Phenotypic Circulating Tumor Cells Heterogeneity Research Articles

Combination of circulating tumor cells and 18F-FDG PET/CT for precision diagnosis in patients with non-small cell lung cancer.

To investigate the value of 2-deoxy-18f-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) and circulating tumor cells (CTCs) for the differential diagnosis of patients with benign lung diseases and those with NSCLC. To explore the phenotypic heterogeneity of CTCs and their correlation with FDG uptake in patients with Stage I-IV NSCLC. Blood specimens from patients with benign lung diseases and patients with primary NSCLC were collected for the detection of CTCs and their subtypes (epithelial, mixed, and mesenchymal) and analyzed for 18F-FDG PET/CT tumor metabolic parameters, including the maximum standardized uptake value (SUVmax), standard uptake value (SUL), metabolic tumor volume of primary lesion (MTV), total lesion glycolysis of primary lesion (TLG). Clinical data including age, gender, smoking history, tumor size, TNM stage and pathology type were also collected. The value of the two method alone and in combination for the differential diagnosis of benign and malignant was comparatively analyzed. Finally, the differences in CTC and its subtypes in different stages of NSCLC were compared, and FDG metabolic parameters were correlated with CTC subtypes. There were a total of 65 patients with pulmonary diseases, including 12 patients with benign pulmonary diseases and 53 patients with NSCLC. The mean age was 67 ± 10 (38-89 years), 27 were females and 38 were males. 31 (22 males and 9 females) had a long history of smoking. The mean size of the largest diameter of all single lesions was 36 ± 22 mm with a range of 10-108 mm. Seven out of 12 benign diseases were inflammatory granulomatous lesions and 5 were inflammatory pseudotumours. Twenty-four out of 53 NSCLC were adenocarcinomas and 29 were squamous carcinomas. Twelve out of 53 patients with NSCLC were in Stage I, 10 were in Stage II, 17 were in Stage III and 14 were in Stage IV. SUVmax, SUL, MTV, TLG, total CTCs, epithelial CTCs, and mixed CTCs were all valuable in the differential diagnosis of benign and malignant. TLG combined with mixed CTCs was statistically different from all other diagnostic methods (p < 0.05) and higher than any other diagnostic criteria. In the differential diagnosis of benign and Stage I NSCLC, only total CTC (Z = -2.188 p = 0.039) and mixed CTCs (Z = -3.020 p = 0.014) had certain diagnostic efficacy, and there was no statistical difference between them (p = 0.480). Only mesenchymal CTCs differed in Stage I-IV NSCLC, with a higher number of those who developed distant metastases than those who had non-distant metastases. Epithelial CTCs correlated with SUVmax (r = 0.333, p = 0.015) and SUL (r = 0.374, p = 0.006). Mmesenchymal CTCs correlated with MTV (r = 0.342, p = 0.018) and TLG (r = 0.319, p = 0.02). Further subgroup analyses revealed epithelial CTCs were correlated with SUVmax (r = 0.543, p = 0.009) and SUL (r = 0.552, p = 0.008), and the total CTCs was correlated with SUVmax (r = 0.622, p = 0.003), SUL (r = 0.652, p = 0.003), MTV (r = 0.460, p = 0.031), and TLG (r = 0.472, p = 0.027) in the early group (Stage I-II). Only mesenchymal CTCs was associated with MTV (r = 0.369, p = 0.041), and TLG (r = 0.415, p = 0.02) in the intermediate-late group (Stage III-IV). Both FDG PET metabolic parameters and CTCs demonstrated diagnostic value for NSCLC, and combining TLG with mixed CTCs could enhance their diagnostic efficacy. The total CTCs and mixed CTCs showed greater diagnostic value than FDG PET in distinguishing benign lesions from Stage I NSCLC. In NSCLC patients, the epithelial CTCs exhibited a positive correlation with SUVmax and SUL, while mesenchymal CTCs correlated with MTV, and TLG. Besides, epithelial CTCs showed stronger correlations with SUVmax and SUL, and total CTCs showed stronger correlations with SUVmax, SUL, MTV, and TLG in Stage I-II NSCLC. Only mesenchymal CTCs in Stage III-IV NSCLC showed correlations with MTV and TLG. Stage IV NSCLC cases displayed a higher number of mesenchymal CTCs.

A novel microfluidic system for enrichment of functional circulating tumor cells in cancer patient blood samples by combining cell size and invasiveness

A highly invasive subpopulation of circulating tumor cells (CTCs) may constitute seeds for metastases, which are therefore considered functional CTCs. However, there are few effective strategies to detect CTCs based on invasive phenotypes. Herein, we focused on functional CTCs with high invasiveness and designed an integrated microfluidic system to differentiate the invasive potential of CTCs for more accurate metastasis prediction. By combining size-based enrichment and invasiveness-based analysis, the system managed to continuously remove most hemocytes by 8 μm gaps and analyze the invasiveness of the enriched CTCs by Matrigel loading. In addition to a device, a single pump and a Petri dish were included to provide an FBS gradient for driving cell invasion and maintain a long-term cell culture. The system successfully identified functional CTCs derived from different types of cancer patients, including colorectal, kidney and bladder cancer patients, using whole blood without any sample pretreatment process. Within 28 cases of colorectal cancer patients, functional CTCs were detected in 61.54% of patients with metastases, along with stronger invasiveness evaluated by migration/invasion distance than those from patients without metastases (P < 0.05). Furthermore, one bladder cancer patient was diagnosed with recurrence six months after detection, indicating the excellent value for cancer metastases prediction. In addition, great phenotypic heterogeneity of CTCs was also observed at the single-cell level, including invasion, proliferation and dormancy, which provided an effective strategy for metastasis prediction based on CTC function as a single cell.

Abstract P4-01-09: Phenotypic profiling of circulating tumor cells (CTCs) in patients with metastatic breast cancer reveals clinically-relevant heterogeneity in CTC morphology and marker expression

Abstract Background: The choice between hormonal therapies and chemotherapy is a frequent decision in the standard of care of metastatic breast cancer (mBCa) patients. We previously developed quantitative measures of phenotypic CTC heterogeneity in metastatic prostate cancer and found that higher heterogeneity was associated with better survival on chemotherapy vs. targeted hormonal therapies in a risk adjusted analysis (Scher et al. 2017 Cancer Research). Here we sought to test the feasibility of measuring CTC heterogeneity in mBCa patients. Methods: 295 blood samples from 165 pts (84 HR+, 19 Her2+, 8 HR+/Her2+, 54 TNBC) from mBCa patients were processed for CTC analysis utilizing the Epic Sciences Functional Cell Profiling platform. Following enumeration, multi-dimensional phenotypic characterization analysis was performed utilizing protein expression and digital pathology features. Features from each CTC (3994 CTCs from 165 pts, were compared by unsupervised clustering to assess intra-patient CTC heterogeneity among mBCa CTC phenotypes. Results: CTCs were detected in 76.9% (227/295) of mBCa patients (med=2.4 CTC/mL, Range 0 - 747). Phenotypic profiling identified distinct CTC subtypes characterized by morphological and protein expression features that correlated with mBCa subtypes. Prevalent CTC features that associated with mBCa were either high cytokeratin (CK) expression or lower nuclear to cytoplasmic (N/C) ratio with lower CK expression. Analysis of mBCa subtypes showed that TNBC pts were characterized by CTCs with a large nuclear area and high CK expression. We observed marked differences in CTC heterogeneity across patients, with some patients having more uniform CTC profiles, and others having higher phenotypic diversity. mBCa subtypes had similar intra-patient heterogeneity level. Conclusions: Distinct CTC phenotypes can be visualized and quantified across patients and associated with specific mBCa subtypes. Our analysis revealed significant heterogeneity between individual CTCs both between and within patients. Identification of patients with high heterogeneity may help to identify patients unlikely to respond to targeted therapies. Studies to link heterogeneity to efficacy of specific therapies and patient outcome are ongoing. Citation Format: Tiziano Pramparo, Adam Jendrisak, Priscilla Ontiveros, Megan Kearney, Lincy Chu, Nadia Ebrahim, Joseph Schonhoft, Rick Wenstrup, Yipeng Wang. Phenotypic profiling of circulating tumor cells (CTCs) in patients with metastatic breast cancer reveals clinically-relevant heterogeneity in CTC morphology and marker expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-09.

Vimentin expression in circulating tumor cells (CTCs) associated with liver metastases predicts poor progression-free survival in patients with advanced lung cancer

ObjectiveTo investigate the presence of vimentin expression in CTCs and its clinical relevance in patients with advanced lung cancer.MethodsPeripheral blood was obtained from 61 treatment-naive patients with advanced lung cancer. Subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) platform was applied to identify, enumerate and characterize CTCs based on cell size, aneuploidy of chromosome 8 (Chr8) and vimentin expression. Quantification and analysis of CTCs were performed on patients before chemotherapy administration and after two cycles of therapy.ResultsBefore treatment, CTCs were detected in 60 (98.4%) patients, small cell CTCs (≤ 5 µm of WBCs) accounted for 52.8% of the absolute CTCs number, while 12 (19.7%) of the included patients had detectable vimentin-positive CTCs (vim+ CTCs). Liver metastases were reported in 7 (11.5%) patients and were significantly correlated to the presence of Vim+ CTCs (p = 0.002), with a high positivity rate of 71.4% (5/7). Vim+ CTCs were mostly in small cell size and Chr8 aneuploidy (77.0% and 82.05%, respectively). Baseline small cell CTCs ≥ 2/6 ml, triploid CTCs ≥ 2/6 ml, Vim+ CTCs ≥ 1/6 ml were found to significantly correlate with poor progression-free survival (PFS) (p = 0.017, p = 0.009 and p = 0.001, respectively). After adjusting for clinically significant factors, baseline Vim+ CTCs ≥ 1/6 ml was the only independent predictor of poor PFS [hazard ratio (HR):2.756, 95% confidence interval (CI): 1.239–6.131; p = 0.013].ConclusionsThis study demonstrates an important morphologic, karyotypic and phenotypic CTCs heterogeneity in advanced lung cancer patients. The majority of Vim+ CTCs are in small size and Chr8 aneuploidy. Baseline presence of Vim+ CTCs is correlated with liver metastases and may help predict poor PFS.

Circulating tumor cells as a response monitor in stage IV non-small cell lung cancer

BackgroundMonitoring circulating tumor cells (CTC) has been shown to be prognostic in most solid malignancies. There is no CTC assay in clinical use for lung cancer therapy monitoring due to inconclusive clinical utility data. Limited data has been published outside of the standard CTC enumerations, regarding clinical significance of phenotypic heterogeneity of CTCs in late stage NSCLC and its ability to correlate with treatment outcomes.MethodsIn 81 patients with stage IV NSCLC, multiple timepoints for CTC analysis were collected after initiation of treatment across 139 lines of therapy using single cell high definition diagnostic pathology imaging of all nucleated cells from 362 peripheral blood samples as a liquid biopsy.ResultsWe analyzed the subset of 25 patients with complete time series data, totaling 117 blood samples, to determine the significance of HD-CTC kinetics during the initiation of treatment. These kinetics follow three distinct patterns: an increase in HD-CTCs with therapy (mean + 118.40 HD-CTCs/mL), unchanged HD-CTCs numbers (stable; mean 0.54 HD-CTCs/mL), and a decrease in HD-CTCs numbers (mean − 81.40 HD-CTCs/mL). Patients with an increasing CTC count during the first 3 months post initiation of new treatment had a better PFS and OS compared to the other groups. There was weak correlation between the absolute number of HD-CTCs at a single time point of therapy and patient outcomes (OS p value = 0.0754). In the whole cohort of 81 patients, HD-CTCs were detected in 51 (63%) patients at initiation of therapy with a median of 2.20 (range 0–509.20) and a mean of 26.21 HD-CTCs/mL (± 15.64).ConclusionsCTCs are identifiable in most patients with stage IV NSCLC. While absolute HD-CTC counts do not correlate with prognosis, the changes in CTC counts were predictive of survival in patients with metastatic lung cancer receiving chemotherapy. The level and dynamics of CTCs indicate very different biological and pharmacological phenomena at different stages of disease and timepoints of treatment, highlighting the complex role of CTCs in cancer research and clinical management.

Imaging features of genomic instability in circulating tumor cells (CTCs) from metastatic triple-negative breast cancer (metTNBC) patients (pts) who received PIKTOR.

e14527 Background: A subset of TNBCs have homologous recombination deficiency (HRD) with upregulation of compensatory DNA repair pathways. A combination of TAK-228 and TAK-117 (PIKTOR), investigational oral TORC1/2 and PI3Kα selective inhibitors, respectively, is hypothesized to increase genomic instability (GI) and decrease double strand DNA repair. CTCs from metTNBC pts are being characterized for imaging features that may correlate GI in blood obtained at baseline (BL) prior to beginning PIKTOR, and at disease progression (PD) on PIKTOR. Pts are treated with a cisplatin-based regimen after PD on PIKTOR. Methods: Following IRB-approved informed consent, metTNBC pts received 4 mg PO TAK-228 and 200 mg PO TAK-117 QDx3d QW until PD. Blood samples collected at BL and at PD were sent to Epic Sciences for CTC analyses including enumeration, cell morphology, phenotypic heterogeneity, and GI analysis via a previously developed GI prediction algorithm based on cell phenotypes (Jendrisak et al AACR 2018). Results: The 9 pts treated with PIKTOR to date had mets in lymph nodes (n = 8), lung (n = 5), chest wall (n = 2), and bone (n = 2). Median number of prior regimens for metTNBC was 2; median duration of PIKTOR treatment was 8 wks (range, 5-15). CTCs were detected in 62.5% (5/8) BL and 100% (9/9) PD samples. 2 pts had high phenotypic CTC heterogeneity at both BL and PD (pt#36: 0.69 to 0.64; pt#80: 0.93 to 1.41), 1 pt had increased heterogeneity (pt#75: 0 to 1.68) and 1 pt had decreased heterogeneity (pt#67: 1.26 to 0) post-PIKTOR. 1 pt had predicted phenotypic GI (pGI+) CTCs detected at both BL and PD (pt#80), 1 pt had pGI+ CTCs at BL only (pt#36) and 1 pt at PD only (pt#75). Higher expression of the epithelial marker, cytokeratin (CK), and larger cell size were observed in the PD samples. Conclusions: Following treatment with and PD on PIKTOR, higher CK expression, higher phenotypic heterogeneity, and more pGI+ CTCs were observed in a subset of pts, suggesting that these TN CTCs may have developed epithelial transition and greater GI with PIKTOR treatment. Accrual continues and CTC GI biomarkers will be correlated with pts’ response to subsequent cisplatin-based therapy.

Anti-EGFR capture mitigates EMT- and chemoresistance-associated heterogeneity in a resistance-profiling CTC platform

Capture and analysis of circulating tumor cells (CTCs) holds promise for diagnosing and guiding treatment of pancreatic cancer. To accurately monitor disease progression, capture platforms must be robust to processes that increase the phenotypic heterogeneity of CTCs. Most CTC-analysis technologies rely on the recognition of epithelial-specific markers for capture and identification, in particular the epithelial cell-adhesion molecule (EpCAM) and cytokeratin. As the epithelial-to-mesenchymal transition (EMT) and the acquisition of chemoresistance are both associated with loss of epithelial markers and characteristics, the effect of these processes on the expression of commonly used CTC markers, specifically EpCAM, EGFR and cytokeratin, requires further exploration. To determine this effect, we developed an in vitro model of EMT and acquired gemcitabine resistance in human pancreatic cancer cell lines. Using this model, we show that EMT-induction and acquired chemoresistance decrease EpCAM expression and microfluidic anti-EpCAM capture performance. Furthermore, we find that EGFR capture is more robust to these processes. By measuring the expression of known mediators of chemoresistance in captured cells using automated imaging and image processing, we demonstrate the ability to resistance-profile cells on-chip. We expect that this approach will allow for the development of improved non-invasive biomarkers of pancreatic cancer progression.

Abstract P3-01-14: Diverse inter- and intra-patient circulating tumor cells (CTCs) phenotypic heterogeneity identified in metastatic breast cancer (MBC) cohort

Abstract Background: There is unmet need for biomarkers to guide the treatment selection in MBC. We described phenotypic CTC heterogeneity in metastatic castrate resistant prostate cancer(mCRPC), and showed that pts with CTC high heterogeneity have improved OS with chemotherapy, while with pts low CTC heterogeneity pts have longer OS with AR inhibitors (Scher et al. 2017 Cancer Research). Here, the same methodology was applied to evaluate the feasibility of CTC heterogeneity analysis in MBC pts. Material and methods: 92 blood samples from MBC pts were processed for HER2 CTC analysis utilizing the Epic Sciences platform. Following enumeration, multi-dimensional phenotypic characterization analysis was performed utilizing protein expression and digital pathology features. 20 features from each CTC were clustered using unsupervised approach (K-means) and the optimal number of clusters was determined using the elbow method with greater than 85% of variance taken into account. Shannon Index was used to score intra-patient CTC heterogeneity. Results: CTCs were detected in 77.2% (71/92) of the MBC pts. 1501 CTCs from 60 pts were clustered into phenotypic subtypes which are distinctively different in nuclear size, circularity, CK and HER2 intensity etc. HER2 expression was enriched in cell types that had higher CK intensity and was more circular morphologies often associated with replicative stress. A wide range of CTC phenotypic heterogeneity was observed across pts, Shannon Index scores from 0 (low heterogeneity) to 1.67 (high heterogeneity) with a median of 0.60. CTC heterogeneity was independent of CTC counts, some low count pts had heterogeneous CTC subtypes while some high count pts had homogeneous CTC subtypes. Conclusions: Diverse inter- and intra-patient phenotypic CTC heterogeneity were observed in this MBC cohort, consistent with that seen in mCRPC and other MBC cohorts (Beverly H et al, ASCO 2018). Studies linking degree and patterns of CTC heterogeneity to therapeutic outcomes are ongoing. Citation Format: Wang Y, Hom B, Ontiveros P, Landaverde C, Roth E, Levin MK, Hippely S, Landers M, Dittamore R, O'Shaughnessy JA. Diverse inter- and intra-patient circulating tumor cells (CTCs) phenotypic heterogeneity identified in metastatic breast cancer (MBC) cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-14.

Bcl-2 expression in circulating tumor cells (CTCs) of patients with small cell lung cancer (SCLC) receiving front-line treatment

IntroductionTo investigate the presence of Bcl-2+CTCs in chemotherapy-naïve SCLC patients and their clinical relevance during front-line treatment. MethodsPeripheral blood was obtained from 66 consecutive-patients before chemotherapy administration, after one-cycle and at relapse. CTCs were detected by CellSearch and immunofluorescence using anti-Bcl-2, anti-M30, anti-cytokeratins(CK), anti-CD45 and anti-vimentin(Vim) antibodies. ResultsBefore treatment, CTCs were detected in 62.1% and 72.7% of patients using the CellSearch and immunofluorescence (Bcl-2+/CD45−), respectively. One-treatment cycle significantly decreased both CTCs’ detection rate(p < 0.001) and their absolute number (p < 0.001). On relapse, both the number of positive-patients and the absolute number of CTC subpopulations were significantly increased, compared to post-1st cycle (CellSearch: p = 0.002 and immunofluorescence: p < 0.001). Immunofluorescence revealed an important CTC heterogeneity (Bcl2+/Vim+, Bcl2+/Vim−, Bcl2+/CK+, Bcl2+/CK− and Bcl2+/M30− CTCs). Moreover, 50.0% of patients without detectable CTCs by CellSearch had detectable Bcl-2+/CD45− cells. Multivariate analysis revealed a significant association between Bcl-2+/CD45−cells at baseline and PFS (HR = 4.5;p = 0.005) and OS (HR: 4.3; p = 0.001). Bcl-2+/CD45−cells after one-treatment cycle were significantly associated with shorter OS (HR: 13.9; p = 0.007). ConclusionsThese results demonstrate an important phenotypic CTCs heterogeneity based on the co-expression of Bcl-2, CK, Vim and M30 in SCLC patients. The changes of Bcl-2+/CD45− CTCs during treatment seem to be a dynamic biomarker associated with treatment efficacy and patients’ clinical outcome.

Analysis of circulating tumor cells (CTCs) in patients across multiple metastatic breast cancer (mBCa) cohorts identifies marked inter- and intra-patient heterogeneity in CTC size, shape, and overall morphology.

1084Background: The choice between hormonal therapies and chemotherapy is a frequent decision in the care of mBCa pts. We previously developed quantitative measures of phenotypic CTC heterogeneity ...

Intra-patient genomic heterogeneity of single circulating tumor cells (CTCs) associated to phenotypic CTC heterogeneity in metastatic castrate resistant prostate cancer (mCRPC).

The disclosure provides methods correlating intra-patient genomic heterogeneity of single CTCs with phenotypic heterogeneity in each of a population of prostate cancer (PCa) patients.

Comparison of three distinct methods for the detection of circulating tumor cells in colorectal cancer patients

The detection of circulating tumor cells (CTCs) has considerable utility in the clinical management of patients with solid cancers. However, the phenotypic heterogeneity of CTCs and their low numbers in the bloodstream of patients means that no standardized detection method currently exists for these cells. This, together with differences in pre-analytical sample processing, has led to the collection and accumulation of inconsistent data among independent studies. Here, we compare the ability of three methods to detect CTCs in the blood of colorectal cancer patients. Specifically, different aliquots of the same blood sample were screened for the presence of CTCs by a multimarker RT-PCR assay, the standardized CellSearch assay and dHPLC-based gene mutation analysis. In the population tested, none of the blood samples analysed appeared to be positive by all three methods. Of the samples, 75% were positive for the presence of CTCs by the RT-PCR method. Only 20% were positive by the CellSearch assay, while 14.3% of samples displayed gene mutations consistent with the presence of CTCs when the dHPLC method was applied. The samples which were positive for CTCs by the CellSearch assay did not overlap with those that were positive by dHPLC. Interestingly, however, all of these samples were positive when assessed by RT-PCR. Conversely, of the samples that resulted negative by RT-PCR analysis, none appeared to be positive by either of the other methods. These data, therefore, indicate that of the three methods tested, the multimarker RT-PCR assay provides maximal probability of CTC detection. Here, we present the preliminary results of an ongoing clinical study. Future follow-up involving detection of CTCs in the blood of colorectal cancer patients using these three distinct methods will allow us to verify whether either a single method, or a combination of different assays, is necessary to uncover further prognostic significance of circulating tumor cells.