Multiparameter Flow Cytometric Research Articles

Artificial intelligence in clinical multiparameter flow cytometry and mass cytometry-key tools and progress.

There are many research studies and emerging tools using artificial intelligence (AI) and machine learning to augment flow and mass cytometry workflows. Emerging AI tools can quickly identify common cell populations with continuous improvement of accuracy, uncover patterns in high-dimensional cytometric data that are undetectable by human analysis, facilitate the discovery of cell subpopulations, perform semi-automated immune cell profiling, and demonstrate potential to automate aspects of clinical multiparameter flow cytometric (MFC) diagnostic workflow. Utilizing AI in the analysis of cytometry samples can reduce subjective variability and assist in breakthroughs in understanding diseases. Here we review the diverse types of AI that are being applied to clinical cytometry data and how AI is driving advances in data analysis to improve diagnostic sensitivity and accuracy. We review supervised and unsupervised clustering algorithms for cell population identification, various dimensionality reduction techniques, and their utilities in visualization and machine learning pipelines, and supervised learning approaches for classifying entire cytometry samples.Understanding the AI landscape will enable pathologists to better utilize open source and commercially available tools, plan exploratory research projects to characterize diseases, and work with machine learning and data scientists to implement clinical data analysis pipelines.

Pre-Transplant Multiparameter Flow Cytometric Measurable Residual Disease in Acute Myeloid Leukemia As an Independent Prognostic Factor for Survival after Myeloablative Allogeneic Hematopoietic Cell Transplantation

Background Growing evidence suggests that multiparameter flow cytometric (MFC) measurable residual disease (MRD) assessment in acute myeloid leukemia (AML) should be used for risk stratification prior to allogeneic hematopoietic stem cell transplantation (alloHCT). We investigated the significance of MFC-MRD status in AML patients treated with myeloablative conditioning (MAC) alloHCT at transplant centers within the Polish Adult Leukemia Group (PALG). Methods All consecutive patients who underwent MAC alloHCT from HLA identical siblings (SD), family haploidentical donor, matched (10/10) unrelated donor (MUD), or mismatched (9/10) unrelated donor (mMUD) between January 2014 and May 2022 and for whom pre-transplant MFC-MRD was assessed were included in the analysis. MFC-MRD assessment has been routinely performed on bone marrow aspirates obtained before alloHCT since 2014 or as part of the PALG-AML1/2016 study protocol (NCT03257241) since 2018. Flow cytometry laboratory protocols were modified and unified by the cooperating laboratories according to the European LeukemiaNet recommendations in 2018. Residual disease at a level ≥0.1% was considered MRD(+). Results We analyzed 302 adult patients (median age 44 years, range 18-68) with AML in first (88%) or subsequent CR (12%) after intensive chemotherapy, including 81 patients (27%) transplanted between 2014-2017 and 221 (73%) transplanted between 2018-2022. Positive MRD status before alloHCT was detected in 143/302 pts (47%). The European LeukemiaNet (ELN) cytogenetic/genetic risk (Dohner 2010) was available in 290 of the 302 patients and was intermediate-II (Int-II)/high in 22%. Patients received MAC based on i.v. busulfan (84%), treosulfan (1%) or total body irradiation (15%) and graft from either SD (29%), haploidentical donor (9%), MUD (55%), or mMUD (7%). GvHD prophylaxis consisted of calcineurin inhibitor (CNI) combined with MTX (with or without ATG) or was based on post-transplant cyclophosphamide in transplants from haploidentical donor or mMUD. After the median follow-up of 30 months (range, 1-97), the 3-year LFS and OS for MRD(-) vs MRD(+) patients was 69% vs 54% (log-rank p=0.007), and 77% vs 62% (log-rank p=0.013), respectively. In multivariate Cox model, the independent adverse prognostic factors for LFS were MRD positive status (HR 1.53, 95%CI 1.03-2.25; p=0.0332), Int-II/high ELN risk (HR 1.87, 95%CI 1.24-2.84; p=0.0031), and age ≥ 45 years (HR 1.65, 95%CI 1.12-2.45; p=0.011). The same factors independently influenced OS [HR 1.59, 95%CI 1.01-2.48; p=0.0411; HR 1.67, 95%CI 1.03-2.71; p=0.0363, and HR 1.79, 95%CI 1.42-2.79; p=0.0109, respectively]. When the patients were classified according to the number of identified independent risk factors, the 3-year LFS and OS for patients with 0, 1, 2 and 3 risk factors was 75%, 68%, 51%, and 27% (p<0.0001) (Figure 1), and 81%, 77%, 57%, and 44%, respectively (p=0.0006). Conclusions Our findings confirm that pre-transplant residual disease at a ≥0.1% level assessed by MFC is an independent poor prognostic factor for both LFS and OS in AML patients treated with MAC alloHCT. As predicted, outcomes after MAC alloHCT were also independently influenced by ELN cytogenetic/genetic risk and age. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular methods for measurable residual disease in acute myeloid leukemia: where are we and where are we going?

Measurable residual disease (MRD) testing has become a standard practice for patients with acute myeloid leukemia (AML) following therapy. However, MRD testing in AML is not straightforward, and both molecular methods and multiparameter flow cytometric (MFC) methods demonstrate clinical utility. While MFC methods are potentially applicable to all AML patients, current molecular MRD methods must be individually tailored to the patient’s AML disease genetics, a strategy that is currently applied for patients with acute promyelocytic leukemia, core binding factor AML, and AML with mutated NPM1. However, there is great interest in next-generation sequencing (NGS) methods for MRD assessment, an approach that could potentially be applied to all patients with AML. Current NGS methods have limited analytic sensitivity when compared with other molecular methods and MFC, but advances in NGS methods and informatic algorithms may improve NGS MRD testing in the near future. In this review, we discuss current recommendations for molecular MRD assessment in AML and discuss opportunities and challenges for MRD assessment by NGS methods.

Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis

Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.

Flow Cytometric-Based Analysis of Defects in Lymphocyte Differentiation and Function Due to Inborn Errors of Immunity.

The advent of flow cytometry has revolutionized the way we approach our research and answer specific scientific questions. The flow cytometer has also become a mainstream diagnostic tool in most hospital and pathology laboratories around the world. In particular the application of flow cytometry has been instrumental to the diagnosis of primary immunodeficiencies (PIDs) that result from monogenic mutations in key genes of the hematopoietic, and occasionally non-hematopoietic, systems. The far-reaching applicability of flow cytometry is in part due to the remarkable sensitivity, down to the single-cell level, of flow-based assays and the extremely user-friendly platforms that enable comprehensive analysis, data interpretation, and importantly, robust and rapid methods for diagnosing PIDs. A prime example is the absence of peripheral blood B cells in patients with agammaglobulinemia due to mutations in BTK or related genes in the BCR signaling pathway. Similarly, the development of intracellular staining protocols to detect expression of SAP, XIAP, or DOCK8 expedites the rapid diagnosis of the X-linked lymphoproliferative diseases or an autosomal recessive form of hyper-IgE syndrome (HIES), respectively. It has also become evident that distinct cohorts of PID patients exhibit unique “lymphocyte phenotypic signatures” that are often diagnostic even prior to identifying the genetic lesion. Flow cytometry-based sorting provides a technique for separating specific subsets of immune cells such that they can be studied in isolation. Thus, flow-based assays can be utilized to measure immune cell function in patients with PIDs, such as degranulation by cytotoxic cells, cytokine expression by many immune cells (i.e., CD4+ and CD8+ T cells, macrophages etc.), B-cell differentiation, and phagocyte respiratory burst in vitro. These assays can also be performed using unfractionated PBMCs, provided the caveat that the composition of lymphocytes between healthy donors and the PID patients under investigation is recognized. These functional deficits can assist not only in the clinical diagnosis of PIDs, but also reveal mechanisms of disease pathogenesis. As we move into the next generation of multiparameter flow cytometers, here we review some of our experiences in the use of flow cytometry in the study, diagnosis, and unraveling the pathophysiology of PIDs.

What Is the Frequency of Transplant-Eligible Multiple Myeloma Patients Being Cured? the Impact of an MGUS-like Signature at Diagnosis and MRD-Negativity

Introduction: Although multiple myeloma (MM) is typically described as an incurable disease, it has been shown in recent years that a small fraction of patients may reach more than 10-years progression-free survival (PFS), which is considered as the minimum threshold to identify patients in "operational cure". However, because of the scarcity of available data there is significant lack of knowledge in MM regarding the frequency of cases attaining operational cure, nor the existence of biomarkers that could prospectively predict such curability.Methods: Herein, we sought to define the frequency as well as the biomarkers predictive of operational cure in a large series of uniformly-treated transplant-eligible patients enrolled in the PETHEMA/GEM2000 protocol (VBMCP/VBAD followed by HDT/ASCT and 2 years of maintenance with interferon and prednisone). Patients' follow-up was updated at the time of abstract submission, and the median follow-up of the series is now of 12-years. We used an automated multiparameter flow cytometric (MFC) classification model focused on the analysis of the bone marrow plasma-cell compartment to identify among newly diagnosed symptomatic MM those with MGUS-like vs. MM-like phenotypic signatures. Minimal residual disease (MRD) was monitored using a first-generation 4-color MFC assay (CD38-FITC / CD56-PE / CD19-PerCPCy5.5 / CD45-APC) with a limit of detection of 10-4. PFS and overall survival (OS) were measured from the time of diagnosis.Results: From a total of 1075 patients enrolled in the GEM2000 protocol, 763 were eligible for this analysis because they either relapsed or died during the first 10-years from diagnosis (n=666; 87%), or remained progression-free and alive for more than 10-yers (n=97; 13%); accordingly, all patients remaining progression-free and alive but for which the follow-up was inferior to 10-years were excluded from the analysis. We then investigated the biomarkers that could help to identify patients reaching operational cure after HDT/ASCT. As compared to the vast majority of cases, patients reaching >10-years PFS (13%) had significantly less frequent anemia (76% vs. 60%, respectively; P=.002), as well as more frequent Durie-Salmon stage IA (14% vs. 6%; P=.004), MGUS-like signature as determined by the automated MFC algorithm (28% vs. 6%; P<.001), complete response (CR) after HDT/ASCT (51% vs. 35%; P=.003), and MRD-negativity by MFC (72% vs. 31%; P<.001). Other biomarkers such as ISS, LDH, ploidy and proliferation were not significantly different among patients reaching >10-years PFS vs. those who relapsed earlier. On multivariate analysis, only the presence of an MGUS-like signature at baseline (P=.04; HR: 3.9) and MRD-negativity at day+100 after HDT/ASCT (P=.006; HR: 6.3) emerged as independent predictive markers for >10-years PFS; anemia, Durie-Salmon and CR status were not retained in the logistic regression model. Accordingly, patients with an MFC-defined baseline MGUS-like signature reaching MRD-negativity after HDT/ASCT (n=14) had a median PFS of 10-years and a 10-year OS rate of 79%, which were significantly superior to those observed among cases with MM-like signatures being MRD-negative (n=54) or positive (n=99) after therapy [median PFS of 6 and 3 years (P<.001); 10-year overall survival rates of 55% and 19% (P<.001)].Conclusions: We demonstrated that operational cure (i.e.: >10-years PFS) was possible for 13% of transplant-eligible MM patients before the era of novel agents. Curability rates were particularly frequent among patients with a benign phenotypic signature at diagnosis and MRD negativity after HDT/ASCT, suggesting a remarkable clinical benefit of attaining deep remissions after intensive treatment for patients with early MM. DisclosuresPaiva:Sanofi: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Onyx: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy; Celgene: Consultancy. Puig:The Binding Site: Consultancy; Janssen: Consultancy. Mateos:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria; Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: Honoraria.

Significance of Peri-Transplant Dynamics of Minimal Residual Disease (MRD) in Adults with Acute Myeloid Leukemia (AML) in Morphological Remission Undergoing Myeloablative Allogeneic Hematopoietic Cell Transplantation

Background: Numerous studies from others and our institution have demonstrated that the presence of minimal residual disease (MRD), detected at the time of hematopoietic cell transplantation (HCT), is strongly and independently associated with increased relapse risk and short survival in adults with acute myeloid leukemia (AML) undergoing myeloablative allogeneic HCT in morphologic complete remission (CR). In contrast, very little information is available regarding the prognostic significance of peri-transplant MRD dynamics in these patients. Since bone marrow staging studies with multiparameter flow cytometric (MFC) assessment for MRD are routinely obtained not only before but also at approximately day +28 following transplantation at our institution, we here retrospectively studied the relationship between peri-HCT MRD dynamics and post-transplant outcomes in a large patient cohort. We asked whether persistence or disappearance of MRD might identify cohorts of patients in whom post-transplant therapy was particularly indicated or unnecessary.Patients and Methods: AML patients ³18 years of age were eligible for this retrospective analysis if they were in first or second morphologic CR or CR with incomplete blood count recovery (CRi) irrespective of the presence of MRD, underwent allogeneic HCT with myeloablative conditioning between 2006 and 2014, received peripheral blood or bone marrow as stem cell source, and had pre-HCT bone marrow staging studies available that included 10-color MFC assessments for MRD. MRD was identified as a cell population showing deviation from normal antigen expression patterns compared with normal or regenerating marrow; any level of residual disease was considered MRDpos. We considered post-HCT MRD assessments in patients in whom bone marrow re-staging with MFC MRD analysis were obtained 28±7 days after transplantation. For this analysis, the primary endpoint of interest was overall survival, which was estimated using the Kaplan-Meier method.Results: 311 patients were identified and included in this study. Consistent with our previous analyses, patients with MRD at the time of HCT (MRDpos; n=76) had significantly shorter survival than MRDneg patients (n=234; estimated 3 year post-HCT survival: 26% [95% confidence interval: 17-37%) vs. 73% [66-78%], P <0.001). 310 patients survived at least 21 days following transplantation; for 279 of these (89.7%), post-HCT MRD assessments were obtained at day +28±7 and available for analysis. 214 patients (76.7%) had no MFC evidence of MRD before and after HCT (MRDneg/MRDneg), 2 (0.7%) were MRDneg/MRDpos, 49 (17.6%) were MRDpos/MRDneg, and 14 (5.0%) were MRDpos/MRDpos. Of the 65 patients who had detectable MRD either before and/or after transplantation, 58 had decreasing levels of MRD (MRDdecr) over the peri-HCT period, whereas 7 patients had increasing MRD levels (MRDincr) around the time of transplantation. As depicted in Figure 1, MRDneg/MRDneg patients had excellent long-term outcomes (survival at 3 years after day +28 MRD assessment: 76% [69-82%]), whereas both MRDneg/MRDpos patients died within 70 days after the day +28 MRD assessment. Interestingly, for patients who were MRDpos before transplantation, outcomes were relatively poor regardless of whether or not they had persistent MRD around day +28 after transplantation (MRDpos/MRDneg patients: 23% [12-36%]; for MRDpos/MRDpos patients: 19% [4-44%]). However, long-term survival was only observed among MRDdecr patients (at 3 years after day +28 MRD assessment: 24% [14-37%]), whereas all MRDincr patients died a median of 97 (range: 15-808) days following the post-HCT MRD assessment (Figure 2).Conclusion: Patients who have no evidence of MRD before and after HCT have excellent long-term outcomes. In contrast, patients who are MRDpos before transplantation have poor survival expectations regardless of whether or not they clear MRD within the first 28 days after transplantation, but long-term survival is only found among some patients with decreasing MRD levels over the peri-transplant period. This finding suggests that patients who are MRDpos at the time of HCT should be considered for pre-emptive therapeutic strategies given their high risk of disease recurrence regardless of the day +28 MRD information. [Display omitted] [Display omitted] DisclosuresRadich:Incyte: Consultancy; Ariad: Consultancy; Gilliad: Consultancy; Novartis: Consultancy, Research Funding. Walter:Amphivena Therapeutics, Inc.: Consultancy, Research Funding; Seattle Genetics, Inc.: Research Funding; Covagen AG: Consultancy; AstraZeneca, Inc.: Consultancy; Pfizer, Inc.: Consultancy; Amgen, Inc.: Research Funding.

Comparison of cross-platform flow cytometry minimal residual disease evaluation in multiple myeloma using a common antibody combination and analysis strategy

A seven-color/eight-antibody approach has recently been proposed for the minimal residual disease (MRD) determination in multiple myeloma (MM), which was developed on FACSCantoII instruments. This strategy should be also applicable on different multiparameter flow cytometers (MFC), but this needs to be demonstrated before moving MRD assessment to local flow cytometry core facilities, nearer to patients, thereby reducing the risk of cell losses induced by sample transportation and delays in cell processing. To evaluate the comparability of testing the same seven-color/eight-antibody single-tube on any instruments, MRD was evaluated concomitantly on two distinct MFC in a cohort of 80 bone marrow MM samples (i.e., 73 patients including seven with two MRD evaluations) in two French centers, Paris-Cochin and Dijon. No significant difference in the MM residual plasma-cells (MM-PCs) quantification was observed. Calculated on the basis of the whole amount of leukocytes assessed, the mean MRD percentage was, respectively, 0.1661% and 0.1458% using FACSCantoII or Navios instruments, with a very high correlation between instruments (r(2) = 0.9798) and a very minimal bias (-0.02). Moreover, there was no difference in MRD interpretation at 10(-4) threshold; whereas three MRD interpretation discordances were observed at 2.5 × 10(-5) threshold. This study demonstrates that this MRD-detection strategy is transposable between harmonized ≥ seven-color instruments. This shows that a homogeneous rapid MRD evaluation can be performed in most MFC platforms, in the near vicinity of clinical wards. However, the clinical validation of this approach needs to be strengthened, as well as its relevance compared to molecular approaches.

Comparison of cross-platform flow cytometry minimal residual disease evaluation in multiple myeloma using a common antibody combination and analysis strategy.

Background: A seven-color/eight-antibody approach has recently been proposed for the minimal residual disease (MRD) determination in multiple myeloma (MM), which was developed on FACSCantoII instruments. This strategy should be also applicable on different multiparameter flow cytometers (MFC), but this needs to be demonstrated before moving MRD assessment to local flow cytometry core facilities, nearer to patients, thereby reducing the risk of cell losses induced by sample transportation and delays in cell processing. Methods: To evaluate the comparability of testing the same 7-color/8-antibody single-tube on any instruments, MRD was evaluated concomitantly on two distinct MFC in a cohort of 80 bone marrow MM samples (ie. 73 patients including 7 with 2 MRD evaluations) in two French centers, Paris-Cochin and Dijon. Results: No significant difference in the MM residual plasma-cells (MM-PCs) quantification was observed. Calculated on the basis of the whole amount of leukocytes assessed, the mean MRD percentage was respectively 0.1661% and 0.1458% using FACSCantoII or Navios instruments, with a very high correlation between instruments (r2 = 0.9798) and a very minimal bias (-0.02). Moreover, there was no difference in MRD interpretation at 10-4 threshold; whereas 3 MRD interpretation discordances were observed at 2.5×10-5 threshold. Conclusion: This study demonstrates that this MRD-detection strategy is transposable between harmonized ≥7-color instruments. This shows that a homogeneous rapid MRD evaluation can be performed in most MFC platforms, in the near vicinity of clinical wards. However, the clinical validation of this approach needs to be strengthened, as well as its relevance compared to molecular approaches. © 2014 Clinical Cytometry Society.

Simultaneous multicolor flow cytometric detection and analysis of IL-17A and IFN-γ mRNA and protein levels expressed in human T lymphocytes (P6364)

Abstract The ongoing development of multiparameter flow cytometers, fluorescent dyes and specific antibodies for surface and intracellular markers provides valuable tools to analyze and define cellular and molecular mechanisms that underlie complex biological systems. These tools have contributed significantly to both basic and clinical research applications including immune system monitoring, drug screening, and diagnostic tests. Recently-developed techniques and reagents allow for the specific, amplified fluorescent detection of different RNA transcript levels expressed in individual cells. In this study, we evaluated the use of multicolor flow cytometry for the simultaneous analysis of differentially coexpressed mRNA and protein levels in primary cells. Using a human Th17-like cell model system, we demonstrate that in vitro-differentiated Th17-like cells can be simultaneously probed for their differentially expressed levels of IL-17A and IFN-γ proteins as well as IL-17A and IFN-γ mRNAs and IL-23 receptor mRNA. These results suggest that numerous potential new mRNA biomarkers can now be defined and used along with conventional immunofluorescent staining of protein biomarkers, to more fully characterize the nature of cells that function in complex biological systems. Simultaneous multicolor flow cytometric analysis of mRNA and protein in individual cells constitutes an important new toolset that can provide novel information from basic and clinical research investigations.

Immunophenotypic Identification and Characterization of Tumor Cells and Infiltrating Cell Populations in Meningiomas

Meningiomas are primary tumors of the central nervous system composed of both neoplastic and other infiltrating cells. We determined the cellular composition of 51 meningioma samples by multiparameter flow cytometric (MFC) immunophenotyping and investigated the potential relationship between mRNA and protein expression levels of neoplastic cells. For immunophenotypic, morphologic, and cytogenetic characterization of individual cell populations, a large panel of markers was used together with phagocytic/endocytic functional assays and MFC sorting. Overall, our results revealed coexistence of CD45(-) neoplastic cells and CD45(+) immune infiltrating cells in all meningiomas. Infiltrating cells included tissue macrophages, with an HLA-DR(+)CD14(+)CD45(+)CD68(+)CD16(-/+)CD33(-/+) phenotype and high phagocytic/endocytic activity, and a small proportion of cytotoxic lymphocytes (mostly T CD8(+) and natural killer cells). Tumor cells expressed multiple cell adhesion proteins, tetraspanins, HLA-I/HLA-DR molecules, complement regulatory proteins, cell surface ectoenzymes, and growth factor receptors. Noteworthy, the relationship between mRNA and protein levels was variable, depending on the proteins evaluated and the level of infiltration by immune cells. In summary, our results indicate that MFC immunophenotyping provides a reliable tool for the characterization of the patterns of protein expression of different cell populations coexisting in meningioma samples, with a more accurate measure of gene expression profiles of tumor cells at the functional/protein level than conventional mRNA microarray, independently of the degree of infiltration of the tumor by immune cells.

P-0200 Multiparameter Flow Cytometric Analysis of CD40 Expression on Circulating Colorectal Cancer Cells

ABSTRACT Introduction Circulating tumour cells in the peripheral blood of colorectal cancer patients may be an important indicator of worse prognosis. They include an heterogeneous population of cells with different biological characteristics. The cell surface costimulatory molecule CD40 (a glycosylated phosphoprotein member of Tumour Necrosis Factor, receptor superfamily), is widely expressed by lymphoid cells and by various tumour types. It has been indicated as prognostic/predictive biomarker and as a potential target for therapy in human tumours. In a previous study we showed that the inhibition of colorectal cancer cell proliferation by the soluble CD40 ligand was mainly due to a slowing down of the cell cycle progression thus confirming the multiple anti-proliferative/growth-inhibitory effects of the CD40 pathway stimulation in this tumor. In the present study we wanted to demonstrate the value of a multiparameter flow cytometric (FCM) approach for a further biological characterization (such as the CD40 expression) of circulating colorectal cancer cells in vivo. Methods In the first part of the study, SW48 human colorectal cancer cells (highly positive for CD40) were serially diluted in normal whole blood in order to evaluate the sensitivity of the method in both the cancer cell identification and in their characterization for CD40 expression. Then, we analyzed peripheral blood samples from 9 patients with metastatic colorectal cancer (before starting the medical treatment for their advanced disease) to detect and characterize circulating cancer cells; 3 normal subjects were utilized as negative controls. The study were performed using a Navios FCM (Instrumentation Laboratories) equipped with a dedicated software for multicolor analyses. Results The method was found to have a sensitivity limit of 10(-5) for the detection of circulating tumor cells. The specificity of the method was 100%. In 8 of our patients we were able to differentiate in vivo circulating cancer cells from normal mononuclear cells based on: increased light scatter parameters, cell marker expression (EpCAM+ CD45-) and nuclear stain with 4'6-diamidino-2-2phenylindole, DAPI. A simultaneous analysis of the CD40 expression was also successfully performed on these cells and in 4 out of 8 patients, circulating tumor cells were found to express detectable levels of CD40. Conclusion Multiparameter FCM can effectively detect circulating tumor cells in colorectal cancer patients and allows to evaluate their CD40 expression. The technique has the potential to be a valuable and sensitive tool for prognosis assessment in these patients and for future studies aimed to evaluate new therapeutic strategies such as antitumor immunotherapy approaches in the metastatic setting.

Flow cytometric detection of circulating endothelial cells and endothelial progenitor cells in healthy subjects

Circulating endothelial cells (CEC) and endothelial progenitor cells (CEP) play an important role in tissue neovascularization. In human tumours, these cells may have clinical implications as prognostic/predictive factors during antiangiogenic therapy. The lack of a standardized assay for the quantification of these rare events has lead to a wide variation in the reported ranges of CEC and CEP. This study aimed to develop a flow cytometric (FCM) method for the immunophenotipic detection and enumeration of these cells in a healthy population. Peripheral blood samples from 32 subjects were analysed. Multiparameter FCM analysis was used to quantify resting and activated CEC and CEP. The mean values of the percentage and of the absolute number were: 0.005 +/- 0.004% and 306 +/- 243 cells/ml for CEC; 0.002 +/- 0.001% and 130 +/- 110 cells/ml for rCEC; 0.003 +/- 0.002% and 176 +/- 150 cells/ml for aCEC; 0.0001 +/- 0.00005% and 6 +/- 2 for CEP. We confirmed that FCM is an accurate and sensitive method for the quantitative analysis of CEC and CEP. The determination of normal ranges of CEC and CEP is helpful in defining their role as surrogate biomarkers of antiangiogenic treatment efficacy during clinical trials in oncology.

In vivo biological effects of panitumumab + chemotherapy in advanced colorectal cancer patients

14576 Background: Panitumumab, a fully human monoclonal antibody against EGFR, has demonstrated efficacy in the treatment of metastatic colorectal carcinoma (mCRC). Multiple links have been found between angiogenesis and both the EGFR pathway and the immunoresponse in human tumors, influencing the properties of these new anticancer agents with molecular targets. Flow cytometric (FCM) determination of circulating endothelial cells (CECs) and progenitor cells (CEPs) has been proposed to monitor anti-angiogenetic drug activity. We have focused on the impact of a first-line Panitumumab-based combination therapy on CECs and CEPs as well as on the both peripheral blood (PB) lymphocyte and dendritic cell (DC) immunophenotype, in order to identify non-invasive biomarkers of efficacy. Methods: We utilized multiparameter FCM to quantify the absolute numbers of CECs (rCEC, resting and aCEC, activated) and CEPs, PB lymphocytes and DC subsets in 11 mCRC pts (M/F:7/4, median age: 71 yrs, range 61–75), before and during a 1st-line, biweekly treatment with Oxaliplatin/5FU/LV + Panitumumab. Baseline data were compared with reference values obtained by 50 healthy subjects (M/F: 25/25, median age:43 yrs, range 21–65). Results: With respect to normal donors, we observed at baseline: a significant decrease of the total lymphocytes, CD4 T-lymphocytes, CD19 and CD20 B-lymphocytes and of NK cells, while DCs (in both DC1 and DC2 subsets) and CECs did not show significant differences. In 8 evaluable pts, after 3 courses of CT + Panitumumab, a trend was shown toward a progressive increase of all (but not CD19 and CD20) lymphocyte subsets, total DCs, DC subsets and total CECs, particularly in their resting form. This trend was confirmed after the 6th course when the increase of both aCECs and rCECs became highly statistically significant vs the baseline. No significant variations were shown on CEPs levels probably due to their extreme rarity. Conclusions: Panitumumab-based, first-line treatment of mCRC has a significant impact on both CECs levels and the in vivo T-cell mediated response. This suggests a potential usefulness of the combined FCM determination of CECs and PB / DC immunophenotype, as predictive biomarkers of efficacy in the clinical setting. No significant financial relationships to disclose.

Predictive Value of Flow Cytometric MRD Analysis in Childhood Acute Lymphoblastic Leukaemia at the End of Remission Induction Therapy.

Patients with acute lymphoblastic leukaemia (ALL) in morphological remission may still have up to 1010 residual malignant cells. Detection of minimal residual disease (MRD) at the end of induction therapy allows better estimation of the leukaemic burden and can help selection of appropriate therapeutic strategies. Flow cytometric (FC) detection of MRD is based on the identification of immunophenotypic combinations expressed on leukaemic cells but not on normal hematopoietic cells - leukaemia associated immunophenotypes (LAIPs). We prospectively analysed bone marrow samples from 77 patients who presented with ALL to our unit between 1999–2003 and attained morphological remission. These patients were treated on a standard protocol. Multiparameter FC identification of LAIPs was performed at various time points, as dictated by the treatment protocol. Our results show that flow cytometric MRD at the end of induction therapy is an independent and the most significant predictor of relapse, both on univariate and multivariate analysis. The relapse risk was 4% if day 28 MRD was <0.01% and 50% if day 28 MRD was >0.01% (p<0.05). We conclude that flow cytometric based MRD assays can be used to assess early response to treatment and predict relapse in a similar way to molecular MRD analysis at the end of induction therapy. Flow cytometric analysis of MRD offers the advantages of being cheaper, more widely available and has quicker turnaround times.

Predictive Role of Flow Cytometry in Assessing Very Early Treatment Response in Childhood ALL.

Acute lymphoblastic leukaemia is the most common childhood malignancy. In the modern treatment of acute leukaemia, advances in supportive care have allowed the intensification of therapy, and survival rates have risen accordingly. Cure rates in childhood acute lymphoblastic leukaemia (ALL) have increased from 10% to 80% over the last four decades. Currently clinical parameters such as white blood cell count and age at diagnosis are used for treatment stratification. This stratification aims to maximise the efficacy and minimize the toxicity of treatment, with the intensity of treatment being adjusted according to the risk of relapse. Unfortunately, the clinical and biologic parameters most commonly used for risk classification fail to identify all patients who relapse, with most patients who relapse falling into the ‘low risk' group. In addition other patients who are actually at low risk of relapse may receive more intensive treatment than is necessary. Morphology is used to assess very early response at day 8/15, but lacks specificity and sensitivity. Many publications have reported marked variability in indivual reports of morphological blast %. Better techniques of assessing very early response are therefore needed. Detection of minimal residual disease (MRD) allows better estimation of the leukaemic burden and can help selection of appropriate therapeutic strategies. Flow cytometric (FC) detection of MRD is based on the identification of immunophenotypic combinations expressed on leukaemic cells but not on normal hematopoietic cells - leukaemia associated immunophenotypes (LAIPs). FC analysis is an attractive option as it is quick and more specific. We prospectively analysed bone marrow samples from 70 patients who presented with ALL to our unit between 1999–2003 and attained morphological remission. These patients were treated on a standard protocol. Multiparameter FC identification of LAIPs was performed at various time points, as dictated by the treatment protocol. We looked at the predictive value of FC at d8/15 on treatment, at different levels of MRD. Our results showed that amongst children with flow MRD< 0.01% (n=5) on day 8/15 of chemotherapy, there were no relapses. Perhaps this cohort of patients could receive less intensive chemotherapy to minimise long-term side effects. The second group was children with flow MRD between 0.01%– 1%(n=33). In this group we saw 14% relapses. The third group was children with flow MRD between 1–10% (n=20), in which we observed 25% relapses. The fourth group was children with flow MRD > 10%, (n=12). 40% of this group suffered relapses. It appears that as early as day 8/15 of treatment, we can identify patients who are at very high risk of relapse, and perhaps these children need intensification of chemotherapy early on or some other novel intervention eg stem cell transplantation. FC appears to offer the ability to identify very early in treatment those at high risk of relapse and those with a high chance of cure. Treatment may therefore be stratified according to these risks with the aim of improving cure rates. These results need to be confirmed in a larger cohort of patients but these preliminary results are very promising in the risk stratification of childhood ALL.

Anaplastic Large Cell Lymphoma

We report our experience with flow cytometric (FC) analysis of 29 cases of anaplastic large cell lymphoma (ALCL). Morphologic analysis of processed cytocentrifuged preparations demonstrated neoplastic cells in 28 cases. In 25 of these, an aberrant lymphoid population was detected by FC analysis. The majority showed high orthogonal light scatter, similar to monocytes or granulocytes. Of the antigens CD2, CD3, CD4, CD5, and CD7, 5 cases expressed 1, 8 expressed 2, 6 expressed 3, 3 expressed 4, and 3 expressed all 5. CD4 was expressed most commonly (20/25 [80%]), followed by CD2 (18/25 [72%]), CD3 (10/25 [40%]), and CD5 and CD7 (8/25 [32%] each). CD45 was expressed in 23 of 25 cases and CD13 in 7 of 9. Of 21 cases, 13 were anaplastic lymphoma kinase (ALK)+, all of which were CD4+, vs 5 of 8 ALK - cases (P = .042). Most ALCLs can be detected and characterized by multiparameter FC analysis. However, light scatter gating on typical lymphoid regions may yield false-negative results in a substantial number of cases.

Single particle high resolution spectral analysis flow cytometry

While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy. Our system utilizes a diffraction grating to disperse the collected fluorescence and side-scattered light from cells or microspheres passing through the interrogation region over a rectangular charge-coupled-device image sensor. The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to assure modularity of the system. Calibration of the prototype spectral analysis flow cytometer included wavelength characterization and calibration of the dispersive optics. Benchmarking of the system demonstrated a single particle/cell intensity sensitivity of 2160 MESF of R-Phycoerythrin. Single particle spectra taken with our instrument were validated against bulk solution fluorimeter and conventional flow cytometer measurements. Coefficients of variation of integrated spectral fluorescence intensity of several sets of standard fluorescent microspheres ranged from 1.4 to 4.8% on the spectral system. Spectral discrimination of free versus PI bound to cells is also demonstrated. It is demonstrated that the flow spectrometer has sufficient sensitivity and wavelength resolution to detect single cells and microspheres, including multi-fluorophore labeled microspheres. The capability to use both standard mathematical deconvolution techniques for data analysis, coupled with the feasibility of integration with existing flow cytometers, will improve the accuracy and precision of ratiometric measurements, enable the analysis of more discrete emission bands within a given wavelength range, and allow more precise resolution of the relative contribution of individual fluorophores in multiply-tagged samples, thereby enabling a range of new applications involving the spectral analysis of single cells and particles.

A New Multi-Parameter Flow Cytometric Assay for Monitoring Lymphoma Growth and Spread in a Pre-Clinical Murine Model for Human Lymphoma.

Murine lymphoma models are commonly used to test the efficacy of idiotype-based vaccines for B-cell lymphoma prior to human trials. Mouse survival has been used as an indirect measure of the tumor response to the vaccine. In this study, we describe a multiparameter flow cytometric (MPFC) assay that enables direct assessment of the tumor at the inoculation site and detection of lymphoma metastases. In initial experiments, we show that 38C13 mouse lymphoma cells expressed an aberrant phenotype compared to normal splenocytes. The combination of CD45, CD19, B220 and an anti-idiotypic antibody (S1C5) along with a sequential gating technique defined the 38C13 cells with high sensitivity and specificity. The specificity of the MPFC assay for 38C13 lymphoma cells was greater than a gating strategy using light scatter and idiotype antibody binding. The MPFC assay was used to monitor 38C13 tumor kinetics in the C3H/HeN mouse and demonstrated tumor growth at the inoculation site and metastases to the lymph nodes and bone marrow. The presence of 38C13 metastases in lymphoid organs and bone marrow predicted by the MPFC assay were confirmed by histology and immunohistochemistry (IHC). The MPFC assay will enable investigators to monitor 38C13 lymphoma tumor response to individual vaccines or other lymphoma therapy prior to clinical trials. Furthermore, the MPFC concept could be readily applied to other models of human B cell lymphoma.

A new multi-parameter flow cytometric assay for monitoring lymphoma growth and spread in a pre-clinical murine model for human lymphoma.

Mouse survival is commonly used as an indirect measure of lymphoma tumor response to anti-idiotype vaccine; however, this gives no information regarding residual lymphoma cells at primary or metastatic sites. We aimed to develop a method with which to monitor lymphoma tumor kinetics in the mouse as an independent measure of vaccine efficacy. We developed a multi-parameter flow cytometric (MPFC) assay for 38C13 mouse lymphoma cells using sequential gating to detect aberrant antigen expression and binding by an anti-idiotype antibody (S1C5). Subsequently, we tested the utility of the MPFC assay in a 38C13 tumor modeling study in the C3H/HeN mouse. The MPFC assay was demonstrated in vitro to have both high specificity and sensitivity for 38C13 lymphoma cells. In tumor kinetic studies in the C3H/HeN mouse, as the tumor enlarged, the MPFC assay showed increasing prevalence of 38C13 cells at the inoculation site in addition to metastases to lymphoid organs and bone marrow. The latter findings were confirmed by both histology and immunohistochemistry. The MPFC assay is an independent parameter for monitoring 38C13 lymphoma kinetics and could be used to monitor tumor response to individual vaccines or other lymphoma therapy prior to clinical trials.