Multicolor Flow Cytometry Analysis Research Articles

Longitudinal analysis of peripheral immune cells in patients with multiple sclerosis treated with anti-CD20 therapy.

Anti-CD20 therapy is a highly effective treatment for multiple sclerosis (MS). In this study, we investigated MS-related changes in peripheral blood mononuclear cell (PBMC) subsets compared to healthy controls and longitudinal changes related to the treatment. Multicolor spectral flow cytometry analysis was performed on 78 samples to characterize disease- and treatment-related PBMC clusters. Blood samples from MS patients were collected at baseline and up to 8 months post-treatment, with three collection points after treatment initiation. Unsupervised clustering tools and manual gating were applied to identify subclusters of interest and quantify changes. B cells were depleted from the periphery after anti-CD20 treatment as expected, and we observed an isolated acute, transitory drop in the proportion of natural killer (NK) and NKTcells among the main populations of PBMC (P = 0.03, P = 0.004). Major affected PBMC subpopulations were cytotoxic immune cells (NK, NKT, and CD8+ Tcells), and we observed a higher proportion of cytotoxic cells with reduced brain-homing ability and a higher regulatory function as a long-term anti-CD20-related effect. Additionally, anti-CD20 therapy altered distributions of memory CD8+ Tcells and reduced exhaustion markers in both CD4+ and CD8+ Tcells. The findings of this study elucidate phenotypic clusters of NK and CD8+ Tcells, which have previously been underexplored in the context of anti-CD20 therapy. Phenotypic modifications towards a more regulatory and controlled phenotype suggest that these subpopulations may play a critical and previously unrecognized role in mediating the therapeutic efficacy of anti-CD20 treatments.

Genomic and immunocyte characterisation of bloodstream infection caused by Klebsiella pneumoniae

ObjectivesThe aim of this study was to evaluate the characteristics of immunocyte associated with bloodstream infection (BSI) caused by Klebsiella pneumoniae (Kpn).MethodsPatients with BSI-Kpn were included from 2015 to 2022 in our hospital. Immunocyte subpopulations of enrolled BSI-Kpn patients were tested on the same day of blood culture using multicolor flow cytometry analysis. Antibiotic susceptibility test was determined by agar dilution or broth dilution method. All included isolates were subjected to whole genome sequencing and comparative genomics analysis. Clinical and genetic data were integrated to investigate the risk factors associated with clinical outcome.ResultsThere were 173 patients with non-duplicate BSI-Kpn, including 81 carbapenem-resistant Kpn (CRKP), 30 extended-spectrum β-lactamases producing Kpn (ESBL-Kpn), 62 none CRKP or ESBL-Kpn (S-Kpn). Among 68 ST11-CRKP isolates, ST11-O2v1:KL64 was the most common serotypes cluster (77.9%, 53/68), followed by ST11-OL101: KL47 (13.2%, 9/68). Compared with CSKP group, subpopulations of immunocyte in patients with CRKP were significantly lower (P < 0.01). In patients with ST11-O2v1:KL64 BSI-Kpn, the level of cytotoxic T lymphocytes (CD3 + CD8 +) is the highest, while the B lymphocytes (CD3-CD19 +) was the least. In addition, the level of immunocyte in patients with Kpn co-harbored clpV-ybtQ-qacE were lower than that in patients with Kpn harbored one of clpV, ybtQ or qacE and without these three genes. Furthermore, co-existence of clpV-ybtQ-qacE was independently associated with a higher risk for 30-day mortality.ConclusionsThe results demonstrate that patients with BSI-CRKP, especially for ST11-O2v1:KL64, exhibit lower leukomonocyte counts. In addition, BSI-Kpn co-harbored clpV-ybtQ-qacE is correlated to higher 30-day mortality.

A potential best-in-class BCMA-CD19 bispecific CART with advanced safety by self-inhibiting IFNG signaling during CRS.

2502 Background: CART cells have demonstrated remarkable clinical efficacy in treating hematological cancers. However, CRS remains as a major challenge in clinical application, with multiple cytokines significantly elevated. IL6 signaling blockade by Tocilizumab has become a standard treatment to relieve CRS in patients. Our previous studies suggested that CAR T secreting IL6 antagonist could effectively maintain IL6 at very low levels during CRS. Furthermore, we observed that a huge quantity of tumor cells in one patient was significantly reduced after CART infusion, but a low level of IFNG with only grade 2 CRS was present. Therefore, we hypothesized that high level of IFNG might not be necessary to CART clinical efficacy and direct blockade of IFNG signaling by CART secreting an IFNG antagonist (designated as SAFET) serves an interesting approach to effectively reduce CRS toxicity. Although IFNG is usually thought important in T cell cytotoxicity, our results indicated that IFNG KO or autonomous secretion of IFNG antagonist did not affect the killing activity of CART cells. Methods: This pilot phase 1 study of SAFET BCMA/CD19 BiCART self-inhibiting IFNG signaling is a single arm study conducted in China, enrolling refractory/relapsed multiple myeloma patients. The patients had received more than 3 lines of prior therapies including at least a proteasome inhibitor and an immunomodulatory agent and were refractory to the last line of treatment. Lymphodepletion was performed with fludarabine and cyclophosphamide prior to the CART infusion. Following 2-14 days of rest, patients received a single infusion of SAFET BCMA/CD19 BiCART, at the dose of 0.4-1.0 × 108 CAR+ T cells/patient. The primary objectives of this study were to evaluate the safety and efficacy of SAFET BCMA/CD19 BiCART. The pharmacokinetics of SAFET BCMA/CD19 BiCART was investigated by quantitative PCR based detection of CAR vector copies in peripheral blood. Minimal residual disease (MRD) negativity was assessed by standardized multicolor flow cytometry analysis of bone marrow aspirate. Results: 10/11 R/R MM patients achieved CR and 1 achieved VGPR. Among the 10 CR patients, 7 remained CR after treatment with a median PFS 858 days; 1 patient showed relapse at Days 215; 2 patients showed MRD positive relapse at Days 637 and 733, and then received KRd and/or Kd treatment to achieve SD and MRD negative CR. Interestingly, the remarkable short and long term remission suggested that CART self-inhibiting IFNG signaling did not impair the CART clinical efficacy. Notably, minimal CRS was observed, including 6 grade 0, 2 grade 1, 2 grade 2, and 1 grade 3 who displayed mild transient hypotension for only 1 day while there were 79.5% blast tumor cells in the bone marrow before CART therapy. Conclusions: These results suggest BCMA-CD19 bispecific CART self-inhibiting IFNG signaling is promising in translation to a best-in-class treatment for MM. Clinical trial information: ChiCTR2000032124.

Enhanced CTLA-4 blockade anti-tumor immunity with APG-157 combination in a murine head and neck cancer.

A phase I clinical study for patients with locally advanced H&N cancer with a new class of botanical drug APG-157 provided hints of potential synergy with immunotherapy. We sought to evaluate the efficacy of the combination of APG-157 and immune checkpoint inhibitors. CCL23, UM-SCC1 (human), and SCCVII (HPV-), MEER (HPV+) (murine) H&N cancer cell lines were utilized for invitro and invivo studies. We measured tumor growth by treating the mice with APG-157, anti-PD-1, and anti-CTLA-4 antibody combinations (8 groups). The tumor microenvironments were assessed by multi-color flow cytometry, immunohistochemistry, and RNA-seq analysis. Fecal microbiome was analyzed by 16S rRNA sequence. Among the eight treatment groups, APG-157 + anti-CTLA-4 demonstrated the best tumor growth suppression (p = 0.0065 compared to the control), followed by anti-PD-1 + anti-CTLA-4 treatment group (p = 0.48 compared to the control). Immunophenotype showed over 30% of CD8+ T cells in APG-157 + anti-CTLA-4 group compared to 4%-5% of CD8+ T cells for the control group. Differential gene expression analysis revealed that APG-157 + anti-CTLA-4 group showed an enriched set of genes for inflammatory response and apoptotic signaling pathways. The fecal microbiome analysis showed a substantial difference of lactobacillus genus among groups, highest for APG-157 + anti-CTLA-4 treatment group. We were unable to perform correlative studies for MEER model as there was tumor growth suppression with all treatment conditions, except for the untreated control group. The results indicate that APG-157 and immune checkpoint inhibitor combination treatment could potentially lead to improved tumor control.

Abstract LB354: A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice

Abstract Oncolytic viruses (OVs) are potent cancer therapeutics that can selectively kill tumor cells and promote anti-tumor immunity. mSJ-650 (Wyeth, K2L-, TK-, murine GM-CSF, human CD55) is the Wyeth strain of vaccinia virus with human complement regulatory protein CD55 incorporated on the intracellular mature virion membrane to evade complement-mediated attack during circulation. The virus also expresses mGM-CSF in replace of thymidine kinase to increase tumor selectivity and enhance immunogenicity. We previously reported that a single dose of mSJ-650 effectively evades complement and shows prominent anti-tumor efficacy in A549 and HCT116 xenograft model. Here, we further investigated the capability of mSJ-650 in promoting anti-tumor response after multiple systemic administration in immunocompetent breast cancer mouse model. In an orthotopic EMT6 breast cancer model, mSJ-650 or control mSJ-612 devoid of CD55 was intravenously injected at low dose (1x106 pfu) and high dose (3x106 pfu), twice weekly. mSJ-650 treatment dose-dependently suppressed tumor growth, showing superior antitumor efficacy compared to treatment of control mSJ-612. Moreover, multiple systemic administration of mSJ-650 was well-tolerated with no apparent signs of toxicity in serum tested via AST and ALT assay. To further elucidate whether mSJ-650 can evade neutralization by antibody and maintain its antitumor potency in vivo, we designed two experimental models. Firstly, endogenous vaccinia virus specific neutralizing antibody was generated by intravenous injection of mSJ-650 prior to tumor cell inoculation in tumor-bearing mouse, followed by systemic administration of mSJ-650 twice weekly. Second, serum containing vaccinia virus specific neutralizing antibody was intravenously injected 24 hours prior to mSJ-650 administration in tumor-bearing mouse. In both experimental models, mSJ-650 treated group significantly inhibited tumor growth despite the presence of neutralizing antibody, while control mSJ-612 treated group failed to suppress tumor growth. These results imply that mSJ-650 is capable of evading neutralization and maintaining its antitumor efficacy. Moreover, we performed multi-color flow cytometry analysis of tumor-infiltrating leukocytes to evaluate immunogenicity of mSJ-650. As a result, we observed a significant increase in recruitment of activated CD4+ and CD8+ T cells by 2-fold and high expression level of cytotoxic molecules (perforin, TNF-α, IFN-γ) in tumor-infiltrating T cells. In summary, multiple systemic administration of mSJ-650 markedly evaded neutralization and suppressed tumor growth by promoting anti-tumor response in immunocompetent breast cancer mouse model, thus implying that mSJ-650 is a promising candidate for cancer immunotherapy. Citation Format: Yeonsoo Yang, Solchan Won, Namhee Lee, Eunhye Kim, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB354.

Characterization of CD34+ Cells from Patients with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS) Using a t-Distributed Stochastic Neighbor Embedding (t-SNE) Protocol.

Using multi-color flow cytometry analysis, we studied the immunophenotypical differences between leukemic cells from patients with AML/MDS and hematopoietic stem and progenitor cells (HSPCs) from patients in complete remission (CR) following their successful treatment. The panel of markers included CD34, CD38, CD45RA, CD123 as representatives for a hierarchical hematopoietic stem and progenitor cell (HSPC) classification as well as programmed death ligand 1 (PD-L1). Rather than restricting the evaluation on a 2- or 3-dimensional analysis, we applied a t-distributed stochastic neighbor embedding (t-SNE) approach to obtain deeper insight and segregation between leukemic cells and normal HPSCs. For that purpose, we created a t-SNE map, which resulted in the visualization of 27 cell clusters based on their similarity concerning the composition and intensity of antigen expression. Two of these clusters were "leukemia-related" containing a great proportion of CD34+/CD38- hematopoietic stem cells (HSCs) or CD34+ cells with a strong co-expression of CD45RA/CD123, respectively. CD34+ cells within the latter cluster were also highly positive for PD-L1 reflecting their immunosuppressive capacity. Beyond this proof of principle study, the inclusion of additional markers will be helpful to refine the differentiation between normal HSPCs and leukemic cells, particularly in the context of minimal disease detection and antigen-targeted therapeutic interventions. Furthermore, we suggest a protocol for the assignment of new cell ensembles in quantitative terms, via a numerical value, the Pearson coefficient, based on a similarity comparison of the t-SNE pattern with a reference.

Imbalance of Lymphocyte Subsets and CD45RA-Expressing Cells in Intrathoracic Lymph Nodes, Alveolar Compartment and Bloodstream of Pulmonary Sarcoidosis Patients.

Sarcoidosis is a systemic granulomatous disease mainly affecting the lungs and hilomediastinal lymph nodes. It is characterized by non-caseating epithelioid cell granulomas in lymph nodes and lungs. Our study aimed to evaluate and compare T, B and NK cell subsets in the alveolar compartment, lymph nodes and the bloodstream simultaneously in the same patients to elucidate the immune responses associated with the development and progression of sarcoidosis. A secondary aim was to evaluate the distribution of CD45RA-expressing cells in the different anatomical compartments. Patients suspected to have sarcoidosis and who underwent bronchoscopy with bronchoalveolar lavage (BAL), lung-draining lymph node (LLN) biopsy by EBUS-TBNA and peripheral blood (PB) sampling were included in the study. They were monitored at the Regional Referral Centre of Siena University Hospital and the Respiratory Diseases Unit of Perugia Hospital. Multicolour flow cytometry analysis through FASCLyric was performed to assess T, B and NK cell subsets. Thirty-two patients (median age (IQR) 57 (52-58) years) were consecutively and prospectively enrolled. Machine learning analysis created a model which selected CD56dim16bright, CD8, Tfc, Th17, Th12, Tfh17, Tfh2, TcemRA, ThemRA, T naïve, Tc naïve, Breg, CD1d+CD5+, Th-reg, Tfh, Th1 and CD4 cells with an accuracy of 0.9500 (kappa 0.8750). Comparative analysis found 18 cell populations that differed significantly between the three anatomical compartments. The bloodstream was enriched in ThemRA (p = 0.0416), Tfh2 (p = 0.0189), Tfh17 (p = 0.0257), Th2 (p = 0.0212), Th17 (p = 0.0177), Th-naïve (p = 0.0368), CD56dimCD16bright (p < 0.0001), CD8 (p = 0.0319), TcemRA (p < 0.0001) and Tfc cells (p = 0.0004) compared with the alveolar compartment, while Th-reg were lower in PB than BAL (p = 0.0329). The alveolar compartment was enriched in Breg (p = 0.0249) and CD1d+CD5+ (p = 0.0013) with respect to LLN samples and PB. Conversely, Tfh (p = 0.0470), Th1 (p = 0.0322), CD4 (p = 0.0486) and Tc-naïve (p = 0.0009) were more abundant in LLN than in BAL and PB. It has been speculated that changes in the relative contents of PB cells could be related to changes in production and to the selective redistribution of PB cells to granulomatous foci. This study further supports the fact that sarcoidosis is multisystemic in nature. However, the low level of immune cells in peripheral blood of patients with sarcoidosis is concerning. A re-expression of CD45RA on CD4+ and CD8+ cells could result in a reduction in peripheral immune activity. Thus, changes in the spectrum of the bloodstream may reflect both pathogenic and compensatory processes.

Abstract 4333: Spatial analysis of tumor-infiltrating lymphocytes in tumor microenvironment as biomarker for immune checkpoint inhibition in biliary tract cancer

Abstract Background: Recently, anti-PD-L1 in combination with cytotoxic chemotherapy has shown significant survival benefit in a randomized phase 3 trial for unresectable or metastatic biliary tract cancer (BTC). However, no biomarker including PD-L1 expression has been established to predict clinical outcomes, and there is an unmet need for a novel predictive biomarker for anti-PD-1 or PD-L1 therapy. Here, we assessed TILs using artificial intelligence (AI)-powered spatial analysis in advanced BTC treated with anti-PD-1 beyond 1st line treatment. Methods: An AI-powered whole-slide image (WSI) analyzer (Lunit SCOPE IO, Lunit, Seoul, Korea) was used to segment tumor epithelium and stroma, and identification of intratumor TIL (iTIL) and stromal TIL (sTIL). H&amp;E stained WSI from pre-treatment samples was acquired from Asan Medical Center (n =166), and a total of 154 samples (92.8%) after quality control were used for the final analysis. Immune phenotypes (IP) were defined as follow: inflamed as high iTIL and sTIL; immune-excluded as low iTIL and high sTIL; immune-desert as low TIL overall. Among them, 20 patients were available for multi-color flow cytometry analysis (FACS) using peripheral blood mononuclear cells, collected at baseline, C1D8, and C2D1. Results: All patients (n=154) were treated with anti-PD-1 (pembrolizumab or nivolumab) monotherapy, and 72 of 154 patients (46.8%) were treated as 2nd line. Gemcitabine plus cisplatin (GemCis) was used prior to anti-PD-1 as first-line therapy in all patients. Overall, 15 (9.7%) patients showed inflamed IP. With median follow-up duration of 15.4 months, the inflamed IP group showed better overall survival (17.2 vs. 6.6 months, P=0.03), and progression-free survival (PFS; 4.5 vs. 2.6 months, P=0.09) along with higher PFS rate at 12 months (33.3% vs. 11.5%, P=0.035), and overall response rate (26. 7% vs. 8.6%, P=0.053) than other phenotype groups. There was no significant difference in median PFS with GemCis among IP groups (P=0.74). In the FACS available subgroup, inflamed IP showed higher baseline central memory T (Tcm)+effector memory T (Tem)/Tnaive ratio than other IPs. With the administration of anti-PD-1, Tcm+Tem/Tnaive ratio was increased, while the proportion of PD1+CD8+T, CD39+CD8+T, CD103+CD8+T and Treg were decreased in the inflamed IP group than other phenotype groups. Conclusions: Immune phenotypes classified by AI-powered spatial TIL analysis was effective to predict the clinical outcomes of patients with advanced BTC treated with anti-PD-1 therapy. Citation Format: Yeong Hak Bang, Kyunghye Bang, Jin Ho Shin, Hyunseok Yoon, Kyu-Pyo Kim, Inkeun Park, Jae Ho Jeong, Heung-Moon Chang, Baek-Yeol Ryoo, Chiyoon Oum, Seulki Kim, Yoojoo Lim, Gahee Park, Chan-Young Ock, Changhoon Yoo. Spatial analysis of tumor-infiltrating lymphocytes in tumor microenvironment as biomarker for immune checkpoint inhibition in biliary tract cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4333.

Flow cell sorting followed by PCR-based clonality testing may assist in questionable diagnosis and monitoring of acute lymphoblastic leukemia.

Multicolor flow cytometry (MFC) has highly reliable and flexible algorithms for diagnosis and monitoring of acute lymphoblastic leukemia (ALL). However, MFC analysis can be affected by poor sample quality or novel therapeutic options (e.g., targeted therapies and immunotherapy). Therefore, an additional confirmation of MFC data may be needed. We propose a simple approach for validation of MFC findings in ALL by sorting questionable cells and analyzing immunoglobulin/T-cell receptor (IG/TR) gene rearrangements via EuroClonality-based multiplex PCR. We obtained questionable MFC results for 38 biological samples from 37 patients. In total, 42 cell populations were isolated by flow cell sorting for downstream multiplex PCR. Most of the patients (n=29) had B-cell precursor ALL and were investigated for measurable residual disease (MRD); 79% of them received CD19-directed therapy (blinatumomab or CAR-T). We established the clonal nature of 40 cell populations (95.2%). By using this technique, we confirmed very low MRD levels (<0.01% MFC-MRD). We also applied it to several ambiguous findings for diagnostic samples, including those with mixed-phenotype acute leukemia, and the results obtained impacted the final diagnosis. We have demonstrated possibilities of a combined approach (cell sorting and PCR-based clonality assessment) to validate MFC findings in ALL. The technique is easy to implement in diagnostic and monitoring workflows, as it does not require isolation of a large number of cells and knowledge of individual clonal rearrangements. We believe it provides important information for further treatment.

Characterization of Dysregulated Transcription in TP53-Mutated AML Subpopulations

Introduction: TP53 is a DNA binding protein that regulates transcription and exerts many functions critical to homeostasis. AML with mutated TP53 (TP53mt) is associated with dismal outcomes and resistance to all currently available therapies. Therefore, delineation of transcriptional dysregulation in TP53mt AML is critical for identification of vulnerabilities in this leukemia subtype. Leukemia cells are functionally heterogeneous, with many cells at diagnosis susceptible to chemotherapy, and a minority of resistant cells that persist despite treatment. Deeper understanding of all leukemia sub-populations is necessary for elucidating mechanisms of resistance. We hypothesized that sub-populations such as leukemia-stem cells (LSCs), and therapy-residual cells possess identifiable, targetable characteristics that drive resistance. We performed RNA-sequencing and compared differences in gene expression between these sub-populations. Methods: We collected 113 bone marrow samples from 39 patients (pts) with adverse risk AML, including 16 pts with TP53mt (41%), 14 pts with FLT3 mutations (FLT3mt), and 10 pts (26%) with other adverse risk. We performed RNA-sequencing on paired pre- and post-treatment, bulk and FACS-sorted samples. Mononuclear cells were flow-sorted for bulk (CD45dim) and LSCs (Lin-CD34+CD38-CD123+) from diagnostic samples. Post-treatment samples were sorted for bulk mononuclear cells and therapy-residual cells, determined based on patient-specific aberrant phenotype using multi-color flow cytometry analysis (Xu J et al., Clinics in laboratory medicine 2017). Samples with low-expression of housekeeping genes were excluded from the analysis. Differential expression was analyzed using DESeq2 and Gene Set Enrichment Analysis (GSEA) was performed using the HALLMARK gene set and considered statistically significant if q<0.1. Results: Regardless of genetic context, we found that LSCs were characterized by down regulation of immune-related pathways such (interferon-g response, allograft rejection, inflammatory response), and upregulation of oxidative phosphorylation and DNA repair among top dysregulated pathways when compared with bulk cells. Unlike FLT3mt, TP53mt LSCs had upregulation of MYC targets when compared with bulk cells. LSCs with TP53mt had a characteristic gene expression signature with a consistent upregulation of 11 genes (Figure 1). Among these genes, TACC3, a transforming acidic coiled-coil (TACC) family member could be a potentially druggable target given its role in protecting microtubule stability and centrosome integrity and recent identification of small molecule TACC3 inhibitors as an anticancer agent (Akbulut et al., Mol Cancer Ther 2020). When comparing differentially expressed pathways in bulk pre-treatment cells of non-responders (post-treatment bone marrow blasts >5%) vs responders (post-treatment bone marrow blasts >5%), we found upregulation of MYC targets, MTORC1 signaling, E2F targets and G2M checkpoint. We also found downregulation of immune-related pathways, namely allograft rejection and interferon-g response. This indicates the possibility of similar transcriptional mechanisms which lead to resistance and immune evasion in LSCs and in bulk populations resistant to therapy. Finally, we analyzed expression of immunotherapy targets with ongoing clinical trials in AML and compared expression of these targets to CD34+ cells from healthy donors as reference (Figure 2). We found differential expression of targets such as CD123, with high expression in bulk and leukemia subpopulations regardless of genotype, therefore implying the possibility of universally targeting AML with a relatively high therapeutic index. However, targets such as FLT3 or KIT had similar expression in AML compared with bone marrow cells from healthy donors therefore indicating the possibility of affecting normal hematopoiesis in therapies against these targets. Targets such as LAG3 and TIM3 were preferentially expressed in samples with FLT3mt. Conclusion: In summary, we show characteristic differences in gene expression associated with TP53mt LSCs and identify novel targets and potential mechanisms of resistance. We demonstrate pervasive downregulation of immune related pathways in LSCs or resistant leukemia. These results could impact future therapeutic strategies for these resistant leukemia subsets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RAS Pathway Alterations in Pediatric AML Patients

RAS pathway alterations are highly prevalent in hematologic malignancies1,2, often involving gain of function KRAS, NRAS or HRAS mutations and have been linked to tumorigenesis and associated with poorer prognosis in different cancer entities3-5. In adult myeloid leukemia, RAS mutations are one of the most common mutations, yet show no clear prognostic value6. In a study of the Japanese pediatric AML cohort, NRAS mutations were associated with a favorable outcome, the effect of KRAS mutations could not be analyzed due to the limited patient size7. Here we report the outcome of 372 pediatric AML patients of the German AML-BFM study between 2016 and 2021 that were characterized by morphology, multicolor flow cytometry, classical cytogenetics and mutation analysis via panel sequencing. In total, 27% (n=102) of the patients showed a RAS pathway alteration, involving n=32 patients (9%) with a KRAS mutation and n=75 (20%) with a NRAS mutation. Notably, no HRAS mutation was detected in our cohort. Age distribution did not differ between the groups (Fig. A). Interestingly, the KRAS cohort consisted mostly of male patients (72%, p=0,0087), while the overall cohort was balanced (48,7% male patients). NRAS and KRAS mutations were strong predictors of therapy response and early death. Only 1/75 (1,3%) of all patients harboring a NRAS mutations showed poor response to induction therapy compared to patients without a NRAS mutation (10,10%, p=0,01). KRAS mutations on the opposite were strongly associated with non-response (21,9%; p<0,01) and subsequent early deaths (n=6/32; p=0,001) compared to patients without KRAS mutations (3,2%). Of note, only 1/75 (1,3%) of patients with NRAS mutation and 17/372 (4,6%) of all AML patients suffered early deaths. Risk stratification taking into account re-stratification after poor therapy response showed a significant higher proportion of high-risk patients in the KRAS-mutated cohort (43,8 %) compared to 20% in the patients with NRAS mutations (p=0,0022). KRAS mutations correlated with KMT2A-MLLT1 (15,6%; p=0,01) translocations, which were significantly rarer in patients without KRAS mutations (3,5%). NRAS mutations occurred frequently with CBFA2T3-RUNX1 (14,7%; p=0,0002) and CBFB-MYH11 translocations (12%; p=0,0068%) as well as KIT mutations (12%; p=0,026). Existence of RAS mutations showed overall no significant impact on event-free survival (EFS=69,6% ±4,8%) and overall survival (OS=82,8% ±4,2%) compared to RAS wildtype (EFS=66,4% ±3,1% ; OS=76,5% ±2,9%) (Fig. B). Multivariate analysis comparing outcome for several subgroups also showed no significant differences. In summary, we show worse therapy response and increased early death rates in patients with KRAS mutations, while NRAS mutations are associated with better therapy response. These findings warrant further analyses and longer follow-up on the cohort to determine long-term significances of RAS pathway alterations in pediatric AML patients. 1.de Luca, A., Maiello, M. R., D'Alessio, A., Pergameno, M. & Normanno, N. The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets16 Suppl 2, (2012). 2.Hobbs, G. A., Der, C. J. & Rossman, K. L. RAS isoforms and mutations in cancer at a glance. Journal of Cell Science129, 1287-1292 (2016). 3.Hsu, H. C. et al. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients. Oncotarget7, 22257 (2016). 4.Vauthey, J., Zimmitti, G., Kopetz, S., ... J. S.-A. of & 2013, undefined. RAS mutation status predicts survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases. ncbi.nlm.nih.gov. 5.Dinu, D. et al. Prognostic significance of KRAS gene mutations in colorectal cancer - preliminary study. Journal of Medicine and Life7, 581 (2014). 6.Neubauer, A. et al. Patients With Acute Myeloid Leukemia and RAS Mutations Benefit Most From Postremission High-Dose Cytarabine: A Cancer and Leukemia Group B Study. Journal of Clinical Oncology26, 4603 (2008). 7.Kaburagi, T. et al. Clinical significance of RAS pathway alterations in pediatric acute myeloid leukemia. Haematologica107, 583-592 (2022). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single Cell Genotypic and Phenotypic Analysis of Measurable Residual Disease in Acute Myeloid Leukemia

Measurable residual disease (MRD) represents the critical population of leukemic cells which persists following anti-leukemic therapy. The presence of MRD has powerful prognostic relevance and represents the reservoir responsible for acute myeloid leukemia (AML) relapse. It is therefore critical to enumerate and molecularly characterize MRD clones in order to better risk stratify AML patients and guide further therapy aimed at preventing relapse and improving outcomes. Current MRD methods, including multi-color flow cytometry (MFC) analysis and bulk NGS sequencing, face challenges in both sensitivity and specificity and are insufficient to distinguish between pre-leukemic and leukemic clones. Consequently, the imperfect predictive value of these techniques hinders meaningful clinical intervention. To overcome these shortcomings associated with conventional methods of MRD detection, we developed a novel single-cell (sc) MRD assay with high sensitivity and specificity by combining precursor/blast population enrichment with integrated scDNA sequencing and immunophenotypic analysis. We reasoned that the MRD clones responsible for relapse largely reside in immature compartments. Enriched CD34+ and/or CD117+ progenitors from 5 unique patient samples were multiplexed followed by integrated single cell analysis on the MissionBio TapestriV2 scDNA+protein platform. We developed a novel computational approach to deconvolute multiplexed sequencing reads and accurately assign cells to individual patients via K-means clustering and UMAP dimension reduction of germline SNP allele frequencies. To demonstrate the sensitivity and specificity of our scMRD assay, 10,000, 1,000, 500, and 200 AML blasts from 3 patients harboring known pathologic mutations (NRAS/RUNX1, JAK2/IDH2, and IDH2 alone) were individually spiked into 10 million normal bone marrow cells to test a sensitivity threshold of 0.1%, 0.01%, 0.005%, and 0.002%. Samples were FACS-enriched for blasts, multiplexed, subjected to the TapestriV2 microfluidics platform, and sequenced. The expected pathologic mutations were reliably identified in patients at a threshold of 0.01%, but not in normal subjects, demonstrating that our assay is nearly 10-fold more sensitive than the current ELN MRD standard [Fig 1]. We then applied our assay to 30 banked post-induction MRD samples from 29 patients (median age 71 years old, 15 male/14 female) who had a diagnosis of AML [Fig 2]. Overall, genomic analysis was concordant in 22/30 (73%) samples between bulk NGS (at MRD stage) and scMRD. The mean VAF of mutations was higher by scMRD than bulk NGS, consistent with our hypothesis that mutations are enriched in immature compartments. 17 mutations were identified by scMRD but missed/unreported by bulk NGS, including RUNX1 (n=5), NPM1 (n=3), KRAS (n=2), IDH2 (n=2), WT-1 (n=2), JAK2, TP53 and SRSF2 mutations, 14 of which were present at relapse. Clonal architecture was illustrated by scMRD data with identification of pre-leukemic and leukemic clones. In 5 post-HSCT samples, scMRD readily distinguished between donor vs. host cells and showed higher host cell chimerism compared to conventional STR testing, suggesting that host cells have higher enrichment in immature cell types and represent an early indicator of relapse. Our scMRD assay also enabled single cell immunophenotypic analysis, and revealed concordant abnormal immunophenotype of leukemic blasts identified by MFC. More importantly, differential immunophenotypic states were identified when comparing pre-leukemic and leukemic clones (i.e. DNMT3A only vs co-mutant DNMT3/NPM1) within and between patients. These data show that integrated genomic/immunophenotypic analysis at the MRD timepoint can distinguish between clonal hematopoiesis and pre-leukemic/leukemic clones with a substantively higher likelihood of relapse. In conclusion, our scMRD assay not only enhances MRD detection, but also provides a molecular glimpse into the clonal architecture of pre-leukemic/leukemic cells surviving AML therapy. Larger studies are needed to comprehensively elucidate the clonal architecture of MRD and study the dynamics of clonal evolution by comparing clonal architecture and immunophenotype at diagnosis and relapse. Our study paves the road for identifying/eradicating high-risk MRD clones and preventing relapse through a better understanding of the molecular drivers of MRD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PL02.2.A Microglia-specific disruption of sialic acid-Siglec-9/E interactions. A novel immunotherapy against glioblastoma?

Abstract Background Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as monocyte-derived macrophages (MdM) or microglia (MG). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in MG-centered immunotherapy against GBM. Material and Methods We employed a CT-2A orthotopic GBM mouse model with MG specific (Sall1CreERT2 x Sigleceflox) and whole innate-compartment (Cx3cr1CreERT2 x Sigleceflox) spatio-temporal deletion of Siglece. We applied multi-color flow cytometry, transcriptomics and proteomics analysis to decipher the immune response upon Siglece knockout. Results TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Siglec-E blockade increased murine MG mediated GBM cell in vitro phagocytosis (normalized phagocytosis of 1.00 in isotype vs. 1.76 in anti-Siglec-E antibody, p &amp;lt; 0.001). In the MG specific spatio-temporal deletion of Siglece (Sall1CreERT2 xSigleceflox), we observed high MG-proliferation upon Siglec-E knockout (Ki-67+ MG 14.8% in Cre- vs. 34.9% in Cre+, p &amp;lt; 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Cre- vs. 12.3% in Cre+, p &amp;lt; 0.001). By extending the Siglece knockout to the MdM compartment in our glioma mouse model (Cx3cr1CreERT2 x Sigleceflox) we observed a significantly prolonged survival in the Cx3cr1Cre+ population (21d in Cre- vs. 27d post-tumor injection in Cre+, p = 0.018), which could be further promoted by combining Siglece knockout with CD47 blockade (30d post-tumor injection in Cre+ + anti-CD47). Unbiased proteomics analysis revealed increased antigen processing and presentation capabilities of Siglece knockout MdMs which was confirmed by ex-vivo OT-1 cross-presentation assays. This bridging of innate and adaptive responses with increased T cell priming upon MdM Siglece knockout was further promoted by addition of anti-PD1 antibody to the combined Siglece knockout and anti-CD47 treatment arm. Animals harboring CT-2A tumors, exhibited a sustained survival benefit under the triple therapy, with 23% of animals experiencing long-term remission, even after tumor re-challenge into the contra-lateral hemisphere. By genetically targeting sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), we observed a strong innate and adaptive immune response with increased GBM-cell phagocytosis by MG and MdMs and less exhausted tumor-infiltrating CD8+ T-cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003). Conclusion These data identify the sialic-acid-Siglec-E pathway as an anti-phagocytic signal in a pre-clinical GBM model, and demonstrate its therapeutic potential in GBM immunotherapy.

Peripheral blood T helper cell subsets in Löfgren’s and non-Löfgren’s syndrome patients

Sarcoidosis is a multisystemic granulomatous disorder of unknown cause, characterized by formation of immune granulomas in various organs, mainly in lungs. Currently, two main phenotypes of pulmonary sarcoidosis are described, i.e., Lofgren’s syndrome (LS) is an acute form with favorable outcome, and non-Lofgren’s syndrome (nLS) is a chronic type of disease with a high risk of pulmonary fibrosis. Our study was aimed to investigate the balance of main “polarized” CD4+central and effector memory T cells from treatment-naive patients with pulmonary sarcoidosis (LS (n = 19) and nLS (n = 63)) compared to healthy volunteers (HC, n = 48). This marker might be used as immunological markers for predicting severity of this disorder. Multicolor flow cytometry analysis demonstrated that the patients with nLS showed significantly low levels of relative and absolute numbers of CD3+CD4+lymphocytes if compared to patients with LS and control group (38.94% (31.33-44.24) versus 48.96% (43.34-53.54) and 47.63% (43.82-52.73), p &lt; 0.001 in both cases). Moreover, patients with nLS had reduced frequencies and absolute numbers of “naive”, CM and EM Th cells if compared with healthy controls. Furthermore, the patients with LS showed increased relative and absolute numbers of peripheral blood EM Th cells, capable for migration to peripheral inflamed tissues, when compared with nLS. Finally, patients with LS had increased frequencies and absolute numbers of effector TEMRA Th cells as compared to HC and nLS. Next, significant differences Th1 and Th2 cells frequencies were shown between the patients with nLS and HC (9.64% (7.06-13.65) versus 13.80% (11.24-18.03) with p &lt; 0.001, and 11.96% (9.86-14.78) versus 10.67% (9.13-12.98) with p = 0.048, respectively). But there were no significant differences in the relative numbers of CXCR5-CCR6+Th17 and CXCR5+follicular T helper cells (Tfh) between the groups. Finally, both groups of patients with pulmonary sarcoidosis contained low proportions of “non-classical” Th17 and DN Th17 cell, but increased levels of DP Th17 cells within total CXCR5-CCR6+ CM Th if compared with HC. Nevertheless, patients with nLS had increased frequency of “classical” Th17 in comparison with healthy controls. A very similar imbalance between different Th17 cell subsets was observed within total CXCR5CCR6+ effector memory Th, that were able to migrate from the bloodstream to the sites of infection, or tissue injury. Taken together, the data suggest that the proportions of Th17 cell subsets in pulmonary sarcoidosis can be evaluated as a diagnostic and/or prognostic marker in clinical practice and these cells could serve as a new therapeutic target.

Abstract 1266: Circulating immune biomarkers in peripheral blood reflect the effect of a CD200 immune checkpoint targeted immunotherapy in pet dogs with high-grade glioma

Abstract The extent to which immune cell phenotypes in the peripheral blood reflect intratumoral immune activity prior to and early in cancer immunotherapy is unclear. Here we studied the population dynamics of immune cells and immune phenotypic changes, using clinical blood samples and a multicolor flow cytometry analysis. These samples were serially obtained from a cohort of high-grade glioma canine patients enrolled in a trial with an immunotherapy that targets the CD200/CD200R immunological synapse. This therapy consists of an alternative checkpoint inhibitor peptide (CD200AR-L) that modulates the CD200 immune checkpoint, currently being used in an adult phase I safety trial for recurrent glioblastoma trial (NCT04642937) and a canine high-grade spontaneous glioma trial (U01CA224160-01). We found evidence of a strong effect of the therapy on peripheral immune cells in responder versus nonresponder dogs. Upon initiation of therapy, immune effector cell proliferation increased in responsive patients, whereas in nonresponders immune cell populations either didn’t change significantly or an increase in immunosuppressive phenotypes was observed. Cell populations such as MDSC, regulatory T cells and exhausted T cells were predominantly higher in nonresponder dogs. These data showed that peripheral immune cell phenotypes were linked to the outcome of the immunotherapy, thus dogs with better immune cell profiles, including increased cytotoxic differentiation, had a better prognosis and longer overall survival rates. Taken together, these results suggest that variation in patient responsiveness can be explained by differences in peripheral immune cell proliferation and differentiation soon after the initiation of immunotherapy. Citation Format: Francisco W. Puerta Martinez, Sathya Schnell, Susan Arnold, Elizabeth G. Pluhar, Michael Olin. Circulating immune biomarkers in peripheral blood reflect the effect of a CD200 immune checkpoint targeted immunotherapy in pet dogs with high-grade glioma [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 1266.

Abstract 2741: A communication theory framework for modeling cytokine-mediated signaling in healthy and breast cancer derived peripheral blood immune cells

Abstract Previous studies have shown that approximately 40% of estrogen receptor positive (ER+) breast cancer (BC) patients harbor immune signaling defects in their blood at diagnosis that predicts overall survival. The aim of this work is to apply communication theory and signal processing methods to model cytokine-mediated signaling errors in immune cells in the peripheral blood. Our approach uses communication theory concepts to develop a communication system model consisting of a transmitter, receiver, and channel, applied to the JAK/STAT signaling pathway and tested using multi-color flow cytometry analysis of peripheral blood mononuclear cells stimulated with IFNγ, IL-2, IL-6, IL-4, IL-10, IL-12, or TGFβ in samples from 2 BC patients and 4 healthy controls. We use our model to estimate the detection error rate when one of the 7 cytokines is being transduced through the JAK/STAT signaling pathway, but its signal is being received on multiple phosphorylated signal transducers and activators of transcription (pSTAT) molecules. In addition, we extend the measured error rate value to an error rate curve by varying the signal-to-noise ratio available to the receiver in our system model. Our results show a trend towards higher detection error rates in immune cells from BC patients as compared to healthy controls, which may indicate altered immune signaling and incorrect cell decisions in these patients. Also, considering different cell subtypes, our results show two orders of magnitude increase in error rate in CD4+ memory T cells compared to CD4+ naïve T cells in BC patients. With further development, we believe that our communication theory model and quantification of optimal signal detection in cells will provide a better understanding into how immune cells can accurately “decode” information to respond properly to various signals and how the immune signaling is altered in BC patients, causing better or worse outcomes. The benefit of our novel approach is that it integrates all the transmitted (cytokines) and received signals (pSTATs) into a single communications framework that can be efficiently analyzed together. Citation Format: Adina Matache, Aleksandra Karolak, Sergio Branciamore, Andrei Rodin, Peter P. Lee, Russell C. Rockne. A communication theory framework for modeling cytokine-mediated signaling in healthy and breast cancer derived peripheral blood immune cells [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 2741.

Enhanced tumor control with a combination of APG-157 and immune checkpoint inhibitors for head and neck cancer.

2652 Background: Natural botanical drugs containing flavonoids are under clinical investigation for the treatment of malignancies. A phase I clinical study for patients with locally advanced head and neck cancer with a new class of botanical drug APG-157 showed excellent tolerability, systemic absorption via sublingual administration, and enhanced immune cell infiltration into the tumor microenvironment and suppression of T cell inhibitory inflammatory cytokine production. We sought to evaluate the efficacy of tumor control for the combination of APG-157 and checkpoint blockade immunotherapy using a head and neck cancer mouse model. Methods: SCCVII (HPV-) and MEER (HPV+) head and neck cancer murine cell line models were utilized in in vivo tumor growth study. After optimization for route and cell number, we measured tumor growth after treating mice with following groups: i) control, ii) APG-157, iii) anti-PD-1, iv) anti-CTLA-4, v) APG-157 + anti-PD-1, vi) APG-157 + anti-CTLA-4, vii) anti-PD-1 and anti-CTLA-4, viii) APG-157 + anti-PD-1 + anti-CTLA-4. Tumors were collected after 2 injections of immune checkpoint inhibitors and characterized for immunophenotype by multi-color flow cytometry, immunohistochemistry, and RNA-sequence analysis. Fecal microbiome was analyzed by 16S rRNA sequence for SCCVII model to assess the impact of APG-157 on the microbiome as we observed the shift of oral microbiome species in the human trial. Results: We confirmed by serum concentration of APG-157 metabolites that the best route of administration is via oral route by mixing with APG-157 with mouse chow. Tumor growth optimization experiments determined that 100,000 cells of SCCVII is the ideal number of cells to inject and day 5-6 is the time to initiate treatment. Among 8 treatment groups, APG-157 + anti-CTLA-4 demonstrated best tumor control (p = 0.0065 compared to control), followed by anti-PD-1 + anti-CTLA-4 treatment group (p = 0.48 compared to control). The immunophenotype assessment by flow cytometry showed over 30% of CD8+ T cell in APG-157 + anti-CTLA-4 group compared to 4-5% of CD8+ T cell for the control group. GSEA analysis from the SCCVII tumors revealed that APG-157 + anti-CTLA-4 group showed an enriched set of genes for inflammatory response, apoptotic signaling pathways. The fecal microbiome analysis showed a substantial difference of lactobacillus genus among groups, highest in the group of APG-157 + anti-CTLA-4 treatment group. The MEER model (HPV positive) tested with same immunotherapy as single or in combination with APG-157 did not grow the tumor, thus we were unable to collect the tumor for immunophenotyping and other studies. Conclusions: These study results indicate that APG-157 is able to modulate the immune system and fecal microbial species that could potentially lead to improved anti-tumor immunity and warrants further studies to define mechanism and the potential for human clinical trial.

1450-P: Identifying Hidden Sources of Tissue Plasticity in Pancreatic Islets with a Novel Multicolor Flow Cytometry Approach

Identifying pancreatic islet cells that acquire a beta-cell like phenotype is important for developing new diabetes therapies. Our studies have revealed a novel serpinB13 protease inhibitor antibody, that augments cathepsin L protease activity, resulting in Notch1 receptor cleavage and mobilization of endocrine progenitor cells to become beta cells in the embryonic pancreas. It is unclear, however, whether a similar induction of additional beta cells occurs with exposure to serpinB13 monoclonal antibody (mAb) in later life. To explore the exact source (s) of new beta cells in response to serpinB13 mAb after birth we developed a novel, multicolor flow cytometry analysis approach to study pancreatic islets. Five-week old normal Balb/c mice were injected with serpinB13 mAb (or control IgG) , and sacrificed at 8 weeks for islet isolation. Islet cell suspensions were stained with a viability dye, and a panel of antibodies to CD31, CD45, cytokeratin 19, EpCAM, insulin (Ins) , glucagon (Glu) , somatostatin (SS) , c-peptide, and four transcription factors, Maf-A, Nkx6.1, NeuroD1, and Pdx-1. Exposure to the serpinB13 mAb caused a significant increase in the number of hormonal-negative (e.g., Ins-Glu-SS-c-peptide-) cells (p=0.0011) , and hormonal negative, but c-peptide positive, cells (p=0.002) . At least one of four transcription factors examined were positive in 5-10% and 10-30% of these populations, respectively. At the same time, there was a trend in the drop of Glu+ and SS+ cells. The average number of pancreatic islets, and the total islet cellularity per mouse, was augmented in animals receiving serpinB13 mAb. Although additional studies are necessary to characterize the hormonal negative cells and assess whether Glu+ and SS+ cells transdifferentiate to beta cells, these findings demonstrate that enhanced tissue plasticity and beta-cell output can be achieved in the pancreas following treatment with antibodies to protease inhibitors. Disclosure Y.Kryvalap: None. S.Z.Meng: None. J.Czyzyk: None.

Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae

Transcription factor-based biosensors represent promising tools in the construction and evaluation of efficient cell factories for the sustainable production of fuels, chemicals and pharmaceuticals. They can notably be designed to follow the production of a target compound or to monitor key cellular properties, such as stress or starvation. In most cases, the biosensors are built with fluorescent protein (FP) genes as reporter genes because of the direct correlation between promoter activity and fluorescence level that can be measured using, for instance, flow cytometry or fluorometry. The expansion of available FPs offers the possibility of using several FPs - and biosensors – in parallel in one host, with simultaneous detection using multicolor flow cytometry. However, the technique is currently limited by the unavailability of combinations of FP whose genes can be successfully expressed in the host and whose fluorescence can be efficiently distinguished from each other.In the present study, the broad collection of available FPs was explored and four different FPs were successfully expressed in the yeast Saccharomyces cerevisiae: yEGFP, mEGFP, CyOFP1opt and mBeRFPopt. After studying their fluorescence signals, population heterogeneity and possible interactions, we recommend two original combinations of FPs for bi-color flow cytometry: mEGFP together with either CyOFP1opt or mBeRFPopt, as well as the combination of all three FPs mEGFP, CyOFP1opt and mBeRFPopt for tri-color flow cytometry. These combinations will allow to perform different types of bi-color or possibly tri-color flow cytometry and FACS experiments with yeast, such as phenotype evaluation, screening or sorting, by single-laser excitation with a standard 488 nm blue laser.

Multiple Myeloma: Nuances of Minimal Residual Disease Diagnosis and Monitoring with the Use of Multicolor Flow Cytometry

The assessment of minimal residual disease (MRD) by multicolor flow cytometry (MFC) is a rapidly growing area of laboratory studies. In recent years, it has become particularly valuable for hematologists. Although the MFC analysis of plasma cells in multiple myeloma patients is sufficiently standardized, there are differences in methods of sample preparation, monoclonal antibody combinations being used as well as in cytometric data evaluation. The present paper summarizes the key international and domestic data on the MFC analysis of plasma cells and documents the authors’ own experience with MFC analysis in multiple myeloma over the last few years.