The aims of this study were to evaluate the prognostic significance of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) in patients with familial ovarian cancer. Clinical and pathological information were retrieved from the Gilda Radner Familial Ovarian Cancer Registry (GRFOCR) in Buffalo, NY. Immunohistochemistry was performed on paraffin-embedded tissue specimens of GRFOCR participants using specific antibodies for CD3+, CD8+, CD25+, FOXP3+, CD68+, and CD163+. The correlation between the frequencies of TILs and TAMs and clinic-pathologic parameters were determined. Overall survival was determined using univariate and multivariate Cox proportional hazards models. High tumor grade correlated with higher frequencies of CD3+ (p = 0.019), CD68+ (p = 0.025), CD163+ (p = 0.018), and Treg (CD25+ FOXP3+) (p = 0.024) cells. Higher stage correlated with higher frequencies of CD163+ cells (p = 0.012). There were correlations between the frequencies of CD68+ and CD3+ (p = 0.029), between Treg and each of CD3+ (p = 0.002), CD8+ (p = 0.018), and CD68+ (p = 0.028) cells. In univariate analysis, age and Treg significantly predicted patient survival. In multivariate survival analysis, Treg frequency was the only significant predictor of prognosis in patients with familial ovarian cancer [HR = 0.92; 95% CI 0.87 – 0.98; p = 0.012]. We concluded that interaction between TILs and TAMs in familial EOC also exists, and tumors with high Treg frequencies have a more favorable outcome. Thus, therapeutic strategies to modulate tumor Treg infiltration could be beneficial for patients with familial ovarian cancer.
This article was published in Cancer Immunity, a Cancer Research Institute journal that ceased publication in 2013 and is now provided online in association with Cancer Immunology Research.
Epithelial ovarian carcinoma (EOC) is the fourth leading cause of cancer mortality among women in western countries (1). The incidence of newly diagnosed EOC in the US is estimated to be 22,430 cases per year, with 15,280 deaths (1). The strongest risk factor for ovarian cancer is the presence of an inherited mutation in one of the two ovarian cancer susceptibility genes, BRCA1 or BRCA2. It is estimated that more than 10% of women in North America with invasive ovarian cancer carry a BRCA1 or BRCA2 mutation (2, 3). The majority of these mutation carriers come from high-risk families where several members are affected by breast and/or ovarian cancer. It is estimated that 30% of families with a strong history of breast and ovarian cancers carry one of these mutations (4), but only one in 300 to 500 people in the general population carry the genes (5). While it is clear that alterations in BRCA1 and BRCA2 genes are important for susceptibility to ovarian cancer, it is also well-known that the immune system has the ability to protect the host from cancer and “sculpt” tumor immunogenicity (6, 7). This interaction between the immune system and cancer is complex, consisting of a very delicate balance between immune activation and immune suppression mechanisms (8).
Immune surveillance plays an important role in tumor outcome in patients diagnosed with EOC (9–17). In the study by Sato et al., intraepithelial CD8+ tumor-infiltrating lymphocytes (TILs) and high CD8+/regulatory T cell (Treg) ratio were shown to be associated with favorable prognosis in ovarian cancer (17). The favorable prognosis of intraepithelial CD8+ cells has been confirmed by other studies (11, 12). In contrast to TILs, tumor-associated macrophages (TAMs) have been reported to be associated with poor outcome in EOC (14), suggesting that they might play an immune suppressive role. Thus, TILs and TAMs could interact to modulate and shape the overall immune environment in EOC.
In an effort to define the role of TILs and TAMs in familial EOC using the Gilda Radner Familial Ovarian Cancer Registry (GRFOCR), the goals of our study were (i) to evaluate the presence of TILs (CD3+, CD8+, Treg) and TAMs (CD68+, CD163+) in patients with familial EOC; (ii) to determine the interaction between TILs and TAMs; and (iii) to define the role of TILs and TAMs in the outcome of patients with familial EOC.
Briefly, the GRFOCR is a self-referred registry of families with two or more ovarian cancer cases in blood relatives. It was established in 1981 at the Roswell Park Cancer Institute (RPCI) by Dr. M. Steven Piver to study the incidence of familial ovarian cancer (18–21). The primary function of the registry is to receive family cancer information voluntarily contributed throughout the US by ovarian cancer patients, referring and concerned physicians, concerned women, and patients of the RPCI Gynecologic Oncology Department. The objectives of the registry include (i) obtaining detailed family histories from individuals who are apparently from families with two or more cases of ovarian cancer, or a syndrome possibly related to ovarian cancer; (ii) documenting through medical records and through pathologist review of tissues the occurrence of cancer; (iii) collecting, processing, and storing biological samples, when possible, from registry participants; and (iv) making the information and biological samples available for research under Institutional Review Board (IRB)-approved research protocols. Registry participants are recruited through probands or index persons, and meet at least one of the following criteria: (i) family history of two or more cases of ovarian cancer; (ii) family history of one case of ovarian cancer and two cases of cancer at any other site; (iii) family history of at least one female with two or more primary tumors with one of the primaries being ovarian cancer; and (iv) family history of two or more cases of cancer with at least one case being ovarian cancer, and the other cancer considered to be of early onset (≤ 45 years old).
Study population and tumor characteristics
Seventy-three patients with adequate tissue material were available for analysis of TILs and TAMs. Patient population and tumor characteristics are presented in Table 1. The age ranged from 26–80 years with a median age of 52. As expected, the majority of patients had FIGO Stage III disease (62%), tumor grade 3 (75%), and serous histology (60%). After a median duration of follow-up of 24.91 months, 30 patients were dead of disease and 43 were alive. The median overall survival (OS) was 58.81 months. Eleven of the 73 patients had BRCA mutation data (five had BRCA1 mutation and six had BRCA2 mutation) and the remaining 62 patients had undefined BRCA mutation status. Therefore, due to the small number of patients with information on BRCA mutation status, we did not further explore any potential association with TILs or TAMs.
TILs and TAMs in familial ovarian cancer
TILs and TAMs in the intraepithelial compartments were counted, as illustrated in Figure 1. The mean cell counts were 55.60±56.13; 62.29±69.77; 27.60±21.46; 30.00±22.95; and 9.56±9.32 for CD3+; CD8+; CD68+; CD163+; and Treg cells, respectively (Figure 2, Supplementary Table 1). Next, we tested the association between the markers of immune cell subsets. As expected, and shown in Table 2, the frequency of CD3+ cells was positively correlated with CD8+ (p < 0.001), CD68+ (p = 0.029), and Treg (p = 0.002) cells. The frequency of CD8+ T cells was also positively correlated with the frequencies of Treg (p = 0.018). Furthermore, CD68+ was positively correlated with CD163+ (p < 0.001) and Treg (p = 0.028). Using the two sample t-tests analysis as shown in Table 3, we found that FIGO stage was negatively associated with CD8+ T cell frequency (p = 0.042) and positively associated with CD163+ cells (p = 0.012). On the other hand, tumor grade was positively associated with CD3+ (p = 0.019), CD68+ (p = 0.025), CD163+ (p = 0.018), and Treg (p = 0.024) cells.
Prognostic significance of TILs and TAMs
With regard to patient outcome, 30 patients were dead of disease. However, information on definite date of death was missing from six of these patients and they were excluded from further analysis. Thus, we ended with 67/73 with adequate follow-up information. In univariate analysis (Table 4), advanced age was found to be significantly associated with poorer overall sur vival (p = 0.002) among the clinicopathological variables. Using TIL and TAM counts, Treg was the only parameter to be significantly associated with overall survival in univariate analysis (p = 0.033, Supplementary Figure 1). The hazard ratio of Treg 0.94, indicated that it was associated with favorable prognosis. In multivariate survival analysis adjusting all clinicopathological covariates (Table 5), the only parameter that appeared to be a significant predictor of poor survival was Treg (p = 0.012). The hazard ratio of Treg 0.92 (95% CI = [0.87, 0.98]), suggested that it was associated with improved survival.
There is a higher risk of ovarian cancer in individuals with a family history of one or more affected first degree relatives with ovarian cancer. While the lifetime risk of EOC in the general population is approximately 1 in 100, this risk increases to 1 in 40 if a first degree relative is affected by ovarian cancer (25). Indeed, an unaffected woman with 2–3 relatives with ovarian cancer had a 4.6 times increased risk compared to the general population risk, i.e., a 7.2% lifetime probability of ovarian cancer (25). Since it is also now clear that the immune system protects against development of cancer (7), our study addressed, for the first time, a potential link between anti-cancer immunity and progression of familial ovarian cancer.
TILs and TAMs are considered major components of the cellular immune response against cancer. While numerous studies have shown infiltration of ovarian cancer by CD8+ TILs to be positively associated with survival (9, 17, 26), multiple immune suppressive mechanisms have also been shown to counteract the beneficial effects of CD8+ T cells. These mechanisms include Treg that mediate homeostatic peripheral tolerance by suppressing autoreactive T cells (27, 28); co-inhibitory molecules such as PD-1 and LAG-3 (8); tolerance-inducing plasmacytoid dendritic cells (pDCs) (18); B7-H4+ macrophages (15, 16, 29); immune suppressive cytokines such as TGF-β (30); and myeloid-derived suppressor cells (MDSCs) (31). The majority of these mechanisms are orchestrated by ovarian cancer cells in order to create a microenvironment that favors escape from immune elimination (32).
In familial EOC, we showed considerable amounts of TILs and TAMs in the intraepithelial compartment, indicating that the immune response is still triggered in this population of ovarian cancer patients. We also showed evidence of a significant relationship among TIL subsets, and between TILs and TAMs. The relationship seen between Treg and CD68+ would suggest an interaction between macrophages and Treg in order to contribute to the profound suppressive environment in ovarian cancer. In support of this observation is a previous report by Kryczek et al. which demonstrated interaction between macrophages and Tregs in sporadic EOC (16). Furthermore, we found that as grade of ovarian tumors increased, the number of intraepithelial tumor-infiltrating CD3+, CD68+, CD163+, and Treg cells also increased. In line with this observation, we found that as tumors became more advanced, there was higher tumor infiltration by CD163+ cells.
Treg, as documented by CD25+ FOXP3+ cells in our study, are a specific population of T cells with immunosuppressive properties. Treg normally function as suppressors of inappropriate immune responses, preventing any unwanted autoimmune reactions (33, 34). Previous studies demonstrated an increase of Treg counts in late-stage and in high-grade EOC (10, 11). Also, infiltration of tumor cells by Treg seemed to suppress tumor-specific T cell immune response, leading to tumor cell growth. Infiltration of tumor cells by Treg was shown to be associated with poor survival in patients with EOC and in other solid tumors such as hepatocellular carcinoma (10, 17, 35). However, this finding was not confirmed by others (13, 34). The difference of results among studies could be mainly due to the methods used in the analysis of T cell subsets. Sato et al. found that high CD8+/Treg count ratio to be the strongest predictive factor for prolonged survival, with a nearly 70% reduction of mortality rate in patients with EOC. In our study on familial EOC, we found that among all immune cell populations and their ratios, Treg was the only independent prognostic factor in predicting favorable outcome in familial EOC. This finding is the first in the literature in familial EOC and it is consistent with the reports by others, where similar results were described in patients with sporadic EOC (13, 14).
Lastly, there are several limitations of the present study—the GRFOCR is a self-referred registry; the documentation of residual disease after optimal debulking (one of the prognostic factors in EOC) was not assessed in this study. The differences in clinical characteristics, together with the relatively small sample size used in this study, might contribute to the difference between prior studies of CD8+ T cells and our study. Also, disease-free survival could not be documented as the registry only knows whether a registry member is alive or dead. However, despite these limitations, our study is the first to shed some light on the role of the immune system in familial ovarian cancer.
In summary, our study supported a role for the immune system in the progression of familial ovarian cancer. Since the frequency of intratumoral Treg was the most significant predictor of outcome in this population of patients, future studies are warranted to examine the relationship between the known ovarian cancer predisposition genes and genes that regulate Treg differentiation and expansion.
Materials and methods
Study subjects for this work derived from the Gilda Radner Familial Ovarian Cancer Registry (formerly referred to as the Familial Ovarian Cancer Registry) under a protocol that has been reviewed and approved by the Roswell Park Cancer Institute IRB.
Patients were included in our study only when a staging procedure was performed at the time of the original surgery, or within the following months using the FIGO (International Federation of Gynecology and Obstetrics) staging criteria (22). All patients received standard care for EOC consisting of cytoreductive surgery, followed by platinum-based chemotherapy. At follow-up, patients had two outcomes: alive (A) or dead of disease (DOD). Information on the status of the residual disease or time to disease relapse was not available.
Hematoxylin and eosin (H&E) slides were available for histologic review. The tumor subtypes and grade were re-reviewed for confirmation by one experienced pathologist (PMF). Histologic subtypes were based on World Health Organization (WHO) classification of tumors (23). The histologic grade was determined according to criteria used by the Silverberg’s grading system (24).
Four μm thick sections were deparaffinized with xylene, and washed with ethanol. Sections were cooled for 20 min and then incubated for 10 min with 3% H2O2 to quench endogenous peroxidase activity. Blocking was performed using serum-free protein block, Dakocytomation (Carpenteria, CA) for 30 min. A pretreatment with Tris Buffer saline solution/EDTA, high pH, for 10 min was used for CD3+, CD8+, CD68+ and steamer for CD163+ staining. Afterwards, sections were incubated with antibodies for 30 min at room temperature for CD3+, CD8+, CD68+, and for 60 min for CD163+. The description of the antibodies used are as follows: CD3+ (ready to use; polyclonal; 1:100; Dakocytomation), CD8+ (clone CD8/114B; monoclonal; 1:200; Dakocytomation), CD68+ (clone PG-M1; monoclonal; 1:3000; Dakocytomation), CD163+ (clone 10D6; monoclonal; 1:50; Novacastra, Cambridge, UK), CD25+ (clone 4C9; monoclonal; 1:50; Vector Labs, Burlingame, CA), and anti-FOXP3 (clone 259D; polyclonal; 1:20; BioLegend, San Diego, CA). Diaminobenzidine tetrahydrochloride was then added for development for 10 min, followed by counterstaining with hematoxylin solution. Treg was defined as CD25+ FOXP3+ cells and, for this purpose, a dual staining procedure for CD25+ FOXP3+ was performed as described previously (17). Negative control slides omitting the primary antibody were included in all assays.
Quantification of TILs and TAMs
Evaluation of the IHC slides was performed by two pathologists independently (PM-F and LA). At first, the entire tumor section was evaluated by using 20X objective lens on an Olympus microscope (Olympus, Holly, MI). Ten independent areas with the most abundant intraepithelial T cell infiltrates (CD3+, CD8+, Treg) and intraepithelial tumor-associated macrophages (CD68+, CD163+) were selected. Intraepithelial tumor infiltrates is defined by macrophages and T cells highlighted by immunohistochemistry that are seen in the epithelial tumor cells. Those seen in the stroma were not counted. The count is done at 40X magnification. Each pathologist counted 10 areas and scored their counts. The scores of the two pathologists were compared and when a discrepancy (count difference of more than 10 cells infiltrates) was noticed, the two pathologists sat down at a double head microscope to solve the issue and reach a final score. Disagreement was not very frequent between the first and the second read and occurred roughly around 5% of the cases. The mean number of tumor infiltrates per 10 high power fields was evaluated as continuous variable.
Statistical analyses were performed by The R Project (36). The clinical parameters used for modeling are age, stage, grade, and counts of TILs and TAMs. To find the association between the CD counts and FIGO stage and tumor grade, two sample t-tests were performed to determine if two groups of cluster of differentiation (CD) counts differ significantly in different categories. Univariate and multivariate Cox proportional hazards models were used to estimate hazard ratio that represents the correlation between relative risk of death among patients and clinical parameters. The p value for the relevant parameter is derived from the Wald test. The proportional hazards assumption for a Cox model fit is tested using the cox.zph function in The R Project. Fair proportional assumption holds for each variable (p > 0.017) except CD163+. CD163+ is not a significant variable using either Cox proportional hazards model (p = 0.835, shown in Table 5) or logistic model (p = 0.594, data not shown). Hence, Cox proportional hazards models were used throughout the study. Pearson correlation coefficients are calculated to access the relationship of TIL and TAM counts. A p value of < 0.05 is considered statistically significant.
We would like to thank Mrs. Cathy Fahey for her help and assistance in providing the data and specimens. Also, we thank Mrs. Mary Vaughn for her expertise in immunohistochemistry. This work was funded by the Gilda Radner Familial Ovarian Cancer Registry, Buffalo, NY.
- Copyright © 2013 by Paulette Mhawech-Fauceglia