Background
The presence of tumor-infiltrating lymphocytes (TILs) reflects an adaptive anti-tumor immune response [
1]. Although the immunogenicity of breast cancer has not been strongly considered in research or clinical practice, TILs are emerging as biomarkers for predicting clinical response to chemotherapy for breast cancer [
2‐
8]. Recent translational studies from large prospective trials suggest that tumors with high TIL levels show a superior prognosis compared to tumors with low TIL levels in breast cancer patients treated with neoadjuvant or adjuvant chemotherapy [
2‐
8]. The correlation between high TIL levels and improved clinical outcome was most prominent in triple-negative breast cancer (TNBC) [
2,
6‐
8]. After the initial report from the BIG2–98 trial, a positive relationship between TIL levels and clinical outcome in TNBC was validated in independent cohorts in two clinical trials [
2,
6].
Interestingly, TIL levels have not been shown to be prognostic in patients with estrogen receptor (ER)–positive tumors receiving adjuvant chemotherapy. However, a recent study reported that patients with a high level of TILs have a greater chance of obtaining a pathological complete response (pCR), even in hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancers [
9]. In addition, a recent meta-analysis showed that HR-positive/HER2-negative tumors with high TIL levels at baseline biopsy, prior to chemotherapy, have a higher probability of pCR (
n = 1366) [
10].
The 21-gene recurrence score (RS) is derived from a polymerase chain reaction-based test that examines 16 tumor related genes and 5 reference genes to analyze cell proliferation, estrogen signaling, and invasion capacity. It has been shown to predict the clinical benefit of chemotherapy for individuals with ER-positive/HER2-negative breast cancer [
11,
12]. Furthermore, previous studies have shown that tumors with a high RS have a higher rate of pCR in the neoadjuvant setting [
13,
14].
Both TIL levels and the RS could serve as biomarkers associated with chemotherapy responsiveness in HR-positive/HER2-negative breast cancer. Moreover, identifying suitable patients among those with HR-positive/HER2-negative cancers who derive clinical benefit from chemotherapy has been a critical issue; however, the relationship between the two markers has not been intensively examined.
In this study, we examined the association between TIL levels and RS in patients with ER-positive/HER2-negative breast cancer who did not receive neoadjuvant chemotherapy (NAC).
Discussion
Many investigators have extensively studied the clinical value of TILs or RS in breast cancer; however, there are only a few studies regarding an association between Oncotype DX-RS and TILs in a specific subset of breast cancer, ER-positive/HER2-negative breast cancer.
We previously hypothesized that tumors with extensive lymphocytic infiltration might have high RS because patients with high TIL levels have a higher rate of pCR to preoperative chemotherapy. Additionally, ER-positive/HER2-negative patients with high RS derive clinical benefit from adjuvant chemotherapy. If a positive correlation could be found between the two markers, further studies testing TIL levels as a biomarker that predicts clinical benefit for adjuvant chemotherapy in ER-positive/HER2-negative cancer would be warranted.
In this study, we found a weak correlation between RS and TIL levels, wherein tumors with high TIL levels tended to have a higher RS. Moreover, high-TIL tumors had significantly higher median RS than low- or intermediate TIL tumors. A weak correlation between the two continuous markers was also observed. However, a close association between categorical RS and TIL levels was not observed. In addition, multivariate analysis revealed that RS was not an independent factor associated with high TIL levels.
These biomarkers are both associated with aggressive tumor biology, but they depend on different pathways in tumor progression. RS mainly reflects the biological characteristics of tumor cells, while TILs are a type of immune cells, reflecting the immune tumor microenvironment. Therefore, it is not surprising that RS was not highly correlated with TIL levels in our analysis.
On examining 21 genes in RS, the CD68 is the only gene obviously associated with immune function in breast cancer. CD68 is a heavily glycosylated glycoprotein that is highly expressed by macrophages and is recognized as a marker for tumor-associated macrophages [
21]. In a hypothesis-generating study using human breast carcinoma treated with or without neoadjuvant chemotherapy, CD68 was found to play a role in the immune microenvironment of breast cancer [
21]. Specifically, several studies using large cohorts of breast cancer patients have revealed that the expression of CD68 in tumor tissue is associated with higher grade [
22,
23], increased angiogenesis [
24‐
26], and reduced disease-free survival [
25,
27,
28]. The impact of CD68 expression on the final 21-gene RS has not been of much interest to researchers. In our study, the relationship between TILs and CD68 levels remained unexplored. Hence, further studies correlating CD68 and TIL levels in ER-positive/HER2-negative cancer will shed light on the tumor-immune interaction in this subset.
There might be an association between HER2 expression and TIL levels because there is solid evidence—from translational studies using tumor samples from prospective trials—that high TIL levels correlate with response to anti-HER2 therapy [
29] and that patients with HER2-positive breast cancer receiving adjuvant trastuzumab showed improved survival [
30]. However, similar to real practice, we only used 21-gene RS for HER2-negative cases; hence, it might be difficult to find a link between TIL levels and HER2 expression in this study.
A growing body of evidence suggests that TIL levels are associated with the response to chemotherapy via several distinct mechanisms of the tumor-immune interaction [
2‐
8]. Chemotherapy can increase the vulnerability of tumor cells to lytic action by cytotoxic CD8+ T cells, and selectively reduce circulating regulatory T cells as well as restore the function of natural killer (NK) cells [
31]. Furthermore, the proliferation of T cells and the lytic function of NK cells were promoted by chemotherapy in breast cancer patients treated with adjuvant taxanes [
32]. These findings are connected with recent studies showing that patients with lymphocyte-predominant breast cancer (LPBC) had a better prognosis compared to non-LPBC among patients treated with chemotherapy [
2,
4,
6]. However, the association between TIL levels and oncologic outcome was reproducibly confirmed in TNBC, but not in ER-positive/HER2-negative breast cancer [
2,
4,
6].
A meta-analysis with 1366 HR-positive/HER2-negative patients receiving NAC found that patients with high TIL levels had a worse prognosis than patients with low TIL levels, but a similar outcome compared to patients with intermediate TIL levels, even though they had a higher rate of pCR [
10]. By contrast, patients with high RS had worse outcome compared to patients with low or intermediate RS [
11,
12]. Taken together with our data, it appears that RS and TIL levels stratify patients into subgroups based on different tumor biology. TIL levels are relatively low in ER-positive/HER2-negative tumors than in other subtypes. Although inconsistent cut-off points (50% or 60%) have been applied to define high TIL levels, the rate of high-TIL tumors is 2.9%–13% in ER-positive/HER2-negative patients [
6,
10]. Our data are in line with these findings, with a rate of high-TIL tumors of only 6.6% (13 of 198; cut-off of 60%).
The relationship between endocrine responsiveness and the degree of TILs needs to be explored. Recently, Dieci et al. evaluated the association between TIL levels in core biopsies and Ki-67 suppression after letrozole treatment with/without lapatinib for 24 weeks [
33]. They found that patients with high TIL levels (cut-off of 10%) more frequently had a relative Ki-67 suppression ≥50% from baseline compared to patients with low TIL levels, without statistical significance (55% vs. 35%). Dunbier et al. showed that an inflammatory signature is associated with a poor response to 2-week aromatase inhibitor treatment, implying that TIL levels might be associated with endocrine resistance [
34]. Further clinical and preclinical research should be performed.
Previously, Krishnamurti et al. compared TIL levels with RS in ER-positive cancer [
35], and their study shows several differences from ours. Krishnamurti et al. did not exclude HER2-positive tumors from ER-positive tumors, whereas we only included ER-positive/HER2-negative tumors because the RS assay is applied clinically for those specific tumors. Moreover, the TIL level cut-offs are different between the studies. Krishnamurti et al. set the high TIL cut-off of as 50%, whereas we set ours at 60%, per the study by Denkert [
10]. In addition, Krishnamurti et al. showed a negative correlation between TIL levels and RS, implying that TIL is a favorable marker that differs from our study. Further studies with larger cohorts are required to determine the prognostic effects of TIL levels in ER-positive/HER2-negative breast cancer.
A major limitation of our study, in addition to the short follow-up duration, is the absence of survival analyses among the groups divided by the two markers. The clinical outcomes of our study population might help refine prognostic discrimination according to these markers. Additionally, evaluation of TIL levels could differ according to the type of sample, such as core biopsies or surgical specimens. In this study, we evaluated the TIL levels using only surgically resected samples, but several studies used samples from core biopsies. Despite these limitations, our analysis comparing RS and TIL levels lays the groundwork for future research on the tumor-immune interaction in ER-positive/HER2-negative breast cancer.
Intriguingly, in our patients receiving RS-guided treatments, chemotherapy was more frequently performed among patients with high TILs and less frequently among those with low or intermediate TILs. Further studies are required considering the clinical outcomes to determine whether TIL levels could clinically benefit chemotherapy for endocrine-sensitive tumors.