In this investigation, we compared CTLA-4 overexpression in cervical carcinomas, a commonly used immune checkpoint marker, with the expression of CD137 in the lymphoplasmacytic infiltrate within the tumor microenvironment. Based on the review of the randomly assembled TMA samples, our findings indicate CTLA-4 overexpression in 31.6% of cervical carcinomas and CD137 expression in over 90% of the lymphoplasmacytic infiltrate within the tumor microenvironment. The expression of CTLA-4 had a propensity for lower grade SCCs, however, the percentages of the “Positive” CTLA-4 expression in SCC, grade I (42.9%), grade II (30.8%) and grade III (26.7%%) did not reach statistical significance (Table 1). Endocervical adenocarcinomas exhibited the lowest “Positive” expression level (15.4%) and adenosquamous carcinoma exhibited the highest “Positive” expression level (62.5%), however, these differences were not statistically significant, likely due to the low number of cases. Naturally, in a randomly constructed tissue microarray, the frequency of low prevalent lesions was also low in the TMA samples. Interestingly, the vast majority of cervical cancers exhibited CD137 expression within the lymphoplasmacytic infiltration, even in cases with no CTLA-4 expression. The expression of CD137 was lowest in grade III SCCs (73.3%) versus grade I (85.7%) and grade II (96.2%). Adenosquamous cell carcinoma had expression of CD137 in 87.5% of cases and all endocervical adenocarcinomas in the TMA had expression of CD137 (100%). In all, more than 30% of the patients with the cervical cancers had overexpression of both CTLA-4 and CD137 and thus may become clinically eligible for the CTLA-4 immunotherapy or possible combination immunotherapy with CD137 expression. We have also used the same TMA in our earlier study to evaluate another check point inhibitor and showed that PD-L1 was positive in 37.8% of the cervical carcinomas .
We have previously devised a uniform systematic assessment for the CTLA-4 reactions by IHC. The scoring system which is introduced in cervical carcinomas in this study is similar to the CTLA-4 scoring system introduced previously in breast carcinomas . Our scoring system is as follows: Cases with no expression had complete negative staining “Negative” with a score of “0”. If the expression was less than 50%, the expression was interpreted as “Low-Positive” with a score of “1”. If 50% or more of the cells had the protein expression, the specimen was interpreted as “Positive” with a score of “2”. Intensity of 1 + adds the suffix of “a” and the intensities of 2 + and/or 3 + gives a suffix of “b” to the score. Although, there are a wide range of antibodies available against the CTLA-4 protein, the ability of these antibodies to detect overexpression might be variable. In several published studies, the reactions have not been classified based on the intensity of the CTLA-4 IHC. We have shown that the 1 + intensity is seen in normal control benign cervical tissue. Therefore, the 1 + intensity reaction has been interpreted as negative in this study as was also suggested in our previous study with 1 + intensity seen in normal breast tissue .
The introduced systematic method, that assigns IHC scores as a percentage of positive tumor cells in relation to the staining intensity, may provide a more objective assessment of the protein expression and a clearer understanding of the roles played by the potential tumor markers in predicting outcome. Most importantly, by evaluating the protein expression quantitatively at the outset, more relevant cutoffs for tumor positivity can be established for the therapeutic agents in different malignancies. In other words, as new agents are introduced, and/or future clinical studies result in changes of the response rates, dynamic cutoff points can be established for each therapeutic agent in each specific malignancy. Therefore, an objective pathology scoring system is needed for a comprehensive and consistent evaluation of the CTLA-4 reactions.
Antibody-based strategies for cancer treatment have dramatically advanced in the past decade. This highlights the importance of understanding the expression levels of these immune checkpoint regulators in different cancers for effective translational and clinical research. CTLA4 is an immune checkpoint inhibitor, best known for its activity and survival effects in metastatic melanoma, as well as other solid tumors [28, 29]. Checkpoint inhibitors such as CTLA-4 trigger inhibitory pathways which dampen T-cell activity when bound to their ligands (CD80/CD86) . Therefore, blocking of this interaction enhances overall immune responses .
Immune checkpoint and costimulatory pathways have different mechanisms with potential for therapeutic synergy approach . The significant point to note is the possible enhanced response rate of immune-checkpoint blockade with combination immunotherapies that promote or block various steps of the immune checkpoint cycle in cancers. This combination strategy improves anti-cancer immunity through the antigen-specific enhancement of CD8 + T-lymphocyte activity and immune memory, but suppresses autoimmunity by increasing the function of regulatory T lymphocytes .
CD137 activation results in recruitment of TNFR factors (TRAF1 and TRAF2) which consequently leads to the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), Jun amino-terminal kinases/Stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein kinases (p38 MAPK) pathways. This generates co-stimulatory signals to induce the activities of both CD4+ and CD8+ T lymphocytes . Two clinical trials had been set for anti-CD137 antibodies namely urelumab (BMS-663513) and utomilumab (PF-05082566) . Urelumab showed a promising cancer treatment potential in preclinical studies, however, there was significant liver toxicity of the antibody which caused the clinical trial to be stopped . Recent studies suggested that this antibody is safe at lower doses for a shorter periods of time , thus combination therapies of low dose anti-CD137 with other FDA-approved immunomodulators and antibody therapeutics can have great potential of anti-tumor activities and minimize the possibility of systemic toxicities. It has already been shown that agonistic CD137 antibody with combination of anti-CTLA-4 therapy increases the survival of mice in the setting of colon cancer, glioblastomas and prostate cancer [33, 38–40]. This immunotherapeutic combination is promising for future clinical development.
We speculate that these immunological features in cervical cancers might be associated with clinical efficacy of the treatment and may help guide immunotherapeutic strategies in the future. The presence of CTLA-4 and CD137 expression as detected by IHC have the potential to be used not only as a prognostic marker in the cervical cancers, but as a potential predictive marker for the immunotherapeutic responses. Percentage scoring should allow a more thorough assessment of the predictive or prognostic significance of these immune check point proteins. However, as with all IHC markers, factors such as tissue fixation (both type and duration), the choice of antibody clone, and the IHC staining methodology can dramatically affect test accuracy and reproducibility so these features must be taken into the account . Finally, more studies are warranted to provide further insights into the association of HPV status and the expression level of CTLA-4 and CD137 in cervical cancers. Combining the level of HPV DNA with the expression of CTLA-4 may also provide a novel predictive biomarker of the efficacy of CTLA-4 inhibitors and the prognosis of patients with cervical cancer.
The results of this study have found a significant expression of CTLA-4 (~ 30%) in cervical cancers. Overall, the vast majority of cervical tumors (> 90%) had expression of CD137 within the lymphoplasmacytic infiltrate. Our findings in this study further support future investigations of anti-CTLA-4 and anti-CD137 immunotherapies in the CTLA-4-positive cervical tumors. This study shows that the cervical cancers may express CTLA-4 in the tumor cells and CD137in tumor infiltrating immune cells. These findings support a possible therapeutic role for CTLA-4 and CD137 inhibitors in a subset of cervical carcinomas.