Single Cell Network Profiling Research Articles

Baseline T cell dysfunction by single cell network profiling in metastatic breast cancer patients

BackgroundWe previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFβ blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFβ blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP).MethodsThe original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression.ResultsAt baseline, a significantly higher level of expression (p < 0.05) of PD-L1 was identified in patient monocytes compared to HD. TCR modulation revealed dysfunction of circulating T-cells in patient baseline samples as compared to HD, and this was more pronounced in PD-1+ cells. Treatment with radiotherapy and fresolimumab did not resolve this dyfunctional signaling. However, in vitro PD-1 blockade enhanced TCR signaling in patient PD-1+ T cells and not in PD-1- T cells or in PD-1+ T cells from HD.ConclusionsFunctional T cell analysis suggests that baseline T cell functionality is hampered in metastatic breast cancer patients, at least in part mediated by the PD-1 signaling pathway. These preliminary data support the rationale for investigating the possible beneficial effects of adding PD-1 blockade to improve responses to TGFβ blockade and radiotherapy.Trial registrationNCT01401062.

Abstract 4987: Role of the PD-1/PDL-1 pathway in resistance of patients with metastatic breast cancer to treatment with radiotherapy and TGFβ neutralization

Abstract Experimental data and clinical observations indicate that local radiation therapy (RT) converts the irradiated tumor into an in situ vaccine and improves responses to immunotherapy. Preclinical breast cancer models show that TGFb neutralization was required to achieve CD8+ T cell priming specific for multiple tumor antigens, and rejection of the irradiated tumor and non-irradiated metastases (Vanpouille-Box et al., Cancer Res 2015). Tumor response and survival were improved by PD-1 blockade. We explored the role of the PD-1/PDL-1 pathway in resistance to combined RT + TGFβ neutralizing antibody (fresolimumab) in metastatic breast cancer patients (pts). 22 pts were treated in a prospective trial at NYU and UCLA (NCT01401062, supported by DOD MTA BC100481). Pts were randomly assigned to 2 doses of fresolimumab (freso) (1mg/kg and 10 mg/kg, q 3 weeks). Image guided RT was delivered to one metastasis on weeks 2 and 7, 7.5 GyX3. 1 pt achieved objective response, with 28% reduction of tumor burden. The response lasted 11 months, anthracycline-induced acute myelogenous leukemia developed. Pts randomized to the higher freso dose had a hazard ratio of 2.17 (95% CI: 0.753-6.272) for risk of death at 1 year follow up. Currently, at a median follow up of 2 years, 20/22 pts have died. To explore reasons for the limited response, peripheral blood mononuclear cells collected at baseline, 5 and 15 weeks into treatment, were analyzed. Responses to survivin, as a tumor antigen, were assessed using tetramers. Single cell network profiling (SCNP) was used to evaluate expression of 6 immunomodulatory receptors plus immune signaling in T cells collected from 7 healthy donors (HD) and 15 breast cancer pts (6 at 1mg/kg, 9 at 10mg/kg fresolimumab). In vitro activation of p-AKT and p-Erk was quantified following in vitro T cell receptor (TCR) modulation with anti-CD3/αnti-CD28. 6/12 HLA-A2.1+ pts had pre-existing survivin-specific CD8+ T cells, and 3 showed an increase over time. 2 pts who were negative generated modest responses after RT and 10 mg/kg freso. Prior to treatment, PDL-1 expression was increased in monocytes (p = 0.01) and CD4+ T cells (p = 0.037) compared to HD. Elevated PD-1 (p = 0.054) and OX-40 (p = 0.014) expression were identified on CD4+ T cells of pts. Upon TCR modulation, CD4+ and CD8+ T cells from pts showed reduced signaling through p-AKT and, to a lesser extent, p-Erk, compared to HD. Signaling was lower in PD-1+ vs PD-1- CD8+ and CD4+ T cells. Addition of anti-PD-1 pembrolizumab partially restored TCR-mediated signaling through p-AKT and p-Erk in PD-1+ but not PD-1- T cells. Overall, data indicate impaired T cell signaling in PD-1+ T cells of metastatic breast cancer pts, which may explain the inability to respond to RT + TGFβ blockade. In vitro addition of anti-PD-1 improved T cell signaling through p-AKT and p-Erk. Together with the preclinical data, this supports adding anti-PD-1 to the combination of RT +TGFβ blockade. Citation Format: Sandra Demaria, Claire Vanpouille-Box, Rachael E. Hawtin, Christie Fanton, Andy Conroy, Erik Evensen, Neha Dixit, Alan Barber, Dorthe Schaue, William H. McBride, Mary Helen Barcellos-Hoff, Silvia C. Formenti. Role of the PD-1/PDL-1 pathway in resistance of patients with metastatic breast cancer to treatment with radiotherapy and TGFβ neutralization. [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 4987.

Identification and characterization of distinct IL-17F expression patterns and signaling pathways in chronic lymphocytic leukemia and normal B lymphocytes.

Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of B lymphocytes. T cell abnormalities are a common feature of CLL and contribute to impaired immune function in these patients. T cells are ineffective in eliminating the leukemic clone and may actually promote tumor growth and survival. Previous work from our laboratory documented elevated circulating levels of IL-17A-producing Th17 cells in CLL patients as compared to healthy age-matched control subjects. These high circulating Th17 levels associated with better prognostic markers and significantly longer overall survival, even among patients whose clones used unmutated IGHVs (U-CLL). Recent studies suggest that Th17 cells are heterogeneous, expressing different profiles of cytokines, and that different subsets of Th17s mediate different biological functions. In the present study, we found significantly higher levels of IL-17F-expressing CD4+ T cells in CLL versus healthy peripheral blood mononuclear cells following in vitro stimulation in the presence of Th17-promoting cytokines. Furthermore, the differentiation of IL-17F-expressing Th17 cells was significantly enhanced when purified CD4+ T cells from CLL patients were cultured in the presence of autologous CLL B cells. Lastly, single-cell network profiling revealed that IL-17F triggers NFκB phosphorylation in T and B cells from patients with CLL, but not age-matched healthy controls. Taken together, our data suggest that the phenotype of Th17 cells in CLL patients is distinct from healthy individuals, expressing higher levels of IL-17F, and that B and T cells from CLL patients are particularly responsive to IL-17F, as compared to healthy age-matched control individuals.Electronic supplementary materialThe online version of this article (doi:10.1007/s12026-015-8722-5) contains supplementary material, which is available to authorized users.

Immune monitoring technology primer: Single Cell Network Profiling (SCNP).

Description of the technology Understanding a patients’ immune status not only from immune cell phenotyping, but also through analysis of functional signaling capacity, enables the generation of a more comprehensive understanding of the complex mechanisms responsible for immunological tolerance in cancer, and generates data that is complementary to other non-functional phenotypic data sets such as immunohistochemical profiling and genomic analyses. Single cell network profiling (SCNP) is a technology that quantifies functional immune signaling capacity and connectivity at a systems biology level. The technology is based on multiparametric flow cytometry that simultaneously quantifies in multiple and rare immune cell subsets, without the need for physical separation, both extracellular surface markers and changes in intracellular signaling proteins in response to extracellular modulators. Quantifying modulated signaling across a panel of modulators (e.g., IFNα, IFNγ, IL-4, IL-10, IL-27, antiCD3 etc.) and intracellular signaling pathways identifies the functional capacity of the signaling network which cannot be assessed by measuring basal (unmodulated) signaling alone. A signaling node is defined as the combination of the extracellular modulator with the intracellular readout. For example TLR4 - > p-Erk defines one signaling node in which TLR4 modulation is quantified through the increase in p-Erk levels as compared to the unmodulated reference. Typically 3 nodes are captured simultaneously per well across multiple immune cell subsets of interest (e.g., TLR4 - > p-Erk, p-S6, IkB). The application of SCNP to clinical decision-making requires the generation of high-content SCNP assays with robust, accurate, quantifiable and reproducible results across time, operators and instruments. Each of the procedural steps associated with an SCNP assay, including pre-analytical sample handling, assay execution and reagents, data acquisition and analysis and the generation of metrics, have been validated [1] (Figs. 1 and 2). Experimental assay setup is performed using proprietary software which enables experimental design/96well plate layouts and data capture to be contiguously linked, ensuring that data from each well is correctly assigned. The laboratory execution can be performed on as many as 30 samples assayed for up to 40 wells (approximately 200–500 SCNP dimensions comprising modulator/ inhibitor/intracellular readout/cell subset combinations) in 2 to 3 days depending on the kinetic time points. A statistical analysis plan (SAP) is drafted for all studies beyond the exploratory phase, based upon clearly stated objectives. For the identification of clinically validated classifiers the time frame for assay development and validation is comparable to that of other technologies (e.g., genomics, IHC) due to the requirements for statistical powering and for verification and validation in independent sample sets.

Cell signaling-based classifier predicts response to induction therapy in elderly patients with acute myeloid leukemia.

Single-cell network profiling (SCNP) data generated from multi-parametric flow cytometry analysis of bone marrow (BM) and peripheral blood (PB) samples collected from patients >55 years old with non-M3 AML were used to train and validate a diagnostic classifier (DXSCNP) for predicting response to standard induction chemotherapy (complete response [CR] or CR with incomplete hematologic recovery [CRi] versus resistant disease [RD]). SCNP-evaluable patients from four SWOG AML trials were randomized between Training (N = 74 patients with CR, CRi or RD; BM set = 43; PB set = 57) and Validation Analysis Sets (N = 71; BM set = 42, PB set = 53). Cell survival, differentiation, and apoptosis pathway signaling were used as potential inputs for DXSCNP. Five DXSCNP classifiers were developed on the SWOG Training set and tested for prediction accuracy in an independent BM verification sample set (N = 24) from ECOG AML trials to select the final classifier, which was a significant predictor of CR/CRi (area under the receiver operating characteristic curve AUROC = 0.76, p = 0.01). The selected classifier was then validated in the SWOG BM Validation Set (AUROC = 0.72, p = 0.02). Importantly, a classifier developed using only clinical and molecular inputs from the same sample set (DXCLINICAL2) lacked prediction accuracy: AUROC = 0.61 (p = 0.18) in the BM Verification Set and 0.53 (p = 0.38) in the BM Validation Set. Notably, the DXSCNP classifier was still significant in predicting response in the BM Validation Analysis Set after controlling for DXCLINICAL2 (p = 0.03), showing that DXSCNP provides information that is independent from that provided by currently used prognostic markers. Taken together, these data show that the proteomic classifier may provide prognostic information relevant to treatment planning beyond genetic mutations and traditional prognostic factors in elderly AML.

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.

FRI0035 Prediction of TNF Inhibitor Response in Rheumatoid Arthritis Patients Using Single Cell Network Profiling of Intracellular Immune Signaling

BackgroundBiomarkers predictive of drug efficacy are lacking in rheumatoid arthritis (RA) and would be useful in clinical practice and clinical trials. Single cell network profiling (SCNP) is a multiparametric flow...

Intracellular signaling as measured by single cell network profiling (SCNP) is stable in longtitudinal samples of peripheral blood mononuclear cells (PBMC) from healthy donors (HD) across multiple immune cell subsets (TECH1P.839)

Abstract Biomarkers of immune function from viable cells would be of high value for many clinical applications. The immune system responds rapidly to a variety of stimuli, so identifying immune measures that are stable, robust, and relevant to clinical features/outcomes can be challenging. SCNP is a multiparametric flow cytometry assay measuring induced changes in intracellular signaling, providing a functional measure of pathway capacity in multiple cell subsets without physical separation. To assess the longitudinal stability of signaling capacity, PBMCs from 11 healthy donors (7 female, age 29 - 60) were collected at 2 time points and characterized by SCNP of 32 nodes (combinations of a modulator and an intracellular signaling readout) in 21 cell subsets. Signaling stability was assessed for each node:subset combination, calculating the ratio of the signaling variability between time points to the mean signaling level. Of 258 node:subset combinations measured: 160 (62%) had a ratio &amp;lt;0.1; 227 (88%) were &amp;lt;0.2 (i.e. the variability between time points is &amp;lt;20% of the average signaling). Memory B cells had the greatest, though modest, variability in signaling between sample times (median ratio 0.15, range 0.04 - 0.43); naive CD4- T cells had the least variation (median ratio 0.066, range 0.03 - 0.13). This study serves as reference for biomarker development for immune monitoring, demonstrating that signaling capacity is a stable, robust phenotype over time and within an individual.

Systems biology analysis of immune signaling in peripheral blood mononuclear cells (PBMC) of melanoma patients receiving ipilimumab; basis for clinical response biomarker identification

Meeting abstracts Ipilimumab, an anti-CTLA-4 monoclonal antibody, is approved for treatment of unresectable/metastatic melanoma. Treatment benefits only a subset of patients (pts) and is associated with significant adverse effects. Biomarkers of pt response are needed. Single cell network profiling

Age-related changes of healthy bone marrow cell signaling in response to growth factors provide insight into low risk MDS

Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that quantifiably and simultaneously measures changes in intracellular signaling proteins in response to in vitro extracellular modulators at the single cell level. Myelodysplastic syndrome (MDS) is a heterogeneous clonal disorder of hematopoietic stem cells that occurs in elderly subjects and is characterized by dysplasia and ineffective hematopoiesis. The functional responsiveness of MDS bone marrow (BM) hematopoietic cells, including functionally distinct myeloid and erythroid precursor subsets, to hematopoietic growth factors (HGF) and the relationship of modulated signaling to disease characteristics is poorly understood. SCNP was used first to examine the effects of age on erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF)-induced signaling in myeloid, nucleated red blood cells (nRBC), and CD34 expressing cell subsets in healthy BM (n = 15). SCNP was then used to map functional signaling profiles in low risk (LR) MDS (n = 7) for comparison to signaling in samples from healthy donors and to probe signaling associations within clinically defined subgroups. In healthy BM samples, signaling responses to HGF were quite homogeneous (i.e., tightly regulated) with age-dependent effects observed in response to EPO but not to GCSF. Despite the relatively small number of samples assayed in the study, LR MDS could be classified into distinct subgroups based on both cell subset frequency and signaling profiles. As a correlate of underlying genetic abnormalities, signal transduction analyses may provide a functional and potentially clinically relevant classification of MDS. Further evaluation in a larger cohort is warranted.

Age-related changes of healthy bone marrow cell signaling in response to growth factors provide insight into low risk MDS.

Background Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that quantifiably and simultaneously measures changes in intracellular signaling proteins in response to in vitro extracellular modulators at the single cell level. Myelodysplastic syndrome (MDS) is a heterogeneous clonal disorder of hematopoietic stem cells that occurs in elderly subjects and is characterized by dysplasia and ineffective hematopoiesis. The functional responsiveness of MDS bone marrow (BM) hematopoietic cells, including functionally distinct myeloid and erythroid precursor subsets, to hematopoietic growth factors (HGF) and the relationship of modulated signaling to disease characteristics is poorly understood. Methods SCNP was used first to examine the effects of age on erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF)-induced signaling in myeloid, nucleated red blood cells (nRBC), and CD34 expressing cell subsets in healthy BM (n=15). SCNP was then used to map functional signaling profiles in low risk (LR) MDS (n=7) for comparison to signaling in samples from healthy donors and to probe signaling associations within clinically defined subgroups. Results In healthy BM samples, signaling responses to HGF were quite homogeneous (i.e. tightly regulated) with age-dependent effects observed in response to EPO but not to GCSF. Despite the relatively small number of samples assayed in the study, LR MDS could be classified into distinct subgroups based on both cell subset frequency and signaling profiles. Conclusion As a correlate of underlying genetic abnormalities, signal transduction analyses may provide a functional and potentially clinically relevant classification of MDS. Further evaluation in a larger cohort is warranted. © 2013 Clinical Cytometry Society.

Development and validation of a single‐cell network profiling assay‐based classifier to predict response to induction therapy in paediatric patients withde novoacute myeloid leukaemia: a report from the Children's Oncology Group

Single cell network profiling (SCNP) is a multi-parameter flow cytometry technique for simultaneous interrogation of intracellular signalling pathways. Diagnostic paediatric acute myeloid leukaemia (AML) bone marrow samples were used to develop a classifier for response to induction therapy in 53 samples and validated in an independent set of 68 samples. The area under the curve of a receiver operating characteristic curve (AUC(ROC)) was calculated to be 0·85 in the training set and after exclusion of induction deaths, the AUC(ROC) of the classifier was 0·70 (P = 0·02) and 0·67 (P = 0·04) in the validation set when induction deaths (intent to treat) were included. The highest predictive accuracy was noted in the cytogenetic intermediate risk patients (AUC(ROC) 0·88, P = 0·002), a subgroup that lacks prognostic/predictive biomarkers for induction response. Only white blood cell count and cytogenetic risk were associated with response to induction therapy in the validation set. After controlling for these variables, the SCNP classifier score was associated with complete remission (P = 0·017), indicating that the classifier provides information independent of other clinical variables that were jointly associated with response. This is the first validation of an SCNP classifier to predict response to induction chemotherapy. Herein we demonstrate the usefulness of quantitative SCNP under modulated conditions to provide independent information on AML disease biology and induction response.

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).

Abstract 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 &amp;gt;1.3, indicating aneuploidy and therefore tumor origin. The majority of epithelial cells were cPARP negative (&amp;gt;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).

Abstract Background: A recent report described an imatinib/dasatinib (Imat/Dasat) resistant MCL patient (pt) with mutations in KIT (V654A) and FcεR1 (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 FcεR1 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 FcεR1 receptor and inhibition by fostamatinib treatment, no basal or α-IgE modulated FcεR1 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 FcεR1 receptor and inhibition by fostamatinib treatment, no basal or α-IgE modulated FcεR1 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 FcεR1 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

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.

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.

AKT Signaling as a Novel Factor Associated with In Vitro Resistance of Human AML to Gemtuzumab Ozogamicin

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ1 derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ1 resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ1 with particularly pronounced effects in otherwise GO or free calicheamicin-γ1 -resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ1. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ1 and, by extrapolation, other DNA damage-based therapeutics.

Comparison of functional immune signaling profiles in peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis (RA) patients versus healthy donors (HD) using Single Cell Network Profiling (SCNP)

Event Abstract Back to Event Comparison of functional immune signaling profiles in peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis (RA) patients versus healthy donors (HD) using Single Cell Network Profiling (SCNP) Jason Ptacek1, Rachael Hawtin1*, Erik Evensen2, James Cordeiro2, Michelle Atallah2, Carol Marimpietri2, Alessandra Cesano2, . TETRAD Investigators3, Peter K. Gregersen4 and S. L. Bridges5 1 Co-first authors, Nodality, Inc., United States 2 Nodality, Inc., United States 3 TETRAD Clinical Sites/UAB Coordinating Center, United States 4 The Feinstein Institute for Medical Research, United States 5 University of Alabama at Birmingham, United States Prognostic and predictive biomarkers are lacking in RA. SCNP is a multiparametric flow cytometry-based assay that simultaneously measures changes in multiple intracellular signaling proteins in response to modulators providing a functional measure of pathway activity in single cells. SCNP of 42 nodes (modulator→intracellular readout) within 21 immune cell subsets was performed on PBMCs from 181 RA patients collected before initiating new treatment, either MTX or biologic agent, within the national Treatment Efficacy and Toxicity in Rheumatoid Arthritis Database and Repository (TETRAD), and 10 age- and gender-matched healthy donors. TETRAD participants were 86% female and 76.5% Caucasian. All met ACR classification criteria for RA and mean Disease Activity Score on 28 joints (DAS28) was 4.77±1.40 [SD]. Using half the donors as a training set, multiple variations in signaling responses in discrete cell subsets associated with donor characteristics (e.g. healthy vs. RA, disease activity) were identified, and will be subsequently confirmed in the remaining donors (test set). Specifically, TNFα signaling was lower in monocytes in most RA samples while analysis of T cell subsets identified significant differences with opposing directionality in IL-6 signaling as compared to healthy: RA helper T cell subsets had decreased IL-6→p-STAT1/3; cytotoxic T cell subsets showed increasing responsiveness to IL-6; central memory cytotoxic T cells had a significant increase in IL-6→p-STAT1. The clinical significance of these observations will be investigated with relation to RA pathophysiology and treatment response. These data reveal the functional biology associated with RA pathophysiology and enable the identification of potential prognostic and predictive biomarkers. Keywords: Rheumatoid arthritis, Autoimmunity, multicolor flow cytometry, Signal Transduction, biomarkers Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Ptacek J, Hawtin R, Evensen E, Cordeiro J, Atallah M, Marimpietri C, Cesano A, TETRAD Investigators ., Gregersen PK and Bridges SL (2013). Comparison of functional immune signaling profiles in peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis (RA) patients versus healthy donors (HD) using Single Cell Network Profiling (SCNP). Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00473 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Rachael Hawtin, Co-first authors, Nodality, Inc., South San Francisco, California, United States, rachael.hawtin@nodality.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jason Ptacek Rachael Hawtin Erik Evensen James Cordeiro Michelle Atallah Carol Marimpietri Alessandra Cesano . TETRAD Investigators Peter K Gregersen S. L Bridges Google Jason Ptacek Rachael Hawtin Erik Evensen James Cordeiro Michelle Atallah Carol Marimpietri Alessandra Cesano . TETRAD Investigators Peter K Gregersen S. L Bridges Google Scholar Jason Ptacek Rachael Hawtin Erik Evensen James Cordeiro Michelle Atallah Carol Marimpietri Alessandra Cesano . TETRAD Investigators Peter K Gregersen S. L Bridges PubMed Jason Ptacek Rachael Hawtin Erik Evensen James Cordeiro Michelle Atallah Carol Marimpietri Alessandra Cesano . TETRAD Investigators Peter K Gregersen S. L Bridges Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Functional proteomic interrogation of immune cell crosstalk and the effects of cytokine-targeted inhibitors using Single Cell Network Profiling (SCNP)

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

Abstract 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.