Xiaoru Chen

Circulating Tumor Cells: Application as a Biomarker for Molecular Characterization and Predictor of Survival in an All-Comer Solid Tumor Phase I Clinical Study

Purpose Clinical development of cancer drugs has a low success rate. Prognostic and predictive biomarkers using minimally invasive approaches hold promise for increasing the probability of success by enabling disease characterization, patient selection and early detection of drug treatment effect. Enumeration and molecular characterization of circulating tumor cells (CTC) may address some of these needs, and thus were evaluated for utility in a Phase I solid tumor clinical study. Experimental Design Blood samples for CTC analysis were obtained from 24 cancer patients in a multi-center all-comer Phase I study of MEDI-575, a novel anti-PDGFRα antibody. Samples were taken at screening and analyzed for enumeration of CTC using the CellSearch® platform and for molecular characterization using a novel quantitative RT-PCR assay. Results Fifty-nine percent of the patients showed at least 1 CTC per 7.5 ml of blood at baseline. Progression-free survival (PFS) and overall survival (OS) of patients with 0 CTCs at baseline were longer than PFS and Os for patients with 1-3 and >3 CTCs (8.8 versus 1.4 and 1.3 months PFS, P = 0.02; 9.0 vs 7.4 and 3.5 months OS, P = 0.20, respectively). Patients with 0 CTC showed a greater percentage of stable disease than the other 2 groups with 1-3 and >3 CTCs (57% vs 29% and 0%). The multimarker qRT-PCR method detected CTC in 40% of the patients, and 80% of these patients were positive for pre-selected drug target genes. Conclusion CTC enumeration of patients in an all-comer study is feasible and may allow for patient stratification for PFS and Os to evaluate the clinical response of investigational agents. Gene expression profiling of isolated CTC may provide a means for molecular characterization of selected tumor targets.

Characterization of the anti‐CD22 targeted therapy, moxetumomab pasudotox, for B‐cell precursor acute lymphoblastic leukemia

Moxetumomab pasudotox is a second‐generation recombinant immunotoxin against CD22 on B‐cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy‐refractory B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient‐derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD‐scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient‐derived xenograft models.

Abstract 1717: Circulating tumor cells from in vitro 3D culture systems have tumor-initiating capacity in vivo

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Circulating tumor cells (CTCs) are cells that have detached from the primary tumor and entered the bloodstream with the potential to seed metastatic tumors in distal sites. High CTC numbers correlate with aggressive disease, increased metastasis, and decreased time to relapse. It has also been shown that cancer stem cells (CSCs) represent a proportion of the CTCs present in patients. Given that CSCs are resistant to chemotherapy and are responsible for tumor initiation, it is posited that the CSCs are the seeds of metastasis. However, direct evidence for this hypothesis is limited because there are few methods for culturing and studying these rare cells. We are using a 3D culture chamber system (RealBio D4™) to establish long-term cultures of human-derived breast and pancreatic tumors. We observed that the systems 3D matrix supported culture development and incorporation of tissue organization and microenvironment. Further, the chamber design allowed CTCs generated within the cultures to migrate out of the cell mass into the circulating nutrient medium where they were collected for characterization. The isolated CTCs displayed CSC properties via CellSearch® (CD44+,CD24-; CD44+,CD24+ for breast and pancreas respectively). In addition, these isolated CTCs displayed tumor-initiating capacity when implanted into mice. Future studies will compare CTCs isolated from 3D culture with cells from tumors, blood, and tissue grown on scaffold through RT-PCR, histology, and gene expression assays. Citation Format: Marina C. Cabrera, Elaine Hurt, Xiaoru Chen, Shi Xiaoqing, Haifeng Bao. Circulating tumor cells from in vitro 3D culture systems have tumor-initiating capacity in vivo. [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 1717.

Abstract 4420: Factors potentially contributing to sensitivities of CD22-targeting agents in B-cell malignancies

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA In order to understand factors which may contribute to in vitro sensitivity of B-cell malignancies to CD22-targeting agents, we evaluated the mRNA expression levels of genes involved with diphthamide biosynthesis, multi-drug resistance, and CD22 (including the splice variant 3 with exon12 deletion) in untreated and 72-hour CD22-targeting agent-treated DLBCL and CLL cell lines. Cell line sensitivity (EC50) to CD22-targeting agent treatment was evaluated by 72 hour exposureto moxetumomab pasudotox (MP).Cell lines were characterized as insensitive if the EC50s were greater than100 ng/mL. The sensitivity to CD22 -targeting agent treatment was found to be significantly associated with baseline CD22 mRNA expression level, as determined by regression analysis. The highly resistant DLBCL cell line RCK8 and the resistant CLL cell line Z138 demonstrated low CD22 expression, corresponding to their elevated EC50′s. However, baseline mRNA was not significantly lower in some other less sensitive cell lines, suggesting that total mRNA CD22 levels may not be the only factor contributing to reduced sensitivity to anti-CD22 exposure. Given the critical roles of dipthamides in CD22 targeting, we evaluated baseline DPH4 levels and their methylation status. There was no correlation between CD22-targeting agent sensitivity and baseline levels of DPH4 expression/promoter methylation. We further explored how CD22, DPH4, WDR85 and MRP1 mRNA expression changed following a 72 hour exposure to MP. Following MP exposure, both mRNA and methylation levels of DPH4 were not significantly changed. However, the mRNA levels of both the diphthamide biosynthesis protein WDR85 and multidrug resistance-associated protein 1 (MRP1) were up-regulated in half of the cell lines following MP exposure. As expression of truncated CD22 has been reported in ALL, we also evaluated CD22 variant expression before and following CD22-targeting agent exposure. All CD22 variants were co-expressed in most cell lines, with significantly increased V3 observed following treatment in some cell lines with lower EC50′s. In conclusion, our results show sensitivity to CD22-targeting agent in the majority of the cell lines tested, and suggest that multiple factors may contribute to sensitivity including CD22 expression. Additionally, our results suggest that factors which may affect continued response to CD22 targeting agents may change with treatment, including increases in CD22 variant 3, MRP1 and WDR85. Ongoing research will evaluate whether these changes are relevant to CD22-targeting agent sensitivity in vivo. Citation Format: Xin Yao, Patricia Burke, Joyce O. Obidi, Xiaoru Chen, Haifeng Bao, Yihong Yao, Jiaqi Huang. Factors potentially contributing to sensitivities of CD22-targeting agents in B-cell malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2015-4420

Abstract 2425: Nucleosomal DNA assay development and utility as PoP biomarker.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Elevated baseline levels of circulating cell-free nucleosomal DNA (nDNA) are observed in some cancer patients and may be associated with clinical outcome and tumor burden. Transient increases in nDNA in response to effective chemotherapy have been observed, indicating nDNA could be a biomarker for apoptosis and could serve as a minimally invasive proof of principal (PoP) biomarker for cancer treatments. The Cell Death Detection ELISA has been used to measure nDNA levels, but the non-quantitative nature of this assay greatly limits its application in clinical studies. Our objectives were to develop a qualified assay for semi-quantitative measurement of nDNA levels in patient plasma samples and utilize the assay in Phase 1 clinical trials to assess its utility. Here we report on nDNA assay development and its application in 2 clinical trials in hematological malignancies. In order to develop a more quantitative assay, we made nDNA standards from healthy-donor blood samples and established their stability using lyophilized plasma. Recovery of freeze-thawed nDNA standard and plasma samples from normal donors and cancer patients were performed. Methods were established for the collection, storage, and analysis of plasma samples. Samples from subjects in Phase 1 dose-escalation studies undergoing treatment with MEDI-551, an anti-CD19 ADCC-enhanced monoclonal antibody (25 subjects, 0.5 - 12 mg/Kg) or moxetumomab pasudotox (CAT-8015), an anti-CD22 immunotoxin (23 subjects, 20 - 60 μg/Kg) were collected at various time points to assess changes in nDNA with treatment. Predose samples were used to assess baseline levels of nDNA for normalizing responses, comparing differences by type of malignancy, and for prognostic purposes. We developed a semi-quantitative, highly reproducible assay with low intra- and inter-plate variability. Although initial sample thaws significantly decreased signal recovery, subsequent thaws did not. Baseline levels of patient nDNA samples were not significantly different across tumor types (DLBCL, FL, CLL, MCL, MM; P=0.1170). The time course of nDNA changes in the first cycle of treatment illustrated potential differences in mechanism of action and dosing for the 2 studies, with short pronounced fold change (FC) increases occurring early in the cycle (Day 2) for MEDI-551 (weekly dosing, cycle 1) compared to prolonged FC increases at Day 5 for CAT-8015 (dosed day 1, 3, 5 every 28 days). A trend of increased nDNA with dose (MEDI-551, P=0.1522; CAT-8015, P=0.2087) and significantly increased FC for end of treatment samples was noted for both studies (MEDI-551, P=0.0438; CAT-8015, P=0.0014). These results indicate that FC increases of nDNA in response to treatment could potentially provide a proof of principle (PoP) biomarker in some clinical oncology studies. In conclusion, we have developed a new semi-quantitative qualified assay for assessing nDNA in blood which has been utilized to support clinical drug development. Citation Format: Xiaoqing (Sarah) Shi, Haifeng Bao, Yuling Wu, Xiaoru Chen, David L. Gold, Theresa M. LaVallee, Patricia A. Burke. Nucleosomal DNA assay development and utility as PoP biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2013-2425

Abstract 3484: CTC enumeration and molecular characterization in a Phase 1 study of IGF-1 and IGF-2 inhibition by MEDI-573.

MEDI-573 selectively binds to human insulin-like growth factor (IGF)-1 and IGF-2 and inhibits ligand-mediated signal transduction via the insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor A isoform (IR-A) pathways, without perturbing glucose homeostasis. As IGF-1, IGF-2, and IGF-1R are overexpressed in bladder cancer, a Phase 1 expansion study including 20 bladder cancer subjects along with 6 additional subjects with other types of cancers from the dose escalation study were included for circulating tumor cell (CTC) analysis as part of a larger 42-subject study (MI-CP184) evaluating PK, safety, and biomarkers. Our objective for the results reported here was to evaluate CTC numbers as a prognostic indicator and as a biomarker of response to MEDI-573 therapy, and to evaluate the expression of IGF-1R on CTC and any correlation with treatment. Subjects in the expansion study were treated with MEDI-573 at 5 mg/Kg weekly (QW) (n=10), 15 mg/Kg QW (n=10), whereas subjects evaluated for CTC in the dose escalation study were treated at 30 or 45 mg/Kg every 3 weeks (Q3W) (n=3 each). CTC collected at screening and pre-dosing each cycle were enumerated and evaluated for IGF-1R expression by CellSearch®. 92% of subjects had at least one CTC at any time during the study and 62.5% had at least one CTC at screening. As has been demonstrated with other tumor types, subjects with ≤3 CTC at screening stayed on study longer than subjects with >3 CTC (2.3 vs. 1.2 months, P=0.0125 [no censoring]). Mean overall survival (OS) was also longer for subjects with ≤3 CTC compared to subjects with >3 CTC (6.37 vs. 2.83 months (P=0.0218, t test), although Kaplan-Myer curves were not different (P=0.0854)). Subjects with decreased numbers of CTC or no change in CTC with treatment stayed on study longer (not significant, P=0.0512, t test) than subjects with increased CTC with treatment (2.95 vs. 1.70 months). 73.3% (11/15) of subjects with positive CTC at screening had at least 1 CTC positive for IGF-1R. In conclusion, our results provide evidence for the utility of CTC as prognostic indicators in a Phase 1 study. Citation Format: Xiaoru Chen, Xiaoqing (Sarah) Shi, Haifeng Bao, Theresa M. LaVallee, Dirk B. Mendel, Patricia A. Burke. CTC enumeration and molecular characterization in a Phase 1 study of IGF-1 and IGF-2 inhibition by MEDI-573. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3484. doi:10.1158/1538-7445.AM2013-3484

Abstract 4587: In vitro and In vivo characterization of circulating tumor cells from breast cancer patients.

Circulating tumor cells (CTCs) are an independent prognostic marker associated with poor clinical outcome in patients with metastatic diseases. However, CTC biology, especially the tumorigenic and metastatic activity of CTCs, is not yet well understood. In this study, we hypothesized that CTCs contain a population of cancer stem cells (CSCs) that are the seed cells responsible for metastases. To test this hypothesis, we sought to assess subpopulations of CTCs in breast cancer patients and to characterize CTCs for CSC properties and tumorigenic potential. For assessment of CTC subsets, blood samples from breast cancer patients were enriched for CTCs by magnetic separation. The enriched CTC samples were immunostained for various cell surface markers and then analyzed by FACS. The phenotypes of CTCs were further confirmed by imaging analysis. To evaluate tumorigenicity of CTCs, CTC samples from patients were cultured in vitro and then implanted into immunodeficient mice by orthotopic injection. Different phenotypes of CTCs were found in breast cancer patients, including EpCAM + CD44 − CD24 −/di m CD45 − , EpCAM + CD44 + CD24 −/di m CD45 − , EpCAM + CD44 + CD24 + CD45 − , and EpCAM + CD44 + CD24 + CD45 dim . Of these phenotypes, EpCAM + CD44 + CD24 −/di m cells have been reported as CSCs in breast cancer. This subset of CSC-like CTCs constituted 4.6% to 71% of the total CTC population. In vitro culture of CTCs resulted in the generation of mammospheres, as is typical of CSCs. During in vitro culture, some of CTCs showed staining positive for Ki-67, indicating that these cells were proliferating. To assess tumorigenic potential of CTCs, cultured CTCs were injected into the mouse mammary fat pads of immunodeficient mice. Human tumors were developed in these mice. Further analysis of the CTC-derived tumor xenografts demonstrated a heterogenous phenotype where EpCAM + CD44 + CD24 dim/ − CSCs accounted for less than 3% of the total tumor cells. Only this subset of cells isolated from CTC-derived xenograft tumors could form secondary tumors after being reimplanted into mice. Our results support the hypothesis that CTCs contain a CSC-like population that may initiate new tumors. As a result, CTCs may be a potential therapeutic target for the prevention of metastasis in addition to serving as a readily accessible source for biomarker evaluation of new therapies in cancer patients. Citation Format: Sanjoo Jalla, Xiaoru Chen, Patricia Burke, Xiaoqing Shi, Meggan Czapiga, Vivekananda Datta, Philip Brohawn, Jiaqi Huang, Charles Brown, Elaine Hurt, Laura Richman, Robert Hollingsworth, Theresa LaVallee, Haifeng Bao. In vitro and In vivo characterization of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4587. doi:10.1158/1538-7445.AM2013-4587

Abstract 796: Molecular characterization of circulating tumor cells using fluidigm biomark dynamic array

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Circulating tumor cells (CTCs) are cancer cells derived from a primary tumor and/or its metastases that circulate in the peripheral blood. The number of CTCs in whole blood of cancer patients has been shown to have clinical relevance with respect to patient prognosis. There is, in addition to CTC enumeration, great interest in the molecular characterization of isolated CTCs. However, there are challenges for molecular characterization of CTCs with commonly used methodologies. The number of CTCs is extremely low and despite the depletion of leukocytes during cell enrichment, the CTC-enriched fractions still include numerous leukocytes which strongly impact CTC-specific gene expression profiling. CellSearch™ CTC test is a FDA approved diagnostic test for enumeration of CTCs. The fixation reagents in the CellSave tube allow processing of samples for several days post-collection, making analysis of clinical samples more practical compared to near-same day processing required for samples collected in EDTA tubes. However, the fixation decreases the mRNA signal and assay sensitivity. We have established a protocol which overcomes these hurdles and allows the profiling of select transcripts of interest in CTCs. By applying a proteinase K treatment and Trizol LS lysis prior to RNA isolation, we were able to overcome the impact of fixation on RNA isolation. RNA was isolated from the treated lysate using a commercially available kit applicable to low cell numbers. Use of this kit allowed us to utilize all of the isolated RNA in the cDNA step. Superscript III cDNA synthesis was then carried out according to the manufacturers protocol. Target genes of interest were then pre-amplified using the Life Technologies established methodology and reagents. The resulting volume of pre-amplified cDNA was concentrated to a set volume to maximize the amount of transcript profiled. Genes of interest were profiled on the Fluidigm Biomark 48.48 Array. mRNA expression analysis was performed on genes which are not expressed in a pure leukocyte population to ensure that any signal detected was from the CTCs, and not from the background leukocyte population. Using this methodology, we succeeded in performing quantitative gene expression analysis on genes such as PDGFRα on as few as five tumors cells (MG63) fixed in CellSave tubes and spiked into a background environment of contaminating leukocytes (typically 1000 or more) pulled down from normal blood using the CellSearch system. These methods could potentially be used not only for greater understanding of the biology and metastatic potentialof CTCs Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 796.

Abstract 3736: Application of circulating tumor cells and circulating cell-free DNA to assess the pharmacodynamic response to chemotherapy in xenograft models

Circulating tumor cells (CTCs) are found in various cancer patients and are a prognostic factor for survival in some tumor indications. Circulating cell-free DNA is elevated in cancer patients and has been shown to decrease in response to treatments that result in clinical benefit in cancer patients. CTCs and circulating DNA have the potential to serve as minimally invasive methods that allow monitoring response of tumor to anticancer treatment. Our objectives were to 1) establish analytical methods for enumeration and characterization of CTCs from human xenografts in mice, and 2) evaluate CTCs and circulating DNA as pharmacodynamic (PD) markers/proof of mechanism biomarkers for anticancer therapy in xenograft models. Methods: the Veridex CellSearch™ method was modified for quantifying CTCs (defined as nucleated cells positive for cytokeratin but negative for mouse CD45) in mouse whole blood. The assay was characterized and validated by spiking humor tumor cells into mouse whole blood. Additional markers of Ki-67 and cleaved caspase 3 were added to the assay for assessment of proliferation and apoptosis of CTCs. Circulating nucleosomal DNA was measured in plasma by anti-histone H3 ELISA assay. Results: CTCs were detectable in blood from mice with tumor xenografts of PC3-M, MDA-MB-468, and H358. The number of CTCs was not correlated with the volume of the primary tumor. To evaluate changes of CTCs and circulating DNA in response to anticancer therapy, mice with tumor xenografts were treated with taxotere (0, 2.5, 7.5 or 15 mg/kg) to assess dose response or following a single dose (15 mg/kg) to assess response kinetics at 1, 3, 5 and 8 days. Treatment of tumor-bearing mice with taxotere increased the total number of CTCs but decreased the percentage of proliferating Ki-67 positive CTCs, which was consistent with decreased levels of Ki-67 positive cells observed in tumor sections. The increase in the number of CTCs was transient, peaking on day 3 and then returning to a low level by day 5 post-treatment. Circulating DNA levels were significantly elevated in tumor-bearing mice as compared with naive mice and were positively correlated with tumor growth. Taxotere treatment further increased the level of circulating DNA in a dose-dependent manner. The elevation of the level of circulating DNA peaked on day 5 and returned to pre-treatment level on day 8. These results suggest that the release of tumor cells into circulation with decreased proliferative potential combined with increased circulating cell-free DNA may represent markers for the early response to effective cancer treatments. Studies in mice support the use of CTCs, particularly if characterized for proliferative potential, in addition to circulating DNA, as potential early response/proof of mechanism biomarkers for anticancer drug candidates and warrant further evaluation in human clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3736.