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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 2  |  Issue : 1  |  Page : 19-24

Evaluation of tumor-infiltrating lymphocytes as a prognostic indicator of head-and-neck squamous cell carcinomas: A prospective study


Department of Head and Neck Surgical Oncology, HCG Cancer Center, Bengaluru, Karnataka, India

Date of Submission17-Nov-2021
Date of Decision15-Feb-2022
Date of Acceptance18-Feb-2022
Date of Web Publication03-May-2022

Correspondence Address:
Prof. (Dr.) U S Vishal Rao
Regional Director, Head and Neck Surgical Oncology and Robotic Surgery, Department of Head and Neck Oncology, HCG Cancer Center, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpo.jpo_4_21

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  Abstract 


Introduction: The incidence of Head-and-Neck Squamous Cell Carcinomas (HNSCC) is on the rise in our country and worldwide, with a worsening prognosis. The abundance of tumor-infiltrating lymphocytes (TILs) is evolving to be a novel prognostic indicator in assessing treatment response of HNSCC. TILs mainly comprise T lymphocytes, which migrate from blood into the tumor as part of the body's immune response. In this context, targeting the tumor microenvironment with the help of immunotherapy may present a potential approach for better cure of head-and-neck cancers. In the present study, we evaluated the potential TILs parameters which can be used in clinical practice to predict treatment outcomes in HNSCC.
Materials and Methods: This is a prospective observational study of 41 patients conducted at a tertiary cancer center between September 2019 and September 2020. All patients with biopsy proven, primary/recurrent HNSCC, without distant metastasis, and complete clinicopathological data were included in the study. Patients with a nonsquamous cell carcinoma, patients who underwent upfront chemotherapy or chemoradiotherapy were excluded from the study. Patients who had follow-up of <1 year or lost to follow-up were excluded from the study. Parameters analyzed include mean age; percentage of cluster of differentiation 3 (CD3) cells; CD4, CD8, and FOXP3cell counts; distribution of CD57 and CD3/FOXP3 ratio.
Results: A total number of 19 patients were grouped under the nonrecurrence TILs and 22 patients under recurrent TILs category. Majority of the patients (90.2%) had oral cavity squamous cell carcinoma. 68.42% of nonrecurrent cases and 36.36% of recurrent cases were found to be harboring Hot tumors. Using odds ratio, it was noted that the odds of having Hot tumor is 3.79 times (95% confidence interval: [1.03,13.91]) higher in nonrecurrent TILs than in recurrent TILs group. CD3 cell count was higher in nonrecurrent cases (54.74 ± 18.37) than in recurrent cases (42.73 ± 16.09) with P = 0.0157. Using two tailed t-test, it was noted that the mean of CD4, CD8, and FOXP3 is not significantly different between nonrecurrent and recurrent tumor TILs groups.
Conclusion: To summarize, we have shown that the CD3/FOXP3 cell ratio, rather than the individual proportion of TILs, is a significant prognostic indicator in HNSCC. It's also been shown that people with Cold tumors have a higher risk of recurrence than those with Hot tumors. Our findings confirm the importance of host immunity in prognosis and suggest that the degree of immune cell infiltration in the tumor microenvironment is a major independent prognostic factor that merits further investigation as a potentially valuable biomarker in HNSCC patients.

Keywords: Cluster of differentiation 3 and FOXP3 in cancer, head-and-neck squamous cell carcinomas, prognostic indicators of head-and-neck squamous cell carcinomas, tumor-infiltrating lymphocytes


How to cite this article:
Reddy N A, Joshna B M, George A, Indu V P, Thakur S, Subash A, Kudpaje A, Vishal Rao U S. Evaluation of tumor-infiltrating lymphocytes as a prognostic indicator of head-and-neck squamous cell carcinomas: A prospective study. J Precis Oncol 2022;2:19-24

How to cite this URL:
Reddy N A, Joshna B M, George A, Indu V P, Thakur S, Subash A, Kudpaje A, Vishal Rao U S. Evaluation of tumor-infiltrating lymphocytes as a prognostic indicator of head-and-neck squamous cell carcinomas: A prospective study. J Precis Oncol [serial online] 2022 [cited 2022 Sep 30];2:19-24. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/1/19/344537




  Introduction Top


The incidence of Head-and-Neck Squamous Cell Carcinomas (HNSCC) is on the raise in our country and worldwide, with a worsening prognosis. It is the sixth most common carcinoma in the world, accounting for about five lakh new cases each year.[1] Immune cell infiltration is a prominent pathologic feature of HNSCC. The abundance of tumor-infiltrating lymphocytes (TILs) is evolving to be a novel prognostic indicator in assessing treatment response of HNSCC. Classification of tumors based on the phenotypes of TILs is rapidly emerging as a good prognostic tool to guide treatment.

TILs mainly comprise T lymphocytes, which migrate from blood into the tumor as part of the body's immune response. TILs are found in the tumor stroma and within the tumor itself. They are a part of the larger category of “tumor-infiltrating immune cells,” which consist of mononuclear and polymorphonuclear immune cells (i.e., T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mastcells, eosinophils, basophils in variable proportions.[2],[3],[4] Their abundance varies with the type and stage of tumor and can relate to disease prognosis. Their functions can dynamically change in response to anticancer therapy and through tumor progression.[5],[6]

Despite improvements in the treatment for HNSCC, the 5-year survival and recurrence rates remain poor.[7] TILs have been analyzed in a range of cancers, demonstrating their value as a supplement to Tumor, Node, Metastasis staging. To explore the relationship between TILs and tumors, formal approaches to evaluate TILs exist in many fields with the exception of HNSCC.[8],[9] The research on TILs in HNSCC gives minimal evidence of their prognostic role.[10] Using immunotherapy to target the tumor microenvironment might be a promising strategy for a better cure of head-and-neck malignancies. In the present study, we evaluated the potential TILs parameters which can be used in clinical practice to predict treatment outcomes in HNSCC.


  Materials and Methods Top


This study was done after obtaining the ethical committee approval from the Institutional Review Board of the HCG hospital. This is a prospective observational study of 41 patients conducted at a tertiary cancer center between September 2019 and September 2020. All patients with biopsy proven, primary/recurrent HNSCC, without distant metastasis, and complete clinicopathological data were included in the study. Patients with a nonsquamous cell carcinoma, patients who underwent upfront chemotherapy or chemoradiotherapy were excluded from the study. Patients who had follow-up of <1 year or lost to follow-up were excluded from the study. Among these patients, 25 patients were newly diagnosed cases and 16 patients had presented with recurrent HNSCC. Out of the 25 newly diagnosed patients, 6 patients were found to have recurrence during the period of follow-up in this study. All the 16 patients with recurrent carcinoma in this study had undergone surgery as the primary modality of treatment for the recurrence. Based on disease recurrence, the data were segregated and analyzed as recurrence and nonrecurrence tumour TILs.

All the histopathology samples were subjected to detailed analysis. Parameters analyzed include mean age; percentage of cluster of differentiation 3 (CD3) cells; CD4, CD8, and FOXP3cell counts; distribution of CD57 and CD3/FOXP3 ratio. Based on the percentage of CD3 cells, patients could be divided into those harboring Hot and Cold tumors. CD3 cell count of <50% is taken as Cold tumors and more than 50% is considered as Hot tumors. Immunohistochemistry staining was done for the subpopulation of TILS and clones and detection kits were used as staining for TILs [Table 1] and [Table 2].
Table 1: Immunohistochemistry staining for tumor infiltrating lymphocytes

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Table 2: Kits used for staining of tumor infiltrating lymphocytes

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Statistical analysis

Data analysis was done using R i386 4.0.3. Categorical data represented by frequency and percentage and continuous data represented using mean ± standard deviation. Unpaired continuous data were compared using t-test/Mann–Whitney U-test and categorical data were compared using Chi-square test/Fisher exact test. P < 0.05 is considered as significant.


  Results Top


A total of 41 cases with HNSCC were considered for the study, with 19 patients falling under the nonrecurrence tumor TILs and 22 patients under recurrent tumor TILs category. Majority of the patients (90.2%) had oral cavity squamous cell carcinoma. The summary of the data is provided in [Table 3].
Table 3: Patient data

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It was observed that the mean age of nonrecurrent and recurrent TILs cases was 58.32 years and 54.09 years, respectively [Figure 1]. Majority of the patients in both the groups were found to be males-14 in nonrecurrent group and 17 in the recurrent group. Using Fisher's exact test, it was seen that recurrent status is not significantly associated with gender. Using t-test, it could be deduced that the mean age of subjects is not significantly different between nonrecurrent and recurrent tumor TILs groups.
Figure 1: Distribution of subjects according to age

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Based on the percentage of CD3 cells, 68.42% of nonrecurrent cases and 36.36% of recurrent cases were found to be harboring Hot tumors. 31.58% of nonrecurrent cases and 63.64% of recurrent patients were found to have Cold tumor. Using Chi-square test, it is noted that there is a significant association of recurrence with Hot and Cold tumors.

[Figure 2] shows majority of the nonrecurrent patients harbor Hot tumor and majority of recurrent patients harbor Cold tumor. Using odds ratio, it is concluded that the odds of having Hot tumor is 3.79 times (95% confidence interval: [1.03, 13.91]) higher in nonrecurrent TILs than in recurrent TILs group. CD3 cell count was found to be higher in nonrecurrent cases (54.74 ± 18.37) than in recurrent cases (42.73 ± 16.09) with P = 0.0157.
Figure 2: Distribution of subjects by nature of tumor in both groups

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Using two tailed t-test, it was noted that the mean of CD4, CD8, and FOXP3 is not significantly different between nonrecurrent and recurrent TILs groups [Table 4]. Analysis with one-tailed t-test showed that the mean of CD3 (in %) is significantly higher in nonrecurrent cases than recurrent TILs cases [Figure 3].
Figure 3: Visualization of comparison of different clinical parameters between recurrent and nonrecurrent cases (part 1)

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Table 4: Comparison of different factors

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The two-tailed Mann–Whitney U-test has shown that the distribution of CD57 (in %) is not significantly different between nonrecurrent and recurrent TILs cases. On using one-tailed Mann–Whitney U-test, it was concluded that the median of CD3/FOXP3 ratio is less for recurrent cases than nonrecurrent TILs cases [Figure 4]. This was found to be statistically significant with P = 0.0432.
Figure 4: Visualization of comparison of different clinical parameter between recurrent and nonrecurrent cases (part 2)

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The overall survival was calculated for the nonrecurrent TILs group [Figure 5]. The Kaplan–Meir curve shows 1-year survival rate of 85.3%.
Figure 5: Overall survival for nonrecurrent patient group

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  Discussion Top


The search for biomarkers that define biologic behavior in HNSCC has intensified with newer modalities paving their way into management. The main aim of this study was to evaluate if TILs were a strong prognostic factor for patients with HNSCC. This was studied prior by a number of investigators and the research extended to include T lymphocytes subsets. CD8 infiltrates that are functionally characterized as cytotoxic/suppressor T lymphocytes have most often been associated with favorable prognosis,[11],[12],[13] whereas FoxP3 infiltrates have been associated with either improved[14],[15] or worse outcomes.[16] CD4 infiltrates less commonly have been associated with favorable outcomes, even though FoxP3 cells are considered a subset of CD4 cells.[17]

Majority of the patients in this study (90.2%) had oral cavity squamous cell carcinoma. The remainder patients had carcinoma oropharynx and larynx. Patients were compared and studied under recurrent and nonrecurrent tumor TILs separately. In this study, a total of 19 patients fell under the nonrecurrent TILs and 22 patients under recurrent TILs category. It was observed that the mean age of nonrecurrent and recurrent TILs cases was 58.32 years and 54.09 years, respectively. Majority of the patients in both the groups were found to be male gender-14 in nonrecurrent group and 17 in the recurrent group. Using Fisher's exact test, it was seen that recurrent status is not significantly associated with gender (P > 0.99). Using t-test, it was seen that the mean age of subjects is not significantly different between nonrecurrent and recurrent TILs groups (P = 0.22).

Based on the percentage of CD3 cells, patients could be divided into tumors harboring Hot and Cold tumors. In our study, we found that majority of the nonrecurrent patients harbored Hot tumor and majority of recurrent patients had Cold tumor. CD3 cell count was found to be higher in nonrecurrent cases (54.74 ± 18.37) than in recurrent cases (42.73 ± 16.09) with P < 0.05. A retrospective study of 200 HNSCC patients done by Qiaoshi Xu et al. identified 70% as a valid threshold for TILs assessment, with TILs levels higher than 70% associated with a better prognosis. They showed that TILs level may serve as an independent predictor for DFS in HNSCC patients. They, however, did not study the effect of T lymphocyte subsets such as CD3+, CD8+, and CD45+ cells.

In our study, we have analyzed each subset in detail and studied its correlation to recurrence. Using two-tailed t-test, it was noted that the mean of CD4, CD8, and FOXP3 was not significantly different between nonrecurrent and recurrent tumor TILs groups. Analysis with one-tailed t-test showed that the mean of CD3 (in %) is significantly higher in nonrecurrent cases than recurrent TILs cases. The two-tailed Mann–Whitney U-test has shown that the distribution of CD57 (in %) is not significantly different between nonrecurrent and recurrent TILs cases.

FOXP3 values in nonrecurrent cases were found to be 10.42 ± 6.13 and in recurrent cases was 13 ± 8.76, with no significant correlation between the two groups (P = 0.2888). Analysis with one-tailed Mann–Whitney U-test concluded that the median of CD3/FOXP3 ratio is significantly less for recurrent cases than nonrecurrent TILs cases. This is an interesting finding because it demonstrates strong correlations of the ratio rather than individual cell percentage values (P = 0.0432).

FoxP3 cells represent a functional subset of regulatory (generally suppressive) T lymphocytes that are a subset of CD4 (helper) T lymphocytes. The original view that FoxP3+ Tregs are associated with poor outcome while CD8+ lymphocytes equal good prognosis is oversimplified. The discrepancies in prognostic value can be attributed to several factors, both biological and technical in nature. In HNSCC, improved survival has been most closely associated with CD8 infiltrates, whereas intratumoral FoxP3 infiltrates have been variably associated with improved,[18] neutral or negative impact on survival.[19]

The prognostic value of different types of TILs in HNSCC differs among studies, as shown in literature. As various factors such as site, stage, and treatment of tumor could confound correlations of TILs with outcomes, it is crucial to perform multivariable analysis. With the evolution of precision and personalized medicine, our current data lend more support to the idea that the immune system plays an important role in outcomes for patients with HNSCC. Our present findings impart additional support to the key role of host immunity in tumor prognosis and show that the degree of immune cells infiltrate in tumor microenvironment is an important independent prognostic factor. A limitation of this study is that it is a single-institution study. Further, there may be subsite differences and the current analysis could have been limited by patient numbers. Subtyping and quantification of TILs reflect the immune response in the tumor microenvironment, contributing to either tumoral attack or escape mechanism, thereby affecting outcome.


  Conclusion Top


We have demonstrated that the ratio of CD3/FOXP3 cells, rather than individual percentage of TILs, is a significant prognostic indicator in HNSCC. It is also shown that patients harboring Cold tumors have more propensity for recurrence than with Hot tumors. Our current findings lend additional support to the important role of host immunity in prognosis and indicate that the degree of immune cell infiltrate in the tumor microenvironment is a significant independent prognostic factor that deserves further study as a potentially useful biomarker in patients with HNSCC.

Ethical committee approval

Ethical committee approval from Institutional Review Board of HCG hospital.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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