Upregulation of PODXL and ITGB1 in pancreatic cancer tissues preoperatively obtained by EUS-FNAB correlates with unfavorable prognosis of postoperative pancreatic cancer patients.

The upregulation of PODXL and ITGB1 in surgically resected pancreatic cancer tissues is correlated with an unfavorable postoperative prognosis. The aim of this study was to investigate whether PODXL and ITGB1 are useful preoperative markers for the prognosis of postoperative pancreatic cancer patients in comparison with the TNM staging system. Immunohistochemistry was performed using anti-PODXL and anti-ITGB1 antibodies on 24 pancreatic cancer tissue samples preoperatively obtained by endoscopic ultrasound-guided fine-needle aspiration biopsy. Cox proportional hazards regression analysis was performed to investigate if the UICC TNM stage and upregulation of PODXL and ITGB1 were correlated with postoperative overall survival rates. Univariate analysis revealed that PODXL, TNM stage, lymphatic invasion and the combination of PODXL with ITGB1 are correlated with postoperative survival. Multivariate analysis demonstrated TNM stage and the combination of PODXL with ITGB1 to be correlated with postoperative survival, and the combination of PODXL with ITGB1 most accurately predicted the postoperative outcomes of pancreatic cancer patients before resection. Therefore, upregulation of PODXL and ITGB1 may indicate preoperative neoadjuvant therapy for pancreatic cancer patients by accurately predicting the postoperative prognosis.

Introduction

Although there have been recent advances in novel cancer therapies, pancreatic ductal adenocarcinoma (PDAC) has the lowest 5-year survival rate [1, 2]. Only 15–20% of PDAC patients are diagnosed with potentially resectable disease, approximately 35% with localized unresectable disease, and approximately 50% with end-stage disease [3, 4]. Surgery is the only potentially curative treatment for PDAC [3]. However, the prognosis of resected PDAC patients remains poor due to the high rate of local recurrence and/or distant metastases, and the 5-year survival rate for these patients is only 20% [5]. The resection margin is the most important factor related to the prognostic outcome of resected PDAC patients, and a positive resection margin usually results in a higher risk of local recurrence and distant metastasis [6], but adjuvant chemotherapy after surgical resection can improve overall survival [7]. Borderline resectable or locally advanced PDAC is radiographically defined with or without vascular involvement, but the definitions for what constitutes borderline resectable and locally advanced PDAC have historically varied across institutions [8]. Recently, neoadjuvant therapy prior to surgical resection for PDAC patients with borderline resectable and locally advanced disease was reported to achieve tumor downstaging with the aim of secondary curative intent surgery [9]. Palliative chemotherapy and best supportive care remain the only options for metastatic PDAC patients [10].

Immunohistochemical analysis of surgical specimens from 102 PDAC patients revealed the upregulation of podocalyxin-like protein (PODXL) in 70.6% of the surgical specimens [11]. PODXL expression significantly correlates with histological grade, but it is not associated with other clinicopathological features such as clinical stage, venous invasion and lymphatic invasion [11]. Similar effects occurred with integrin β1 (ITGB1), as ITGB1 is upregulated in 67.6% of the surgical specimens of PDAC, and ITGB1 is not associated with any clinicopathological features [12]. Importantly, the combination of PODXL with ITGB1 immunohistochemically scored using resected PDAC tissues accurately predicts the postoperative prognosis of PDAC patients better than the Union for International Cancer Control (UICC) tumor node metastasis (TNM) staging system [12]. TNM staging was reported as an independent prognostic factor affecting the prognosis of patients with PDAC [13]. In addition, 86 of 102 PDAC patients received adjuvant chemotherapy, which does not correlate with the postoperative prognosis [12]. PODXL functions in promoting the invasion of PDAC cells by binding to the cytoskeletal protein gelsolin [11]. ITGB1 was reported to promote cell invasion and tumor metastasis of PDAC cells [14], thus ITGB1 is a therapeutic target for the treatment of PDAC [15]. These studies suggested that PODXL and ITGB1 play important roles in the invasiveness and/or metastasis of PDAC cells, and that increased expression of PODXL and ITGB1 is associated with the poor prognosis of PDAC.

Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) is widely available and is the gold-standard technique for the preoperative pathological diagnosis of PDAC [16]. If increased expression of PODXL and ITGB1 in PDAC tissues preoperatively obtained by EUS-FNAB is correlated with the postoperative poor prognosis, it may be useful to select which PDAC patients should receive preoperative neoadjuvant therapy to improve their postoperative prognosis. In the present study, we investigated the preoperative use of PODXL and ITGB1 in PDAC tissue samples from EUS-FNAB as useful markers for the prognosis of postoperative PDAC patients in comparison with the TNM staging system. The combination of PODXL with ITGB1 predicted the postoperative outcomes of PDAC patients better than the TNM staging system prior to surgery.

Results

PODXL expression in PDAC tissue samples obtained by EUS-FNAB

Immunohistochemical analysis of the expression levels of PODXL and ITGB1 was performed for 24 PDAC tissue samples obtained by EUS-FNAB before resection (Fig 1A), and we used the immunostaining scores of PODXL and ITGB1. The background information of PDAC patients is shown in Table 1. The tumor diameters were greater than 20 mm for 20/24 PDAC patients, and the numbers of patients with each UICC stage were as follows: IA (n = 5); IB (n = 4); IIA (n = 9); IIB (n = 5); and III (n = 1). Five patients (20.9%) received neoadjuvant therapy consisting of gemcitabine plus oral fluoropyrimidine (S-1) or gemcitabine plus nab-paclitaxel. All PDAC patients were surgically treated, and 18 (75.0%) had R0 and 6 (25.0%) had R1 resections. Seventeen patients (87.5%) received adjuvant chemotherapy consisting of S-1 or gemcitabine.

Fig 1

Expression of PODXL in PDAC tissues obtained by EUS-FNAB.

(A) Study flow chart. (B, C) Representative immunohistochemical staining of PDAC tissue samples using anti-PODXL antibody showing (B) high and (C) low expression of PODXL. Arrows, PDAC cells; arrowheads, normal pancreatic duct epithelium. Magnification: ×400.

Table 1

Summary of characteristics of 24 patients with pancreatic cancer.

Characteristics	Percentage (%)		Charasteristics	Percentage (%)
Age at surgery			Resection margin status
50–60	20.9	[n = 5]	R0	75.0	[n = 18]
60–70	33.3	[n = 8]	R1	25.0	[n = 6]
70–80	33.3	[n = 8]	R2	0	[n = 0]
> 80	12.5	[n = 3]	CA19-9
Gender			Upregulated	79.2	[n = 20]
Male	50.0	[n = 12]	Normal range	20.8	[n = 4]
Female	50.0	[n = 12]	Tumor size
Stage *			> = 2.0 cm	79.2	[n = 20]
IA	20.8	[n = 4]	< 2.0cm	20.8	[n = 4]
IB	16.7	[n = 5]	Neoadjuvant treatment
IIA	37.5	[n = 9]	Yes	20.9	[n = 5]
IIB	20.8	[n = 5]	No	79.1	[n = 19]
III	4.2	[n = 1]	Adjuvant treatment
Extent of the tumor *			Yes	87.5	[n = 21]
T1	29.2	[n = 7]	No	12.5	[n = 3]
T2	33.3	[n = 8]	PODXL expression
T3	37.5	[n = 9]	Low	45.8	[n = 11]
T4	0	[n = 0]	High	54.2	[n = 13]
Regional lymph nodes *			ITGB1 expression
N0	75.0	[n = 18]	Low	70.8	[n = 17]
N1	25.0	[n = 6]	High	29.2	[n = 7]
			PODXL and ITGB1 expression
Distant metastasis *			Others	79.1	[n = 19]
M0	100	[n = 24]	Both high expression	20.9	[n = 5]
M1	0	[n = 0]

*, Classified according to the classification of International Union against Cancer.

The immunostaining of PODXL in EUS-FNAB samples is shown in Fig 1B and 1C. PODXL was mainly stained in the cytoplasm and cell membranes of PDAC cells. The scores of immunostaining were classified into a high-expressing PODXL group (score ≥ 4, Fig 1B) and low-expressing PODXL group (≤ 3, Fig 1C), and 13/24 PDAC tissues exhibited strong PODXL staining (Table 1). The intensity of PODXL staining was weak in normal pancreatic ducts of all EUS-FNAB samples (Fig 1B and 1C).

ITGB1 expression in PDAC tissue samples obtained by EUS-FNAB

ITGB1 was stained in the cytoplasm of PDAC cells in EUS-FNAB samples (Fig 2A and 2B). The scores of immunostaining were classified into a high-expressing ITGB1 group (score ≥ 4, Fig 2A) and low-expressing ITGB1 group (≤ 3, Fig 2B), and 7/24 PDAC tissues exhibited strong ITGB1 staining (Table 1). ITGB1 staining was also weak in normal pancreatic ducts of all EUS-FNAB samples (Fig 2A and 2B).

Fig 2

Expression of ITGB1 in PDAC tissues obtained by EUS-FNAB.

(A, B) Representative immunohistochemical staining of PDAC tissue samples using anti-ITGB1 antibody showing (A) high and (B) low expression of ITGB1. Arrows, PDAC cells; arrowheads, normal pancreatic duct epithelium. Magnification: ×400.

Association of the overexpression of PODXL and ITGB1 with prognosis

The postoperative overall survival (OS) and relapse-free survival (RFS) rates for PDAC patients using immunostaining scores of PODXL and ITGB1 in EUS-FNAB samples before surgery were calculated by Kaplan-Meier curves. The 1-year and 2-year OS rates of the high-expressing group of PODXL were 0.61 [95% confidence intervals (CI): 0.40–0.94] and 0.46 (95% CI: 0.25–0.83), respectively, and those of the low-expressing group were 1.00 and 0.90 (95% CI: 0.75–1.00) (Fig 3A). The 1-year and 2-year OS rates of the high-expressing group of ITGB1 were 0.42 (95% CI: 0.18–1.00) and 0.28 (95% CI: 0.08–0.92), respectively, and those of the low-expressing group were 0.94 (95% CI: 0.83–1.00) and 0.82 (95% CI: 0.66–1.00) (Fig 3B). The 2-year RFS rate decreased from 0.63 (95% CI: 0.40–0.99) in the low-expressing PODXL group to 0.15 (95% CI: 0.04–0.55) in the high-expressing PODXL group (Fig 4A). The 2-year RFS rate decreased from 0.47 (95% CI: 0.28–0.77) in the low-expressing ITGB1 group to 0.14 (95% CI: 0.02–0.87) in the high-expressing ITGB1 group (Fig 4B). The postoperative OS and RFS rates for postoperative PDAC patients according to UICC TNM stage are shown in Fig 4C and 4D. These were consistent with the clinical experience that PDAC patients diagnosed with advanced stage have poorer outcomes than those with an earlier stage.

Fig 3

Correlation between high expression of PODXL and ITGB1 and poor outcomes of PDAC patients.

(A, B) Kaplan-Meier analysis of postoperative OS rates according to (A) PODXL expression and (B) ITGB1 expression in PDAC tissues obtained by EUS-FNAB.

Fig 4

Relapse-free survival rates of high expression of PODXL and ITGB1, and UICC TNM stage.

(A, B) Kaplan-Meier analysis of postoperative RFS rates according to (A) PODXL expression and (B) ITGB1 expression. (C, D) Kaplan-Meier analysis of postoperative (C) OS rate and (D) RFS rate according to UICC TNM stage (early: UICC stage IA-IIA; late: UICC stage IIB-III).

Univariate Cox regression analysis revealed that three clinicopathological features, including PODXL, UICC TNM stage and lymphatic invasion, are significantly associated with OS (P<0.05) (Table 2). Other clinicopathological variables, including resectability, neoadjuvant treatment and adjuvant treatment, were not significant preoperative predictors for prognosis in resected PDAC. Among single clinicopathological variables, the highest hazard ratio (HR) for OS rate was PODXL (12.45, 95% CI: 1.59–97.69), followed by lymphatic invasion (3.78, 95% CI: 1.09–13.23), UICC TNM stage (3.78, 95% CI: 1.09–13.12) and ITGB1 (3.04, 95% CI: 0.89–10.38) (Table 2). In the Japanese guideline of adjuvant chemotherapy for resected PDAC patients, S-1 monotherapy is recommended and gemcitabine monotherapy is used for patients with low tolerability for S-1 [17, 18]. As S-1 or gemcitabine was used for all resected PDAC patients except for those with jaundice or common bile duct stenosis in this study, the HR of adjuvant treatment was unable to be calculated statistically (Table 2). Of note, the HR for the combination of PODXL with ITGB1 was higher (14.37, 95% CI: 3.23–63.99) than that of PODXL and ITGB1. Furthermore, multivariate Cox regression analysis revealed that UICC TNM stage and the combination of PODXL with ITGB1 are significantly associated with OS (P<0.05) (Table 2). The combination of PODXL with ITGB1 predicted the prognosis most accurately (HR: 31.16, 95% CI: 4.36–222.45), and HR of UICC TNM stage was 7.36 (95% CI: 1.32–40.93). This suggested that the combination of PODXL with ITGB1 is a useful preoperative predictor of poorer postoperative survival for PDAC. The Akaike information criterion (AIC) revealed that other clinicopathological variables, including extent of the tumor, lymphatic invasion, resectability, neoadjuvant treatment and serum cancer antigen 19–9 (CA19-9) concentration, were not able to predict the prognosis of postoperative PDAC patients as accurately as the combination of PODXL with ITGB1. CA19-9 is a tumor marker commonly associated with PDAC.

Table 2

Univariate and multivariate analysis of prognostic factors for overall survival.

	Overall survival
	Univariate**		Multivariate***
	HR (95% CI)	P	HR (95% CI)	P
Stage *
IA + IB + IIA	Reference		Reference
IIB + III	3.78 (1.09–13.12)	0.036	7.36(1.32–40.93)	0.023
Age at surgery	1.03 (0.94–1.08)	0.93
Gender
Female	Reference
Male	0.58 (0.16–2.05)	0.39
PODXL expression
Low	Reference
High	12.45 (1.59–97.69)	0.017
ITGB1 expression
Low	Reference
High	3.04 (0.89–10.38)	0.076
PODXL and ITGB1 expression
Others	Reference		Reference
Both high expression	14.37 (3.23–63.99)	< 0.01	31.16(4.36–222.45)	< 0.01
Extent of the tumor *
T1 + T2	Reference		Reference
T3 + T4	3.27 (0.69–15.42)	0.135	2.01(0.87–4.91)	0.100
Lymphatic invasion *
N0	Reference
N1	3.78 (1.09–13.23)	0.036
Resectability
R0	Reference
R1	1.70 (0.49–5.93)	0.405
Neoadjuvant treatment
No	Reference
Yes	1.31 (0.34–5.10)	0.693
Adjuvant treatment
No	Reference
Yes	0 (0- infinity)	0.99
CA19-9
Normal range	Reference
Upregulated	1.49 (0.31–7.07)	0.617

*, Classified according to the classification of International Union against Cancer.

**, Univariate analysis was performed for variables including age, sex, UICC TNM stage, PODXL, ITGB1, the combination of PODXL with ITGB1, extent of the tumor, regional lymph nodes, resection margin status, neoadjuvant treatment, adjuvant treatment and CA19-9.

***, Stepwise model selection using the Akaike information criterion (AIC) and multivariate analysis were performed for variables including age, sex, UICC TNM stage, the combination of PODXL with ITGB1, extent of the tumor, regional lymph nodes, resection margin status, neoadjuvant treatment and CA19-9.

Ability of the combination of PODXL and ITGB1 to predict the prognosis of PDAC patients

We investigated the potential of using a combination of PODXL and ITGB1 to predict the postoperative prognosis in comparison with PODXL, ITGB1 and UICC TNM stage prior to resection separately. Based on Kaplan-Meier curves, the postoperative OS rate (Fig 5A) and RFS rate (Fig 5B) for PDAC patients with overexpression of both PODXL and ITGB1 (n = 5) in EUS-FNAB samples before surgery were significantly shorter than those for PDAC patients without high expression of both PODXL and ITGB1 (n = 19) (P<0.01 for OS rate and RFS rate).

Fig 5

Correlation between the combination of PODXL with ITGB1 and poor outcomes of PDAC patients.

(A, B) Kaplan-Meier analysis of (A) OS and (B) RFS rates according to the combination of PODXL with ITGB1.

The 1-year and 2-year OS rates of the group highly expressing both PODXL and ITGB1 were 0.20 (95% CI: 0.03–1.00) and 0.00, respectively, and those of the group with low expression of both were 0.94 (95% CI: 0.85–1.00) and 0.84 (95% CI: 0.69–1.00) (Table 3). The OS rates of UICC TNM stage IA-IIA and IIB-III were 0.83 (95% CI: 0.67–1.00) and 0.66 (95% CI: 0.37–1.00) for 1 year, and 0.72 (95% CI: 0.54–0.96) and 0.50 (95% CI: 0.22–1.00) for 2 years, respectively (Table 3). This suggests that the combination of PODXL with ITGB1 is a useful predictor of postoperative outcomes for PDAC patients before resection.

Table 3

Survival rates and median survival times of the combination of PODXL with ITGB1.

	n	Survival rate (95% CI) (%)		Median survival time (95% CI) (month)
		1-year	2-year
PODXL and ITGB1 expression
Others	19	0.94 (0.85–1.00)	0.84 (0.69–1.00)	47 (47-NA)
Both high expression	5	0.20 (0.03–1.00)	0.00 (NA-NA)	8 (5-NA)
UICC TNM stage
Stage IA-IIA	18	0.83 (0.67–1.00)	0.72 (0.54–0.96)	47 (47-NA)
Stage IIB-III	6	0.66 (0.37–1.00)	0.50 (0.22–1.00)	23 (8-NA)

Ability of the combination of PODXL and ITGB1 to predict the prognosis of PDAC patients with UICC TNM stage IA-IIA

The increasing shift towards neoadjuvant treatments for both resectable and borderline PDAC, and the use of conversion therapy for locally advanced disease suggest the need for biological predictors in addition to the UICC TNM stage [19]. As these predictors are not currently clinically employed, we focused on the ability of the combination of PODXL and ITGB1 to predict the postoperative prognosis of PDAC patients with UICC TNM stage IA-IIA, including both resectable and borderline PDAC before surgery. Based on Kaplan-Meier curves, the postoperative OS rate for UICC TNM stage IA-IIA PDAC patients with high expression of both PODXL and ITGB1 (n = 3) in EUS-FNAB samples before surgery was significantly shorter than that of UICC TNM stage IA-IIA PDAC patients without high expression of both PODXL and ITGB1 (n = 15) (P<0.01) (Fig 6A). The 1-year and 2-year OS rates of UICC TNM stage IA-IIA patients highly expressing both PODXL and ITGB1 compared with other PDAC patients were 0.33 (95% CI: 0.06–1.00) and 0.93 (95% CI: 0.81–1.00), and 0.00 and 0.86 (95% CI: 0.71–1), respectively (Table 4). The prognostic analysis using messenger RNA (mRNA)‑seq data of 42 resected UICC TNM stage IA-IIA PDAC samples from The Cancer Genome Atlas (TCGA) database revealed that the postoperative OS rate for PDAC patients with UICC TNM stage IA-IIA with upregulated mRNA levels of both PODXL and ITGB1 (n = 7) was significantly lower than that for PDAC patients without high expression of both PODXL and ITGB1 (n = 35) (P = 0.006) (Fig 6B). This suggests that the combination of PODXL with ITGB1 is useful to predict the postoperative outcomes of PDAC patients with UICC TNM stage IA-IIA prior to surgery.

Fig 6

Correlation between the combination of PODXL with ITGB1 and poor outcomes of PDAC patients at stage IA-IIA.

(A) Kaplan-Meier analysis of OS rate according to the immunohistochemical scores of the combination of PODXL with ITGB1 in PDAC patients at stage IA-IIA. (B) Kaplan-Meier analysis of OS rate according to the mRNA expression levels of the combination of PODXL with ITGB1 in the TCGA set.

Table 4

Survival rates and median survival times of the combination of PODXL with ITGB1 at UICC stage IA-IIA.

	n	Survival rate (95% CI) (%)		Median survival time (95% CI) (month)
		1-year	2-year
PODXL and ITGB1 expression
Others	15	0.93 (0.81–1.00)	0.86 (0.71–1.00)	47 (47-NA)
Both high expression	3	0.33 (0.06–1.00)	0.00 (NA-NA)	10 (5-NA)

Discussion

Preoperative biomarkers were demonstrated to have preliminary value in predicting the prognosis of PDAC patients. Among them, preoperatively high serum levels of both CEA and CA19-9 are associated with a poor postoperative prognosis of resected PDAC patients [20]. Serum KRAS mutations, especially the KRAS G12D mutation, in preoperative cell-free circulating tumor DNA are associated with the poor prognosis of resected PDAC patients [21]. Preoperative serum complement factor B accurately predicts the prognosis of resected PDAC compared with high CEA and CA19-9 [22]. Furthermore, few predictive biomarkers for the prediction of responsiveness to neoadjuvant chemotherapy have been identified in PDAC. A microRNA miRNA-320c, which is associated with the invasiveness of PDAC cells, was reported as a prognostic factor for PDAC to predict the clinical response of gemcitabine [23]. Gemcitabine-based chemotherapy is commonly utilized as a first-line treatment to treat advanced PDAC patients [24]. As radical resection with a negative margin (R0 resection) is the key factor for long-term survival of PDAC [6], potential prognostic factors that are available before resection are necessary for PDAC patients who are commonly considered operable to prepare a neoadjuvant strategy. The present study demonstrated that the high expression of both PODXL and ITGB1 in preoperatively extracted PDAC tissues obtained by EUS-FNAB is closely associated with the poor prognosis of PDAC patients after resection (P<0.01), even though there was no notable difference in prognosis between R0 resection and R1 resection (P = 0.405).

PODXL, which binds to the cytoskeletal protein gelsolin, promotes PDAC cell invasion by increasing membrane protrusions with abundant peripheral actin structures [11]. A retrospective clinical study revealed that serum PODXL can significantly distinguish PDAC patients with UICC stage 0/I/II from control individuals compared with serum CA19-9 [25]. The difficulty of detecting the presence of localized retroperitoneal invasion and micro-metastasis in PDAC patients by diagnostic imaging is the main reason for the high rate of local recurrence and/or distant metastasis after resection [6, 26]. The recurrence rates after curative surgery for PDAC are 56.7, 76.6 and 84.1% at 1, 2 and 5 years, respectively [27]. Over 90% of resected PDAC patients develop recurrence in the abdominal cavity [28]. The immunohistochemically high expression of both PODXL and ITGB1 in resected early-stage (UICC TNM stage 0-IIA) PDAC tissues is associated with a poorer prognosis [12], suggesting that PODXL and ITGB1 play important roles in the invasiveness and early recurrence of PDAC, and this correlates with the function of PODXL in promoting the invasiveness of PDAC cells in in vitro experiments [11]. In this study, the combination of PODXL with ITGB1 most accurately predicted the postoperative outcomes of PDAC patients before resection according to univariate and multivariate analyses. Importantly, it is possible that the combination of PODXL with ITGB1 is a useful predictor of postoperative outcomes for PDAC patients at an earlier stage before resection.

Evidence of improved OS with neoadjuvant therapy for borderline resectable or locally advanced PDAC is supported by results from large cancer databases and meta-analyses of non-randomized trials, and a meta-analysis of non-randomized cohorts suggested the utility of FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan and oxaliplatin), which is currently used many institutions [29]. However, as clinical evidence from randomized phase III trials using neoadjuvant therapy for borderline resectable or locally advanced PDAC is limited [8, 27], guidelines for its use are not well defined. The most effective regimens, FOLFIRINOX or gemcitabine plus nab-paclitaxel, have been expanded into neoadjuvant treatments for resectable PDAC because of their potential benefits, including for the early treatment of occult micro-metastasis [30, 31]. A randomized phase II/III trial (Prep-02/JSAP05) performed in Japan demonstrated that gemcitabine plus S-1 with upfront surgery improves the postoperative overall survival of resectable PDAC patients [32]. In the present study, 5 patients (20.9%) received neoadjuvant chemotherapy before surgery, and there was no notable difference in prognosis between resected PDAC patients with neoadjuvant chemotherapy and resected PDAC patients without neoadjuvant chemotherapy (P = 0.693). The development of neoadjuvant therapeutic approaches that are more beneficial than FOLFIRINOX, gemcitabine plus nab-paclitaxel or gemcitabine plus S-1 is important to increase survival compared with upfront surgery.

As no reliable biomarkers can gauge the response to neoadjuvant therapy prior to the initiation of treatment [33], it is difficult to discriminate patients with operative PDAC in whom neoadjuvant therapy may be effective and suitable. Most patients received upfront surgery without neoadjuvant therapy in this study. Therefore, PDAC patients with the overexpression of PODXL and ITGB1 need to be scrutinized closely for incomplete resectability or operability if patients with early-stage PDAC do not receive neoadjuvant chemotherapy. Further studies are needed to determine whether prognostic predictors, including PODXL and ITGB1, can be used to optimize the selection of PDAC patients who will benefit from neoadjuvant treatment and eventually improve outcomes. Toward this end, we started a prospective clinical study (UMIN000034022) to clarify the association of the overexpression of PODXL and ITGB1 with the benefit of neoadjuvant therapies in resectable and borderline resectable PDAC patients.

In conclusion, immunohistochemical scores of PODXL and ITGB1 in preoperative EUS-FNAB samples accurately predicted the postoperative prognosis of PDAC patients better than the UICC TNM stage. The combination of PODXL with ITGB1 can discriminate PDAC patients with a poorer prognosis who have early-stage PDAC prior to surgery. Patients with PDAC that overexpresses both PODXL and ITGB1 should be considered for neoadjuvant therapy first instead of undergoing upfront surgery. However, the use of PODXL and ITGB1 overexpression to optimize neoadjuvant therapy for resectable PDAC patients requires further testing in prospective studies.

Materials and methods

Primary human PDAC samples

We retrospectively enrolled 24 PDAC patients who were histologically diagnosed with PDAC from tissue samples obtained by EUS-FNAB before resection, and having UICC TNM stage 0-III tumors without distant metastasis between March 2015 and October 2017 at Kochi Medical School Hospital and Kanagawa Cancer Center. These patients were classified into three resectability groups: resectable, borderline resectable or locally unresectable, according to the 7th edition of the General Rules for the Study of Pancreatic Cancer by the Japan Pancreas Society [34]. Five of 24 patients received neoadjuvant chemotherapy with gemcitabine plus S-1 or gemcitabine plus nab-paclitaxel before resection of PDAC. All patients underwent resection, and 17 of 24 patients received adjuvant chemotherapy with S-1 or gemcitabine after the resection of PDAC. Pre-treatment serum CA19-9 was measured at certified laboratories associated with the hospital where the patients were treated. Postoperative follow-up methods and acquisition of the medical records were described previously [12]. Observation was censored at PDAC-related death or at the end of observation at least 2 years after resection.

This study was registered in UMIN-CTR (UMIN000032835) and approved by the ethics review boards of Kochi Medical School (approval number: ERB-104012) and Kanagawa Cancer Center (approval number: 2018–131). Obtaining written informed consent from PDAC patients was waived because of the retrospective and observational nature of the analyses, and the opt-out method was approved by the ethics review boards of Kochi Medical School and Kanagawa Cancer Center. The information regarding this study was provided to patients through the institutional website of Kochi Medical School and Kanagawa Cancer Center for obtaining consent. PDAC patients who did not want to participate in this study were able to request to opt-out to prevent trial enrollment.

Immunohistochemical staining

Immunohistochemistry was performed as described previously [11, 35]. The staining intensity of PODXL and ITGB1 in PDAC cells was scored and compared with the normal pancreatic duct epithelium, as described previously [12]. The expression levels were classified into low and high groups based on the score with reference to previous reports [12, 36].

Statistical analysis

All statistical analyses were performed using R (version 3.3.3; The R Foundation, Wien, Austria) with the packages “KMsurv”, “rms” and “survival” as described previously [37]. The survival time was from the date of EUS-FNAB and the analysis was timed to PDAC-related death. We used arbitrary UICC stage categories (IA, IB, IIA vs. IIB and III) and extent of the tumor (T1 and T2 vs. T3 and T4). Estimates of survival probabilities were performed by the Kaplan-Meier method and evaluated by the Gehan-Wilcoxon test. Univariate and multivariate analyses for the chosen explanatory variables were performed by the Cox proportional hazards model to estimate the HR with 95% CI, and P-values were calculated by the z test. The Efron parameter approach method was used in the Cox proportional hazards model. Univariate analysis was performed for variables including age, sex, UICC TNM stage, PODXL, ITGB1, the combination of PODXL with ITGB1, extent of the tumor, regional lymph nodes, resection margin status, neoadjuvant treatment, adjuvant treatment and CA19-9. We were unable to build a multi-variate regression model including all the variables due to the risk of overfitting. Instead, forward-backward stepwise model selection using the AIC and multivariate analysis was performed for variables including age, sex, UICC TNM stage, the combination of PODXL with ITGB1, extent of the tumor, regional lymph nodes, resection margin status, neoadjuvant treatment and CA19-9. All statistical tests were two-tailed and p<0.05 was considered significant.

Prognostic analysis using the public PDAC gene expression profile data

TCGA data were retrieved from UCSC Xena [38]. The clinical data obtained from 196 resected PDAC patients and RNA-seq expression data obtained from 183 resected PDAC patients were analyzed. Expression data included the gene-level transcription estimates, as in log2(x+1) transformed RNA-Seq by Expectation-Maximization (RSEM) normalized count (cBioinformatics Co., LTD., Tokyo, Japan). We obtained expression data from 42 resected PDAC patients with UICC TNM stage IA, IB and IIA. These 42 patients were divided into two groups by PODXL and ITGB1 expression in the PDAC biopsy samples obtained prior to surgery. The cut-off value was exploratorily identified using percentile values referring to the ratio of UICC TNM stage IA-IIA PDAC patients without high expression of PODXL or ITGB1 in the immunohistochemical analysis of this study (11/18 for PODXL and 13/18 for ITGB1). The 61st percentile for PODXL and 72nd percentile for ITGB1 were used to analyze the TCGA dataset. We defined patients with high expression of both genes as the “Both high expression group”. Other patients were defined as the “Other group”. OS rates of“Both high expression group” and “Other group” were calculated according to the Kaplan-Meier method and evaluated by the Gehan-Wilcoxon test. Regardless of the statistical test performed, differences with p<0.05 were considered significant.

8 Jul 2021

PONE-D-21-14241

Upregulation of PODXL and ITGB1 in pancreatic cancer tissues preoperatively obtained by EUS-FNAB correlates with unfavorable prognosis of postoperative pancreatic cancer patients

PLOS ONE

Dear Dr. Taniuchi,

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Surinder K. Batra

Academic Editor

PLOS ONE

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3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

[ We thank Yuri Jobu, Chiharu Tanaka, Miki Nishigawa, Hitomi Seki, and Shunichi Manabe for their excellent technical assistance. This study was supported by Grants - in - Aid for Scientific Research (KAKENHI: 17K09463, 19K08446, and 20K07699), and AMED under Grant Number JP19lm0203007. ]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

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4. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

: Taniuchi K, Furihata M, Naganuma S, Sakaguchi M, Saibara T (2019) Overexpression of PODXL/ITGB1 and BCL7B/ITGB1 accurately predicts unfavorable prognosis compared to the TNM staging system in postoperative pancreatic cancer patients. PLoS ONE 14(6): e0217920. https://doi.org/10.1371/journal.pone.0217920

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #1: The authors in the manuscript entitled ‘Upregulation of PODXL and ITGB1 in pancreatic cancer tissues preoperatively obtained by EUS-FNAB correlates with unfavorable prognosis of postoperative pancreatic cancer patients’ investigated the expressions of PODXL and ITGB1 as preoperative markers for extrapolating the postoperative prognosis of pancreatic cancer patients. The authors claim that prior to surgery, a higher expression of PODXL and ITGB1 significantly predicted the postoperative outcomes of pancreatic cancer patients as compared to the TNM staging system. The investigators also suggest that the expression status of PODXL and ITGB1 will direct the patient stratification to receive preoperative neoadjuvant therapy to improve their postoperative prognosis. The overall idea of the work is interesting and seems promising to benefit the pancreatic cancer biomarker field of research, and further improve the patient prognostication. However, there are some major concerns that need to be addressed before considering this manuscript for publication:

1. The rationale of selecting these two markers for a “directed approach” of biomarker selection wasn’t very clear from the introduction and discussion parts. With the accumulating evidence in biomarker research over the past few years, several markers have been studied of which many showed promise in pilot studies but eventually failed to recapitulate to the clinics. Hence, it is essential to have a strong rationale in terms of functional requirement of the protein (and not merely the expression) during the particular cancer stage under investigation.

2. In Table 4, the authors have compared the survival kinetics of TNM stage IA-IIA patients with high expression of PODXL and ITGB1 and those with low expression of both these markers. The number of patients in the high expressor category is too less (n=3), while those on the other arm is n=15. It is understandable that it is a longitudinal study, hence patient recruitment might be difficult. The authors should try to increase the patient number in the first category, or at least try to derive transcriptomic analysis from the patient information available in TCGA or other bioinformatics platforms. This will support their claims.

3. The authors at multiple places claimed that the PODXL and ITGB1 can independently perform as pre-operative predictors, better than TNM staging. However, I don’t see any analysis where the authors do a blinded study on the survival statistics of PDAC patients based on the expression of PODXL and ITGB1, and then look for their TNM stage. The authors segregated patients in stage IA-IIA, and then correlated their survival with expression of these markers. A blinded study on a validation cohort will be helpful, even if it is on a small group of patients.

4. There are some grammar and typographical mistakes throughout the text. Authors are encouraged to correct them in the revised manuscript after a professional English editing.

5. The manuscript has some tables with elaborate information and logistics. The authors are encouraged to incorporate table legends or footnotes, explaining the critical terminologies and providing a statement on the overall observation from the analysis.

Reviewer #2: In their article “Upregulation of PODXL and ITGB1 in pancreatic cancer tissues preoperatively

obtained by EUS-FNAB correlates with unfavorable prognosis of postoperative

pancreatic cancer patients”. Taniuchi and colleagues present there working regarding the ability of PODXL and ITGB1 immunostaining in EUS biopsies to predict post-operative prognosis. Overall, the work is interesting and well written. The samples included, while small in number, are appropriate for this investigation. Moreover, the scientific framing of the article, in the intro and discussion sections, makes the potential clinical relevance of this study clear. Finally, the author’s assertions regarding the utility of these findings are, in this reviewer’s opinion, well-grounded and not overstated. There are, however, several points for further consideration that may further enhance the readability and strength the author’s claims.

Minor Comments

1. Image quality for figures 1 and 2 is limited and makes assessment of the IHC quality and histology more difficult

2. Font in Figures 3-6 are difficult to read. Clarity would be enhanced by improving the legibility in these figures.

3. Within the statistical analysis section of the methods and materials, it is important to state which factors were used in the generation of the cox proportional hazard models. The inclusion/exclusion of various patient and disease factors can have marked impacts on the assessment of single factor as being a

4. The lack of p-values present in the figures as well as the text manuscript and figure legends make it slightly unwieldy for the reader to easily assess the statistical significance of the various comparisons present in this study

5. Please indicate the statistic used for calculating univariate survival. There are numerous different statistics that can be used within the overall framework of Kaplan-Meier analysis. These varying statistics weigh early and late event differently and can have notable effects on the outcomes of such analyses.

6. In table 2 under the univariate column, the HR for stage is listed as 3.78 with a 95% confidence interval that does not include 1, yet the p-values is not significant. By definition, this is significant. Please explain.

Major Comments

1. The inclusion of only PODXL and ITGB1 staining profiles in multivariate models while excluding/ not reporting the results of other significant univariate factors (such as Lymphatic invasion or the very closely numerically related factor of stage) seems to be an inappropriate use of cox proportional hazards. These other factors must be included in the model and the results reported for such an analysis; without this the findings are difficult to interpret and may be misleading.

2. Given that this study is conducted in a subset of patients who have undergone surgery, this reviewer feels that it is imperative to report the R-status (R0, R1, R2) of the resection. Moreover, this reviewer feels that this will be an important factor to include in the univariate survival analysis and the multivariate analysis if significant due to the fact that differences in the R value can have a major impact on survival independent of the staging. Previous literature supports this notion indicating that patients who undergo an R2 resection do not have improved survival over patients with unresectable disease.

3. Similarly, it is odd that the adjuvant and neoadjuvant chemotherapy were not analyzed in univariate survival analyses. This reviewer understands that in the previous paper these authors did not find that adjuvant therapy and a significant effect on survival. Nonetheless, the authors mention that well controlled trials have demonstrated benefits to adjuvant treatments. Consequently, this reviewer feels that it is important to address the adjuvant and neo adjuvant therapy in the univariate survival analysis and multivariate analyses should it prove to be a significant factor.

**********

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Reviewer #1: No

Reviewer #2: No

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21 Aug 2021

Dr. Surinder Batra,

August 19, 2021

Submission ID: PONE-D-21-14241

Upregulation of PODXL and ITGB1 in pancreatic cancer tissues preoperatively obtained by EUS-FNAB correlates with unfavorable prognosis of postoperative pancreatic cancer patients

Dear Dr. Surinder Batra,

I am returning the above manuscript that was revised according to your letter dated the 8th of July. Our responses to the reviewers’ comments are provided below.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

Response : The manuscript was revised according to PLOS ONE's style requirements, including those for file naming.

2. Please provide additional information about the opt out mechanism employed in your study. In your ethics statement in the manuscript and in the online submission form, please ensure that you have discussed whether all data/samples were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data/samples from their medical records used in research, please include this information.

Response : Additional information about the opt-out system was added to the section of “Primary human PDAC samples” of the Materials and Methods section in the revised manuscript.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

[We thank Yuri Jobu, Chiharu Tanaka, Miki Nishigawa, Hitomi Seki,and Shunichi Manabe fortheir excellent technical assistance. This study was supported by Grants-in-Aid for Scientific Research (KAKENHI: 17K09463, 19K08446, and 20K07699), and AMED under Grant Number JP19lm0203007.]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

[Grants-in-Aid for Scientific Research (KAKENHI): 17K09463 (kt), 19K08446 (kt), and 20K07699 (mo)]

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: The funding-related text was removed from the revised manuscript. The Funding Statement is as follows: Grants-in-Aid for Scientific Research (KAKENHI): 17K09463 (kt), 19K08446 (kt), 19K07461 (mf), 20K07699 (mo), 20K07806 (ry) and 20K08359 (kt), and Japan Agency for Medical Research and Development (AMED) under Grant Number JP19lm0203007 (kt).

4. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: Taniuchi K, Furihata M, Naganuma S, Sakaguchi M, Saibara T (2019) Overexpression of PODXL/ITGB1 and BCL7B/ITGB1 accurately predicts unfavorable prognosis compared to the TNM staging system in postoperative pancreatic cancer patients. PLoS ONE 14(6): e0217920. https://doi.org/10.1371/journal.pone.0217920.

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Response: The overlapping text with the previous publication (PLoS ONE 14(6): e0217920) was addressed in the revised manuscript.

Reviewer #1:

We appreciate the Reviewer’s useful comments.

Comments:

1. The rationale of selecting these two markers for a “directed approach” of biomarker selection wasn’t very clear from the introduction and discussion parts. With the accumulating evidence in biomarker research over the past few years, several markers have been studied of which many showed promise in pilot studies but eventually failed to recapitulate to the clinics. Hence, it is essential to have a strong rationale in terms of functional requirement of the protein (and not merely the expression) during the particular cancer stage under investigation.

Response: In response to the Reviewer’s comments, the Abstract section, 2nd paragraph of the Introduction section, and the 1st and 2nd paragraphs of the Discussion section were revised.

2. In Table 4, the authors have compared the survival kinetics of TNM stage IA-IIA patients with high expression of PODXL and ITGB1 and those with low expression of both these markers. The number of patients in the high expressor category is too less (n=3), while those on the other arm is n=15. It is understandable that it is a longitudinal study, hence patient recruitment might be difficult. The authors should try to increase the patient number in the first category, or at least try to derive transcriptomic analysis from the patient information available in TCGA or other bioinformatics platforms. This will support their claims.

Response: In response to the Reviewer’s comments, the prognostic analysis of TNM stage IA-IIA patients with high expression of PODXL and ITGB1 using the TCGA bioinformatics platform was added in Fig. 6B.

3. The authors at multiple places claimed that the PODXL and ITGB1 can independently perform as pre-operative predictors, better than TNM staging. However, I don’t see any analysis where the authors do a blinded study on the survival statistics of PDAC patients based on the expression of PODXL and ITGB1, and then look for their TNM stage. The authors segregated patients in stage IA-IIA, and then correlated their survival with expression of these markers. A blinded study on a validation cohort will be helpful, even if it is on a small group of patients.

Response: As the Reviewer mentioned, a blinded study on the survival statistics of pancreatic cancer patients based on the expression of PODXL and ITGB1 was not performed. To better clarify this, the sentences about the results of multivariate Cox regression analysis in “Associations of overexpression of PODXL and ITGB1 with prognosis” in the Results were revised in the revised manuscript. Concerning the prognostic analysis of TNM stage IA-IIA patients with high expression of PODXL and ITGB1, the results of the TCGA bioinformatics platform were added to Fig. 6B.

4. There are some grammar and typographical mistakes throughout the text. Authors are encouraged to correct them in the revised manuscript after a professional English editing.

Response: Grammar mistakes were corrected in the revised manuscript.

5. The manuscript has some tables with elaborate information and logistics. The authors are encouraged to incorporate table legends or footnotes, explaining the critical terminologies and providing a statement on the overall observation from the analysis.

Response: In response to the Reviewer’s comments, table footnotes were incorporated into Table 2.

Reviewer #2:

We appreciate the Reviewer’s useful comments.

1. Image quality for Figures 1 and 2 is limited and makes assessment of the IHC quality and histology more difficult.

Response: The images of Figures 1 and 2 were exchanged, which improved the quality of the revised manuscript.

2. Font in Figures 3-6 are difficult to read. Clarity would be enhanced by improving the legibility in these figures.

Response: Font size in Figures 3-6 was enlarged in the revised manuscript.

3. Within the statistical analysis section of the methods and materials, it is important to state which factors were used in the generation of the cox proportional hazard models. The inclusion/exclusion of various patient and disease factors can have marked impacts on the assessment of single factor as being a

Response: In response to the Reviewer’s comments, information about variables for the univariate and multivariate analysis were added to the statistical analysis section of the Methods and Materials and footnote in Table 2.

4. The lack of p-values present in the figures as well as the text manuscript and figure legends make it slightly unwieldy for the reader to easily assess the statistical significance of the various comparisons present in this study.

Response: In response to the Reviewer’s comments, the p-values for the Kaplan-Meier survival analysis were added to Figures 3-6, and p-values for the univariate and multivariate analysis were added in the text and Table 2. Stepwise model selection from the variables including age, sex, UICC TNM stage, extent of the tumor, regional lymph nodes, resection margin status, neoadjuvant treatment, CA19-9, and the combination of PODXL with ITGB1 using the Akaike information criterion (AIC) was performed in multivariate analysis; therefore, the hazard ratio and p-values of significant variables were added to Table 2.

5. Please indicate the statistic used for calculating univariate survival. There are numerous different statistics that can be used within the overall framework of Kaplan-Meier analysis. These varying statistics weigh early and late event differently and can have notable effects on the outcomes of such analyses.

Response: In response to the Reviewer’s comments, information about the statistical methods used in the univariate analysis and Kaplan-Meier survival analysis was added to “Statistical analysis” in the Materials and Methods section.

6. In table 2 under the univariate column, the HR for stage is listed as 3.78 with a 95% confidence interval that does not include 1, yet the p-values is not significant. By definition, this is significant. Please explain.

Response: The erroneous p-value for TNM stage was corrected in Table 2 of the revised manuscript.

Major Comments

1. The inclusion of only PODXL and ITGB1 staining profiles in multivariate models while excluding/ not reporting the results of other significant univariate factors (such as Lymphatic invasion or the very closely numerically related factor of stage) seems to be an inappropriate use of cox proportional hazards. These other factors must be included in the model and the results reported for such an analysis; without this the findings are difficult to interpret and may be misleading.

Response: Information about variables for the multivariate analysis was added to the statistical analysis section of the Methods and Materials and footnote in Table 2. In the multivariate analysis, variables included age, sex, UICC TNM stage, the combination of PODXL with ITGB1, extent of the tumor, regional lymph nodes, resection margin status, neoadjuvant treatment and CA19-9, and stepwise model selection using the Akaike information criterion and multivariate analysis were performed.

2. Given that this study is conducted in a subset of patients who have undergone surgery, this reviewer feels that it is imperative to report the R-status (R0, R1, R2) of the resection. Moreover, this reviewer feels that this will be an important factor to include in the univariate survival analysis and the multivariate analysis if significant due to the fact that differences in the R value can have a major impact on survival independent of the staging. Previous literature supports this notion indicating that patients who undergo an R2 resection do not have improved survival over patients with unresectable disease.

Response: Information about the resection margin status (R-status) was added to Table 1-2, and the R-status was included as one of the variables in the univariate and multivariate analyses. R-status failed to predict the prognosis of postoperative pancreatic cancer patients accurately compared with the combination of PODXL with ITGB1. This result is described in “Associations of overexpression of PODXL and ITGB1 with prognosis” in the Results section of the revised manuscript.

3. Similarly, it is odd that the adjuvant and neoadjuvant chemotherapy were not analyzed in univariate survival analyses. This reviewer understands that in the previous paper these authors did not find that adjuvant therapy and a significant effect on survival. Nonetheless, the authors mention that well controlled trials have demonstrated benefits to adjuvant treatments. Consequently, this reviewer feels that it is important to address the adjuvant and neo adjuvant therapy in the univariate survival analysis and multivariate analyses should it prove to be a significant factor.

Response: The adjuvant and neoadjuvant treatments were included as variables for the univariate analysis. As S-1 or gemcitabine was used for all resected PDAC patients except for those with jaundice or common bile duct stenosis in this study, the hazard ratio of adjuvant treatment was unable to be calculated statistically, as described in Table 2. The adjuvant and neoadjuvant treatments were not associated with the prognosis of postoperative pancreatic cancer patients compared with the combination of PODXL with ITGB1. This suggests that the development of neoadjuvant therapeutic approaches that are more beneficial than currently available chemotherapy regimens, such as FOLFIRINOX, gemcitabine plus nab-paclitaxel or gemcitabine plus S-1, is important to improve survival.

I hope that the revised manuscript is now acceptable for publication.

Yours sincerely,

Keisuke Taniuchi, MD, PhD.

Department of Gastroenterology and Hepatology

Kochi Medical School, Kochi University

Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan

Phone: +81-88-880- 2338; Fax: +81-88-880- 2338

Email: ktaniuchi@kochi-u.ac.jp

Submitted filename: TANIUCHI et al_ResponseToReviewer2.pdf

Click here for additional data file.

9 Nov 2021

7 Jan 2022

Comments:

1. However, the use of TCGA data, while a meaningful addition, is not executed appropriately in this manuscript. In the TCGA PAAD dataset, there a number of benign and malignant entities which comprise the the 183 samples in the full data set. To indicate that these are all PDAC samples is misleading and incorrect. Moreover, the inclusion of pancreatic neuroendocrine tumors (included in the 183 samples) in the analysis has the potential to bias or confound the meaning of outcomes studies. Please repeat this analysis using only the samples in the TCGA PAAD dataset that are confirmed to be PDAC.

Response:

In response to the Reviewer’s comments, we excluded neuroendocrine tumors and repeated the prognostic analysis of 42 PDACs that were confirmed to be TNM stage IA-IIA using the TCGA bioinformatics platform. Based on Kaplan-Meier curves, the postoperative overall survival rate for PDAC patients with UICC TNM stage IA-IIA with upregulated mRNA levels of both PODXL and ITGB1 (n = 7) was significantly lower than that for PDAC patients without high expression of both PODXL and ITGB1 (n = 35) (P=0.006), as shown in Fig. 6B.

Submitted filename: (revise2)TANIUCHI et al_ResponseToReviewer.pdf

Click here for additional data file.

28 Feb 2022

Upregulation of PODXL and ITGB1 in pancreatic cancer tissues preoperatively obtained by EUS-FNAB correlates with unfavorable prognosis of postoperative pancreatic cancer patients

PONE-D-21-14241R2

Dear Dr. Taniuchi,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Surinder K. Batra

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

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: Yes

**********

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: You have addressed all previous comments and I feel that the manuscript is now acceptable for publication

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

3 Mar 2022

PONE-D-21-14241R2

Upregulation of PODXL and ITGB1 in pancreatic cancer tissues preoperatively obtained by EUS-FNAB correlates with unfavorable prognosis of postoperative pancreatic cancer patients

Dear Dr. Taniuchi:

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.

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on behalf of

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