*2.2.1 Selection of gating markers*

CD19-CAR-T is the most used immunotherapy, and MRD detection after CD19- CAR-T in B-ALL is also the focus of researchers. Because all or part of CD19 expression is lost or weak in 7.4–62.5% of B-cell malignancies after CD19-CAR-T treatment, CD19 gating cannot be the only rough B gating marker [3, 19, 20, 36, 37]. As for the selection of alternative gate markers, some laboratories chose CD22+ and/or CD24+/CD66b− [19], while we and some laboratories chose multiparameter synchronous gate setting by cytoplasmatic (c) CD79a combined with CD19 and lymphoblast markers [1–3, 20]. The reasons are as follows: 10–20% of B-ALL cases do not express CD24, especially in cases with MLL-related fusion genes [3, 19]. After the failure of CD19-CAR-T therapy, the choice of CD19/CD22 bi-specific CAR-T or CD22-CAR-T, coupled with the weak

*Recent Developments in Application of Multiparametric Flow Cytometry in CAR-T… DOI: http://dx.doi.org/10.5772/intechopen.108836*

expression of CD22 in B lymphoblasts, all determine that this is not an ideal rough B gating marker [3, 38]. Although studies have found that CD22dim/-MRD did not appear after CD22-CAR-T treatment [37], further studies are needed because of the small number of cases. Therefore, we used cCD79a as the main B marker in MRD detection after CD19-CAR-T or CD19/CD22 combined CAR-T treatment, and achieved good clinical evaluation results [3]. Besides the biggest advantage of cCD79a panel is that we can use the same panel for all MRD detection after any B marker CAR-T treatment in the future because it is an intracellular antigen not for CAR-T target [51].

The same idea was adopted in CD7-CAR-T for T-ALL, other lineage markers are added in the MRD panel, such as cCD3, CD5, and CD2, as well as blast markers, such as TdT, CD34, CD99bri, and CD1a [4].

#### *2.2.2 Changes in phenotype and observation methods*

When selecting cCD79a combined panel to detect MRD after CD19 and/or CD22-CAR-T therapy in B-ALL, the following should be noted: (1) the expression intensity of some antigens may change after the intracellular operation. (2) Recognize the immunophenotype of normal CD19-negative hematogones, most of which are the earliest stage of CD34+ B progenitor cells. They are different from the CD19 positive counterpart in that weaker CD10 expression and larger SSC, and may be misdiagnosed as MRD; (3) in fact, CD19-negative hematogones exist in normal BM but are ignored, because most of them are rare and CD19 is routinely used for gating. A significant decrease in the proportion of CD19 positive B progenitors with CD19- CAR-T results in a relative increase in the proportion of CD19-negative B progenitors, which together with changes in gate setting and most importantly the focus on CD19 negative B cells, made this population prominent [3, 19, 20].

Given that the heterogeneity of tumor cells is obvious after CAR-T therapy, even with the use of alternative gate markers, detection will be difficult, not to say the rare use of cytoplasmatic markers in cerebrospinal fluid (CSF) specimens. After CAR-T, multiple gates by multiple markers will be helpful in MRD detection by MFC. For example, SSC/cCD79a, SSC/CD19, SSC/CD10,SSC/CD34, and SSC/TdT in B-ALL, CD99bri/SSC, cCD3/CD45dim, CD5/CD45dim, CD34and/or CD1a/SSC, and TdT/ SSC in T-ALL, CD229/CD45dim and CD138/CD45dim in MM. CD45dim/CD10 positive and/or CD34 positive and/or CD38 positive cells are not present in normal CSF samples, so CD34 and/or CD10 and/or CD38 combined with CD45 gate method was used for identification of CD19-negative B-ALL MRD. See **Figure 1**.

In addition, special attention should be paid to myeloid conversion after CAR-T in ALL patients [66]. After CD7-CAR-T cell therapy, MRD detection may be affected due to interference of CAR-T cells. In the case of targeted therapy with CD38, CD123, and other markers of progenitors, attention should be paid to the phenotypic changes of the normal blast caused by the loss of these markers during MRD detection.

#### **2.3 Kinetics of target antigen recovery**

After injection, CAR-T cells expand more than 10,000 times *in vivo*. The number of amplifications and duration of presence *in vivo* largely determine the efficacy and side effects of CAR-T. The recovery of cells expressing the target antigen can indirectly reflect the recovery kinetics of CAR-T [3, 19, 20, 34, 35, 67]. Target antigen recovery is not evaluated alone, generally detected as part of MRD or immunoassay [3, 19, 20, 34, 35, 67].

#### **Figure 1.**

*BM from one B-ALL patient for MRD detection by MFC, the title of each dot plot was the gate where the subplot showed the cell. A, before CD19-CAR-T immunotherapy. The cells in red color were malignant B lymphoblasts with 7.66% of live cells. They were positive for CD19, CD10, CD81, TdT, and CD34part, negative for CD45. B, 30d after CD19-CAR-T. No CD10 or cytoplasmatic (c)CD79a-positive cells were observed. C, relapsed 4 months after CD19-CAR-T. the blast was 3.22% of live cells and consisted of four subsets. The cells in red color were the major subset, positive for CD10, CD38, CD81, TdT, and cCD79a part, negative for CD45, CD34, and CD19. The cells in dark green color were the minor subset 1, positive for CD10, CD19, CD38, and cCD79a, negative for CD45and CD34, not known for CD81 and TdT. The cells in dark brown color were the minor subset 2, positive for CD10, CD38, and CD81, negative for CD45, CD34, TdT, cCD79a, and CD19. The cells in sapphire color were the minor subset 3, positive for CD10, CD34, TdT, and cCD79a, negative for CD38, CD81, CD45, and CD19. Normal B cells (fluorescent green color) and plasma cells (magenta color) were all CD19 partially positive.*

The recovery dynamics decide the choice of detection time points. Besides CAR-T products, the efficacy depends largely on *in vivo* CAR-T cell proliferation. The target expression cells begin to recover 1–3 months after treatment, and commonly BM, CSF, or other involved sites are selected as specimens. BM should ideally be tested once a month for the first 6 months and at least once every 3 months for the first 6 to 12 months [1–4, 19–22, 32]. Generally, PB or CSF of patients is selected as specimens for CAR-T cell assay and cytokines detection. Before infusion on day 0, and after infusion on other time points, PB-related assays are frequent in the first month, such as 4 d, 7 d (optional 10 d or 11 d), 14 d, 28 d, 2 m, 3 m, and 6 m [1–4, 32–37]. The detection of CAR-expressing cells can be stopped after the detected values are lower than the limit of detection (LOD) for two consecutive times. MRD and CAR-T cell detection of CSF are performed at the appropriate time point. PB is selected for immune assay, at least once a month for the first 6 months, and once every 3–6 months after 6 months [32–37].

Taking CD19-CAR-T treatment for B-ALL as an example, the duration of B-cell deficiency varies greatly among different studies, generally lasting 2–3 months. The recovery of B cells in PB is a signal of CD19-CAR-T dysfunction. In pediatric B-ALL, recovery of B cells at 3 months suggests a high risk of relapse, possibly due to CAR-T depletion [36, 37, 40–43].
