1. CD19 expression in DLBCL patients treated with CART.
In this study, one DLBCL patient who received CD19 CART therapy and obtained CR, was observed to be CD19 negative in her pleural effusion lymphoma cells at initial diagnostic IHC assay (Fig. 1A). To further investigate these results, the sample was evaluated using 7 IHC antibodies targeting different clones, and found that the sample was positive in antibodies OTI1F9 and 3B10 detection, low positive in antibodies LE-CD19 and D4V4B detection, and negative in antibodies OTI1F2, EPR5906 and SP291 detection (Fig. 1B). To determine the results, we evaluated CD19 expression in the pleural effusion cells by FCM, which identified that CD19 expression in the lymphoma cells was positive (Fig. 1C). To further investigate the relevance of CD19 expression and patients’ response to CART therapy, IHC assays of CD19 expression in other 7 DLBCL patients treated with CD19 CART therapy were performed. The patients’ characteristics were showed in Table 1. 6 out of 8 DLBCL patients who received CD19 CART therapy obtained complete or partial remission (response rate, 75%). It is observed that 6 DLBCL tumors were identified as CD19-negative by at least one antibody. Among the 6 patients, 5 objective responses were seen (response rate, 83.3%). 2-year progression-free survival rate 38% (95% CI 4.68%-71.32%), 2-year overall survival 50% (95% CI 4.68%-71.325). As the data were too small to analyze IHC staining bias, additional 90 DLBCL sample were reviewed by IHC with 7 antibodies.
2. CD19 expression in DLBCL tissues varies with different IHC antibodies.
To compare the performance of different CD19 IHC antibodies, we tested 90 formalin-fixed and paraffin-embedded (FFPE) DLBCL samples with 7 CD19 IHC antibodies by uIHC (Fig. 2A). 68 DLBCL qualified samples were involved in the analysis, samples showed different CD19 expression status among different antibodies. 57 out of 68 (83.8%) samples were assessed as CD19 negative by at least one antibody. The results are displayed by a heat map (Fig. 2B) The percentage of positive cells were found to differ when detected by different antibodies. Antibody LE-CD19 had highest percent of CD19 positive cells, 1F2, D4V4B, 1F9 and 3B10 had moderate percent of CD19 positive cells, and EPR5906 and SP291 had the lowest percent of CD19 positive cells (P < 0.05) (Fig. 2C). CD19 IHC showed a poor consistency among different antibodies.
3. RT-PCR, Western blotting and IHC analysis of CD19 mRNA and CD19 protein expression in human DLBCL cell lines.
As CD19 IHC showed low concordance among antibodies, to find out a method with good consistency, we compared IHC and FCM in 6 DLBCL cell lines, including TMD-8, OCI-LY1, HBL-1, SUDHL-2, SUDHL-4 and DB. CD19 mRNA levels in the 6 cell lines was determined by RT-PCR, and all of these cell lines were detected CD19 mRNA. TMD-8 (0.201%±0.019%) and SUDHL-4 (0.246%±0.016%) showed high levels of CD19 mRNA, OCI-LY1(0.142%±0.012%), HBL-1(0.0605 ± 0.004%) and SUDHL-2 (0.121%±0.002%) showed moderate levels of CD19 mRNA, while DB (3.65×10− 5%±1.195×10− 5%) cell line showed the lowest level of CD19 mRNA (Fig. 3A). As the protein level, CD19 expression was detected by Western Blotting analysis(Fig. 3B). TMD-8 (41.6235%±2.2582%), OCI-LY1 (42.1468%±0.0683%) and SUDHL-2 (38.1858%±2.4609%) showed high CD19 protein expression, HBL-1 (30.2584%±3.4691%) and SUDHL-4 (28.6253%±2.5132%) showed moderate CD19 protein expression, and DB cell line showed undetectable CD19 expression༈Fig. 3C༉. IHC assay with 7 different CD19 antibodies was performed in the 6 cell lines and results were displayed by a heat map (Fig. 3D). CD19 expression in cell lines TMD-8, SUDHL-2 and SUDHL-4 were identically strong positive (Fig. 3E,H-I). Cell line OCI-LY1 showed CD19 positive in antibodies OTI1F9, 3B10, EPR5906 and SP291, while showed CD19-negative in antibodies LE-CD19 and D4V4B (Fig. 3F). Cell line HBL-1 showed low positive in antibodies LE-CD19 and OTI1F2, and strong positive in remain antibodies (Fig. 3G). Cell line DB showed strong positive in antibodies 3B10 and low positive in antibodies LE-CD19 while negative in the remaining antibodies (Fig. 3J). The discrepancy of CD19 expression in cell lines OCI-LY1, HBL-1 and DB indicated that CD19 expression could be false negative due to single assay with one antibody, emphasizing the importance of developing a complementary method to assess the CD19 expression level following CART therapy.
4. FCM had high sensitivity and good concordance for CD19 detection.
Unlike IHC assay which was used FFPE samples, FCM was applied in living cells. CD19 expression was detected using FCM with 3 antibodies targeting different CD19 epitopes of SJ25-C1, HIB19 and 4G7. All the 6 cell lines tested CD19 positive expression. For the SJ25-C1 and HIB19 antibodies, the TMD-8, SUDHL-2 and SUDHL-4 cell lines were observed high CD19-Mean fluorescence intensity (MFI), OCI-LY1 and HBL-1 cell lines showed moderate CD19-MFI and cell line DB showed low CD19-MFI (Fig. 4A-B). For the 4G7 antibody, TMD-8 cell line was observed high CD19-MFI, while OCI-LY1 and HBL-1, SUDHL-2 and SUDHL-4 cell lines showed moderate CD19-MFI and DB cell line showed low CD19-MFI (Fig. 4C). A positive correlation was observed between CD19 expression level determined by Western blotting assay and that of CD19-MFI measured by FCM with the HIB19 antibody (R2 = 0.8125, P = 0.0137) (Fig. 4D). FCM and Western blotting showed good concordance in measuring the expression of CD19 expression.