Abstract
Objective: To find a strong and applicable panel for T-ALL MRD detection by flow cytometry and find the relation with genetics. Methods: From February 28, 2024 to May 29, 2025, 1025 T-ALL patients were tested for MRD in our Hospital using full-spectrum flow cytometry. A total of 1570 tests were completed. All patients were Chinese, without disabilities, with a male-to-female ratio of 768:257, and a median age of 16 years (1-68). There was 1 Uyghur, 2 Tibetans, 5 Hui people, and 1017 Han people. The detectionpanel was CD99 FITC/cCD3 PE/CD3 BV785/CD48 PECy7/CD4 APC Cy7/CD5 APC R700/CD2 BV605/CD7 APC/CD16 efluor 450/CD56 BV711/TdT BV421/CD45 V500/CD34 PerCP Cy5.5/CD94 BV650/CD8a BV570/TCRγδ BV480. A total of 612 people had detectable genes. Ten cases of complete remission and ten cases of MRD positive were selected for correlation testing between the traditional panel and the full-spectrum panel. Data analysis was performed using Kaluza 2.3.0. Statistical analysis was conducted using SPSS 17.0. 68 cases were implemented in Python. Results: (1) The correlation between the full-spectrum flow cytometry and the traditional flow cytometry was good. (2) Among the 1570 tests, 155 tests from 103 individuals were positive. Among the positive patients, the ratio of male to female was 79:24, with a median age of 21 years (ranging from 4 to 68 years). There was 1 Tibetan, 1 Hui, and 101 Han. The median tumor burden was 1.66% (ranging from 0.002% to 94.15%). (3) 68 MRD positive cases had genetics results, 33 had WT1, 2 had EVI, 13 were positive for SIL::TAL1, 1 for PCM1::JAK2, 4 for SET::CAN, 6 for MLL::AF6, 1 for MBNL1::TAL1, 1 for KMT2A::PAP1GDS1, 2 for DIAPH1::PDGFB, and 3 for CALM::AF10. One case of EVI and 6 cases of WT1 had values below the reference range. (4) Among the 52 cases with CD7 negative after CD7 CAR-T, 43 were MRD negative and 9 were MRD positive. Among the MRD positive patients, 2 were cCD3 negative. Among 52 cases with CD7 negative,cases negative for cCD3, CD2, and CD5 was 0. (5) Among the 6 cases with lost cCD3, 2 cases were negative for both CD2 and CD5. (6) The relationship between genetics and immunophenotype: the probability of co-expression of CD7 and CD99 was the highest in the MLL::AF6, CALM::AF10, and EVI1 genetic abnormality groups, which was 100% (P < 0.001). The genotypes that were prone to cCD3 weakening or partial loss were PCM1::JAK2 (100%, 2/2), SET::CAN (75%, 3/4), MLL::AF6 (67%, 2/3), DIAPH1::PDGFB (50%, 1/2), EVI1 (50%, 1/2), WT1 (40%, 14/35), and CALM::AF10 (33%, 1/3). On the MRD recognition markers, strong expression of CD99 and weakened CD48 were common abnormalities. PCM1::JAK2, SET::CAN, and WT1 were prone to losing the characteristic of strong expression of CD99. The probability of weakened or negative CD48 was 100% for MLL::AF6 (6/6), SET::CAN (4/4), CALM::AF10 (3/3), MBNL1::TAL1 (1/1),and KMT2A::PAP1GDS1 (1/1). It was 64% for SIL::TAL1, and 40% for WT1. The positive rate of CD34 in all T-ALL cases was only 19%, while CALM::AF10 and EVI1 was 100% positive, MLL::AF6 was 50%, WT1 was 23%, SET::CAN was 25%. CD45dim/CD7bri is the most commonly used gating method, but should exclude NK cell interference. The probability of CD56 positivity was 67% for CALM::AF10 and 20% for WT1. The positive rate of CD3 was 79% (11/14) for SIL::TAL1, 50% for DIAPH1::PDGFB (1/2) and EVI1 (1/2), 46% (16/35) for WT1, and 25% (1/4) for SET::CAN. MLL::AF6 has a 100% positive rate for CD7briCD99briCD48-CD56-CD34+, theoretically providing the highest degree of identification. Next is EVI1, which is 100% positive for CD7briCD99briCD48+CD56-CD34+ because CD48 could not provide contribution. CALM::AF10 has a 100% positive rate for CD7briCD99briCD48-CD34+, but the CD56 positive rate is 67% (2/3), and it also needs to be differentiated from NK cells, making it an easily identifiable subtype. PCM1::JAK2 and some WT1 subtypes may be relatively more difficult to identify. Conclusion: Full-spectrum flow cytometry can simultaneously perform more than four pan-system markers, theoretically meeting the gating requirements for MRD detection after CAR-T therapy at 98.51%. However, to increase detection coverage, pan-T markers need to be added. There is a certain correlation between genetics and immunophenotype. Different genotypes benefit from different markers, and full-spectrum flow cytometry can simultaneously perform more marker combinations to maximize coverage of common abnormalities in various genetic subtypes.
