The Quick Antigen Identification By Tissue Flow Cytometry for CAR-T Therapy in Relapsed/Refractory B-Cell Malignancies without Bone Marrow Involvement or Serous Cavity Effusions
Authors: Yu Xinjian , Li Pan , Feng Heyuan , Kang Jian , Zheng Xichang , Zhang Yunmei , Yang Wenhong , Wu Tong , Liu Shuangyou 
Published: February 2021

In recent years, chimeric antigen receptor (CAR) T-cell therapy has been widely used to treat relapsed/refractory B-cell malignancies. Before CAR-T, the identification of target antigens on tumor cells is needed for choosing a suitable antigen-specific CAR, which is usually performed by multiparameter flow cytometry (FCM) for bone marrow (BM) samples or immunohistochemical (IHC) staining for tissue samples. However, some patients with tumor masses only and without BM involvement or serous cavity effusions (SCE) may not have much time to wait the results from IHC staining owing to rapidly growing tumor cells, therefore, we established a quick antigen identification by FCM using tissue samples.
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 &gt;80%, 20-80% and &lt;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 &lt;5000 and 17 cases &gt;100,000), the percentages of tumor cells in all cells available for analysis varied from 0.03% to 99.44% (5 cases &lt;5% and 13 cases &gt;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 (&lt; 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.