Clinicopathological significance of androgen receptor expression and tumor infiltrating lymphocytes in triple-negative breast cancer: a retrospective cohort study

doi:10.21203/rs.3.rs-5018547/v1

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Clinicopathological significance of androgen receptor expression and tumor infiltrating lymphocytes in triple-negative breast cancer: a retrospective cohort study

https://doi.org/10.21203/rs.3.rs-5018547/v1

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Background

Triple-negative breast cancer (TNBC) is aggressive and has limited treatment options. This study explored the clinical significance of androgen receptor (AR) expression and tumor-infiltrating lymphocytes (TILs) in predicting neoadjuvant chemotherapy (NAC) resistance in TNBC, hypothesizing that AR/TIL classification can identify NAC-resistant subgroups and improve the understanding of apocrine differentiation.

Methods

This retrospective study included 96 consecutive patients with TNBC treated with NAC. AR immunostaining was defined as positive if ≥ 1% of the tumor cell nuclei were stained and stromal TIL levels were assessed, with high levels defined as ≥ 50%. Apocrine differentiation was detected using an anti-15-PGDH antibody. Pathological response to NAC was also evaluated.

Results

Overall, 38% (n = 36) of the patients achieved pathological complete response (pCR). AR⁺/TIL^low tumors had a significantly higher non-pCR rate (79%, 23 of 29 patients) and were resistant to NAC. Kaplan-Meier plots showed significant differences in overall survival (OS) and distant metastasis-free survival (DMFS) among the four AR/TIL subgroups (OS, P = 0.047; DMFS, P = 0.0053). All seven cases with some degree of apocrine differentiation were AR⁺/TIL^low, 15-PGDH-positive, and showed NAC resistance. AR⁺/TIL^low status as an independent predictor of non-pCR (OR 0.32, P = 0.032). pCR predicted better prognosis (OS, HR 0.14, P = 0.010; DMFS, HR 0.11, P = 0.003), whereas AR⁺/TIL^low status was not significantly associated with OS or DMFS.

Conclusions

AR/TIL classification was used to identify TNBC subgroups with distinct NAC responses and prognoses. AR⁺/TIL^low TNBC, including cases with some degree of apocrine differentiation, are NAC-resistant, suggesting the need for alternative therapies.

Pathology

Oncology

Triple-negative breast cancer

androgen receptor

tumor-infiltrating lymphocytes

apocrine

15-prostaglandin dehydrogenase

Triple-negative breast cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen receptor (ER) and progesterone receptor (PR) expression, along with the lack of overexpression or amplification of human epidermal growth factor receptor 2 (HER2). TNBC is associated with rapid progression and poor prognosis. Compared with other breast cancer subtypes, TNBC has fewer targeted therapeutic options and is often managed with neoadjuvant chemotherapy (NAC). NAC responses are evaluated histopathologically, and pathological complete response (pCR) is associated with improved survival outcomes[1].

TNBCs have high pCR rates, but some are resistant to NAC[1, 2]. Predicting the therapeutic effect of NAC by pre-treatment needle biopsy may avoid the adverse effects of treatment and improve prognosis. Androgen receptors (ARs) have been the focus of attention in the development of novel treatment strategies for NAC-resistant TNBCs[3]. AR expression in TNBC is closely associated with the molecular subtype of luminal androgen receptor (LAR)[3–5]. LAR is a subtype that is resistant to NAC but has a good prognosis[6]. However, the association between the AR expression status in TNBC and NAC responsiveness or prognosis is inconclusive.

Tumor-infiltrating lymphocytes (TILs) are well-known histological features that can predict chemotherapy response and prognosis[7–9]. To date, no study has examined whether combined immunostaining of AR expression and TILs status in TNBC is predictive of NAC resistance. Among these AR/TIL classifications, AR-positive and low TILs may share features of apocrine carcinoma (AC)[10, 11]. According to the World Health Organization tumor classification, pure AC is defined as an apocrine morphology of > 90% tumor cells and a characteristic steroid receptor profile (ER−/PR−/AR+)[12].

Pure AC with triple-negative status, recently termed triple-negative apocrine carcinomas (TNAC), tends to have lower TIL levels and does not completely respond to NAC[13]. TNAC is clinically less aggressive than TNBC; therefore, breast surgeons and oncologists should consider surgical treatment rather than NAC[13]. However, patients with TNBC who exhibit only focal or some degree of apocrine differentiation cannot be clearly classified as TNAC and can thus be treated as TNBC of no special type (TNBC-NST). The therapeutic response and prognosis of TNBC with some degree of apocrine differentiation have not yet been elucidated.

Therefore, this study examined the clinical significance of the AR/TIL classification in TNBC. We hypothesized that AR/TIL classification would stratify the risk of TNBC and identify subgroups with NAC resistance. We also assessed the clinicopathological features of the TNBCs exhibiting apocrine differentiation in relation to this classification.

Patient selection

After obtaining approval from the institutional ethics review board, we retrospectively searched for patients with TNBC who underwent surgery at St. Luke’s International Hospital between January 2010 and December 2017. This study included only patients with primary disease who were treated with NAC. Patients with multiple concurrent cases were excluded from this study. Clinical information was collected from medical charts.

Histopathological evaluation

Histological diagnoses were reviewed for all cases. Hematoxylin and eosin (HE) staining and immunohistochemistry were performed on formalin-fixed paraffin-embedded specimens. ER, PR, HER2, and Ki67 assays performed at diagnosis were reviewed. The Ki67 index was digitally measured at the hotspots. Digital images of the tumors were captured by a trained pathologist and scored digitally using Definiens Tissue Studio® (v3.6.1; Definiens AG, Munich, Germany). Nottingham histological grade was routinely reported. The pathologist double-checked and manually counted the index values when necessary. ER, PR, and HER2 results were reported according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines[14, 15]. HER2 fluorescence in situ hybridization assay was performed on HER2 IHC 2+ (equivocal) samples to confirm HER2 status.

Stromal TILs were assessed in biopsy specimens according to the guidelines of the International TIL Working Group[7]. A high stromal TILs level was defined as ≥ 50%. The pathological response to NAC was evaluated in the surgically resected specimens. pCR was defined as the absence of invasive carcinoma in the breast and axillary lymph nodes, ypT0/is ypN0 in the Union for International Cancer Control TNM staging system[16].

In this study, we defined TNBC with some degree of apocrine differentiation (TNBC-Apo) as having apocrine differentiation in ≤ 90% of tumors. To detect partial or minimal apocrine differentiation, we performed immunostaining with an anti-15-PGDH antibody, which has been reported to be a specific marker for apocrine carcinoma[17]. All cases were immunohistochemically analyzed for AR and 15-PGDH expression in the biopsy specimens. AR immunostaining was defined as positive if ≥ 1% of tumor cell nuclei were stained, according to the Allred score. 15-PGDH was defined as positive if ≥ 1% of tumor cells were positive. The antibodies and staining protocols used are listed in Online Resource 1. Staining was performed using the ultraView DAB Universal Kit (Ventana, Tucson, AZ, USA) and BenchMark Ultra (Ventana, Tucson, AZ, USA).

Statistical analysis

Logistic regression analysis was used to identify the predictors of pCR. A multivariate logistic regression model was constructed by selecting variables using a stepwise method based on univariate p values (threshold of 0.1). Overall survival (OS) was defined as the time from the date of primary surgery to the date of death, regardless of the cause, whereas distant metastasis-free survival (DMFS) was defined as the time from the date of primary surgery to the date of the first distant metastasis. Kaplan-Meier plots and log-rank tests were used to assess OS and DMFS. A Cox proportional hazard regression model was constructed by considering the effects of events per variable. Statistical differences were analyzed using Fisher’s exact test for categorical data and the Kruskal-Wallis test for continuous data. Statistical significance was set at P < 0.05. Statistical analyses were performed using R Statistical Software (v4.4.0; R Foundation for Statistical Computing, Vienna, Austria).

Patient demographics

In total, 180 consecutive patients with TNBC were identified. Biopsy specimens were obtained from 102 patients. Six patients were excluded after applying the exclusion criteria. Two patients, initially diagnosed with triple-negative status on needle biopsy, were found to be HER2 positive in the surgical specimens. The two others had multiple concurrent cases. One patient had NAC interrupted too early for the pathological response to be determined, and the remaining patient had a special histological type (spindle cell carcinoma). Finally, 96 patients were included in this study (Fig. 1). Among the 96 patients, 36 (38%) achieved pCR. Four cases with no residual invasive cancer in the primary tumor but residual cancer metastasis in the lymph nodes (ypT0N1 in one case, ypTisN1 in one case, and ypT0N2 in two cases) were all considered non-pCR.

Patient characteristics according to the AR/TIL classification are shown in Table 1. Significant differences were observed in the Ki67 proliferation index (P < 0.001), 15-PGDH status (P = 0.001), distant metastatic recurrence (P = 0.013), and the follow-up period (P = 0.040). All seven 15-PGDH-positive cases were AR⁺/TIL^low. The AR⁺/TIL^low group tended to have a relatively low Ki67 index compared to the other groups (median 60.5%, IQR 48.0–76.3%). In terms of pathological response, the non-pCR rate tended to be higher in the low TILs group (44/63), particularly in the AR⁺/TIL^low group, which had the highest non-pCR rate (23/29, 79%). Although not statistically significant, there was a trend toward differences in mortality rates among the four groups (P = 0.082). Histological grade 2 was most common in the AR⁺/TIL^low group (9/29, 31%) (P = 0.064). No statistically significant differences were observed in age or menopausal status between groups. NAC regimens included anthracycline- and taxane-based (97%, 93/96), anthracycline-based (1%, 1/96), and taxane-based (2%, 2/96) regimens. None of the patients in this study had received anti-androgen therapy or immune checkpoint inhibitors. Germline BRCA gene mutations were evaluated in 46 of 96 patients. Among these, six patients harbored BRCA1 mutations and two patients harbored BRCA2 mutations. One patient had a BRCA2 variant of unknown significance, which was considered negative. Most patients with germline BRCA mutations were AR-negative, but one patient with germline BRCA1 mutation had AR-positive TNBC.

Table 1

Patient characteristics according to the AR/TIL classification
Characteristic	AR⁺/TIL^high, n = 13¹	AR⁺/TIL^low, n = 29¹	AR^–/TIL^high, n = 20¹	AR^–/TIL^low, n = 34¹	p-value²
Age (y)					0.55
Median (IQR)	45 (40–51)	50 (41–63)	44 (37–63)	48 (40–57)
Menopausal status					0.48
Pre-menopause	10 (77%)	15 (52%)	11 (55%)	21 (62%)
Post-menopause	3 (23%)	14 (48%)	9 (45%)	13 (38%)
Clinical tumor size (cT)					0.57
T1	3 (23%)	7 (24%)	7 (35%)	10 (29%)
T2	8 (62%)	16 (55%)	11 (55%)	18 (53%)
T3	2 (15%)	1 (3.4%)	0 (0%)	4 (12%)
T4	0 (0%)	5 (17%)	2 (10%)	2 (5.9%)
Clinical nodal status (cN)					> 0.99
N0	9 (69%)	17 (59%)	12 (60%)	20 (59%)
N1	1 (7.7%)	5 (17%)	3 (15%)	4 (12%)
N2	2 (15%)	3 (10%)	2 (10%)	5 (15%)
N3	1 (7.7%)	4 (14%)	3 (15%)	5 (15%)
Clinical stage					0.96
Stage I	1 (7.7%)	5 (17%)	3 (15%)	7 (21%)
Stage II	9 (69%)	15 (52%)	11 (55%)	17 (50%)
Stage III	3 (23%)	9 (31%)	6 (30%)	10 (29%)
Histological grade					0.064
Grade 2	2 (15%)	9 (31%)	3 (15%)	2 (5.9%)
Grade 3	11 (85%)	20 (69%)	17 (85%)	32 (94%)
Ki67 index (%)					< 0.001*
Median (IQR)	63.6 (54.8–71.0)	60.5 (48.0–76.3)	74.8 (62.4–81.6)	80.6 (75.0–89.9)
HER2 Score					0.12
Score 0	5 (38%)	16 (55%)	12 (60%)	25 (74%)
Score 1+	8 (62%)	13 (45%)	7 (35%)	9 (26%)
Score 2+/ISH-negative	0 (0%)	0 (0%)	1 (5.0%)	0 (0%)
15-PGDH					0.001*
Positive	0 (0%)	7 (24%)	0 (0%)	0 (0%)
Negative	13 (100%)	22 (76%)	20 (100%)	34 (100%)
Surgery					0.056
Conservative surgery	12 (92%)	21 (72%)	12 (60%)	18 (53%)
Mastectomy	1 (7.7%)	8 (28%)	8 (40%)	16 (47%)
Pathological response					0.064
pCR	8 (62%)	6 (21%)	9 (45%)	13 (38%)
Non-pCR	5 (38%)	23 (79%)	11 (55%)	21 (62%)
NAC regimen					0.67
Anthracycline and taxane based	13 (100%)	29 (100%)	20 (100%)	31 (91%)
Anthracycline based	0 (0%)	0 (0%)	0 (0%)	1 (2.9%)
Taxane based	0 (0%)	0 (0%)	0 (0%)	2 (5.9%)
Adjuvant radiation therapy	12 (92%)	27 (93%)	18 (90%)	26 (76%)	0.27
Adjuvant chemotherapy	1 (7.7%)	1 (3.4%)	2 (10%)	6 (18%)	0.33
Germline BRCA mutation					0.59
BRCA1 mutation	1(7.7%)	0 (0%)	2 (10%)	3 (8.8%)
BRCA2 mutation	0 (0%)	0 (0%)	1 (5.0%)	1 (2.9%)
Negative	6 (46%)	11 (38%)	6 (30%)	15 (44%)
Unknown	6 (46%)	18 (62%)	11 (55%)	15 (44%)
Mortality	1 (7.7%)	5 (17%)	2 (10%)	12 (35%)	0.082
Distant metastatic recurrence	1 (7.7%)	6 (21%)	2 (10%)	15 (44%)	0.013*
Follow-up (mo)					0.040*
Median (IQR)	92 (82–119)	88 (79–110)	121 (90–130)	85 (39–99)
¹n (%)
²Kruskal-Wallis rank sum test; Fisher's exact test
*Statistically significant (< 0.05)

Survival plot according to the AR/TIL classification

For survival analysis, the median follow-up period was 93 months (IQR 79–120 months). Kaplan-Meier plots of OS and DMFS according to the AR/TIL classification are shown in Fig. 2. The log-rank test for the four categories showed significant differences in OS (P = 0.047) and DMFS (P = 0.0053). Mortality and distant metastasis rates were highest in the AR^–/TIL^low group (mortality rate: 35% [12/34]; distant metastatic rate: 44% [15/34]), followed by the AR⁺/TIL^low group (mortality rate: 17% [5/29]; distant metastatic rate: 21% [6/29]) (Table 1). Although pairwise comparisons showed no significant differences between the subgroups, this tendency was also observed in the survival plots (Fig. 2). AR-positive TNBC also demonstrated a tendency to improve both OS and DMFS in Kaplan-Meier plots. However, the AR status alone did not show statistically significant differences (OS, P = 0.17; DMFS, P = 0.089) (Online Resource 2).

TNBC-Apo: TNBC with some degree of apocrine differentiation

Seven of the 96 cases in the present cohort had apocrine differentiation, which was in perfect agreement with the 15-PGDH-positive cases, and showed diffuse strong positivity for AR (≥ 95% positivity in all cases) and low TILs levels (≤ 10% in 71%, 5/7; 30% in 29%, 2/7). All cases expressing 15-PGDH showed varying degrees of apocrine morphology in biopsy tissue. All seven cases were consistent with those of TNBC-Apo. Clinicopathological information on the seven TNBC-Apo is presented in Table 2. The Ki67 proliferation index was > 30% in all cases. Most patients were aged > 50 years and had histological grade 2 carcinomas. Postoperative evaluation of the pathologic response to chemotherapy revealed that six patients did not achieve pCR (non-pCR). One patient who achieved pCR (case 5) had residual cancer in situ (ypTisN0). One patient (case 2) had local and contralateral axillary lymph node metastasis recurrence 4 months after surgery and died of breast cancer 48 months later. The remaining six patients were alive without distant metastasis during the follow-up period of 55.5–138.5 months. One case was negative for germline BRCA mutations (case 3), whereas the remaining six cases had unknown status. A representative histology (case 7 in Table 2) is shown in Fig. 3, where AR was diffusely positive in the nuclei, 15-PGDH staining was positive in the cytoplasm, and some cells were weakly positive in the plasma membrane and nuclei.

Table 2

Case list of TNBC with some degree of apocrine differentiation
Case#	Age	Stage	gBRCA	HG	Ki67(%)	TILs	AR	15-PGDH	NAC Regimen	Response	Follow-up (mo)	Survival events	Distant metastatic recurrence
1	50	cT2N0M0 StageII	n/a	3	73.6	Low	+	+	Anthracycline and taxane based	Non-pCR	138.5	Alive	No
2	65	cT2N3M0 StageIII	n/a	2	35.5	Low	+	+	Anthracycline and taxane based	Non-pCR	51.6	Dead	Yes
3	63	cT4N3M0 StageIII	‒	2	35.3	Low	+	+	Anthracycline and taxane based	Non-pCR	120.5	Alive	Yes
4	73	cT2N0M0 StageII	n/a	2	36.4	Low	+	+	Anthracycline and taxane based	Non-pCR	108.1	Alive	No
5	68	cT2N0M0 StageII	n/a	2	60.0	Low	+	+	Anthracycline and taxane based	pCR¹	98.5	Alive	No
6	74	cT4N2M0 StageIII	n/a	2	67.0	Low	+	+	Anthracycline and taxane based	Non-pCR	78.9	Alive	No
7	48	cT2N0M0 StageII	n/a	2	50.0	Low	+	+	Anthracycline and taxane based	Non-pCR	55.5	Alive	No
¹Residual in situ carcinoma (ypTisN0M0)
gBRCA, germline BRCA mutation; HG, histological grade; n/a, not available

Logistic regression model for pCR

Univariate logistic regression analysis showed a statistically significant difference in the AR⁺/TIL^low status (OR 0.32, 95% CI 0.11–0.85; P = 0.029). Multivariate modeling using a stepwise method revealed that AR⁺/TIL^low status was significantly associated with non-pCR (OR 0.32, 95% CI 0.10–0.86; P = 0.032) (Table 3). Logistic regression analysis was performed with AR positivity and low TILs levels as separate variables. However, no significant correlation with pathological response was found for these variables in multivariate analysis (Online Resource 3).

Table 3

Logistic regression model for pathological complete response
	Univariate			Multivariate
Characteristic	OR	95% CI	p-value	OR	95% CI	p-value
Age (≥ median, y)	0.98	0.43–2.25	0.96
Post-menopause	1.29	0.55–2.98	0.56
cT3–4	0.33	0.07–1.12	0.10	0.40	0.08–1.51	0.21
cN1–3	0.44	0.18–1.05	0.070	0.46	0.17–1.17	0.11
Histological grade 3	2.00	0.63–7.65	0.26
Ki67 (≥ median, %)	1.00	0.44–2.29	> 0.99
Low-HER2	1.38	0.59–3.22	0.45
Apocrine differentiation	0.26	0.01–1.59	0.22
AR⁺/TIL^low	0.32	0.11–0.85	0.029*	0.32	0.10–0.86	0.032*
*Statistically significant (< 0.05)

Cox proportional hazard regression model for survival

The multivariate Cox proportional hazard regression model indicated that pCR was an independent predictor of better prognosis (OS, HR 0.14, 95% CI 0.03–0.63, P = 0.010; DMFS, HR 0.11, 95% CI 0.02–0.46, P = 0.003), whereas the AR⁺/TIL^low profile was not (Table 4). Furthermore, a multivariate model was constructed using apocrine differentiation (immunostaining 15-PGDH-poitive) status instead of the AR⁺/TIL^low, which showed no significant association with prognosis (Online Resource 4).

Table 4

Cox proportional hazard regression model for survival
		OS			DMFS
Characteristic	HR	95% CI	p-value	HR	95% CI	p-value
AR⁺/TIL^low	0.54	0.20–1.51	0.24	0.49	0.19–1.24	0.13
pCR	0.14	0.03–0.63	0.010*	0.11	0.02–0.46	0.003*
*Statistically significant (< 0.05)

Our study is the first to report the association between preoperative needle biopsy AR/TIL classification and NAC responsiveness and prognosis in consecutive TNBC patients. Among these subgroups, the AR⁺/TIL^low subgroup is resistant to NAC. Simultaneously, we examined the distribution of cancers showing apocrine differentiation according to AR/TIL classifications. Patients with TNBC-Apo (TNBC with some degree of apocrine differentiation) were included in this cohort, all of whom had AR⁺/TIL^low profiles. 15-PGDH immunostaining was specifically positive in these apocrine tumors.

TNBCs are heterogeneous tumors that respond differently to chemotherapy[6]. The tumor microenvironment (TME) significantly impacts systemic chemotherapy. TILs are important histological factors that reflect TME[8]. The molecular subtype classification of TNBC identified by Lehmann et al. provides important insights into the differences in TME and the therapeutic response of TNBC[4, 6]. Among the molecular subtypes identified by Lehmann et al., the LAR subtype, characterized by high AR expression, is similar to hormone receptor-positive breast cancer, particularly in its dependence on AR signaling pathway[4]. AR signaling pathway in LAR creates an immunosuppressive TME characterized by reduced TILs levels[5]. Masuda et al. compared the response to NAC in TNBC based on Lehmann’s molecular subtypes and reported that basal-like 2 and LAR were the subtypes with the lowest pCR rates [6]. Our research confirmed these previous studies by immunostaining for AR expression and TILs with HE staining. When TNBC was classified by AR/TIL, the AR⁺/TIL^low subgroup, which resembles the LAR profile, was found to be resistant to NAC.

Among the AR/TIL subgroups, Kaplan-Meier plots of OS and DMFS consistently showed that the AR⁻/TIL^low group tended to have the worst prognosis; the AR⁻/TIL^low group had the second lowest pCR rate (38%) after the AR⁺/TIL^low group (21%). It has been suggested that TME differs between LAR and non-LAR. Thompson et al. suggested that LAR and non-LAR have different TME composition profiles based on RNA-seq analysis using various public databases[5]. They suggested that differences in TME composition profiles may affect treatment response to NAC[5]. Different TME mechanisms may account for the differences in NAC responses and Kaplan-Meier plots in the two low TILs subgroups (AR⁺/TIL^low and AR⁻/TIL^low).

The AR/TIL classification may serve as a predictive tool for NAC resistance and identify patients who may benefit from targeted therapy. Subgroups with high TILs levels may benefit from NAC and immune checkpoint inhibitors (ICIs)[8, 9]. In early TNBC, high TILs levels indicate a good prognosis, suggesting the benefits of omitting or de-escalating NAC [18]. Subgroups with positive AR results showed potential for effective anti-androgen therapy[19–21]. The LAR molecular subtype is similar to the luminal intrinsic subtype, which is NAC resistant and prone to late recurrence, suggesting a long-term adjuvant therapy targeting the AR signaling pathway[6]. Since our cohort did not include patients receiving antiandrogen therapy, future studies should investigate the impact of TIL on the efficacy of antiandrogen therapy in AR-positive TNBC. Moreover, similar to the AR⁺/TIL^low subgroup, the AR⁻/TIL^low subgroup should demonstrate a restricted response to NAC. Our study provides preliminary evidence to support this possibility; however, further investigation is required, particularly considering specific therapeutic targets for this AR-negative subgroup.

Molecular subtyping is ideal for the practice of precision medicine because it allows for detailed characterization of TNBC[22]. Although cancer genomic analysis is becoming more common, the cost of performing next-generation sequencing analysis for all TNBC cases remains an issue. AR immunostaining is a low-cost method that can be performed routinely in general pathology laboratories without the need for new equipment. This method can be implemented in any institution to determine treatment strategies for TNBC.

Recent pathological studies have suggested that TNAC is a morphologically, immunophenotypically, and genetically distinct tumor[11, 13]. In addition to triple-negative status and apocrine morphology, TNAC is characterized by diffusely positive AR immunostaining and low TILs levels (< 15–20%)[11, 13]. Our NAC-treated TNBC cohort included seven cases of TNBC-Apo rather than pure TNAC. All seven patients with TNBC-Apo belonged to the AR⁺/TIL^low subgroup, with diffusely positive ARs (≥ 95%) and low TILs (≤ 30%), a profile consistent with AR/TILs in TNAC. We hypothesized that, similar to TNAC, TNBC-Apo may also exhibit NAC resistance[23]. We found that patients with TNBC-Apo were more likely to develop residual disease after NAC. However, due to the small number of cases, it is unclear whether this observation can be generalized to all TNBC-Apo cases. In addition, this study did not show any particular trend regarding prognosis or distant metastatic recurrence in nonpure apocrine TNBC.

Apocrine differentiation is determined by characteristic morphological features. Morphological judgments can be subjective and suffer from reproducibility issues among pathologists. An interesting study proposed a more objective system for assessing apocrine morphology using a semiquantitative scoring method, but there is not enough data available for subsequent validation[24]. Celis et al. reported that immunostaining for 15-PGDH was specifically expressed in apocrine tumors[25]. 15-PGDH is involved in prostaglandin metabolism and acts as a tumor suppressor[26]. It is one of the few apocrine-specific markers, and its specificity was validated in a cohort of 47 Japanese patients with invasive apocrine carcinomas[17]. All 47 patients had pure AC, and it was unclear what the staining results would be in a population that did not contain pure AC[17, 25]. In this study, 15-PGDH was detected only in apocrine differentiation. Therefore, 15-PGDH may provide reproducibility and objectivity in morphological assessments. However, whether 15-PGDH is exclusively expressed in TNBC-Apo is inconclusive due to the limited number of cases.

This study had some limitations. First, although AR/TIL was examined histopathologically by immunostaining, the extent to which AR⁺/TIL^low shares characteristics with the LAR subtype is unclear. Future studies should investigate whether AR⁺/TIL^low shares a genomic profile with the LAR subtype. Second, we adopted 1% as the cutoff for AR positivity, which is consistent with the Allred score used to assess ER and PR status. The effect of AR positivity on NAC responsiveness in TNBC is not well understood in studies reported to date. Some studies have reported that AR-positive TNBCs have better NAC response, while others have reported no significant difference between the AR status and NAC response[3, 28, 29]. Furthermore, there is much debate, with some studies reporting AR positivity as a poor prognostic factor for TNBC, while others have reported it as a good prognostic factor[30–35]. The cutoff value for AR in these studies varied from study to study, such as 0%, 1%, or 10%[19–21, 29, 30, 33]. In this study, we found a trend for AR positivity to improve OS and DMFS; however, the difference was not statistically significant. Mangia et al. reported that AR positivity was associated with poor disease-free survival (DFS) under an AR cutoff value of 10%, and the AR⁺/TIL^low subgroup had the worst DFS among the AR/TIL subgroups[34]. Consensus criteria for AR positivity are needed to better understand how AR expression affects prognosis and treatment strategies for TNBC. Further studies are required to determine the most clinically appropriate AR cutoff values for immunostaining.

In conclusion, this study highlights the significant association between the AR/TIL classification and NAC response in TNBC. We identified the AR⁺/TIL^low subgroup as resistant to NAC and found that tumors with partial apocrine differentiation predominantly belonged to this category. The AR/TIL classification provides valuable prognostic and predictive insights, paving the way for personalized treatments. Further research is needed to validate these findings and to explore targeted therapies for these subgroups.

Acknowledgments

We want to thank all patients, clinicians, and pathologists who participated in this study at the Breast Center, St. Luke’s International Hospital. We are grateful for the exceptional technical assistance provided by Naoto Tsuneda, Hiromi Ishiguro, and Kouki Yoshida at the Department of Pathology, St. Luke’s International Hospital. Additionally, we thank Editage (www.editage.com) for English language editing.

Funding

This study was supported by JSPS KAKENHI Grant Number 22H04343 and Supplementary Research Support Program of the St. Luke’s Health Science Research Fund (ZK2022-04).

Author contributions

TU designed the study, evaluated the histology, scored the cases, performed data analysis, interpreted the data, and wrote the first draft. NK conceived and planned the study, contributed to the evaluation of histology and data interpretation, critically reviewed the draft, and approved the final manuscript. NH contributed to the pathology review, critically reviewed the draft, and approved the final manuscript. YK, AY, and HY contributed to the collection of clinical data, critically reviewed the draft, and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest related to this article.

Ethical approval

The study was approved by the Institutional Review Board (22R010). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Patient consent

Informed consent was obtained from all individual participants included in the study.

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The authors declare no competing interests.

ESM2.pdf
Supplementary Fig. 1 Kaplan–Meier plots of OS (a) and DMFS (b) according to the AR/TIL classification
ESM1.xlsx
Supplementary Table S1. Immunohistochemistry antibody information
ESM3.xlsx
Supplementary Table S2. Logistic regression model for pathological complete response
ESM4.xlsx
Supplementary Table S3. Cox proportional hazard regression model for survival

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Clinicopathological significance of androgen receptor expression and tumor infiltrating lymphocytes in triple-negative breast cancer: a retrospective cohort study

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Abstract

Background

Methods

Results

Conclusions

Figures

INTRODUCTION

PATIENTS AND METHODS

Patient selection

Histopathological evaluation

Statistical analysis

RESULTS

Patient demographics

Survival plot according to the AR/TIL classification

TNBC-Apo: TNBC with some degree of apocrine differentiation

Logistic regression model for pCR

Cox proportional hazard regression model for survival

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

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