Angiotensin II receptor blockers and oral squamous cell carcinoma survival: A propensity-score-matched cohort study.

OBJECTIVES: Angiotensin II receptor blockers (ARBs) improve the survival rates of patients with various cancers. However, it remains unclear whether ARBs confer a survival benefit on patients with oral squamous cell carcinoma (OSCC). Here, we assessed the associations between ARB use and survival in patients with OSCC of different stages.
MATERIALS AND METHODS: This was a 10-year retrospective cohort study of OSCC patients. We enrolled 7,558 patients diagnosed with oral cancer between January 2007 and December 2017 whose details had been entered into the Chang Gung Research Database. Seven hundred and fourteen patients were recruited from the Chang Gung Research Database after performing 1:1 propensity score-matching between ARB users and non-users. Cox's regression models with adjusted covariates were employed to detect factors influencing the survival rates of patients with OSCC.
RESULTS: Kaplan-Meier analysis revealed that the overall survival (OS) rate of 180-day ARB users increased (p = 0.038). Cox's regression models indicated that ARB use, younger patients, early-stage OSCC, and patients without diabetes mellitus were independently prognostic of improved OS. Increased OS was more prominent in 180-day ARB users in stage III, Iva, and IVb categories.
CONCLUSIONS: ARB use for more than 180 days is associated with an increased survival rate and is a positive, independent prognostic factor in patients with OSCC. A further two-arm study should be conducted to confirm the clinical usefulness of ARBs in OSCC patients.

Introduction

Oral cancer is one of the most frequently occurring cancers worldwide. Oral squamous cell carcinoma (OSCC) represents the most common type of oral cancer, constituting approximately 90% of all oral cancers [1]. In 2018, more than 355,000 individuals were diagnosed with oral cancer worldwide, and approximately 177,000 oral cancer-related deaths were reported [2]. Despite advances in surgical techniques and chemoradiotherapy, the prognosis of patients with OSCC remains unsatisfactory, especially for those diagnosed with advanced disease. Therefore, the identification of novel therapeutic targets in OSCC is of high clinical importance.

The renin-angiotensin system (RAS) is involved in the regulation of blood pressure. Therefore, angiotensin I-converting enzyme inhibitors (ACEIs) and angiotensin II type 1 receptor blockers (ARBs) are the most widely used anti-hypertensive drugs. A retrospective cohort study conducted by Lever and colleagues showed that the long-term ACEI use protected against cancer [3], suggesting that the local RAS played roles in tumor development and progression. Additionally, the RAS has been implicated in most human cancers; thus, the use of ACEIs/ARBs has been proposed as a promising anti-tumor strategy, which could potentially suppress tumor progression through inhibition of cancer cell proliferation and neovascularization [4]. Indeed, the combination of ACEIs/ARBs with conventional anti-cancer therapies has been shown to improve clinical outcomes of patients with various types of cancer, including breast, urothelial, and gastrointestinal tract cancers [5-8].

However, the clinical usefulness of RAS inhibitors in patients with OSCC remains unclear. Also, most previous studies did not separately evaluate the anti-neoplastic effects of ACEIs and ARBs; the drug classes were combined when exploring the clinical outcomes of cancer patients. The impacts of ARBs alone were inconsistent [9, 10], suggesting that the ACEIs exerted all of the observed anti-neoplastic effects. Thus, we investigated the efficacy of ARBs in patients with OSCC. We used the Chang Gung Memorial Hospital database to perform a 10-year, retrospective cohort study. Furthermore, we explored the effects of ARBs on patients with advanced-stage OSCC.

Material and methods

Study cohort

We enrolled 7558 patients diagnosed with oral cancer between January 2007 and December 2017 whose details had been entered into the Chang Gung Research Database [11-13]. Fig 1 is a flow chart of the cohort study design for statistical analysis. Patients with non-squamous cell carcinoma or unclear staging data were excluded. Patients who did not have surgery or distant metastasis were also excluded because survival in these two groups was markedly different from patients who received surgery [14]. Patients with ARB use < 180 days were excluded to prevent possible partial effects on survival. Non-ARB users with survival < 180 days were also excluded to make the two groups comparable. Hence, 5673 OSCC patients remained after applying the above exclusion criteria (Table 1). We performed propensity score-matching (PSM) to balance covariates between ARB users and non-users. Hence, data from 714 patients were analyzed in our study, including 357 patients treated with ARBs and 357 matched patients who did not receive ARBs (Table 2). This retrospective cohort study was approved by the Institutional Review Board (IRB) of Kaohsiung and Chiayi Chang Gung Memorial Hospital (Approval Nos. 202001463B0 and 201700253B0C602), and all experiments were performed in accordance with relevant guidelines and regulations. The Chang Gung Medical Foundation IRB (Approval No. 202001463B0) approved the waiver of participant consent.

Fig 1

Flow diagram illustrating propensity score matching in patients with oral cancer.

ARBs, angiotensin II receptor blockers.

Table 1

Demographic and clinical characteristics of OSCC patients before matching.

Variables	OSCC patients n = 5673	ARBs ≥ 180 days n = 362	Non-Users n = 5311	p value
Median age at diagnosis, years (IQR)	52(45–60)	58(51.7–66)	52(45–59)	*<0.001
Gender				0.501
Female	477(8.4%)	27(7.5%)	450(8.5%)
Male	5196(91.6%)	335(92.5%)	4861(91.5%)
Tumor sites				0.352
Lip	292(5.1%)	22(6.1%)	270(5.1%)
Oral tongue	2105(37.1%)	126(34.8%)	1979(37.3%)
Upper/lower Gum	652(11.5%)	51(14.1%)	601(11.3%)
Floor of mouth	229(4.0%)	12(3.3%)	217(4.1%)
Buccal mucosa	1894(33.4%)	125(34.5%)	1769(33.3%)
Hard palate	86(1.5%)	2(0.6%)	84(1.6%)
Retromolar trigone	269(4.7%)	18(5.0%)	251(4.7%)
Unidentified	146(2.6%)	6(1.7%)	140(2.5%)
Lifestyle Risk Factors
Smoking (n = 5647)				*<0.001
No	1309(23.2%)	112(30.9%)	1197(22.6%)
Yes	4338(76.8%)	250(69.1%)	4088(77.4%)
Betel nuts consumption (n = 5647)				*0.001
No	2084(36.9%)	162(44.8%)	1922(36.4%)
Yes	3563(63.1%)	200(55.2%)	3363(63.6%)
Alcoholic beverages (n = 5646)				0.194
No	2235(39.6%)	155(42.8%)	2080(39.4%)
Yes	3411(60.4%)	207(57.2%)	3204(60.6%)
Comorbidities
Diabetes mellitus				*<0.001
No	4816(84.9%)	161(44.5%)	4655(87.6%)
Yes	857(15.1%)	201(55.5%)	656(12.4%)
Hypertension				*<0.001
No	4614(81.3%)	0(0.0%)	4614(86.9%)
Yes	1059(18.7%)	362(100.0%)	697(13.1%)
Hyperlipidemia				*<0.001
No	5081(89.6%)	174(48.1%)	4907(92.4%)
Yes	592(10.4%)	188(51.9%)	404(07.6%)
Clinical AJCC 7th staging				*0.002
I	1324(23.3%)	107(29.6%)	1217(22.9%)
II	1436(25.3%)	104(28.7%)	1332(25.1%)
III	781(13.8%)	42(11.6%)	739(13.9%)
IVa & IVb	2132(37.6%)	109(30.1%)	2023(38.1%)
Pathological AJCC 7th staging				*<0.001
I	1555(27.4%)	122(33.7%)	1433(27.0%)
II	1318(23.2%)	106(29.3%)	1212(22.8%)
III	765(13.5%)	45(12.4%)	720(13.6%)
IVa & IVb	2035(35.9%)	89(24.6%)	1946(36.6%)
Treatment				*0.001
Operation alone	3108(54.8%)	229(63.3%)	2879(54.2%)
Operation plus RT/CCRT	2565(45.2%)	133(36.7%)	2432(45.8%)
Recurrence				*0.030
No	4545(80.1%)	306(84.5%)	4239(79.8%)
Yes	1128(19.9%)	56(15.5%)	1072(20.2%)
Survival				*0.005
Alive	4135(72.9%)	288(79.6%)	3847(72.4%)
Primary OSCC related death	981(17.3%)	41(11.3%)	940(17.7%)
Die of other reasons	557(9.8%)	33(9.1%)	524(9.9%)

Abbreviations: AJCC, American Joint Committee on Cancer; ARBs, angiotensin II receptor blockers; CCRT, concurrent chemoradiotherapy; IQR, interquartile range; OSCC, oral squamous cell carcinoma; RT, radiotherapy.

Table 2

Demographic and clinical characteristics of the study cohort.

Variables	Cohort n = 714	ARBs ≥ 180 days n = 357	Non-Users n = 357	p value
Median age at diagnosis, years (IQR)	58(52–66)	58(51–66)	59(52–66)	0.426
Gender				0.888
Female	55(7.7%)	27(7.6%)	28(7.8%)
Male	659(92.3%)	330(92.4%)	329(92.2%)
Tumor sites				0.932
Lip	43(6.0%)	21(5.9%)	22(6.2%)
Oral tongue	249(34.9%)	125(35.0%)	124(34.7%)
Upper/lower Gum	99(13.9%)	50(14.0%)	49(13.7%)
Floor of mouth	21(2.9%)	12(3.4%)	9(2.5%)
Buccal mucosa	245(34.3%)	124(34.7%)	121(33.9%)
Hard palate	5(0.7%)	2(0.6%)	3(0.8%)
Retromolar trigone	37(5.2%)	18(5.0%)	19(5.3%)
Unidentified	15(2.1%)	5(1.4%)	10(2.8%)
Lifestyle Risk Factors
Smoking				0.324
No	210(29.4%)	111(31.1%)	99(27.7%)
Yes	504(70.6%)	246(68.9%)	258(72.3%)
Betel nuts consumption				0.598
No	315(44.1%)	161(45.1%)	154(43.1%)
Yes	399(55.9%)	196(54.9%)	203(56.9%)
Alcoholic beverages				0.084
No	329(46.1%)	153(42.9%)	176(49.3%)
Yes	385(53.9%)	204(57.1%)	181(50.7%)
Comorbidities
Diabetes mellitus				0.598
No	315(44.1%)	161(45.1%)	154(43.1%)
Yes	399(55.9%)	196(54.9%)	203(56.9%)
Hypertension				*<0.001
No	223(31.2%)	0(0.0%)	223(62.5%)
Yes	491(68.8%)	357(100.0%)	134(37.5%)
Hyperlipidemia				0.369
No	360(50.4%)	174(48.7%)	186(52.1%)
Yes	354(49.6%)	183(51.3%)	171(47.9%)
Clinical AJCC 7th staging				0.110
I	192(26.9%)	105(29.4%)	87(24.4%)
II	215(30.1%)	101(28.3%)	114(31.9%)
III	101(14.1%)	42(11.8%)	59(16.5%)
IVa & IVb	206(28.9%)	109(30.5%)	97(27.2%)
Pathological AJCC 7th staging				0.927
I	231(32.4%)	119(33.3%)	112(31.4%)
II	212(29.7%)	104(29.1%)	108(30.3%)
III	94(13.2%)	45(12.6%)	49(13.7%)
IVa & IVb	177(24.8%)	89(24.9%)	88(24.6%)
Treatment				0.348
Operation alone	460(64.4%)	224(62.7%)	236(66.1%)
Operation plus RT/CCRT	254(35.6%)	133(37.3%)	121(33.9%)
Recurrence				0.758
No	601(84.2%)	302(84.6%)	299(83.8%)
Yes	113(15.8%)	55(15.4%)	58(16.2%)
Survival				0.176
Alive	549(76.9%)	285(79.8%)	264(73.9%)
Primary OSCC related death	94(13.2%)	41(11.5%)	53(14.8%)
Die of other reasons	71(09.9%)	31(08.7%)	40(11.2%)

Abbreviations: AJCC, American Joint Committee on Cancer; ARBs, angiotensin II receptor blockers; CCRT, concurrent chemoradiotherapy; IQR, interquartile range; OSCC, oral squamous cell carcinoma; RT, radiotherapy.

Statistical analyses

Categorical data (e.g., sex, comorbidities, lifestyle risk factors, cancer sites, and AJCC stage) were analyzed using a two-sided Fisher’s exact test or a two-sided Pearson’s chi-squared test. Parametric and non-parametric continuous data were analyzed using Student’s t-test and the Mann–Whitney U test, respectively. To minimize the confounding effects due to non-randomized allocation, data were analyzed from a 1:1 propensity score-matched cohort (ARBs vs. nil), which had been identified by the Greedy method with a 0.25 caliper width using NCSS software, version 10 (NCSS Statistical Software, Kaysville, UT, USA). Propensity scores were calculated using a logistic regression model with sex, age, pathological AJCC stage, comorbidities, and the diagnostic year of OSCC as covariates (S1 Table). In ARB users, we calculated the survival time from the day of OSCC diagnosis if ABR was already used or from the day of starting ARB use if the patient had not used it after OSCC diagnosis. Because the diagnostic years were matched in both groups, the calculated survival time in non-users started from the same day as its match to make the comparison between the two groups fair. The Kaplan–Meier method and log-rank test were used to evaluate the effects of ARBs on the primary outcome. The correlations between variables were evaluated using Pearson’s correlation coefficient to prevent multicollinearity before building a regression model. Several models were built and tested as a sensitivity analysis, and the Cox proportional hazards model was used if the model met the criteria of the smallest Akaike information criterion (AIC). The Cox proportional hazards model tested the dependence of primary factors on other prognostic factors in multivariate survival modeling. Stratified analysis was performed and adjusted to analyze the efficacy of ARBs in patients at different pathological stages. All statistical analyses were performed using SAS software, version 9.4 of the SAS System for Windows (SAS Institute Inc., Cary, NC, USA). P-values < 0.05 were considered statistically significant.

Results

Among the 7558 oral cancer patients, 5673 OSCC patients remained after applying the exclusion criteria (Table 1). In brief, age, lifestyle risk factors, and AJCC stages of cancer markedly differed between ARB users and non-users. In addition, there were significantly higher numbers of ARB users with comorbidities, such as hypertension, diabetes mellitus (DM), and hyperlipidemia, compared to non-users. Treatments were very different between the two groups as well.

A total of 714 patients were recruited for this cohort study after performing 1:1 PSM to balance covariates between the two groups, of which 357 were identified as ARB users and 357 as non-users after their diagnosis of cancer. Only ARB administration was significantly associated with hypertension. Otherwise, there were no statistically significant differences in clinical features between ARB-treated patients and those who did not receive ARB. Baseline clinicopathological characteristics of the study cohort are summarized in Table 2. Of the 714 patients with OSCC, 92.3% (n = 659) were men and 7.7% (n = 55) were women. The median age at diagnosis was 58 years. Tongue (34.9%) and buccal mucosa (34.3%) were the most common tumor sites. In all, 443 patients (62.0%) had early-stage cancer (stage I or II), while the remaining 271 patients (38.0%) had advanced-stage tumors (stage III, IVA, or IVB). Also, 460 patients (64.4%) underwent surgery alone, and 254 (35.6%) underwent surgery plus adjuvant radiotherapy (RT) or concurrent chemoradiotherapy (CCRT). At the end of the study period, 165 (23.1%) patients had died; 94 of these (13.2%) had died of primary head and neck cancer.

Regarding the influence of prognostic factors on survival, univariate Cox regression analysis showed that various clinical variables, including age, pathological AJCC stages of cancer, treatments, DM, and ARB use, were significantly associated with overall survival (OS), while sex and hypertension were not significantly correlated with survival rate. On the other hand, only pathological AJCC stages of cancer and treatments were statistically significantly associated with disease-specific survival (DSS) (Table 3). In this 10-year cohort study, patients receiving ARBs for more than 180 days exhibited a significantly higher OS rate than those who did not receive ARBs (Fig 2). However, the DSS rate was not statistically significantly different between patients receiving ARBs and those not receiving ARBs (Fig 3).

Table 3

Univariate analyses of prognostic factors for OS and DSS in patients with oral cancer.

Factor	Cohort	OS		DSS
		Hazard ratio (95% CI)	p value	Hazard ratio (95% CI)	p value
Age (year (IQR))	58(52–66)	1.04(1.02–1.05)	*<0.001	1.01(0.99–1.03)	0.403
Gender			0.707		0.822
Female	55(07.7%)	1		1
Male	659(92.3%)	0.90(0.53–1.54)		0.92(0.45–1.90)
Pathological AJCC 7th staging			*<0.001		*<0.001
I	231(32.4%)	1		1
II	212(29.7%)	1.06(0.69–1.65)		1.37(0.72–2.60)
III	94(13.2%)	1.22(0.71–2.10)		1.49(0.68–3.26)
IVa & IVb	177(24.8%)	2.83(1.91–4.18)		4.57(2.62–7.98)
Treatment			*<0.001		*<0.001
Operation alone	460(64.4%)	1		1
Operation plus RT/CRT	254(35.6%)	1.85(1.36–2.51)		2.58(1.72–3.87)
Diabetes mellitus			*0.011		0.175
No	315(44.1%)	1		1
Yes	399(55.9%)	1.52(1.10–2.10)		1.34(0.88–2.03)
Hypertension			0.097		0.207
No	223(31.2%)	1		1
Yes	491(68.8%)	0.76(0.56–1.05)		0.76(0.50–1.16)
ARBs use			*0.038		0.121
No	357(50.0%)	1		1
≥ 180 days	357(50.0%)	0.72(0.53–0.98)		0.72(0.48–1.09)

Abbreviations: AJCC, American Joint Committee on Cancer; ARBs, angiotensin II receptor blockers; CRT, chemoradiotherapy; DSS, disease specific survival; OS, overall survival; RT, radiotherapy.

* p ≤ 0.05.

Fig 2

Kaplan–Meier survival curve of OS rates between ARBs users (≥180 days) and non-users.

The estimated 5- and 10-year OS rates of ARB non-users (None) were 70.6% and 52.1%, respectively. The estimated 5- and 10-year OS rates of ARB users (≥180 days) were 77.7% and 57.9%, respectively. ARBs, angiotensin II receptor blocker; OS, overall survival.

Fig 3

Kaplan–Meier survival curve of DSS rates between ARB users (≥180 days) and non-users.

The estimated 5- and 10-year DSS rates of ARB non-users (None) were 81.8% and 75.3%, respectively. The estimated 5- and 10-year DSS rates of ARB users (≥180 days) were 85.4% and 82.7%, respectively. ARBs, angiotensin II receptor blockers; DSS, disease-specific survival.

Several regression models were built before a final Cox regression model could be determined (Table 4). Model 1 was adjusted for all potential confounders, including age, sex, pathological AJCC 7th staging, treatment, and diabetes mellitus. Model 2 was adjusted for suspected confounders according to the crude associations in univariate analyses. Model 3 was built according to the stepwise solution in statistical software. The stable effect size was found across different models in either OS or DSS. The final Cox proportional hazards models were chosen for OS and DSS if the models met the criteria of the smallest AIC. Therefore, models 2 and 3 were selected for OS and DSS, respectively.

Table 4

Modeling for the effects of ARBs on OS and DSS in patients with resectable OSCC.

Outcomes	ARBs ≥ 180 days (n = 357)	Non-Users (n = 357)	Adjusted Hazard Ratio (95% CI)
			*Model 1	**Model 2	***Model 3
OS	79.8%	73.9%	0.73(0.53–0.99)	0.73(0.53–0.99)	0.74(0.54–1.01)
			AIC = 1860.9	AIC = 1858.9	AIC = 1864.7
DSS	88.5%	85.2%	0.72(0.48–1.09)	0.71(0.47–1.07)	0.73(0.48–1.10)
			AIC = 1106.0	AIC = 1107.2	AIC = 1103.5

Abbreviations: AIC, Akaike information criterion; ARBs, angiotensin II receptor blockers; DSS, disease-specific survival; OS, overall survival; OSCC, oral squamous cell carcinoma.

*Model 1 was adjusted for all potential confounders, including age, sex, pathological AJCC 7th staging, treatment, and diabetes mellitus.

**Model 2 was adjusted for suspected confounders according to the crude associations in Table 3. For OS, age, pathological AJCC 7th staging, treatment, and diabetes mellitus were adjusted; for DSS, pathological AJCC 7th staging and treatment were adjusted.

***Model 3 was built with age, pathological AJCC 7th staging, and diabetes mellitus adjustment according to the statistical software (stepwise solution).

According to the chosen models, multivariate analyses revealed that only advanced disease was associated with reduced OS and DSS. In addition, aging and diabetes mellitus were related to poor OS. Notably, 180-day ARB use was associated with improved OS (HRARB users vs. non-users = 0.73, 95% CI = 0.53–0.99) but was not statistically significantly associated with improved DSS (HRARB users vs. non-users = 0.73, 95% CI = 0.48–1.10) in patients with resectable oral cancer (Table 5). Furthermore, a survival benefit with at least 180 days of ARB use was observed in patients with stages III and IV OSCC (HRARB users vs. non-users = 0.61, 95% CI = 0.39–0.94), but no statistical significance was observed in patients with stages I and II OSCC in advanced analysis (Table 6). Overall, these analyses suggest that patients with late-stage OSCC are the most likely to benefit from ARB use for more than 180 days after OSCC diagnosis.

Table 5

Multivariate analyses of prognostic factors for OS and DSS in patients with oral cancer.

Factor	Cohort	**OS		***DSS
		Hazard ratio (95% CI)	p value	Hazard ratio (95% CI)	p value
Age (year (IQR))	58(52–66)	1.05(1.03–1.06)	*<0.001	1.02(0.99–1.04)	0.058
Pathological AJCC 7th staging			*<0.001		*<0.001
I	231(32.4%)	1		1
II	212(29.7%)	1.09(0.70–1.71)		1.40(0.73–2.65)
III	94(13.2%)	1.19(0.66–2.13)		1.50(0.68–3.30)
IVa & IVb	177(24.8%)	3.21(1.93–5.34)		5.08(2.88–8.95)
Treatment			0.355	===	===
Operation alone	460(64.4%)	1
Operation plus RT/CRT	254(35.6%)	1.21(0.80–1.84)
Diabetes mellitus			*0.003		0.113
No	315(44.1%)	1		1
Yes	399(55.9%)	1.63(1.17–2.25)		1.40(0.92–2.14)
ARBs use			*0.049		0.142
No	357(50.0%)	1		1
≥ 180 days	357(50.0%)	0.73(0.53–0.99)		0.73(0.48–1.10)

Abbreviations: AJCC, American Joint Committee on Cancer; ARBs, angiotensin II receptor blockers; CRT, chemoradiotherapy; DSS, disease specific survival; OS, overall survival; RT, radiotherapy.

* p ≤ 0.05.

** Model for OS was adjusted for age, pathological AJCC 7th staging, treatment and diabetes mellitus according to the smallest AIC in Table 4.

** Model for DSS was adjusted for age, pathological AJCC 7th staging and diabetes mellitus according to the smallest AIC in Table 4.

Table 6

Effects of ARBs on OS in patients with early and advanced OSCC.

Pathological AJCC staging	Variables	Death	Alive	Crude HR (95% CI)	p-value	**Adjusted HR (95% CI)	p-value
Stage I & II	None	41(18.6%)	179(81.4%)	1	0.619	1	0.684
	ARBs	39(17.5%)	184(82.5%)	0.90(0.58–1.39)		0.92(0.59–1.42)
Stage III & IV	None	52(38.0%)	85(62.0%)	1	*0.018	1	*0.026
	ARBs	33(24.6%)	101(75.4%)	0.59(0.38–0.91)		0.61(0.39–0.94)

Abbreviations: AJCC, American Joint Committee on Cancer; ARBs, angiotensin II receptor blockers; HR, Hazard Ratio; OSCC, oral squamous cell carcinoma.

* p ≤ 0.05.

** Model was adjusted for age, treatment and diabetes mellitus.

Discussion

To the best of our knowledge, this was the first study to investigate the potential clinical benefit of ARBs in patients with OSCC receiving surgery. In this 10-year retrospective cohort study, patients who received ARBs for at least 180 days had improved OS compared to patients who did not receive ARBs. In addition, patients with locally advanced OSCC experienced the most significant benefit from ARBs.

The RAS consists of several enzymatic and non-enzymatic protein components and is essential for the maintenance of vascular homeostasis. Angiotensinogen is produced in the liver and cleaved by the aspartyl protease renin to angiotensin I. Angiotensin I is subsequently cleaved by the angiotensin I-converting enzyme to produce angiotensin II (Ang II). Ang II is a key component of the RAS, which exerts its actions by binding to two G protein-coupled receptors: angiotensin receptor 1 (AT1R) and the lesser known angiotensin receptor 2 [15]. It is increasingly evident that, in addition to systemic effects on blood pressure and fluid homeostasis, AT1R and Ang II have important roles at the local tissue level. AT1R overexpression has been reported in numerous cancers, including ovarian, breast, and bladder cancer [16, 17]. Consistent with these findings, ARBs and ACEIs have been reported to reduce tumor growth and vascularization in a wide range of cancers, suggesting a role for Ang II in cancer development and progression [4]. We summarized the studies containing ARBs in the last ten years and revealed the effects on OS and DSS across different malignancies (Table 7) [5–8, 18–23]. However, there was no consistent conclusion on whether ARBs have a survival benefit in patients coexisting with malignancies. One of the main reasons was the discrepancy in confounding control across these studies. Unknown confounding and selection bias might exist in these retrospective studies with different study design, indicating the findings are only the association between ARBs use and survival. Future two-arm controlled study should be conducted for strengthening the causal relationship. In addition, the varying extent of concurrent ACEI and ARB use among previous studies may confound their effects. Therefore, we evaluated patients who had received ARBs only in our study (S2 Table).

Table 7

Summary of previous research on the effects of ARBs on OS and DSS across different malignancies.

Cancer	Studies	Medication	Sample size	Outcomes	Study design	Notes
Breast	Holmes et al. (2013)	ACEi/ARBs	Exp: 880Non-E: 2310	OS aHR (95% CI): 1.22 (1.04, 1.44)	Retrospective cohort	Not control comorbidities
	Cardwell et al. (2014)	ARBs	Cases: 648Controls: 3193	OS aOR (95% CI): 0.79 (0.60, 1.03)DSS aOR (95% CI): 0.94 (0.65, 1.37)	Nested case–control	===
Esophageal	Busby et al. (2018)	ARBs	Exp: 168Non-E: 2565	DSS aHR (95% CI): 0.89 (0.71, 1.10)	Retrospective cohort	Not control stages
Gastric	Kim et al. (2012)	ACEi/ARBs	Exp: 30Non-E: 33	OS aHR (95% CI): 0.54 (0.30, 0.97)	Retrospective cohort	Advanced stages
	Busby et al. (2018)	ARBs	Exp: 168Non-E: 2565	DSS aHR (95% CI): 0.79 (0.62, 1.00)	Retrospective cohort	Not control stages
Liver	Facciorusso et al. (2015)	ARBs	Exp: 43Non-E: 113	OS HR (95% CI): 0.71 (0.46–1.10)	Retrospective cohort	Not adjust for HR
Pancreas	Nakai et al. (2010)	ACEi/ARBs	Exp: 27Non-E: 103	OS aHR (95% CI): 0.52 (0.29, 0.88)	Retrospective cohort	===
	Cerullo et al. (2017)	ARBs	Exp: 479Non-E: 3820	OS aHR (95% CI): 0.76 (0.67, 0.87)	Retrospective cohort	===
Colorectal	Holmes et al. (2013)	ACEi/ARBs	Exp: 1187Non-E: 1864	OS aHR (95% CI): 1.03 (0.93, 1.15)	Retrospective cohort	Not control comorbidities
	Cardwell et al. (2014)	ARBs	Cases: 1093Controls: 5231	OS aOR (95% CI): 1.02 (0.82, 1.26)DSS aOR (95% CI): 0.80 (0.59, 1.09)	Nested case–control	===
	Morris et al. (2016)	ACEi/ARBs	Exp: 25Non-E: 90	OS aOR (95% CI): 0.73 (0.45–1.20)	Retrospective cohort	===
Renal	Asgharzadeh et al. (2020)	ARBs	===	OS aHR (95% CI): 0.81 (0.69, 0.96)	Meta-analysis	===
Prostate	Cardwell et al. (2014)	ARBs	Cases: 766Controls: 3777	OS aOR (95% CI): 0.92 (0.75, 1.12)DSS aOR (95% CI): 0.82 (0.61, 1.11)	Nested case–control	===
	Mao et al. (2016)	ACEi/ARBs	===	OS aRR (95% CI): 0.92 (0.87, 0.98)	Meta-analysis	===

Abbreviations: ACEi, Angiotensin converting enzyme inhibitors; aHR, adjusted Hazard ratio; aOR, adjusted Odds ratio; aRR, adjusted risk ratio; ARBs, angiotensin II receptor blockers; DSS, disease specific survival; Exp, exposure; HR, Hazard ratio; Non-E, non-exposure; OS, overall survival; OSCC, oral squamous cell carcinoma.

Head and neck cancer is the sixth most common cancer worldwide [24]. The critical role of RAS in head and neck cancer has been shown in various tissues, including the oral mucosa [25]. Additionally, Ang II also has been found to promote HNSCC cell migration and invasion [26]. The effects of Ang II on autocrine and paracrine signaling pathways are mediated by AT1R, suggesting that ARBs might provide a clinical benefit in patients with HNSCC. This was approved by Lin et al., which ARBs were found to exert antiproliferative and antiangiogenesis effects by inducing apoptosis in nasopharyngeal carcinoma (NPC). In addition, improved 5-year OS and DSS were found among patients with NPC using ARBs [27]. Interestingly, we found that ARB use for at least 180 days improved the OS rate of patients with OSCC statistically, yet not DSS rate (Table 5). Accordingly, RAS is a major regulator of blood pressure (BP) and vascular response to injury. There is large evidence that RAS inhibition provides end-organ protection independent of BP lowering [28]. That probably explained the survival benefit of using ARBs is mainly through as end-organ protective effect, which further reduced overall mortality [29]. As for the anti-cancer effect of ARBs in oral cancer patients, it still remained controversial in our current study. Notably, the survival effects were most pronounced for patients with late-stage resectable OSCC (Table 6), suggesting that pathological staging served as an effect modifier.

We included ARB users for at least 180 days, as these medications are unlikely to have immediate effects on cancer progression [20]. In addition, the best lag-time to be applied in studies accordingly was around 6 months, which was the most appropriate period for the assessment of drug exposure [30]. On the other hand, we excluded patients without ARB use surviving < 180 days after the index date to make the comparison between the two groups fair. Although ARB use was not associated with a statistically improved DSS rate in our study, the hazard rate was similar to OS after adjusting for potential confounding factors (Table 4). The possible reason for the lack of statistical power was that our sample size was not large enough. The use of PSM to reduce the bias due to confounding variables was the highlight of our study, although the matched sample size was limited. When we used a 1:2 or larger ratio of matching, unmatched cases dramatically increased. Therefore, we created 1:1 PSM in our study, which only dropped five patients in the group of ARB users (S1 Table). As major comorbidity, hypertension was not selected to be a matched variable in our study because of its high correlation with ARB users (Pearson’s correlation coefficient = 0.673, p<0.001). If we input hypertension into the PSM model, the matched non-ARB users would be highly associated with hypertension. The matched candidates would be scarce, and the generalizability would be limited.

Our study had a few limitations. First, medical records were incomplete for some patients. Therefore, some critical clinicopathological characteristics (e.g., surgical margin, extranodal extension, and depth of tumor invasion) could not be analyzed in our study. Second, although our study included only data from patients treated with ARBs, a considerable proportion of the patients were receiving concurrent treatment with other agents to control hypertension, which might have influenced our findings. Several patients received amlodipine and hydrochlorothiazide (S2 Table); however, there is no evidence that these agents suppress cancer development or progression [31, 32]. Therefore, we presume that treatment with these agents had a minimal influence on our findings. Third, various ARBs were included; therefore, the standardized effective dosage was difficult to calculate, and the dose-response relationship could not be measured in our study. Lastly, blood pressure, a crucial variate in an anti-hypertensive medication study, was not taken into account in our research. The main reasons were that complete data of blood pressure was not available in our database, and many confounding factors existed in the residual database as well. However, taking representative blood pressure data into analysis should be considered in future studies. In conclusion, a future prospective controlled study should be conducted to overcome these limitations.

Conclusion

This was the first study to investigate the clinical usefulness of ARBs in patients with OSCC receiving surgery. Patients who received ARBs for at least 180 days exhibited improved OS. Additionally, ARBs use was associated with a more significant survival benefit in patients with operable stage III, IVa, and IVb OSCC. A further two-arm study should be conducted to confirm the clinical usefulness of ARBs in OSCC patients.

Baseline characteristics of OSCC patients before and after propensity-score matching.

(DOCX)

Click here for additional data file.

The included angiotensin II receptor blockers and numbers in our study.

(DOCX)

Click here for additional data file.

14 Sep 2021

PONE-D-21-16892Angiotensin II receptor blockers and oral squamous cell carcinoma survival: A propensity-score-matched cohort studyPLOS ONE

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Reviewer #1: The authors reported that ARB use for more than 180 days is associated with an increased survival rate and is a positive, independent prognostic factor in patients with oral squamous cell carcinoma(OSCC) in their propensity-score-matched cohort from registry database.

It is interesting, but also arguable results. As the authors commented and listed other studies reporting the association between the ARBs and cancers (Table 7), it is still unclear whether the ARBs have survival benefit in most cancers. For now it is evident that treatment with an ARB medication does not increase the risk of cancer (FDA report https://www.fda.gov/drugs/drug-safety-and-availability/fda-drug-safety-communication-no-increase-risk-cancer-certain-blood-pressure-drugs-angiotensin) .

Even though they found through the skilled analysis from their propensity-score-matched cohort, their findings are the association between ARBs use and survival in OSCC patients NOT the causal relationship. So they may not conclude “ARBs have survival benefit in OSCC patients”. It should be very prudent to this conclusion. In this regard, I recommend to erase the final parenthesis “These findings highlight the clinical usefulness of ARBs in OSCC patients with advanced disease” in conclusion. And the authors had better to add some comments on this critical issue regarding the causal relationship.

Reviewer #2: The authors described that long use of ARB is associated with an increased survival

rate and is a positive, independent prognostic factor in patients with oral squamous cell carcinoma (OSCC).

The point of this observation is very interesting, and while the manuscript is well written and are well

performed, I want you to revise the manuscript because of the following questions.

Major questions

1. There is no data of blood pressure. Whether the patients take ARB or not, whether the patients have

hypertension or not, whether the patients have cancer or not, hypertension is the very important

factor of mortality. Therefore, the data of blood pressure of two groups is very important and if

authors prove the usefulness of ARB in the context of increasing survival rate, the authors must

show the data of blood pressure and when the authors analyze the data, the authors must correct

the data of blood pressure.

2. The authors reveal that all patients using ARB have hypertension, but only 37.5 % of the patients not

using ARB have hypertension. If the authors discuss the usefulness of ARB in the context of increasing

survival rate of OSCC patients, the authors need to demonstrate whether the reason of the usefulness

of ARB is class effect or antihypertensive effect.

3. There are various pathological types in oral cancer. Why do the authors choose only squamous cell

carcinoma? Why do the authors exclude non-squamous cell carcinoma during the first stage? I can

understand that the authors analyze by type of pathological findings, however I cannot understand

excluding the patient of non-squamous cell carcinoma. I think the authors must also analyze for the

patients of non-squamous cell carcinoma.

4. Can’t the cancer duration at the start of medication of ARB or antihypertensive agents be a

confounding factor?

5. Why do the authors decide the cut off period of receiving ARB to more than 180 days or not?

**********

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13 Oct 2021

RE: “Angiotensin II receptor blockers and oral squamous cell carcinoma survival: A propensity-score-matched cohort study.”

We would like to thank the reviewers for the thorough reading of our manuscript as well as their valuable comments. We have followed their comments closely and feel that their suggestions have further strengthened the manuscript. Below are our point-by-point responses.

Response to Reviewer’s Comments:

Review 1:

The authors reported that ARB use for more than 180 days is associated with an increased survival rate and is a positive, independent prognostic factor in patients with oral squamous cell carcinoma (OSCC) in their propensity-score-matched cohort from the registry database. It is interesting but also arguable results. As the authors commented and listed other studies reporting the association between the ARBs and cancers (Table 7), it is still unclear whether the ARBs have a survival benefit in most cancers. For now, it is evident that treatment with an ARB medication does not increase the risk of cancer (FDA report https://www.fda.gov/drugs/drug-safety-and-availability/fda-drug-safety-communication-no-increase-risk-cancer-certain-blood-pressure-drugs-angiotensin). Even though they found through the skilled analysis from their propensity-score-matched cohort, their findings are the association between ARBs use and survival in OSCC patients NOT the causal relationship. So they may not conclude “ARBs have survival benefit in OSCC patients”. It should be very prudent to this conclusion. In this regard, I recommend to erase the final parenthesis “These findings highlight the clinical usefulness of ARBs in OSCC patients with advanced disease” in conclusion. And the authors had better to add some comments on this critical issue regarding the causal relationship.

Response: Dear reviewer, I agree with your precise comment. I deleted the final parenthesis according to your opinion and changed it to “A further two-arm study should be conducted to confirm the clinical usefulness of ARBs in OSCC patients.” (Page 2 Abstract; Page 22 Conclusion). We also added some comments on this issue in the Discussion section as the following: “However, there was no consistent conclusion on whether ARBs have a survival benefit in patients coexisting with malignancies. One of the main reasons was the discrepancy in confounding control across these studies. Unknown confounding and selection bias might exist in these retrospective studies with different study design, indicating the findings are only the association between ARBs use and survival. Future two-arm controlled study should be conducted for strengthening the causal relationship.” (Page 17)

Review 2:

1. There is no data of blood pressure. Whether the patients take ARB or not, whether the patients have hypertension or not, whether the patients have cancer or not, hypertension is the very important factor of mortality. Therefore, the data of blood pressure of two groups is very important and if authors prove the usefulness of ARB in the context of increasing survival rate, the authors must show the data of blood pressure and when the authors analyze the data, the authors must correct the data of blood pressure.

Response: Dear reviewer, I agree with your opinion very well, and I think what you are concerned about is very important. However, I am sorry that we didn’t have the complete blood pressure (BP) data for each person in our database. The most representative BP comes from the daily measurement by the patient at home, or the BP measured in the clinic at least. However, we only had the BP measured during the hospitalization, which was available for 616 patients. The most crucial limitation from the hospitalization BP was the incomplete data for all patients and many confounding factors existing in this data. For example, patients staying in the hospital might suffer from acute disease, which induced unstable BP measurement. Some patients might even be in a critical status, which caused a lower BP result. Because of the reasons above, I deem it as a major limitation in our study, and put it into our discussion. I hope you could accept my explanation.

We agree your comment is crucial for future study. Therefore, I analyzed the hospitalization BP regardless of the reasons we mentioned above. The data units of systemic blood pressure (SBP) level below 80 mmHg were deleted first, which I deemed probable data of critical status. Then I collected and averaged all the SBP data for each person (n=616). Afterward, the SBP data were classified into normal and abnormal by BP=140 mmHg. We put the variate into our Cox regression model and found no statistical effect on overall survival (HR high BP vs. normal BP = 0.79, 95% CI = 0.54-1.14). A similar result was found if we changed the divide by BP=120 mmHg (HR high BP vs. normal BP = 0.73, 95% CI = 0.48-1.11). However, we can’t jump to the conclusion that BP was not associated with survival because the above analysis was based on an incomplete and unrepresentative hospitalization BP database.

In conclusion, a complete representative BP measurement should be taken into account in a future study. Therefore, I summarized the above explanation and added it into the discussion section: “Lastly, blood pressure, a crucial variate in an anti-hypertensive medication study, was not taken into account in our research. The main reasons were that complete data of blood pressure was not available in our database, and many confounding factors existed in the residual database as well. However, taking representative blood pressure data into analysis should be considered in future studies.” (Page 21)

2. The authors reveal that all patients using ARB have hypertension, but only 37.5 % of the patients not using ARB have hypertension. If the authors discuss the usefulness of ARB in the context of increasing survival rate of OSCC patients, the authors need to demonstrate whether the reason of the usefulness of ARB is class effect or antihypertensive effect.

Response: Dear reviewer, your comment about survival benefit might be from ARB use probably from the antihypertensive effect instead of the class effect. It is a question not easy to answer. Here, I assumed that the survival of patients with ARB use would not be superior to those without hypertension if the antihypertensive effect plays a significant role in survival. This assumption is based on the previous research (Ref 1), in which patients with hypertension had poor long-term survival than patients without hypertension. In other words, the class effect of ARBs on the survival benefit in oral cancer patients could be approved if the survival of patients with ARB use (hypertension group) is superior to those without hypertension.

We tested this hypothesis and calculated the crude and adjusted hazard ratio, including a 95% confidence interval for overall survival in patients with ARBs use (n=357) relative to patients without hypertension (n=223). Notably, 180-day ARB use was consistently associated with improved OS with or without adjustment. (Crude HR ARB users vs. non-HTN = 0.70, 95% CI = 0.50-0.99; adjusted HR ARB users vs. non-HTN = 0.67, 95% CI = 0.48-0.95). The above findings displayed the possible anti-cancer effect of ARBs in oral cancer patients. In addition, there is no evidence that other antihypertensive agents suppress cancer development or progression, which had been described in the Limitation section. The class effect of ARBs was therefore indirectly approved.

We appreciate your incisive comments. The above statements were concisely added into our Discussion section as the following:” Some might argue that the survival benefit from ARB use was probably from the antihypertensive effect instead of the anti-cancer effects. According to a previous study, patients with hypertension had poor long-term survival than those without hypertension [28]. However, in our study, 180-day ARB use in patients with hypertension was consistently associated with improved OS compared to patients without hypertension. (Crude HRARB users vs. non-HTN = 0.70, 95% CI = 0.50-0.99; adjusted HRARB users vs. non-HTN = 0.67, 95% CI = 0.48-0.95). The above findings displayed the possible anti-cancer effect of ARBs in oral cancer patients.” (Page 19)

Ref 1. Andersson OK, Almgren T, Persson B, Samuelsson O, Hedner T, Wilhelmsen L. Survival in treated hypertension: follow up study after two decades. BMJ. 1998;317(7152):167-171. doi:10.1136/bmj.317.7152.167

3. There are various pathological types of oral cancer. Why do the authors choose only squamous cell carcinoma? Why do the authors exclude non-squamous cell carcinoma during the first stage? I can understand that the authors analyze by type of pathological findings. However, I cannot understand excluding the patient of non-squamous cell carcinoma. I think the authors must also analyze for the patients of non-squamous cell carcinoma.

Response: Dear reviewer, what you had concerned about is reasonable. However, according to the past epidemiology study of oral cancer in Asia, the majority (84-97%) of oral cancer is squamous cell carcinoma (Ref 1). One native study in Taiwan also showed that squamous cell carcinoma type occupied 93.1% of oral cancer (Ref 2). Because of the epidemiologic characteristics in Taiwan, we focused on the major squamous cell carcinoma type to prevent accidental confounding from other types. I hope you can accept this explanation.

Ref 1. Krishna Rao SV, Mejia G, Roberts-Thomson K, Logan R. Epidemiology of oral cancer in Asia in the past decade--an update (2000-2012). Asian Pac J Cancer Prev. 2013;14(10):5567-77. doi: 10.7314/apjcp.2013.14.10.5567.

Ref 2. Huang CC, Ou CY, Lee WT, Hsiao JR, Tsai ST, Wang JD. Life expectancy and expected years of life lost to oral cancer in Taiwan: a nation-wide analysis of 22,024 cases followed for 10 years. Oral Oncol. 2015 Apr;51(4):349-54. doi: 10.1016/j.oraloncology.2015.01.001. Epub 2015 Jan 17.

4. Can’t the cancer duration at the start of medication of ARB or antihypertensive agents be a confounding factor?

Response: Yes, we agree with your opinion. The duration between initial cancer diagnosis and the start point of ARB use could be a confounding factor, which was often considered an immortal time bias. To prevent such a bias, we had considered “the diagnostic year of oral cancer” as a covariate in both groups when performing propensity score matching. We had mentioned this part in the “Statistical Analyses” section as the following:” Propensity scores were calculated using a logistic regression model with sex, age, pathological AJCC stage, comorbidities, and the diagnostic year of OSCC as covariates (S1 Table). … Because the diagnostic years were matched in both groups, the calculated survival time in non-users started from the same day as its match to make the comparison between the two groups fair.” (Page 6 Statistical Analyses)

5. Why do the authors decide the cut off period of receiving ARB to more than 180 days or not?

Response: Thank you for the excellent comment. Why we decided the cut-off period of 180 days was based on the previous research. The main reason is to control for protopathic bias, and therefore some studies have incorporated the concept of lag-time into their exposure definition. The best lag-time to be applied was also studied, which around six months were the most appropriate period for the assessment of drug exposure (Ref 1). We didn’t explain it clearly in the original manuscript, and therefore, we now added the supplement as the followings: “In addition, the best lag-time to be applied in studies accordingly was around six months, which was the most appropriate period for the assessment of drug exposure [29].” (Page 20)

There was another direct and practical reason. We had run a pre-analysis Kaplan-Meier Method, which considers the possible effects of at least three months (90days) or six months (180 days). The figures are shown below, and the preliminary result showed that only receiving ARBs for more than 180 days had a statistical survival benefit.

Ref 1. Tamim H, Monfared AA, LeLorier J. Application of lag-time into exposure definitions to control protopathic bias. Pharmacoepidemiol Drug Saf. 2007 Mar;16(3):250-8. doi: 10.1002/pds.1360. PMID: 17245804.

Submitted filename: 20210927_Rebutal-letter opinions.docx

Click here for additional data file.

3 Nov 2021

PONE-D-21-16892R1Angiotensin II receptor blockers and oral squamous cell carcinoma survival: A propensity-score-matched cohort studyPLOS ONE

Dear Dr. Chiu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The revised manuscript is well assessed by the two reviewers; however, several minor revisions are still necessary in the present form.

See the reviewer's comments carefully and respond them appropriately.

==============================

Please submit your revised manuscript by Dec 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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

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Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #2: Yes

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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 #1: The authors addressed adequately my comments. I think the paper revised properly with adequate answers to reviewer's comments.

Reviewer #2: Thank you for your response to my question.

Most of your response of the authors can be understanded, however, some responses seem to be arguable.

About my question No.1-2, your responses are very well-formed, and your analysis about 616 patients of available information of BP measurements is very important data, however it seems that the reason of survival benefit of using ARB in oral cancer patients is not always anti-cancer effect of ARBs. Even if the effect of survival benefit of using ARB is not dependent on blood pressure, the reasons of survival benefit of using ARB are thought of as several organ protective effect, such as cardioprotective effect or renal protective effect. Therefore, the author should delete enrollment of possible anti-cancer effect of ARBs, and should describe another consideration about survival benefit of using ARB in oral cancer patients.

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Reviewer #1: No

Reviewer #2: No

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8 Nov 2021

RE: “Angiotensin II receptor blockers and oral squamous cell carcinoma survival: A propensity-score-matched cohort study.”

We would like to thank the reviewers for the thorough reading of our manuscript as well as their valuable comments. We have followed their comments closely and feel that their suggestions have further strengthened the manuscript. Below are our point-by-point responses.

Response to Reviewer’s Comments:

Review 1:

The authors addressed adequately my comments. I think the paper revised properly with adequate answers to reviewer's comments.

Response: Dear reviewer, we thanked again for your thorough reading of our manuscript as well as the valuable comments.

Review 2:

Most of your response of the authors can be understanded, however, some responses seem to be arguable. About my question No.1-2, your responses are very well-formed, and your analysis about 616 patients of available information of BP measurements is very important data, however it seems that the reason of survival benefit of using ARB in oral cancer patients is not always anti-cancer effect of ARBs. Even if the effect of survival benefit of using ARB is not dependent on blood pressure, the reasons of survival benefit of using ARB are thought of as several organ protective effect, such as cardioprotective effect or renal protective effect. Therefore, the author should delete enrollment of possible anti-cancer effect of ARBs, and should describe another consideration about survival benefit of using ARB in oral cancer patients.

Response: Dear reviewer, I agree with your opinion addressed” Even if the effect of survival benefit of using ARB is not dependent on blood pressure, the reasons of survival benefit of using ARB are thought of as several organ protective effect, such as cardioprotective effect or renal protective effect.” I think what you mentioned explained the main findings in our result, which the survival benefit only showed statistically significant in OS, but not in DSS. Therefore, some discussion and reference about this issue were added accordingly.

However, directly deleting possible anti-cancer effect of ARBs concerns me. In our discussion, the evidence of anti-cancer mechanism did exist in the molecular level in head and neck cancer (Ref 1,2). In addition, a newly published article in Cancer (Ref 3) also proved that ARB use provided clinical survival benefits (both OS and DSS) with specific anti-tumor molecular signaling in nasopharyngeal carcinoma, which indicated that ARB might have anti-cancer effects on HNSCC. In our article, the anti-cancer effect didn’t reach a statistically significance (DSS, HRARB users vs. non-users = 0.73, 95% CI = 0.48–1.10), but the effect size was nevertheless similar to OS (HRARB users vs. non-users = 0.73, 95% CI = 0.53-0.99). I would rather interpret that there might probably exist an anti-cancer effect, yet with more variability, which the conclusion of anti-cancer effect can’t be reached in our study.

Therefore, I changed to use more conservative wording in the text, deleted some arbitrary sentences, and revised relevant reference of anti-cancer in HNSCC. I consider that making a balanced discussion between the effects of ARBs would be better than presenting only one side. I hope you could accept my explanation.

The followings are some changes made in our text:

1. We deleted the sentence” The effectiveness of anti-cancer therapies has been supported in many clinical studies.” (Page 17 Discussion).

2. We deleted the sentence” The results of our clinical study particularly highlight the anti-cancer effects on HNSCC;” (Page 19 Discussion).

3. We deleted the sentence” the ACEI perindopril has been shown to reduce the growth of head and neck squamous cell carcinoma (HNSCC) in vivo [26], suggesting a role for Ang II in HNSCC.” (Page 19 Discussion).

4. We deleted the sentences” Some might argue that the survival benefit from ARB use was probably from the anti-hypertensive effect instead of the anti-cancer effects. According to a previous study, patients with hypertension had poor long-term survival than those without hypertension [31]. However, in our study, 180-day ARB use in patients with hypertension was consistently associated with improved OS compared to patients without hypertension. (Crude HRARB users vs. non-HTN = 0.70, 95% CI = 0.50-0.99; adjusted HRARB users vs. non-HTN = 0.67, 95% CI = 0.48-0.95). The above findings displayed the possible anti-cancer effect of ARBs in oral cancer patients.” (Page 20 Discussion).

5. We balanced the discussion as followings: “…This was approved by Lin et al., which ARBs were found to exert antiproliferative and antiangiogenesis effects by inducing apoptosis in nasopharyngeal carcinoma (NPC). In addition, improved 5-year OS and DSS were found among patients with NPC using ARBs [27]. Interestingly, we found that ARB use for at least 180 days improved the OS rate of patients with OSCC statistically, yet not DSS rate (Table 5). Accordingly, RAS is a major regulator of blood pressure (BP) and vascular response to injury. There is large evidence that RAS inhibition provides end-organ protection independent of BP lowering [28]. That probably explained the survival benefit of using ARBs is mainly through as end-organ protective effect, which further reduced overall mortality [29].As for the anti-cancer effect of ARBs in oral cancer patients, it still remained controversial in our current study.” (Page 19 Discussion).

Ref 1. George, A.J., W.G. Thomas, and R.D. Hannan, The renin-angiotensin system and cancer: old dog, new tricks. Nat Rev Cancer, 2010. 10(11): p. 745-59.

Ref 2. Hinsley, E.E., et al., Angiotensin 1-7 inhibits angiotensin II-stimulated head and neck cancer progression. Eur J Oral Sci, 2017. 125(4): p. 247-257.

Ref 3. Lin, Y.-T., et al., Angiotensin II receptor blockers valsartan and losartan improve survival rate clinically and suppress tumor growth via apoptosis related to PI3K/AKT signaling in nasopharyngeal carcinoma. Cancer, 2021. 127(10): p. 1606-1619.

Submitted filename: 20211104_Rebutal-letter opinions.docx

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17 Nov 2021

Angiotensin II receptor blockers and oral squamous cell carcinoma survival: A propensity-score-matched cohort study

PONE-D-21-16892R2

Dear Dr. Chiu,

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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Masaki Mogi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

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Comments to the Author

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Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: (No Response)

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Reviewer #2: No

22 Nov 2021

PONE-D-21-16892R2

Angiotensin II receptor blockers and oral squamous cell carcinoma survival: A propensity-score-matched cohort study

Dear Dr. Chiu:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

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on behalf of

Dr. Masaki Mogi

Academic Editor

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