Scott Z. Fields

Epratuzumab, a Humanized Anti-CD22 Antibody, in Aggressive Non-Hodgkin’s Lymphoma: Phase I/II Clinical Trial Results

Purpose: We conducted a single-center, dose-escalation study evaluating the safety, pharmacokinetics, and efficacy of epratuzumab, an anti-CD22 humanized monoclonal antibody, in patients with aggressive non-Hodgkin’s lymphoma. Experimental Design: Epratuzumab was administered once weekly for 4 weeks at 120-1000-mg/m2 doses to 56 patients [most (n = 35) with diffuse large B-cell lymphoma]. Results: Patients were heavily pretreated (median, 4 prior therapies), 25% received prior high-dose chemotherapy with stem cell transplant, and 84% had bulky disease (≥5 cm). Epratuzumab was well tolerated, with no dose-limiting toxicity. Most (95%) infusions were completed within 1 h. The mean serum half-life was 23.9 days. Across all dose levels and histologies, objective responses (ORs) were observed in five patients (10%; 95% confidence interval, 3–21%), including three complete responses. In patients with diffuse large B-cell lymphoma, 15% had ORs. Overall, 11 (20%) patients experienced some tumor mass reduction. Median duration of OR was 26.3 weeks, and median time to progression for responders was 35 weeks. Two responses are ongoing at ≥34 months, including one rituximab-refractory patient. Conclusions: These data demonstrate that epratuzumab has a good safety profile and exerts antitumor activity in aggressive non-Hodgkin’s lymphoma at doses of ≥240 mg/m2, thus warranting further evaluation in this clinical setting.

A Phase I Study of 7-t-Butyldimethylsilyl-10-Hydroxycamptothecin in Adult Patients with Refractory or Metastatic Solid Malignancies

Purpose: 7-t-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67) is a novel third generation camptothecin selected for development based on the blood stability of its pharmacologically active lactone form and its high potency in preclinical models. Here, we report the initial phase I experience with i.v. AR-67 in adults with refractory solid tumors. Experimental Design and Methods: AR-67 was infused over 1 hour daily five times, every 21 days, using an accelerated titration trial design. Plasma was collected on the 1st and 4th day of cycle 1 to determine pharmacokinetic parameters. Results: Twenty-six patients were treated at nine dosage levels (1.2-12.4 mg/m2/d). Dose-limiting toxicities were observed in five patients and consisted of grade 4 febrile neutropenia, grade 3 fatigue, and grade 4 thrombocytopenia. Common toxicities included leukopenia (23%), thrombocytopenia (15.4%), fatigue (15.4%), neutropenia (11.5%), and anemia (11.5%). No diarrhea was observed. The maximum tolerated dosage was 7.5 mg/m2/d. The lactone form was the predominant species in plasma (>87% of area under the plasma concentration-time curve) at all dosages. No drug accumulation was observed on day 4. Clearance was constant with increasing dosage and hematologic toxicities correlated with exposure (P < 0.001). A prolonged partial response was observed in one subject with non–small cell lung cancer. Stable disease was noted in patients with small cell lung cancer, non–small cell lung cancer, and duodenal cancer. Conclusions: AR-67 is a novel, blood-stable camptothecin with a predictable toxicity profile and linear pharmacokinetics. The recommended phase II dosage is 7.5 mg/m2/d five times every 21 days. Clin Cancer Res; 16(2); 673–80

Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP)

BackgroundHomologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity.MethodsFlow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates.ResultsEtoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples.ConclusionsSCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.

Single cell network profiling assay in bladder cancer

The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved‐PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p‐AKT and p‐ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the “evaluable” criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor‐induced p‐AKT and p‐ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC‐0941 reduced or completely inhibited basal and epidermal growth factor‐induced p‐AKT but, as expected, had no effect on p‐ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve as a tool for disease characterization and drug development in bladder cancer and other solid tumors.

Abstract 3523: Quantitative measurements of EGFR pathway signaling and modulation in pleural effusion samples from non-small cell lung cancer (NSCLC) patients using single cell network profiling (SCNP).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: SCNP is a multiparametric flow cytometry-based assay that quantitatively and simultaneously measures, at the single cell level, both extracellular surface markers and activation levels of intracellular signaling proteins in response to modulation. SCNP has been successfully applied to hematologic malignancies in both prognostic and predictive analyses. Previous work using bladder washes from bladder cancer patients (pts) established the feasibility of applying SCNP to the functional characterization of epithelial cell signaling and response to modulation and targeted kinase inhibition in non-conventional samples. Objectives: Assess the feasibility of applying SCNP to detect and functionally characterize epithelial cells, including sensitivity to targeted kinase inhibition, in pleural effusion (PE) samples from NSCLC pts. Methods: PE from confirmed NSCLC pts (n=4) were collected at the NSUH and shipped overnight to Nodality for processing on day of receipt. Antibodies to CD45, CytoKeratin (CK), EpCAM, and cPARP were used to differentiate non-apoptotic epithelial cells from leukocytes. DNA aneuploidy was evaluated using the lymphoid population in the patient samples as a diploid G0/G1 reference. Signaling in PI3K and MAPK pathways was quantified through measurement of p-AKT and p-ERK levels at baseline and after in vitro exposure to EGF +/- PI3K inhibitor GDC-0941. The epithelial bladder carcinoma cell line HT-1376 was used as a positive control for epithelial phenotypic staining and EGF-induced signaling. Results: In 3 of 4 PE samples, epithelial cells (DAPI+, CD45-, CK+, and EpCAM+) were identified (range 0.35% to 15.87% of all nucleated cells). Epithelial cells had a DNA index >1.3, indicating aneuploidy and therefore tumor origin. The majority of epithelial cells were cPARP negative (>80%) and suitable for SCNP analysis. In all 3 samples with epithelial cells: 1) EGF modulation induced increased p-AKT and p-ERK; 2) Pre-treatment with GDC-0941 inhibited EGF induced p-AKT but not p-ERK; 3) Constitutive activation of the PI3K pathway in the tumor epithelial cells was suggested by reduction in p-AKT levels compared to basal (no EGF exposure) following GDC-0941 treatment. Conclusion: This study demonstrates the feasibility of applying SCNP to the functional characterization of PE samples from NSCLC pts. Ongoing analyses will expand upon these data, including both additional NSCLC PE samples and the evaluation of compounds targeting alternative signaling pathways relevant to NSCLC (eg: MAPK, EGFR). The feasibility of extending such SCNP analyses to other solid tumor indications, using alternative tissue sources and circulating tumor cells, is under further investigation for both prognostic and response predictive utility. [S.Z. Fields and H. Raftopoulos are senior co-authors.] Citation Format: Matt Westfall, Rachael Hawtin, Todd Covey, Michael Gulrajani, Michelle Atallah, Michelle Cholankeril, Reena Vora, Alessandra Cesano, Scott Z. Fields, Haralambos Raftopoulos. Quantitative measurements of EGFR pathway signaling and modulation in pleural effusion samples from non-small cell lung cancer (NSCLC) patients using single cell network profiling (SCNP). [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 3523. doi:10.1158/1538-7445.AM2013-3523

Abstract 3524: Functional characterization of KIT and FcεR1 receptor mutations in Mast Cell Leukemia using Single Cell Network Profiling (SCNP).

Background: A recent report described an imatinib/dasatinib (Imat/Dasat) resistant MCL patient (pt) with mutations in KIT (V654A) and FceR1 (L188F) receptors [Spector et al, Leukemia 2011]. The pt did not respond to cytarabine-based induction therapy combined with Dasat or to post-induction Imat. The functional consequence of the receptor mutations on downstream signaling networks, and sensitivity to alternative therapeutics, was unknown. SCNP is a flow cytometry-based assay that quantitatively and simultaneously measures, in single cells, extracellular surface markers and activation of intracellular signaling proteins in response to modulation. SCNP was applied to interrogate downstream signaling networks and network sensitivity to targeted therapeutics by examining: 1) Basal and modulated signaling using stem cell factor (SCF) or α-IgE 2) Effect on basal and modulated signaling of: a) KIT inhibitors Imat, Dasat, and Nilotinib b) PI3K inhibitor GDC-0941 c) SYK inhibitor fostamatinib R406 Methods: Cryopreserved MCL BMMCs and healthy donor BMMCs were processed alongside fresh healthy donor basophils (FHDB) as controls. BMMCs were modulated with SCF for 5 and 15 min +/-KIT or PI3K inhib. MCL BMMCs and FHDB were modulated with α-IgE for 5min +/- fostamatinib. Signaling in the KIT and FceR1 pathways was quantified through measurement of p-AKT/ p-ERK/ p-S6 and p-ERK/ p-PLCγ2 / p-SYK levels, respectively, in the MCL population: CD45+, CD34+, CD33+, and CD117+. Results: As previous reported, the V654A KIT mutation did not result in constitutive activation of the PI3K or MAPK pathways in MCL blasts, but was associated with dysfunctional SCF modulated signaling. Specifically, SCNP identified SCF induced p-AKT levels at 5min, higher (2X) compared to CD34+/CD117+ FHDB, and sustained to 15min with no simultaneous induction of p-ERK or p-S6. Consistent with the clinically observed Imat/Dasat resistance of the MCL case, in vitro AKT induction was unaffected by the presence of KIT inhibitors but sensitive to the PI3K inhib GDC-0941. KIT inhibitors and GDC-0941 blocked SCF induced signaling in the healthy BMMC control. Despite robust p-ERK induction in the FHDB control after α-IgE modulation of the FceR1 receptor and inhibition by fostamatinib treatment, no basal or α-IgE modulated FceR1 receptor signaling was detected in MCL BMMC cells. Conclusions: SCNP functionally characterizes signaling and drug resistance profiles in MCL BMMCs and can potentially inform on therapeutic selection. Despite robust p-ERK induction in the FHDB control after α-IgE modulation of the FceR1 receptor and inhibition by fostamatinib treatment, no basal or α-IgE modulated FceR1 receptor signaling was detected in the cryopreserved MCL BMMCs. Further studies will elucidate whether the lack of detected signal could be related to the freeze/thaw process and/or to cell-type specific differences. Citation Format: Matt Westfall, Mona S. Spector, Michael Gulrajani, Carol Marimpietri, Athena Kritharis, Scott Z. Fields, Steven L. Allen, Rachael Hawtin. Functional characterization of KIT and FceR1 receptor mutations in Mast Cell Leukemia using Single Cell Network Profiling (SCNP). [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 3524. doi:10.1158/1538-7445.AM2013-3524

Abstract P6-07-31: Assessing germline Homologous Recombination pathway deficiency in BRCA1 mutation carriers using Single Cell Network Profiling.

Background: Inherited alterations in BRCA1/2 genes increase genomic instability and cancer susceptibility. DNA sequencing detects BRCA1/2 mutations, but has the following limitations; 1) mutations may have unknown functional significance, 2) epigenetic alterations and mutations in other Homologous Recombination (HR) pathway components are not detected, and 3) the combined effects of pathway mutations are not understood. Thus a functional assessment of HR competence at the single cell level remains an unmet need as BRCA1/2 sequencing does not holistically inform on functionality of the HR pathway. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, at the single cell level, extracellular surface markers and functional changes in intracellular signaling in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). In this study, we tested the ability of SCNP to detect and quantify functional changes in HR signaling using peripheral blood mononuclear cell (PBMC) samples from BRCA1 mutation carriers (MUT) and wild type (WT) subjects. Methods: HR pathway activity was examined in PBMCs from BRCA1 MUT (n = 21) or WT (n = 20) subjects. Cell lines carrying BRCA1 MUTor WT genes were used as controls. PBMCs were stimulated with anti-CD3 and anti-CD28 for 24 hours to induce T cell proliferation then treated with PARP inhibitor (PARPi) AZD2281 +/− Temozolomide (TMZ) for 48h or 72h to induce DNA damage. DNA damage response (DDR) readouts were measured in both CyclinA2- and CyclinA2+ T cell subsets. Measurements included induced levels of p21, p53 and phosphorylation (p−) of p-H2AX, p-DNA-PKcs, p-RPA2/32, and p-BRCA1. Results: As expected based on the mechanism of action of PARPi, higher levels of induced p-H2AX and p53 were observed in CyclinA2+ cells of BRCA1 MUT versus WT cell line controls. In PBMCs, T cell proliferation (%CyclinA2+) was positively associated with PARPi induced DDR readouts. After controlling for proliferation, statistically significant differences in PARPi induced DDR signaling were observed between BRCA1 MUT and WT samples in many simultaneously assessed readouts including p-H2AX, p53 and p21 (increased in MUT), particularly in CyclinA2+ cells. Additionally, BRCA1 MUT samples displayed lower basal p-BRCA1 but higher induced p-BRCA1 levels compared to BRCA1 WT samples. Conclusions: SCNP was able to detect and quantify functional differences between PBMC samples from BRCA1 MUT (haploinsufficient) and WT donors by quantitatively assessing DDR signaling in CyclinA2+ T cells. Once verified on a larger data set, the assay could form the basis for the development of screening tests to identify subjects at higher risk of developing cancer or stratification tests to inform on cancer patient selection for treatment with PARP inhibitors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-31.

Abstract C92: Single cell network profiling (SCNP) assay application to bladder cancer (BC) samples: Identifying rare epidermal growth factor (EGF) responsive epithelial cancer cells sensitive to PI3K inhibition from bladder washings.

Background: SCNP is a multiparametric flow cytometry-based assay that simultaneously provides measurements, at the single cell level, of extracellular surface markers and quantitative changes in the activation levels of intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Studies in hematologic malignancies have shown the value of quantitatively measuring single cell signaling networks under modulated conditions as a basis for the development of prognostic and predictive tests. The aim of this study was to assess the feasibility of applying SCNP to the examination of signaling networks in an epithelial cancer, using bladder washings as a BC sample source and EGF +/− the PI3K inhibitor GDC-0941 as modulators. Method: Bladder washes from non-cancer (NC) patients (pts) (n=8) and confirmed/suspected BC pts (n=20) were collected using standard practice and shipped overnight on ice for processing within 24 hours. Antibodies against CD45, CytoKeratin (CK), EpCam, and cleaved-PARP (cPARP) were used to differentiate non-apoptotic epithelial cells from leukocytes, while measurements of DNA aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK both at baseline and in response to pathway modulation. Upon delivery cells were pelleted, counted and incubated for 1 hr at 37°C. GDC-0941 (200nM) or vehicle control was added for 1 hr prior to stimulation with EGF (5 min) or vehicle control. After cell fixation and permeabilization, samples were stained with fluorophore-conjugated antibodies/DAPI cocktail, and data acquired using multi-parametric flow cytometry. The epithelial bladder carcinoma cell line HT-1376 was used as a control for the epithelial phenotype and EGF signaling. DNA index was determined using the lymphoid population in the pt samples as a diploid G0/G1 reference. Results: 50% (N=10) of BC samples and 25% (N=2) of NC samples met the “evaluable” criteria i.e., at least 400,000 total cells upon sample receipt and >2% of cells acquired containing an epithelial phenotype (DAPI+, CD45Low, CK+, and EpCam+). The majority of epithelial cells detected in BC samples were non-apoptotic (i.e. >77% cPARPneg) and therefore suitable for functional pathway analysis. In 3/10 BC samples a quantifiable increase in EGF-induced p-AKT and p-ERK signaling was identified (2–3 fold over baseline) and was preventable by GDC-0941 pre-incubation, whereas no EGF-induced signaling was observed in the NC specimens. Conclusion: This study demonstrates the feasibility of applying SCNP, using multi-parametric flow cytometry, to the functional characterization of BC. Ongoing studies are currently focused on correlation of the individual patient in vitro network signaling profile with clinical outcome to develop prognostic and predictive tools for improved BC management to inform treatment options. The feasibility of extending such SCNP analyses to other solid tumor indications, using alternative tissue sources is also under investigation, e.g., using circulating tumor cells or cells obtained from body fluids such as pleural effusions, ascites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C92.