Trastuzumab is currently used in combination with cytotoxic chemotherapy (Heinemann et al. 2011; Harbeck et al. 2016) or a trastuzumab drug conjugate (TDC) for the treatment of HER2-positive BC (von et al. 2023). Trastuzumab is believed to have a diverse and complex mechanism of action. As trastuzumab is an HER2-targeted monoclonal antibody that directs growth inhibition leading to apoptosis of tumor cells, it has been postulated that antibody-dependent cytotoxicity may play an important role in the mechanism of action of this drug (Arnould et al. 2006; Tóth et al. 2016).
In the current study, we investigated the association between immune genetic polymorphism and trastuzumab-mediated cytotoxicity in vitro. To the best of our knowledge, we are the first to explore the appropriate target BC cell, E/T ratio, and antibody conc. In our experimental system, BT474 cell lines showed high reactivity with trastuzumab compared to MCF7, and then the use of BT474 as target cell is beneficial in ensuring a sufficient cytotoxicity result for the next study and analysis since the coculture time, E/T ratio and antibody concentration requirements are not too high.
In contrast to some studies that showed that cytotoxicity was lower in BC patients compared to healthy donors (Petricevic et al. 2013), our data indicate that trastuzumab stimulates cytotoxicity in BC patients as effectively as in healthy donors. However, for BC patients we observed higher basal cytotoxicity activity compared to healthy donors. These difference results may be due to the fact that PBMC in these studies comes from patients who have received trastuzumab treatment, and the treatment could potentially lead to an inhibition of cytotoxicity. The finding that cytotoxicity levels and TNF releases in the trastuzumab treatment group were significantly higher than in the basal group supports the postulate of cytotoxicity in the action of trastuzumab. At the same time, we observed a wide interindividual variability in trastuzumab-mediated cytotoxicity in 148 healthy donors. Some donors had up to 80%, while some others had a low of 20%. In fact, this interindividual variability has been reflected in many of previous studies (Petricevic et al. 2013; Boero et al. 2013).
Immune function genes in the antibody-dependent cytotoxicity pathway have previously been shown to predict the clinical outcome of some immunotherapies or therapies in immune disease (Dávila-Fajardo et al. 2015; Perez et al. 2015; Kjersem et al. 2014; Li et al. 2014; Dong et al. 2014; Chai et al. 2012). And the wide interindividual variability of cytotoxicity levels among individuals that might be related to genetic polymorphisms of some immune function genes. In this study, we investigated in vitro the cytotoxicity elicited by PBMCs from 148 healthy donors and correlated its extent with these immune function gene genotypes. We found that the CD247 rs16859030 T genotypes could elicit stronger cytotoxicity compared to the CD247 rs16859030 CC genotype, which trend is also maintained in the low-concentration trastuzumab group. To explain why there is no statistical significance at the low concentration of trastuzumab, it is conceivable that the overall effect of cytotoxicity is so low that it cannot be distinguished by genotype. Our findings may be consistent with the results of the genomic analysis, which suggested that CD247 was related to the increased relapse-free survival (RFS) of patients treated with trastuzumab (Perez et al. 2015). Rs16859030 was reported to be significantly associated with systemic lupus erythematosus (SLE) in African ancestry, but not with SLE in the other three populations (Martins et al. 2015). Furthermore, the anti-inflammatory cytokine IL10 was released more in the TT genotype than in the CC genotype. Increased IL10 release can occur upon stimulation of Fc receptor pathways (Saraiva et al. 2010), which is in line with cytotoxicity results.
Although our data also observed a significant P value between ZAP70 rs13420683 and basal cytotoxicity, the cytotoxicity of this group was almost too low. Only one study reported a genetic association between ZAP70 rs13420683 and inflammatory disease (Bouzid et al. 2013). The addition of our sample size is limited, so the difference between basal cytotoxicity was not considered in the analysis of trastuzumab-mediated cytotoxicity.
We chose PBMCs to evaluate the overall cytolytic activity in order to better mimic the cytotoxicity condition that may occur in vivo. Considering that NK is the main effector cell within PBMC that can engage trastuzumab, we also evaluate the cytolytic activity of NK cells. As expected, we found that NK cells could elicit a stronger cytotoxicity compared to PBMC.
Although our entire study cohort consisted of 148 healthy donors and was sufficient for our candidate SNPs, the NK cell and patient PBMC results were relatively small. We are further evaluating whether these genetic polymorphisms correlate with cytotoxicity in PBMC of BC patients or NK cells, but we cannot address this point in the present study. The exact molecular mechanism of CD247 rs16859030 affects cytotoxicity mediated by trastuzumab remains unclear and should be further investigated.
In conclusion, our results suggest that trastuzumab-mediated cytotoxicity is similar in BC patients compared to healthy donors. Furthermore, the results of our work suggest that the CD247 rs16859030 polymorphism affects trastuzumab-mediated cytotoxicity and IL10 release in in vitro assays. Our findings have potential clinical implications that could be useful for the selection of HER2-positive patients who are the best candidates for trastuzumab treatment.
Table 1
Detailed information of candidate SNPs.
Gene | Chr | SNP | Position | Call rate | Allele | MAF | P-value |
Bas | T3 | T6 |
CD247 | 1 | rs864537 | intron | 98.66% | A > G | 0.219 | 0.167 | 0.741 | 0.367 |
| | rs1214611 | intron | 99.33% | G > A | 0.429 | 0.417 | 0.437 | 0.976 |
| | rs12144621 | intron | 98.66% | C > G | 0.429 | 0.496 | 0.772 | 0.632 |
| | rs858545 | intron | 100% | C > A | 0.3 | 0.209 | 0.366 | 0.501 |
| | rs2949655 | intron | 99.33% | G > A | 0.486 | 0.556 | 0.933 | 0.676 |
| | rs1723015 | intron | 99.33% | T > C | 0.443 | 0.521 | 0.733 | 0.969 |
| | rs16859030 | intron | 99.33% | C > T | 0.21 | 0.415 | 0.139 | 0.012* |
| | rs1052230 | UTR | 99.33% | G > C | 0.219 | 0.871 | 0.912 | 0.629 |
ZAP-70 | 2 | rs13420683 | intron | 100% | C > A | 0.224 | 0.03* | 0.246 | 0.502 |
| | rs1020396 | intron | 98.66% | C > T | 0.257 | 0.29 | 0.563 | 0.56 |
| | rs2278699 | UTR | 99.33% | G > C | 0.157 | 0.062 | 0.259 | 0.543 |
FCGR2A | 1 | rs1801274 | exon | 99.33% | C > G | 0.3 | 0.637 | 0.962 | 0.679 |
FCGR2C | 1 | rs114945036 | intron | 98.66% | T > C | 0.1714 | 0.57 | 0.186 | 0.09 |
FCGR3A | 1 | rs396991 | intron | 98.66% | A > C | 0.2739 | 0.205 | 0.805 | 0.965 |
TNF | 6 | rs1799964 | promoter | 98.66% | T > C | 0.1905 | 0.158 | 0.723 | 0.582 |
IFNG | 12 | rs2430561 | intron | 100% | T > A | 0.1619 | 0.087 | 0.961 | 0.354 |
MAF, minor allele frequency; Bas, basal; T3, trastuzumab 103 pg/ml; T6, trastuzumab 106 pg/ml; ZAP70, zeta chain of T-cell receptor associated protein kinase 70; FCGR2A, Fc fragment of IgG receptor IIa; FCGR2C, Fc fragment of IgG receptor IIc; FCGR3A, Fc fragment of IgG receptor IIIa; TNF, tumor necrosis factor; IFNG, interferon gamma.