Circulating Tumor Cell Burden Research Articles

An indocyanine green-based liquid biopsy test for circulating tumor cells for pediatric liver cancer.

Hepatoblastoma and HCC are the most common malignant hepatocellular tumors seen in children. The aim of this study was to develop a liquid biopsy test for circulating tumor cells (CTCs) for these tumors that would be less invasive and provide real-time information about tumor response to therapy. For this test, we utilized indocyanine green (ICG), a far-red fluorescent dye used clinically to identify malignant liver cells during surgery. We assessed ICG accumulation in cell lines using fluorescence microscopy and flow cytometry. For our CTC test, we developed a panel of liver tumor-specific markers, including ICG, Glypican-3, and DAPI, and tested it with cell lines and noncancer control blood samples. We then used this panel to analyze whole-blood samples for CTC burden with a cohort of 15 patients with hepatoblastoma and HCC and correlated with patient characteristics and outcomes. We showed that ICG accumulation is specific to liver cancer cells, compared to nonmalignant liver cells, non-liver solid tumor cells, and other nonmalignant cells, and can be used to identify liver tumor cells in a mixed population of cells. Experiments with the ICG/Glypican-3/DAPI panel showed that it specifically tagged malignant liver cells. Using patient samples, we found that CTC burden from sequential blood samples from the same patients mirrored the patients' responses to therapy. Our novel ICG-based liquid biopsy test for CTCs can be used to specifically detect and quantify CTCs in the blood of pediatric patients with liver cancer.

Abstract 3696: Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer

Abstract Background: Prostate-Specific Membrane Antigen (PSMA) is a highly expressed prostate cancer (PCa) cell surface protein regulated by androgen receptor signaling and the target of the recently approved radioligand therapy 177Lu-PSMA-617. While some patients have dramatic responses to this therapy, rates of upfront or acquired resistance are high even in patients with PSMA-PET-avid disease, and better biomarkers of 177Lu PSMA-617 response and resistance are needed to select patients more effectively for this therapy. To address this need, we evaluated the relationship between longitudinal circulating tumor cell (CTC) androgen signaling and PSMA expression to 177Lu-PSMA-617 response or resistance in patients with metastatic prostate cancer. We leveraged a CTC androgen signaling signature that we have previously shown is prognostic for patients with metastatic prostate cancer treated with AR signaling inhibitors. Methods: Longitudinal blood samples were collected from 20 patients for circulating tumor cell (CTC) analysis at C1D1, C2D1, C4D1, and C6D1. CTCs were isolated with antibodies to EpCAM followed by parallel enumeration, analysis of cell surface expression of PSMA and RNA extraction for AR or neuroendocrine marker transcriptional profiling. Results: CTC samples were assessed for PSMA cell surface protein expression prior to treatment and on treatment. FOLH1 (PSMA) gene expression was detected from CTCs at baseline that persisted through treatment. Gene expression representative of AR pathway activation was present in 70% of the cohort at baseline and 50% of patients at C2D1. Concordant with these observations, no significant decrease in CTC number or PSMA protein expression was seen from baseline to C2D1. However, increased CTC AR pathway activation at C2D1 was associated with increased mean serum PSA (155 vs 21.93 ng/ml) and was associated with decreased duration of response. Conclusions: Improved tools to predict who will respond to 177Lu-PSMA-617 and detect early treatment resistance and disease progression are needed. Here, we show that persistent CTC burden, CTC AR signaling, and CTC PSMA expression are common early in treatment with 177Lu-PSMA-617 and may be associated with lower likelihood of clinical response. Further ongoing studies are needed to elucidate the relationship between these liquid biopsy results and clinical outcomes. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Luke Nunamaker, Viridiana Carreno, Alex H. Chang, Shannon R. Reese, Charlotte Linebarger, Amy K. Taylor, Grace C. Bliitzer, John Floberg, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3696.

Effect of circulating tumor cells distribution in treatment naive and treated patients with advance stage breast cancer on disease burden.

e12517 Background: Breast malignancies is a leading cause of cancer-related mortalities and show ascending incidence rate. Despite advancements in our understanding of the disease, its clinical outcome is often dismal. This largely remains owing to the characteristic wide window of relapse, spanning months to decades after primary treatment. Therefore, continuous monitoring of the disease is an offered choice to detect metastatic progression and recurrence. Circulating tumor cells (CTCs) have emerged as a powerful prognostic tool to predict the disease outcome in many epithelial cancers. CTCs are a real-time surrogate biomarker accounting for minimal residual disease (MRD) which is often missed in CT PET scanning. This leads to a progression of metastasis when the patient is often considered as ‘clinically disease free’. Here, we analyzed the presence of CTCs in treatment-naïve and chemo-recipient breast cancer patients. Methods: In this retrospective study on 417 breast cancer patients, CTCs were isolated from 1.5 ml of blood using the Drug Controller General of India (DCGI) 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 based on automated digital imaging platform. Results: 42.6 % (n=178) of patients were clinically at progressive stage (stage II and III) and treatment naïve. On the other hand, 47.4 % of patients received treatment including surgery and chemotherapy. CTCs were not observed in 5.6 % (n=10) of the treatment naïve population while 32% (n=75) of patients who received therapy did not show CTCs. The mean CTC number in treatment naïve patients was 15, while the mean CTC count in patients receiving therapy was drastically reduced to 2. This implied that therapy effectively countered the tumor progression and reduced the shedding of tumor cells in circulation. The distribution of CTC in treatment naïve patients exhibited a bimodal trend centered at values of 10 and 50, suggesting two distinct populations of patients with respect to CTC count. CTC count did not show any correlation with the age in both population groups. Surprisingly CTC count in younger patients (20-50 years) was 50% higher compared to the older population (50-75 years). Conclusions: The presence of CTCs in treatment naïve, progressive breast cancer patients indicated the biologically aggravated disease. Although therapeutic intervention drastically reduced the CTC burden, their presence in a large population was suggestive of an MRD and the likelihood of recurrence after discontinuation of therapy. A distinct pattern of CTC occurrences in Tx naïve and Tx recipient patient suggested that CTCs can be an important clinical indicator to monitor the therapy response, progression and residual disease.

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 .

Abstract 3370: Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer

Abstract Aggressive variants of prostate cancer (AVPC) emerge from prostate adenocarcinoma following enduring therapeutic pressure. They grow independent of the androgen receptor, progress rapidly and can show traits of neuroendocrine (NE) differentiation. As progression to AVPC is not well detected by prostate specific antigen monitoring, new biomarkers are urgently needed. The aim of this study is to evaluate the detection of circulating tumor cells (CTCs) in combination with gene expression analysis of NE genes to identify AVPC patients with neuroendocrine traits. Blood samples were collected from patients suffering from AVPC (n=78, including 25 patients with histological evidence of NEPC) as well as from patients with hormone-sensitive prostate cancer (HSPC) as controls (n=12) at the University Medical Center Hamburg-Eppendorf. CTC counts were determined by CellSearch analysis. In parallel, CTCs were immuno-magnetically enriched using the AdnaTest and bulk gene expression of prostate-specific and NE genes was analyzed by semi-quantitative PCR. From a subset of patients, CTCs were additionally enriched by Parsortix to compare gene expression of CTCs with EpCAM-based or size-based CTC enrichment. Using CellSearch analysis, CTCs were found in 88.9 % of AVPC patients. CTC counts ranged between 0 – 20,000 CTCs/7.5 ml of blood with a median of 32 CTCs/7.5 ml. Similarly, 81.8 % of NEPC patients were positive for CTCs with a median count of 35 CTCs/7.5 ml (range 0-13,000). In contrast, CTCs were found in only 50 % of HSPC patients at lower concentrations (median count of 0.5 CTCs/7.5 ml, range 0-34). CTC counts were significantly elevated in AVPC and NEPC patients compared to HSPC (p = 0.002, p = 0.012), but no significant differences were found between AVPC and NEPC samples.In 93.8 % of AVPC samples, CTCs expressed prostate-specific genes such as KLK3 or PSMA. At least one of the NE transcripts was detected in 37.5 % of AVPC patients. In CTCs from NEPC patients, the positivity for prostate markers was significantly reduced to 66.6 % (p = 0.01) and the positivity for NE markers was increased to 61.1 %. A random Forrest model trained on all analyzed transcripts allowed for a distinction of HSPC and NEPC with an AUC of 79.5 %. Comparison of CTC enrichment strategies in a subset of 13 patients showed similar NE marker detection by size-based enrichment (53.8 % NE-positive) compared to the AdnaTest (61.5 % NE-positive). AVPC and NEPC patients show a high CTC burden that will allow subsequent molecular analyses, and CTC counts allowed a distinction between blood samples from HSPC and NEPC patients. Gene expression analysis revealed a high degree of inter-patient heterogeneity for neuroendocrine-specific transcripts with a reduction of prostate markers in NEPC patients. Future molecular characterization and longitudinal monitoring of CTCs will shed more light on the evolution of AVPC and NEPC. Citation Format: Lina Merkens, Sarah Greimeier, Sabine Riethdorf, Klaus Pantel, Gunhild von Amsberg, Stefan Werner. Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3370.

Abstract A008: An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma

Abstract Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Tumor progression is monitored with MRI, chest CT, and serum levels of Alpha fetoprotein. Unfortunately, these assays involve exposing children to radiation and anesthesia and are not always accurate. The goal of this work is to develop and validate a liquid biopsy test for circulating tumor cells (CTCs) with a panel of HB-specific markers to provide a less invasive and more accurate way of assessing disease. Methods: We first explored accumulation of the far-red fluorescent dye indocyanine green (ICG) in widely available cell lines (HepG2, Huh-6, Hep3B, HepRG, 293T, SH-SY5Y, A549), our own HB patient-derived cell line (HB17), and primary fibroblasts. We assessed ICG accumulation with fluorescence microscopy and flow cytometry, including standard flow cytometry on a BD Symphony instrument and imaging flow cytometry on the Amnis ImageStream X MK II instrument. Based on this data, we then developed a new method for quantifying CTC burden from patient whole blood samples using HB-specific markers ICG, Glypican-3 (GPC3), and DAPI with fluorescence microscopy and flow cytometry readouts. We tested this panel with cell lines and non-cancer control blood samples, as well as blood samples from mice harboring intrahepatic patient-derived xenograft (PDX) tumors representing HB. Finally, we used this panel to analyze whole blood samples for CTC burden with a cohort of HB patients and correlated with patient characteristics and outcomes. Results: First, we showed that ICG accumulation is specific to liver cancer cells (HepG2, Huh-6, Hep3B, HB17), compared to non-malignant liver cells (HepRG), non-liver solid tumor cells (SH-SY5Y, A549), and non-malignant cells (293T, primary fibroblasts). In addition, we found that ICG+ liver tumor cells remain positive for at least 96 hours after uptake. We also showed that ICG accumulation can be used to identify HepG2 HB cells in a mixed population of cells down to a dilution of 1:100 (2.7% ICG+ in the HepG2/SH-SY5Y mix versus 1.9% ICG+ in the SH-SY5Y control). Flow cytometry with HepG2 and A549 cell lines with the ICG/GPC3/DAPI panel showed 94.4% of HepG2 cells were triple ICG+/GPC3+/DAPI+ while 0% of A549 cells were triple ICG+/GPC3+/DAPI+. With non-cancer control blood samples, we identified less than 1 ICG+ cell per ml of blood. We showed the presence of triple ICG+/GPC3+/DAPI+ cells in the blood of animals harboring PDX tumors. Most importantly, with a low-risk HB patient sample, we found 2.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood; with a high-risk sample we counted 17.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood. Conclusions: This work shows the translation from the use of ICG to identify HB lesions in patients during surgery to its role in a novel liquid biopsy test for CTCs, which has the real potential to improve the standard of care by offering a more accurate and less invasive means of monitoring disease. Citation Format: Andres F. Espinoza, Roma H. Patel, Bryan W. Armbruster, Pavan Kureti, Saiabhiroop R. Govindu, Sanjeev A. Vasudevan, Sarah E. Woodfield. An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A008.

Monitoring Plasma Cells and Clonal Emergence through Genome Sequencing of Circulating Tumor Cells for Minimally Invasive Molecular Characterization of Multiple Myeloma

Introduction Multiple Myeloma (MM) develops from well-defined precursor stages, however, bone marrow (BM) biopsy limits screening and monitoring strategies for patients. Precursor conditions are incidentally diagnosed and require invasive BM biopsies for complete characterization, highlighting the need for improved early detection methods. Minimally invasive blood biopsies to measure circulating tumor cells (CTCs) as markers of MM disease development are a promising solution to this need. CTCs are amenable to repeatable enrichment, enumeration, and molecular characterization for early detection, real-time monitoring, and assessment of treatment response and clonal evolution. We present our novel method, MinimuMM-seq, for enumeration and whole genome sequencing (WGS) of CTCs that enables genomic profiling and WGS-based cytogenetic analyses from enriched liquid biopsy samples. CTCs were enumerated from 241 patients, with 50 patients undergoing WGS that revealed that CTCs were of tumor origin and could faithfully match BM sequencing results, detecting 100% of clinically reported events. Longitudinal sampling of CTCs tracked clonal dynamics over time and detected the emergence of high-risk genetic subclones. Methods Peripheral blood from 241 precursor patients (82 MGUS, 159 SMM) from the PCROWD observational study (IRB #14-174) was collected in CellRescuepreservation tubes and processed on the CellSearch® system (Menarini Silicon Biosystems) using the Circulating Multiple Myeloma Cell (CMMC) assay kit with 4mL of blood. Enrichment and enumeration of CTCs was based on CD138+38+CD45-19- immunophenotype. Nucleated cells were identified using DAPI staining. In 50 patients, CTCs were sorted for library construction, quantification, and WGS on Illumina NovaSeq6000. Mutation analyses were performed with the cancer genome analysis pipelines of the Broad Institute. Tumor phylogeny was reconstructed with the PhylogicNDT algorithm in patients with matched CTC and BM sequencing (n=27), as well as serial CTCs derived from PB only (n=10). Results CTCs were detected in 75% of MGUS and 86% of SMM patients enrolled in the study, with a median count of 2.5 (range 0 to 1328) and 13 CTCs (range 0 to 43836), respectively. An increased number of CTCs correlated with progressive disease staging from MGUS, to low, intermediate and high-risk SMM defined by IMWG 2/20/20 risk stratification. MinimuMM-seq yielded a median tumor purity of 98% (range 45 to 100%) in CTCs. Our method detected 100% of clinically reported BM biopsy events by fluorescence in situ hybridization (FISH), including translocations and copy number abnormalities. It could replace molecular cytogenetics for diagnostic yield and risk classification in patients with paired BM and CTC samples (n=27). Additional diagnostic yield compared to clinical BM FISH was found in 88% of patients. Our method detected novel events and unknown/cryptic translocations, such as t(8;14) and t(14;20), and MM driver mutations, including KRAS, NRAS, and TP53. As peripheral blood continuously circulates, liquid biopsy sampling may be affected by the phenomena of spatial and temporal heterogeneity in CTC burden. In 10 patients longitudinal CTC sampling was carried out to monitor clonal evolution of disease. We validated stability of the major clone and described shifting dynamics of subclones over time with the emergence of potentially high-risk CTC subclones. Expansion of subclones harboring MM events (such as 13q deletion) and driver mutations (such as KRAS and DIS3) was observed, indicating their potential to confer selective advantage for clonal fitness and independence for circulation. Anecdotally, one SMM patient received early interventional treatment, and we identified the emergence of the new fittest clone (bearing KRAS p.G12S) before any potential resistance was clinically observed. Conclusion We have established an approach for WGS of CTCs that can replace standard molecular cytogenetics. CTCs harbored the same pathognomonic MM abnormalities as BM plasma cells. We tracked clonal dynamics over time and detected the emergence of high-risk CTC subclones. Our findings show that capture and genomic profiling of CTCs could be a robust surrogate for BM biopsy, allowing minimally invasive detection and monitoring of disease and unlocking the clinical potential of liquid biopsies for MM diagnostics.

Increased Levels of Circulating Plasma Cells in Patients with Newly Diagnosed Multiple Myeloma Are Independently Associated with Poor Prognosis

The presence of circulating tumor cells (CTCs) has long been suggested as a valuable prognostic biomarker in Multiple Myeloma (MM). Recently, increased baseline CTC levels in patients with newly-diagnosed MM (NDMM) have been associated with adverse outcomes in the GEM2012MENOS65/GEM2014MAIN and FORTE trials. Since January 2017, we have been evaluating the presence of CTCs utilizing Next-generation Flow cytometry (NGF) with a median limit of detection (LOD) reaching 2x10-6. NGF assessment has been performed according to the EuroFlow guidelines for sample preparation, antibodies, cytometer settings and analysis of data. Currently, we have analyzed 525 matched BM and PB samples from NDMM patients and have found clonal plasma cells in 100% of BM and 89.1% (468/525) of PB samples respectively. Patients presented with ISS III/R-ISS III, high-risk cytogenetics, serum β2 microglobulin above median and hemoglobin levels ≤ 8.5 g/dL had increased CTC levels. Inversely, the absence of CTCs or their low prevalence (<10-5) was associated with an increased normal plasma cell compartment within the BM (R2=0.82, P < .0001). No association was detected between the CTC number and the therapeutic response to induction treatment in accordance with previous reports. Nevertheless, over a median follow-up monitoring of 42 months (range: 3-66 months), a running log-rank test with a step increase of 0.1% CTCs, highlighted an optimal cut-off point for PFS risk assessment at the level of 2x10-4. In the multivariable analysis, NDMM patients with CTCs above this cut-off showed a 2.2-fold higher risk of progression which was independent from ISS, induction scheme and baseline cytogenetics (HR: 2.2, 95% CI: 1.24 - 3.28, P< .001). Our large number of matched samples allowed for a phenotypic comparison between BM clonal cells and CTCs. The majority of patients with detectable CTCs (86%) showed a matched phenotypic profile of aberrant plasma cell in the two sites. However, 66 patients showed phenotypic discrepancies based on one of the following patterns: i) all phenotypic subsets were present in both BM and PB but with significantly altered ratios (≥20% of relative prevalence) for at least two concomitant subsets; ii) presence of ≥ 1 phenotypic subsets (with a minimum relative prevalence of 20% of all clonal cells) only in the BM; and iii) presence of ≥ 1 phenotypic subsets only in the PB. Remarkably, patients with phenotypic discrepancies had significantly higher CTC levels than those with a phenotypic agreement on the two sites (P < .001), together with signs of a more diffuse disease pattern, as evidenced by the imaging results. It is likely that these patients may share a patchy BM infiltration (and/or even extramedullary disease) and accumulate circulating clonal cells from various sites (thus leading to increased tumor burden) with a spatial clonal heterogeneity, which, as a total, may differ from a specific phenotype derived from a particular BM site. The biological differences among patients with differential baseline CTCs expand beyond the tumor cells. Performing a deep phenotypic approach for the identification of 32 distinct immune subsets, we analyzed the immune profile of both ΒΜ and PB in NDMM patients differing to their CTC levels by two log decimals. Patients with CTCs ≥ 5x10-3 had substantially increased levels of total T cells (especially CD8+), NK cells, tumor associated macrophages (TAMs) and an elevated ratio of memory to naïve B cells (P < .0001 for all subsets) in their BM microenvironment than those with less than 5x10-5 CTCs. The same differences, though with weaker statistical significance, applied also in PB, where, we could also identify a clear immune signature with characteristics of tumor suppression and T cell exhaustion for NDMM with the high CTC burden. Altogether, our results confirm the negative and independent prognostic impact of increased CTC levels in the NDMM and further imply its relevance in the real-world setting to establish improved risk-stratification models. Moreover, since the liquid biopsy is a better representative of the entire tumor load than a tissue biopsy sample, the analysis of CTCs may serve as the new hallmark for the real-time evaluation of a patient's disease and/or immune status.

Abstract 2401: Investigating the role of type II IL4 receptor in breast cancer brain metastasis

Abstract Breast-to-brain metastasis is a devastating disease complication for which there are currently few treatment options. Among breast cancer subtypes, patients with triple-negative breast cancer (TNBC) and HER2+ breast cancer experience elevated rates of brain metastasis. The cytokines IL4 and IL13 are overexpressed by many human solid tumors. Both are associated with invasive and metastatic phenotypes; IL13 is additionally consequential in primary brain tumors. These cytokines signal through type I (IL4 only) and type II (IL4/IL13) IL4 receptors (IL4R). Importantly, expression of type II IL4R correlates with poor patient prognosis in basal-like (i.e., triple-negative) breast cancer, and HER2+ tumors display elevated type II IL4R expression. Our laboratory has shown that genetic loss of IL4R signaling in tumor cells attenuates lung and liver metastasis in mice. Additionally, we recently identified a role for IL4R signaling in modulating glycosylation in tumor cells. We hypothesize that IL4R signaling influences TNBC brain metastasis at least in part via modulating glycosylation. In a pilot mouse study, pharmacological blockade of IL4R after intracardiac injection of syngeneic TNBC cells resulted in attenuation of brain metastatic growth and circulating tumor cell burden. We have also observed significant changes in the expression of a sialyltransferase (ST8SIA1) that has been implicated in primary brain tumors, as well as in the abundance of several sialic acid-containing protein species in breast cancer cells following treatment with IL4 or IL13. Further, IL4 stimulation increases the overall glycosylation of several proteins of interest, including a glucose transporter and a glutamine transporter, without altering gene expression. Overall, our data suggest that type II IL4R signaling promotes metastasis, potentially through a mechanism involving protein glycosylation. Blockade of this receptor may be a useful therapeutic approach for patients with breast cancer brain metastases. Citation Format: Wendy Bindeman, Demond Williams, Ebony Hargrove-Wiley, Barbara Fingleton. Investigating the role of type II IL4 receptor in breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2401.

Abstract P2-02-03: Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC)

Abstract Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC)BackgroundThe presence of CTCs has been shown to be a poor prognostic factor in patients with MBC. Studies in prostate cancer have shown patients with distinct morphological subtypes of CTCs have worse outcomes than high CTC burden alone and could have treatment implications. In this study, we report a comprehensive analysis of CTC phenotypes in MBC and their impact on survival.MethodsA total of 148 blood samples from 90 patients with MBC whose disease was progressing on therapy were enrolled for CTC analysis. Blood was sent overnight to Epic Sciences, processed onto glass slides and biobanked. Replicate slides were stained with antibodies targeting the androgen receptor (AR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2) in addition to cytokeratin, CD45, and DAPI. Total CTCs were quantified per sample from approximately 3 mL of blood. A threshold defining positive AR, ER, and HER2 protein expression was set using cultured cancer cell lines expressing the individual proteins. CTC morphology subtypes (heterogeneity [Scher et al Cancer Res 2017], small cell/neuroendocrine-like [SC/NE; Brown et al CCR 2021] and chromosomal instability [CIN+; Schonhoft et al Cancer Res 2020]) were quantified as previously described. High heterogeneity samples (Het+) were defined as having a Shannon Index (SI; a measure of morphologic diversity) of ≥1. CIN positivity was defined as having ≥0.7 CIN+ CTC/mL. Association with these CTC subtypes and survival were evaluated with a univariate COX proportional hazard model. ResultsOver 90% of samples and patients had CTCs (Table 1). The percentage of patients with the CTC subtypes evaluated are below. Table 1. Percentage of blood samples with CTCs and CTC subtypes in MBC patients, by MBC subtype The presence of CTCs was associated with worse survival, with a median survival of 15.3 months (mos) vs 24.0 mos with no CTCs (p=0.028). The presence of AR+ or HER2+ CTCs was not associated with survival. Analysis of ER is ongoing and will be presented. High morphologic heterogeneity or the presence of the SC/NE or CIN+ CTC subtypes, predicted for a worse survival outcome (Table 2). Table 2. Overall survival of patients with and without distinct CTC subtypes CTC morphological subtypes did not correlate with each other, except CTC heterogeneity by SI which correlated weakly with SC/NE and CIN+. HER2+ CTCs strongly correlated with a higher SI while AR+ CTCs did not. AR and HER2 expression did not associate with the CIN+ or SC/NE subtypes. ConclusionsDigital pathology analysis of CTCs identifies distinct CTC morphological subtypes that predict for worse prognosis in MBC patients. To our knowledge, this is the first report of SC/NE CTCs in MBC patients and that MBC patients with SC/NE and CIN+ CTCs have worse outcomes. Longitudinal characterization of CTC subtypes across the MBC continuum could potentially identify effective therapies against these more lethal cancers. Citation Format: Joyce A O'Shaughnessy, Maren K Levin, Esther San Roman Rodriguez, James Lu, Joshua T Jones, Amanda KL Anderson, Alisa Tubbs, Joseph D Schonhoft, Rick Wenstrup. Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-03.

A phase (Ph) 1b/2 study of ribociclib (R) in combination with docetaxel (D) plus prednisone (P) in metastatic castration-resistant prostate cancer (mCRPC).

5043 Background: The survival benefit of D in mCRPC is modest. CDK4/6 inhibitors such as R have shown synergistic activity with taxanes in pre-clinical cancer models. We sought to determine the safety and efficacy of R + D + P in mCRPC patients (pts). Methods: This was a Ph 1b/2 multicenter, open-label single arm trial of mCRPC pts with progression (PD) on ≥ 1 prior androgen receptor signaling inhibitor (ARSi) who had not previously received D for mCRPC (NCT02494921). Pts were treated with escalating doses of R in combination with D + P for 6-9 cycles, followed by single agent maintenance R until radiographic or clinical PD. The Ph 2 primary endpoint was 6-month (mo) radiographic progression-free survival (rPFS) rate by PCWG2 criteria, with a target rate of 55% and null hypothesis of 35%. Ph 2 pts underwent baseline circulating tumor cell (CTC) enumeration and genome sequencing (Epic Sciences). Cox proportional hazard model and log-rank test were used to test for associations between rPFS and CTC burden and copy number (CN) variants, respectively. Results: 43 pts were enrolled from 11/2015 to 6/2019. Median age was 68 (range 55-84). 20.9% of pts had visceral metastases. 33 (77%) had PD on prior abiraterone, 27 (63%) on enzalutamide, and 17 (40%) on both. In Ph 1b, 19 pts were enrolled. In the first cohort (D 75 mg/m2 day [d] 1, R 200 mg/d d2-14 of every 21d cycle), 2 pts experienced DLTs (febrile neutropenia [FN] and grade 4 neutropenia). With an alternative dosing schema of D 60 mg/m2 on d1, and R daily on d1-4 and 8-15 of cycle, with daily G-CSF support on d5-7, the MTD was not reached and D 60 mg/m2 + R 400 mg/d was chosen as the recommended Ph 2 dose (RP2D). In total, 30 pts were treated at RP2D; median number of D cycles was 8.5 and 60% went on to receive maintenance R. The Ph 2 primary endpoint was met with a 6-mo rPFS rate of 65% (95% CI 50-85%). Median rPFS was 8.0 mos (95% CI 4.1-10.0). PSA response rate (RR) defined as ≥50% reduction was 27.6% (95% CI 12.7-47.2%) and objective RR was 30.8% (95% CI 9.1-61.4%). Among pts treated at RP2D, the most common grade ≥3 treatment-related adverse events were neutropenia (n= 11, 36.7%), lymphocytopenia (n=3, 10%); no cases of FN were observed. Baseline CTC burden was associated with an increased risk of radiographic PD or death (HR 1.038, 95% CI 1.001-1.074, p = 0.038). Pts harboring CTCs without MYC (4/11 pts) or CDK6 CN gain (7/11 pts) had prolonged rPFS compared to those with gene amplification (median rPFS 10.76 vs 4.11 mos, p = 0.03, and 7.01 vs 1.92 mos, p = 0.053, respectively). Conclusions: The combination of R + D was well tolerated and showed promising activity in mCRPC pts who had progressed on an ARSi. The Ph 2 study met its primary endpoint, with an encouraging 6-mo rPFS rate of 65%. Lack of MYC or CDK6 amplification on CTC sequencing was associated with longer rPFS. Funding: Novartis Pharmaceuticals, PCF YIA. Managed by the PCCTC. Clinical trial information: NCT02494921.

Circulating Tumor Cell Subtypes and T-cell Populations as Prognostic Biomarkers to Combination Immunotherapy in Patients with Metastatic Genitourinary Cancer.

Circulating tumor cells (CTC) are under investigation as a minimally invasive liquid biopsy that may improve risk stratification and treatment selection. CTCs uniquely allow for digital pathology of individual malignant cell morphology and marker expression. We compared CTC features and T-cell counts with survival endpoints in a cohort of patients with metastatic genitourinary cancer treated with combination immunotherapy. Markers evaluated included pan-CK/CD45/PD-L1/DAPI for CTCs and CD4/CD8/Ki-67/DAPI for T cells. ANOVA was used to compare CTC burden and T-cell populations across timepoints. Differences in survival and disease progression were evaluated using the maximum log-rank test. From December 2016 to January 2019, 183 samples from 81 patients were tested. CTCs were found in 75% of patients at baseline. CTC burden was associated with shorter overall survival (OS) at baseline (P = 0.022), but not on-therapy. Five morphologic subtypes were detected, and the presence of two specific subtypes with unique cellular features at baseline and on-therapy was associated with worse OS (0.9-2.3 vs. 28.2 months; P < 0.0001-0.013). Increasing CTC heterogeneity on-therapy had a trend toward worse OS (P = 0.045). PD-L1+ CTCs on-therapy were associated with worse OS (P < 0.01, cycle 2). Low baseline and on-therapy CD4/CD8 counts were also associated with poor OS and response category. Shorter survival may be associated with high CTC counts at baseline, presence of specific CTC morphologic subtypes, PD-L1+ CTCs, and low %CD4/8 T cells in patients with metastatic genitourinary cancer. A future study is warranted to validate the prognostic utility of CTC heterogeneity and detection of specific CTC morphologies.

In Vivo Enrichment and Elimination of Circulating Tumor Cells by Using a Black Phosphorus and Antibody Functionalized Intravenous Catheter.

The circulating tumor cell (CTC) count is closely related to cancer recurrence and metastasis. The technology that can in vivo destroy CTCs may bring great benefits to patients, which is an urgent clinical demand. Here, a minimally invasive therapeutic intravenous catheter for in vivo enriching and photothermal killing of CTCs is developed. The surface of catheter is modified with anti‐EpCAM antibody and the interior is filled with black phosphorus nanosheets (BPNSs). CTCs in the peripheral blood are captured by the catheter continually with the aid of circulation. The captured CTCs are used for downstream analyses or in vivo eliminated by the near‐infrared (NIR) photothermal effect of BPNSs. A capture efficiency of 2.1% is obtained during the 5 min of treatment, and 100% of the captured CTCs are killed by following NIR light irradiation in both an in vitro closed‐loop circulation system and an in vivo rabbit model. This cost‐effective modality for lowering the CTCs burden can be a good supplement to traditional therapies, which holds great promise as an effective clinical intervention for cancer patients.

Postoperative circulating tumor cells: An early predictor of extrahepatic metastases in patients with hepatocellular carcinoma undergoing curative surgical resection.

Postoperative extrahepatic metastases (EHM) contribute to a grim outcome in patients with hepatocellular carcinoma (HCC) who are undergoing curative surgical resection. The current study investigated the clinical value of circulating tumor cells (CTCs) in predicting EHM after curative surgery. A total of 197 patients with HCC who were undergoing curative surgical resection were assigned to a retrospective training cohort (144 patients) or a prospective validation cohort (53 patients). The CELLSEARCH system was used for the detection of CTCs prior to surgical resection and 1month thereafter. The cutoff value of CTCs was estimated using receiver operating characteristic analysis. Bonferroni correction was applied for multiple testing in a Cox proportional hazards regression model. In the training cohort, EHM was found to be associated with a higher postoperative CTC burden compared with no EHM (mean: 4.33 vs 0.52; P < .001). Receiver operating characteristic analysis demonstrated a postoperative CTC count ≥3 as the optimal cutoff value for the prediction of EHM. Patients with a postoperative CTC count ≥3 experienced a higher EHM risk (56.3% vs 5.5%) and a shorter median overall survival (31.25months vs not reached) (all P<.001). The prognostic significance of a postoperative CTC count ≥3 also applied to patient subgroups with a low EHM risk, such as those with an α-fetoprotein level ≤400ng/mL, absence of vascular invasion, well differentiation, and early tumor stage, and its predictive value was retained in patients with a continuous normal α-fetoprotein level during postoperative follow-up (all P<.05). The results were confirmed in the validation cohort. A postoperative CTC count ≥3 appears to be a surrogate marker for the prediction of EHM after curative surgical resection of HCC. More careful surveillance should be recommended to patients with a high CTC load to ensure the greater possibility of early interventions for postoperative EHM.

Identification and Characterization of Circulating Tumor Cells in Men Who have Undergone Prostatectomy for Clinically Localized, High Risk Prostate Cancer.

Approximately 15% of men with newly diagnosed prostate cancer have high risk features which increase the risk of recurrence and metastasis. Better predictive biomarkers could allow for earlier detection of biochemical recurrence and change surveillance and adjuvant treatment paradigms. Circulating tumor cells are thought to represent the earliest form of metastases. However, their role as biomarkers in men with high risk, localized prostate cancer is not well defined. Two to 5 months after prostatectomy we obtained blood samples from 37 patients with high risk, localized prostate cancer, defined as stage T3a or higher, Gleason score 8 or greater, or prostate specific antigen 20 ng/ml or greater. Circulating tumor cells were enumerated using a commercial platform. Matched tumor and single circulating tumor cell sequencing was performed. Circulating tumor cells were detected in 30 of 37 samples (81.1%) with a median of 2.4 circulating tumor cells per ml (range 0 to 22.9). Patients with detectable circulating tumor cells showed a trend toward shorter recurrence time (p=0.12). All patients with biochemical recurrence had detectable circulating tumor cells. Androgen receptor over expression was detected in 7 of 37 patients (18.9%). Patients with biochemical recurrence had more circulating tumor cell copy number aberrations (p=0.027). Matched tumor tissue and single circulating tumor cell sequencing revealed heterogeneity. We noted a high incidence of circulating tumor cell detection after radical prostatectomy and shorter time to biochemical recurrence in men with a higher circulating tumor cell burden and more circulating tumor cell copy number aberrations. Genomic alterations consistent with established copy number aberrations in prostate cancer were detectable in circulating tumor cells but often discordant with cells analyzed in bulk from primary lesions. With further testing in appropriately powered cohorts early circulating tumor cell detection could be an informative biomarker to assist with adjuvant treatment decisions.

Circulating tumor cells dynamics in pancreatic adenocarcinoma correlate with disease status: Results of the prospective cluster study

OBJECTIVES Previous retrospective studies demonstrated that circulating tumor cells (CTCs) subtypes correlate with overall survival in patients with pancreatic ductal adenocarcinoma (PDAC). Herein, we report results of a prospective observational study on CTCs dynamics to assess their clinical significance. METHODS The CLUSTER study is a prospective longitudinal study on PDAC CTCs dynamics (NCT02974764). Multiple peripheral blood samples were collected from 200 consecutively enrolled patients with presumed PDAC diagnosis. CTCs were isolated and characterized by immunofluorescence. RESULTS Two major CTCs subtypes were identified in PDAC patients: epithelial CTCs (eCTCs) and epithelial/mesenchymal CTCs (mCTCs). Patients who received neoadjuvant chemotherapy had significantly lower total CTCs (tCTCs, P = 0.007), eCTCs (P = 0.007), and mCTCs (P = 0.034), compared with untreated patients eligible for upfront resection. Surgical resection of the primary tumor resulted in significant reduction, but not disappearance, of CTCs burden across all cell subtypes (P < 0.001). In multivariable logistic regression analysis, preoperative numbers of all CTCs subpopulations were the only predictors of early recurrence within 12 months from surgery in both chemo-naive and post-neoadjuvant patients (odds ratio 5.9 to 11.0). Alterations in CTCs were also observed longitudinally, before disease recurrence. A risk assessment score based on the difference of tCTCs increase accurately identified disease recurrence within the next 2 months, with an accuracy of 75% and 84% for chemo-naive and post-neoadjuvant patients, respectively. CONCLUSION We report novel findings regarding CTCs from a large prospective cohort of PDAC patients. CTCs dynamics reflect progression of disease and response to treatment, providing important information on clinical outcomes, not available by current tumor markers and imaging.

Abstract P6-18-22: Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2-negative metastatic breast cancer (MBC)

Abstract BACKGROUND: TMEM (Tumor Microenvironment of Metastasis) are the portal for tumor cell intravasation into the circulation and subsequent metastasis (Harney et al Cancer Discov 2015). The potent Tie2 kinase inhibitor rebastinib inhibits intravasation at TMEM sites, reduces circulating tumor cell (CTC) burden, increases angiopoietin (Ang) 1/2 levels, prevents distant metastases, and improves survival in breast cancer animal models when added to either P or E (Harney et al MCT 2017), and circumvent chemotherapy-induced pro-metastatic changes in the tumor microenvironment mediated by TMEM (Karagiannis et al STM 2017). We sought to determine the safety of rebastinib combined with antitubulin therapy (P or E) in patients with HER2- MBC. We also hypothesized that addition of rebastinib would reduce CTC burden and increase Ang levels by blocking Ang-mediated stimulation of VEGF release from TMEM-associated macrophages. METHODS: We aimed to determine the safety and recommended phase 2 dose (RP2D) of rebastinib (2 dose levels: 50 mg or 100 mg PO BID) in combination with P (80 mg/m2 x 12 weeks) or E (1.4 mg/m2 on day 1 &amp; 8 q 21 days) using a standard 3+3 design (1 cycle = 21 days). Secondary objectives included evaluating the effect of the P/E + rebastinib combination on CTCs (TelomeScan) and Ang levels. Dose limiting toxicity (DLT) was defined as grade 3-4 febrile neutropenia, thrombocytopenia, and non-hematologic toxicity during the first 6 weeks of therapy. Eligibility included HER2- MBC, ECOG PS 0-1, CDK4/6 inhibitor progression if ER+. Patients with ≤ 2 prior non-taxane chemotherapy regimens received P+ rebastinib, whereas those with ≥ 2 chemo regimens (including a taxane) received E+ rebastinib. RESULTS: Of 11 treated patients, 6 received rebastinib + P and 5 received rebastinib + E (2 non-evaluable due to rapid disease progression and non-compliance). Among 11 patients who received 60 treatment cycles, only 1 patient (treated with eribulin) had grade 3 events (anemia and neuropathy after week 6) potentially related to treatment. When combined with P, the RP2D of rebastinib was 100 mg PO BID, with DLT occurring in 0/6 patients. When combined with E, 0/3 evaluable patients had a DLT at 50 mg BID of rebastinib (accrual ongoing for 100mg BID). Best response included partial response/stable disease in 4(2PR/2SD) of 6 treated with P+ rebastinib, and 1(1PR) of 5 treated with E+ rebastinib. CTCs decreased during therapy (median decrease 99.7 %) and 4/8 patients converted from CTC+ to CTC-. Ang1 levels increased during therapy in 8 patients (0.2-7.0 fold), while Ang2 levels were also increased in 8 patients (0.2-1.4 fold). CONCLUSIONS: When combined with P x 12 weeks, the RP2D of rebastinib is 100 mg PO BID. When combined with E, the RP2D of rebastinib is at least 50mg PO BID; however, the 100 mg PO BID dose level is still accruing patients. The P/E + rebastinib combinations are associated with antitumor activity and exhibit pharmacodynamic evidence indicating blockade of Tie2 (increased Ang) and TMEM function (reduced CTCs) We plan to further evaluate the P+ rebastinib combination as neoadjuvant therapy in the I-SPY program, and continue further evaluation of P/E + rebastinib combinations in MBC. Citation Format: Anampa JD, Xue X, Oh S-y, Kornblum N, Sadan S, Oktay MH, Condeelis J, Sparano JA. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2-negative metastatic breast cancer (MBC) [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 P6-18-22.

Circulating Tumor Cells Dynamics in Pancreatic Adenocarcinoma Correlate With Disease Status: Results of the Prospective CLUSTER Study.

Previous retrospective studies demonstrated that circulating tumor cells (CTCs) subtypes correlate with overall survival in patients with pancreatic ductal adenocarcinoma (PDAC). Herein, we report results of a prospective observational study on CTCs dynamics to assess their clinical significance. The CLUSTER study is a prospective longitudinal study on PDAC CTCs dynamics (NCT02974764). Multiple peripheral blood samples were collected from 200 consecutively enrolled patients with presumed PDAC diagnosis. CTCs were isolated and characterized by immunofluorescence. Two major CTCs subtypes were identified in PDAC patients: epithelial CTCs (eCTCs) and epithelial/mesenchymal CTCs (mCTCs). Patients who received neoadjuvant chemotherapy had significantly lower total CTCs (tCTCs, P = 0.007), eCTCs (P = 0.007), and mCTCs (P = 0.034), compared with untreated patients eligible for upfront resection. Surgical resection of the primary tumor resulted in significant reduction, but not disappearance, of CTCs burden across all cell subtypes (P < 0.001). In multivariable logistic regression analysis, preoperative numbers of all CTCs subpopulations were the only predictors of early recurrence within 12 months from surgery in both chemo-naive and post-neoadjuvant patients (odds ratio 5.9 to 11.0). Alterations in CTCs were also observed longitudinally, before disease recurrence. A risk assessment score based on the difference of tCTCs increase accurately identified disease recurrence within the next 2 months, with an accuracy of 75% and 84% for chemo-naive and post-neoadjuvant patients, respectively. We report novel findings regarding CTCs from a large prospective cohort of PDAC patients. CTCs dynamics reflect progression of disease and response to treatment, providing important information on clinical outcomes, not available by current tumor markers and imaging.

Abstract CT021: Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2 negative metastatic breast cancer (MBC)

Abstract BACKGROUND: Metastasis is the primary cause of death in breast cancer, yet no current therapies specifically inhibit metastasis. TMEM (Tumor Microenvironment of Metastasis) are the portal for tumor cell intravasation into the circulation and subsequent metastasis (Harney et al Cancer Discov 2015 [PMCID:4560669). The potent Tie2 kinase inhibitor rebastinib inhibits intravasation at TMEM sites, reduces circulating tumor cell (CTC) burden, prevents distant metastases, and improves survival in breast cancer animal models when added to either P or E (Karagiannis et al STM 2017 [PMCID:5592784]; Harney et al MCT 2017 [PMCID:28838996]). We hypothesized that the addition of rebastinib to antitubulin therapy (P or E) in patients with HER2- MBC would be safe, and by inhibiting TMEM function, would also reduce CTC burden by blocking hematogenous dissemination. METHODS: The primary objective was to determine the safety and recommended phase 2 dose (RP2D) of rebastinib (2 dose levels: 50 mg or 100 mg PO BID) in combination with P (80 mg/m2 x 12 weeks) or E (1.4 mg/m2 on day 1 &amp; 8 q 21 days) using a standard 3+3 design (1 cycle = 21 days). Secondary objectives included evaluating the effect of the P/E + rebastinib combination on CTCs (TelomeScan). Dose limiting toxicity (DLT) was defined as grade 3-4 febrile neutropenia, thrombocytopenia, and non-hematologic toxicity during the first 6 weeks of therapy. Eligibility included HER2- MBC, ECOG PS 0-1, progression after a CDK4/6 inhibitor if ER+. Patients with ≤ 2 prior non-taxane chemotherapy regimens received P+ rebastinib, whereas those with ≥ 2 chemo regimens (including a taxane) received E+ rebastinib. RESULTS: Of 10 treated patients, 6 received rebastinib + P and 4 received rebastinib + E (2 non-evaluable due to rapid disease progression [n=1] and non-compliance [n=1]). Among 10 patients who received 48 treatment cycles, only 1 patient (treated with eribulin) had grade 3 events (anemia and neuropathy after week 6) potentially related to treatment. When combined with P, the RP2D of rebastinib was 100 mg PO BID, with DLT occurring in 0/6 patients. When combined with E, 0/2 evaluable patients had a DLT at 50 mg BID of rebastinib (accrual ongoing). Best response included partial response/stable disease in 4(2PR/2SD) of 5 treated with P+ rebastinib (1 too early), and 2(1PR/1SD) of 4 treated with E+ rebastinib. CTCs decreased during therapy (baseline vs. day 43, median decrease 99.7 %) and 4/8 patients converted from CTC+ to CTC-. CONCLUSIONS: When combined with P x 12 weeks, the RP2D of rebastinib is 100 mg PO BID. The P/E + rebastinib combinations are associated with antitumor activity and exhibit pharmacodynamic evidence suggesting TMEM function blockade. We plan to further evaluate the P+ rebastinib combination as neoadjuvant therapy in the I-SPY program, and continue further evaluation of P/E + rebastinib combinations in MBC. Citation Format: Jesus D. Anampa, Xiaonan Xue, Sun-young Oh, Noah Kornblum, Sara Sadan, Maja Oktay, John Condeelis, Joseph A. Sparano. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2 negative metastatic breast cancer (MBC) [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 CT021.

Identification and characterization of circulating tumor cells in post prostatectomy patients with localized high risk prostate cancer.

69 Background: Over 15% of men with newly diagnosed prostate cancer (PCa) have high-risk features that raise the recurrence risk. Better biomarkers could allow for even earlier detection of biochemical recurrence (BCR) and inform adjuvant treatment decisions. Circulating tumor cells (CTCs) may represent the earliest form of metastases, however their role as biomarkers in men with localized PCa is not well defined. Here, we aim to enumerate and molecularly and genomically analyze CTCs using an enrichment-free, unbiased CTC identification technology from men with high-risk, localized PCa after radical prostatectomy (RP) and correlate the analysis with clinical outcomes. Methods: Blood samples from 37 patients with high-risk, localized PCa were obtained 2-5 mos post RP and shipped to Epic. All nucleated cells were subjected to immunofluorescent staining for cytokeratin (CK), CD45, and AR. CTCs were identified using algorithmic analysis. CK+ CTCs were enumerated and subsequently analyzed for AR expression and individually sequenced for copy number alterations (CNA). Patients were followed for BCR, defined as detectable PSA &gt; 0.2ng/dL. Progression free survival (PFS) was calculated using Kaplan-Meier and Cox proportional hazards. Results: CTCs were detected in 81.1%(30/37) of patients with an average of 5.2 CTCs/ml (range: 0 – 22.9) detected per patient. AR expression was detected in 18.9% (7/37) of patients. Ninety nine CTCs from 14 patients were picked and sequenced. CNAs were identified in CTCs in commonly mutated genes in PCa, including MYC amplification and CHD1 deletions. Patients with higher traditional CTC (CK+) burdens exhibited a trend towards shorter PFS (hazard ratio: 1.65; 95% confidence interval: 0.7-3.86; p = 0.13). Conclusions: There was a high incidence of CTC detection after RP in patients with high-risk, localized PCa. A trend toward shorter PFS was seen in those with higher CTC burden. Genomic alterations were detectable in CTCs and consistent with established CNAs in PCa. With further testing in appropriately powered cohorts early CTC detection after primary therapy could represent an informative biomarker to stratify patients with high risk PCa.