Adipophilin expression is an independent marker for poor prognosis of patients with triple-negative breast cancer: An immunohistochemical study.

Adipophilin is a lipid droplet-associated protein whose expression can act as a prognostic marker for specific cancers. Using immunohistochemical staining and tissue microarrays, we assayed the expression of adipophilin in 61 patients with triple-negative breast cancer (TNBC) who underwent surgery from January 2006-December 2018. Relapse-free survival (RFS) and its risk factors were analyzed based on adipophilin expression. Fourteen (23.0%) patients expressed adipophilin. As compared to the adipophilin-negative TNBC patients, adipophilin-positive patients exhibited poor RFS (p = 0.032). Among the TNBC patients with a high Ki-67 labeling index, patients negative for adipophilin exhibited better RFS than patients positive for adipophilin (p = 0.032). Moreover, among patients who did not undergo adjuvant chemotherapy, patients negative for adipophilin expression exhibited better RFS than adipophilin-positive patients (p = 0.080). Multivariate analysis showed that adipophilin expression correlated with a high rate of relapse (hazard ratio, 4.89; 95% confidence interval, 1.04-23.0; p = 0.044). Taken together, these results indicate that adipophilin is a novel marker for the poor prognosis of patients with TNBC.

Introduction

Triple-negative breast cancer (TNBC), a high-grade breast cancer with poor prognosis, is characterized by the lack of estrogen and progesterone receptors and human epidermal growth factor receptor 2 (HER2) [1, 2]. The Nottingham Prognostic Index, lymph node status, tumor size, and Ki-67 labeling index (LI) are useful prognostic markers, albeit non-specific for TNBC [3, 4]. The risk of cancer recurrence in patients with hormone receptor-positive breast cancer can be predicted using multigene assays, such as Oncotype DX®. This helps determine which patients should receive chemotherapy [5-7]. However, these tests cannot predict the prognosis of TNBC. Most TNBC patients undergo chemotherapy owing to the lack of a method to evaluate the risk of recurrence. Therefore, there is an urgent need to identify novel biomarkers for TNBC.

Adipophilin, also known as perilipin 2, is a lipid-regulating protein of the perilipin/adipophilin/tail interacting protein of 47 kDa (PAT) family that coats the surfaces of cytoplasmic lipid droplets [8, 9]. Lipids are essential for tumor cell proliferation [10]. PAT family proteins are expressed in various types of cancer cells [11-16]. Recent studies have demonstrated the correlation between adipophilin expression and poor prognosis of some types of cancers, including lung adenocarcinoma [14] and pancreatic ductal adenocarcinoma [16]. Expression of adipophilin has been reported in breast cancer tissues [17]; however, the prognostic value of adipophilin expression in TNBC remains to be elucidated. Thus, the aim of this study is to determine the correlation between adipophilin expression and prognosis of patients with TNBC.

Materials & methods

Patient selection

We selected 165 consecutive patients with TNBC who underwent surgical resection at the Department of Surgery of the Kansai Medical University Hospital in January 2006–December 2018. Patients who were administered for neoadjuvant chemotherapy or those who had a special type of invasive carcinoma were excluded from the study; the study cohort comprised 61 TNBC patients (S1 Fig).

This study was conducted in accordance with the principles embodied in the Declaration of Helsinki and the study protocol was approved by the Institutional Review Board of the Kansai Medical University Hospital (Approval #2019041, #2019234). Informed consent was obtained from patients by opt-out methodology owing to the retrospective design of the study, with no risk for the participants. Information regarding this study, such as the inclusion criteria and opportunity to opt out, was provided through the institutional website. (http://www.kmu.ac.jp/hirakata/hospital/2671t800000135f8-att/a1582783385210.pdf).

Histopathological analysis

Surgically resected specimens were fixed with formalin, sectioned, and stained with hematoxylin and eosin. All histopathological diagnoses were evaluated independently by more than two experienced diagnostic pathologists. We used the AJCC/UICC TNM classification and stage groupings. Histopathological grading was based on the Nottingham histological grade evaluated in the tumor tissue using tissue microarray [18]. We also evaluated the presence of clear cytoplasm, eosinophilic cytoplasm, and prominent nucleoli in the tumor tissue using tissue microarray. Clear or eosinophilic cytoplasm was considered to be present when these histological changes were identified in >5% of the tumour cells, as described earlier [14].

The Ki-67 LI, which was also evaluated in the tissue microarray, was considered high when it was 40% or more, according to a meta-analysis of TNBC patients [19]. The Ki-67 LI of two patients were not evaluated because less than 1,000 carcinoma cells were present in the tissue microarray.

Tissue microarray

Hematoxylin and eosin-stained slides were used to select the most morphologically representative carcinoma regions. Three tissue cores of 2 mm diameter were punched out from the paraffin-embedded blocks of each patient sample. Tissue cores were arrayed in the recipient paraffin block.

Immunohistochemistry

Immunohistochemical analyses were performed using an autostainer (Discovery ULTRA System; Roche Diagnostics, Basel, Switzerland) according to the kit instructions. A primary mouse monoclonal antibody was used to detect adipophilin (AP125, Progen Biotechnik, Heidelberg, Germany; diluted at 1:100). For antigen retrieval, tissue sections were autoclaved at 100°C for 20 minutes in a tris-based buffer, pH 8.5, (Conditioning Solution CC1, Ventana Medical System). Thereafter, the automated protocol steps for immunostaining were followed. In addition, 3,3’-diaminobenzidine (DAB) was used as a colorimetric agent. Human sebaceous glands tissues were used as positive controls for adipophilin immunoreactivity. At least two researchers evaluated the immunohistochemical staining independently. Adipophilin expression was considered positive when the neoplastic cells showed granular and/or globular cytoplasmic expression as reported previously (Granular staining pattern is defined as staining of round smaller size, while globular staining pattern is defined as staining of round to oval larger size.) [14, 16]. The patient was considered adipophilin-positive when one or more cores from the same patient showed positive immunoreactivity as described previously [14, 16]. In order to determine the cut-off value for adipophilin positivity, analysis was performed with positive cut-off values of 10, 20, 30, 40, and 50%.

Statistical analyses

All analyses were performed using SPSS Statistics 25.0 (IBM, Armonk, NY, USA). Correlation between two groups was determined using Fisher’s exact test for categorical variables and Mann–Whitney U test for continuous variables. The rate of relapse-free survival (RFS) was evaluated using Kaplan–Meier analysis; log-rank tests were used to compare between groups. The Cox proportional-hazards model was used to examine the correlation between clinicopathological parameters and survival. p<0.05 was considered statistically significant.

Results

Patient characteristics

This study comprised 61 female patients. The median age at the time of initial diagnosis was 58 years (range: 31–93 years). All patients were diagnosed with TNBC based on biopsy results, and all samples were invasive carcinoma, no special type. No discrepancy was found in the pathological diagnosis and molecular subtype between the pre-operative biopsy specimens and operative specimens. The median tumor diameter was 20 mm (range: 2–55 mm). Patients were staged as I (25 patients), IIA (23 patients), IIB (5 patients), IIIA (4 patients), IIIB (3 patients), and IIIC (1 patient). Based on the histopathology, 2, 27, and 32 patients had tumor grades 1, 2, and 3, respectively. The Ki-67 LI was high, low, and not evaluated in 26, 33, and 2 patients, respectively. The observation period was up to 36 months in all patients. Eight (13.1%) patients experienced relapse (all had distant metastasis, and none experienced local recurrence), and five (8.2%) patients died of the disease. Clinical characteristics of all 61 patients can be found online in S1 Table.

Correlation between adipophilin expression and clinicopathological factors

Table 1 shows the correlation between adipophilin expression and clinicopathological factors in the study cohort. Only cut-off value of 30% was significantly associated with RFS (p = 0.032), while cut-off values of 10, 20, 40, and 50% were not significantly associated with RFS (p = 0.280, 0.072, 0.07, and 0.07, respectively). Therefore, in the present study, the cut-off value was set at 30% for the subsequent analyses. Fourteen patients (23.0%) were adipophilin-positive and 47 (77.0%) were adipophilin-negative. Most cases exhibited a pan-cytoplasmic globular pattern of staining (Fig 1). Adipophilin expression did not correlate with any clinical factors including age, menopausal status, body mass index, or adjuvant chemotherapy. A high Ki-67 LI was significantly correlated with adipophilin expression (p = 0.005), but not with any other factors including tumor diameter, pathological stage, histological grade, lymphatic invasion, venous invasion, and lymph node status. Moreover, no differences were observed between the morphological features of adipophilin-positive and -negative cases, including the presence of clear cytoplasm, eosinophilic cytoplasm, and prominent nucleoli.

Table 1

Correlation between adipophilin expression and clinicopathological factors.

Factors	ADP-positive (n = 14)	ADP-negative (n = 47)	P-value
Age (years; mean ± standard deviation)	66 ± 15	65 ± 16	0.850
Menopausal status
Premenopausal	2	7	1.000
Postmenopausal	11	40
Unknown	1	0
Body mass index	23.7 ± 3.6	23.4 ± 3.8	0.674
Tumor size (mm)
≦20	7	24	1.000
>20	7	23
Pathological stage
I+II	12	41	1.000
III	2	6
Lymph node status
Positive	5	9	0.263
Negative	6	27
Not tested	3	11
Lymphatic invasion
Positive	13	40	0.668
Negative	1	7
Venous invasion
Positive	10	27	0.534
Negative	4	20
Nottingham histological grade
1 + 2	5	24	0.372
3	9	23
Ki-67 labeling index
High	11	15	0.005
Low	3	30
Not evaluated	0	2
Clear cytoplasm
Present	1	5	1.000
Absent	13	42
Eosinophilic cytoplasm
Present	1	11	0.264
Absent	13	36
Prominent nucleoli
Present	4	7	0.256
Absent	10	40
Adjuvant chemotherapy
Performed	7	28	0.749
Not performed	6	17
Undetermined	1	2

Fig 1

Representative images of the immunohistochemical staining for adipophilin in triple-negative breast cancer (×200 magnification).

Correlation between adipophilin expression and postoperative RFS

Fig 2A shows the RFS curves of adipophilin-positive and -negative patients. The frequency of 36-month RFS was 71.4% and 91.5% for adipophilin-positive and -negative patients, respectively. Adipophilin expression significantly correlated with poor RFS (p = 0.032) of TNBC patients. Among TNBC patients with a high Ki-67 LI, those negative for adipophilin exhibited better RFS than those positive for adipophilin (p = 0.032; Fig 2B). Among the adipophilin-negative patients, there was no difference in RFS between patients with high and low Ki-67 LI (p = 0.215). In TNBC patients without adjuvant chemotherapy, adipophilin-negative patients showed a trend for better RFS than adipophilin-positive patients (p = 0.080; Fig 2C).

Fig 2

Kaplan–Meier curves for the relapse-free survival (RFS) of triple-negative breast cancer patients.

(A) RFS curves in adipophilin-positive (red line) and -negative (blue line) patients. (B) RFS curves of adipophilin-negative patients with a low Ki-67 labeling index (LI; blue line), adipophilin-negative patients with a high Ki-67 LI (red line), and adipophilin-positive patients with a high Ki-67 LI (green line). (C) RFS curves in TNBC patients (adipophilin-positive [red line] and -negative [blue line]) without adjuvant chemotherapy.

Prognostic potential of adipophilin expression

Univariate and multivariate analyses were used to determine the effects of clinicopathological factors on RFS (Table 2). Based on the univariate analysis, advanced pathological stage (stage III), lymph node metastasis, and adipophilin expression significantly correlated with poor RFS (p = 0.036, 0.025, and 0.048, respectively). In contrast, multivariate Cox hazard analyses showed that adipophilin expression was an independent factor for poor prognosis of patients with TNBC (hazard ratio: 4.89; 95% confidence interval; 1.04–23.0; p = 0.044). Moreover, adipophilin expression was independent from adjuvant chemotherapy in RFS (hazard ratio: 4.12; 95% confidence interval; 1.02–16.6; p = 0.047).

Table 2

Univariate and multivariate analysis of relapse-free survival.

Factor		Univariate analysis			Multivariate analysis
	HR	95% CI	P-value	HR	95% CI	P-value
Tumor size (mm)
20 < vs ≦ 20	0.98	0.25–3.92	0.981
Pathological stage
III vs I + II	4.64	1.11–19.5	0.036	2.47	0.44–14.0	0.306
Lymph node status
positive vs negative	6.56	1.27–33.8	0.025	3.65	0.55–24.5	0.182
Lymphatic invasion
positive vs negative	24.5	0.02–3.3×105	0.510
Venous invasion
positive vs negative	4.46	0.55–36.2	0.162
Nottingham histological grade
3 vs. 1+2	1.65	0.39–6.92	0.493
Ki-67 labeling index (LI)
high vs. low	0.973	0.22–4.35	0.971
Adjuvant chemotherapy
not perform vs. perform	4.72	0.95–23.5	0.058
Adipophilin expression
positive vs. negative	4.05	1.01–16.3	0.048	4.89	1.04–23.0	0.044

Discussion

In this study, we examined the clinicopathological significance of adipophilin expression in TNBC patients. We demonstrated the following findings: expression of adipophilin was an independent factor for determining the prognosis of patients with TNBC using multivariate analysis; adipophilin-negative patients with a high Ki-67 LI had a significantly better prognosis compared to that of adipophilin-positive patients with a high Ki-67 LI; and among TNBC patients without adjuvant chemotherapy, those negative for adipophilin exhibited a better prognosis compared to those positive for adipophilin. To the best of our knowledge, this is the first study addressing the prognostic significance of adipophilin expression in TNBC, although a previous study reported that adipophilin expression was frequently associated with TNBC and HER2 subtypes compared to that in luminal subtypes and higher Ki-67 LI [17].

Many studies have attempted to identify prognostic factors in patients with TNBC [3]. The Nottingham Prognostic Index, lymph node status, tumor size, pathological stage, and Ki-67 LI are known prognostic markers [3, 4]. In this study, univariate analysis for RFS showed that pathological stage (III vs I+II) and lymph node status were significant prognostic factors (p = 0.036 and 0.025, respectively), but Nottingham histological grade and Ki-67 LI were not. Multivariate analysis showed that pathological stage and lymph node status were not associated with RFS whereas adipophilin expression was a prognostic factor for poor RFS in patients with TNBC (p = 0.044). These results indicate that adipophilin expression is a useful prognostic marker for RFS in TNBC patients. Moreover, the presence of clear cytoplasm, eosinophilic cytoplasm, and prominent nucleoli was not associated with adipophilin expression in TNBC. Some previous reports showed that the presence of clear cytoplasm was not associated with adipophilin expression in lung adenocarcinoma and pancreatic ductal adenocarcinoma [14, 16].

Interestingly, adipophilin expression was a significant marker for poor prognosis in patients with a high Ki-67 (p = 0.032); in contrast, Ki-67 LI (high or low) did not correlate with RFS in adipophilin-negative patients (p = 0.215). These results indicate that adipophilin is a superior prognostic marker in patients with TNBC, and analysis of adipophilin might better identify patients with a favorable prognosis when combined with Ki-67. Moreover, adipophilin-negative patients without adjuvant chemotherapy showed better prognosis. To the best of our knowledge, there are no established markers for determining whether adjuvant chemotherapy should be administered to patients. Therefore, adipophilin-negative patients may be subjected to de-escalation treatment for TNBC. More prospective clinical studies are required to validate this hypothesis.

Consistent with the results in this study, previous studies demonstrated that adipophilin expression was a factor for poor prognosis for several cancers [14, 16]. The mechanism involved in adipophilin expression in these cancers remains unclear. Adipophilin expression is associated with upregulated lipid synthesis in neoplastic cells [14, 16]. Aerobic glycolysis is the primary energy-generating pathway in cancer cells; this is known as the Warburg effect [10] that results in increased concentrations of intracellular lipids. Increased cancer cell proliferation requires large amounts of lipids to produce cell membranes [20, 21]. Lipid metabolism is associated with TNBC growth [22], and adipophilin expression correlates with higher Ki‐67 [17]. Moreover, hypoxia drives the activation of adipophilin in breast cancer cell lines [23], and highly proliferative breast cancer cells thrive in hypoxic conditions [24, 25]. Thus, adipophilin expression in TNBC reflects upregulated lipid metabolism that correlates with a higher proliferative capacity of cancer cells in hypoxic tumor microenvironments [23].

It is important to note the limitations associated with this study. First, this was a retrospective single-institution study with a small sample size that could have led to selection bias. Second, because TMA cores of 2 mm in diameter were used to determine adipophilin expression in patients, there could be heterogeneous expression of adipophilin in the cancer tissues even though we selected regions that were most representative (morphologically) of cancer. Further studies based on adipophilin immunostaining using whole tissue sections are required to validate our results. Moreover, differential adipophilin expression in operative and biopsy specimens needs to be evaluated. Third, since chemotherapy affects the expression of adipophilin [14], this study excluded patients who were administered neoadjuvant chemotherapy. Further experiments are needed to clarify whether adipophilin is an independent prognostic factor for TNBC patients with and without neoadjuvant chemotherapy. Fourth, we used a follow-up period of more than 36 months based on published data that showed the risk of recurrence in TNBC patients’ peaks within 3 years [2, 26]. However, recurrence at a later period in this cohort is possible, and the significance of adipophilin expression in RFS might be different during this period. Finally, this study focused on the expression of adipophilin in TNBC patients. Because adipophilin expression differs among molecular subtypes of breast cancer [17], its prognostic value might be different in patients with the luminal and HER2 subtypes. Thus, further analyses are needed to investigate the prognostic value of adipophilin expression in patients with breast cancers in addition to TNBC.

In conclusion, this study demonstrates that adipophilin expression is an independent factor for poor prognosis of patients with TNBC. Additional studies are needed to elucidate the molecular mechanisms involved in the expression of adipophilin in TNBC, and to develop therapeutic interventions for adipophilin-positive TNBC patients with a high risk of recurrence.

Clinical characteristics of patients with triple-negative breast cancer.

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Flowchart of the exclusion criteria in this study.

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7 Aug 2020

PONE-D-20-22115

Adipophilin expression is an independent marker for poor prognosis of patients with triple-negative breast cancer: An immunohistochemical study

PLOS ONE

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Reviewer #1: The manuscript by Yoshikawa et al. evaluated the prognostic value of adipophilin, as evaluated by IHC, in a cohort of 61 TNBC. Obtained results indicated that adipophilin-positive cases have worse prognosis than negative ones.

Overall this work is interesting and timing but there are some points that need the authors’ attention:

Major points:

a. The cut-off used (30% of positive cells) to define adipophilin positivity has never been used in other publications. The authors have to define why they chose this cut-off. A distribution of the % of positive cells in the cohort should be shown. Moreover, they have 23% positivity that is quite different from the 50% positivity in ER- tumors of Kuniyoshi et al paper. Discussion on this point has to be added.

b. Based on the year of patient recruitment (2006-2018), it would be interesting to extend survival analysis beyond 3 years of follow-up.

c. The authors have to demonstrate that adipophilin prognostic value in untreated TNBC patients is independent from other clinico-pathological features. Is the prognostic value of adipophilin independent from adjuvant chemotherapy treatment in multivariate analysis?

Minor points:

d. Please include details in Table S1 of the type of TNBC included in the analysis and specify among exclusion criteria in M&M the special types excluded.

e. Details on IHC method to evaluate adipophilin need to be included.

Reviewer #2: The authors studied expression of adipophilin (ADP) in 61 patients with triple-negative breast cancer (TNBC), and proposed that ADP may be a novel marker for the poor prognosis of patients with TNBC. This paper contains novel information and results which may be valuable for the readers. However, there are some major issues to be revised.

1) Authors describe that the tumor cells exhibited globular ADP staining pattern. Globular staining reminds the reviewer of sebaceous carcinoma. What is the criteria between globular and granular staining pattern?

2) Previously, ADP expression has been reported in apocrine carcinoma and lipid rich carcinoma of the breast (PMID: 21566511). Was their any morphologic characteristics in your ADP-positive breast carcinoma?

3) In the manuscript, the authors describe that "All patients were diagnosed with TNBC based on

108 biopsy results, and all samples were invasive carcinoma, no special type". Is their any possibility of discrepancy in the status between biopsy and surgically resected samples? Please refer to the current treatment guidlines.

4) Was histopathological grading performed on biopsy, surgical specimen, or TMA?

5) Since ADP staining was assessed on TMA, Ki67 index should be also analyzed on TMA instead of the preoperative biopsy specimens in order to investigate their relationship. Expression of ADP and Ki67 may be hetwrogeneous. Thus the sainings are better assessed at the same tumor site.

6) Regarding ADP expression and patients' prognosis, what is possible reasons for the discrepant results between TNBC with high and low Ki67 index? Again, ADP and Ki67 are better to be assessed on the same section. Study design should be reconsidered.

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Reviewer #2: No

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8 Oct 2020

September 29, 2020

Dr. Joerg Heber

Editor-in-Chief

PLoS ONE

Resubmission - Manuscript ID: PONE-D-20-22115

Dear Dr. Heber:

I would like to resubmit an article for publication in PLoS ONE, titled “Adipophilin expression is an independent marker for poor prognosis of patients with triple-negative breast cancer: An immunohistochemical study”.

Your comments, as well as those of the reviewers, were highly insightful and enabled us to greatly improve the quality of our manuscript. In the following pages, I have provided our point-by-point response to all comments and have highlighted the revised portions of the manuscript.

I hope that you will find our revised manuscript suitable for publication in PLoS ONE. I look forward to hearing from you at your earliest convenience.

Sincerely,

Mitsuaki Ishida

Kansai Medical University

2-5-1, Shinmachi, Hirakata City

Osaka, 573-1010, Japan

Tel: +81-72-804-2794

Fax: +81-72-804-2794

E-mail: ishidamt@hirakata.kmu.ac.jp

Response to the comments of Reviewer #1

Thank you very much for reviewing our manuscript. We appreciate your constructive comments. We have made the following revisions in response to the issues raised by you.

a. The cut-off used (30% of positive cells) to define adipophilin positivity has never been used in other publications. The authors have to define why they chose this cut-off. A distribution of the % of positive cells in the cohort should be shown. Moreover, they have 23% positivity that is quite different from the 50% positivity in ER- tumors of Kuniyoshi et al paper. Discussion on this point has to be added.

Response: Kaplan–Meier curves with cut-off values of 10, 20, 30, 40, and 50%, respectively, were generated and evaluated. As cut-off values of 30% significantly correlated with relapse-free survival, they were adopted in the present study. Kuniyoshi et al. showed that positive rate of adipophilin (perilipin 2) in basal type (=triple negative breast cancer) was 27% (cut-off value of 50%), and this corresponded with the results of the present study (23% of TNBC was adipophilin-positive in the present study).

“In order to determine the cut-off value for adipophilin positivity, analysis was performed with positive cut-off values of 10, 20, 30, 40, and 50%” (page 8, lines 108-110)

“Only cut-off value of 30% was significantly associated with relapse-free survival (p = 0.032), while cut-off values of 10%, 20%, 40%, and 50% were not significantly associated with relapse-free survival (p = 0.280, 0.072, 0.07, and 0.07, respectively). Therefore, in the present study, the cut-off value was set at 30% for the subsequent analyses” (page 9, lines 140-143)

b. Based on the year of patient recruitment (2006-2018), it would be interesting to extend survival analysis beyond 3 years of follow-up.

Response: As you pointed out, it would be interesting to analyze survival data beyond the 3-years of follow-up period. However, the number of patients whose clinical information was available for more than 3 years was not high in this present cohort. Thus, we did not show the survival data beyond 3 years of follow-up.

c. The authors have to demonstrate that adipophilin prognostic value in untreated TNBC patients is independent from other clinico-pathological features. Is the prognostic value of adipophilin independent from adjuvant chemotherapy treatment in multivariate analysis?

Response: As you suggested, we analyzed adipophilin expression and adjuvant chemotherapy in relapse-free survival. Adipophilin expression was found to be independent from adjuvant chemotherapy (hazard ratio: 4.118; 95% confidence interval; 1.02–16.6; p = 0.047). We added this information in the manuscript as follows:

“Moreover, adipophilin expression was independent from adjuvant chemotherapy in RFS (hazard ratio: 4.12; 95% confidence interval; 1.02–16.6; p = 0.047)” (page 14, lines 185-187)

Minor point

d. Please include details in Table S1 of the type of TNBC included in the analysis and specify among exclusion criteria in M&M the special types excluded.

Response: Following your instructions, we have created a flowchart depicting the exclusion criteria with details of the histological type (Figure S1).

e. Details on IHC method to evaluate adipophilin need to be included.

Response: Based on your suggestion, the details of immunostaining were added.

“A primary mouse monoclonal antibody was used to detect adipophilin (AP125, Progen Biotechnik, Heidelberg, Germany; diluted at 1:100). For antigen retrieval, tissue sections were autoclaved at 100°C for 20 minutes in a tris-based buffer, pH 8.5, (Conditioning Solution CC1, Ventana Medical System). Thereafter, the automated protocol steps for immunostaining were followed. In addition, 3,3’-diaminobenzidine (DAB) was used as a colorimetric agent. Human sebaceous glands tissues were used as positive controls for adipophilin immunoreactivity” (page 7, lines 95-101)

Response to the comments of Reviewer #2

Thank you very much for reviewing our manuscript. We appreciate your constructive comments. We have made the following revisions in response to the issues raised by you.

1) Authors describe that the tumor cells exhibited globular ADP staining pattern. Globular staining reminds the reviewer of sebaceous carcinoma. What is the criteria between globular and granular staining pattern?

Response: The criteria for adipophilin staining patterns were based on reference 14.

Granular staining pattern is defined as staining of smaller subnuclear structure regions, while globular staining is defined as staining of larger pan-cytoplasmic regions.

“Adipophilin expression was considered positive when the neoplastic cells showed granular and/or globular cytoplasmic expression as reported previously (Granular staining pattern is defined as staining of smaller subnuclear structure regions, while globular staining pattern is defined as staining of larger pan-cytoplasmic regions).” (page 7, lines 104-106)

2)Previously, ADP expression has been reported in apocrine carcinoma and lipid rich carcinoma of the breast (PMID: 21566511). Was their any morphologic characteristics in your ADP-positive breast carcinoma?

Response: This study did not contain any cases of apocrine carcinoma and lipid-rich carcinoma. Moreover, no differences were observed between the morphological features of adipophilin-positive and -negative cases.

“Moreover, no differences were observed between the morphological features of adipophilin-positive and -negative cases” (page 10, lines 150-152)

3)In the manuscript, the authors describe that "All patients were diagnosed with TNBC based on biopsy results, and all samples were invasive carcinoma, no special type". Is their any possibility of discrepancy in the status between biopsy and surgically resected samples? Please refer to the current treatment guidlines.

Response: No discrepancy was found in pathological diagnosis and molecular subtype between pre-operative biopsy specimens and operative specimens in our cohort, which is similar to the previous reports that have shown high concordance between biopsy and surgically results, especially in patients with hormone-negative and HER2-negative cancer (Ann Oncol. 2009; 20(12):1948-52).

“No discrepancy was found in the pathological diagnosis and molecular subtype between the pre-operative biopsy specimens and operative specimens” (page 8, line 125 – Page 9, 127)

4)Was histopathological grading performed on biopsy, surgical specimen, or TMA?

Response: We evaluated the histopathological grading using TMA. I added some sentences in the manuscript to make it easier to understand.

“Histopathological grading was based on the Nottingham histological grade evaluated in the tumor tissue using tissue microarray [18]” (page 6, lines 79-81)

5)Since ADP staining was assessed on TMA, Ki67 index should be also analyzed on TMA instead of the preoperative biopsy specimens in order to investigate their relationship. Expression of ADP and Ki67 may be heterogeneous. Thus the stainingsare better assessed at the same tumor site.

Response: Following your recommendation, we re-analyzed all statistical data and determined Ki-67 LI of TMA; the statistical data associated with Ki-67 LI were all changed.

“The Ki-67 LI, which was also evaluated in the tissue microarray, was considered high when it was 40% or more, according to a meta-analysis of TNBC patients with tissue microarray [19]. The Ki-67 LI of two patients were not evaluated because less than 1,000 carcinoma cells were present in the tissue microarray” (page 6, lines 81-84)

“A high Ki-67 LI was significantly correlated with adipophilin expression (p = 0.005),” (page 10, lines 147-148)

6) Regarding ADP expression and patients' prognosis, what is possible reasons for the discrepant results between TNBC with high and low Ki67 index? Again, ADP and Ki67 are better to be assessed on the same section. Study design should be reconsidered.

Response: As you suggested, we assessed the immunohistochemical staining of Ki-67 LI and adipophilin on the same section using tissue microarray. All statistical analyses were re-performed using new Ki-67 LI data. However, the results remained unchanged; adipophilin expression was a significantly worse prognostic marker for patients with TNBC.

As you mentioned, Ki-67 has been a well-known prognostic marker for patients with TNBC, and the present study clearly showed that adipophilin expression was a significantly worse prognostic marker and it might be superior to Ki-67 because among TNBC patients with a high Ki-67 LI, those negative for adipophilin exhibited better prognosis than those positive for adipophilin. Among adipophilin-negative patients, those with higher Ki-67 LI showed slightly better prognosis compared to those with lower Ki-67 LI (not significant). As you suggested, additional studies with larger number of patients are needed to clarify the significance of Ki-67 and adipophilin in prognosis of patients with TNBC.

22 Oct 2020

PONE-D-20-22115R1

Adipophilin expression is an independent marker for poor prognosis of patients with triple-negative breast cancer: An immunohistochemical study

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #1: (No Response)

Reviewer #2: 1. Regarding the criteria for adipophilin staining patterns, reference 14 does not clearly define granular and globular staining patterns, but I presume that granular and globular staining patterns are defined by the size and shape of adipophilin positive substance instead of the stained region in the cells.

2. You mentioned that morphologic characteristics of ADP-positive and -negative breast carcinoma did not differ. Please specify the parameters you compared between the 2 groups; foamy cytoplasm, eosinophilic cytoplasm, prominent nucleoli, etc.

3. I would drop the following parts in the abstract and conclusion; "and might be superior to Ki-67 as a prognostic marker" and "Adipophilin may be a superior prognostic marker compared to Ki-67".

**********

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Reviewer #1: No

Reviewer #2: No

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3 Nov 2020

November 3, 2020

Dr. Joerg Heber

Editor-in-Chief

PLoS ONE

Resubmission - Manuscript ID: PONE-D-20-22115 R1

Dear Dr. Heber:

I would like to resubmit an article for publication in PLoS ONE, titled “Adipophilin expression is an independent marker for poor prognosis of patients with triple-negative breast cancer: An immunohistochemical study”.

Your comments, as well as those of the reviewer, were highly insightful and enabled us to greatly improve the quality of our manuscript. In the following pages, I have provided our point-by-point response to all comments and have highlighted the revised portions of the manuscript.

I hope that you will find our revised manuscript suitable for publication in PLoS ONE. I look forward to hearing from you at your earliest convenience.

Sincerely,

Mitsuaki Ishida

Kansai Medical University

2-5-1, Shinmachi, Hirakata City

Osaka, 573-1010, Japan

Tel: +81-72-804-2794

Fax: +81-72-804-2794

E-mail: ishidamt@hirakata.kmu.ac.jp

Response to the comments of Reviewer #2

Thank you very much for reviewing our manuscript. We appreciate your constructive comments. We have made the following revisions in response to the issues raised by you.

1. Regarding the criteria for adipophilin staining patterns, reference 14 does not clearly define granular and globular staining patterns, but I presume that granular and globular staining patterns are defined by the size and shape of adipophilin positive substance instead of the stained region in the cells.

Response: Based on your suggestion, we have clarified the definition of adipophilin staining.

“Granular staining pattern is defined as staining of round smaller size, while globular staining pattern is defined as staining of round to oval larger size.” (page 8, line 109 - 111)

2. You mentioned that morphologic characteristics of ADP-positive and -negative breast carcinoma did not differ. Please specify the parameters you compared between the 2 groups; foamy cytoplasm, eosinophilic cytoplasm, prominent nucleoli, etc.

Response: Based on your suggestion, we evaluated the relationship between ADP and some morphologic features, including clear cytoplasm, eosinophilic cytoplasm, and prominent nucleoli. These features were not correlated with ADP expression. We added the results in Results and Table 1.

“We also evaluated the presence of clear cytoplasm, eosinophilic cytoplasm, and prominent nucleoli in the tumor tissue using tissue microarray. Clear or eosinophilic cytoplasm was considered to be present when these histological changes were identified in >5% of the tumour cells, as described earlier [14].” (page 6, line 82 – 85)

“Moreover, no differences were observed between the morphological features of adipophilin-positive and -negative cases, including the presence of clear cytoplasm, eosinophilic cytoplasm, and prominent nucleoli.” (page 10, line 155 - 158)

3. I would drop the following parts in the abstract and conclusion; "and might be superior to Ki-67 as a prognostic marker" and "Adipophilin may be a superior prognostic marker compared to Ki-67".

Response: Based on your suggestion, we have dropped the specified part in the Abstract and Conclusion.

5 Nov 2020

Adipophilin expression is an independent marker for poor prognosis of patients with triple-negative breast cancer: An immunohistochemical study

PONE-D-20-22115R2

Dear Dr. Ishida,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Elda Tagliabue

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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9 Nov 2020

PONE-D-20-22115R2

Adipophilin expression is an independent marker for poor prognosis of patients with triple-negative breast cancer: An immunohistochemical study

Dear Dr. Ishida:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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PLOS ONE