Session: 803. Emerging Diagnostic Tools and Techniques: Poster I
Hematology Disease Topics & Pathways:
Technology and Procedures, flow cytometry
Fresh tumor tissues taken by fine or coarse needle puncture from different body parts were put in saline with 2% fetal calf serum and sent to the FCM lab as quickly as possible, once in lab, samples were processed immediately. Tissue strips were placed into a 60 mm petri dish, gently pressed by a syringe piston and repeatedly flushed with saline to separate cells from tissues; then the liquid with cells in petri dish were transferred into a 100μm cell strainer on top of 50 mL conical tube to exclude non-cellular tissue components, the strainer was washed with saline for 2-3 times. The strained cell suspension in conical tube was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded by pipetting, cells were counted and resuspended with saline at a concentration of 104-105/ml, then the single cell suspension was ready for flow cytometry assay. 7-AAD and antibodies CD19/CD22/CD7/CD45 were added into the first tube to quantify viable cells and identify cells available for analysis, if dead cells were more than 30% of all cells, 7-AAD was needed for all next tubes, otherwise, it could be omitted. The standard antibody panels consist of CD10/CD20/CD34/CD19/CD38/CD45 for precursor B-cells and kappa/lambda/CD10/CD19/CD20/CD38/CD45 for mature B-cells, extra antibodies could be added according to patient’s situations. Samples were performed on the 8-color BD FACSCanto II flow cytometer, and analyzed by BD FACS Diva software or Kaluza software from Beckman Coulter. Antigen expression, partial expression and no expression were defined as >80%, 20-80% and <20% of gated cells displaying interested antigen, respectively. Dim expression was defined as more than one log weaker than the normal counterpart‘s mean fluorescence intensity (MFI).
From October 2017 through June 2020, a total of 63 patients with relapsed/refractory B-cell malignancies who had tumor masses only and no BM involvement or malignant SCE were hospitalized in our center for CAR-T treatment after failure of multiple-line therapies, including 11 (17.5%) acute lymphoblastic leukemia (ALL) and 52 (82.5%) non-Hodgkin's lymphoma (NHL), the median age was 41 years (range, 1-71) with both adults (n=47, 74.6%) and children younger than 18 years (n=16, 25.4%). Acquired events from tissue samples varied from 1000 to more than 100, 000 each tube (5 cases <5000 and 17 cases >100,000), the percentages of tumor cells in all cells available for analysis varied from 0.03% to 99.44% (5 cases <5% and 13 cases >90%). We have CD19-, CD20- and CD22-specific CAR-T cells for B-ALL and B-NHL, the choice of a CAR-T product depends on the antigen expression on tumor cells, patients with partial or no or dim expression (PND expression) of certain antigen are not suitable for the relevant antigen-specific CAR-T therapy. Among this cohort of patients, CD19 expressed in all but 1 ALL case who had accepted CD19 CAR-T; PND expression of CD20 occurred in 19.2% (10/52) of NHL patients since they were repeatedly administrated anti-CD20 antibodies in previous treatments which resulted in antigen diminishment or loss, all B-ALL patients had no or partial CD20 expression; PND expression of CD22 was seen in 18.2% (2/11) of ALL and 13.5% (7/52) of NHL. All flow results came back within hours (< 4-6 hours), much quicker than results from IHC staining which takes days.
In conclusion, the correct selection of a target antigen is the basis of effective CAR-T treatment in relapsed/refractory B-cell malignancies especially for patients having accepted antibody or CAR-T therapies, our antigen detection by tissue flow cytometry provided a quick and accurate antigen identification for patients with no BM or SCE samples.
Disclosures: No relevant conflicts of interest to declare.
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