Single Cell Network Profiling as a Tool to Identify AML Chemotherapy Resistant Cell Phenotypes Under In Vivo Therapeutic Pressure.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 397-397
Author(s):  
Alessandra Cesano ◽  
John Woronicz ◽  
Aileen Cleary Cohen ◽  
Todd Covey ◽  
Santosh Putta ◽  
...  
Keyword(s):  
Single Cell ◽  
Induction Chemotherapy ◽  
Intracellular Signaling ◽  
Cell Subpopulation ◽  
Disease Relapse ◽  
Employment Equity ◽  
Cell Network ◽  
Diagnostic Samples ◽  
Equity Ownership

Abstract Abstract 397 Background: Although most patients with acute myeloid leukemia (AML) have disease that responds initially to standard cytarabine based induction chemotherapy, approximately 2/3 experience leukemia relapse and succumb to the disease. A reliable and reproducible methodology which could identify individual patients at high risk of disease relapse could be used to selectively triage those patients to more intensive post-remission therapies and thereby improve their outcome while minimizing overall toxicity. Objectives: his study evaluated whether single cell network profiling (SCNP), in which cells are perturbed with a modulator and their response ascertained by multiparametric flow cytometry, could be used to characterize specific signaling network profiles associated with in vivo AML blast chemotherapy resistance. The initial hypothesis was to determine whether: a) intracellular signaling profiles dominant at relapse could be identified in subpopulations of cells present at diagnosis and b) whether the presence of blasts with these intracellular signaling profiles at diagnosis could predict for disease relapse. Methods: Modulated SCNP was evaluated after sample incubation with 14 cytokines (e.g. interleukins and G-CSF), growth factors (e.g. FLT3L, SCF), chemotherapeutic agents (e.g. cytarabine, etoposide), and other modulators. The use of fluorochrome-conjugated antibodies that recognized leukemic blasts and intracellular phospho-epitopes allowed signaling to be measured in specific cell types at the single cell level. In addition, drug transporters and surface receptor levels were measured. Results: Four pairs of AML bone marrow or peripheral blood samples were studied. Intra-patient comparison of intracellular signaling profiles were made between diagnostic samples and with either a primary refractory sample after induction chemotherapy or with a sample obtained at first relapse. High morphologic and functional heterogeneity of myeloblasts was observed in all of the samples both at diagnosis and after induction therapy. Notably, a subpopulation of leukemic cells characterized by simultaneous SCF-mediated increases in the levels of phosphorylated (p-) Akt and p-S6, herein defined as “SCF functional signature”, was identified. This functionally defined leukemic cell subpopulation, although dominant in the post induction samples, was present and detectable at a much lower frequency in the diagnostic samples. Cell surface markers, including c-kit expression, were unable to capture these functionally defined cellular subgroups in single defined subpopulations. The value of the newly identified SCF functional signature in predicting early relapse was then assessed in a larger, independent AML sample set of clinically annotated diagnostic samples. This set included 52 AML samples collected from patients whose leukemia achieved a complete remission after induction chemotherapy, 29 of whom experienced disease relapse within 2 years. In seven of the 52 diagnostic AML samples a subpopulation of blasts displaying the SCF functional signature identified above was detected. Six of these patients experienced disease relapse (five patients within ∼1 year and one patient within 2 years from initial CR). The seventh patient of the group, whose AML blasts carried the t(8;21) translocation (a marker of good prognosis), remained in complete continuous remission at 2 years. Noteworthy, the prognostic value of the SCF functional signature was independent of FLT3 mutational status: of the five patients who relapsed in only one case the leukemic blasts carried the FLT3 ITD mutation. Conclusions SCNP offers a novel approach to identify subpopulations of cells present at diagnosis with characteristics predictive of higher rates of relapse and this information can be used for monitoring or to guide therapy. Disclosures: Cesano: Nodality, Inc.: Employment, Equity Ownership. Woronicz:Nodality, Inc.: Employment, Equity Ownership. Cleary Cohen:Nodality Inc.: Employment, Equity Ownership. Covey:Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Gayko:Nodality, Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership. Kornblau:Nodality, Inc.: Consultancy.

Single Cell Network Profiles in Non-M3 AML Associated with Patient Response to Standard Induction Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1582-1582
Author(s):  
Steven Kornblau ◽  
M. D. Minden ◽  
David B. Rosen ◽  
Santosh Putta ◽  
Aileen C. Cohen ◽  
...  
Keyword(s):  
Single Cell ◽  
Signaling Pathways ◽  
Induction Chemotherapy ◽  
Intracellular Signaling ◽  
Independent Set ◽  
Training Set ◽  
Employment Equity ◽  
Disease Response ◽  
Cell Network ◽  
Equity Ownership

Abstract Abstract 1582 Poster Board I-608 Background Traditional AML prognostic markers are based on clinical characterization (e.g. age) or static measurements of leukemia biology present at diagnosis, such as cytogenetics and isolated molecular events (e.g. presence of FLT3 ITD mutation). No validated methods currently exist to predict the disease response to standard AML induction chemotherapy for individual patients. Objectives: Single Cell Network Profiling (SCNP) was used to measure intracellular signaling in response to extracellular modulators in order to develop a new proteomic tool to characterize and monitor AML biology in the context of therapeutic applications. Methods Modulated SCNP using a multiparametric flow cytometry platform was performed evaluating the phosphorylation of intracellular signaling molecules in their basal states and after treatment with modulators in specific cell populations (e.g. leukemic cells). Since multiple signaling pathways may be dysregulated in AML and contribute to the likelihood of response to a given therapy, pathways that affect proliferation, apoptosis, and DNA damage were analyzed. Analyses were aimed to assess assay reproducibility, identify a signaling profile associated with likelihood of response to standard induction chemotherapy (first training set, n=34), and test extrapolation of the identified profile to a fully independent set of AML samples (second training set; n=88). Results High assay reproducibility (Pearson correlation coefficients ≥ 0.8) was observed. In the first training study univariate analysis revealed multiple “nodes” (modulated read outs of proteins in signaling pathways) associated with disease response to conventional induction therapy (i.e. AUC of ROC >0.66; p<0.05). Importantly combination of some of the independently predictive nodes improved disease response stratification (AUC of ROC up to 1.0; p<0.05). Extrapolation of the assay to a second independent set of samples revealed similar findings after accounting for clinical covariates. Specifically, for patients <60 years, the presence of intact apoptotic pathways was correlated with complete response (CR) while in samples from patients ≥60 years increased p-Akt and p-Erk levels in response to FLT3L stimulation correlated with non response (NR). Importantly, the predictive values of these nodes was independent from cytogenetic and FLT3 mutational status. Conclusions The two studies reported here show that AML biology characterization in individual patients using modulated SCNP can be performed with high technical accuracy and reproducibility to quantitatively characterize the biology of AML. This approach can be used to generate highly predictive tests for therapeutic response independently of classic prognostic factors. Disclosures Kornblau: Nodality, Inc.: Consultancy. Rosen:Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Cohen:Nodality, Inc.: Employment, Equity Ownership. Covey:Nodality, Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership. Gayko:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc.: Employment, Equity Ownership.

Characterization of FLT3 Pathway Deregulation by Single Cell Network Profiling (SCNP) Stratifies AML Patients Beyond Molecular Profiling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2739-2739
Author(s):  
David B. Rosen ◽  
Santosh Putta ◽  
Urte Gayko ◽  
Alessandra Cesano ◽  
Larry Cripe ◽  
...  
Keyword(s):  
Single Cell ◽  
Intracellular Signaling ◽  
Kinase Inhibitors ◽  
Flt3 Ligand ◽  
Employment Equity ◽  
Mutation Status ◽  
Cell Network ◽  
Equity Ownership ◽  
Pathway Deregulation

Abstract Abstract 2739 Background: Internal tandem duplication mutations of the FMS-like tyrosine kinase 3 receptor (FLT3-ITD) are among the most frequent mutations in de novo acute myeloblastic leukemia (AML) and result in constitutive activation of the receptor tyrosine kinase. Based on data showing that these mutations negatively affect outcome in AML, FLT3 receptor kinase activity is currently being targeted in the clinic. To date, only FLT3 receptor mutant AML patients have been selected for trials involving FLT3 receptor kinase inhibitors, with surprisingly variable clinical responses. Downstream targets of FLT3 receptor activation, whether by receptor mutation or FLT3 ligand binding, involve Stat5, PI3-kinase/Akt and the Ras/Raf/Erk kinase signal transduction pathways which ultimately affect cell survival and proliferation. Functional characterization of those signaling pathways in mutated versus non-mutated FLT3 receptor (FLT3 -WT) myeloblasts has significant potential to reveal heterogeneity among these genetically defined subgroups and to predict for response to kinase inhibition, independent of FLT3 receptor mutation status. Methods: Modulated single cell network profiling (SCNP) was used to evaluate the activation state of intracellular signaling molecules (i.e. nodes), including phosphorylated (p)-Akt, p-Erk, p-S6, p-Stat5 and cleaved-PARP, at baseline and after treatment with specific modulators [including cytokines (such as IL-27) growth factors (such as FLT3 ligand) and drugs (such as cytosine arabinoside)] in 7 healthy bone marrow mononuclear blasts (BMMb) and leukemic myeloblasts, characterized for FLT3 receptor mutation status, from 44 AML patients (38 FLT-WT and 6 FLT3-ITD), aged >60 years (ECOG trial E3999). A total of 64 node-metrics were analyzed. Results: Signaling profiles differed significantly in FLT3-ITD vs. FLT3-WT AML, and in FLT3-WT vs. BMMb (shown in Figure 1 for a representative node, FLT3 ligand induced p-S6). Specifically, compared to BMMb, FLT3-ITD blasts uniformly showed increased basal p-Stat5 levels, decreased FLT3 ligand-induced activation of P13K and Raf/Ras/Erk pathways, minimal IL-27 induced activation of the Jak/Stat pathway, and higher apoptotic responses to DNA-damaging agents. Two AML samples harboring a low FLT3-ITD mutant burden, however, exhibited a signaling pattern similar to FLT3-WT AML samples. By contrast, FLT3-WT samples displayed heterogeneous signaling profiles, overlapping both with those of FLT3-ITD and BMMb samples, suggesting that a fraction of FLT3-WT AML samples exhibit FLT3 receptor pathway deregulation even without FLT3-ITD. Conclusions This study showed that SCNP, which provides a detailed view of intracellular signaling networks at the single-cell level, subclassified patients with AML beyond their molecularly determined FLT3 mutation status. In particular, a fraction of FLT3-WT AML samples signaled as if containing a FLT3 receptor length mutation while FLT3-ITD samples with low mutational load signaled like FLT3-WT AML samples. The clinical relevance of this observation, both for disease prognosis and response to kinase inhibitors, will be revealed only if AML patients are accrued to kinase inhibition trials irrespective of FLT3 receptor mutation status. The wide range of signaling responses observed in FLT3-WT AML suggests that disease across FLT3-WT patients is heterogeneous, likely promoted through distinct mutations and alterations, giving rise to distinct signaling profiles in individual patients Our data also provide evidence for the co-existence of differentially signaling blast populations in individual patients. The potential impact of signaling heterogeneity on clinical response needs to be assessed and may require an individualized combination of treatment modalities. Disclosure: Rosen: Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership.

Single-Cell Network Profiling (SCNP)-Based Classifier to Predict Response to Induction Therapy in Elderly Patients with Acute Myeloid Leukemia (AML): Validation in Two Independent Sample Sets From ECOG and SWOG Trials.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2489-2489
Author(s):  
Alessandra Cesano ◽  
Cheryl L Willman ◽  
Kenneth J. Kopecky ◽  
Urte Gayko ◽  
Santosh Putta ◽  
...  
Keyword(s):  
Elderly Patients ◽  
Single Cell ◽  
Induction Chemotherapy ◽  
Induction Therapy ◽  
De Novo ◽  
Training Set ◽  
Employment Equity ◽  
Cell Network ◽  
Disease Characteristics ◽  
Equity Ownership

Abstract Abstract 2489 Purpose: Standard Induction chemotherapy (Ara-C/daunorubicin, 3+7 regimen) in elderly patients (pts) with AML results in approximately 35–45% complete remission (CR) rate, and pts with resistant disease (RD) have a median survival of only 1–3 months. Developing a test for accurate prediction of response to standard induction therapy at the time of diagnosis may help inform treatment selection and improve clinical trial design. Methods: Single cell network profiling (SCNP) is a multi-parameter flow cytometry based approach for simultaneous interrogation of intracellular signaling pathways at a single cell level. SCNP was used to evaluate signaling profiles in leukemic blasts and to develop a classifier (DXSCNP) of response to induction therapy (CR/CRi, i=incomplete) in a Training Set of cryopreserved diagnostic samples (57 PB and 43 BM) collected from 74 non-M3 AML pts, aged 56+ treated with 3+7-based regimens on 4 SWOG clinical trials. SCNP intracellular readouts quantifying apoptotic response after 24 hrs in vitro treatment with Ara-C/Daunorubicin formed the inputs for DXSCNP. Pt and disease characteristics available either at diagnosis, i.e. relevant to induction therapy choices (e.g., age, WBC counts, FAB class, secondary AML, performance status – CLINICAL1), or available after start of induction therapy (CLINICAL2) were used to develop 2 clinical predictors (DXCLINICAL1 and DXCLINICAL2) of CR/CRi in the Training Set. The performance characteristics of these 3 classifiers were then tested separately in BM and PB sample sets. Specifically, classifier validation was performed in 2 independent BM sample sets (A: n=24 BM samples from ECOG E3999 trial; B: n=42 independent BM samples from the same 4 SWOG trials from which the Training samples were derived) and in 1 PB sample set (C: n=53, from the 4 SWOG trials; notably only 24 patients were shared between Set B and C)). The area under the receiver operating characteristic curve (AUROC) was used to measure each classifier's ability to predict response to 3+7 induction therapy. Out of bag estimates (OOB) of AUROC were calculated using the Training Set, and H0:AUROC=0.5 was tested against HA:AUROC>0.5 for each classifier in the Validation Sets. Results: As shown in Table 1, DXSCNP was a significant predictor of CR/CRi in BM samples. The AUROC for the DXSCNP classifier was 0.81 in the Training Set and 0.76 (p=0.01) and 0.72 (p=0.02) in Validation Sets A and B, respectively. No significant DXCLINICAL1 was identified from the 74 pts in the Training Set(OOB AUROC∼0.5). By contrast the AUROC of DXCLINICAL2 (which included inputs for cytogenetics, Flt3ITD, and NPM1 mutational status) was 0.63 (OOB) in the Training Set, and 0.61 (p=0.18) and 0.53 (p=0.38) in Validation Sets A and B, respectively. Of note, DXSCNP remained significant (p=0.03 and 0.04) when controlling for DXCLINICAL2 in both sample sets. Similar performance of DXSCNPwas observed in different pre-specified subgroups (although of small sample sizes) defined by pt, sample or disease characteristics. Using PB samples, the AUROCs for the DXSCNP classifier were, 0.87 (OOB) in the Training Set and 0.53 (p=ns) in the Validation Set C. However, the performance of DXSCNP in PB samples differed significantly between secondary vs. De novo AML i.e. AUROC= 0.24 vs 0.8 respectively. Of note, DXSCNPperformed similarly for the subset of de novo patients for whom paired BM with PB samples were tested (AUC=0.78 (PB) and 0.74 (BM)). Conclusion: This is the first study describing the successful validation of an SCNP-based classifier to predict response to standard induction chemotherapy in elderly AML pts with performance superior to clinical variables available at diagnosis. A similar SCNP classifier with overlapping cellular biology inputs was previously validated for pediatric AML. These results confirm the ability of quantitative SCNP readouts to provide independent and actionable information on disease biology and pt treatment choices. Independent validation in prospective studies is warranted. Disclosures: Cesano: Nodality, Inc: Employment, Equity Ownership. Gayko:Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Louie:Nodality, Inc.: Employment, Equity Ownership. Westfall:Nodality, Inc.: Employment, Equity Ownership. Purvis:Nodality, Inc.: Employment, Equity Ownership. Spellmeyer:Nodality, Inc.: Employment, Equity Ownership. Marimpietri:Nodality, Inc.: Employment, Equity Ownership. Hackett:Nodality, Inc.: Employment, Equity Ownership. Shi:Nodality, Inc.: Employment, Equity Ownership.

Specimen Source (BM or PB) Does Not Affect Proteomic Signaling In Patients with AML and Circulating Blasts

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2693-2693
Author(s):  
Alessandra Cesano ◽  
David B. Rosen ◽  
Santosh Putta ◽  
Urte Gayko ◽  
Larry Cripe ◽  
...  
Keyword(s):  
Linear Regression ◽  
Single Cell ◽  
Intracellular Signaling ◽  
Goodness Of Fit ◽  
Kinase Inhibitors ◽  
Mononuclear Cells ◽  
Linear Regression Analysis ◽  
Employment Equity ◽  
Weak Response ◽  
Equity Ownership

Abstract Abstract 2693 Background: Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using this technology, ECOG in collaboration with Nodality is developing several novel biomarker assays with the aim to find blast functional signaling profiles predictive of response to induction therapy and risk of relapse in AML patients. To date, such assays utilized patient bone marrow (BM) as the sample source of blasts. However, in about 65% of patients with AML, circulating peripheral blasts are detected and peripheral blood (PB) sampling is easier and less invasive for patients than BM sampling. Objectives: The objective of this study was to compare by SCNP the functional effects of a panel of compounds simultaneously on different signaling pathways (such as the phosphoinositide 3-kinase (P13K )and the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) pathway) relevant both to the biology of the disease and the development of new therapeutics, in paired, diagnostic, cryopreserved PB mononuclear cells (PBMC) and BMMC samples from 44 AML patients. A paired sample was defined as a BMMC and PBMC specimen collected from the same patient on the same day. Methods: Modulated SCNP using a multiparametric flow cytometry platform was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with specific modulators (Table 1). The SCNP phosphoflow assay was performed on 88 BMMC/PBMC pairs from ECOG trial, E3999. The relationship between readouts of modulated intracellular proteins (“nodes”) between BMMC and PBMC was assessed using linear regression, Bland-Altman method or Lin's concordance correlation coefficient. Results: Table 1 shows the goodness of fit (R2) values from the linear regression analysis for both the basal levels and the modulated levels of intracellular signaling proteins. Most of the signaling nodes show strong correlations (R2 >0.64) with several of the exceptions belonging to nodes with weak response to modulation (e.g. SCF -> p-Akt) or antibodies with dim fluorphores (i.e. Alexa 647). The lack of response is however, consistent between the tissue types for the weak response nodes. Using a rank based metric that is less sensitive to the absolute intensity levels seem to perform better for the antibodies with dim fluorophores. Results from other methods; Bland Altman and Lin's Concordance also show good concordance between the tissue types. Conclusions: The data presented here demonstrate: 1) Specimen source (BM or PB) does not affect proteomic signaling in patients with AML and circulating blasts. 2) PB myeloblasts can be used as a sample source for Nodality SCNP assays to identify functionally distinct leukemic blats cell populations with distinct sensitivities to therapy. 3) The ability to use PB as a sample source will greatly improve the utility of these assays. In particular, our results will facilitate the monitoring of cellular signaling effects following the administration of targeted therapies, e.g., kinase inhibitors, at time-points when BM aspirates are not clinically justifiable. Disclosures: Cesano: Nodality Inc.: Employment, Equity Ownership. Rosen:Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership.

Single Cell Network Profiling (SCNP) Functionally Characterizes FLT3 Pathway Deregulation in Non-M3 Acute Myeloid Leukemia (AML) and Provides Prognostic Value Independent From Mutational Status

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2512-2512
Author(s):  
Alessandra Cesano ◽  
Santosh Putta ◽  
Kavita Mathi ◽  
David B. Rosen ◽  
Urte Gayko ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Induction Therapy ◽  
Mutational Load ◽  
Receptor Signaling ◽  
Employment Equity ◽  
Cell Network ◽  
Mutational Status ◽  
Equity Ownership

Abstract Abstract 2512 Background: FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations (FLT3 ITD+) result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients (pts) with AML; however, substantial heterogeneity in clinical outcomes still exists within both the FLT3 ITD+ and FLT3 ITD- AML subgroups, suggesting alternative mechanisms of disease relapse not accounted for 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 (Kornblau et al. Clin Cancer Res 2010). Previously, we reported the use of this assay to functionally characterize FLT3 receptor signaling in healthy bone marrow and AML samples (Rosen et al. PLoS One 2010). By applying it to a separate cohort of samples collected from elderly non-M3 AML pts at diagnosis, a subclassification of AML samples beyond their “static” molecular FLT3 ITD status was generated (Rosen et al. ASH 2010 Abstr 2739). Specifically, FLT3 ITD- AML samples displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD+ AML samples. Conversely, FLT3 ITD+ AML samples displayed more homogeneous induced signaling, with the exception of those with low mutational load, which had profiles more analogous to FLT3 ITD- AML samples. Due to the small numbers of pts in that exploratory study (n=44 [38 FLT3 ITD- and 6 FLT3 ITD+ pts]), an independent study was undertaken to confirm the observations, as well as to evaluate their clinical relevance (i.e., association with disease free survival (DFS) following anthracycline/cytarabine-based induction therapy). Methods: SCNP was performed as previously described on cryopreserved bone marrow or peripheral blood samples collected prior to anthracycline/cytarabine-based induction therapy from 104 elderly (>60y) non-M3 AML pts enrolled on ECOG trial 3999 or 3993 for whom ITD mutational status (including % mutational load), response and DFS data were available. Samples included 85 FLT3 ITD- and 19 FLT3 ITD+ AML, 30 and 8 of which, respectively, were collected from patients who achieved complete remission (CR). Objectives: The primary study objective was to confirm that levels of FLT3 ligand (FLT3L)-induced signaling (as measured by changes in intracellular phospho-S6 level) are more homogeneous in FLT3 ITD+ than in FLT3 ITD- myeloblasts. Four FLT3 ITD+ groups were pre-defined based on % mutation load (>0, 30%, 40%, or 50%). In addition, FLT3 ITD mutational status and signaling data from the SCNP assay (FLT3L and stem cell factor-induced phospho-S6 signaling and cytarabine/daunorubicin-induced apoptosis [cleaved PARP]) were combined to mathematically model their association with DFS among pts who achieved CR. DFS was defined as time from date of confirmed CR to date of relapse or death. Results: As shown in Figure 1a, our previous observations that variance in FLT3L-induced signaling is higher in FLT3 ITD- AML samples than in FLT3 ITD+ ones and that variance is decreased with increasing mutational load were verified in this study (Levene Test for FLT3 ITD- vs FLT3 ITD+ 50 p value=0.023). Further, when the association of DFS with FLT3 ITD mutational status and signaling data from the SCNP assay was measured using a Cox Proportional-Hazards model, the SCNP data were shown to provide independent information from FLT3 ITD mutational status (p =0.0115 for FLT3L-induced phospho-S6 signaling, Figure 1b). Conclusions: These data add to the growing body of evidence that, even within currently accepted risk stratification groups, AML is a heterogeneous disease. Functional characterization of FLT3 receptor signaling deregulation using SCNP provides prognostic information independent from FLT3 ITD mutational status and allows for more accurate pt stratification by functionally defining DFS risk sub-groups. Characterization of FLT3 signaling deregulation by SCNP could ultimately aid in the improved clinical management of AML pts and help identify candidates for FLT3 receptor inhibitor studies. Disclosures: Cesano: Nodality: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Mathi:Nodality: Employment. Rosen:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership.

Single-Cell Network Profiliing (SCNP) Signatures Independently Predict Response to Induction Therapy In Older Patients with Acute Myeloid Leukemia (AML)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2695-2695
Author(s):  
Alessandra Cesano ◽  
Santosh Putta ◽  
Urte Gayko ◽  
James Hackett ◽  
David B. Rosen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induction Therapy ◽  
Older Patients ◽  
De Novo ◽  
Employment Equity ◽  
Cell Network ◽  
Independent Validation ◽  
Damage Response ◽  
Apoptosis Pathways ◽  
Equity Ownership

Abstract Abstract 2695 Background: Age and cytogenetics are major determinants of outcome in older patients with AML. Complete response (CR) to induction chemotherapy is observed in less than half of de novo AML patients (excluding APL) older than 60 years. Because unfavorable treatment results are often obtained despite intensive chemotherapy, which carries the potential for significant treatment-induced morbidity and mortality, it would be valuable to be able to predict with high accuracy at the individual patient level who will achieve a CR. Prospective identification of patients unlikely to benefit from anthracycline/cytarabine-based chemotherapy could spare patients from unnecessary treatment-related toxicities and allow consideration for alternative therapeutic strategies. Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using SCNP technology we have developed a set of classifiers that predict for likelihood of response to anthracycline/cytarabine-based induction therapy in older patients with AML. Methods: SCNP assays were performed on paired, bone marrow (BM) and peripheral blood (PB), samples from 44 AML patients (de novo, evolved from an antecedent MDS or MPN or treatment related), >60 years old, enrolled on ECOG trial E3999. Based on two previous training studies, 38 combinations of modulators and intra-cellular proteins (signaling nodes along the phosphoinositide 3-kinase (P13K ), the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) and the DNA damage response and apoptosis pathways) were investigated. Basal and modulated protein levels and the effect of modulation on proteins levels in the leukemic blast cells were expressed using a variety of metrics. A total of 64 node/metric combinations (dimensions) were used to build multi-parametric classifiers (ranging from 2 to 10 nodes/metrics) using different modeling methodologies (including random forest, boosting, lasso and a bootstrapped best subsets logistic modeling approach that shrinks regression coefficients (BBLRS)) able to predict the likelihood of response to induction therapy. The performance characteristics of the classifiers built on the BM samples were then evaluated independently on the paired PB samples. Results: Several promising models with high area under the operator/receiver curve (AUROC) values (indicating strong agreement between actual clinical responses and responses as predicted by the model) were developed based on SCNP proteomic read outs for BM samples. The observed and predicted values from the current best BBLRS model are shown in Figure 1. The unadjusted AUROC of this model is 0.98 and the expected AUROC for the model when applied to an independent (validation) sample is 0.84. Five signaling nodes are represented in this model; they include nodes belonging to growth factor- induced survival pathways (PI3K, RAS/MAPK) as well as DNA damage response and apoptosis pathways. When the predictive accuracy of the lead SCNP classifier was compared to that of a model based on traditional clinical/molecular predictors (i.e. the combination of age, therapy-related AML, and karyotype) the adjusted AUROC of the SCNP classifier far surpassed that of the clinical predictors (adjusted AUROC=0.61 for clinical/molecular predictors vs. adjusted AUROC= 0.84 for the SCNP classifier). Finally, when the nodes in the best BBLRS model developed on data from BM samples were used to model read outs from the paired PB samples, the adjusted AUROC of the resulting BBLRS model was comparable to that of the model fit to BM samples. Conclusion: This training set data show the value of performing quantitative SCNP under modulated conditions as the basis for developing highly predictive tests for response to induction chemotherapy. Most importantly, the predictions made by the SCNP classifier are independent of established prognostic factors, such as age and cytogenetics The ability of one set of nodes to accurately predict response in paired BM or PB samples from individual patients suggests that the predictive power of the SCNP assay is independent of sample source, further improving the practicality of the test. Independent validation studies are ongoing. Disclosures: Cesano: Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hackett:Nodality Inc.: Consultancy. Rosen:Nodality Inc.: Employment, Equity Ownership.

Single Cell Network Profiling as a Platform to Reveal Leukemia-Specific Signaling Signatures and Sensitivity to Kinase Inhibitor Therapies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2753-2753
Author(s):  
Todd M. Covey ◽  
Michael Gulrajani ◽  
Heiko Becker ◽  
Jason C. Chandler ◽  
Sebastian Schwind ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Clinical Trials ◽  
Growth Factor ◽  
Single Cell ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Employment Equity ◽  
Cell Network ◽  
Healthy Donors ◽  
Equity Ownership

Abstract Abstract 2753 Aberration in kinase activity by either the gain-of-function mutations or overexpression of the encoding genes plays a pivotal role in myeloid leukemogenesis. An increasing number of kinase inhibitors are being developed as “targeted therapies” for the treatment of acute myeloid leukemia (AML) and other myeloproliferative disorders. However, given the biologic and clinical heterogeneity inherent to these diseases, an unmet medical need exists for tools to guide the choice of inhibitor(s) most relevant for individual patients. With the aim of developing a platform for the biological characterization of patient-specific tumors, which could assist patient stratification strategies for clinical trials, we combined signaling pathway analysis and drug response profiling in AML samples using Single Cell Network Profiling (SCNP) assays. This technology allows for the simultaneous measurement of the activation state of multiple signaling proteins at the single cell level. Cryopreserved mononuclear cells from blood leukapheresis of patients with AML (N=6) were analyzed in two experimental arms. #1 Signaling Arm: A panel of kinase inhibitors targeting FLT3, cKit, PI3 kinase, mTor, MEK, and JAK proteins was added at varying concentrations to the AML cells followed by stimulation with G-CSF, IL-27, cKit ligand (SCF), FLT3 ligand (FLT3L), or a vehicle control. Using multiparameter flow cytometry, the phosphoylation status of AKT, ERK, S6 Ribosome, STAT1, STAT3, and STAT5 were measured in multiple leukemia cell subsets defined by expression of CD34, cKit, CD3, and light scatter properties. Per sample, there were a total of 68 treatments measuring 3 phospho-proteins in 3 cell subsets. #2 Apoptosis/Cytostasis Arm: The leukemic cells were driven into cell cycle by exposure to IL-3, SCF, and FLT3L, followed by a 48-hr incubation with a combination of 1 to 5 kinase inhibitors targeting the same pathways referred to previously. The kinase inhibitor impact was measured on distal functional readouts, including apoptosis (cleaved PARP) and cell cycle (CyclinB1-S/G2 phase; p-Histone H3-M phase). These results were compared with results using bone marrow samples from healthy donors (N=6). Results: Each patient's sample generated a unique signaling profile. A broad range of protein-specific phosphorylation status of AKT, ERK, S6 Ribosome, STAT1, STAT3, and STAT5 was observed in response to growth factor stimulation. Response was measured by setting a region gate that captures the overall percentage of cells with fluorescence above the unstimulated level. The percentage of SCF, G-CSF and FLT3L responsive cells ranged between 6%-49%, 3%-56%, and 3%-22%, respectively. Overall, patient samples could be grouped based on their signaling profile, proliferative potential, and sensitivity to kinase inhibitor treatment. Specifically, the 2 samples with the greatest SCF and G-CSF signaling response also showed the most robust in vitro proliferation and were most sensitive to the JAK inhibitor, CP-690,550 (1μM) (as measured by cytostasis readouts). Whereas, 2 other samples that displayed only modest SCF and G-CSF signaling, but robust FLT3L signaling expanded slowly in culture and were particularly sensitive to the cytostatic effects of the PI3K inhibitor, GDC-0941, (1uM) or the Flt3 inhibitor, tandutinib, (1uM). Finally, the last 2 AML samples had weak growth factor signaling and did not proliferate in culture and therefore could not be tested for kinase inhibitor-induced cytostasis. While the successfully tested patient samples showed variable sensitivity (as measured by cytostasis and apoptosis) to different drug combinations, the samples from healthy donors showed considerable similarity in response across all inhibitor combinations. Conclusions: This study provides preliminary proof-of-concept on the utility of SCNP to dissect the pathophysiologic heterogeneity of hematologic tumors and assess their differential response to single and combination therapies. Ultimately, this functional pathway profiling and drug sensitivity assay may be useful to stratify patients to different kinase combination treatments tested in clinical trials. Disclosures: Covey: Nodality Inc.: Employment, Equity Ownership. Gulrajani:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership.

scholarly journals Single-Cell Mutational Profiling Describes the Molecular Heterogeneity of Clonal Evolution in MDS during Therapy and Relapse

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5503-5503
Author(s):  
Alexey Aleshin ◽  
Robert Durruthy-Durruthy ◽  
Bruno C. Medeiros ◽  
Dennis J. Eastburn ◽  
Peter L Greenberg
Keyword(s):  
Single Cell ◽  
Clonal Evolution ◽  
Next Generation Sequencing Data ◽  
Molecular Heterogeneity ◽  
Disease Relapse ◽  
Sequencing Data ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Single Cell Sequencing ◽  
Equity Ownership

Abstract Background: Myelodysplastic syndromes (MDS) are a collection of clonal diseases of dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. Increased understanding of the mutational landscape of MDS has led to initial improvements in prognostic models based on clinical and cytogenetic variables. However, bulk sequencing techniques are limited in their ability to delineate clonal complexity and identify rare drug resistant subclones. To better understand clonal heterogeneity and clonal evolution of MDS we applied a high-throughput single cell sequencing technique to both diagnostic and longitudinal MDS samples. Methods: Samples were examined for 5 patients with MDS at diagnosis and, when available, progression. Mutational bulk sequencing was performed by NGS panel sequencing and exon sequencing was available in select cases. Single cell processing was performed using the Tapestri (Mission Bio) platform. Briefly, individual cells were isolated using a microfluidic approach, followed by barcoding and genomic DNA amplification for individual cancer cells confined to droplets. Barcodes are then used to reassemble the genetic profiles of cells from next generation sequencing data. We applied this approach to individual MDS samples, genotyping the most clinically relevant loci across upwards of 10,000 individual cells. Results: Single-cell sequencing was able to be performed successfully on all samples tested and recapitulated bulk sequencing data. We observed high concordance between bulk variant allele frequencies (VAFs) and sample level VAFs derived from single cell sequencing data (r2 = 0.98). Additionally, single cell analysis allowed for resolution of subclonal architecture and tumor phylogenetic evolution beyond what was predicted from bulk sequencing alone. Single-cell SNVs were able to resolve host and donor cell populations after bone marrow transplant and accurately predict chimerism and disease relapse. Furthermore, we were able to resolve the co-occurance of molecular alterations within subclones and establish zygosity of individual mutations at a single cell level. Rare subclones associated with disease relapse, were able to be identified in initial diagnostic samples that were frequently under the limit of detection of bulk NGS. Conclusions: Our results suggest more molecular complexity in MDS tumor samples than implied from bulk sequencing methods alone and indicates utility of single-cell sequencing for identification of resistant clones and longitudinal therapy monitoring. Disclosures Aleshin: Mission Bio, Inc.: Consultancy; Natera, Inc.: Employment. Durruthy-Durruthy:Mission Bio, Inc.: Employment, Equity Ownership. Medeiros:Genentech: Employment; Celgene: Consultancy, Research Funding. Eastburn:Mission Bio, Inc.: Employment, Equity Ownership.

scholarly journals Single Cell Signaling Pharmacodynamics in a Phase 1b Trial of the Axl Inhibitor BGB324 in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3995-3995 ◽  
Author(s):  
Monica Hellesøy ◽  
Stein-Erik Gullaksen ◽  
Benedicte Sjo Tislevoll ◽  
Oda Helen Eck Fagerholt ◽  
Hakon Reikvam ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Flow Cytometry ◽  
Single Cell ◽  
Peripheral Blood ◽  
Intracellular Signaling ◽  
Research Funding ◽  
Prognostic Information ◽  
Employment Equity ◽  
Phase 1B ◽  
Equity Ownership

Abstract Axl is a receptor tyrosine kinase that has been shown to have a strong oncogenic potential in many cancer types. Overexpression and activation of Axl is found in many cancers, and is linked to increased proliferation, migration/invasion and resistance to apoptosis. Axl overexpression has been shown to be a poor prognostic marker, and recently overexpression of Axl has also been linked to the acquired resistance to chemotherapy and other anticancer therapies in many malignancies, including AML. BGB324 (BerGenBio AS) is a first-in-class highly specific small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in a clinical safety trial in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in a phase 1b clinical trial in patients with refractory/relapsed AML and MDS (BGBC003, ClinicalTrials.gov Identifier:NCT02488408; Loges S et al. J Clin Oncol 34, 2016 suppl; abstr 2561). 20 AML and 4 MDS patients have been treated at the following dose levels (loading dose/continuation dose): 400/100mg, 600/200mg and 900/300mg. Objective responses were observed in 2/4 MDS patients and 2/20 AML patients including one CR (AML). Enrollment continues to define MTD. The effect of BGB324 on intracellular signaling and the immune profile of leukemic blasts in patients treated in the clinical study was investigated using phospho-flow cytometry. Blasts were identified using surface markers (CD45low, CD66b-, CD38-, and CD117+ and/or CD34+), and the following direct and indirect downstream targets of Axl were explored: phosphorylated (p)-Akt(S473 and T308), pErk(T202/Y204), pp38(T180/Y182), pPLCγ1(Y783), pNFκB(S529), pCREB(S113) and pSTAT1(Y701), 3(Y705), 5(Y694)and 6(Y641). Preliminary analyses of blood samples from six patients show very rapid responses in signaling pathways downstream of Axl (including Akt, Erk, NFκB and PLCγ1) within hours or days of ingestion of the first dose, although the response patterns varies from patient to patient (Figure 1A). Two distinct blast populations were identified: one CD117+/CD34- and one CD117+/CD34+. In most patients the CD117+/CD34- population displayed the most extensive signaling changes during treatment, and this population also decreased during treatment with BGB324. In contrast, the CD117+/CD34+ population expanded during the course of the treatment (Figure 1B). White cell differential counts of peripheral blood from two patients treated with BGB324 for a prolonged period of time (15 weeks or more) showed a decrease in peripheral blast count, and a corresponding increase in granulocyte and monocyte counts, suggesting that Axl inhibition may push the blasts towards differentiation. The clinical trial is ongoing, and the signaling profile of leukemic blasts in blood and bone marrow of treated patients will be further examined by conventional phosphoflow cytometry and mass cytometry searching for signaling profiles with prognostic information. In conclusion, BGB324 has unique pharmacodynamic properties and molecular responses to exposure can be observed in peripheral blood leukemic blasts by phospho-flow cytometry within hours of ingestion of the first treatment dose. Further studies may establish whether single cell signal profiling can discriminate responders from non-responders and provide information about dose-response in a clinically meaningful way. Disclosures Cortes: Astellas: Research Funding; Arog: Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Ambit: Research Funding. Heuser:Novartis: Consultancy, Research Funding; Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Pfizer: Research Funding. Lorens:BerGenBio AS: Employment, Equity Ownership, Research Funding. Gausdal:BerGenBio AS: Employment. Micklem:BerGenBio AS: Employment, Equity Ownership. Gjertsen:BerGenBio AS: Consultancy, Research Funding.

BCR Responsiveness is Associated with Time to First Treatment (TTFT) in B-Cell Chronic Lymphocytic Leukemia (B-CLL): Results From a Single Cell Network Profiling (SCNP) Verification Study

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2834-2834
Author(s):  
Alessandra Cesano ◽  
Erik Evensen ◽  
Jason Ptacek ◽  
James Cordeiro ◽  
Rachael E. Hawtin ◽  
...  
Keyword(s):  
B Cell ◽  
Single Cell ◽  
Lymphocytic Leukemia ◽  
Erk Signaling ◽  
Employment Equity ◽  
Cell Network ◽  
Bcr Signaling ◽  
Binet Stage ◽  
Equity Ownership

Abstract Abstract 2834 Background: B-cell chronic lymphocytic leukemia (B-CLL) follows a highly variable clinical course, with some patients having indolent disease and not requiring treatment, whereas others experience rapid disease progression, treatment resistance, and death within 2 to 3 years. Widely accepted staging systems, biological parameters, and prognostic indices help stratify patients into risk groups, yet there remains substantial intragroup heterogeneity. Single cell network profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, quantitatively at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Previously, we reported the use of this assay to functionally characterize BCR signaling in 21 cryopreserved samples from Binet Stage A B-CLL patients. In that study, a panel of signaling nodes (a node is defined as the combination of extracellular modulator and corresponding intracellular readout and is abbreviated node→readout) was examined and increased anti-IgM-induced phospho (p)-Erk signaling (anti-IgM→p-Erk) was associated with shorter time from diagnosis to first treatment (TTFT) (Scupoli et al. EHA 2010). Objectives: The present study was undertaken to independently verify the association between BCR responsiveness and shorter TTFT, to assess repeatability of SCNP measurements in B-CLL cells, and to explore additional signaling nodes relevant to B-CLL biology. Methods: SCNP was performed as previously described on cryopreserved peripheral blood mononuclear cells (PBMC) collected between 2004 and 2009 from 32 patients with untreated Binet Stage A B-CLL at various points during their clinical follow up; at the time of the analysis, 9 (28%) progressed to active disease, requiring treatment. Median follow-up was 47 months (range 4 to 179 months). SCNP analysis of 21 signaling nodes investigating B cell receptor, survival and NFkB signaling in B-CLL cells was performed in replicate, on separate plates run on the same day. Repeatability was assessed by regressing paired induced signaling measurements from experimental replicates. Associations between SCNP measurements and TTFT were determined using Cox Proportional Hazards regression. Results: Excellent repeatability was observed for SCNP signaling measurements in B-CLL cells (e.g., anti-IgM→p-Erk: R2=0.97, slope=1.01). Consistent with the previous study, an association between BCR responsiveness, measured by anti-IgM→p-Erk, and shorter TTFT was also observed in this study, although the pre-specified significance criteria were not met (likelihood ratio (LR) Chi square (χ2) test p=0.07). A post hoc analysis excluding two samples with low viability (low % aqua negative cells) was also performed (Figure 1a) showing a statistically significant association between anti-IgM→p-Erk signaling and TTFT (p=0.03, Figure 1b). Among the new nodes investigated in this study, an additional significant association between increased anti-IgD→p-NFkB and shorter TTFT was identified (p=0.02). Conclusions: This study confirms, in an independent data set, the association between increased anti-IgM→p-Erk signaling and shorter TTFT in B-CLL that was observed previously in a separate study. In addition, a newly identified association between anti-IgD→p-NFkB and TTFT was observed, thus supporting the role of BCR signaling in the pathogenesis of the disease and the utility of SCNP assay in elucidating these signaling deregulations with the potential for the development of prognostic and predictive tests. Disclosures: Cesano: Nodality: Employment, Equity Ownership. Evensen:Nodality: Employment, Equity Ownership. Ptacek:Nodality Inc.: Employment, Equity Ownership. Cordeiro:Nodality Inc.: Employment, Equity Ownership. Hawtin:Nodality Inc.: Employment, Equity Ownership. Ware:Nodality Inc.: Employment, Equity Ownership.

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