Reduction Of Circulating Tumor Cells Research Articles

Analyzing circulating tumor cells and epithelial-mesenchymal transition status of papillary thyroid carcinoma patients following thyroidectomy: a prospective cohort study.

This study investigated the prevalence and subtype distribution of circulating tumor cells (CTCs) in patients with papillary thyroid cancer (PTC) before and after thyroidectomy to determine the potential of CTC count as a non-invasive marker of the efficacy of surgical treatment in PTC. Between January 2021 and January 2022, 62 PTC patients who underwent thyroidectomy at OOOOOOO Hospital were prospectively evaluated. Peripheral blood samples (7.5ml) were collected from each patient for CTC analysis before surgery and at 2 weeks and 3 months after surgery. CTC count and the distribution of CTC subtypes, including epithelial, epithelial-mesenchymal, and mesenchymal phenotypes, were analyzed using the negative selection method and immunofluorescence staining. The relationship between CTC count and clinicopathological characteristics was analyzed before and after surgery. Before surgery, CTCs were detected in 87% (54/62) of patients; the mean CTC count was 8.0 and the median was 5.0 in 7.5ml of peripheral blood. The mesenchymal or epithelial-mesenchymal phenotypes were predominant. After thyroidectomy, the mean and median CTC count values decreased to 5.3 and 2.5, respectively, at 2 weeks and to 4.3 and 3.0, respectively, at 3 months. This postoperative reduction in CTCs was more pronounced in patients with lymphatic invasion, lymph node metastasis, or BRAF V600E mutation. CTCs were detected in patients with PTC with a predominance of cells undergoing epithelial-mesenchymal transition. The CTC count decreased postoperatively, suggesting that liquid biopsy with CTC detection could be a valuable non-invasive tool for monitoring the efficacy of surgery in PTC.

Abstract B024: Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression

Abstract Very small nuclear (vsn) circulating tumor cells (CTCs) are detected in a subgroup of metastatic, castration resistant prostate cancer (mCRPC) patients who develop visceral metastasis following intensive androgen receptor suppression. In addition, we have previously observed that nuclear shape instability (characterized by the loss and/or mis-localization of the nuclear envelop protein emerin) is present in human prostate cancer tissues, and in CTCs from patients with metastatic disease. Herein, we investigated the biological and clinical implications of the vsn phenotype in prostate CTCs. Patients with vsnCTCs had poor overall survival and low PSA response to androgen receptor suppression compared with non-vsnCTC patients. The vsnCTCs exhibited a loss/downregulation of emerin (EMD) and we found a direct correlation between emerin loss and nuclear size reduction in CTC from mCRPC patients. Further, in vitro studies revealed that the vsn phenotype was characterized by high invasive capacity, enhanced cellular plasticity, and resistance to AR target therapy. Finally, depletion of EMD in prostate tumor cells resulted in the vsn phenotype. Together, these findings demonstrate that the vsnCTC phenotype is characterized by deregulation of EMD expression, and the presence of these cells result in poor clinical outcomes in prostate cancer. Citation Format: Karen Cavassani, Edwin Posadas, Pai-Chi Teng, Sadaf Pustchi Pustchi, Minhyung Kim, Neil Bhowmick, Sungyong You, Michael Freeman, Leigh Ellis, Dolores Di Vizio, Hsian-Rong Tseng, Leland W. Chung, Susan Yao, Catherine Grasso. Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B024.

In vivo detection demonstrates circulating tumor cell reduction instead of baseline number has prognostic value in bladder cancer patients receiving neoadjuvant chemotherapy

Previous studies have suggested the potential prognostic value of circulating tumor cells (CTCs) in bladder cancer (BC) patients. This study aims to validate the prognostic value of in vivo detection of CTCs in muscle invasive bladder cancer (MIBC) patients receiving neoadjuvant chemotherapy (NAC). A total of 107 MIBC patients were enrolled in this study. All patients had one in vivo detection of CTCs before initial treatment as baseline, and those who received neoadjuvant chemotherapy (NAC) had a second detection after NAC and before radical cystectomy. CTCs dynamic change after NAC was analyzed. Prognostic value of in vivo CTCs detection was investigated. Among 68 patients who received NAC, 45 patients (66%) had a CTC reduction after NAC. CTC reduction instead of baseline CTC positivity was a key prognostic factor for better progression free survival (PFS) among all MIBC patients receiving NAC in Kaplan-Meier analysis (P < 0.01) and in both crude (HR 6.14, 95%CI 1.63-23.21) and adjusted regression model (HR 6.76, 95% CI 1.59-28.88). The AUC was 0.85. Our study demonstrated the prognostic value of in vivo detection of CTCs. The dynamic change of CTCs count may help evaluate the efficacy of NAC.

Role of Circulating Tumor Cells in Determining Prognosis in Metastatic Breast Cancer.

Dinesh Chandra DovalBackground Circulating tumor cells (CTCs) in the peripheral blood may play a major role in the metastatic spread of breast cancer. This study was conducted to assess the role of CTCs to determine the prognosis in terms of survival in metastatic breast cancer patients. Methods This prospective study of 36 patients was conducted at the Hospital from April 2016 to May 2018. Details of each patient related to the demographic profile, tumor type, treatment, and follow-up information were recorded. The number of CTCs in the peripheral blood was measured by Celsee PREP 400 sample processing system and Celsee Analyzer imaging station. Results There was a positive correlation between the number of site of metastasis with number of CTCs ( p -value < 0.001). In the patients with clinical/partial response, a significant reduction in the number of CTCs after 1 month of therapy was observed ( p -value = 0.003). When the number of CTCs at baseline and 6 months were compared with the positron emission tomography response at 6 months, a statistically significant difference in CTCs in patients having partial response after 6 months was observed ( p -value = 0.001). On comparison with the responder groups, a statistically significant reduction in CTCs at baseline and 6 months was observed ( p -value = 0.001). Patients with CTCs less than 5 and more than or equal to 5 after 1 month of treatment had a mean progression-free survival of 11.1 months and 7.5 months ( p -value = 0.04) and a mean overall survival of 11.6 and 9.6 months ( p -value = 0.08), respectively. Conclusion Assessment of CTCs provides a more quantifiable response than radiographic evaluation and at a much earlier time point and is also a better predictor of survival.

Abstract PO-063: Extracellular vesicle miRNAs and autophagic CTCs: Predictive and prognostic biomarkers in radiotherapy treated NSCLC patients

Abstract Introduction: Most non-small cell lung carcinoma (NSCLC) patients are diagnosed at advanced stages, when the prognosis is dismal. Thoracic irradiation in combination with chemotherapy have demonstrated to increase the survival of these patients, however, progression is still developed early. The failure of the treatment may be caused by inaccurate patient stratification due to the heterogeneous and evolving nature of tumors that tissue biopsy fails to reflect. Therefore, real-time predictive and prognostic biomarkers are needed to improve the survival of NSCLC patients. In this context, miRNAs are potential markers as they regulate the expression of cancer genes and can be selectively encapsulated into extracellular vesicles (EVs) by cancer cells and found in blood circulation. Similarly, circulating tumor cells (CTCs) are a key event in the metastatic process that shredded into the circulation resemble the features of the tissue of origin. Thus, CTCs can act as tissue surrogates and potential predictive and prognostic factors in these irradiated patients. Recent evidence has associated autophagy activation to an increased resistance to radiotherapy and chemotherapy. However, controversial reports showed a dual role of autophagy during tumor cell survival, thus requiring further research to be done. In this work, we aimed to identify the predictive and prognostic role of specific EV miRNAs and autophagic CTCs in advanced NSCLC patients under concomitant radio-chemotherapy. Methods: This study prospectively enrolled 38 locally advanced NSCLC patients under concomitant radio-chemotherapy (cisplatin-vinorelbine/carboplatin-taxol). Peripheral blood samples were drawn before, during, and after the treatment. CTCs were immunomagnetically isolated and autophagic activity was characterized by immunofluorescence. EVs were isolated by ultracentrifugation methods and specific miRNAs were analyzed by RT-PCR. Internal validation of the predictive model was performed by bootstrap approach. Results: The increase of EV-miR-375 and miR-200c was associated with the reduction of autophagic CTCs along the treatment (p=0.013 &amp; p=0.025). Moreover, baseline levels of EV-miR-375, miR-200c, and miR-30c predicted the response to the treatment with an area under the curve (AUC) of 86%. The presence of autophagic CTCs and low EV-miR-30c were independent prognostic biomarkers for shorter relapse-free survival (p=0.002 &amp; p=0.002) and overall survival (p=0.012 &amp; p&amp;lt;0.001). The analysis of miRNA targets showed that miR-375, miR-200c, and mir30c regulate genes involved in autophagy and cell division such as PIK3CA, potentially modulating radio-chemotherapy resistance. Conclusion: This study reports for the first time that EV-miRNAs and autophagic CTCs are predictive and prognostic biomarkers in advanced NSCLC patients under concomitant radio- chemotherapy. Their identification could serve as tumor surrogates for a real-time treatment stratification of NSCLC patients and might also benefit new combinatory strategies based on autophagy inhibition or immunotherapy. Citation Format: Diego de Miguel Perez, Rosario Guerrero Tejada, Alessandro Russo, Francisco Gabriel Ortega, Antonio Martínez-Única, Muthukumar Gunasekaran, Jose Antonio Lorente, Jose Exposito, Maria Jose Serrano Fernandez, Christian Rolfo. Extracellular vesicle miRNAs and autophagic CTCs: Predictive and prognostic biomarkers in radiotherapy treated NSCLC patients [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-063.

Cut-Off Analysis of CTC Change under Systemic Therapy for Defining Early Therapy Response in Metastatic Breast Cancer.

Detection of circulating tumor cells (CTC) can distinguish between aggressive and indolent metastatic disease in breast cancer patients and is thus considered an independent, negative prognostic factor. A clear decline in CTCs is observed in patients who respond to systemic therapy. Nevertheless, CTCs can decrease in patients experiencing disease progression during systemic therapy, too. This study aims to determine the differences between CTC decline in patients responding to therapy and those in whom disease is progressing. Therefore, CTC values were compared at the start and after one cycle of a new line of systemic therapy. In all, 108 initially CTC-positive patients (with ≥5 intact CTCs in 7.5 mL blood) were enrolled in this study and intact and apoptotic CTCs were measured via the CellSearch® system. A cut-off analysis was performed using Youden’s J statistics to differentiate between CTC change in the two groups. Here, 64 (59.3%) patients showed stable disease or partial response vs. 44 (40.7%) presenting disease progression. Median overall survival was 23 (range: 4–92) vs. 7 (2–43) months (p < 0.001). Median intact CTC count at enrollment was 15.0 (5–2760) vs. 30.5 (5–200000) cells (p = 0.39) and 2.5 (0–420) vs. 8.5 (0–15000) cells after one cycle of systemic therapy (p = 0.001). Median apoptotic CTC count at enrollment was 10.5 (0–1500) vs. 9 (0–800) cells (p = 0.475) and 1 (0–200) vs. 3 (0–250) cells after one cycle of systemic therapy (p = 0.01). A 50% reduction in baseline apoptotic CTC count represents the optimal cut-off to differentiate between therapy response and disease progression. An apoptotic CTC reduction of ≤10% is 74% specific for early disease progression.

Pivotal Role of AKT2 during Dynamic Phenotypic Change of Breast Cancer Stem Cells.

Therapeutic resistance seen in aggressive forms of breast cancer remains challenging for current treatments. More than half of the patients suffer from a disease relapse, most of them with distant metastases. Cancer maintenance, resistance to therapy, and metastatic disease seem to be sustained by the presence of cancer stem cells (CSC) within a tumor. The difficulty in targeting this subpopulation derives from their dynamic interconversion process, where CSC can differentiate to non-CSC, which in turn de-differentiate into cells with CSC properties. Using fluorescent CSC models driven by the expression of ALDH1A 1(aldehyde dehydrogenase 1A1), we confirmed this dynamic phenotypic change in MDA-MB-231 breast cancer cells and to identify Serine/Threonine Kinase 2 (AKT2) as an important player in the process. To confirm the central role of AKT2, we silenced AKT2 expression via small interfering RNA and using a chemical inhibitor (CCT128930), in both CSC and non-CSC from different cancer cell lines. Our results revealed that AKT2 inhibition effectively prevents non-CSC reversion through mesenchymal to epithelial transition, reducing invasion and colony formation ability of both, non-CSC and CSC. Further, AKT2 inhibition reduced CSC survival in low attachment conditions. Interestingly, in orthotopic tumor mouse models, high expression levels of AKT2 were detected in circulating tumor cells (CTC). These findings suggest AKT2 as a promising target for future anti-cancer therapies at three important levels: (i) Epithelial-to-mesenchymal transition (EMT) reversion and maintenance of CSC subpopulation in primary tumors, (ii) reduction of CTC and the likelihood of metastatic spread, and (iii) prevention of tumor recurrence through inhibition of CSC tumorigenic and metastatic potential.

CTC enumeration and characterization as a pharmacodynamic marker in the phase I clinical study of APX3330, an APE1/Ref-1 inhibitor, in patients with advanced solid tumors.

e14531 Background: APE1/Ref-1 is a dual-function protein with a pleiotropic role in regulating transcription factors involved in cancer cell signaling via redox control, as well as responding to oxidative and base DNA damage. APX3330 is a highly selective inhibitor of the Ref-1 mediated redox function in tumors, while enhancing the neuronal protective function of APE1. APX3330 is undergoing clinical development as an anti-tumor agent that also protects and reverses oxidative damage to neurons. We now report on the CTC analysis conducted as part of the study (NCT0337508). Results of the clinical study are reported in a separate abstract. Methods: A total of 19 patients with 10 various solid tumors, including rectal, pancreatic, colon, endometrial, gall bladder, hepatocellular, prostate, melanoma, bladder, and ovarian cancers received APX3330 in escalating divided daily doses of 240, 360, 480, 600, and 720mg. Blood samples were collected from all patients prior to receiving APX3330 and after achieving steady-state serum concentrations of the drug. Samples were sent to Epic Sciences to analyze circulating tumor cells (CTCs) in peripheral blood via their CTC Platform. Results: Cumulatively, 37 samples from 19 patients were received. 35/37 (95%) samples passed technical quality control and were feasible for downstream analysis. 9/17 (53%) baseline samples (BL) had CTCs detected, while 9/18 (50%) on-treatment (OTx) draws had CTCs detected. 16 patients had BL and OTx samples that were further evaluated with the longitudinal analysis. Of these, 7/16 (44%), 6/16 (38%), and 3/16 (19%) patients showed a reduction trend, an increase trend, and no change in all CTC populations (delta greater than or equal to 0 CTC/ml), respectively. Patient follow-up is ongoing and the correlation of CTC biomarkers with clinical outcomes is pending. Conclusions: APX3330 is undergoing clinical evaluation as an anti-tumor agent that protects against and reverses CIPN. In this phase I study, 44% of evaluable blood samples showed a reduction in CTCs after initiation of treatment with APX3330. Additional studies are now being planned. Clinical trial information: NCT0337508.

Preliminary Study of Clinical Role of Circulating Tumor Cells in Patients with Small-Cell Lung Cancer Treated By Prophylactic Cranial Irradiation

Prophylactic cranial irradiation (PCI) decreases the rate of brain metastases, and improves the overall survival (OS) of small-cell lung cancer (SCLC). However, the improvement of OS is uncertain under the era of MRI. The circulating tumor cells (CTCs) play a vital role in recurrence, prognosis, efficacy evaluation, and individualized therapy of cancer patients. Our aims to investigate the preliminary clinical value of CTCs in SCLC patients treated by PCI. In this prospective study comparing two different thoracic radiotherapy regimens, SCLC patients received chemo-radiotherapy and subsequent PCI after brain metastases were excluded by MRI screening. The blood samples for CTCs analysis were obtained from 20 SCLC patients before and after PCI. Instead of the CellSearch system, CTCs were assessed with a new detection method based on telomerase-specific, replication-selective oncolytic herpes simplex virus-1 that targets telomerase reverse transcriptase-positive cancer cells and expresses a green fluorescent protein that identifies CTCs (oHSV1-hTERT-GFP), with high sensitivity and specificity in lung cancer patients. The endpoints were progression-free survival (PFS) and OS. Eleven patients were limited-stage SCLC, and 9 were extensive-stage SCLC. The CTCs detection rate was 100%. The follow-up time was 39.2 months for the whole group. Seven patients failed with distant metastases. The median CTCs count were 12 (range, 1-43) before PCI, and 4 (range, 0-34) after PCI. Logistic regression showed the CTCs count was not associated with the clinical parameters. Patients with ≥12 CTCs before PCI tended to show worse PFS and OS than those with <12 CTCs (31.8 months vs not reached, p = 0.084; not reached vs not reached, p = 0.457). Six in 12 patients with ≥12 CTCs failed, but only 1 relapsed in 8 patients with <12 CTCs before PCI. PFS and OS were significantly different in patients with ≥4 CTCs after PCI and those with <4 CTCs (28.1 months vs not reached, p = 0.001; not reached vs not reached, p = 0.029). All 10 patients were alive without disease in the group with <4 CTCs, however, 7 relapsed in 10 patients with ≥4 CTCs after PCI. Significantly better PFS and OS were found in 10 patients with the reduction rate of more than 58% than other 10 patients with less than 58% (not reached vs 26.4 months, p = 0.006; not reached vs not reached, p = 0.029). CTCs counts after PCI and CTCs reduction rate are valuable prognostic markers of better survival of SCLC patients. Patients with the high CTCs reduction rate could benefit from PCI. However, the prognostic significance of CTCs needs to be evaluated in large-scale clinical trials.

Phase II trial of SM 88 in non-metastatic biochemical recurrent prostate cancer.

175 Background: Despite toxicity and no clear survival benefit, non-metastatic recurrent prostate cancer (nmPC) is typically treated with androgen deprivation therapy (ADT). SM88 is a relatively non-toxic novel combination therapy (amino acid analogue, CYP3a4 inducer, mTOR inhibitor and catalyst) based on the Warburg effect, with activity in a variety of cancers including prostate (JCO 2017 35, e16567). Phase I and II results demonstrated stable or rising testosterone levels while achieving a reduction of CTCs (circulating tumor cells) and no radiographic progression events (Annal Oncol 2017, 28(5):274-5). We now report results for toxicities typically seen with ADT. Methods: Prospective ongoing Phase Ib/II of SM88 (230mg po bid) in recurrent nmPC with rising PSA (PCWG3 definition), detectable CTCs, and no radiographically identified metastases at baseline. Results: Since Sept 2016, there have been 17 consented (of 34 planned) with 10 evaluable (completed &gt; 1 cycle) (median 7, range 1-13). Mean age was 71.2 (53-84); all had prior ADT after curative intent RT (50%) or surgery (50%); with no patient currently on ADT. Mean testosterone level was 307.6 ng/dL before SM88 and 349.2 after (p = 0.07, one-tail T test), rising in (5/10) or remained stable except for one patient who entered the trial castrate ( &lt; 2.5). Overall 70% had some grade 1-2 adverse event (AE) but no drug related serious AE. EORTC-QLQ-C30 relating to mental function domain (Q20-28) and QLQ-PR25 intimacy domains (Q44-50) remained stable, including 9 (90%) reporting no or resolved hot flashes; 7 (70%) reporting an improved or stable “interest in sex”; and 6 (60%) having “excellent” “overall health” and “QOL”. Weight and osteoporosis (measured by Urinary NTX) did not worsen; mean EKG QTc (422 ms); mean arterial pressure (95.1 mmHg); glucose (115.2 mg/dl) and hematocrit (41.5%) were not affected. Conclusions: Toxicities typically associated with ADT were not seen with SM88, a novel non-hormonal anti-neoplastic. Despite the limited follow-up, these data suggest that ADT may be avoided or delayed without progression in selected patients with nmPC. A phase III RCT of SM88 vs patient choice of hormonal or active surveillance is planned for confirmation of these results. Clinical trial information: NCT02796898.

SM88 in non-metastatic rising PSA-recurrent prostate cancer.

e16567 Background: SM88 is a novel combination of substituted amino acid ( AA) and repurposed agents with non-toxic activity in a variety of cancers including prostate (PC). Non-metastatic biochemically recurrent PC (nmPC) is ideal for such a well-tolerated, non-androgen based treatment. We present biomarker activity including CTCs (circulating tumor cells) after completion of the Ib dose escalating pharmacokinetic portion of the SM88 development plan. Methods: Planned analysis of an ongoing prospective Phase Ib/II open label study of nmPC (PCWG3 definition). Subject 1 received the 1stdose level of AA, and others received 2x dose. All subjects also received a combination of low dose re-purposed CYP3a4 inducer, oxidative stress catalyst, and mTOR inhibitor. Results: Four subjects completed at least 1 cycle (28d) with all PK sampling as prespecified. Median age was 73(70-80); all had prior ADT that was discontinued 3-6 yrs earlier, and either had curative intent prostatectomy (n = 2) or RT (n = 2); Testosterone level was castrate in one after prior RT. CTCs fell (p &lt; 0.01) to undetectable (n = 1) or by &gt; 25% (n = 3) (see table) while PSA remained stable (PCWG3 criteria). Preliminary LDH, neutrophil/lymphocyte ratio (N:L), urinary NTx, bone specific AlkPhos (bAP) trends are in the table below. There were no drug related serious adverse events (2 grade: 1- pigmentation, 1- vasomotor). EORTC-QLQ30 and PR25 scores were either improved or stable. Conclusions: Treatment with SM88 in patients with nmPC in the completed phase Ib cohort was associated with CTC reduction, and stable biomarker trends including PSA and LDH. There was no significant toxicity or adverse patient reported outcomes. Based on these results, the dose escalation was stopped at the second level and a cohort expansion phase II initiated. A phase III RCT is planned for confirmation of these results. Clinical trial information: NCT02796898. [Table: see text]

Abstract 589: Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model

Abstract Osteosarcoma (OS) is a malignancy of bone and five-year survival rates are still at a low 20% if metastatic disease is present. Metastatic OSs most frequently metastasize to the lung. TNF-α is a powerful cytokine already shown to be active against many cancers. Nevertheless, TNF-α is highly toxic at clinically relevant concentrations. To overcome this limitation, targeted forms of TNF-α can be used. In this study, targeting is achieved through linking TNF-α to the antibody fragment F8, targeting the EDA-domain of fibronectin. To test the efficacy of F8-TNF-α against OS, we sought to investigate the effects of F8-TNF-α in an orthotopic syngeneic OS model. In addition to evaluating compound efficacy, we also tested whether the route of administration (systemic versus local application) can even further increase treatment efficacy. Ultimately, we aimed at measuring treatment success by assessing different stages of OS progression, especially metastatic disease. To show clinical relevance, we performed immunofluorescence (IF) of EDA on primary human OS samples. In a second step, we evaluated drug efficacy in a syngeneic OS mouse model. K7M2L2 OS cells, stably infected to express the marker proteins lacZ and mCherry, were orthotopically injected into the tibia of mice. Once primary tumors were detectable, the mice were treated with either PBS or F8-TNF-α. The compound was given either systemically (intravenously (i.v.), tail vein) or locally (intraarterial (i.a.), femoral artery). Four days after the last treatment, all mice were sacrificed, terminal blood sampling was performed and blood samples were analyzed for the presence of circulating tumor cells (CTCs) via FACS/mCherry fluorescence. Following X-gal staining, metastases on the surface of lungs were counted. EDA and infiltration of immune cells (CD4+, CD8+, natural killer (NK) cells, CD19+) were assessed histologically. Using IF, we detected strong expression of EDA in primary tumors as well as bone and lung metastases from human OS patients. Preliminary results from our treatment study showed a significant decrease in the number of lung metastases as well as the number of CTCs in mice treated with i.a. F8-TNF-α while the treatment was well tolerated by the mice. The reduction of lung metastases could, at least in part, be explained by a strong expression of EDA in the lung metastases. In addition, significant increases in lung-infiltrating immune cells were observed after administration of F8-TNF-α (i.v. F8- TNF-α: increased CD4+ T-cells; i.a. F8-TNF-α: increased NK cells). Although significant reductions in numbers of metastases and CTCs were detected, F8-TNF-α did not significantly impair the growth of the primary tumors. In conclusion, our study demonstrates a reduction of CTCs as well as lung metastases after administration of F8-TNF-α and thus, provides a rationale for further studying the anti-metastatic effects of this compound in a clinically relevant model of OS. Citation Format: Bernhard Robl, Sander Martijn Botter, Aleksandar Boro, Dario Neri, Bruno Fuchs. Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model. [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 589.

Neoadjuvant Therapy of DOF Regimen Plus Bevacizumab Can Increase Surgical Resection Ratein Locally Advanced Gastric Cancer: A Randomized, Controlled Study.

Locally advanced gastric cancer (LAGC) is best treated with surgical resection. Bevacizumab in combination with chemotherapy has shown promising results in treating advanced gastric cancer. This study aimed to investigate the efficacy of neoadjuvant chemotherapy using the docetaxel/oxaliplatin/5-FU (DOF) regimen and bevacizumab in LAGC patients.Eighty LAGC patients were randomized to receive DOF alone (n = 40) or DOF plus bevacizumab (n = 40) as neoadjuvant therapy before surgery. The lesions were evaluated at baseline and during treatment. Circulating tumor cells (CTCs) were counted using the FISH test. Patients were followed up for 3 years to analyze the disease-free survival (DFS) and overall survival (OS).The total response rate was significantly higher in the DOF plus bevacizumab group than the DOF group (65% vs 42.5%, P = 0.0436). The addition of bevacizumab significantly increased the surgical resection rate and the R0 resection rate (P < 0.05). The DOF plus bevacizumab group showed significantly greater reduction in CTC counts after neoadjuvant therapy in comparison with the DOF group (P = 0.0335). Although the DOF plus bevacizumab group had significantly improved DFS than the DOF group (15.2 months vs 12.3 months, P = 0.013), the 2 groups did not differ significantly in OS (17.6 ± 1.8 months vs 16.4 ± 1.9 months, P = 0.776. Cox proportional model analysis showed that number of metastatic lymph nodes, CTC reduction, R0 resection, and neoadjuvant therapy are independent prognostic factors for patients with LAGC.Neoadjuvant of DOF regimen plus bevacizumab can improve the R0 resection rate and DFS in LAGC. These beneficial effects might be associated with the reduction in CTC counts.

Clinico-pathological correlation of serial measurement of circulating tumor cells in 24 metastatic colorectal cancer patients receiving chemotherapy reveals interpatient heterogeneity correlated with CEA levels but independent of KRAS and BRAF mutation

Background: The Veridex CellSearch is an FDA-approved technology for enumerating circulating tumor cells in blood samples of metastatic colorectal cancer mCRC) patients and has prognostic value. It is important to understand how counts of circulating tumor cells (CTCs), which are advocated to be tools for “liquid biopsy” of tumors, correlate with clinical and pathologic variables of significance in these patients. In this study, we have attempted to make such correlations along with evaluating how CTC counts change during the course of chemotherapy. Patients and methods: Following an IRB-approved protocol, blood samples were collected from 24 patients with mCRC along with relevant clinico-pathological data. Blood was collected at defined time-points both prior to as well as during the course of treatment with combination chemotherapy, and CTC counts were enumerated from7.5 ml of blood. Results: Seventeen out of 24 patients with mCRC showed a CTC count of 2 or less cells in 7.5 ml of blood at base-line assessment before chemotherapy while 7 patients showed 3 or more cells in 7.5 ml of blood at that point. A correlation was found between high carcino-embryonic antigen (CEA) levels and high CTC counts (P = 0.018) although it was also found that some patients had elevated CTCs without an elevated CEA. No correlation with the time interval between detection of primary tumor and appearance of secondary (metastatic) tumor(s) was found. CTC counts did not correlate with the presence of lung or liver metastases, i.e. a number of mCRC patients with lung or liver metastases had a count of zero CTCs at baseline. We also noted no correlation between CTC number and the status of KRAS or BRAF mutation. CTC counts dropped immediately after the start of chemotherapy in 11 out of 21 patients, and also reduced from the baseline at the end of chemotherapy in 5 out of 10 patients. Six of 7 patients who started with 3 or more CTCs in 7.5 ml at baseline also showed a final CTC reduction at the end of the therapy assessment. Conclusions: Analysis of circulating tumor cells may be of use in monitoring response to therapy in mCRC, either in combination with CEA monitoring or alone when CTCs are elevated but CEA level is not.

Quantification and molecular profiling of circulating tumor cells (CTCs) in urothelial cancer (UC) before and during systemic treatment: Implications across the clinical stages.

319 Background: CTCs from patients (pts) at different clinical stages were analyzed by a never explored experimental approach based on a combination of two techniques. Provision of this information may contribute to optimize tailored therapies. Methods: 3 cohorts were analyzed: pts with muscle-invasive bladder cancer receiving neoadjuvant (NA) sorafenib + chemotherapy (CT) (NCT01222676), and metastatic (M1) pts receiving 1st-line MVAC, and 2nd-line (M2) anti-TGFβ receptor ALK1 PF-03446962 (NCT01620970). 5 ml of whole blood were filtered by ScreenCell Cyto devices and CTC status was assessed with centralized scoring by referral pathologists. Additional 5 ml of whole blood were processed by immunomagnetic beads (AdnaTestSelect kit) and the expression level of a panel of markers (including EPCAM and MUC1) was studied using RT-multiplex PCR. The objective was the association with clinical response/relapse. Results: From 07/2012 to 08/2013, 48 pts (17 NA, 17 M1, and 14 M2) were enrolled. Rates of baseline CTC+ were: 91, 77, 75% (ScreenCell), and 36, 41, 69% with AdnaTest, respectively. NA setting: from baseline to post-CT, all pts had a stepwise reduction of CTC count/5 ml blood by ScreenCell (median baseline of 15 [0-40] to 0 [0-3]). Increase in circulating EPCAM±MUC1 levels by CTC was seen in accordance with the 3 disease progressions (PD). M1 setting: Pts who relapsed had a median of 25 CTC/5 ml (IQR: 12-47) at the end of CT, while all the others had levels &lt;10. EPCAM profile was concordant in all cases (median 1.10 vs 0.42 ng/µl). Despite classified as partial/complete responders by RECIST criteria, 5/7 cases with &gt;6 month follow-up displayed an increase in EPCAM levels and CTC number which anticipated a relapse. M2 setting: increase in CTC signals from baseline to +2 months was observed in 8/9 evaluable cases by AdnaTest and 4/5 evaluable cases by ScreenCell, in accordance with PD. Conclusions: This combined technique showed a promising ability to anticipate the detection of clinical relapse, although it still needs a larger sample size. Refining molecular characterization will help designing informed clinical trials.

Circulating tumor cells (CTC) as a biomarker of response in patients with extensive-stage small cell lung cancer (EX-SCLC).

e22048 Background: SCLC, a variant of lung cancer marked by early metastases, accounts for 13% of all lung cancers diagnosed in US. Despite high response rates to treatment, it is an aggressive disease with a median survival of 9-11 months for patients with EX-SCLC. Detection of CTCs is a novel laboratory technique currently in use to determine response to therapy and to predict prognosis in breast, colorectal and prostate cancer. We initiated a study to study the role of CTC as a biomarker of response and relapse in patients with EX-SCLC. Methods: We collected blood sample from chemotherapy naïve patients with EX-SCLC prior to initiation of therapy, following completion of systemic therapy, follow up every 6-8 weeks and at relapse. The CTC was determined using the Cell Search system in a central laboratory. The study was conducted in 4 different sites and it was reviewed and approved by respective Research Review Committee and IRBs. Results: We enrolled 27 patients with EX-SCLC, 1 was excluded due ineligibility, all patients were treated with platinum and etoposide. We observed partial response in 16 patients (61%), stable disease in 3 patients, 1 with progression of disease and not assessed in 7 patients (5 deceased, 2 not available); the baseline CTC of 2 patient were not measured (one due instrument failure and one due insufficient blood). The overall median number of CTCs in 24 patients measured at baseline and post-tx was 75 (range 0 to 3430) and 2 (range 0 to 526), respectively. A significant reduction in CTCs from baseline to post-tx was identified; for the 14 subjects with pre and post treatment CTC, the median reduction was 51.5% (51.5% decrease, range -2904% to +5%, p&lt; 0.001). Conclusions: We were able to demonstrate feasibility of using CTC as a biomarker of response in patients with EX-SCLC in clinical setting. CTC could be a useful biomarker in the management of SCLC to predict response to therapy.

Phase II trial of intravenous carboplatin (C), oral everolimus (E), and prednisone (P) in docetaxel-pretreated (DP) metastatic castrate-resistant prostate cancer (mCRPC): A Prostate Cancer Clinical Trials Consortium study.

5041 Background: A phase II clinical trial was conducted of the combination of C and E due to the synergy noted. Methods: The primary endpoint was time to progression (TTP). Intravenous C at a target AUC of 5 on day 1, and oral E 5mg once daily and P 5mg twice daily were administered in 21 day cycles. PSA was assessed every 21 days and radiologic response was assessed every 3 cycles. Secondary endpoints included overall survival (OS), the correlation of TTP with phosphorylated (p) mTOR, pAKT, p70S6, and circulating tumor cells (CTC). A 1-stage study design assumed: a reference median TTP = 1.5 months; 1-sided alpha = 0.15; and power = 0.90, requiring 26 patients (pts). Results: 26 pts enrolled; median age 69 years (range 54-86) ;8 African American and 18 Caucasians. Median pretherapy PSA was 190 ng/ml (range 13 - 2174). 18 pts (69%) each had bone pain and Gleason score &gt; 8. 125 cycles have been administered; median 3 cycles (range 1 - 16). Predominant grade 3 or 4 toxicities were thrombocytopenia in 8 pts, pulmonary embolism in 2 and neutropenia in 3. No treatment related deaths occurred. 4 (15%) had a &gt; 30% PSA decline and 1 had a &gt;90% PSA decline. 8/19 pts had stable disease but no objective responses in MD. The median TTP and OS were 2.5 months (90% CI: 1.8 - 4.3), and 12.5 months (90% CI: 6.7 - 16.1), respectively. Median area under curves were 5.9 (range, 4.3 – 11.0) and 4.5 (range, 4.1 – 7.1) mg/mL*min with C given alone and in combination with E, respectively. E did not influence pharmacokinetics of C. Median baseline CTC (n=18) was 30 (range 0-2372). 5/18 pts had favorable CTC (CTC&lt;5/7.5 mL) pretherapy. Patients with TTP &gt;18 weeks had reduction in post-therapy CTC with a median decrease of 63% (range 11%-100%). Lack of IHC staining for pAKT was noted in 2/2 pts on therapy for &gt; 30 weeks vs increased expression was noted in 8/8 pts on therapy for &lt; 9 weeks. Testing for TSC1 mutation is planned and will be reported. Conclusions: The combination was tolerable but revealed modest clinical efficacy. Biomarker evaluations such as pAKT may help identify a subset likely to benefit from mTOR inhibitor strategy in mCRPC. Clinical trial information: NCT01051570.

Circulating tumor cells (CTCs) in patients (pts) with small cell lung cancer (SCLC) as a marker of disease burden and therapeutic response, and predictor of disease relapse.

7092 Background: Currently there are no validated biomarkers for assessment or prediction of disease burden or activity in SCLC. Enumeration of CTCs by CellSearch is FDA approved, highly reproducible and validated in other malignancies. Application as a prognostic and predictive marker in SCLC is limited due to a lack of studies documenting serial monitoring in pts on therapy. Methods: We are conducting a prospective study serially enumerating CTCs in pts with newly diagnosed SCLC. CTC number (per 7.5 ml peripheral blood) and percentage of CTCs demonstrating DNA damage and apoptosis based on γH2AX and M30 staining respectively, are being assessed prior to initiation of chemotherapy, during each cycle and at relapse. We are correlating CTC number with disease stage, number of metastatic sites, response to therapy, and time to progression (TTP). Results: 21 SCLC pts are evaluable. 9 pts with limited disease (LD) had median baseline CTC value of 1 (0-8); only 2 of 9 pts had &gt;5 CTCs. 12 pts with extensive disease (ED) had median baseline CTC value of 80.5 (0-37780); 8 of 12 pts had &gt;5 detectable CTCs (p value 0.02). Amongst the 12 pts with ED, median baseline CTC count was higher in pts with ≥3 (n=3) compared to 1-2 sites of metastatic disease (n=9) (2668 vs 71; p value 0.52). Median percentage of CTCs positive for γH2AX and M30 was also higher for pts with ≥3 compared to 1-2 metastatic sites (83, 164.5 vs. 2, 7.5) (p-value 0.40, 0.69). Serial CTC data are available on 3 pts with ED; all had responsive disease and reduction in CTC number to 0-1 after 2 cycles of chemotherapy. As this protocol is ongoing, correlation of CTCs with updated response status and TTP will be presented at the ASCO meeting. Conclusions: CTCs can be isolated and serially enumerated in pts with SCLC. Baseline CTCs correlate directly with disease stage. In pts with ≥ 3 metastatic sites, baseline CTCs tend to be greater and show higher levels of DNA damage and apoptosis compared to those with 1-2 metastatic sites. Reduction in CTCs is associated with radiographic response to therapy. Correlation between absolute number at baseline and TTP is not yet known.

Abstract 3411: A pilot feasibility study to evaluate the efficacy of lapatinib in the elimination of HER2-positive circulating tumor cells in peripheral blood of women with metastatic breast cancer

Abstract Background: The monitoring of circulating tumor cells (CTCs) detected in peripheral blood (PB) of patients (pts) with metastatic breast cancer (BC) has been proven a potent prognostic tool. CTCs detected after the completion of therapy are theoretically chemo- and/or hormone-resistant and could lead to disease progression. EGFR and HER2 expression has been demonstrated in CTCs from pts with BC. In the present trial we aimed to evaluate the efficacy of lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, in eliminating HER2 positive (+) CTCs of women with metastatic BC. Pts and methods: Pts with metastatic BC and HER2+ CTCs detected despite disease stabilization or response after the completion of prior therapy were eligible. Pts received lapatinib, 1500 mg/day orally till disease or CTC progression, whichever occurred first. HER2 expression was evaluated in cytospins of peripheral blood mononuclear cells’ (PBMCs) prepared from PB obtained at baseline and monthly thereafter. Cytospins were stained with HER2 along with cytokeratin antibody; a total of 10^6 PBMCs/pt were analyzed by double immunofluorescence. Results: Nineteen pts were enrolled from September 2008 to October 2011. Median age was 62 years, 12 (63.2%) pts were post-menopausal and 2 had HER2+ tumors. Twelve (63.2%) pts had visceral disease and 7 (36.8%) had received β3 lines of treatment. One pt discontinued treatment prior to the completion of the first cycle due to toxicity and 2 pts are not evaluable yet. The median duration of treatment was 4.0 (range, 0.5 - 34.0) months (mo). CTC counts declined in 15 out of 16 pts during treatment with a maximum percentage of decrease of 100% in all of them. The median time to and the median duration of nadir CTC counts were 1 mo (range, 1 - 3 mo) and 1 mo (range, 0 - 7 mo), respectively. Prior to the second treatment cycle, a decrease in CTC counts was observed in 14 (88%) pts, in 1 (7%) the CTC count increased by 33% and in 1 (7%) remained stable. Median percentage of CTC count decrease per pt was 100% (range, 45-100%). At baseline, a total of 2642 HER2+ CTCs were detected (median: 79/pt, range 2 - 617), whereas after the completion of first cycle a total of 430 HER2+ CTCs were identified (median: 0, range 0 - 223). Disease evaluation revealed stable disease in 5 pts and progressive disease in 11. In pts with stable disease a sustained decrease in CTC counts over 2 to 7 mo was observed. On the contrary, in 9 out of 11 pts with disease progression, the decrease in CTC counts was short-lasting (median 1.0 mo, range 0 - 4). Conclusions: Lapatinib is effective in decreasing HER2+ CTCs in pts with metastatic BC, irrespective of the HER2 status of the primary tumor. Sustained CTC reduction was associated with disease stabilization. The above results suggest that tailoring therapy according to targets present on CTCs could be an effective therapeutic strategy in BC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3411. doi:1538-7445.AM2012-3411

Quantification and Phenotypic Characterization of Circulating Tumor Cells for Monitoring Response to a Preventive HER2/neu Vaccine-Based Immunotherapy for Breast Cancer: A Pilot Study

Ongoing cancer vaccine trials are limited by the inability of immunologic assays to monitor clinically relevant surrogates of response. Recent advances in the ability to quantify and phenotype circulating tumor cells (CTCs) in breast cancer patients may lead to a role for CTCs in monitoring response to vaccine-based immunotherapy. The CellSearch System (Veridex-LLC, Warren, NJ) was used to enumerate total and HER2/neu+ CTCs in 20 mL of blood from all 16 node-positive (NP) breast cancer patients active in our NP HER2/neu E75 peptide vaccine trial at the initiation of this pilot study. These patients were vaccinated with E75 (1000 microg)/GM-CSF (250 microg) monthly x 6 after completion of multimodality therapy. Mean (+/-SEM) number of CTCs and HER2/neu+ CTCs were compared in unmatched (n = 16) and matched (n = 9) prevaccination and postvaccination cases. CTCs were detected in 14 of 16 (88%) patients (mean: 3.4 +/- 0.2 CTC/20 mL). After vaccination, a reduction in CTC/20 mL (prevaccination 3.9 +/- 1.5 vs postvaccination 0.7 +/- 0.4, P = .077) and HER2/neu+ CTC/20 mL (prevaccination 2.8 +/- 1.0 vs postvaccination 0.5 +/- 0.2, P = .048) was demonstrated. A significant delayed-type hypersensitivity (DTH) response suggesting that vaccination was effective in eliciting a peptide-specific immune response was confirmed (22.3 +/- 4.1 vs 3.0 +/- 2.2 [controls] mm, P < .01). All nine patients followed throughout the vaccination series also showed significant reduction in CTCs (4.8 +/- 1.5 vs 0.3 +/- 0.2, P < .01) and HER2/neu+ CTCs (3.0 +/- 0.9 vs 0.4 +/- 0.2, P = .013). CTCs are readily demonstrated in posttreatment, clinically disease-free NP breast cancer patients. E75+GM-CSF vaccination appears to reduce the number of CTCs. These data suggest a potential role for this clinically validated CTC assay in assessing response to preventive vaccine-based immunotherapy, and further validation studies are underway.