Introduction
Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide, and its incidence continues to rise [
1]. Currently, surgery followed by radiotherapy or concurrent chemoradiotherapy is the treatment of choice for HNSCC [
1]. However, the prognosis of HNSCC is still unsatisfactory because of the high incidence of tumor recurrence and/or metastasis [
2]. Recurrent and/or metastatic (R/M) HNSCC generally has a poor clinical outcome and has therapeutic challenges [
3,
4]. Although immune checkpoint inhibitors (ICIs) demonstrated efficacy and safety for R/M HNSCC and have recently been approved by the FDA, application of ICI therapy remains limited to a subset of tumors with programmed death-ligand 1 (PD-L1) expression [
1,
4]. In the era of precision cancer therapy, a new therapeutic approach is urgently needed to provide additional treatment options and improve the clinical outcomes of R/M HNSCC patients.
An innovative strategy, known as tumor-infiltrating lymphocyte (TIL) therapy, is based on adoptive cell therapy (ACT) with the application of TILs for the treatment of cancer [
5,
6]. For TIL therapy, TILs are isolated from the resected tumor specimens, expanded in culture with interleukin-2 to achieve a clinically relevant number of cells, and subsequently infused back into the patients. TIL therapy presents several advantages for treating solid tumors: (1) TILs possess diverse T-cell receptor (TCR) repertoires capable of recognizing variable tumor antigens, effectively overcoming the intratumoral heterogeneity that often leads to resistance against targeted therapy. (2) TILs, primarily composed of effector memory T cells that have been stimulated by tumor antigens in vivo, harbor chemokine receptors on their cell surfaces and thereby have better tumor-homing ability. (3) To-date, limited reports exist regarding the off-target toxicity of TIL therapy, and the process of negative selection of TCRs within TILs may contribute to the safety of this therapeutic approach.
Several trials have demonstrated the clinical benefits of TIL therapy, mainly in metastatic melanoma [
7,
8], but also with other solid tumors, including breast cancer, cervical cancer, colorectal cancer, and non-small cell lung cancer [
9‐
12]. Also, a recent study reported that TIL therapy in combination with pembrolizumab achieved a high overall response rate in R/M HNSCC [
13]. The application of novel TIL therapy can address a clinical unmet need in R/M HNSCC patients who are refractory to conventional chemotherapy or immunotherapy.
Despite the promising results, the detailed process of TIL culture for preparation of the treatment and the clinicopathological characteristics associated with successful TIL culture of HNSCC specimens have not been documented previously. Here in this study, we cultured TILs from 47 specimens of primary tumors and metastatic lymph nodes (LNs) of HNSCC obtained from the oral cavity, oropharynx, and larynx of 33 patients. Next, we comprehensively investigated their clinicopathological characteristics, including stromal TIL (sTIL) percentage, human papillomavirus (HPV) infection status, and PD-L1 combined positive score (CPS), which could affect successful TIL expansion. Additionally, we analyzed the compositions of the expanded TIL using flow cytometry and performed a second round of TIL culture using a standard rapid expansion protocol (REP).
Discussion
In this study, we demonstrated that TILs can be obtained from HNSCC with varying clinicopathological features. The sTIL percentage was found to be significantly correlated with successful TIL expansion, and could serve as a useful indicator for estimating the yield of initial TIL culture. Additionally, our findings showed that after initial expansion, TILs are mostly composed of CD4 + T cells with a predominant effector memory cell subset.
HNSCC is a histologically, molecularly, and immunologically heterogeneous disease [
1]. Its pathophysiology varies according to its location, and its prognosis differs based on HPV infection status, sTIL percentage, and PD-L1 expression status [
19‐
21]. Furthermore, sTIL and PD-L1 expression are recognized as predictive factors of immune checkpoint inhibitor responses [
22]. We analyzed whether these prognostic and predictive clinicopathological parameters affect TIL expansion, but found that only sTIL percentage was a factor associated with successful TIL culture. Additionally, HPV infection, tumor location, sample type, and smoking history had no significant influence on the expanded TIL numbers. These findings suggest that TIL culture from HNSCC samples is not confined to specific subtypes and is feasible across a diverse spectrum of HNSCC. This also indicates the potential applicability of TIL therapy to HNSCCs with different biologic behaviors.
Few studies have described successful TIL expansion with delineation of their proportions from real world HNSCC samples. Zenga et al. investigated the functionality and tumor specificity of TILs obtained from surgically resected 31 HPV-negative HNSCCs [
23]. TIL culture was successful in 77% of patients, with the expanded tumor-specific T cells demonstrating anti-tumor effect in co-cultures with patient-matched malignant cells. The density of T cell subset revealed a predominance of CD4 + T cells, which was in line with our study. Knochelmann et al. conducted TIL expansion using 9 oral cavity squamous cell carcinomas [
24]. The expanded TILs exhibited varying populations of CD4 + and CD8 + T cells, depending on the individual patient, and demonstrated heterogeneous expression of inhibitory receptors.
We set the cutoff of essential TIL number per fragment after initial culture to be 0.8 × 10
5 cells based on our previous study using breast cancer samples [
18]. Excluding the contaminated 13 samples, the success rate of sufficient TIL expansion was 50% (17/34) of the samples. Although the success rate is slightly lower than previous reports of 60–91% across various cancer types [
25‐
28], our findings indicate that an adequate number of TILs can be obtained from HNSCC samples.
We found that microbial contamination is a notable issue for TIL culture of HNSCC samples. Thirteen of 47 (27.5%) samples had to be discarded due to contamination during initial TIL culture. It is known that a higher frequency of microbiological contamination is present in head and neck cancer samples compared to other organs [
29]. Antibiotics, such as penicillin, streptomycin, and gentamicin, have been utilized to prevent microbe contamination in head and neck cancer cell and organoid cultures [
29‐
31]. Similarly, TIL culture methods incorporate antimicrobial agents in its protocol in order to limit the risk of contamination [
32]. In our study, we added gentamicin to the media, but failed to prevent contamination in about one-fourth of the samples. Establishing a prophylactic or timely application of suitable antimicrobial agents is essential to enhance the success rate of TIL culture of HNSCC samples in future studies.
In our study, we observed that approximately 70% of cases had CD4 + T cells as a major lymphocyte subset after initial TIL expansions. Predominant CD4 + T cells in the TILs was reported for breast cancer (76%), with lower levels in gastrointestinal cancers (54%) and malignant melanoma (21%) [
33‐
36]. While CD8 + T cells are traditionally recognized as key anti-cancer effectors, the use of ACT of CD4 + TILs showed a remarkable regression of tumor burden in patients with metastatic tumors [
33,
35‐
37]. The elevated prevalence of CD4 + TILs in our study suggests that MHC-class II restricted neoantigens and reactive T cells are frequent in HNSCC. This finding also underscores the significant therapeutic potential of CD4 + TILs in ACT for HNSCC patients. We also noted that T
em had the highest proportion among the T cell subtypes in all cases. Considering the significance of effector memory cells in enhancing the therapeutic impact of ACT [
38‐
40], a high proportion of the T
em phenotype from expanded TILs from HNSCC tissue indicates its adequacy for successful TIL therapy.
Post-REP TILs were evaluated in 3 available cases, and the proportion of T cell subsets were similar after REP. One case showed noticeable shift in the CD8 + T cell subset from T
em to T
eff. The expected fold change following REP is over 1,000 since at least 1.0 × 10
10 cells per patient are known to be essential for use in ACT [
41,
42]. Although we were able to examine post-REP TILs and fold change in only three cases, two cases using G-Rex exceeded the fold change of 1,000 and one case using T-flask obtained slightly lower (960) results than the cutoff. Our findings suggest that sufficient number of TILs could be obtained and this indicates the potential viability of TIL therapy applications in HNSCC.
Our study has several limitations. First, the efficacy of the expanded TILs was not demonstrated through in vitro experiments or in vivo xenograft mouse models. The validation of the tumor reactivity of autologous TILs, and assessments of the long-term effects of expanded TILs, including in vitro phenotypic changes and in vivo TIL persistence after TIL infusion, requires further evaluation in future studies. Second, the REP process was available in a subset of cases after the initial two weeks of TIL expansion, preventing the observation of adequate fold changes in all cases. Although a shift in the CD8 + T cell subset was observed in one case, inferring the frequency of T cell subset changes and identifying the factors leading to this shift was difficult, given that not all cases underwent REP. Thirdly, p16, PD-L1 IHC and HPV ISH studies were only performed in a subset of cases, leading to an incomplete correlation analysis between these variables and successful TIL expansion. Despite these limitations, our study represents the comprehensive analysis of clinicopathological features associated with successful TIL expansion, conducted with large numbers of HNSCC tissue samples. Furthermore, we presented the detailed TIL phenotype following initial expansion and the subsequent REP processes, confirming that the expanded TILs are suitable for ACT, both quantitatively and qualitatively. We anticipate that our comprehensive study will contribute to expanding the current knowledge about TIL culture of HNSCC, and ultimately provide valuable insights for future research regarding TIL therapies.
In summary, we conducted TIL culture using HNSCC samples and analyzed their clinicopathological features. Despite antibiotic usage, HNSCC has an inherent susceptibility to contamination. Higher sTIL levels correlate with increased success rates and quantities of cultured TILs. The composition of the expanded TILs is predominantly CD4 + T cells with an effector memory phenotype.
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