Presence Of Circulating Tumor Cells Clusters Research Articles

Association of Circulating Tumor Cells, Megakaryocytes and a High Immune-Inflammatory Environment in Metastatic Breast Cancer.

Liquid biopsy is becoming an important source of new biomarkers during the treatment of metastatic cancer patients. Using size-based microfluid technology, we isolated circulating tumor cells (CTCs) from metastatic breast cancer patients to evaluate their presence and cluster formation, as well as the presence of megakaryocytes and immune-inflammatory blood cells, and to correlate their presence with clinicopathological data and overall survival (OS). In total, 59 patients (median age 60.4 years) were included in the study: 62.7% luminal A/B-like, 20.3% HER2-positive, and 17% triple-negative. Our results showed that at least one CTC was present in 79.7% and ≥5 CTCs in 35.2% of the patients. CTC clusters were present in patients with ≥5 CTCs only (in 19.2% of them), and megakaryocytes were present in 52% of all patients. The presence of CTC clusters and megakaryocytes was positively associated with the CTC count. Patients with low pan-inflammatory value (PIV), low systemic immune-inflammatory index (SII), and low relative change from baseline (ΔPIV%, ΔSII%) were associated with significantly higher OS than their counterparts. ΔPIV%, the presence of infection in the last month, and a long duration of metastatic disease were identified as independent prognostic factors for OS. The interplay of CTCs, CTC clusters, megakaryocytes, and PIV needs to be further explored.

Circulating tumor cells (CTCs) detection and isolation in different subtypes of early-stage breast cancer patients from Bangladesh.

e12529 Background: Breast cancer is a highly heterogeneous pathophysiology characterized by poor outcomes. Due to the increasing incidence and disease progression rates and undefined relapse periods, reliable disease monitoring is a challenge and has remained an unmet need. Advancements in liquid biopsy have significantly enhanced our understanding of clinical oncology. CTC-based liquid biopsy is emerging as a reliable prognostic tool to predict various clinical indicators. Although extensively investigated in metastatic breast cancers, little is known about CTCs in early-stage breast cancers. CTCs with respect to different molecular subtypes of breast cancer in early-stage breast cancer patients is evaluated. Methods: In this prospective clinical trial (CMC 59.27.0000.013.19 PG.009.2022/262) 40 early-stage patients with luminal (A +B, 33.33%), HER 2 positive (12.8%), triple-negative (12.8%) and undetermined (41.07%) subtype were recruited. CTCs were isolated in 1.5 ml blood using the Drug Controller General of India approved OncoDiscover CTC test. This platform contains affinity-based magnetic nanoparticles to mediate EpCAM-based CTC isolation. CTCs were detected as CK18+, DAPI+, and CD45- cells using a fluorescence detection-based automated digital imaging platform. Results: CTCs were detected in 60 % of patients with a mean CTC count of 1 cell / 1.5ml blood. Among total positive patients, the luminal subtype was the least positive (46 %), followed by TNBC (60 %) and undetermined (62.5 %) subgroup, while all HER2-positive patients showed the presence of CTCs. Besides individual cells, CTC clusters were detected in 12.5 % of patients and they were equally distributed in luminal and HER2-positive subpopulations. When analyzed on the scale of tumor grade, grade I patients did not show the presence of CTC, while 58.33 % of grade II patients had ≥ 1 CTC. All grade III patients showed the presence of ≥ 1 CTC. CTC count was high among CTC-positive grade II patients (average 2 CTCs) and correlated well with the presence of CTC clusters in these patients. Patients who had surgical intervention had a low CTC burden compared to patients who did not have a surgical resection. 75 % of treatment naïve patients showed the presence of CTC while 58 % of patients receiving chemotherapy alone showed the presence of 1 CTC. 50 % of patients who had surgery followed by CT+RT showed the presence of 1 CTC. Conclusions: The presence of CTCs may suggest for biological progression of disease in early-stage BC patients. CTC detected in all HER2-positive patients suggested the high shedding nature of these tumors, which correlates well with their reported migratory tendency. The presence of CTCs did not show a clear correlation with the treatment regimen. However, this data is based on a single time point and needs longitudinal correlation with CTC on a larger sample size. Clinical trial information: 59.27.0000.013.19 PG.009.2022/262 .

Clinical Relevancy of Circulating Tumor Cells in Breast Cancer: Epithelial or Mesenchymal Characteristics, Single Cells or Clusters?

Metastatic breast cancer (MBC) is typically an incurable disease with high mortality rates; thus, early identification of metastatic features and disease recurrence through precise biomarkers is crucial. Circulating tumor cells (CTCs) consisting of heterogeneous subpopulations with different morphology and genetic, epigenetic, and gene expression profiles represent promising candidate biomarkers for metastatic potential. The experimentally verified role of epithelial-to-mesenchymal transition in cancer dissemination has not been clearly described in BC patients, but the stemness features of CTCs strongly contributes to metastatic potency. Single CTCs have been shown to be protected in the bloodstream against recognition by the immune system through impaired interactions with T lymphocytes and NK cells, while associations of heterotypic CTC clusters with platelets, leucocytes, neutrophils, tumor-associated macrophages, and fibroblasts improve their tumorigenic behavior. In addition to single CTC and CTC cluster characteristics, we reviewed CTC evaluation methods and clinical studies in early and metastatic BCs. The variable CTC tests were developed based on specific principles and strategies. However, CTC count and the presence of CTC clusters were shown to be most clinically relevant in existing clinical trials. Despite the known progress in CTC research and sampling of BC patients, implementation of CTCs and CTC clusters in routine diagnostic and treatment strategies still requires improvement in detection sensitivity and precise molecular characterizations, focused predominantly on the role of CTC clusters for their higher metastatic potency.

Abstract A03: Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers

Abstract Metastasis in head and neck cancer (HNC) patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in HNC. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. n=300 head and neck cancer patients were recruited to investigate the prognostic role of CTCs. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples, which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification). The presence of CTC clusters was associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression-free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence interval [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof-of-principle study, we were able to demonstrate for the first time short-term patient-derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggest that PD-L1 is frequently expressed on CTCs in HNC and an immunoscore may be able to identify patients likely to benefit from immunotherapy—a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression, and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean Paul Thiery, Ken O’Byrne, Chamindie Punyadeera. Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A03.

Abstract P4-01-04: The distribution and prognostic significance of circulating tumor cells are different in invasive lobular carcinoma compared to invasive ductal carcinoma of the breast

Abstract Background: Circulating tumor cells (CTCs) have repeatedly been shown to carry independent prognostic information in metastatic breast cancer (MBC). A CTC count of 5 cells per 7.5 mL blood is a generally accepted cut-off, where MBCs with <5 CTCs are classified as MBCindolent and ≥5 CTCs as MBCaggressive. Furthermore, several studies have shown that the presence of CTC clusters add prognostic information to CTC count alone. Invasive lobular carcinoma (ILC) is the second most common histological breast cancer (BC) type (~10%). The vast majority of all BCs (~80%) are classified as invasive ductal carcinoma (IDC). Many distinguishing clinicopathological and genomic features have been identified between these two types, but in spite of this, current clinical management strategies and treatments are similar and mainly based on studies dominated by IDCs. The aim of this study was to explore potential differences in the distribution and prognostic significance of CTC count and CTC clusters in metastatic ILC vs. IDC, in a prospective observational study. Patients and methods: Between April 2011 and June 2016, 139 women with newly diagnosed metastatic ILC (N=28) and IDC (N=111) were included. CTCs and CTC clusters were detected, using CellSearch technology (Menarini Silicon Biosystems), at baseline (BL) before first-line systemic therapy, and during the first 6 months of follow-up (FU). The primary endpoint was progression-free survival (PFS) and the secondary endpoint overall survival (OS). Median FU time was 49 (27-93) months. Results: There was a highly significant (P<0.001) difference in the median CTC count between ILC (70, range 0-2598) and IDC (2, range 0-668) at BL, and presence of CTC clusters was also higher (36% vs. 18%, P=0.07). These differences between ILC and IDC persisted in the luminal A-like subgroup. The CTC count and CTC clusters declined in both ILC (median count 4, range 0-85; clusters: 4%) and IDC (median count 0, range 0-263; clusters: 12%) after 1 month of systemic treatment, but the decline was most pronounced in ILC. Seventy-nine percent of the ILCs and 46% of the IDCs were classified as MBCaggressive (CTC ≥5) at BL (P=0.003). The prognostic value of CTC ≥5 on PFS (HR 1.5, 95% CI 0.55-4.0, P=0.44) and OS (HR 2.4, 95% CI 0.71-8.3, P=0.16) in ILC was weak, whereas significant prognostic effects were seen in IDC (PFS: HR 1.7, 95% CI 1.2-2.6, P=0.007; OS: HR 2.1, 95% CI 1.3-3.3, P=0.002). With higher cut-offs the prognostic impact of CTC count on PFS/OS was significant also in ILC (CTC ≥20: HR 3.0, 95% CI 1.3-6.8, P=0.01 / HR 3.1, 95% CI 1.2-8.3, P=0.02) (CTC ≥80: HR 3.6, 95% CI 1.5-8.8, P=0.004 / HR 5.9, 95% CI 2.0-17.8, P=0.002) and the prognostic effect in IDC remained. The presence of ≥1 CTC cluster was a negative prognostic factor significantly associated with impaired survival in ILC (PFS: HR 4.6, 95% CI 1.7-12.4, P=0.003; OS: HR 4.9, 95% CI 1.7-13.8, P=0.003), whereas the effect was weaker in IDC (PFS: HR 1.2, 95% CI 0.69-2.0, P=0.55; OS: HR 1.9, 95% CI 1.1-3.3, P=0.02). First-line systemic treatment was similar in ILC vs. IDC (endocrine 46% vs. 39% and chemotherapy 54% vs. 61%) and the overall prognosis did not differ (PFS: HR 0.89, 95% CI 0.57-1.4, P=0.59; OS: HR 0.99, 95% CI 0.60-1.6, P=0.96). Conclusions: In this study of metastatic ILC and IDC, the CTC count at BL was remarkably higher in ILCs and the presence of CTC clusters was also more common, in spite of the fact that no difference in prognosis was seen. The prognostic value of the generally accepted CTC cut-off (≥5) was clearly weaker and the presence of CTC clusters stronger in ILC vs. IDC. A higher cut-off might be more suitable in ILCs in order to better discriminate between MBCindolent and MBCaggressive forms, and CTC clusters could potentially add prognostic information. Citation Format: Ulrik Christoffer Narbe, Pär-Ola Bendahl, Kristina Aaltonen, Mårten Fernö, Carina Forsare, Charlotte Levin Tykjær Jørgensen, Anna-Maria Larsson, Lisa Rydén. The distribution and prognostic significance of circulating tumor cells are different in invasive lobular carcinoma compared to invasive ductal carcinoma of the breast [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 P4-01-04.

Abstract 1333: Isolation, characterization and expansion of circulating tumor cells in solid cancers

Abstract Metastasis in cancer patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in a number of solid tumor types. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. Head and Neck cancer (n=300) and lung cancer (n=80) patients were recruited to investigate the prognostic role of CTCs. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification, ALK-translocations). In HNC, the presence of CTC clusters associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence internal [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof of principle study, we were able to demonstrate for the first time, short-term patient derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggests that PD-L1 is frequently expressed on CTCs in HNC and lung cancer and an immunoscore may be able to identify patients likely to benefit from immunotherapy-a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean-Paul Thiery, Ken O'Byrne, Chamindie Punyadeera. Isolation, characterization and expansion of circulating tumor cells in solid cancers [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 1333.

Abstract P3-01-19: HER2-positive circulating tumor cells (CTCs) in advanced breast cancer (BC): A feature independent of BC subtype

Abstract Introduction: HER2 overexpression is observed on CTCs in advanced BC (ABC), but their significance is not known. We aimed to describe clinical, pathologic, and molecular associations with HER2 overexpression on CTCs in ABC patients (pts). Methods: We conducted a retrospective analysis of data from ABC pts treated at Thomas Jefferson University and Northwestern University who had evaluation of CTCs and circulating tumor DNA (ctDNA). CTCs were enumerated with the CellSearch immunomagnetic kit (Menarini Silicon Biosystems), HER2 expression on CTCs was evaluated using the CellSearch CXC Kit, and ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Associations with the presence of HER2+ CTCs were explored through univariate and multivariate logistic regression. Kruskal-Wallis testing evaluating HER2+ CTCs as a continuous variable was also conducted to confirm consistency of findings. Time to development of HER2+ CTCs was evaluated using Cox proportional hazards regression analysis. Results: Baseline CTCs were evaluated in 209 pts (10% stage III, 90% stage IV) of whom 41% had no detectable CTCs, 23% had 1-4 CTCs, and 36% had >5 CTCs (stage IV aggressive). Twelve percent had CTC clusters. At least 1 HER2+ CTC was seen in 33% of pts at baseline draw. Of 39 patients with HER2+ BC, only 18% had HER2+ CTCs. Of patients with HER2+ CTCs, 55% had hormone receptor positive BC, 28% had triple negative BC, and 18% had HER2+ BC. On univariate logistic analysis, BC subtype or HER2 status was not associated with the presence of HER2+ CTCs. IBC pts represented 52% of pts and were less likely to have HER2+ CTCs (OR 0.40 95% CI 0.19-0.84). Bone metastases were associated with an increased likelihood of HER2+ CTCs (OR 2.46, 95% CI 1.12-5.38); however, other sites of metastases and number of metastatic sites were not correlated with HER2+ CTCs. Aggressive disease features including >5 CTCs and presence of CTC clusters were strongly associated with HER2+ CTCs (OR 15.72, 95% CI 6.89-35.8 and 8.97, 95% CI 3.23-24.89, respectively). Of 168 pts with ctDNA analysis, ERRB2 aberrations were seen in 22% of pts and were significantly associated with HER2+ CTCs (OR of 3.74, 95% CI 1.45-9.63). On multivariate analysis, the associations with >5 CTCs and ERBB2 alterations in ctDNA remained statistically significant. The associations of HER2+ CTCs with bone disease, >5 CTCs, CTC clusters, and ERBB2 alterations in ctDNA, and the inverse relationship with IBC were consistent when HER2+ CTCs were evaluated as a continuous variable with Kruskal-Wallis testing. Among pts without HER2+ CTCs at baseline, the time to detection of HER2+ CTCs correlated with the presence of bone metastases (HR 3.40, 95% CI 1.14-10.19), >5 CTCs (3.77, 95% CI 1.33-10.70), and visceral disease (HR 3.00, 95% CI 1.07-8.44). Conclusions: HER2+ CTCs are common in ABC, independent of HER2 status of the tumor, and, in fact, common in the luminal BC. HER2+ CTCs were also strongly associated with CTC characteristics of aggressive disease with poor survival (CTCs clusters and >5 CTCs) and ERBB2 aberrations in ctDNA. Further studies will be investigating the role of HER2+ CTCs in endocrine resistance and the potential of anti-HER2 therapy in this unique CTC-defined setting. Citation Format: Shah AN, Gerratana L, Davis AA, Zhang Q, Zhang Y, Rossi G, Wang C, Strickland K, Yang H, Flaum L, Abu-Khalaf M, Behdad A, Ye Z, Platanias L, Gradishar WJ, Cristofanilli M. HER2-positive circulating tumor cells (CTCs) in advanced breast cancer (BC): A feature independent of BC subtype [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 P3-01-19.

Abstract P2-01-03: Improved prognostic information by serial monitoring of CTC enumeration and CTC-clusters from baseline to six months in patients with metastatic breast cancer scheduled for 1st line systemic therapy

Abstract Background: Detection and enumeration of circulating tumor cells (CTCs) allows real time monitoring of disease evolvement. In women with metastatic breast cancer (MBC), a CTC count of ≥5 CTCs is associated with decreased progression-free survival (PFS) and overall survival (OS). Serial sampling after therapy initiation has indicated that longitudinal CTC enumeration adds prognostic information, but data from long time sampling is sparse. The aim of this study was to evaluate if prospective longitudinal detection of CTC count and CTC clusters in women with newly diagnosed MBC can improve prognostication and monitoring of patients in the clinical setting. Methods: Longitudinal blood samples were collected at baseline (BL) and after 1, 3 and 6 months in 156 women with MBC scheduled for 1st line systemic therapy. CTC enumeration and cluster detection were performed by the CellSearch® system in a prospective monitoring trial (NCT01322893). 115 patients had evaluable samples at all time-points. Primary endpoint was PFS and secondary endpoint was OS at BL in relation to CTC count and as landmark analyses during treatment. In addition, change in CTC count during therapy was compared to progressive disease (PD) versus non-PD. Structured clinical and radiological evaluation for PD was performed every 3rd month. Results: Seventy-nine (52%) of 152 evaluable patients had ≥5 CTC and 14/79 patients had CTC-clusters (33 clustered CTC) at BL. Median follow-up time was 25 (7-69) months. Patients with ≥5 CTCs had inferior PFS and OS in uni-(data not shown) and multivariable analysis (HRPFS 1.91 (1.26-2.91), P=0.003) (HROS 3.57 (2.02-6.31), P<0.001) at BL. Presence of clusters at BL was prognostic for OS (HROS 2.37 (1.25-4.51), P=0.008). Longitudinal landmark analysis of number of CTCs and presence of CTC clusters showed a time-dependent increase in HR during treatment for CTCs and CTC-clusters and predicted worse PFS and OS at all time-points. Stratifying patients based on CTC count and presence of clusters revealed four risk groups (0, 1-4, ≥5 CTC, ≥5 CTC + clusters) where patients with clusters had inferior PFS and OS at all time points. Change in CTC count from BL to 1 and 3 months, and from 3 to 6 months was significantly related to evaluation at 3 and 6 months (PD vs non-PD, P=0.013 (3 months), P=0.016 (6 months)) and change in CTC count from BL to 1, 3 and 6 months was also significantly predictive of both PFS and OS. Notably, survival was significantly inferior for patients with persistent CTC ≥5 during treatment. Discussion: CTC is an independent prognostic factor for MBC patients scheduled for 1st line systemic therapy. By longitudinal monitoring during treatment, the prognostic information by presence of ≥5 CTC and clusters increases over time and supports long time monitoring of patients. Importantly, detection of CTC-clusters identifies a subgroup of patients with dismal prognosis at all time-points indicating that CTC-clusters renders important clinical information. Change in CTC count during systemic therapy is related to outcome of evaluation and prognosis at all time-points. Citation Format: Larsson A-M, Jansson S, Bendahl P-O, Baker S, Graffman C, Lundgren C, Loman N, Aaltonen KE, Rydén L. Improved prognostic information by serial monitoring of CTC enumeration and CTC-clusters from baseline to six months in patients with metastatic breast cancer scheduled for 1st line systemic therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-03.

Abstract 1727: Circulating Tumor Cells (CTCs) in patients with extensive stage small cell lung cancer and their association with clinical outcome

Abstract Background: The NOTCH pathway has been identified as a key therapeutic pathway in SCLC. Tarextumab (TRXT, anti-Notch2/3, OMP-59R5) is a fully human monoclonal antibody that targets the Notch2 and Notch3 receptors. PINNACLE is a Phase 1b/2 trial of TRXT in combination with etoposide and platinum therapy (EP) in patients with untreated extensive stage small cell lung cancer (ES-SCLC). Baseline CTCs and post treatment changes in CTCs have previously been shown to predict the response to chemotherapy in SCLC. CTCs may also serve as pharmacodynamic biomarkers. Here we describe a study of baseline and longitudinal CTCs in ES-SCLC patients from the PINNACLE phase 1b trial (clinicaltrials.gov:NCT01859741). Materials and methods: CTCs, CTC clusters, apoptotic CTCs and N-Cadherin+ CTCs were identified and enumerated from patient blood samples using Epic Sciences CTC technology. Baseline CTCs from 26 patients were correlated with clinical outcome: progression-free survival (PFS), overall survival (OS) and best overall response, as well as metastatic status. A mixed effects model was used to investigate the post treatment changes in CTCs among the dose groups. Association of CTCs with PFS/OS and best overall response, including CTCs at each time point, as well as temporal changes of CTC status, were studied. Multivariate analysis was performed to identify CTC numbers in a subset of time points to correlate with response to treatment. Results: CTCs were present in 81% of the patients (21/26). CTC clusters and apoptotic CTCs were detected in 38% and 77% of the patients, respectively. At baseline, CTC counts ≥ 5/mL were significantly associated with poor OS (p=0.04). There was a trend that the presence of CTC clusters was associated with worse OS. With a cut-off of 3.4/mL, apoptotic CTCs showed a trend in association with overall survival. CTC numbers in patients with liver metastasis were significantly higher than in patients without liver metastasis. CTCs were also found to be correlated significantly with the number of metastatic sites. When measuring at Day 7 post dosing, CTC numbers were significantly decreased. Conclusions: Our findings suggest that CTCs are frequently detectable in patients and are a prognostic factor in ES-SCLC. CTCs decrease with TRXT and platinum-based chemotherapy. Updated results will be presented. CTCs will be further evaluated in the phase 2 portion of the PINNACLE trial. Citation Format: Chun Zhang, Ryon Graf, Adam Jendrisak, Amanda K. Anderson, Priscilla Ontiveros, Sarah Orr, Anne Chiang, David Spigel, Charles Rudin, Eric Holmgren, Jakob Dupont, Gretchen Argast, Leonardo Faoro, Lei Zhou, John Lewicki, Ann M. Kapoun. Circulating Tumor Cells (CTCs) in patients with extensive stage small cell lung cancer and their association with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1727. doi:10.1158/1538-7445.AM2017-1727

Abstract P4-01-06: Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis

Abstract Introduction: Real-time monitoring of biological changes in tumors that metastasize may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in patients with localized disease pose major hurdles. The goal of this study was to determine if breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes. Methods: CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counter staining of RBC-lysed blood and bone marrow fractions, respectively. The lung metastasis (LM) rate was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed using the gene expression arrays of primary tumors from different PDX lines and were subsequently overlapped to identify common genes. Results: A total of 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present either as single cells or as clusters, were detected in 83% (15/18) and 62.5% (10/16) of the lines, respectively. There was a positive association between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 out of 18 (50%) lines, of which all 9 had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the 2 genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified 4 genes (HLA-DP1A, GJA1, PEG3, and XIST). This 4-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients. Conclusion: This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases. Citation Format: Mario Giuliano, Sabrina Herrera, Pavel Christiny, Chad Shaw, Chad J Creighton, Tamika Mitchell, Raksha Bhat, Xiaomei Zhang, Sufeng Mao, Lacey Dobrolecki, Ahmed Al-rawi, Fengju Chen, Bianca M Veneziani, Xiang H Zhang, Susan G Hilsenbeck, Alejandro Contreras, Carolina Gutierrez, Rinath Jeselsohn, Mothaffar F Rimawi, C Kent Osborne, Michael T Lewis, Rachel Schiff, Meghana V Trivedi. Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-06.

Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis.

IntroductionReal-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.MethodsCTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.ResultsIn total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.ConclusionThis study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0508-5) contains supplementary material, which is available to authorized users.

Circulating Tumor Cells Detection and Counting in Uveal Melanomas by a Filtration-Based Method

Uveal melanoma is one of the most deadly diseases in ophthalmology for which markers able to predict the appearance of metastasis are needed. The study investigates the role of circulating tumor cells (CTC) as a prognostic factor in this disease. We report the detection of circulating tumor cells by Isolation by Size of Epithelial Tumor cells (ISET) in a cohort of 31 uveal melanoma patients: we identified single CTCs or clusters of cells in 17 patients, while the control population, subjects with choroidal nevi, showed no CTC in peripheral blood. The presence of CTCs did not correlate with any clinical and pathological parameter, such as tumor larger basal diameter (LBD), tumor height and TNM. By stratifying patients in groups on the basis of the number of CTC (lower or higher than 10 CTC per 10 mL blood) and the presence of CTC clusters we found a significant difference in LBD (p = 0.019), Tumor height (p = 0.048), disease-free and overall survival (p < 0.05). In conclusion, we confirm the role of CTC as a negative prognostic marker in uveal melanoma patients after a long follow-up period. Further characterization of CTC will help understanding uveal melanoma metastasization and improve patient management.