Abstract
Background/Aim: Prognostic factors, including CD8-positive tumor-infiltrating lymphocytes (CD8+TILs), in definitive radiotherapy (RT) for squamous cell carcinoma (SqCC) of the uterine cervix need to be studied. This study aimed to explore these factors in a retrospective cohort. Patients and Methods: Patients with SqCC who underwent definitive RT comprising external beam RT and intracavitary brachytherapy at our facility between April 2006 and November 2013 were evaluated. CD8 immunohistochemistry was performed in pre-treatment biopsy samples to analyze the prognostic significance of CD8+TILs in the tumor nest. Positive staining was defined as at least one CD8+ lymphocyte infiltrating the tumor area in the specimen. Results: In total, 150 consecutive patients were included. Among them, 66 (43.7%) patients had International Federation of Gynecology and Obstetrics (FIGO, 2008 edition) stage IIIA or higher progressive disease. The median follow-up period was 61 months. In the entire cohort, the 5-year cumulative rates of overall survival (OS), progression-free survival (PFS), and pelvic recurrence-free rate (PRFR) were 75.6%, 69.6%, and 84.8%, respectively. Of the 150 patients, 120 (80.0%) patients were CD8+TIL positive. The independent favorable prognostic factors were FIGO stage I or II disease, administration of concurrent chemotherapy, and CD8+TILs for OS (p=0.028, 0.005, and 0.038, respectively); FIGO stage I or II disease and CD8+TILs for PFS (p=0.015 and <0.001, respectively); and CD8+TILs for PRFR (p=0.017). Conclusion: The presence of CD8+TILs in the tumor nest may be a favorable prognostic factor of survival after definitive RT in patients with SqCC of the uterine cervix.
Radiotherapy (RT) comprising external beam radiotherapy (EBRT) and intracavitary brachytherapy (ICBT) with or without platinum-based chemotherapy is the standard treatment modality for locally advanced uterine cervical cancer (1). Previous studies showed that age, race, histology, histological grade, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor size, and lymph node metastasis potentially influence prognosis after definitive RT (2-8). CD8 is a glycoprotein expressed on the surface of cytotoxic T cells, and the presence of CD8-positive tumor-infiltrating lymphocytes (CD8+TILs) was also reported to be associated with a favorable prognosis (9-11). Moreover, it is noteworthy that a previous study showed that the presence of CD8+TILs in the tumor nest was associated with a favorable prognosis in adenocarcinoma of the uterine cervix. In the study, the positivity of CD8+TILs was defined as at least one positively stained lymphocyte inside the tumor nest in the pre-treatment biopsy specimens (12). It seems to be a simple and clear definition that could be used for prognostic prediction. However, this has not been examined in squamous cell carcinoma (SqCC). Therefore, this study aimed to explore the prognostic significance of CD8+TILs in the tumor nest, in patients with SqCC of the uterine cervix treated with definitive RT.
Patients and Methods
Study design and patients. This retrospective study was approved by the appropriate Institutional Ethics Review Board (ID: 1109) and performed according to the principles outlined in the Declaration of Helsinki and the Guidelines for Good Clinical Practices. The need for written informed consent for participation was waived owing to the retrospective observational nature of the study, but all patients or their relatives had the opportunity to opt out.
Patients with SqCC who underwent definitive RT between April 2006 and November 2013 were identified in the institutional database. The inclusion criteria were (i) untreated and pathologically diagnosed SCC and (ii) available pretreatment biopsy specimens. Patients with distant metastasis, except to the para-aortic lymph node metastasis, were excluded. Staging was according to the International Federation of Gynecology and Obstetrics (FIGO) staging criteria (2008 edition). All pre-treatment formalin-fixed paraffin-embedded biopsy specimens were sectioned to obtain 4-μm samples and stained with hematoxylin and eosin. The samples were reviewed by two independent researchers, and a diagnostic pathologist confirmed the results.
Treatment and follow-up. Definitive RT consisting of EBRT and ICBT was performed in all patients. EBRT was delivered at a dose of 2 Gy per fraction 5 times per week. Whole pelvic irradiation of 20 Gy for stage IB-II tumors measuring ≤4 cm, 40 Gy for bulky tumors, and 30 Gy for the others was delivered, followed by pelvic irradiation with a 3-cm-wide central shield. The total dose administered to the pelvic region was 50 Gy in 25 fractions. For patients with para-aortic lymph node metastases, the pelvic irradiation field was extended to include the gross metastatic region, and reduction of a fractional dose to 1.8 Gy with a total dose to the extended pelvic region of 50.4 Gy was considered. In patients with gross lymph node metastases, another 6-10 Gy in 3-5 fractions was administered to boost the external dose to the metastases.
After completing whole-pelvic irradiation, ICBT using a high-dose rate 192Ir remote after-loading system (microSelectron, Elekta, Stockholm, Sweden) was performed once weekly. EBRT was skipped on the day of ICBT. In principle, ICBT of a total dose of 24 Gy in 4 fractions was delivered to point A. Additional ICBT was considered when the tumor did not shrink well. In cases of bulky and/or asymmetric tumors, interstitial brachytherapy along with ICBT was considered. Concurrent chemotherapy was administered to patients with tumors larger than 4 cm, lymph node metastasis, and FIGO stage III or higher progressive disease unless they were aged ≥75 years or had comorbidities restricting the use of chemotherapy (e.g., renal dysfunction, severe diabetes, or ischemic heart disease). Cisplatin (40 mg/m2) was administered on a weekly basis. Dose reduction up to 30 mg/m2 was permitted for patients with extended-field treatment.
The patients were followed-up every 1-3 months for the first 2 years and every 3-6 months for the next 3 years. Disease status was evaluated at each follow-up examination with respect to overall survival (OS), progression-free survival (PFS), and the pelvic recurrence-free rate (PRFR).
Immunohistochemical analysis. Immunohistochemical studies were performed to detect CD8 expression in pretreatment biopsy specimens excised from cervical tumors. The details have been previously reported (12). Paraffin sections (4 μm) were dewaxed in xylene and rehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked by a 10-min incubation in 0.3% hydrogen peroxide. After pretreatment according to the manufacturer’s instructions, the sections were incubated overnight with a primary antibody [mouse monoclonal anti-CD8 (1:200 dilution, M7103 (C8/144B); Dako, Carpinteria, CA, USA)] at 4°C. A commercially available biotin–streptavidin immunoperoxidase kit (Histofine, Nichirei, Tokyo, Japan) and diaminobenzidine were used for coloration. Positive staining was defined as more than one lymphocyte positive for CD8 in the tumor nest. The tumor nest included cancer cells, but with stromal and invasive margins. Representative images are shown in Figure 1.
Representative images of immunohistochemistry. (A) Positive and (B) negative staining for CD8 (magnification: ×400; scale bar, 100 μm).
Statistical analysis. OS, PFS, and PRFR curves were generated using the Kaplan–Meier method and compared between subgroups using the log-rank test. Cox regression analysis was performed to estimate the prognostic significance of the clinicopathological factors. The covariates were chosen based on the results of previous studies (2-8). The increased risk of a type I error due to multiple testing was not adjusted as this was an exploring study. All statistical analyses and visualizations were conducted using R 3.6.2 (R Core Team, Vienna, Austria) (13). Statistical significance was set at p<0.05.
Results
Patient characteristics and oncologic outcomes. A total of 150 patients were identified. The patient characteristics are summarized in Table I. All patients underwent EBRT and ICBT. Concurrent chemotherapy was administered in 62% (93/150) of the patients. The median follow-up interval was 61 months (range=4-131 months). The 3- and 5-year post-RT follow-up rates were 97.3% (146/150) and 90% (135/150), respectively. In total, 80% (120/150) of the patients were positive for CD8+TILs in the tumor nest, and 20% (30/150) were negative.
Patient characteristics.
The Kaplan–Meier curves of the entire cohort are shown in Figure 2. The 2-, 3-, and 5-year cumulative OS rates were 81.1% (95%CI=75.1-87.7), 79.8% (95%CI=73.5-86.5), and 75.6% (95%CI=69.0-82.9), respectively. Meanwhile, the 2-, 3-, and 5-year cumulative PFS rates were 74.5% (95%CI=67.8-81.9), 71.0% (95%CI=64.1-78.7), and 69.6% (95%CI=62.6-77.5), respectively. The 2-year cumulative PRFR rate was 85.5% (95%CI=80.0-91.5), and those at 3 and 5 years were both 84.8% (95%CI=79.1-90.9).
Clinical outcomes of all the patients (n=150). Kaplan–Meier curves of overall survival (OS), progression-free survival (PFS), and pelvic recurrence-free rate (PRFR) are shown. Filled regions indicate the 95% confidence interval.
Clinicopathological factors and prognoses. Results of univariate analyses are summarized in Table II. FIGO stage I or II was associated with favorable OS, PFS, and PRFR (p≤0.001, <0.001, and 0.039, respectively). Absence of pelvic lymph node metastasis was associated with favorable OS and PFS (p=0.0067 and 0.034, respectively). The presence of CD8+TILs was associated with favorable PFS and PRFR (p=0.0027 and 0.037, respectively). The Kaplan–Meier curves according to the status of CD8+TILs are described in Figure 3. Results of multivariate analyses are shown in Table III. FIGO stage, maximum tumor diameter, lymph node metastasis, use of concurrent chemotherapy, and CD8+TILs were included in the analyses. The results showed that the independent favorable prognostic factors were FIGO stage I or II disease, administration of concurrent chemotherapy, and CD8+TILs for OS (p=0.028, 0.0052, and 0.038, respectively); FIGO stage I or II disease and CD8+TILs for PFS (p=0.015 and <0.001, respectively); and CD8+TILs for PRFR (p=0.017).
Summary of univariate analyses.
Kaplan–Meier curves of (A) overall survival, (B) progression-free survival, and (C) pelvic recurrence-free rate in patients with or without CD8-positive tumor-infiltrating lymphocytes (CD8+TILs) (n=150). Filled regions indicate the 95% confidence interval.
Summary of multivariate analyses.
Discussion
In this study of patients with SqCC of the uterine cervix treated with definitive RT, our findings showed that the presence of CD8+TILs in the tumor nest was a favorable prognostic factor for OS, PFS, and PRFR; FIGO stage I or II was a favorable prognostic factor for OS and PFS; and the administration of concurrent chemotherapy was a favorable prognostic factor for OS.
Previous studies on cervical cancer demonstrated that a higher FIGO stage was associated with a worse prognosis after definitive RT (2, 6-8). Consistent results were observed in the current study. A larger tumor size and lymph node metastasis have also been suggested to be independent prognostic factors (3-8). However, although the current study observed some tendencies of better survival in patients with a smaller tumor size and those without lymph node metastasis, prognosis did not significantly differ according to these factors. This might be because our sample size was not sufficient to detect a statistical significance.
Host anti-tumor immunity in the treatment of cervical cancer has attracted attention in recent years (14-18), and furthermore, the associations between TILs and prognosis after RT have been previously reported. Nakano et al. reported favorable OS in patients with intratumoral infiltration of immune cells such as dendritic cells and T cells (19). Since CD8+ T cells have been found to be one of the most important immune cells correlated with prognosis (20), CD8+ TILs have been the focus of prognostic evaluation studies in recent years (Table IV) (9-12, 21). Enwere et al. evaluated the pre-treatment biopsies of PD-L1-positive cervical cancer patients and found that the presence of intratumoral CD8+ cells was associated with favorable PFS [HR=0.43 (95%CI=0.18-1.01)] (9). Ohno et al. also reported better prognosis in patients with a high level of CD8+, as well as of CD3+ and CD4+, lymphocyte infiltration into intratumoral lesions than in patients with a low-level infiltration (21). Consistent findings were observed in the current study.
Summary of studies regarding CD8-positive tumor-infiltrating lymphocytes (CD8+ TILs) in patients with cervical cancer treated with radiotherapy.
However, Someya et al. reported no superior survival when intratumoral CD8+ lymphocyte were evaluated separately in the stroma and tumor regions (in stroma, p=0.454; in tumors, p=0.519) (11). These results are in contrast to our findings that intratumoral CD8+TILs are associated with a favorable prognosis. However, it is noteworthy that cut-off values for evaluating CD8+ lymphocytes were different between their study and the present study. Someya et al. divided their cohort by the median value of the number of CD8+TILs, whereas we defined the presence of CD8+TILs as at least one CD8+lymphocyte in the tumor nest. Enwere et al. and Ohno et al. also used cut-off values lower than the median values in the aforementioned studies.
Someya et al. also reported post-RT clinical outcomes for cervical cancer stratified by the distribution of CD8-positive cells (22). They reported that approximately 10% of patients with cold-type tumors, which indicated poor infiltration of CD8-positive cells in both the tumor and stroma (less than 30 in the high-power field), had extremely worse disease-specific survival than did the others (p<0.001). The cold-type tumors in their study would be similar to the intratumoral CD8+TIL-negative tumors in the present study. There is currently no established method for measurement of intratumoral CD8+TILs as prognostic predictors. However, a previous study on colorectal cancer showed that CD8+TILs in tumor nests were more predictive of survival than were CD8+TILs in the stromal regions and invasive margins (23). The present study also showed that CD8+TILs in tumor nests were significantly associated with a favorable PFS.
This study has some limitations. First, because clinical data in this study were based on a chart review at a single institution, there are biases that cannot be eliminated. Second, some potential influencing factors, such as age, anemia status, total duration of RT, and total dose to the tumor, could not be sufficiently analyzed owing to the limited number of patients. The effects of these factors should be considered in future studies. Our findings should be interpreted considering these limitations and be validated in further studies.
In conclusion, this retrospective study of 150 patients with SqCC of the uterine cervix showed that, in addition to FIGO stage I or II disease and concurrent chemotherapy, CD8+TILs in tumor nests could influence prognosis after definitive RT.
Acknowledgements
The Authors thank Mr. Koji Isoda (Gunma University) for technical assistance in performing the immunohistochemical analysis.
Footnotes
Authors’ Contributions
All the Authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by Yuhei Miyasaka, Yuya Yoshimoto, and Kazutoshi Murata, respectively. The pathological methods and evaluations were confirmed by Hayato Ikota and Hideaki Yokoo. Yuhei Miyasaka wrote the first draft of the manuscript. The previous manuscript was revised by Yuya Yoshimoto Ken Ando, Kazutoshi Murata, Daisuke Irie, Hiro Sato, Shin-Ei Noda, Takashi Nakano, and Tatsuya Ohno. Supervision was performed by Tatsuya Ohno. All Authors have read and approved the final manuscript.
Conflicts of Interest
Yuya Yoshimoto is an endowed chair funded by Okinawa PCR Rinsyo Center.
Funding
This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) for Scientific Research (Grant number JP22K15840).
- Received February 8, 2023.
- Revision received February 23, 2023.
- Accepted February 24, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
