Multiparameter Flow Analysis Research Articles

Abstract 246: Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib

Abstract Background: The receptor tyrosine kinase inhibitor (TKI) Sunitinib has proven useful in the treatment of a variety of tumors. Sunitinib has shown therapeutic activity in patients with metastatic renal cell carcinoma and currently represents a frontline therapy for this disease. The immune modulatory effect of Sunitinib on immune cells within the tumor microenvironment has been well documented. Here, we developed a novel 3D ex-vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal components and tumor immune microenvironment to assess the therapeutic efficacy of Sunitinib. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma using a proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with Sunitinib for 48h to detect treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, NK cells, and macrophages. Additionally, we analyzed treatment-mediated changes in T-cell activation and phagocytic activity of myeloid cells. Results: Multiparameter flow analysis revealed that Sunitinib treatment led to an increase in CD8+CD25+ central memory-like phenotype and increased granzyme B expression in CD4 and CD8+ T cells indicating T cell activation. Furthermore, multiparameter flow cytometry analysis showed increased EdU uptake by CD8 and NK cells with sunitinib treatment, while cytokine release assays demonstrated increased IFN-γ and IL-2 production accompanied by downregulation of IL-10 and IL-6 cytokines. We observed Sunitinib mediated phagocytotic activity by tumor resident myeloid macrophages, monocytes, and dendritic cells using a quantitative phagocytosis assay. Conclusions: These results demonstrate that 3D-EXplore using fresh tumor samples provides a clinically relevant platform to assess drug response and serves as a critical tool for reflecting the true intact tumor microenvironment for novel immune-oncology drug development. Citation Format: Brittany Bunch, Krithika N. Kodumudi, Jared Ehrhart, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. Application of the 3d fresh patient tumoroid platform 3d-explore to assess the immunomodulatory and phagocytic activity of the receptor tyrosine kinase inhibitor, Sunitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 246.

Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via...

Peripheral Blood As a Substitute for Bone Marrow for Assessing the Immune Profile in AML

Background: Immunotherapy has become a major strategy for cancer therapy and has dramatically improved patients' outcomes, especially in hematologic malignancies, but little is known about the functional capacity of T cells in Acute Myeloid Leukemia (AML) patients. The AML Bone Marrow (BM) microenvironment is a dynamic structure that plays a crucial role in initiating and maintaining leukemogenesis and contributing to AML drug resistance and immune suppression, and it is likely that T cells extant in the AML environment are functionally impaired. For example, pre-clinical and clinical studies have identified the upregulation of PD-1/PD-L1 axis as a potential mechanism of resistance to T-cell therapy suggesting that a better understanding of the expression of immune regulatory molecules in AML will help guide future therapeutic interventions, as well as serve as potential predictive/prognostic biomarkers for T-cell therapies. Frequent bone marrow sampling is not feasible in AML patients and so it is important to understand whether peripheral blood T-cells reflect T-cell phenotypes in the BM, including expression of inhibitory molecules. The objective of this study is to evaluate the use of Peripheral Blood (PB) as a surrogate for BM to support periodic immunophenotype monitoring in a clinical trial setting.Methods: Patient matched cryopreserved BM and PB primary AML samples (n=10 pairs) were surveyed by multiparameter flow cytometry to quantitate parameters including CD4 and CD8 effector T cell populations (Naïve: CD45RAhiCCR7hi, Central Memory: CD45RAloCCR7hi, Effector Memory: CD45RAloCCR7lo, Terminal Effector CD45RAhiCCR7lo), regulatory T cells (CD4+CD25+CD127-), expression of various immune checkpoint molecules (PD-1, TIM3, LAG3 and TIGIT), and leukemia cell phenotypes such as CD33, CD34, CD38, PD-1 and TIGIT ligands. All parameters collected for T cells and AML cells were analyzed using the Bland-Altman and Lin's concordance methods to evaluate the similarities between BM and PB. For eight samples, primary AML mononuclear cells (MNCs) were treated ex vivo for 4 days with Control or AMG 330 (CD33/CD3 BiTE®) and phenotyped to monitor response to BiTE® treatment and changes in checkpoint receptors.Results: PB is an appropriate surrogate for BM for many of the T cell and AML cell attributes analyzed. CD33 expression on blasts and the distribution of CD4 effector subsets in PB and BM were concordant based on Lin's concordance correlation, while CD4:CD8 ratio and T cell:blasts ratio were qualitatively similar (Spearman Rank). We also observed that expression of CD25 on CD4 and CD8 T cells and the AML progenitor population CD34+/CD38+ in PB and BM were linearly correlated. CD69 was consistently expressed on a higher percentage of CD4 and CD8 T cells in the BM compared to PB, consistent with its role in memory T cell retention in the BM. Checkpoint receptor expression was similar PB and BM (see Figure 1) but did not meet the statistical test for concordance; analysis of a larger number of samples will provide greater insights into non-correlated samples. We observed that most of the AML samples had a high median frequency of T cells expressing PD-1 (CD4+: 36%; CD8+: 46%) and TIGIT (CD4+: 22%; CD8+: 52%). TIM3 expression was lower (CD4+: 18%; CD8+: 17%) while LAG3 was rarely expressed on AML T cells (2/9 AML samples tested had >5% LAG3+). We also observed that PD-1 was expressed more frequently on CD8+ T cells from R/R AML BM compared to ND (medians: 43% vs 53%, respectively, p=0.022). TIGIT ligands, PVR and PVRL2, were present on AML blasts in the majority of patients (7/9 and 5/9 had >20% PVR+ or PVRL2+ respectively), while PD-L1 and PD-L2 were not expressed on blasts in any of the AML samples tested. To understand if these immunophenotypes relate to BiTE® responsiveness, primary AML MNCs (n=9) were treated ex vivo for 4 days with AMG 330. Response of these samples to treatment was dependent on CD33 expression and T cell:target cell ratio. Changes in PD-1 and TIGIT induced by ex vivo AMG 330 treatment will also be presented.Conclusion: Using multiparameter flow analysis, we observed a strong correlation between BM and PB for several biomarkers tested including CD4:CD8 ratios, T cell effector subsets, and leukemia cell phenotypic markers. Our results suggest that PB immunophenotype is representative of the BM in AML and can be used in a clinical trial setting to gain relevant biomarker data. [Display omitted] DisclosuresLesley:Amgen Inc.: Employment, Equity Ownership. Beutner:Amgen Inc.: Employment, Equity Ownership. Anderson:Amgen Inc.: Employment, Equity Ownership. Fitzpatrick:Amgen Inc.: Employment, Equity Ownership. Loberg:Amgen Inc.: Employment, Equity Ownership. Mehta:Amgen Inc.: Employment, Equity Ownership. Dos Santos:Amgen Inc.: Employment.

Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples.

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

3134 – CHARACTERIZATION OF THE HEMATOPOIETIC STEM CELLS AND BONE MARROW MICROENVIRONMENT IN SICKLE CELL DISEASE PATIENTS

Little is known about the status of the bone marrow (BM) in Sickle Cell Disease (SCD) patients. In this study, we characterized the hematopoietic stem and progenitors (HSPC) and the stromal cells from BM aspirates of eight SCD patients before transplant in comparison with age-matched BM aspirates from healthy individuals. By using multiparametric flow analysis, we observed a group of SCD patients (n=4) with characteristics similar to controls, and a distinct group of SCD patients (n=4) that exhibited lower average frequency of CD34+ cells, HSCs, multipotent and lymphoid progenitors. Methylcellulose colony assays showed a decreased ability of the BM cells of these patients to form colonies compared to control BM. scRNA-seq analysis of sorted BM CD34+ cells from SCD patients indicated changes in the DNA damage and cell cycle regulatory pathways compared to controls. Interestingly, all SCD patients displayed an increase in the BM non-hematopoietic cells including CD45-/CD31+ and CD45-/CD105+ subsets compared to controls, suggesting a change in their BM stromal niche. In vitro, SCD BM-derived stromal cells revealed distinct gene expression patterns including an increase in inflammatory cytokine (IL-6) & cell death-related genes (p16 and Bax) but decrease expression of a pluripotent gene (Oct4). We also detected an increase of inflammatory regulators, miR-155, and miR-146 in all BM-derived stromal cells from SCD patients, which correlates with increased inflammatory gene expression in these cells. Finally, analysis of the BM biopsies revealed alterations in the stromal components as evidenced by an overall decrease in cellularity in some patients, and increased iron deposits and activated macrophages. Taken together, these preliminary data suggest that the BM cells and BM microenvironment of SCD patients are phenotypically & molecularly different from control BM. Further studies will clarify whether there are correlations between the severity of the disease and BM alterations and whether the BM status at pre-transplant may help in predicting engraftment and outcomes of BM transplants in SCD patients.

Multiparametric flow analysis using four-dimensional flow magnetic resonance imaging can detect cerebral hemodynamic impairment in patients with internal carotid artery stenosis.

MRI-based risk stratification should be established to identify patients with internal carotid artery stenosis (ICS) who require further PET or SPECT evaluation. This study assessed whether multiparametric flow analysis using time-resolved 3D phase-contrast (4D flow) MRI can detect cerebral hemodynamic impairment in patients with ICS. This retrospective study analyzed 26 consecutive patients with unilateral ICS (21 men; mean age, 71years) who underwent 4D flow MRI and acetazolamide-stress brain perfusion SPECT. Collateral flow via the Willis ring was visually evaluated. Temporal mean flow volume rate (Net), pulsatile flow volume (ΔV), and pulsatility index (PI) at the middle cerebral artery were measured. Cerebral vascular reserve (CVR) was calculated from the SPECT dataset. Patients were assigned to the misery perfusion group if the CVR was < 10% and to the nonmisery perfusion group if the CVR was ≥ 10%. Parameters showing a significant difference in both groups were statistically evaluated. Affected side ΔV, ratio of affected to contralateral side Net (rNet), and ratio of affected to contralateral side ΔV were significantly correlated to CVR (p = 0.030, p = 0.010, p = 0.015, respectively). Absence of retrograde flow at the posterior communicating artery was observed in the misery perfusion group (p = 0.020). Combined cut-off values of the affected side ΔV (0.18ml) and rNet (0.64) showed a sensitivity and specificity of 100% and 77.8%, respectively. Multiparametric flow analysis using 4D flow MRI can detect misery perfusion by comprehensively assessing blood flow data, including blood flow volume, pulsation, and collateral flow.

Abstract LB-B06: A novel ex vivo 3D tumor organoid model of fresh patient tumors (3D-ACT) to assess efficacy of cellular therapy in immuno-oncology

Abstract Introduction: The spatial organization and dynamic interplay of the complex cell-to-cell interactions in patient tumors play an important role in cellular phenotypes that can result in permanent alterations in cellular functions and response to immuno-oncology (IO) treatments. Therefore, to assess the therapeutic efficacy of IO treatments, including cellular therapeutics, it is imperative to develop models that preserve the stromal-stoichiometry of the tumor microenvironment. Here we describe a patient-derived high content confocal imaging approach using an unpropagated tumor organoid model with intact tumor stroma for quantitative assessment of infiltration and target tumor cell killing activity of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancer. Materials and Methods: All human tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D tumoroids measuring 150 micron in size were prepared and cryopreserved during the process of ex vivo propagation of autologous TILs. Ex vivo propagated TILs were characterized using multiparameter flow analysis, fluorescently labeled and exposed to fresh tumor organoids. Computational confocal image analysis was used to quantify TILs infiltration into the tumor organoids and target tumor cell killing. Multiplex cytokine assays and flow cytometry analysis were performed to assess activation of TILs upon exposure to tumor organoids. Results: We successfully prepared matched autologous TILs and unpropagated 3D tumor organoids from 75% of NSCLC patient tumors. The characteristics of tumor immune microenvironment and tumor cell viability was evaluated in previously cryopreserved organoids using a custom image analysis algorithm that was developed for the collection of data in a structurally relevant environment on quantification of marker-specific cell number, cell viability and apoptosis in addition to structural and functional analysis of cells in intact 3D tumor organoids. High content confocal imaging analysis demonstrated that CD3/CD28 pre-activated TILs with increased activation phenotypes and enhanced pro-inflammatory cytokine release had marked infiltration into the 3D organoids compared to untreated TILs and PBMCs. The data was correlated with quantitative tumor cell killing assessment for each tumor organoid. Conclusion: These results demonstrate that our 3D-ACT model using ex vivo expanded TILs and 3D tumor organoids is an effective tool for the therapeutic assessment of autologous TILs and indicate that it can also be used to assess efficacy of other cellular therapy applications in Immuno-oncology. Furthermore, implementation of this platform in the clinical studies may also allow determining the most effective combinatorial cellular therapy strategies for individual patients. Citation Format: Vijayendra Agrawal, Mibel Pabon, Melba Marie Page, Jenny Kreahling, Soner Altiok. A novel ex vivo 3D tumor organoid model of fresh patient tumors (3D-ACT) to assess efficacy of cellular therapy in immuno-oncology [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B06. doi:10.1158/1535-7163.TARG-19-LB-B06

Immune Mediated Mechanisms of Resistance to Daratumumab

Introduction: Daratumumab (Dara) is a human monoclonal antibody targeting the highly expressed multiple myeloma (MM) surface receptor CD38, with significant activity in relapsed MM. However, resistance to Dara develops in virtually all patients (pts). Thus, in order to maximize its clinical activity and prevent resistance, it is imperative to understand why pts stop responding. Our group recently reported that, in addition to the malignant cells, several immune-effector cells also express CD38. Thus, we hypothesized that the expression of CD38 or lack of it on non-cancer immune cell-subsets may also play a role in both clinical response and resistance to Dara.Results: To address our hypothesis, we analyzed CD38 surface expression in CD138+ MM cells isolated from 5 Dara-naïve (D-naïve) pts and from 8 Dara progressing pts (D-prg) who had discontinued Dara therapy for at least 4 weeks prior to analysis. We observed that the CD138+ MM-cells of D-prg displayed a lower median fluorescence intensity (MFI) of CD38 compared to the MFI of MM cells isolated from the D-naïve (p=0.04); no significant difference was however observed in the percentage of CD38+ MM cells between the two groups. In fact, all of the MM cells expressed CD38 in both D-naïve and D-prg, supporting the hypothesis that resistance to Dara was not mediated by loss of CD38 expression on MM cells. Next, we looked at whether CD38 on the surface of MM cells from D-prg was still recognizable by Dara. Thus, we treated MM cells ex-vivo with Dara at the clinically achievable concentration of 100 μg/ml. We showed that Dara was still able to bind CD38 on the surface of the D-prg CD138+ MM cells, since, after Dara treatment, no CD38+ signal was detected upon staining with a CD38 monoclonal antibody sharing the same targeting CD38 epitope (clone IB6) (Fig.1a). Conversely, we detected a CD38+ signal when a different anti-human CD38 multi-epitope antibody was used in a similar experiment (Cytognos) (Fig.1b). CD38 mRNA expression was observed in all MM cells isolated from D-prg, and we were not able to identify any missense mutations in CD38 mRNA from MM cells of D-prg for whom material was available for analysis.Thus, having demonstrated that resistance to Dara was not related to the expression of CD38 on the MM-cells, we turned our attention to the MM bone marrow (BM) microenvironment (ME). We performed RNA-sequencing of the BM-ME depleted of the CD138+ MM cells obtained from the D-prg (n=5) and D-naïve (n=5). A gene expression signature differentiated the two groups. In D-prg, we observed a significant downregulation (fold-change<0.5, p<0.05) of 193 genes. DAVID functional annotation showed that the immune response-regulating genes, including TLR8, CD47, CXCL10 and CXCL4, were the most downregulated gene pathways in D-prg vs D-naïve (p<0.0001) (Fig.1c), supporting the notion that changes in the immune compartment of the ME may play a role in Dara resistance. Multi-parametric flow analysis showed that surface CD38 was significantly downregulated in terms of both MFI and percent in several immune cell subsets within the BM and peripheral blood (PB) of D-prg (n=14), in contrast to the BM and PB of the D-naïve (n=20) and the PB of the responding pts (n=6). Single cell RNA sequencing of PBMCs from naïve pts and D-prg is currently ongoing. Cell killing assays show that total PBMCs obtained from the D-prg, which have low/null CD38 expression in contrast to D-naïve and healthy donors, have no capability to kill CD38+ MM cells in the presence of Dara. However, total PBMCs obtained from D-naïve maintain an intact MM killing ability. Cell separation of the PBMC CD38+ and CD38- fractions obtained from healthy donors shows that Dara induced effector-mediated MM cell killing only in the CD38+ PBMCs treated with Dara; no killing effect was observed when the PBMCs CD38-negative fraction was used in the same experimental conditions. Moreover, killing assays show that Dara activates PBMCs to target CD38+ MM cells even when MM cells are not directly exposed to the antibody and only the effectors are treated.Conclusions: Our data are supportive of the hypothesis that loss of Dara binding to MM-cell surface CD38 or the complete loss of CD38 on MM cells may not necessarily be the cause of Dara treatment resistance. We instead propose that Dara binding on CD38+ immune effector cells may be critical and that the loss of CD38 expression on these cells can lead to mechanisms of Dara-acquired resistance. [Display omitted] DisclosuresRosenzweig:Celgene: Speakers Bureau. Krishnan:Celgene: Consultancy, Equity Ownership, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Sutro: Speakers Bureau; Takeda: Speakers Bureau; Onyx: Speakers Bureau.

Exploring the Possibility of Using Herpes Simplex Virus in Oncolytic Virotherapy of Multiple Myeloma

Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Approximately, 80,000 people have died of the disease in the United States and 25,000 new cases are registered every year. Majority of patients develop resistance to current therapeutic treatments and die within 5-10 years of diagnosis. Thus, need of novel therapeutic intervention is extremely urgent. Although the field of oncolytic virotherapy (OV) based on using viruses with natural or engineered tumor selective replication to intentionally infect and kill tumor cells has been extensively explored for the treatment of solid tumors, only few data are available for the treatment of hematopoietic malignancies. Our laboratory was one of the first to show that OV using Reovirus can be an effective therapeutic strategy for the treatment of MM in vitro and in MM patients. In this work we aim at exploring the possibility of using genetically engineered HSV1 (Herpes Simplex Virus) for the treatment of MM. HSV1 is an enveloped, double stranded DNA virus. Engineered HSV1 (HSVQ) has both copies of viral gene important in viral replication in normal cells viz., ICP 34.5 gene deleted and has one copy of GFP inserted into viral ICP6 gene. Such engineered virus has been used for cancer cell selective killing in preclinical and clinical studies for the treatment of several types of solid tumors including melanoma and glioblastoma multiforme. In this study, we investigated the biological and preclinical impact of HSVQ on MM cell in vitro and in vivo.Method: Recombinant HSVQ was amplified in African green monkey kidney epithelial Vero cells, purified by sucrose density gradient centrifugation and titrated by plaque assay on Vero cells. Several MM cell lines (MM1.S, U266, RPMI8226, L363, NIH-H929) were infected with HSVQ at Multiplicity of Infection (MOI) 0.01 to 5. Fluorescence microscopy and flow cytometry analysis were used to assess MM cell infectivity with the virus. RT-PCR was performed to detect presence of viral genome in MM cell lines. Viral replication assays were also performed. Cell proliferation and apoptotic assays including MTT Assay, Tryphan Blue exclusion test, LIVE/DEAD cell viability staining and Annexin/7-AAD assays were done to determine viability of virus infected MM cells. Western Blot analysis was carried out to determine endoplasmic reticulum (ER) stress response mediated by ERK, Hsp90, Bip/GRP78, Hsp40 and apoptosis in HSVQ treated MM cells. Total bone marrow (BM) cells obtained from MM patients were infected with HSVQ and multi parametric flow analysis was performed to determine infectivity and specific killing of CD138+ MM cells by the virus.To study in vivo anti-tumorigenic properties of HSVQ, 12.5 x106 GFP/Luc + MM1.S or NIH-H929 cells were subcutaneously injected into the right flank of 20 NOD-SCID mice. Two weeks after injection, mice with comparable size tumors were randomly divided (5 animal for each treatment group) and treated twice with 1x107PFU (Plaque Forming Unit) HSVQ for 2 weeks or with saline. Tumor growth was measured to determine anti tumorigenic effect of HSVQ on MM tumors.Results and Conclusion: Fluorescence microscopy and flow cytometry revealed that MM cell lines can be effectively infected with and killed by HSVQ even at MOI as low as 0.1. Under such conditions, Western Blot analysis revealed increased BAX expression, decreased BCL2 expression and cleavage of Caspase 3 and PARP indicating apoptosis of virus infected cells. Interestingly, multi parametric flow analysis revealed that HSVQ specifically infects and kills CD138+ MM plasma cells in a total population of BM cellular fraction isolated from MM patients. Moreover in vivo preclinical data show that HSVQ dramatically reduces tumor volume (p<0.001) in both MM.1S and NIH-H929 xenograft mouse models. Thus, from the preliminary observations, it can be concluded that HSVQ can selectively infect and induce apoptosis in myeloma cells. Mechanisms of HSVQ replication in MM cells and induced MM cell killing are being currently investigated. Here for the first time we are providing clear evidences that HSVQ can infect and specifically kill MM cells supporting the idea of the use of HSV for the treatment of MM. Moreover, since the backbone of HSVQ can be further engineered, it can be used to specifically deliver anti-angiogenic and anti-inflammatory genes to MM cells for the treatment of MM. DisclosuresHofmeister:Arno Therapeutics, Inc.: Research Funding; Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Incyte, Corp: Membership on an entity’s Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Signal Genetics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity’s Board of Directors or advisory committees.

Abstract 495: The impact of preoperative hydroxyprogesterone on serial levels of circulating tumor cells [CTCs] in patients undergoing surgery for operable breast cancer

Abstract Introduction: Assessment of Circulating Tumour Cells [CTCs] from the peripheral blood of patients diagnosed with cancer is emerging as a valuable biomarker for prognostication and tailoring therapy. A previous study demonstrated that a single depot injection of hydroxyprogesterone prior to surgery improved the DFS of patients with large tumours or positive nodes, forming the basis for this investigation, designed to assess effects of this drug on serial levels of CTCs on patients with operable breast cancer. Methods: Seventy patients were enrolled, randomized, and as previously described a single depot injection of hydroxyprogesterone was administered prior to surgery in patients in the experimental arm. Peripheral blood samples were obtained at three time points during surgery; prior to induction of anaesthesia, intra operative and at completion. Peripheral Blood Mononuclear Cell [PBMC] fractions separated on Ficoll hypaque were enriched for tumour cells with double positive immunomagnetic selection for EpCAM [Epithelial Cell Adhesion Molecule]. Half the enriched fraction was routed for multistep sequential labelling of cell-surface markers; EpCAM and CD45, cytoplasmic Cytokeratin and staining with DAPI for the nucleus, facilitating acquisition with multi parametric flow cytometry and imaging using confocal microscopy. With pre-amplification of other half of the enriched fraction, a quantitative expression analysis for a panel of epithelial genes was performed. Results:With multi parametric flow analysis, events that were CD45-/ EpCAM+/ CK+ with DAPI positivity were defined as CTCs, reported as events/ml blood and in all samples were in the range of 0 to 155 /ml of blood with no significant difference observed between the two arms. Detailed analysis with pooled data of all time points, in premenopausal patients, using the Mann Whitney test, showed that the mean number of CTCs is significantly lower[p = 0.005] in the experimental arm[n = 30] as compared to the control arm[n = 36] .Analysis within the hydroxyprogesterone group using the Wilcoxon signed rank test showed that the mean number of CTCs is lower in the post-surgery time point when compared to the pre surgery time point, in patients with positive nodes[n = 16][p = 0.044], large tumours [n = 18] [NS; p = 0.085] and post-menopausal node positive group [n = 7] [NS; p = 0.063] The expression analysis for the selected markers and confocal images validated the identity of tumour cells. Conclusions: Patient follow-up will ascertain the benefits of reduced numbers observed and establish CTCs as a surrogate marker for prognostication. Citation Format: Ujjwala M. Warawdekar, Vani Parmar, Akshita Singh, Swapnil Aher, Abhay Kulkarni, Meenal Chaudhari, Sudeep Gupta, Rajendra A. Badwe. The impact of preoperative hydroxyprogesterone on serial levels of circulating tumor cells [CTCs] in patients undergoing surgery for operable breast cancer. [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 495.

Abstract 4701: NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming

Abstract Adoptive immunotherapy for cancer has been limited by a lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We hypothesized that infusion of genetically modified tumor-specific T cells following autologous stem cell transplant (ASCT) may overcome these barriers for multiple myeloma (MM). To test this, we conducted a phase I/II clinical trial (NCT01352286) in which T cells engineered with an HLA-A*0201 restricted, affinity-enhanced TCR recognizing NY-ESO-1 / LAGE-1 peptides (NY-ESOc259-T), were infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy given with ASCT. HLA-A*0201 MM patients eligible for ASCT, with antigen positive tumor were enrolled. NY-ESOc259-T was manufactured in a 10 day process using anti-CD3/CD28 microbeads and lentiviral vector, and was administered two days following ASCT. IMWG criteria were used to assess response at day 100 with the addition of a near complete response category (nCR) due to the common occurrence of oligoclonal banding observed following rapid post-ASCT immune reconstitution. Blood and marrow samples were taken at multiple timepoints for serum cytokine analysis, NY-ESOc259-T persistence and trafficking, multiparameter flow analysis to examine the phenotype and function of NY-ESOc259-T, and tumor biomarker analysis. 25 of 29 enrolled patients were infused. A mean of 2.8 × 109 engineered cells were administered (range 8.3 × 108-4.2 × 109), and the average transduction efficiency was 33% (range 30%-45%). Patients tended to have advanced disease (64% chromosomal abnormalities, and 24% prior ASCT). At 3 months, 67% (16/24) and 58% (14/24) of patients were in VGPR and nCR or better, respectively. Infusions were well-tolerated and no cytokine release syndrome was reported. NY-ESOc259-T persisted at 6 months in all but one patient, and in a subset of patients at 2 years; marrow infiltration was consistently observed from day 7 through day 180. NY-ESOc259-T initially displayed a dominant activated effector phenotype which converted towards a dominant effector memory phenotype by 1 year post infusion, in a pattern that mirrored clinical responses. Persisting cells demonstrated a polyfunctional response (IFN-γ and TNF-α) with a cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Tumor biomarker analysis is ongoing. MM relapse occurred in 13/25 patients. This data show that NY-ESOc259-T cells exhibit robust trafficking and expansion, durable persistence without exhaustion, and follow a natural immune expansion and contraction pattern consistent with an antigen-driven mechanism of action. Relapse correlated with a loss of persistence or tumor antigen escape, suggesting that targeting multiple antigens and maintenance infusions may increase durable remissions. Citation Format: Aaron Rapoport, Edward Stadtmauer, Luca Melchiori, Ryan Wong, Eduardo Davila, Gwendolyn Binder-Scholl, Tom Holdich, Dan Vogl, Brendan Weiss, Jeffrey Finkelstein, Simon Lacey, Sarah Bond, Marylene Fortin, Yoav Peretz, Joanna Brewer, Alan Bennett, Andrew Gerry, Nick Pumphrey, Helen Tayton-Martin, Lilliam Ribeiro, Ashraf Badros, Saul Yanovich, Nancy Hardy, Jean Yared, Naseem Kerr, Sunita Philip, Sandra Wesphal, Bruce L. Levine, Carl June, Michael Kalos, Bent Jakobsen. NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2015-4701

Abstract PR05: Combination therapy with anti-CTLA4 and anti-PD1 leads to distinct immunologic changes in-vivo

Abstract Therapies targeting T cell immune checkpoints such as CTLA4 and PD1/PDL1 axis have shown considerable promise in the therapy of human cancer. Combination therapy with dual immune checkpoint blockade (ICB) was recently shown to be highly active in melanoma. While signaling via both PD1 and CTLA4 is known to converge downstream and dampen T cell function, data comparing in vivo effects of blockade of these immune checkpoints either alone or in combination in vivo in humans are limited. Here we have analyzed paired pre/post therapy samples from patients treated either anti-CTLA4 (n=5) or anti-PD1 (n=6) alone, or a combination of anti-CTLA4 and anti-PD1 (n=8), using several methodologies including multi-parameter flow cytometry, single-cell mass-cytometry (CyTOF), Luminex and analysis of transcriptome of purified immune cells with exon-level arrays. We show that blockade of CTLA4, PD1 or combination blockade leads to distinct immunologic, genomic and cytokine signatures in vivo. CTLA4 blockade leads to a prominent proliferation signature in vivo, manifest as an increase in Ki-67 expression in a subset of T cells with transitional memory phenotype. PD1 blockade does not induce this phenotype and instead leads to marked changes in T cells expressing NK and cytolysis associated genes, as exemplified by Granzyme+ T cells. Combination blockade leads to non-overlapping changes in gene expression including proliferation-associated and chemokine genes and leads to an increase in both Ki67+and Granzyme+ T cells. Overall, therapy-induced changes are more prominent in T cells than in monocytes include also involve non-overlapping changes in several alternatively-spliced transcripts, and non-coding RNAs. Each of the ICB therapies also leads to a distinct cytokine profile with differential effects on systemic levels of sIL2R and IL1a. Changes seen in the peripheral blood T cells can also be seen in the tumor infiltrating lymphocytes. Combination therapy leads to an increase in interferon-gamma; producing T cells in both circulation as well as tumor bed. PD1 expression is higher on tumor infiltrating T cells when compared to T cells in circulation. Importantly, PD1 receptor occupancy following anti-PD1 therapy may be incomplete in the tumor infiltrating T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that blockade of PD1, CTLA4 alone or in combination have distinct immunologic effects in vivo and each strategy serves as a unique immune-therapeutic. Improved understanding of the in vivo effects of ICB is needed for rational development of future immune-based combinations against cancer. This abstract is also being presented as Poster B06. Citation Format: Rituparna Das, Rakesh Verma, Mario Sznol, Chadra Sekhar Boddupalli, Scott Gettinger, Harriet Kluger, Madhav Dhodapkar, Kavita Dhodapkar. Combination therapy with anti-CTLA4 and anti-PD1 leads to distinct immunologic changes in-vivo. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR05.

Stable phenotype of B-cell subsets following cryopreservation and thawing of normal human lymphocytes stored in a tissue biobank.

Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that "overall, there are no phenotypic differences between cryopreserved and fresh B-cell subsets." Subsequently, we performed an uncontrolled comparison of tonsil tissue samples. By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue-specific comparative analysis.

Stable Phenotype Of B-Cell Subsets Following Cryopreservation and Thawing of Normal Human Lymphocytes Stored in a Tissue Biobank.

Background Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. Methods We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that "overall, there are phenotypic differences between cryopreserved and fresh B-cell subsets". Subsequently, we performed a consecutive uncontrolled comparison of tonsil tissue samples. Results By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. Conclusions We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue specific comparative analysis. © 2014 Clinical Cytometry Society.

Multiparameter DNA content analysis identifies distinct groups in primary breast cancer

Background:Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique.Methods:The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis.Results:Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76–1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18–1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI⩾1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and ‘multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (⩾10%) independently associated with poor overall survival (P=0.027, CR).Conclusion:Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.

Sensitive Detection of Plasma Cells by Integrated Enrichment in-Column Staining and Analysis

Abstract 4968In Multiple Myeloma immunophenotyping by multiparameter flow cytometry is a well-established method in clinical research to identify, characterize and enumerate neoplastic plasma cells. In addition, recent clinical studies demonstrated prognostic relevance of MRD detection by multiparameter flow analysis. The high applicability of the flow cytometric analysis compared to patient-specific RT-PCR holds the potential to become an important tool in MRD assessment.For the identification of plasma cells the detection of more than one antigen is required and for an unequivocal discrimination between normal and neoplastic plasma cells the combination with clonality assessment is mandatory. Here we established a combination of five antibodies for surface staining: CD138-PE, CD38-FITC, CD56-APC, CD19-PE-Vio770, CD45-APC-Vio770 and two antibodies for intracellular staining: Anti Igκ-PerCP, Anti Igλ-VioBlue. The combination of fluorochromes for this 7-color panel was optimized for high resolution of the target population.To minimize variability among flow cytometric data, it is crucial to standardize the various procedures – in particular in clinical settings. Pre-defined antibody panels and simplified sample handling are major factors in the reduction of variability. Furthermore, in order to obtain maximal sensitivity processing of large cell numbers is required, which is a major obstacle for conventional flow cytometers.Here we demonstrate that magnetic pre-enrichment of CD138+ in combination with a new protocol for in-column intracellular staining for κ/λ light chains of retained plasma cells allows handling of up 108 cells (fig. 1). Together with the optimized antibody panel this approach offers a unique and valuable tool for characterization of rare plasma cells and offers increased sensitivity during MRD assessment. The MACSQuant® Analyzer offers the possibility to combine magnetic pre-enrichment with intracellular staining and flow cytometric analysis. This minimizes manual handling steps and reduces cell loss due to omission of centrifugation steps usually required for intracellular staining procedures.The approach of combined cell enrichment, in-column intracellular staining and usage of standardized antibody panels on the MACSQuant Analyzer provides an automated workflow, which is highly suitable for routine detection and characterization of rare plasma cells. [Display omitted] Disclosures:Kühbach:MiltenyiBiotec GmbH: Employment. Herber:Miltenyi Biotec GmbH: Employment. Koehler:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment.

Preclinical Characterization of Mitochondria-Targeted Small Molecule Hsp90 Inhibitors, Gamitrinibs, in Advanced Prostate Cancer

This study aimed to characterize the preclinical activity of the first class of combinatorial, mitochondria-targeted, small molecule heat shock protein-90 (Hsp90) inhibitors, gamitrinibs, in models of hormone-refractory, drug-resistant, localized, and bone metastatic prostate cancer in vivo. Mitochondrial permeability transition, apoptosis, and changes in metabolic activity were examined by time-lapse videomicroscopy, multiparametric flow cytometry, MTT, and analysis of isolated mitochondria. Drug-resistant prostate cancer cells were generated by chronic exposure of hormone-refractory PC3 cells to the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG). The effect of gamitrinibs on s.c. or intratibial prostate cancer growth was studied in xenograft models. Bone metastatic tumor growth and bone parameters were quantified by micro-computed tomography imaging. In the NCI 60-cell line screening, gamitrinibs were active against all tumor cell types tested, and efficiently killed metastatic, hormone-refractory, and multidrug-resistant prostate cancer cells characterized by overexpression of the ATP binding cassette transporter P-glycoprotein. Mechanistically, gamitrinibs, but not 17-AAG, induced acute mitochondrial dysfunction in prostate cancer cells with loss of organelle membrane potential, release of cytochrome c, and caspase activity, independently of proapoptotic Bcl-2 proteins Bax and Bak. Systemic administration of gamitrinibs to mice was well tolerated, and inhibited s.c. or bone metastatic prostate cancer growth in vivo. Gamitrinibs have preclinical activity and favorable safety in models of drug-resistant and bone metastatic prostate cancer in vivo.

Cdx4 Is Dispensable for Murine Hematopoiesis and MLL-AF9 Mediated Leukemogenesis.

Caudal-related (Cdx) genes are transcription factors that function as master homeobox regulators of HOX genes. Three Cdx genes (Cdx1, Cdx2 and Cdx4) have been identified in mammals, and function in part as mediators anterior-posterior patterning during development. However, accumulating data suggests a central role for Cdx genes in vertebrate hematopoiesis. This link was first established in the zebrafish model whereby Cdx4-deficient fish exhibited severe anaemia within the first day of development and fail to specify blood progenitors (Davidson et al, Nature 2003). Given the high degree of conservation of Cdx genes between species, we anticipated that the loss of Cdx4 in the mouse would result in a phenotype similar to the lethal bloodless phenotype observed in zebrafish. This initial hypothesis was supported by reports that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells or embryoid bodies (McKinney-Freeman et al, Blood 2008). To establish the function of the Cdx4 gene in mammalian hematopoiesis, we analyzed the hematopoietic system during development and in the adult hematopoietic compartment, using mice in which Cdx4 was either deleted in the germline, or conditionally deleted in the adult hematopoietic system using Mx-Cre mediated excision. Germline deletion resulted in fertile and viable animals born with expected Mendelian rations. Surprisingly, there were no apparent gross phenotypic manifestations of Cdx4 deletion in either context despite complete excision of the Cdx4 gene in the hematopoietic compartment. Peripheral blood counts were normal, as was flow cytometric analysis for the myeloid and lymphoid compartments as assessed using Mac1/Gr1 and various B- & T-cell markers. Using multi-parameter flow analysis and methylcellulose plating assays, we observed that loss of Cdx4 in both mouse models only caused subtle hematopoietic perturbations, such as an increase in thymus weight and a modest decrease in pre-B colony formation ability in vitro. However, there were no other apparent defects in the hematopoietic stem and progenitor compartments including GMP, CMP & MEP. There were also no marked differences in the expression of Hox genes in the hematopoietic compartment, even though striking dysregulation of various Hox genes was observed in Cdx4-deficient zebrafish model. We also assessed the effect of Cdx4 deletion on the development of leukemia mediated by MLL fusion genes. Using in vivo retroviral transduction/bone marrow transplantations and in vitro serial-replating assays, we observed that Cdx4 null mice remain susceptible to leukemic transformation by MLL-AF9, indicating that Cdx4 is not necessary for MLL-AF9 mediated leukemias. The only subtle differences observed were that Cdx4−/− progenitors transduced with MLLAF9 generated more differentiated transformed blast colony-forming units in vitro, and induced leukemias with longer disease latencies compared to their wild-type littermate controls. Thus, in contrast with the zebrafish system, we found that Cdx4 is dispensable for normal mammalian hematopoiesis, as well as for MLL-AF9 mediated leukemia. The striking differences we observed between the studies of the same gene in the two different vertebrate systems emphasizes the importance of not solely relying on a single model system to assess the role of transcription factors in development, and that the deficiency of even a highly conserved gene might perturb function to variable extents in different animal models.

Simultaneous Isolation of Human Bone Marrow Hematopoietic, Endothelial, and Mesenchymal Progenitor Cells by Flow Sorting Based on Aldehyde Dehydrogenase Activity.

ALDH bright [ALDHbr] cells exhibit low side scatter and high activity of the intracellular enzyme aldehyde dehydrogenase [ALDH] in flow cytometric analysis. ALDH regulates gene expression in many developing systems by producing retinoic acids. ALDHbr populations from human umbilical cord [UCB] and mobilized peripheral blood preparations [PBSC] are highly enriched for cells that express CD34 and CD133 and for hematopoeitic progenitor cells. Because human bone marrow [BM] contains a highly heterogeneous population of stem and progenitor cells, we used multiparameter flow analysis and cell sorting to measure expression of cell surface markers and lineage specific developmental potential of BM ALDHbr populations. The ALDHbr population comprised 1.2 ± 0.8% (mean ± SD, n=39) of the nucleated BM cells and was significantly (p<0.05) enriched in cells expressing CD34, CD117, CD105, CD127, CD133, and CD166. ALDHbr populations were also significantly enriched in cells expressing primitive progenitor phenotypes including CD34+CD38− and CD34+CD133+ cells. The cell surface antigen expression of BM ALDHbr populations differed significantly from UCB and PBSC populations. In particular, the BM ALDHbr populations had more CD34− cells and CD45− cells and fewer CD133+ cells than the UCB and PBSC ALDHbr populations. Next, we compared the ability of sorted ALDHbr cells and ALDHdim cells derived from each bone marrow to develop into erythroid and myeloid cells, megakaryocytes, endothelial cells, and mesenchymal cells in culture. The ALDHdim population was the residual cell population produced when we sorted ALDHbr cells out of BM. ALDHbr populations were 144-fold more active in primary hematopoietic colony forming activity than ALDHdim cells, and all detectable BM cells that could form megakaryocyte colonies were ALDHbr. Even though hematopoietic progenitor activity was concentrated in the ALDHbr population, the majority of CD34+ and CD133+ cells in BM were ALDHdim. Cultures seeded with 50,000 ALDHbr cells gave rise to confluent growth of endothelial cells that expressed surface Ulex lectin receptors and von Willebrand factor, formed tubules on Matrigel, and took up acetylated LDL in 34 of 39 BM tested. In contrast, only 18 of 33 ALDHdim BM populations tested gave rise to endothelial cultures, and 500,000 ALDHdim cells were required to generate confluent cultures in 21 days. Cells that gave rise to early endothelial growth colonies at day 7 were 435-fold more frequent in the ALDHbr than the ALDHdim population. Confluent cultures of cells expressing cell surface antigens and functional attributes of multipotential mesenchymal cells were also routinely established with 50,000 ALDHbr cells. Again, ten-fold more ALDHdim cells than ALDHbr cells were needed to establish mesenchymal cultures. The mesenchymal cultures could be driven to differentiate into adipocytes, chondrocytes, osteoblasts, and neural lineage cells in standard culture assays. These results show that most of the hematopoietic, endothelial, and mesenchymal progenitor cell activities in human BM can be simultaneously isolated by cell sorting based on ALDH activity. Such ALDHbr populations may be useful in a variety of cell therapy indications.

Rapid and Sensitive Detection of Minimal Plasma Cell Populations Using Six-Colour Flow Cytometry.

Flow cytometry is a powerful tool for diagnosis and detection of minimal residual disease (MRD) in patients with monoclonal gammopathies. Multi-parameter MRD flow analysis is more sensitive than serum immunofixation for the detection of residual disease and is as effective as PCR analysis for predicting outcome after high-dose therapy. Attaining a limit of detection of 0.01% abnormal plasma cells in every patient requires analysis of several neoplastic markers. However, basic immunophenotyping coupled with clonality assessment can be informative in many patients. This approach can also be helpful at diagnosis in MGUS patients to determine whether the primary abnormal component is plasmacytoid or lymphoid. The aim of this study was to develop a single six-colour assay that could be used as a rapid and sensitive screen for the presence of neoplastic plasma cells. Bone marrow aspirate samples were assessed from 83 patients at diagnosis with a suspected plasma cell disorder (median age 69.1 years, 48 male and 35 female) and from 62 patients with myeloma after treatment (median age 61.0 years, 40 male and 22 female). Cells were washed and surface-labelled with CD19 PE, CD38 PE-Cy7, CD138 APC and CD45 APC-Cy7 then fixed, permeabilised and incubated with Kappa FITC and Lambda PE-Cy5. The assay allows assessment of B-cell surface-Ig and plasma cell cytoplasmic-Ig light chain restriction and abnormal plasma cell phenotype according to CD19 and CD45 expression. In presentation cases with more than 10% plasma cells by morphology, neoplastic plasma cells were present in 48/50 samples with one reactive plasmacytosis (32% plasma cells) and one patient with Castlemans disease (11% PC). In the remaining 33 cases with <10% plasma cells, 16/33 (48%) had monoclonal plasma cells, 4/33 (12.1%) showed a low-level of monoclonal B-cells and the remainder had normal B-cells and plasma cells. The assay provided a quantitative MRD result in 44/62 (70%) cases: neoplastic plasma cells represented <1% of leucocytes in 26/44 of the samples (59%) and the assay could detect neoplastic plasma cells when they represented as few as 0.01% of total leucocytes in several patients (4/44, 9.1%). Using this screening approach an extended MRD analysis panel was only required in 18/62 (30%) of cases where CD19- plasma cells were detected on a polyclonal background. Marrow quality was compared with a paired trephine biopsy in 68 samples. Using a algorithm based on the levels of normal plasma cells, B-progenitors, nucleated red cells and myeloid progenitors it was possible to determine whether the samples were representative bone marrow and a quantitative result could be provided. This single tube six-colour approach allows simultaneous assessment of B-cell and plasma cell immunophenotype and clonality. The incorporation of all the relevant markers into a single test makes analysis more straightforward and the gating strategy more reproducible. The assay is more rapid and cost-effective than four-colour analysis as fewer total markers are required. It can be applied to presentation samples to exclude reactive plasmacytoses and B-lymphoproliferative disorders, and to follow up samples to reduce the need for further flow cytometric investigations and optimise any downstream testing that is necessary. In approximately 70% of post-treatment myeloma samples this assay is capable of providing a definitive MRD result up to a maximum sensitivity of 0.01%.