specificity. The vimentin high expression in CTCs showed both that is a possible marker to CTCs identification and isolation and the importance of EMT mechanism transition in CTCs. Citation Format: Marianna Scrima, Donatella Malanga, Carmela De Marco, Giuseppe Viglietto. Isolation and characterization of circulating tumor cells (CTCs) in non small cell lung carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2239.

Abstract 3371: Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer

Abstract Background: Despite a good initial response to chemotherapy, most small cell lung cancer (SCLC) patients relapse with drug resistant disease. Targeting tumor vasculature in SCLC with anti-angiogenic drugs produced disappointing results, therefore angiogenesis-independent tumor vascularisation pathways warrant further investigation. Vasculogenic mimicry (VM) is the ability of tumor cells to mimic endothelial cells by trans-endothelial differentiation, characterised by increased expression of vascular markers including VE-Cadherin. We previously demonstrated that VM correlates with poor Overall Survival in Limited Stage SCLC patients and sought to phenotypically and genetically characterise VM vessels using SCLC Circulating Tumour Cells (CTCs) and CTC-Derived eXplant (CDX) models1 and to explore the functional significance of VE-Cadherin for VM formation in vitro and in vivo.Methods: VM was evaluated using CD31/periodic acid-Schiff (PAS) staining in tumors from 10 CDX models. Laser Capture Microdissection (LCM) and Copy Number Alteration (CNA) analysis was performed on CDX regions with high and low levels of VM. VE-Cadherin expression in SCLC CTCs was evaluated following ISET microfiltration of patients’ blood and Immunofluorescence staining for DAPI, CD45, pan Cytokeratin and VE-Cadherin. VE-Cadherin function was evaluated in vitro by Matrigel network assay using H446 SCLC cells and H446 shRNA VE-Cadherin knockdown (KD) cell lines, and in vivo by growth as xenografts, further treated with Cisplatin. Results: VM was present in CDX models and LCM followed by CNA analysis of VM vessels confirmed their SCLC origin. ISET microfiltration and immunofluorescent staining of CTCs from 37/38 SCLC patients revealed VE-Cadherin as a putative VM biomarker in SCLC CTCs. VE-Cadherin shRNA KD in VM competent H446 SCLC cells abrogated their ability to form VM networks in vitro and in vivo. Cisplatin treatment of mice bearing H446 VE-Cadherin KD tumors resulted in reduced cisplatin binding compared to parental H446 tumors. Conclusions: VM is present in CDX models and co-localises with VE-Cadherin expression. CNA confirms that VM vessels originate from tumour, and these VM-enriched regions bear a unique chromosomal signature compared to the low-VM regions. VE-Cadherin is required for network formation in vitro and VE-Cadherin levels and VM vessel numbers are positively correlated in vivo. Moreover, levels of VM in tumors had significant impact on both tumour growth kinetics and cisplatin delivery which has implications for drug senstivity. Our future research will interrogate the VM signaling pathway in SCLC and its role in chemosensitivity and establish feasibility for therapeutic targeting. Citation Format: Francesca Trapani, Stuart Williamson, Robert L. Metcalf, Hui Sun Leong, Benjamin Abbott, Jenny Antonello, Cassandra Hodgkinson, Lynsey Franklin, Mary J. Hendrix, Richard E b Seftor, Elisabeth Seftor, Dominic Rothwell, Ged Brady, Crispin Miller, Fiona H. Blackhall, Kathryn L. Simpson, Caroline Dive. Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3371.

Potential role of nuclear PD-L1 expression in cell-surface vimentin positive circulating tumor cells as a prognostic marker in cancer patients.

Although circulating tumor cells (CTCs) have potential as diagnostic biomarkers for cancer, determining their prognostic role in cancer patients undergoing treatment is a challenge. We evaluated the prognostic value of programmed death-ligand 1 (PD-L1) expression in CTCs in colorectal and prostate cancer patients undergoing treatment. Peripheral blood samples were collected from 62 metastatic colorectal cancer patients and 30 metastatic prostate cancer patients. CTCs were isolated from the samples using magnetic separation with the cell-surface vimentin(CSV)-specific 84-1 monoclonal antibody that detects epithelial-mesenchymal transitioned (EMT) CTCs. CTCs were enumerated and analyzed for PD-L1 expression using confocal microscopy. PD-L1 expression was detectable in CTCs and was localized in the membrane and/or cytoplasm and nucleus. CTC detection alone was not associated with poor progression-free or overall survival in colorectal cancer or prostate cancer patients, but nuclear PD-L1 (nPD-L1) expression in these patients was significantly associated with short survival durations. These results demonstrated that nPD-L1 has potential as a clinically relevant prognostic biomarker for colorectal and prostate cancer. Our data thus suggested that use of CTC-based models of cancer for risk assessment can improve the standard cancer staging criteria and supported the incorporation of nPD-L1 expression detection in CTCs detection in such models.

Circulating tumor cells (CTCs) are associated with abnormalities in peripheral blood dendritic cells in patients with inflammatory breast cancer.

CTCs are involved in tumor dissemination and are an independent prognostic factor in primary and metastatic breast cancer patients. Dendritic cells (DCs) are the most efficient antigen presenting cells and are comprised of plasmacytoid-(pDC) and myeloid-(mDC) derived DC subsets. This study aimed to correlate CTC counts with the peripheral blood DC immunophenotypes and functions of inflammatory breast cancer (IBC) patients. This study included 65 IBC patients. Peripheral blood (PB) was obtained from patients prior to starting a new line of chemotherapy for CTCs enumeration by CellSearch® and DC phenotype and function by flow cytometry; the characteristics of DCs were then correlated with CTC counts and clinical outcome. Twenty-one (32.3%) patients with CTCs ≥5 had a significantly inferior overall survival (OS) compared to patients with <5 CTCs (p=0.045). In addition, patients with ≥5 CTCs had a lower percentage of mDCs capable of producing TNF-α before or after activation through the toll-like receptor (TLR), as well as a lower percentage of mDCs producing IL-12 after TLR-activation. There was a positive correlation between CTCs counts and expression of the activation (CCR7) and costimulatory (CD86) receptors on TLR-activated mDCs and pDCs, respectively. Moreover, presence of high percentage of mDC capable to produce increased levels of TNF-α was independently associated with inferior OS (p = 0.0006). An increase in the percentage of mDC producing TNF-α might induce a pro-inflammatory environment that could play a role in determining the poor clinical outcome in IBC patients and could add further prognostic value to CTCs.

AFP mRNA level in enriched circulating tumor cells from hepatocellular carcinoma patient blood samples is a pivotal predictive marker for metastasis

Circulating tumor cells (CTCs) quantification may be helpful for evaluating cancer dissemination, predicting prognosis and assessing therapeutic effectiveness and safety. In the present study, CTCs from blood samples of 72 patients with hepatocellular carcinoma (HCC) were enriched with anti-EpCAM nanoparticles. AFP mRNA level was detected by nested polymerase chain reaction (PCR) after enrichment of CTCs from HCC blood samples at 0, 3, 6, 9 and 12 months after hepatectomy, respectively. AFP mRNA expression in CTCs was positive in 43 patients (59.7%) and negative in 29 patients (40.3%) before hepatectomy. Among 43 patients with positive AFP mRNA expression in CTCs before hepatectomy, 10 and 11 were diagnosed as intrahepatic/extrahepatic metastasis before and after hepatectomy, respectively. In addition, these 21 patients with metastasis had persisting positive AFP mRNA of CTCs during the whole tested year. Specifically, 3 patients with AFP mRNA negative in CTCs before hepatectomy changed to be positive at 6 and 9 months, and 2 of them were diagnosed as metastasis 12 months after hepatectomy. We conclude that the positive AFP mRNA of CTCs can be a pivotal predictor for HCC metastasis before and after hepatectomy. The release of AFP expression from hepatocellular carcinoma cells into circulation must be a major source of HCC metastasis.

Triple fluorescence analysis of vitamin D receptor status of circulating tumor cells (CTC) in breast cancer: from cell models to metastatic patients

In recent years, several trials suggested that optimal vitamin D levels have a protective effect against development of breast cancer. Vitamin D exerts its activity by binding to the vitamin D receptor (VDR). In addition to steroid hormone receptors (eg estrogen and progesterone receptor), VDR is also expressed in normal breast tissue and breast tumors. It may thus have the potential to be a new prognostic biomarker for breast cancer. Increasing evidence supports the view that the detection of circulating tumor cells (CTCs) predicts outcomes of early and metastatic breast cancers. To our knowledge, the evaluation of VDR expression in CTCs of breast cancers patients has never been addressed so far and may contribute to better characterize CTCs.

PD32-11 GENE EXPRESSION ANALYSIS OF BONE METASTASIS AND CIRCULATING TUMOR CELLS FROM METASTATIC CASTRATE-RESISTANT PROSTATE CANCER PATIENTS

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-11 GENE EXPRESSION ANALYSIS OF BONE METASTASIS AND CIRCULATING TUMOR CELLS FROM METASTATIC CASTRATE-RESISTANT PROSTATE CANCER PATIENTS Won-Jin Cho, Michael Cher, Daniel Oliveira, Abdo Najy, Leandro Mainetti, Elisabeth Heath, Kim Hyeong-Reh, and R. Daniel Bonfil Won-Jin ChoWon-Jin Cho More articles by this author , Michael CherMichael Cher More articles by this author , Daniel OliveiraDaniel Oliveira More articles by this author , Abdo NajyAbdo Najy More articles by this author , Leandro MainettiLeandro Mainetti More articles by this author , Elisabeth HeathElisabeth Heath More articles by this author , Kim Hyeong-RehKim Hyeong-Reh More articles by this author , and R. Daniel BonfilR. Daniel Bonfil More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.687AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Characterization of gene expression in bone metastasis is critical to the identification of therapeutic targets and prognostic/predictive biomarkers in metastatic castrate-resistant prostate cancer (mCRPC). Although bone marrow tissue can be obtained for molecular analysis, bone marrow biopsy (BMBx) is relatively invasive. In contrast, circulating tumor cells (CTCs) can be obtained by simple blood draw, and in mCRPC, CTCs likely emanate from bone metastases. Herein, we wished to establish a sensitive method to analyze gene expression in BMBxs and CTCs and to determine whether potential biomarkers present in bone lesions are mirrored in CTCs. METHODS Areas of relatively pure prostate cancer tissue were collected from BMBxs using laser capture microdissection. CTCs were enriched from blood using the CellSearch® platform. RNA from these two sources was amplified linearly using an Eberwine-based procedure to obtain antisense mRNA (aRNA). In this pilot study, the expression of 8 genes was assessed using RT-PCR. In control experiments, the efficiency of CTC isolation and linearity of RNA amplification were determined by comparing relative gene expression levels in non-amplified RNAs from large numbers of cultured PC3 cells and amplified aRNAs from small numbers of PC3 cells spiked in normal blood and recovered using the CellSearch® system. RESULTS RNAs were successfully extracted from as few as 1-5 CTCs in blood samples. The relative expression levels of reference genes were maintained after RNA amplification. The integrity of the amplified RNA was demonstrated by RT-PCR analysis using primer sets that target the 5′-end, middle, and 3′-end of reference mRNAs. Besides epithelial- and prostate-specific genes, Interleukin-6 was the only gene expressed in all CTCs analyzed. In this pilot study, we found partial concordance between gene expression patterns in CTCs and microdissected prostate cancer tissue from bone metastases of the same patients. CONCLUSIONS aRNA amplification through in vitro transcription may serve as a useful method for gene analysis in small numbers of CTCs and tumor cells microdissected from BMBxs. To our knowledge, this is the first comparative study of gene expression in CTCs and BMBxs from individual mCRPC patients. Our exploratory study in this small cohort of patients suggests that molecular analysis of CTCs might reveal gene expression patterns that might be missed by analysis of single BMBx sites. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e764-e765 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Won-Jin Cho More articles by this author Michael Cher More articles by this author Daniel Oliveira More articles by this author Abdo Najy More articles by this author Leandro Mainetti More articles by this author Elisabeth Heath More articles by this author Kim Hyeong-Reh More articles by this author R. Daniel Bonfil More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...