266 Tissue antigen expression levels control the fundamental decision between tolerance and autoimmunity

Abstract Abstract 1436 Background: The distinction in antigen expression between normal T cells and T-ALL is currently limited by a narrow breadth of clinically useful antibodies. Our study aimed to enhance the diagnostic utility of flow cytometry in T-ALL diagnosis and minimal residual disease (MRD) detection by identifying novel aberrant antigen expression in T-ALL. Method: A high throughput flow cytometric screening method containing 242 antibodies (Lyoplate, Becton Dickinson) was used to evaluate 3 normal peripheral blood specimens and 9 diagnostic paediatric T-ALL cases. An LSRII (Becton Dickinson) was used for all flow acquisition, and Woodlist software for all analysis. The initial screening panel comprised a 9 colour assay (1 detection and 8 gating fluorochromes). All Lyoplate antibodies were analysed for differences in median fluorescence intensity between normal T cells and T-ALL populations. Subsequently, 9 specific novel antibodies were chosen for expanded testing in prospectively enrolled paediatric T-ALL cases on Children's Oncology Group protocols, using 3 additional tubes. A 9 color flow cytometric assay was designed with the following backbone antibody combination: CD3 PE-TR, CD16+CD56 PE-Cy5, CD5 PE-Cy7, CD7 V450, CD45 KO. Additionally, in each tube one of the following combinations was utilized: CD226 FITC, CD184 PE, CD229 APC or CD6 FITC, CD53 PE, CD200 APC or CD244 FITC, CD165 PE, CD27 APC. Median fluorescence intensites were compared between T-ALL populations and normal donor T cells, and the ratio of abnormal T-ALL: normal donor T cells calculated for each antibody. Results: A total of 59 patients with T-ALL were analyzed median age 8 (2–29) with 38 males, including 6 early thymic precursor (ETP) cases by St Jude's criteria. In total, 94 episodes were included: 59 pretreatment, and 35 post treatment cases at Day 29. In the T-ALL, CD27 expression was significantly reduced with a median blast/normal T cell ratio of 0.54 at diagnosis, and 0.52 post treatment. 48/53 (91%) patients had a reduced ratio <0.75 at diagnosis and 18/31 (58%) post treatment. Additionally CD6, CD226 and CD200 showed a mild reduction at diagnosis of 0.75, 0.81 and 0.84 respectively, with similar ratios seen with these 3 antibodies post treatment. The ETP cases showed a significant reduction in CD27 expression, median ratio 0.44, while reduced expression was also seen with CD6 (0.72), CD165 & CD184 (0.77), CD53 (0.78) and CD226 (0.80). There was no difference in the ratios calculated when using internal patient T cells versus normal donor T cell expression levels. Conclusion: We have demonstrated that expression levels of CD27 are consistently reduced in the majority of T-ALL and ETP patients at diagnosis and expression levels are stable at post treatment assessment. This marker has potential utility in clinical practice for diagnosis and MRD assessment. A spectrum of additional antibodies also demonstrated reduced expression, especially in ETP cases, however further patient recruitment and evaluation is required for more definitive evaluation of the utility of these antigens. Disclosures: No relevant conflicts of interest to declare.

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Improved Recognition of Hematogones from B Lymphoblastic Leukemia By 5-Color Flow Cytometric Analysis

Blood ◽

10.1182/blood.v128.22.1732.1732 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1732-1732

Author(s):

Amanda Lo ◽

Matthew Miller ◽

Melissa Cervania ◽

Quanlin Li ◽

Mark David Ewalt ◽

...

Keyword(s):

Bone Marrow ◽

Lymphoblastic Leukemia ◽

Antigen Expression ◽

Internal Controls ◽

Mean Fluorescent Intensity ◽

Color Flow ◽

Fluorescent Intensity ◽

Single Tube ◽

Expression Levels ◽

Antigenic Expression

Abstract Background: Hematogones (HG) are benign precursor B-cells that are seen in increased numbers in the bone marrow during childhood, following chemotherapy or bone marrow transplant, in certain immune deficiencies, and in autoimmune disorders. Flow cytometry typically shows expression of TdT, CD10, CD19, variable CD20, dim CD22, CD34 (early HG), and dim CD45. As this phenotype is also seen in patients with B lymphoblastic leukemia (B-ALL), it causes a significant problem in distinguishing leukemic blasts from HG, particularly in a regenerating marrow. Furthermore, hematogones are usually more numerous at baseline in younger patient populations, the same age group with a relatively higher incidence of B-ALL. Despite guidance by earlier studies using the above markers for differentiating HG from B-ALL, these markers are not always sufficient and may hinder correct interpretation. Previous studies demonstrate CD58 is commonly expressed in B-ALL but not in hematogones; however, CD58 as a single marker is somewhat limited when expressed at lower levels. In order to improve diagnostic accuracy, we generated a five color antibody panel including CD10, CD19, CD45, CD38, and CD58 to assess the utility of a single tube panel in distinguishing B-ALL from HG. Design: A total of 35 cases with immature B-cell populations, 16 B-ALL (diagnostic and residual/relapsed cases) and 19 HG, were analyzed by 5-color flow cytometry. 32/35 cases were bone marrow aspirates and 3/35 cases were peripheral blood. A single tube containing CD10 FITC/CD58 PE/CD19 ECD/CD38 PC5/CD45 PC7 was analyzed together with the standard acute leukemia panels. To eliminate technical and fluorochrome variability in expression level analysis, relative antigenic expression was determined through comparison with appropriate internal controls. Antigen expression, as measured by the geometric mean fluorescent intensity (MFI), was then compared between B-ALL and HG using the Mann-Whitney U-test to assess for significant difference. Results were correlated with the morphologic, immunohistochemical, cytogenetic, and molecular findings for precise diagnostic classification as HG or leukemia. Results: HG demonstrated significantly brighter expression of CD38 (p<0.01) than that seen in B-ALL. In contrast, B-ALL expressed significantly brighter CD58 (p <0.01) than HG, which showed dim to no expression of the antigen. HG also showed significantly bright expression of CD10 relative to internal control granulocytes; however, this level of expression was similar to that seen in B-ALL. Median antigen expression. Hematogones show bright CD38, but dim to no CD58. Conversely, B-ALL expresses very dim CD38 and variable CD58. CD10 expression, though, demonstrates overlap between the two populations. B-ALL = B lymphoblastic leukemia, MFI = mean fluorescent intensity Comparative antigenic expression levels in hematogones and B-ALL. Select representative histograms showing HG and B-ALL blasts for the antigens CD38, CD58, and CD10 were selected from various patients studied based on those with the closest relative MFI to the overall median detected for that population in the study. The far right column shows the distribution in MFI of relative antigen expression exhibited the populations studied. HG show significantly brighter CD38 expression than B-ALL does (p<0.01). While B-ALL generally expresses brighter CD58 relative to internal controls, expression levels are variable. HG, though, show significantly dimmer CD58 to essentially no CD58 expression, compared to B-ALL (p<0.01). Similar to CD38, HG demonstrate significantly bright CD10, while B-ALL shows overall bright CD10 but variable expression levels amongst studied cases. These expression levels for CD10 overlap between HG and B-ALL and show no real statistical difference. Conclusions: The combination of CD38 and CD58 in a single tube increases the diagnostic accuracy in differentiation of HG from B-ALL. Without utilizing both antigens together, certain cases would have been difficult to interpret. Based on this analysis, we recommend that these markers be utilized in the routine evaluation for acute lymphoblastic leukemia. This is especially critical in post treatment cases in order to avoid misdiagnosis. Furthermore, the use this single tube panel would cut costs while at the same time improve patient care. Table Table. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Quantitating Antibody Uptake In Vivo: Conditional Dependence on Antigen Expression Levels

Molecular Imaging and Biology ◽

10.1007/s11307-010-0397-7 ◽

2010 ◽

Vol 13 (4) ◽

pp. 623-632 ◽

Cited By ~ 33

Author(s):

Greg M. Thurber ◽

Ralph Weissleder

Keyword(s):

Antigen Expression ◽

Expression Levels ◽

Conditional Dependence ◽

Antibody Uptake

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Alterations in phenotype and cell-surface antigen expression levels of human monocytes: Differential response to in vivo administration of rhm-csf or rhgm-csf

Cytometry ◽

10.1002/cyto.990220205 ◽

1995 ◽

Vol 22 (2) ◽

pp. 103-110 ◽

Cited By ~ 25

Author(s):

Ingrid Schmid ◽

Gayle C. Baldwin ◽

Edwin L. Jacobs ◽

Valentin Isacescu ◽

Negoita Neagos ◽

...

Keyword(s):

Cell Surface ◽

Surface Antigen ◽

Antigen Expression ◽

Differential Response ◽

Cell Surface Antigen ◽

Human Monocytes ◽

Expression Levels ◽

Surface Antigen Expression ◽

In Vivo Administration

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Effectivity Of Complement-Dependent Cytotoxicity Induced By Therapeutic Antibodies In Acute Lymphoblastic Leukemia Is Dictated By Antigen Expression Levels and Curtailed By Membrane-Bound Complement Regulatory Proteins

Blood ◽

10.1182/blood.v122.21.3909.3909 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3909-3909

Author(s):

Floris C Loeff ◽

Esther H.M. van Egmond ◽

Judith Olde Wolbers ◽

J.H. Frederik Falkenburg ◽

Constantijn J.M. Halkes ◽

...

Keyword(s):

Cell Lines ◽

Target Cell ◽

Lymphoblastic Leukemia ◽

Antigen Expression ◽

Target Antigen ◽

Therapeutic Antibodies ◽

Expression Levels ◽

Maximum Decrease ◽

Anti Cd20 ◽

Cd59 Expression

Abstract Membrane-bound complement regulatory proteins (mCRPs) are known to protect cells from bystander lysis and spontaneous activation of complement. In the case of antibody therapy these mCRP’s may down-modulate antibody-induced complement-dependent cytotoxicity (CDC). Two of these mCRPs, CD55 (DAF) and CD59 (MIRL), have been suggested as modulators of CDC. However, no strong correlation between their expression level and target cell lysis has been demonstrated thus far. Our goal was to elucidate the role of these mCRPs in the efficacy of antibody-induced CDC. We tested the effectivity of CDC induced by the therapeutic antibodies Alemtuzumab (Alt, anti-CD52), Rituximab (Rtx, anti-CD20), and Ofatumumab (Ofa, anti-CD20) in 16 cell lines showing differential expression of target antigens and mCRPs. These cell lines were generated in our laboratory from primary acute lymphoblastic leukemia (ALL) cells and their phenotype and karyotype greatly resembles the primary malignancy. CDC was induced by incubation of target cells with a serial dilution of the respective antibody in the absence or presence of human serum for a period of 18 hours. Subsequent target cell death was determined using flow cytometry by measuring the absolute number of surviving cells (propidium iodide negative, CD19 positive) per sample, using a fixed number of CytoCount™ beads as an internal reference, and calculating the magnitude of cell loss compared to untreated control samples. A strong correlation was found between antigen expression level and CDC induction by Alt, Rtx, and Ofa. Interestingly, considerable deviation from this correlation was observed especially in cell lines with intermediate antigen expression levels. To test whether this was due to differential expression of mCRPs, surface expression levels of CD55 and CD59 were measured by flowcytometry. The magnitude of CDC strongly correlated to the mCRP expression in the cell lines with intermediate antigen expression (MFI 1128±104 (CD52, n=5) 796±132 (CD20, n=3)), resulting in 51% (Alt) and 75% (Ofa) reduction of antibody-induced lysis in cell lines with high CD55/CD59 expression compared to cell lines with low CD55/CD59 expression. In the cell lines with high antigen expression (MFI 2802±67 (CD52) 2136±692 (CD20)), the protective effect of co-expression of mCRPs appeared to be overpowered by the high target antigen expression levels. To study the individual contribution of CD55 and CD59 to the protective effect against CDC, we induced the expression of CD55 and CD59 separately in the cohort of target cell lines with intermediate target antigen expression. Cell lines were transduced, using the bicistronic LZRS vector and the ϕ-NX-A retrovirus packaging cell line, with a construct coding for CD55 or CD59, respectively, coupled to the truncated nerve growth factor receptor (tNGFR) via an internal ribosome entry site (IRES)-sequence. Cells positive for membrane expression of the tNGFR marker and CD55 or CD59 were sorted using flowcytometric cell sorting. This resulted in cell lines with significantly increased expression levels of the respective mCRP compared to their MOCK transduced counterparts, yet never exceeding physiological levels as found in the original ALL cell lines. When compared to MOCK transduced cell lines, all CD55 transduced cell lines (n=4) showed a decreased sensitivity for Alt-induced CDC with a maximum decrease of 69%. Similarly, forced induction of CD59 expression significantly decreased the sensitivity to CDC compared to MOCK transduced cell lines when using Alt (maximum decrease 52%), Rtx (maximum decrease 56%), or Ofa (maximum decrease 30%) (n=5). In conclusion, we show that efficacy of CDC induction by therapeutic antibodies is dictated by the level of target antigen expression, but is dominantly influenced by expression of the mCRPs CD55 and CD59. This restraining effect on sensitivity to antibody-induced CDC was observed using Alemtuzumab as well as Rituximab and Ofatumumab, suggesting a general mechanism independent of target antigen or type of antibody. Therefore, mCRP expression on malignant cells is likely to be an important factor in the sensitivity to antibody treatment, especially when cells with intermediate antigen expression are targeted. Disclosures: No relevant conflicts of interest to declare.

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