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Contemporary Oncology/Współczesna Onkologia
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Original paper

High tumour-infiltrating lymphocytes correlate with distinct gene expression profile and favourable survival in single hormone receptor-positive breast cancer

Aleksandra Ciarka
1
,
Michał Kunc
1
,
Marta Popęda
1
,
Aleksandra Łacko
2, 3
,
Barbara Radecka
4, 5
,
Marcin Braun
6
,
Joanna Pikiel
7
,
Maria Litwiniuk
8
,
Katarzyna Pogoda
9
,
Ewa Iżycka-Świeszewska
10
,
Anna Zeller
11
,
Magdalena Niemira
11
,
Rafał Pęksa
1
,
Wojciech Biernat
1
,
Elżbieta Senkus
12

  1. Department of Pathology, Medical University of Gdańsk, Gdańsk, Poland
  2. Department of Oncology, Wrocław Medical University, Wrocław, Poland
  3. Department of Oncology, Breast Unit, Lower Silesian Oncology Centre, Wroclaw, Poland
  4. Department of Oncology, Institute of Medical Sciences, University of Opole, Opole, Poland
  5. Department of Clinical Oncology, Tadeusz Koszarowski Cancer Centre in Opole, Opole, Poland
  6. Department of Pathology, Chair of Oncology, Medical University of Łódź, Łódź, Poland
  7. Department of Oncology, Morski Hospital, Gdynia, Poland
  8. Department of Clinical Oncology, Greater Poland Cancer Centre, Poznań, Poland
  9. Department of Breast Cancer and Reconstructive Surgery, Maria Sklodowska Curie National Research Institute of Oncology, Warsaw, Poland
  10. Department of Pathology and Neuropathology, Medical University of Gdańsk, Gdańsk, Poland
  11. Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, Medical University of Gdańsk, Gdańsk, Poland
  12. Department of Oncology and Radiotherapy, Medical University of Gdańsk, Gdańsk, Poland
Contemp Oncol (Pozn) 2024; 28 (1): 75–83
Online publish date: 2024/05/03
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Introduction

Mutations in cancer cells generate new cancer cell-specific antigens (neoantigens), eliciting a host immune response against cancer cells [1]. In a favourable scenario, activated innate and adaptive immune cells may effectively eliminate neoplastic cells. However, tumours are capable of immune escape facilitating cancer development. The biomarker of the persistent immune response against cancer is the presence of tumour-infiltrating lymphocytes (TILs) inside the tumour (intratumoural TILs) and on its borders (stromal TILs). According to the recommendations of the International TILs Working Group TILs assessment should include the percentage of lymphocytes in the stroma within the invasive tumour borders, excluding areas of crush artifacts, necrosis, reactive stromal hyalinisation, and the site after a previous biopsy. Tumour-infiltrating lymphocytes should be evaluated as an averaged continuous parameter, without focusing on hotspots [2]. The abundance of TILs reflects the tumour mutational burden (TMB), which in breast cancer (BC) is average compared to other cancers, with only 5% of BC considered TMB high [3, 4]. The main component of TILs are T cells, B cells, monocytes, and NK cells, which usually account for about 75%, < 20%, < 10%, and < 5%, respectively [5]. The cellular composition and prevalence of TILs vary in different types of BCs. Some subpopulations promote whereas others suppress tumour progression. Inflammatory infiltration already occurs in benign ductal hyperplasia and ductal carcinoma in situ (DCIS) [6]. Interestingly, in DCIS dense TILs are associated with negative oestrogen receptor (ER) status and shorter recurrence-free survival (RFS) [7, 8]. Number of TILs, cellular composition of the inflammatory infiltrate, and expression of immune-related genes have predictive and prognostic significance in patients with HER2– amplified/overexpressing (HER2+ BC) and triple-negative breast cancer (TNBC). In the group of 294 BC patients, higher number of TILs was linked to ER– and HER2+ phenotype, larger tumour size, higher grade, higher Ki-67, and metastases to lymph nodes [9]. In the neoadjuvant setting high TILs in biopsy specimen predict favourable response to chemotherapy, and the presence of TILs in residual disease correlates with better outcomes in TNBC [10]. Similar trends were observed in HER2+ BC [11]. Much less is known about the role of TILs in hormone-dependent BC, especially single hormone receptor-positive BC (i.e. ER+/PgR– and ER–/PgR+). In a large study on ER+/HER2+ and ER+/HER2– tumours, high TILs were associated with unfavourable clinicopathological features but in patients treated with adjuvant chemotherapy correlated with longer distant disease-free survival (DFS) [12]. Interestingly, in the metasta- tic lesions, TILs are usually less abundant [13]. In a group of 94 patients with TILs assessed in BC metastases, lower abundance of TILs was found in younger patients, while higher TILs were correlated with prolonged overall survival (OS). Moreover, the composition of TILs differed depending on the location of the metastasis [14].

In BC the abundance of TILs correlates with immune-related gene expression profiles [15]. Interestingly, specific immunological profiles of BC microenvironment correlate with steroid hormone receptor status. Among luminal BCs, 3 subgroups were distinguished based on the expression of immune-related genes, one of which was characterised by higher amount of TILs and lower expression of ESR1/ESR2, supporting the relationship between weaker hormone receptor expression and enhanced immune reaction in BC [16].

In the current study, we aimed to investigate the role of TILs in single hormone receptor-positive BCs. We analysed associations with clinicopathological features, gene expression profiles, and long-term outcomes.

Material and methods

Study group

Tumours from 212 female patients diagnosed with single hormone receptor-positive BC were collected. Basic clinicopathological data (ER, PgR and HER2 status, Ki-67 proliferation index, grade, histological type, pathological tumour-node-metastasis [pTNM], use of neoadjuvant systemic treatment, age at diagnosis, date of diagnosis, date of relapse, and date of death) were retrieved from local databases. All ER–/PgR+ BC cases incorporated in the study had their phenotype adequately confirmed as described previously [17]. Subsequently, available microscopic slides were collected and subjected to analysis. Six patients were excluded from the subsequent analyses due to lack of slides for the assessment of TILs, leaving 206 patients in the study.

Evaluation of tumour-infiltrating lymphocytes

Assessment of stromal TILs was performed according to the International TILs Working Group guidelines by 2 pathologists (MK and RP) [2]. In the case of discrepant opinion, the final score was established after a discussion between pathologists. In general, one selected representative haematoxylin and eosin section from each case was analysed under 200–400× magnification. The percentage of the stromal TILs was assessed only within the invasive tumour borders. Tumour-infiltrating lymphocytes were scored as continuous variables and later subcategorised as low (0–5% of stromal TILs), and high (> 5% of stromal TILs).

RNA extraction

Total RNA was extracted from 52 samples prepared as formalin-fixed paraffin-embedded primary tumour tissue cores (4 cores of 0.6 mm diameter per patient, sampled from representative tumour areas) using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, US) with an Agilent RNA 6000 Pico Kit (Agilent Technologies).

nCounter gene expression assay

nCounter gene expression analysis technology was employed to decipher transcriptomic differences between single hormone receptor-positive BCs according to TILs abundance. Extracted RNA (4 µl) was pre-amplified using nCounter Low RNA Input Kit (NanoString Technologies, USA) with a dedicated Primer Pool covering sequences of 758 genes included in nCounter Breast Cancer 360 Panel (NanoString Technologies, USA). Pre-amplified samples were analysed using NanoString nCounter Analysis System (NanoString Technologies, USA) according to the manufacturer’s procedures for hybridisation, detection, and scanning, with BC 360 Panel Standard included in each run.

Background correction and normalisation were performed using nSolver 4.0 software (NanoString Technologies, USA). In brief, the background level was estimated by thresholding over the mean plus 2 standard deviations of negative control counts. Subsequently, the data were normalised according to the global mean of the counts of positive controls and the 3 most stably expressed housekeeping genes – OAZ1, TFRC, and PUM1. Breast Cancer 360 Panel Standard was used for batch calibration. The negative and positive control probes were included in the assay.

Following normalisation, low-expression genes (log2 me-- dian count in all samples < 6) were excluded, leaving 533 target genes for analysis. The collected RNA expression results were then analysed according to the TILs score of the corresponding primary tumour. Differences in gene expression between the TILs groups were presented as log2 fold change (log2FC). Genes with median-based log2FC ≥ 1 were considered as up-regulated and genes with log2FC ≤ –1 as down-regulated. Selected genes were associated with GO and Reactome terms using the STRING database [18].

Statistics

Statistical analyses were performed with the use of Statistica 13 (RRID:SCR_014213, Tibco, CA, USA) licensed to the Medical University of Gdańsk, and R statistical environment [19]. Categorical variables were compared by Fisher’s exact test or χ2 test with Yates correction. Continuous variables were analysed utilising the Wilcoxon test or Kruskal-Wallis test when applicable. Overall survival was defined as the time from the diagnosis to the date of death from any cause. Disease-free survival was defined as the time from the diagnosis to the date of relapse (either local or distant) or death and was analysed only in M0 cases (199 cases). Event-free survival (EFS) was calculated from the date of diagnosis to the earliest date of disease progression, relapse, or death from any cause. Kaplan-Meier curves were plotted to calculate the survival rates. The log rank test was used to compare survival between groups. Hazard ratios (HRs) were estimated using Cox regression analysis. A p-value < 0.05 was considered significant; in cases of multiple comparisons, p-values were adjusted with Benjamini-Hochberg correction. Boxplots and scatterplots were produced using the ‘ggplot2’ and ‘ggstatsplot’ packages [20, 21]. Kaplan-Meier curves were plotted using the ‘survminer’ and ‘ggsci’ packages [22, 23].

Data availability statement

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Results

In the study group, 130 (63.1%) patients were classified as ER+/PgR– and 76 (36.9%) as ER–/PgR+ (Table 1). The frequency of HER2 overexpression/amplification did not differ between groups (p = 0.718, χ2). The mean age of the patients was 61 years (range 28–91, interquartile range, IQR 53–69). Patients in ER+/PgR– were significantly older (p = 0.0218, Wilcoxon test), and their tumours had lower Ki-67 index (p < 0.001, Wilcoxon test). Tumours with ER–/PgR+ phenotype more commonly displayed grade 3 histology when compared to ER+/PgR– cancers that were predominantly grade 2 (p < 0.001, χ2). Neoadjuvant treatment was administered to 34 (44.7%) patients in the ER–/PgR+ group and 9 patients (7%) in the ER+/PgR– group (p < 0.001, χ2). ER–/PgR+ tumours were significantly larger (p < 0.001, Wilcoxon test) and had higher pT descriptors in TNM (p = 0.044, χ2). Moreover, ER–/PgR+ tumours were more commonly associated with the presence of distant metastases (p = 0.048, χ2). However, there was no significant difference in the frequency of nodal metastases (p = 0.187, χ2). Median follow-up was 38.5 months (IQR 21.5–76 months). Twenty-four (18.5%) ER+/PgR– patients died and 22 (16.9%) had a relapse/progression during follow-up, compared to 12 (15.8%) and 11 (14.5%) ER–/PgR+ patients, respectively. There were no significant differences in OS, DFS, and EFS between ER+/PgR– and ER–/PgR+ patients. When subcategorised according to HER2 status, ER–/PgR+/HER2+ patients had the best OS, followed by ER+/PgR–/HER2– patients, while ER+/PgR–/HER2+ and ER–/PgR+/HER2– patients had poorer OS (p = 0.006, log rank).

Table 1

Main clinicopathological features of the study groups

ParametersTILs analysis population,
N = 206
Gene expression analysis population,
N = 52
Featuren%n%
Phenotype        
  ER+/PgR–/HER2–9244.51426.9
  ER+/PgR/HER2+3818.41121.2
  ER–/PgR+/HER2–5426.31732.7
  ER+/PgR/HER2+2210.71019.2
T        
  18842.72038.5
  2–411455.33261.5
  n/a41.900
N        
  012359.73465.4
  1–37938.31834.6
  n/a41.900
M        
  019594.752100
  173.400
  n/a41.900
Grade        
  1209.723.8
  29244.71528.8
  39445.63567.3
Neoadjuvant treatment      
  No16278.64688.5
  Yes4320.9611.5
  n/a10.500

[i] n/a – not applicable, TILs – tumour-infiltrating lymphocytes * The percentages may not add up to 100% due to rounding.

Tumour-infiltrating lymphocytes score

Fifty-three (25.7%) cases were scored as TILs negative. The remaining cases showed at least 1% of stromal TILs (median 4%, mean 16.3%, IQR 0–20%). ER–/PgR+ tumours displayed significantly denser TILs than ER+/PgR– cases (p < 0.001, Wilcoxon test), irrespectively of HER2 status (Fig. 1). HER2 overexpression was associated with higher TILs but only in the ER+/PgR– subgroup (as a dichotomised variable – p = 0.006, χ2 and as a continuous variable – p = 0.003, Wilcoxon test). The abundance of TILs positively correlated with higher grade (p < 0.001, Kruskal-Wallis), higher Ki-67 (p < 0.001, R = 0.36, Spearman) (Fig. 2), and younger age (p = 0.008, R = –0.19, Spearman) but not with tumour size (p = 0.54, R = –0.04, Spearman), status of regional lymph nodes (p = 0.983, Wilcoxon test), and distant metastases at the time of diagnosis (p = 0.841, Wilcoxon test).

Fig. 1

Tumour-infiltrating lymphocytes density according to joint ER/PgR/HER2 phenotype

/f/fulltexts/WO/54090/WO-28-54090-g001_min.jpg
Fig. 2

Correlation between Ki-67 and tumour-infiltrating lymphocytes percentage in the whole cohort

/f/fulltexts/WO/54090/WO-28-54090-g002_min.jpg

Association of tumour-infiltrating lymphocytes status with long-term outcomes

Tumour-infiltrating lymphocytes percentage as a continuous variable was associated with better EFS with borderline statistical significance (HR 0.98, 95% CI: 0.96–0.99, p = 0.043, Cox), and it had no impact on DFS (HR 0.98, 95% CI: 0.96–1.0, p = 0.083, Cox) and OS (HR 0.98, 0.97–1.0, p = 0.102, Cox). When dichotomised, high-TILs (i.e. > 5%) correlated with better EFS in the whole cohort, as well as in the ER+/PgR– and ER–/PgR+ subgroups (Figs. 3–5). The 5-year EFS rate in the whole cohort was 87% in the high TILs group, and 67% in the low TILs group (p = 0.001, log rank). Significant differences in OS were noted in the whole cohort and in the ER–/PgR+ subgroup, whereas significant differences in DFS were observed in the whole cohort and in the ER+/PgR– subgroup (Suppl. Figs. 16).

Fig. 3

Kaplan-Meier curves for event-free survival in the whole co-hort according to tumour-infiltrating lymphocytes status

/f/fulltexts/WO/54090/WO-28-54090-g003_min.jpg
Fig. 4

Kaplan-Meier curves for event-free survival in ER+/PgR– patients according to tumour-infiltrating lymphocytes status

/f/fulltexts/WO/54090/WO-28-54090-g004_min.jpg
Fig. 5

Kaplan-Meier curves for event-free survival in ER–/PgR+ patients according to tumour-infiltrating lymphocytes status

/f/fulltexts/WO/54090/WO-28-54090-g005_min.jpg

Multivariate analysis was restricted to EFS due to insufficient numbers of DFS and OS events. In the whole cohort nodal status, the presence of distant metastases, TILs, and Ki-67 index were associated with EFS in multivariate analysis (Table 2). In the analysis restricted to ER–/PgR+ patients, only TILs status and distant metastases were associated with EFS (Table 3), whereas in ER+/PgR– patients, T-descriptor, nodal status, HER2, and TILs correlated with EFS (Table 4).

Table 2

Multivariate Cox regression analysis for event-free survival in the whole cohort

CharacteristicHR95% CIp-value
T (low vs. high)0.7540.328–1.7340.506
N (0–1 vs. 2–3)0.3280.134–0.8040.014
M (0 vs. 1)0.0690.017–0.289< 0.001
Grade (low vs. high)0.5890.117–0.2980.522
HER2 (neg vs. pos)0.9100.409–2.0280.819
Ki-67 (high vs. low)2.5271.024–6.2350.044
TILs (low vs. high)6.5492.536–16.914< 0.001

[i] HR – hazard ratio

Table 3

Multivariate Cox regression analysis for event-free survival in ER–/PgR+ cohort

CharacteristicHR95% CIp-value
T (low vs. high)1.8360.506–6.6610.355
N (low vs. high)0.4880.114–2.0840.333
M (0 vs. 1)0.0850.011–0.6540.018
Grade (low vs. high)n/an/an/a
HER2 (neg vs. pos)5.3080.844–33.3930.075
Ki-67 (high vs. low)0.6340.065–7.7720.712
TILs (low vs. high)5.2591.346–20.5440.018

[i] HR – hazard ratio, n/a – not applicable, TILs – tumour-infiltrating lymphocytes

Table 4

Multivariate Cox regression analysis for event-free survival in ER+/PgR– cohort

CharacteristicHR95% CIp-value
T (low vs. high)0.2530.076–0.8470.025
N (0–1 vs. 2–3)0.1890.050–0.7230.015
M (0 vs. 1)n/an/an/a
Grade (low vs. high)0.5480.097–3.090.994
HER2 (neg vs. pos)0.2640.089–0.7780.016
Ki-67 (high vs. low)2.450.804–7.4620.115
TILs (low vs. high)7.2371.768–29.6160.005

[i] HR – hazard ratio, n/a – not applicable, TILs – tumour-infiltrating lymphocytes

Association of tumour-infiltrating lymphocyte status with gene expression profile

The analysis covered 758 genes associated with BC biology, microenvironment, and immune response. When ER+/PgR– and ER–/PgR+ cases were analysed together, 50 DEGs were identified, including 39 up- and 11 down-regulated genes in the high TILs group (Table 5). The most upregulated genes included CXCL13 (C-X-C Motif Chemokine Ligand 13), BCL11A (BCL11 Transcription Factor A), CD27 (CD27 Molecule), GABRP (γ-aminobutyric acid type A receptor subunit Pi), GNLY (granulysin), and KLRK1 (killer cell lectin-like receptor K1). The genes with higher expression in high TILs tumours were associated with immune response, including NK chemotaxis regulation, antigen presentation, chemokine receptor binding, or interleukin and interferon signalling (Suppl. Table 1). Importantly, the upregulated expression of immune-related genes was consistent also across single hormone receptor-positive groups analysed separately (ER+/PgR–, ER–/PgR+) (Fig. 6). On the other hand, TILs-high tumours showed reduced expression of genes involved in tight-junction formation (CLDN3, OCLN), most probably resulting from lower abundance of epithelial cells in tissue cores sampled from high-TILs tumours.

Table 5

Differently expressed genes identified for tumour-infiltrating lymphocytes high (n = 25) vs. tumour-infiltrating lymphocytes low (n = 27) comparison in single hormone-receptor positive breast cancers (ER+/PgR– and ER–/PgR+ cases analysed together)

GeneTILs low (median)TILs high (median)Log2FCp-value
CCL56739181271.430.000
CCL249912501.320.000
LTB1113251.540.000
TAP1521331.350.000
ENPP22486891.470.000
NKG71394201.590.000
MARCO1948282.090.000
B3GNT321511802.450.000
CD270826.370.000
TGFB2781881.250.000
CXCL1302127.740.000
CXCL9591881.640.000
S100A7242043.060.000
GNLY0696.130.000
MFNG431111.350.000
CXCL10381191.610.000
BMP7231572.710.000
KRT6B481871.930.000
MIA592311.950.000
RARRES3371041.480.000
HLA-A283462691.150.000
CCL8161383.000.001
GABRP0746.230.001
IL2RB1855061.450.001
LAG3381041.440.001
HLA-B84219291.190.001
IDO1461741.900.001
IKZF32477911.680.002
PSMB9501341.410.002
PTGDS30881.540.002
JAK345951.060.003
EGFR31581549–1.030.003
PSAT1260108–1.260.003
KLRK10656.040.004
CMKLR122482–1.430.007
STAT142871.030.008
HLA-DRA396169–1.220.008
CD8A34721.050.010
HLA-DPB120289–1.180.010
PYCARD401176–1.180.013
BCL11A0926.540.016
IL7R321051.660.016
EDN19746–1.050.018
SOCS1421001.230.019
CD27417986–1.050.029
RASGRP1114330321.410.033
HLA-DQA113057–1.180.034
RUNX342981.190.037
CHI3L111653–1.100.038
PLA2G2A19387–1.140.049

[i] TILs – tumour-infiltrating lymphocytes Statistical significance was evaluated with the Mann-Whitney-Wilcoxon test.

Fig. 6

Venn diagram presenting the overlap of upregulated genes identified for tumour-infiltrating lymphocytes high vs. tumourinfiltrating lymphocytes low comparison in single receptor-positive breast cancers analysed together and separately

/f/fulltexts/WO/54090/WO-28-54090-g006_min.jpg

The only gene that had significantly different (higher) expression among TILs-high ER–/PgR+ (n = 14), comparing to TILs-high ER+/PgR– tumours (n = 11) was SOX10 (log2FC = 7.75, p = 0.0201, Wilcoxon test).

Discussion

Single hormone receptor-positive BC accounts for about 15–20% of BCs, and the vast majority display the ER+/PgR– phenotype, whereas ER–/PgR+ tumours are very rare [24]. The latter is a controversial group, and many authors disregard ER–/PgR+ cancers as technical artifacts. Moreover, given the infrequency of ER–/PgR+ tumours, there is a significant scarcity of studies dedicated to them, and we were able to include a relatively substantial number of such cases in our study. Nevertheless, multiple studies have confirmed their existence and demonstrated similarities to TNBC [25]. Therefore, the assessment of predictive and prognostic factors in this subpopulation is of great clinical importance. ER–/PgR+ BC are more common in younger women, they are more often poorly differentiated, larger, more frequently have high proliferation index, positive lymph node status, distant metastases, and high glucose metabolism, and carry worse prognosis compared to ER+/PgR+ tumours [26, 27]. Similarly, in the current study, ER+/PgR– BCs occurred in older patients and had lower Ki-67 index, while ER–/PgR+ BCs were larger, had higher grade, were more likely to have high TILs, and were more often associated with distant metastases. In one study survival in both groups of single hormone receptor-positive BCs was similar, although in ER–/PgR+ cancers higher expression of the PgR was associated with longer RFS and DFS [28]. On the contrary, in our cohort of ER–/PgR+/HER2– BCs, a negative prognostic significance of higher PgR expression was demonstrated [17]. In ER+ BCs a lack of PgR expression is predictive for higher incidence of pCR after neoadjuvant chemotherapy and poorer response to anti-ER treatment [26].

ER-positive BCs are less immunogenic compared to hormone receptor-negative BCs; therefore, they are less likely to have high TILs [8, 29], which may explain the lower prevalence of intermediate/high TILs in ER+/PgR– cancers in our study. Data on the prognostic and predictive value of TILs in BC are inconsistent. Moreover, there are few data on the prognostic significance of TILs in single hormone receptor-positive BCs. Most of the available data relate to triple-negative and HER2+ cancers, in which higher TILs are associated with better response to neoadjuvant chemotherapy and improved prognosis; however, in luminal BC high TILs seem to be a negative prognostic factor [3032], including worse OS [9]. This unexpected correlation may be explained by confounding effects of age, grade, and stage on the prognostic significance of TILs [32]. In the current study high TILs as a continuous parameter correlated positively with EFS in both groups of single hormone receptor-positive BCs. Alike in TNBC, we demonstrated that also in single hormone receptor-positive BCs, higher TILs are associated with higher tumour grade [7].

There are differences in gene expression among high and low TILs BCs. In the group of 200 HER2+ BCs, high TILs were associated with up-regulation of immune genes, down-regulation of stromal and ER genes, and better response to trastuzumab [33]. Similarly, in our group of single hormone receptor-positive BCs, we identified a relationship between high TILs and higher expression of immune-related genes, and lower expression of stromal genes. One of the immune-related genes which were up-regulated in our cohort is CXCL13, which plays an important role in the formation of organised structures composed of B, T lymphocytes, and dendritic cells – tertiary lymphoid structures (TLSs), which are involved in the immune response against cancer cells. CXCL13 has a prognostic significance in BC, especially in HER2+ BC, positively affecting DFS [5]. In addition, CXCL13/CXCR5 signalling is of significant prognostic importance in TNBC immunotherapy with atezolizumab [34].

Another interesting finding is the difference in SOX10 expression between TILs-high ER–/PgR+ and ER+/PgR– tumours. SOX10 is a useful immunohistochemical marker of both primary and metastatic TNBC even if standard breast lineage markers are reduced [35]. In a study on HER2+/ER– BC SOX10 expression was associated with features typically seen in TNBC, i.e. large acellular zone or prominent TILs [36]. On the other hand, HER2+/ER+ tumours are almost exclusively SOX10-negative [37]. Thus, higher expression of SOX10 mRNA in TILs-high ER–/PgR+ tumours may indicate their phenotypic resemblance to other ER– BCs, and future studies should elucidate the role of SOX10 as a potential biomarker of ER–/PgR+ BC.

The optimal cut-offs for TILs in various types of BC can vary depending on the specific subtype and the assessment method used. It is important to note that the field of TILs and their prognostic or predictive value in BC is still evolving, and there are no universally accepted cut-offs for all subtypes. It ranges from 5 to 75% in different research studies or clinical trials, and the cut-offs are generally higher in HER2+ BCs and TNBC compared to luminal BCs [12, 3841]. In our study we did not predefine the cut-off for TILs assessment, and long-term outcomes analysis was based on the median TILs percentage identified in our cohort, and this is a potential limitation of our study.

Other major limitations are the study’s retrospective nature and the small size of the cohort. Moreover, while we assessed only chemo-naïve cases, in some instances, TILs were assessed in biopsy material or surgical specimens, depending on the availability of specimens, which might have impacted the results. Thus, our findings should be analysed with caution.

Conclusions

Our study confirms that single hormone receptor-positive BCs and both their subgroups (ER+/PgR– and ER–/PgR+) are biologically distinct groups of BCs in which the assessment of TILs carries prognostic significance. Additionally, in patients with single hormone receptor-positive BC the presence of TILs is associated with distinct gene expression profiles and better outcomes. Our findings suggest that the assessment of TILs should be considered as part of routine assessment of single hormone receptor-positive BC, although the role of TILs in clinical decision-making in these BC subtypes warrants further research. Future studies should validate these findings in larger cohorts and address the potential role of TILs as a predictive factor in these tumours.

Disclosures

The study was retrospective in nature, it contains no direct personal data or personally identifiable information; patients’ consent was not required in accordance with local or national guidelines. This study protocol was reviewed and approved by Bioethics Committee for Scientific Research of Medical University of Gdansk, approval number NKBBN/119-620/2021 and NKBBN/119/2018.

Assistance with the article

None.

This research was funded by the National Science Centre, Poland (grant 2017/25/B/NZ5/00656).

Conflicts of interest

None.

References

1 

Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science 2015; 348: 69-74.

2 

Salgado R, Denkert C, Demaria S, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol 2015; 26: 259-271.

3 

Barroso-Sousa R, Jain E, Cohen O, et al. Prevalence and mutational determinants of high tumor mutation burden in breast cancer. Ann Oncol 2020; 31: 387-394.

4 

Thomas A, Routh ED, Pullikuth A, et al. Tumor mutational burden is a determinant of immune-mediated survival in breast cancer. Oncoimmunology 2018; 7: e1490854.

5 

Gu-Trantien C, Loi S, Garaud S, et al. CD4+ follicular helper T cell infiltration predicts breast cancer survival. J Clin Invest 2013; 123: 2873-2892.

6 

Hussein MR, Hassan HI. Analysis of the mononuclear inflammatory cell infiltrate in the normal breast, benign proliferative breast disease, in situ and infiltrating ductal breast carcinomas: preliminary observations. J Clin Pathol 2006; 59: 972-977.

7 

Toss MS, Miligy I, Al-Kawaz A, et al. Prognostic significance of tumor-infiltrating lymphocytes in ductal carcinoma in situ of the breast. Mod Pathol 2018; 31: 1226-1236.

8 

Hussein MR, Hassan HI. Analysis of the mononuclear inflammatory cell infiltrate in the normal breast, benign proliferative breast disease, in situ and infiltrating ductal breast carcinomas: preliminary observations. J Clin Pathol 2006; 59: 972-977.

9 

Kurozumi S, Matsumoto H, Kurosumi M, et al. Prognostic significance of tumour-infiltrating lymphocytes for oestrogen receptor-negative breast cancer without lymph node metastasis. Oncol Lett 2019; 17: 2647.

10 

Loi S, Dushyanthen S, Beavis PA, et al. RAS/MAPK activation is associated with reduced tumor-infiltrating lymphocytes in triple-negative breast cancer: therapeutic cooperation between MEK and PD-1/PD-L1 immune checkpoint inhibitors. Clin Cancer Res 2016; 22: 1499-1509.

11 

Liu S, Lachapelle J, Leung S, Gao D, Foulkes WD, Nielsen TO. CD8+ lymphocyte infiltration is an independent favorable prognostic indicator in basal-like breast cancer. Breast Cancer Res 2012; 14: R48.

12 

Criscitiello C, Vingiani A, Maisonneuve P, Viale G, Viale G, Curigliano G. Tumor-infiltrating lymphocytes (TILs) in ER+/HER2-breast cancer. Breast Cancer Res Treat 2020; 183: 347-354.

13 

Cimino-Mathews A, Ye X, Meeker A, Argani P, Emens LA. Metastatic triple-negative breast cancers at first relapse have fewer tumor-infiltrating lymphocytes than their matched primary breast tumors: a pilot study. Hum Pathol 2013; 44: 2055-2063.

14 

Dieci MV, Tsvetkova V, Orvieto E, et al. Immune characterization of breast cancer metastases: prognostic implications. Breast Cancer Res 2018; 20: 62.

15 

Li B, Geng R, Wu Q, et al. Alterations in immune-related genes as potential marker of prognosis in breast cancer. Front Oncol 2020; 10: 333.

16 

Zhu B, Tse LA, Wang D, et al. Immune gene expression profiling reveals heterogeneity in luminal breast tumors. Breast Cancer Res 2019; 21: 147.

17 

Kunc M, Pęksa R, Cserni G, et al. High expression of progesterone receptor may be an adverse prognostic factor in oestrogen receptor-negative/progesterone receptor-positive breast cancer: results of comprehensive re-evaluation of multi-institutional case series. Pathology 2022; 54: 269-278.

18 

Szklarczyk D, Gable AL, Lyon D, et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 2019; 47: D607-D613.

19 

RCT. R: A language and environment for statistical computing. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna 2020.

20 

Patil I. Visualizations with statistical details: the “ggstatsplot” approach. J Open Source Softw 2021; 6: 3167.

21 

Wickham H. ggplot2: elegant graphics for data analysis. Springer 2016.

22 

Kassambara A., Kosinski M., Biecek P. Survminer: drawing survival curves using “ggplot2”. R package version 0.4.8. Available from: https://cran.r-project.org/web/packages/survminer/. 2020.

23 

Xiao N. ggsci: Scientific Journal and Sci-Fi Themed Color Palettes for “ggplot2. R package version 2.7. 2017.

24 

Rakha EA, El-Sayed ME, Green AR, et al. Biologic and clinical characteristics of breast cancer with single hormone receptor positive phenotype. J Clin Oncol 2007; 25: 4772-4778.

25 

Loi S, Michiels S, Salgado R, et al. Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial. Ann Oncol 2014; 25: 1544-1550.

26 

Kunc M, Popęda M, Biernat W, Senkus E. Lost but not least-novel insights into progesterone receptor loss in estrogen receptor-positive breast cancer. Cancers (Basel) 2021; 13: 4755.

27 

Bae SY, Kim S, Lee JH, et al. Poor prognosis of single hormone receptor-positive breast cancer: similar outcome as triple-negative breast cancer. BMC Cancer 2015; 15:138.

28 

Chan M, Chang MC, González R, et al. Outcomes of estrogen receptor negative and progesterone receptor positive breast cancer. PLoS One 2015; 10: e0132449.

29 

Dieci MV, Mathieu MC, Guarneri V, et al. Prognostic and predictive value of tumor-infiltrating lymphocytes in two phase III randomized adjuvant breast cancer trials. Ann Oncol 2015; 26: 1698-1704.

30 

Mao Y, Qu Q, Zhang Y, Liu J, Chen X, Shen K. The value of tumor infiltrating lymphocytes (TILs) for predicting response to neoadjuvant chemotherapy in breast cancer: a systematic review and meta-analysis. PLoS One 2014; 9: e115103.

31 

Denkert C, Loibl S, Noske A, et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010; 28: 105-113.

32 

Denkert C, von Minckwitz G, Darb-Esfahani S, et al. Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy. Lancet Oncol 2018; 19: 40-50.

33 

Kim SR, Gavin PG, Pogue-Geile KL, et al. Abstract 2837: a surrogate gene expression signature of tumor infiltrating lymphocytes (TILs) predicts degree of benefit from trastuzumab added to standard adjuvant chemotherapy in NSABP (NRG) trial B-31 for HER2+ breast cancer. Cancer Res 2015; 75(15_Suppl.): 2837-2837.

34 

Zhang Y, Chen H, Mo H, et al. Single-cell analyses reveal key immune cell subsets associated with response to PD-L1 blockade in triple-negative breast cancer. Cancer Cell 2021; 39: 1578-1593.e8.

35 

Tozbikian GH, Zynger DL. A combination of GATA3 and SOX10 is useful for the diagnosis of metastatic triple-negative breast cancer. Hum Pathol 2019; 85: 221-227.

36 

Weisman P, Yu Q, Xu J. Clinicopathological characteristics of the SOX10+ subset of HER2+ breast cancer. Ann Diagn Pathol 2023; 63: 152087.

37 

Qazi MS, McGregor SM. Combined use of SOX10 and GATA3 in mammary carcinoma. Pathol Res Pract 2020; 216: 152801.

38 

El Bairi K, Haynes HR, Blackley E, et al. The tale of TILs in breast cancer: a report from The International Immuno-Oncology Biomarker Working Group. NPJ Breast Cancer 2021; 7: 150.

39 

Dieci MV, Tsvetkova V, Griguolo G, et al. Integration of tumour infiltrating lymphocytes, programmed cell-death ligand-1, CD8 and FOXP3 in prognostic models for triple-negative breast cancer: analysis of 244 stage I–III patients treated with standard therapy. Eur J Cancer 2020; 136: 7-15.

40 

Iseki Y, Shibutani M, Maeda K, et al. A new method for evaluating tumor-infiltrating lymphocytes (TILs) in colorectal cancer using hematoxylin and eosin (H-E)-stained tumor sections. PLoS One 2018; 13: e0192744.

41 

De Jong VMT, Wang Y, Ter Hoeve ND, et al. Prognostic value of stromal tumor-infiltrating lymphocytes in young, node-negative, triple-negative breast cancer patients who did not receive (neo) adjuvant systemic therapy. J Clin Oncol 2022; 40: 2361-2374.

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