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.