It is well established that the expression of AR differs according to molecular subtypes with more frequent expression in ER negative cancers. The prevalence of AR + expressing tumors is generally ranging from 10 to 41% in TNBC cases 1,7−13, with rare reports showing rates up to 79% 14,15. In accordance with most published reports, our rate of AR expression in TNBC was 21.2%.
Whether clinico-pathologic characteristics of TNBC vary based on AR expression status has been extensively studied 7,12,14,16,17. Some studies showed that patients with AR + tumors were significantly older, exhibited tumors with significantly lower grades (I-II), more frequent nodal involvement, non-ductal histology, and lower Ki67 13,14,16,17. Other reports described reduced lymph node metastases in AR + TNBCs 7, or just similar clinico-pathologic profile between AR + and AR-TNBC 12. Herein, there was no statistical significant difference in the clinico-pathological parameters according to AR expression. However, AR + cases were older in age and exhibit more regional nodal spread. Despite statistically insignificant, this profile was analogous to the LAR subtype described by Lehmann and colleagues 2.
Available evidence about the prognostic value of AR in TNBC is controversial. Some reports suggested that AR-positivity was associated with good outcomes 7,12. Whereas, others concluded that AR status conferred worse prognosis18 or had no significant impact on disease prognosis 4,19,20. Many factors may explain these discrepant results across studies including the sample size limited cohorts, differences in the ethnic origin, the anti-AR antibodies used for staining, staining/scoring method, as well as variability in the thresholds used to define AR positivity 4. A meta-analysis published in 2017, demonstrated that AR-positive status was associated with better DFS and OS in TNBC (Hazard ratio (HR) = 0.64, 95% CI = 0.51–0.81, p < 0.001 and HR = 0.64, 95%CI = 0.49–0.88, p < 0.001, respectively), in univariate analysis5. Of note, no multivariate analysis was provided and this meta-analysis included heterogeneous studies in terms of methods of AR scoring, clinical cohorts’ characteristics, therapies received and length of follow up. A large multi-institutional study including about 1,407 TNBC tumors issued after this meta-analysis concluded that the AR-positivity was a marker of good prognosis in US and Nigerian cohorts, whereas it conferred poor prognosis in Norway, Ireland and Indian cohorts, and was neutral in UK cohort 4.Whereas a more recent meta-analysis (2020), 20 demonstrated that AR expression in TNBC was not associated with DFS (HR = 0.92; 95% CI = 0.67–1.27; p = 0.63), OS (HR = 0.91; 95% CI = 0.67–1.22; p = 0.53), distant- DFS (HR = 1.02; 95% CI = 0.96–1.08; p = 0.48), or recurrence-free survival (HR = 0.95; 95% CI = 0.46–1.98; p = 0.90), regardless of confounding factors and heterogeneity that existed among included studies. Our study results had matched the latter meta-analysis results, where no statistical difference in median OS (31.5vs. 25 months, p = 0.77) or relapse/progression rate (26.9% vs. 14.3%, p = 0.48) was found between AR- and AR + groups.
Compared to other subtypes, TNBC was shown to exhibit higher levels of TILs 21. There is heterogeneity of TILs cut-off used in published studies in order to distinguish between LPBC and LDBC. Some studies defined LPBC as showing more than 50% of lymphocyte infiltration 22,23, whereas others used different cut-offs 24. In our cohort, median TILs was 30% (range: 1–70%), with a LPBC prevalence of 32.2%, which is not in full agreement with other reports. Adams et al. 22 reported much lower median TILs percentage (10%), and using the same cut-off of ≥ 50% TILs, only 4.4% were LPBC; whereas, Pruneri et al. 23 described a median TILs level of 20%, with LPBC prevalence of 22% of cases.
Little is known about the association between TNBC clinico-pathologic features and lymphocytic predominance. A pooled analysis of nine large studies by Loi et al. 24 demonstrated that TILs were significantly lower in older age. Whilst, Adams et al.22 reported no strong associations between TILs scores and age or menopausal status. Despite not statistically significant, we showed lower median TILs in patients ≥ 60 years vs < 60 years old (10% vs. 38 %, p = 0.45).
Interestingly, we found that patients with lymph node positive tumors were significantly more likely to be LDBC, where a total TILs expression < 50% (LDBC) was associated with higher risk of lymph node involvement (OR = 6, 95%CI = 1.05–34.21, p = 0.04). This is in agreement with Loi et al.24, but in contrast with a recent meta-analysis that concluded no significant association between increased TILs and lymph node metastasis risk 25.
Our knowledge about the association between TILs and AR is still limited. In a large cohort study about non-metastatic TNBC of LAR subtype, this tumor subset was found to exhibit lower median stromal TILs and to be less likely LPBC (≥ 50% TILs) compared to non LAR, although this not reached statistical significance 26, similarly to our study. However, we did not examine the genetic profiles of our AR + tumors to classify them into the LAR subtype. Other reports using IHC, described significant association between AR expression and lower levels of stromal TILs 10,16.
Studies about the cells subsets composition of TILs according to AR expression are very scarce. We found that median CD20 was significantly higher in AR- tumors compared to those AR+ (20% vs 7.5% respectively, p = 0.008). Whereas, mean CD3 was significantly lower in AR- vs. AR+ (80.7% vs 93.3% respectively, p = 0.007). Previous publications reported that CD8 + were more frequent in AR + than AR-tumors 11,27,28. In contrast, neither CD8 nor CD4 were statistically different between AR + and AR- tumors, in our study.
Based on two large-scale BC genomics data, evidence from a comprehensive analysis of 26 immune gene-sets including 15 immune cell type and function suggested that TNBC had the strongest tumor immunogenicity. Comparison of the immune infiltrate densities of different immune cell subpopulations demonstrated higher degree of infiltration in TNBC than non-TNBC, including CD3, CD8 and CD20 and others29.
T-lymphocytes represent the main lymphocytes type in the tumor microenvironment, and the majority of T lymphocytes express a cytotoxic effector phenotype (CD8+). Intra-tumoral and adjacent stromal CD8 + T-cell infiltration have been found to be significantly associated with ER negativity and basal phenotype 30,31. Infiltrating CD8 + T-cells have been reported in more than 60% of TNBC cases 31,32. In our study, CD8 was expressed in 100% of the cases with the mean of its expression was 73.4%.
The role of tumor-infiltrating B cells (CD20) as components of TILs in BC subtypes is still unclear. A positive correlation between higher numbers of total CD20 + B cells and ER and PR negativity, and basal phenotype has been reported 33. In our study, CD20 was expressed in 90.3% of the tumors and its median expression was significantly higher in AR- vs. AR + TNBC (20% vs. 7.5% respectively, p = 0.008)
Using a digital pathology computational workflow to quantify the spatial patterns of five immune markers (CD3, CD4, CD8, CD20, and FoxP3) in TNBC, Mi et al.34 demonstrated positive correlations between CD3 and CD8 cells. Similarly, we also showed a significant positive correlation between CD3 and CD8. Data from a study that used multiplexed ion beam imaging to simultaneously quantify in situ expression of 36 proteins in 41 TNBC patients suggested that all patients with B cells had also CD4 T cells and CD8 T cells 35. In contrast, we found in our study significant inverse correlations between CD20 and CD8, CD20 and CD3 as well as CD8 and CD4.
Immune cellular subpopulations in BC representing the innate immunity (natural killer, CD68 + and CD11c + cells) and adaptive immunity (CD3+ (CD8+, CD4+) cells and CD20 + cells) 36, worth thorough evaluation in TNBC, with the aim of understanding its clinical implications in BC management.
This study had comprehensively described the expression patterns of AR and TILs in TNBC. Moreover, the correlation between AR and the total and different TILs subpopulations was illustrated. TILs were evaluated by one pathologist who was blinded to the clinical characteristics and according to the International Working Group. However, our findings should be interpreted carefully. The limitations of our study include: i) the retrospective nature, ii) the small sample size, and iii) our survival data was not mature due to the short follow-up duration (median: 39 months).