Quantification of expression of antigens targeted by antibody-based therapy in chronic lymphocytic leukemia.

OBJECTIVES: Anti-CD20 (rituximab), anti-CD52 (alemtuzumab), anti-CD22 (BL22, HA22), and anti-CD25 (Oncotac) are therapeutic options that are the mainstay or in preclinical development for the treatment of chronic lymphocytic leukemia (CLL). Studies suggest that levels of surface antigen expression may affect response to monoclonal antibody-based therapy. METHODS: Using the flow cytometric Quantibrite method (BD Biosciences, San Jose, CA) to determine antibodies bound per cell, we quantified the levels of surface expression of CD20, CD22, CD25, and CD52 in CLL cells from 28 untreated patients. RESULTS: The CLL cells in all cases expressed CD20, CD22, and CD52 but 4 (14%) cases were negative for CD25. Although the ranking of levels of expression from highest to lowest was CD52, CD20, CD22, and CD25, the level of antigen expression on any specific case could not be accurately predicted. CONCLUSIONS: Quantification of antigens might be useful in evaluating new antigens to target for therapy and may provide a systematic approach to selecting individualized therapy in CLL.

Variables affecting the quantitation of CD22 in neoplastic B cells.

BACKGROUND: Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory for diagnosis, prognosis, and assessment of patients receiving antibody-based therapy. ABC values and the effect of technical variables on CD22 quantitation in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FCL), hairy cell leukemia (HCL) and normal B cells were studied. METHODS: The QuantiBrite System® was used to determine the level of CD22 expression (mean antibody bound per cell, ABC) by malignant and normal B cells. The intra-assay variability, number of cells required for precision, effect of delayed processing as well as shipment of peripheral blood specimens (delayed processing and exposure to noncontrolled environments), and the effect of paraformaldehyde fixation on assay results were studied. RESULTS: The QuantiBRITE method of measuring CD22 ABC is precise (median CV 1.6%, 95% confidence interval, 1.2-2.3%) but a threshold of 250 malignant cells is required for reliable CD22 ABC values. Delayed processing and overnight shipment of specimens resulted in significantly different ABC values whereas fixation for up to 12 h had no significant effect. ABC measurements determined that CD22 expression is lower than normal in ALL, CLL, FCL, and MCL but higher than normal in HCL. CONCLUSIONS: CD22 expression was atypical in the hematolymphoid malignancies studied and may have diagnostic utility. Technical variables such as cell number analyzed and delayed processing or overnight shipment of specimens impact significantly on the measurement of antigen expression by QFCM in the clinical laboratory.

Variables in the quantification of CD4 in normals and hairy cell leukemia patients.

BACKGROUND: Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory. Quantitative normal T-cell CD4 expression represents a biologic standard and quality control agent. However, low levels of CD4 expression were detected in normal T-cells in Hairy Cell Leukemia (HCL) samples. METHODS: The QuantiBrite System® was used to determine the level of CD4 expression (mean antibody bound per cell, ABC) in fresh and shipped HCL blood and fresh normal donor blood (NDB). The effects of shipping, lysing reagent, cell preparation method, and antibody lot were evaluated. RESULTS: Shipped HCL specimens (n = 69) had a significantly lower mean CD4 ABC of 38,788 (CV = 9.1%) compared to fresh specimens (n = 105) CD4 value of 40,330 (CV = 8.4%) (P < 0.05). In NDB, significant differences were seen for fresh versus shipped specimens using the stain/lyse method but not for lyse/stain method. Consistent differences in CD4 ABC based upon antibody lot were observed in fresh HCL and NDB samples. Stain/lyse and lyse/stain methods using NH(4) Cl lyse were compared in NDB using identical samples and antibodies. The NDB CD4 ABC values obtained with the lyse (NH(4) Cl)/stain method (45,562, 3.7% CV) were lower than those obtained with the stain/lyse (NH(4) Cl) method (49,955, 3.3% CV) with P < 0.001. CONCLUSIONS: CD4 expression in HCL patient samples is not inherently different from that observed in NDB and therefore may serve as a biological control in clinical QFCM. Technical variables impact significantly on QFCM of CD4.

Visual inspection versus quantitative flow cytometry to detect aberrant CD2 expression in malignant T cells.

BACKGROUND: Abnormal levels of T-cell antigen expression occur in T-cell neoplasia. We examined CD2 expression in malignant and normal T cells to determine if the level of CD2 expression differed significantly and if quantitation assisted in detecting this difference. METHOD: Flow cytometric immunophenotypic (FCI) evaluation was performed on specimens from 36 patients with mature T-cell neoplasia. Abnormal T cells were identified based upon the abnormal FCI and morphology. Levels of CD2 expression were quantitated using 1:1 PE conjugates of anti-CD2 and QuantiBRITE bead standards to calculate the antibodies bound per cell (ABC). The efficacy of ABC measurement versus simple examination of dots plots was compared. RESULTS: Abnormal levels of CD2 expression were frequently observed in mature T-cell malignancies. The CD2 ABC values were highly sensitive in detecting differences between malignant and normal T cells (P = 0.0028). In most cases (24/32 specimens, 75%), CD2 ABCs differed by >20%. CD2 ABCs had high variability in normal T cells. CONCLUSIONS: CD2 expression by malignant T cells differed significantly from that of normal T-cells by CD2 ABC quantitation. The high variability in normal T-cell CD2 ABCs limited the determination of normal reference ranges and, thus, its utility in the diagnosis of T-cell neoplasia. However, examination of CD2 can help in detection of tumor cells when residual normal T cells are present for comparison. Moreover, the increased sensitivity of CD2 quantitation is valuable in confirming FCI cases where abnormalities in CD2 expression are difficult to appreciate by visual inspection alone.

Variable CD52 expression in mature T cell and NK cell malignancies: implications for alemtuzumab therapy.

The anti-CD52 antibody alemtuzumab has been explored as a novel targeted therapy in T cell malignancies. To assess the suitability of alemtuzumab therapy, we carried out a comprehensive study of CD52 expression using flow cytometry (FC) in 78 untreated patients diagnosed with mature T/natural killer (NK) cell neoplasms, including 34 adult T cell leukaemia/lymphomas (ATLL), two anaplastic large cell lymphomas (ALCL), three angioimmunoblastic T cell lymphomas (AITL), 16 cutaneous T cell lymphomas (CTCL), four extra-nodal T/NK cell lymphomas (ENT/NKCL), four hepatosplenic T cell lymphomas (HSTCL), 13 peripheral T cell lymphomas, not otherwise specified (PTCL-NOS) and two T-prolymphocytic leukaemia (T-PLL). The level of CD52 expression was quantified using QuantiBRITE standard beads. The level of CD52 expression varied widely within each diagnostic category. All AITL, HSTCL and T-PLL cases were CD52-positive and the frequency of CD52 expression was high in PTCL-NOS (92.3%), ATLL (94.1%) and CTCL (87.5%), implying a rational role for alemtuzumab in the treatment of these diseases; however, CD52 expression was low in ALCL (50%) and ENT/NKCL (25%). FC testing for cell surface expression of CD52 is indicated in patients with T/NK cell malignancies being considered for alemtuzumab therapy. Further studies are necessary to determine if the level of CD52 expression correlates with response to therapy.