Discussion
Cancer immunotherapy harnessing the immune system of patients to attack tumors has achieved promising clinical benefits in patients with advanced cancers. However, most tumors, especially solid tumors, remain largely unresponsive to current therapies(24), thus leaving still unmet demands in the clinic. TIL is a type of adoptive cellular therapy with distinctive advantages in the treatment of solid tumors, including but not limited to its diverse TCR clones capable of recognizing heterogeneous tumor antigens, its superior homing ability to the tumor site and low off-target toxicity. So far, TIL therapy has shown promising clinical results in several types of solid tumors, such as metastatic melanoma and cervical cancer. Vulvar cancer is a serious gynecological malignancy with increasing incidence in the last few decades. Effective therapeutic strategy is still an urgent need, especially for advanced vulvar cancer. Lymphocyte infiltration in the tumor site, together with its positive association with overall and recurrence-free survival, provides a solid theoretical and practical foundation for applying TIL therapy to vulvar cancer patients. Therefore, we explored the feasibility of isolating and expanding TILs in vitro for vulvar cancer.
We used a manufacturing process with optimized culture conditions based on previous TIL studies, and we have successfully produced CC TILs displaying desirable properties for therapeutic uses. In this study, we showed that VC TILs can be amplified from both in situ tumor samples and lymph node metastases. Even for samples with a low percentage of T cells in the preREP stage, it should be noted that VC TILs can still be successfully amplified in subsequent expansion with around 95% CD3+ T cells in the final product. Although T cells in the tumor environment usually showed an exhausted phenotype with attenuated functions, the high CD28 and low PD-1 expression on VC and CC TILs after REP indicated that TILs may be able to mount antitumor activities in response to tumor antigens. Besides, the percentage of Tregs in the final product is very low (below 5% percent).
The uCDR3 number and diversity analysis revealed the expansion and enrichment of certain TCR clonotypes in the TIL product. The relative abundance of Top10 and Top100 ranked uCDR3 in TILs is much higher than that in the PBMC samples. Most clonotypes were detected only in one sample, and most shared clonotypes were clones with low frequency, suggesting that most of the TCR repertoires in TIL product are patient-specific. As reported by Bobisse et al., this patient-specific TCR can be identified even in some patients with tumors harboring a relatively low mutational load such as ovarian cancer. The patient-specific TCR may be generated by differential expression of tumor antigens, such as the expression of patient-specific neoantigens(25) or patient-specific responding process against autologous tumor(26).
Functional analysis showed that our TIL product, especially VC TILs, can upregulate CD107a and produce IFN-γ and TNF-α upon tumor or antibody stimulation and mediate cytotoxicity to HeLa cells. The phenotype and function of our TIL product is similar to those of TIL from melanoma as reported before(27), indicating that our cell production process is able to enrich effector T cells with antitumor responses. A major challenge to evaluate the tumor specificity of TIL is the availability of autologous tumor samples. Currently, we are developing methods to culture patient-derived cancer cells (PDCs) and organoids from vulvar cancer samples in vitro, which could be used to test the specific responses of VC TILs to vulvar tumor antigens in the future.
Compared with cervical cancer, the number of cells obtained from vulvar cancer samples after preREP is similar, but the average amplification rate during the REP stage is twice that of cervical cancer, demonstrating the good amplification capacity of VC TILs in culture. VC and CC TILs have similar T and NK cell composition, but the expression of activation marker CD28 and stem cell marker TCF1 is higher on VC TILs. Moreover, VC TILs showed better cytokine secretion and tumor-killing capability compared with that of CC TILs, indicating better effector function of VC TILs. Additional studies, such as profiling of T cell receptor repertoire and analysis of TCR specificity to shared tumor antigens or neoantigens, will facilitate a better understanding of VC TILs.
Taken together, we have demonstrated in this report that TILs can be successfully harvested and expanded from tumor and lymph nodes of vulvar cancer and characterized by an activation phenotype with stemness and good effector function in response to tumor or antibody stimulation. Therefore, as a promising new therapeutic strategy for vulvar tumor, the clinical application of TIL therapy should be further investigated for vulvar cancer patients.