High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnos... more High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnosis at advanced stage compounded with its excessive genomic and cellular heterogeneity make curative treatment challenging. Two critical therapeutic challenges to overcome are carboplatin resistance and lack of response to immunotherapy. Carboplatin resistance results from diverse cell autonomous mechanisms which operate in different combinations within and across tumors. The lack of response to immunotherapy is highly likely to be related to an immunosuppressive HGSOC tumor microenvironment which overrides any clinical benefit. Results from a number of studies, mainly using transcriptomics, indicate that the immune tumor microenvironment (iTME) plays a role in carboplatin response. However, in patients receiving treatment, the exact mechanistic details are unclear. During the past decade, multiplex single-cell proteomic technologies have come to the forefront of biomedical research. Mass...
To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon ... more To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial c...
To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon ... more To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial c...
Objectives: The recent FDA approval of pembrolizumab for the treatment of recurrent, tissue agnos... more Objectives: The recent FDA approval of pembrolizumab for the treatment of recurrent, tissue agnostic cancers with Microsatellite Instability-High (MSI-H) or Mismatch Repair Deficient (dMMR) has led to the treatment of a selected cohort of endometrial cancer (EC) patients. Methods: We designed a study to ascertain tumor immune modulatory effects of pembrolizumab in the front-line setting for advanced stage III/IV surgically-resectable endometrial cancers regardless of MSI-H or dMMR status. The primary objectives were to determine the safety of treatment with pembrolizumab by radiographic imaging and to determine progression-free survival at 6 months. Exploratory objective was to compare the immune response before and after treatment. In an open label, single-arm Phase I trial, 8 EC patients were treated with 2 doses of preoperative pembrolizumab IV prior to surgery followed by carboplatin and paclitaxel and 4 doses of pembrolizumab 200mg/kg q3 weeks IV. Peripheral blood was collected...
397 Background: Although most patients with acute myeloid leukemia (AML) have disease that respon... more 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 diagno...
325 Background: Acute Myeloid Leukemia (AML) is the most common myeloid malignancy in adults and ... more 325 Background: Acute Myeloid Leukemia (AML) is the most common myeloid malignancy in adults and represents an aggressive disease with significant biological and clinical heterogeneity. Currently, cytogenetics and molecular changes are used to inform treatment strategies. However a wide range of clinical responses are observed in these patient subgroups necessitating alternative methodologies to provide information that could inform clinical decisions for AML disease management. Since the net result of the cytogenetic and molecular changes is necessarily a functional alteration of proteins within signal transduction networks the current study was undertaken to understand the diversity of signaling responses in AML patient samples. Critically, in these studies treatment of samples with a variety of input stimuli allowed intracellular phospho-signaling and apoptosis network properties to be revealed that would otherwise remain unseen in resting cells. Objectives: Single cell network p...
1582 Poster Board I-608 Background Traditional AML prognostic markers are based on clinical chara... more 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 cont...
1588 Poster Board I-614 Background Mutations in the receptor tyrosine kinase (RTK) Fms-like tyros... more 1588 Poster Board I-614 Background Mutations in the receptor tyrosine kinase (RTK) Fms-like tyrosine kinase 3 (FLT3) gene are among the most common somatic mutations in AML with FLT3 internal tandem duplications (ITDs) occurring in 20-35% of adult and 5-15% of pediatric AML. While the presence of FLT3 ITD mutation does not appear to influence outcome to induction chemotherapy, this mutation has been shown to confer a poor prognosis with significantly shorter disease free and relapse free survival. For patients with intermediate risk cytogenetically normal AML, molecular testing for FLT3 ITD has recently been incorporated into the National Comprehensive Cancer Network (NCCN) guidelines for clinical practice. However, while molecular testing can identify a subset of patients at high risk for relapse, there remains clinical heterogeneity likely due to differences in activation of signal transduction networks. Objectives This study tested the ability to use single cell network profiling...
Purpose: We sought to enhance the cytometric analysis of MDS by performing a pilot study of a sin... more Purpose: We sought to enhance the cytometric analysis of MDS by performing a pilot study of a single cell mass cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS. Experimental Design: Twenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter mass cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering. Results: This high-dimensional assay provided three major benefits relative to traditional cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. ...
Insight into the cancer cell populations that are responsible for relapsed disease is needed to i... more Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'deve...
We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovaria... more We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a foc...
High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnos... more High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnosis at advanced stage compounded with its excessive genomic and cellular heterogeneity make curative treatment challenging. Two critical therapeutic challenges to overcome are carboplatin resistance and lack of response to immunotherapy. Carboplatin resistance results from diverse cell autonomous mechanisms which operate in different combinations within and across tumors. The lack of response to immunotherapy is highly likely to be related to an immunosuppressive HGSOC tumor microenvironment which overrides any clinical benefit. Results from a number of studies, mainly using transcriptomics, indicate that the immune tumor microenvironment (iTME) plays a role in carboplatin response. However, in patients receiving treatment, the exact mechanistic details are unclear. During the past decade, multiplex single-cell proteomic technologies have come to the forefront of biomedical research. Mass...
To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon ... more To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial c...
To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon ... more To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial c...
Objectives: The recent FDA approval of pembrolizumab for the treatment of recurrent, tissue agnos... more Objectives: The recent FDA approval of pembrolizumab for the treatment of recurrent, tissue agnostic cancers with Microsatellite Instability-High (MSI-H) or Mismatch Repair Deficient (dMMR) has led to the treatment of a selected cohort of endometrial cancer (EC) patients. Methods: We designed a study to ascertain tumor immune modulatory effects of pembrolizumab in the front-line setting for advanced stage III/IV surgically-resectable endometrial cancers regardless of MSI-H or dMMR status. The primary objectives were to determine the safety of treatment with pembrolizumab by radiographic imaging and to determine progression-free survival at 6 months. Exploratory objective was to compare the immune response before and after treatment. In an open label, single-arm Phase I trial, 8 EC patients were treated with 2 doses of preoperative pembrolizumab IV prior to surgery followed by carboplatin and paclitaxel and 4 doses of pembrolizumab 200mg/kg q3 weeks IV. Peripheral blood was collected...
397 Background: Although most patients with acute myeloid leukemia (AML) have disease that respon... more 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 diagno...
325 Background: Acute Myeloid Leukemia (AML) is the most common myeloid malignancy in adults and ... more 325 Background: Acute Myeloid Leukemia (AML) is the most common myeloid malignancy in adults and represents an aggressive disease with significant biological and clinical heterogeneity. Currently, cytogenetics and molecular changes are used to inform treatment strategies. However a wide range of clinical responses are observed in these patient subgroups necessitating alternative methodologies to provide information that could inform clinical decisions for AML disease management. Since the net result of the cytogenetic and molecular changes is necessarily a functional alteration of proteins within signal transduction networks the current study was undertaken to understand the diversity of signaling responses in AML patient samples. Critically, in these studies treatment of samples with a variety of input stimuli allowed intracellular phospho-signaling and apoptosis network properties to be revealed that would otherwise remain unseen in resting cells. Objectives: Single cell network p...
1582 Poster Board I-608 Background Traditional AML prognostic markers are based on clinical chara... more 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 cont...
1588 Poster Board I-614 Background Mutations in the receptor tyrosine kinase (RTK) Fms-like tyros... more 1588 Poster Board I-614 Background Mutations in the receptor tyrosine kinase (RTK) Fms-like tyrosine kinase 3 (FLT3) gene are among the most common somatic mutations in AML with FLT3 internal tandem duplications (ITDs) occurring in 20-35% of adult and 5-15% of pediatric AML. While the presence of FLT3 ITD mutation does not appear to influence outcome to induction chemotherapy, this mutation has been shown to confer a poor prognosis with significantly shorter disease free and relapse free survival. For patients with intermediate risk cytogenetically normal AML, molecular testing for FLT3 ITD has recently been incorporated into the National Comprehensive Cancer Network (NCCN) guidelines for clinical practice. However, while molecular testing can identify a subset of patients at high risk for relapse, there remains clinical heterogeneity likely due to differences in activation of signal transduction networks. Objectives This study tested the ability to use single cell network profiling...
Purpose: We sought to enhance the cytometric analysis of MDS by performing a pilot study of a sin... more Purpose: We sought to enhance the cytometric analysis of MDS by performing a pilot study of a single cell mass cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS. Experimental Design: Twenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter mass cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering. Results: This high-dimensional assay provided three major benefits relative to traditional cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. ...
Insight into the cancer cell populations that are responsible for relapsed disease is needed to i... more Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'deve...
We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovaria... more We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a foc...
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