Prevalence Of Circulating Tumor Cells Research Articles

Association Between Obesity and Circulating Tumor Cells in Early Breast Cancer Patients

BackgroundObesity and the presence of circulating tumor cells (CTCs) before and/or after chemotherapy are associated with poor outcome in breast cancer (BC) patients. The activation of oncogenic pathways in fatty tissue leads to cell proliferation, suggesting a possible link between obesity and CTCs. Materials and MethodsIn the phase III SUCCESS A trial, 3754 patients with early BC were randomized to 3 cycles of fluorouracil, epirubicin and cyclophosphamide followed by 3 cycles of docetaxel with or without gemcitabine. Data of 1088 patients with CTC assessments (CellSearch-System; Menarini Silicon Biosystems, Italy) and body mass index (BMI) measurements both before and after chemotherapy were available. Patients were classified according to the WHO's international definitions as underweight, normal weight, overweight, or obese, and according to their weight-change during chemotherapy into a weight-loss group (> 5% decrease), stable-weight group (≤ 5% weight-change) or weight-gain group (>5% increase). Associations between CTC positivity and, BMI or weight-change group were analyzed using frequency-table methods. ResultsAt study entry, 47.4% patients were underweight or normal weight, 33.6% were overweight and 18.9% were obese. Before and after chemotherapy, CTCs were detected in 20.1% and 22.6% of patients, respectively. There was no association between CTC positivity and BMI before (P = 0.104) or after (P = 0.051) chemotherapy. Furthermore, there was no association between weight-change group and CTC status before/after chemotherapy (P = 0.332). ConclusionsAccording to our analysis, the risk factors obesity and prevalence of CTCs are not associated and may represent independent prognostic factors.

Fusion Cell Markers in Circulating Tumor Cells from Patients with High-Grade Ovarian Serous Carcinoma

Cancer is primarily a disease in which late diagnosis is linked to poor prognosis, and unfortunately, detection and management are still challenging. Circulating tumor cells (CTCs) are a potential resource to address this disease. Cell fusion, an event discovered recently in CTCs expressing carcinoma and leukocyte markers, occurs when ≥2 cells become a single entity (hybrid cell) after the merging of their plasma membranes. Cell fusion is still poorly understood despite continuous evaluations in in vitro/in vivo studies. Blood samples from 14 patients with high-grade serous ovarian cancer (A.C. Camargo Cancer Center, São Paulo, Brazil) were collected with the aim to analyze the CTCs/hybrid cells and their correlation to clinical outcome. The EDTA collected blood (6 mL) from patients was used to isolate/identify CTCs/hybrid cells by ISET. We used markers with possible correlation with the phenomenon of cell fusion, such as MC1-R, EpCAM and CD45, as well as CEN8 expression by CISH analysis. Samples were collected at three timepoints: baseline, after one month (first follow-up) and after three months (second follow-up) of treatment with olaparib (total sample = 38). Fourteen patients were included and in baseline and first follow-up all patients showed at least one CTC. We found expression of MC1-R, EpCAM and CD45 in cells (hybrid) in at least one of the collection moments. Membrane staining with CD45 was found in CTCs from the other cohort, from the other center, evaluated by the CellSearch® system. The presence of circulating tumor microemboli (CTM) in the first follow-up was associated with a poor recurrence-free survival (RFS) (5.2 vs. 12.2 months; p = 0.005). The MC1-R expression in CTM in the first and second follow-ups was associated with a shorter RFS (p = 0.005). CEN8 expression in CTCs was also related to shorter RFS (p = 0.035). Our study identified a high prevalence of CTCs in ovarian cancer patients, as well as hybrid cells. Both cell subtypes demonstrate utility in prognosis and in the assessment of response to treatment. In addition, the expression of MC1-R and EpCAM in hybrid cells brings new perspectives as a possible marker for this phenomenon in ovarian cancer.

P-040: CD138-independent strategy to predict relapse in Multiple Myeloma patients

<h3>Background</h3> CD138 has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug resistant minimal-residual disease (MRD) and circulating tumor cells (CTCs) were shown to have lower expression of this marker. We previously published that residual MM cells following treatment in vivo were hypoxic and the combination of hypoxia and chemotherapy such as bortezomib downregulated CD138 expression, thereby making this marker unsuitable for MM detection. Needless to say, accurate number of MM cells is critical in diagnosis, autologous transplantation, MRD and drug efficiency assessment. Moreover, CTCs are considered an unfavorable prognostic factor and indicate an aggressive form of the disease, and therefore detecting CTCs can be used as a powerful prognostic tool for MM. <h3>Methods</h3> We used an alternative biomarker-set using flow cytometry defining MM cells as any cell that expresses CD38 but excluding T cell (CD3), B cell (CD19), NK cell (CD16), monocyte/macrophage (CD14), neutrophil/eosinophil (CD16), and basophil/dendritic cell (CD123). We demonstrated previously that this approach it widely available due to accessibility of flow cytometry, inexpensive, and works independently of hypoxic-, CD138 expression- and treatment status. We analyzed primary patient samples (n=50) with complete response or very good partial response for MRD and CTCs and correlated the numbers of detected MM with patients' time-to-progression (TTP) obtained from a clinical data base. <h3>Results</h3> We found that the alternative biomarker-set identifies MM cells more precisely and at higher numbers than CD138 marker by flow or histology. Moreover, we found a correlation between the number of MM cells detected and TTP in these patients: the amount of MM cells detected by the new method ranged between 0.5-7.3%, and patients who progressed sooner than two years had 2.5-fold higher percentage of MM cells compared to patients who relapsed later than 2 years. Similarly, patients who relapsed sooner than 3 years had 4-fold higher number of MM cells than patients who relapsed later than 3 years. We further found that, among all patients who had more than 2% of MM cells detected by the new method had a mean TPP of about 20 months, while patients who had less than 2% had a mean TPP of about 38 months. Testing the prevalence of CTCs in MM patients with progressive disease using CD138 or the new method, demonstrated that CD138 detected minimal amounts of MM cells in all patients (less than 0.1%), while the new method detected a range between 0.1 - 1.8% of MM cells in the peripheral blood. <h3>Conclusion</h3> These results suggest that the alternative biomarker-set detected MM cells which correlated with relapse in MM patients. Therefore, the amount of residual cells in the bone marrow and circulating myeloma cells can be used as a prognostic marker in MM patients. Further examination to characterize this population and its role in MM relapse is warranted.

Circulating Tumor Cells in Desmoid Tumors: New Perspectives.

IntroductionDesmoid tumor (DT) is a rare neoplasm with high local recurrence rates, composed of fibroblastic cells that are characterized by the expression of key molecules, including the intermediate filament vimentin, cyclooxygenase-2 (COX-2), and nuclear β-catenin, and lack of epithelial markers. Circulating tumor cells (CTCs) isolated from the peripheral blood of patients with sarcomas and other neoplasms can be used as early biomarkers of tumor invasion and dissemination. Moreover, CTCs can also re-colonize their tumors of origin through a process of “tumor self-seeding.”ObjectivesWe aimed to identify CTCs in the peripheral blood of patients with DT and evaluate their expression of β-catenin, transforming growth factor receptor I (TGF-βRI), COX-2, and vimentin proteins.Material and MethodsWe conducted a prospective study of patients with initial diagnosis or relapsed DT with measurable disease. Blood samples from each patient were processed and filtered by ISET® (Rarecells, France) for CTC isolation and quantification. The CTC expression of β-catenin, COX-2, TGF-βRI, and vimentin was analyzed by immunocytochemistry (ICC).ResultsA total of 18 patients were included, and all had detectable CTCs. We found a concordance of β-catenin expression in both CTCs and primary tumors in 42.8% (6/14) of cases by using ICC and immunohistochemistry, respectively.ConclusionsOur study identified a high prevalence of CTCs in DT patients. Concordance of β-catenin expression between primary tumor and CTCs brings new perspectives to assess the dynamics of CTCs in the blood compartment, opening new avenues for studying the biology and behavior of DT. In addition, these results open the possibility of using CTCs to predict DT dynamics at the time of disease progression and treatment. Further studies with larger sample sizes are needed to validate our findings.

Abstract LB060: Circulating tumor cells in desmoid tumors

Abstract Introduction: Desmoid tumor (DT) is a rare neoplasm with high local recurrence rates. CTNNB1 mutations are found in most sporadic cases and are located in exon 3, usually in codons 41 or 45 (p.T41A, p.S45F and p.S45P). Circulating tumor cells (CTCs) isolated from the peripheral blood of patients with sarcomas and other neoplasms can be early biomarkers of tumor invasion and dissemination. Moreover, CTCs can also re-colonize their tumors of origin through a process of “tumor self-seeding.” Objectives: To identify CTCs in the peripheral blood of patients with DT; to evaluate their expressions of β- catenin and vimentin proteins. Materials and methods: We conducted a prospective study of patients with initially diagnosed and relapsed DT with measurable disease. Blood samples from each patient were processed and filtered by ISET® (Rarecells, France) for the isolation and quantification of CTCs. The expressions of β -catenin and vimentin in CTCs were analyzed by immunocytochemistry (ICC). Results: A total of 18 patients were included, and all had detectable CTCs. We found a concordance of β-catenin expression in CTCs and in primary tumors in 42.85% of cases by using ICC and immunohistochemistry respectively. Conclusions: Our study identified a high prevalence of CTCs in DT patients. Concordance of β-catenin expression between primary tumor and CTCs bring new perspectives to assess the dynamics of CTCs in the blood microenvironment, opening new avenues for studying the biology and behavior of DT. In addition, these results open the possibility of using CTCs to predict DT dynamics at the time of progression and during treatment. Further studies with larger sample sizes are needed to validate our findings. Citation Format: Alexcia Camila Braun, Fernando Campos, Emne Ali Abdallah, Anna Paula Ruano, Milena Tariki, Fabio Pinto, Celso Abdon de Mello, Vilma Regina Martins, Ludmilla Thome Chinen. Circulating tumor cells in desmoid tumors [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 LB060.

Circulating tumour cells at baseline and late phase of treatment provide prognostic value in breast cancer

To determine the prognostic value of the timing of circulating breast tumour cell measurement during treatment, peripheral blood from 164 patients with breast disease was collected. Circulating tumour cells (CTCs) were enriched by using immunomagnetic nanospheres (IMNs) and were identified by using immunofluorescent staining. The CTC shows nuclear-positive, EpCAM-positive, CK19-positive, and CD45-negative. Patients with CTC positivity (> 19/7.5 mL blood) had shorter progression-free survival (PFS) and overall survival (OS) than those with negative results (≤ 19/7.5 mL blood) at baseline. Surgery caused an increase in the number and prevalence of CTCs, and the effect disappeared on day 14 after surgery. During adjuvant chemotherapy, CTCs detected before therapy was only correlated with PFS; however, CTCs at the end of adjuvant chemotherapy were correlated with both PFS and OS. The PFS and OS of the CTC-positive group were significantly shorter than those of the CTC-negative group at the end-point follow-up visit. The prognostic value of CTCs at different measurement time points was demonstrated during breast cancer treatment. Surgery and chemotherapy affected the prevalence of CTCs, leading to different prognostic relevance of CTCs at different treatment stages. CTCs detected at baseline or in the late phase of treatment are preferable for prognosis.

Evaluation of a marker independent isolation method for circulating tumor cells in esophageal adenocarcinoma.

The enrichment of circulating tumor cells (CTCs) from blood provides a minimally invasive method for biomarker discovery in cancer. Longitudinal interrogation allows monitoring or prediction of therapy response, detection of minimal residual disease or progression, and determination of prognosis. Despite inherent phenotypic heterogeneity and differences in cell surface marker expression, most CTC isolation technologies typically use positive selection. This necessitates the optimization of marker-independent CTC methods, enabling the capture of heterogenous CTCs. The aim of this report is to compare a size-dependent and a marker-dependent CTC-isolation method, using spiked esophageal cells in healthy donor blood and blood from patients diagnosed with esophageal adenocarcinoma. Using esophageal cancer cell lines (OE19 and OE33) spiked into blood of a healthy donor, we investigated tumor cell isolation by Parsortix post cell fixation, immunostaining and transfer to a glass slide, and benchmarked its performance against the CellSearch system. Additionally, we performed DEPArray cell sorting to infer the feasibility to select and isolate cells of interest, aiming towards downstream single-cell molecular characterization in future studies. Finally, we measured CTC prevalence by Parsortix in venous blood samples from patients with various esophageal adenocarcinoma tumor stages. OE19 and OE33 cells were spiked in healthy donor blood and subsequently processed using CellSearch (n = 16) or Parsortix (n = 16). Upon tumor cell enrichment and enumeration, the recovery rate ranged from 76.3 ± 23.2% to 21.3 ± 9.2% for CellSearch and Parsortix, respectively. Parsortix-enriched and stained cell fractions were successfully transferred to the DEPArray instrument with preservation of cell morphology, allowing isolation of cells of interest. Finally, despite low CTC prevalence and abundance, Parsortix detected traditional CTCs (i.e. cytokeratin+/CD45-) in 8/29 (27.6%) of patients with esophageal adenocarcinoma, of whom 50% had early stage (I-II) disease. We refined an epitope-independent isolation workflow to study CTCs in patients with esophageal adenocarcinoma. CTC recovery using Parsortix was substantially lower compared to CellSearch when focusing on the traditional CTC phenotype with CD45-negative and cytokeratin-positive staining characteristics. Future research could determine if this method allows downstream molecular interrogation of CTCs to infer new prognostic and predictive biomarkers on a single-cell level.

10]-Gingerol improves doxorubicin anticancer activity and decreases its side effects in triple negative breast cancer models.

Although doxorubicin is widely used to treat cancer, severe side effects limit its clinical use. Combination of standard chemotherapy with natural products can increase the efficacy and attenuate the side effects of current therapies. Here we studied the anticancer effects of a combined regimen comprising doxorubicin and [10]-gingerol against triple-negative breast cancer, which does not respond to hormonal or targeted therapies. Cytotoxicity was evaluated by MTT assay, cell cycle progression and apoptosis were analyzed by flow cytometry and signaling pathways were analyzed by Western blotting in human and murine triple negative breast cancer cell systems. The anticancer/antimetastatic and toxic effects of the combined regimen was evaluated using syngeneic and xenograft orthotopic models. The combination of doxorubicin and [10]-gingerol significantly increased the number of apoptotic cells, compared to each compound alone. In 4T1Br4 cells, the combined regimen was the only condition able to increase the levels of active caspase 3 and γH2AX and to decrease the level of Cdk-6 cyclin. In vivo, doxorubicin (3 mg/Kg, D3) and [10]-gingerol (10 mg/Kg, G10) resulted in a significant reduction in the volume of primary tumors and a decrease in the number of circulating tumor cells (CTCs). Interestingly, only the combined regimen led to decreased tumor burdens to distant organs (i.e., metastasis) and reduced chemotherapy-induced weight loss and hepatotoxicity in tumor-bearing animals. Likewise, in a xenograft model, only the combined regimen was effective in significantly reducing the primary tumor volume and the prevalence of CTCs. Our data indicate that [10]-gingerol has potential to be used as a neoadjuvant or in combined therapy with doxorubicin, to improve its anticancer activity.

Abstract IA19: Multiple uses of circulating tumor cells in lung cancer?

Abstract Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA. Citation Format: Caroline Dive. Multiple uses of circulating tumor cells in lung cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA19.

Abstract PD8-03: Clinico-pathological and molecular features in young women with metastatic breast cancer

Abstract Introduction: Advanced breast cancer can be more aggressive and portend a worse prognosis in the younger population (age &amp;lt; 50). While several studies have outlined clinico-pathologic features and molecular features of primary disease in young women with breast cancer, data in the metastatic setting remain scarce. Thus, the goal of this study was to explore the underlying clinic-pathologic and molecular profile of young women with metastatic breast cancer. Methods: We conducted a retrospective analysis of 138 females with metastatic breast cancer treated at Northwestern Medicine who provided consent for serial evaluation of circulating biomarkers. Patient were divided into two cohorts based on age at the time of metastasis, namely premenopausal (defined as age &amp;lt; 50) and postmenopausal (age ≥ 50). CellSearch™ immunomagnetic kit (Menarini Silicon Biosystems) was utilized to enumerate circulating tumor cells (CTCs), and the CellSearch™ CXC Kit (in 7.5 cc whole blood) characterized CTC HER2 expression. Circulating tumor DNA (ctDNA) was sequenced using the Guardant360 next-generation sequencing (NGS) assay (Guardant Health). When available, tissue samples from the primary and metastatic site(s) were sequenced using FoundationOne and/or Tempus xT NGS assays. Associations were drawn using Pearson’s χ2 test, independent samples T-tests, and multivariate logistic regression. Results: Of the 138 women, 54 (39%) were premenopausal with median age of 42 (range: 28-49), and 84 (61%) were postmenopausal with median age of 57 (range: 50-81). For the premenopausal cohort, 96% had invasive ductal carcinoma, 2% invasive lobular carcinoma and 2% mixed/unknown, compared to postmenopausal with 74%, 19% and 7% respectively, p=0.003. No statistically significant association was observed based on disease subtype (HR+/HER2-, HR+/HER2+, HR-/HER2+, TNBC), correspondingly stratified as 46%, 17%, 9%, 28% (premenopausal) and 57%, 10%, 13% 20% (postmenopausal). In total, 39% of the premenopausal and 40% of the postmenopausal patients had inflammatory breast cancer (IBC). Among patients initially diagnosed with non-metastatic disease, time (years) to metastasis was 2.76 (95% CI 2.11 to 3.41) for the premenopausal and 6.08 (95% CI 4.89 to 7.27) for the postmenopausal cohort, p=0.0001. Statistically significant associations were found when comparing the NGS datasets, derived from serial collection of ctDNA +/- tissue samples. Specifically, the premenopausal group had a higher incidence of GATA3 (11 vs 6 cases; p=0.017) alterations, but a lower incidence of NF1 (1 vs 18; p&amp;lt;0.001) and RB1 (1 vs 9; p=0.049) alterations. Most common gene alterations included TP53 (67%), PIK3CA (45%), ERBB2 (26%), GATA3 (26%), MYC (24%), FGFR1 (19%) and ESR1 (17%) for the younger cohort, versus TP53(67%), PIK3CA (39%), ESR1 (27%), NF1 (27%), MYC (23%), ERBB2 (23%) and FGFR1 (20%) for the postmenopausal cohort. There were no statistically significant differences between the cohorts in terms of total number of ctDNA alterations at baseline draw (median[IQR]: 3[1-7] premenopausal group; 5[2-7] postmenopausal group), presence of ≥5 CTCs (54%; 46% of total cases, respectively), occurrence of CTC clusters (26%; 24%), or HER2+ CTC expression (44%; 45%). Conclusion: Our data reveal that premenopausal women diagnosed with metastatic breast cancer had a more rapid progression to metastasis and differ from their postmenopausal counterparts in both their pathologic profile (almost exclusively invasive ductal carcinoma) and molecular profile (notably, gene alteration frequencies of NF1, RB1 and GATA3), which could have significant implications in developing targeted treatment paradigms for younger women. Additionally, CTC prevalence in the metastatic setting differs from earlier stage breast cancer data showing a higher proportion of ≥5 CTCs in postmenopausal patients. Citation Format: Kristen Carroll, Ami M Shah, Lorenzo Gerratana, Chenyu Lin, Andrew A Davis, Qiang Zhang, Youbin Zhang, Lisa Flaum, Amir Behdad, Leonidas C Platanias, William J Gradishar, Massimo Cristofanilli. Clinico-pathological and molecular features in young women with metastatic breast cancer [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 PD8-03.

Detection and prognostic relevance of circulating tumour cells (CTCs) in Asian breast cancers using a label-free microfluidic platform.

ObjectivesWe aimed to study the prevalence of CTCs in breast cancer (BC) patients undergoing neoadjuvant or palliative therapy with a label-free microfluidic platform (ClearCell FX), and its prognostic relevance in metastatic BC (mBC).Materials and methodsPeripheral blood samples were collected from 108 BC patients before starting a new line of treatment (“baseline”), majority of whom had mBC (76/108; 70.4%). CTCs were retrieved by dean flow fractionation that enriched for larger cells, and enumerated using immunofluorescence-based staining. Progression-free survival (PFS) in mBC patients was analysed using Kaplan-Meier method; cox proportional hazard models were used for univariable and multivariable analyses.ResultsThe detection rate of CTCs before starting a new line of treatment was 75.9% (n = 108; median: 8 CTCs/7.5 ml blood) at a cut off of ≥2 CTCs. PFS was inferior for mBC patients with baseline CTC count ≥5 CTCs/7.5 ml blood vs. those with < 5 CTCs/7.5 ml blood (median PFS: 4.3 vs. 7.0 months; p-value: 0.037). The prognostic relevance of CTCs was most significant in patients with HER2- mBC (median PFS: 4.1 vs. 8.3 months; p-value: 0.032), luminal (HR+HER2-) subtype (median PFS: 4.2 vs. 8.3 months; p-value: 0.048), and patients who had one or more prior treatments (median PFS: 4.2 vs. 7.0 months; p-value: 0.02). On multivariable analysis, baseline CTC level (hazard ratio (HR): 1.84, p-value: 0.02) and pre-treatment status (HR: 1.87, p-value: 0.05) were independent predictors of PFS.ConclusionsThis work demonstrates the prognostic significance of CTCs in mBC detected using a label-free size-based enrichment platform.

Abstract 457: HER2, AR protein expression and chromosomal instability in circulating tumor cells (CTCs) of metastatic breast cancer (MBC) patients (pts)

Abstract Background: Upregulation of HER2 and AR protein expression, and increase in chromosomal instability, are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these biomarkers requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which precludes their utility to guide treatment in late stage metastatic pts. A CTC-based test could expand the clinical utility. Using the Epic Sciences platform, MBC blood samples were characterized for CTC prevalence, HER2 and AR expression on CTCs, and CTC chromosomal instability at time of disease progression. Material and methods: Blood samples were acquired from 127 total pts with HR+/HER2-, HR-/HER2+ and TNBC metastatic breast cancer pts. 114 samples were analyzed for HER2 and 110 were analyzed for AR using the Epic CTC Platform. Single-cell whole genome sequencing was performed on 157 CTCs from 19 pts to assess large scale transitions (a surrogate of chromosome instability) and gene copy number alterations. Results: 87/114 (76.3%) of pts had detectable CTCs, with a median of 1.1 CTC/mL. 17/114 (14.9%) had at least one HER2(+) CTC, 21/110 (19.1%) had at least one AR(+) CTC, and 10/110 (9%) had both AR(+) and HER2(+) CTCs detected in technical replicates. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. The majority of HER2+ and AR+ CTCs had high cytokeratin expression. Most pt samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. CTCs with larger nuclear area and higher N/C ratio had higher frequency of chromosomal instability. ERBB2, FGFR1 gain, PTEN, CDH1, BRCA1, BRCA2 loss were frequently observed and the prevalence were associated with CTC phenotypes. Conclusions: CTCs are detected in most MBC pts upon disease progression. CTC based biomarkers assessing HER2, AR and chromosomal instability status could guide therapy selection. The results warrant prospective evaluation of these biomarkers on MBC pts’ CTCs as a resistance marker for targeted therapies. Citation Format: Priscilla Ontiveros, Connie Landaverde, Maren K. Levin, Sarah Hippely, Mark Landers, Yipeng Wang, Ryan Dittamore, Joyce A. O'Shaughnessy. HER2, AR protein expression and chromosomal instability in circulating tumor cells (CTCs) of metastatic breast cancer (MBC) patients (pts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 457.

Abstract P3-01-07: Enabling HER2 and androgen receptor (AR) protein expression and localization in circulating tumor cells (CTCs) of ER(+/-)HER2(-) metastatic breast cancer (MBC) patients (pts)

Abstract Background: Upregulation of HER2 and AR are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these proteins and their localization requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which limits their utility to guide treatment in late stage metastatic patients. A CTC-based test could expand the clinical utility of these biomarkers. Here we utilized the Epic Sciences CTC platform for CTC detection and characterization. MBC blood samples were characterized for CTC prevalence, HER2 and AR expression at time of disease progression. Material and methods: HER2 and AR expression levels were determined based on model cell lines. A total of 72 blood samples were acquired from ER(+/-)/HER2(-) patients (by standard tissue pathology) at disease progression. 72 samples were analyzed for HER2 and 64 were analyzed for AR using the Epic Platform. Single-cell whole genome sequencing was performed to assess clonality and inter-patient heterogeneity of CTCs detected. Results: 55/72 (76.4%) of patients had CTCs detected across two slides. 13/72 (18.1%) had at least one HER2(+) CTC, 14/64 (21.9%) had at least one AR(+) CTC, and 7/64 (10.9%) had at both AR(+) and HER2(+) CTCs detected on replicate slides. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. Most patient samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. Subsequently, these cells will be individually sequenced to better determine the clonality of resistance. Conclusions: CTCs are detected in most MBC pts upon disease progression, with expression of known endocrine therapy resistance markers, HER2 and AR, observed that CTCs could guide subsequent therapy selection. Prospective evaluation of HER2 and AR on MBC pts' CTCs as predictive biomarkers of benefit from inhibitors of these proteins is needed. Citation Format: Ontiveros P, Landaverde C, Graf R, Levin MK, Hippely S, Wang Y, Landers M, Dittamore R, O'Shaughnessy JA. Enabling HER2 and androgen receptor (AR) protein expression and localization in circulating tumor cells (CTCs) of ER(+/-)HER2(-) metastatic breast cancer (MBC) patients (pts) [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 P3-01-07.

Abstract 5702: Characterization of circulating tumor cells during immune checkpoint inhibition in metastatic renal cell carcinoma and malignant melanoma

Abstract Background: Although immune checkpoint inhibitors (ICIs) have shown efficacy in a range of solid tumors, there are no established circulating biomarkers able to identify early responders to treatment. Circulating tumor cells (CTCs) allow a non-invasive peripheral assessment of disease burden over time. We evaluated the prevalence of CTCs before and during treatment with ICIs. Methods: Patients were enrolled prospectively at Duke Cancer Center and UNC Lineberger Cancer Center and selected for metastatic renal cell carcinoma (mRCC) and metastatic malignant melanoma (mMM) receiving standard of care immunotherapy. Using E-selectin for slowing cell rolling and a nanoparticle dendrimer based technology for cell capture with EpCAM, HER2, and EGFR, the CapioCyte platform was used for CTC capture at baseline, during treatment, and upon disease progression. Results: We enrolled 5 subjects with mRCC and 4 subjects with mMM. Prior to treatment, subjects had a median of 293 CTCs/mL (all healthy volunteers (HVs, n=10) had &amp;lt;8 CTCs/mL). All subjects had more CTCs at baseline than HVs. One subject had CTCs rise within the first 4 weeks on ICI treatment, which corresponded to clinical disease progression. Several other subjects showed a rapid decrease in CTCs within 4 weeks but increased at the time of disease progression. In most cases of disease control, CTCs decreased by at least 50% at the 4-week timepoint and increased at time of disease progression. Conclusions: CapioCyte is a sensitive platform to detect CTCs in mRCC and mMM. Based on CTC detection, many patients had early response to ICI treatment, but rapid progression corresponding to clinical progression. This pilot data suggest that CTCs can be a valuable biomarker for monitoring the clinical benefit of cancer immunotherapies. Citation Format: Tian Zhang, SinJung Park, Daniella Runyambo, Michael J. Poellmann, Marco Reyes-Martinez, Zahra Mahbooba, Rebecca Green, Carrie Lee, Daniel J. George, Andrew J. Armstrong, Seungpyo Hong, Andrew Z. Wang. Characterization of circulating tumor cells during immune checkpoint inhibition in metastatic renal cell carcinoma and malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5702.

Prevalence of circulating tumor cells in early breast cancer patients 2 and 5years after adjuvant treatment.

Several studies have provided evidence on the prognostic relevance of circulating tumor cells (CTCs) detected before and after chemotherapy regarding overall survival (OS) and progression-free survival (PFS) in early breast cancer (EBC). We provide data on the prevalence of CTCs 2 and 5years after primary diagnosis in a cohort of patients with EBC. The SUCCESS study is a multicenter, prospective, randomized trial comparing PFS in primary breast cancer patients undergoing one of two adjuvant chemotherapy regimens followed by 2 versus 5years of treatment with zoledronate. CTCs from patients without signs of breast cancer recurrence were analyzed in peripheral blood using the FDA cleared CellSearch® System (Veridex, USA) 2 and 5years after primary diagnosis. CTCs were detected at 2 and 5years after primary diagnosis in 96 (16.7%) and 47 (8.2%) of the 574 patients, respectively. There were no associations between CTC status and patient and tumor characteristics or treatment regimens. In 442 (77.0%) patients, no CTCs were detected at either of the two time points, and in 11 patients (1.9%), CTCs were found at both 2 and 5years after primary diagnosis. In 85 (14.8%) patients, CTCs were present 2years after primary diagnosis but not after 5years, while 36 (6.3%) patients had CTCs in their blood only at the 5-year follow-up. In patients with EBC, CTCs can be detected even 5years after primary diagnosis without clinical signs of disease recurrence.

Role of regulatory T cells and CD8+ T lymphocytes in the dissemination of circulating tumor cells in primary invasive breast cancer.

Circulating tumor cells (CTCs) dissemination is involved in tumor metastasis and is an independent prognostic factor in patients with primary and metastatic breast cancer. Regulatory T cells (Tregs) and cluster of differentiation (CD)8+ T lymphocytes are the main types of immune cells in the tumor microenvironment and exert opposite roles on the progression and outcome of breast cancer. The aim of the present study was to evaluate the associations between CTCs and intratumoral/peritumoral Tregs and CD8+ T lymphocytes in breast cancer. Peripheral CTCs were detected by a multi-marker quantitative polymerase chain reaction platform in 167 patients with invasive breast cancer. Intratumoral/peritumoral Tregs and CD8+ T lymphocytes were assessed by immunohistochemistry in 167 patients with invasive breast cancer to establish an association between these cell types and detection of peripheral CTCs. CTCs were detected in 55% of the patients with breast cancer. The prevalence of CTCs was positively associated with the number of intratumoral (P=0.002) and peritumoral Tregs (P=0.045), and the primary tumor size (P=0.012). This result was verified by analyzing intratumoral Tregs (P=0.044) and primary tumor size (P=0.044) with multivariate analysis, which indicated that the CTC-positive rate increased with an increasing number of intratumoral Tregs and a larger tumor size In the multivariate analysis, other variables including menopause, tumor-node-metastasis stage and peritumoral Tregs were not associated with the prevalence of CTCs. The prevalence of CTCs was inversely and weakly associated with the number of peritumoral CD8+ T lymphocytes using the univariate analysis, however this result was not statistically significant (P=0.470). In conclusion, regulatory T cells and CD8+ T lymphocytes may be involved, at least in part, in the CTCs dissemination of breast cancer, whereby Tregs appear to exert the dominant effect. Furthermore, the role of Tregs in the progression of breast cancer may be mediated by suppressing the dissemination of CTCs, which is primarily determined by intratumoral Tregs.

Presence of circulating tumor cells is associated with metabolic-related variables in postoperative patients with early-stage breast cancer.

Although circulating tumor cells (CTCs) have been well-established as promising prognostic biomarkers in both early breast cancer and metastatic settings, little is known regarding the prognostic relevance of CTCs in the long-term postoperative monitoring of patients with non-metastatic breast cancer (non-MBC). In this study, we investigated the associations of CTCs with clinicopathological features and metabolic-related variables, such as obesity and hyperglycemia. In this retrospective study, we recruited 264 patients with postoperative stage I-III breast cancer at Guangdong General Hospital from January 2009 to December 2015. The prevalence and number of CTCs were assessed using the CellSearch System at a median time of 19.0 months [interquartile range (IQR), 7.8-33.0] after surgery. The CTC assay results were correlated with the clinicopathological features and metabolic-related variables. A multivariate logistic regression analysis was performed to further determine the independent predictors of CTCs. CTCs were detected in 10.6% of all patients. The positive rate of CTCs in patients with infiltrating ductal carcinoma was lower than that in patients with other pathological types (9.0% vs. 28.6%, P=0.020). More importantly, the presence of CTCs was correlated with blood glucose level (P=0.015) and high-density lipoprotein level (P=0.030). The multivariate logistic regression analysis showed that the pathological type [odds ratio (OR): 1.757, 95% CI: 1.021-3.023; P=0.042] and blood glucose level (OR: 1.218, 95% CI: 1.014-1.465; P=0.035) were independent predictors of the presence of CTCs. This study revealed potential associations between CTCs and metabolic-related factors in Chinese patients with non-MBC and supports the hypothesis that metabolic dysfunction in breast cancer patients might influence the biological activity of metastatic breast cancer, leading to a higher prevalence of CTCs.

Abstract P1-01-03: Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial

Abstract Background: The presence of circulating tumor cells (CTCs) before chemotherapy is known to be associated with reduced disease free survival (DFS) and overall survival (OS) in early breast cancer (EBC). In addition, recent findings suggest that CTCs persisting after adjuvant chemotherapy indicate poor prognosis. In an explorative analysis of the SUCCESS A trial, we evaluated the prognostic relevance of changes in CTC counts during the course of adjuvant chemotherapy across molecular subtypes to assess whether the prognostic role of persisting CTCs varies according to tumor biology. Methods: The SUCCESS A trial is a phase III study, where patients with high-risk EBC (stage pN1-3 or pT2-4 or grade 3 or age ≤ 35 or hormone-receptor negative) were randomized to adjuvant chemotherapy with 3 cycles of epirubicin-fluorouracil-cyclophosphamide followed by either 3 cycles of docetaxel or 3 cycles of gemcitabine-docetaxel. CTC enumeration was performed before and after chemotherapy using the FDA-approved CellSearch® System (Janssen Diagnostics, LLC), and CTC positivity was defined as ≥ 1 CTC in 23 ml blood. Molecular subtypes were defined as luminal A like (hormone-receptor positive, grading 1 or 2), luminal B like (hormone-receptor positive, grading 3), triple-negative or HER2-positive. Patient outcome in terms of DFS and OS was analyzed using univariate log-rank tests and Cox regression models (median follow-up time 65.2 months). Results: Data on both molecular subtypes and CTC status before and after chemotherapy were available for 1485 (39.6%) of 3754 patients randomized. This cohort contained 577 (38.9%) luminal A like, 236 (15.9%) luminal B like, 379 (25.5%) HER2-positive and 293 (19.7%) triple negative tumors. Overall, 917 (61.8%) patients were CTC negative before and after chemotherapy (neg/neg), 260 (17.5%) patients had a negative CTC status before and a positive CTC status after chemotherapy (neg/pos), 229 (15.4%) patients converted from positive to negative CTC status (pos/neg), and 79 (5.3%) patients were positive for CTCs at both time points (pos/pos). There were significant differences in DFS and OS among these four groups in patients with luminal A like tumors (log rank test, both p &amp;lt; 0.003) and patients with luminal B like tumors (log rank test, both p &amp;lt; 0.001). In both patients with luminal A like or luminal B like tumors, persistently CTC positive patients had the worst outcome (relative to persistently CTC-negative patients) in terms of DFS and OS. In contrast to luminal-like tumors, no significant differences with regard to DFS or OS were found among the four groups (neg/neg, neg/pos, pos/neg, pos/pos) in patients with HER2-positive or triple-negative tumors (log rank test, all p &amp;gt; 0.13). Conclusion: The presence of CTCs both before and after adjuvant chemotherapy was associated with poor survival in luminal A like and luminal B like tumors, but not in HER2-positive or triple-negative tumors. Further research is needed to evaluate the effect of chemotherapy on CTC prevalence in different molecular subtypes of EBC. Citation Format: Tzschaschel MLJ, Rack B, Andergassen U, Friedl TWP, Schneeweiss A, Mueller V, Tanja F, Pantel K, Gade J, Lorenz R, Rezai M, Tesch H, Soeling U, Polasik A, Alunni-Fabbroni M, Trapp EK, Mahner S, Schindlbeck C, Lichtenegger W, Beckmann MW, Fasching PA, Janni W. Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-03.

Prevalence of Circulating Tumor Cells After Adjuvant Chemotherapy With or Without Anthracyclines in Patients With HER2-negative, Hormone Receptor-positive Early Breast Cancer

BackgroundUse of anthracycline-based chemotherapy in patients with early breast cancer (EBC) has been well-established but is often associated with cardiotoxicity. Based on data suggesting a limited benefit of anthracyclines in human epidermal growth factor receptor 2 (HER2)-negative patients, the Simultaneous Study of Docetaxel Based Anthracycline Free Adjuvant Treatment Evaluation, as well as Life Style Intervention Strategies (SUCCESS) C study randomized patients to either anthracycline-containing or anthracycline-free chemotherapy. Given the proven prognostic value of circulating tumor cells (CTCs) in EBC, we compared the prevalence of CTCs after chemotherapy between both treatment arms for a preliminary efficacy assessment. MethodsThe SUCCESS C trial (NCT00847444) is an open-label, phase III study randomizing 3547 patients with HER2-negative EBC to either 3 cycles of epirubicin, 5-fluorouracil, and cyclophosphamide followed by 3 cycles of docetaxel (FEC-DOC) or 6 cycles of docetaxel and cyclophosphamide (DOC-C). CTC status was prospectively evaluated in hormone receptor-positive patients at the time of last chemotherapy cycle using the US Food and Drug Administration-approved CellSearch System (Janssen Diagnostics). ResultsData on CTC status were available for 1766 patients. Overall, CTCs were found in 221 (12.5%) patients. Univariate analyses revealed that presence of CTCs at time of last chemotherapy cycle was not significantly associated with tumor or patient characteristics (all P > .1). There was no significant difference with respect to presence of CTCs between patients randomized to FEC-DOC or DOC-C (11.5% vs. 13.6%; P = .18). ConclusionsThe comparable prevalence of CTCs at the time of last chemotherapy cycle may indicate that anthracycline-free chemotherapy is equally effective to anthracycline-containing chemotherapy in HER2-negative, hormone receptor-positive EBC. However, efficacy data from the final survival analysis of SUCCESS C have to be awaited to confirm these preliminary findings.

Abstract PL04-04: Developing circulating tumor cell derived explant models (CDX)

Abstract Serial biopsies of solid tumors can be challenging, not always without risk to the patient and do not capture intratumoral heterogeneity. ‘Liquid Biopsies’ in the form of Circulating Tumor DNA (ctDNA) and Circulating Tumor Cells (CTCs) offer minimally invasive means to stratify patients for therapy and to routinely monitor therapy responses and anticipate emergent therapy resistance. Whilst technically more challenging than ctDNA, CTCs offer a wider range of biomarker application allowing RNA and protein measurements within single and pools of CTCs following enrichment from the blood. In addition, we have focussed on the development of CTC derived explant models (CDX), particularly in small cell lung cancer (SCLC), where biopsies are particularly difficult to obtain (and often small and necrotic) to study the biology of this aggressive disease and to test new therapies. We have generated 17 SCLC CDX models to date where CTCs, enriched using negative depletion of blood cells, are grown subcutaneously in immune-compromised mice. The take rate is ∼45% and is broadly correlated to the number of CellSearch detected EpCam positive CTCs measured in a paired blood sample. CDX models are derived from SCLC patients at presentation before chemotherapy, where most patients are initially chemosensitive but ∼20% are chemorefractory. For some patients, a matched CDX has been derived following chemotherapy response and subsequent relapse with progressive disease and where a biopsy is rarely acquired. CDX histopathology mimics the diagnostic biopsy of the donor patient and recapitulates the donor patient's response to chemotherapy. CDX can be passaged with maintained growth characteristics and as few as 5-10 CDX cells can re-establish the tumor consistent with a high frequency of tumor initiating cells. We are exploiting CDX models to explore the biology of SCLC, mechanisms of tumor cell dissemination and chemotherapy resistance. The CDX models are also being used to facilitate drug development. Therapies that show promise in CDX models can be rapidly translated to the clinic where the high prevalence of CTCs in SCLC patients provide minimally invasive, proof of mechanism pharmacodynamic biomarkers and CTC number as a surrogate of tumor response. Once passaged in vivo, tumor cells from disaggregated CDX can be cultured ex vivo over 2-3 weeks for initial and more rapid drug screening. SCLC CDX are also being used to assess the importance of vascular mimicry (VM) - a manifestation of tumor cell plasticity that endows endothelial cell behaviour, and we have shown that VE-Cadherin plays a functional role in SCLC VM, altering tumor growth kinetics and intratumor delivery of cisplatin. The generation of s.c CDX from NSCLC patients is proving more challenging. We have generated a CDX from a NSCLC patient with no CellSearch EpCam positive CTCs in their 7.5ml blood sample but whose blood did contain &amp;gt;150 CTCs/ml blood detected by filtration using an immunofluorescence assay for Epithelial to Mesenchymal Transition. The resultant CDX and the majority of this patient's CTCs were mesenchymal. Alternative approaches including intrathoracic implantation of enriched NSCLC CTCs are being explored. Subcutaneous CDX have been successfully derived from advanced melanoma patients and can now complement other approaches and extend studies where metastatic melanoma biopsies are unforthcoming. In summary, the development of CDX models yields new opportunities to study advanced disease and examine treatment response and resistance. We are currently developing approaches that will allow the genetic manipulation of CDX cultures to underpin mechanistic studies and we are exploring the generation of CDX across other tumor types. Citation Format: Caroline Dive, Kris Frese, Melanie Galvin, Alice Lallo, Cassandra Hodgkinson, Christopher Morrow, Kathryn Simpson, Francesca Trappani, Stuart Williamson, Lynsey Priest, Romina Girotti, Crispin Miller, Ged Brady, Richard Marais, Fiona Blackhall. Developing circulating tumor cell derived explant models (CDX). [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 PL04-04.