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Abstract

BACKGROUND:

The recognition of high-risk colon cancer patients prone to chemoresistant and recurrent disease is a challenge.

OBJECTIVES:

We aimed to assess the immunohistochemical expression of ERCC1, PARP-1, and AQP1 in 60 cases of stage II and III colon cancer who underwent curative resection and adjuvant chemotherapy. Their predictive role of tumor progression and disease-free survival (DFS) was analyzed.

METHODS:

The immunohistochemical expression of ERCC1, PARP-1, and AQP1 in 60 cases of stage II and III colon cancer who underwent curative resection and adjuvant chemotherapy was studied. The collected data on the overall survival (OS), disease-free survival (DFS), and the response to the chemotherapy were analyzed.

RESULTS:

Positive nuclear ERCC1 expression was identified in 58.3% of the patients, ERCC1 expression was significantly associated with left-sided tumors ( $P<$ 0.01). Moreover, its expression was significantly associated with the aggressive tumor characteristics including high grade, lymph node metastasis and advanced tumor stage ( $P<$ 0.001 for each). High nuclear PARP-1 expression was observed in 63.3% of the cases, and its expression was significantly associated with tumor grade and lymph node metastasis ( $P=$ 0.003 for each). Positive membranous AQP1 expression was identified in 41.7% of patients, and it was associated with high grade, lymph node metastasis and advanced tumor stage ( $P<$ 0.001 for each). During the follow-up period, 23 patients (38.3%) exhibited a tumor progression; this was significantly associated with positive ERCC1, high PARP-1, and negative AQP1 expression. Statistics of the survival data revealed that shorter DFS was significantly associated with positive ERCC1, high PARP-1, and positive AQP1 expression ( $P=$ 0.005, 0.016, 0.002, respectively).

CONCLUSIONS:

ERCC1, PARP1, and AQP1 are adverse prognostic biomarkers in stage II–III colon cancer. Moreover, adjuvant chemotherapy may not be beneficial for patients with positive ERCC1, high PARP1, and AQP1-negative tumors. Therefore, we recommend that ERCC1, PARP-1, and AQP1 should be assessed during the selection of the treatment strategy for stage II–III colon cancer patients.

Keywords

Colon cancer ERCC1 PARP-1 AQP1 immunohistochemistry

1. Introduction

Colon cancer (CC) is the third most common cancer worldwide, with a high mortality rate [1, 2]. In Egypt, it represents about 33.8% of whole GIT tumors and about 6.5% of all diagnosed cancer with a tendency toward younger ages [3]. Although surgical resection is effective in localized cases, nearly 20% of stage II and 40% of stage III progress within 5 years after surgery [4]. This gives the rationale for adjuvant chemotherapy after surgical resection. The 5-fluorouracil (5-FU)-based chemotherapy represents the standard adjuvant protocol for stage III patients [5]. A subset of stage II patients with high-risk of relapse may benefit from adjuvant therapy, however, its routine use in standard risk stage II is is not recommended. As well, there is a considerable heterogeneity amongst colon cancer patients leading to different prognosis and variable chemotherapeutic response. Therefore, it is essential to identify predictive biomarkers of recurrence and chemoresistance [6].

Oxaliplatin is a platinum-based chemotherapeutic agent that has efficacy against various tumor cell lines and became a part of the classical therapeutic protocol of CC [7]. Adding oxaliplatin to 5FU (FOLFOX regimen) improved the adjuvant treatment by reducing the risk of relapse and improving OS [8]. Oxaliplatin exerts its effect by forming DNA-platinum mono-adducts, inhibiting both DNA replication and transcription, and inducing apoptosis in actively dividing cells. The nucleotide excision repair (NER) pathway; a major DNA repair system in the mammalian cells; removes these bulky adducts produced by oxaliplatin. DNA repair efficiency may contribute to platinum-based cytotoxic drug resistance. The ERCC1 protein, which is a key component of NER pathway [9] repair the interstrand cross-links in the DNA beside the recognition and removal of cytotoxic agents as oxaliplatin [10]. The correlation of ERCC1 expression with the prognosis has been investigated in various tumors like gastric cancer [11], ovarian cancer [12], and lung cancer [13].

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is activated and engaged in the repair of DNA single-strand breaks through base excision repair pathway (BER), in addition to regulation of DNA transcription and cell cycle progression. PARP-1 inhibitors interrupt BER, rendering it inactive causing increased levels of persisting single-strand breaks, with DNA double-strand breaks (DSB) upon replication finally [14]. PARP-1 has a central role in CC carcinogenesis. Based on the preclinical studies which verified the improvement of radiosensitivity and chemotherapy response in experimental models of CC by PARP-1 inhibitors, a number of clinical trials have been initiated with the use of four PARP inhibitors in CC patients [15]. Thus, it is important to investigate the PARP-1 protein expression in clinical samples of CC. However, little is known about that [16].

Aquaporins (AQPs) are a group of basic membrane proteins that assist in water transport through the cell membrane. They are reported to play a major role in cellular proliferation, angiogenesis, apoptosis, cell migration, and mitochondrial metabolism. Although the role of AQPs in human pathology has been studied broadly, lately their roles in cancer have become an area of interest. Numerous studies have revealed that AQPs are overexpressed in different malignant tumors [17]. An in vitro research confirmed that AQP1-mediated plasma membrane water permeability is fundamental for colon cancer cell migration and may be associated with tumor invasion and metastasis [18]. Although the clinical and biological relevance of AQP1 has been investigated in numerous types of cancer, including CC, only a small number of studies have focused on its prognostic role, and these results were conflicting. Furthermore, it remains to be assessed whether the AQP1 expression is predictive of response to chemotherapy or not [19].

Several prognostic parameters have been analyzed in colon cancer. However, they are still inadequate to recognize the high-risk patients prone to chemoresistant and progressive tumor. Therefore, there is an actual need to identify new predictive biomarkers for relapse and chemotherapeutic resistance. These predictive markers can tailor the management of each individual patient, and consequently enhance the benefit from a specific therapeutic agent. Thus, the current study evaluated the potential clinical utility of ERCC1, PARP-1, and AQP1 as predictive and prognostic molecular markers in stage II and III CC patients receiving 5-FU-based adjuvant chemotherapy after curative surgery.

2. Materials and methods

2.1 Patients’ selection

The current prospective study was carried out at Pathology, Clinical Oncology, Medical Oncology, and General Surgery Departments, Faculty of Medicine, Zagazig University hospitals after written informed consent was taken from our participants. A total of 60 consecutive patients of stage II or III primary CC that underwent curative resection between July 2015 and December 2017 were enrolled in the study. All the hematoxylin and eosin-stained slides were revised to confirm the diagnosis, histological grade, and tumor stage. The tumor stage was pathologically confirmed according to TNM staging system (American Joint Committee on Cancer (AJCC), the 8th edition). Patients with distant metastases at the time of operation, preoperative chemotherapy and/or radiotherapy, or patients with rectal cancer were excluded from the study. Patients’ clinical data, including survival and follow-up information, were retrieved from the hospital registry, and the clinical patients’ records. Approval of the local research ethics committee in Zagazig University hospitals was obtained.

2.2 Treatment schedule and follow-up

All the patients were admitted to the General Surgery department and diagnosed by the clinical symptoms (e.g., bleeding per-rectum, obstructive manifestations), abdominal ultrasonography, computed tomography with oral contrast, and colonoscopy biopsies. For the twenty patients of right-sided colon cancer, right hemicolectomy and ileotransverse anastomosis was done. For the forty patients of left-sided colon cancer, left hemicolectomy with primary anastomosis was done after preoperative colonic preparation. All the patients were followed-up until stitches removal and then completed their treatment in the Oncology department.

Curative resection was done for all patients and followed by the chemotherapy regimen in 50 cases only (high risk stage II and stage III). The fifty cases were treated with the standard mFOLOFOX6 consisting of oxaliplatin (85 mg/m ${}^{2}$ ) intravenous infusion over 2-hour on day 1, Leucovorin (400 mg/m ${}^{2}$ ) intravenous infusion over 2-hour on days 1, followed by intravenous injection of 5-FU (400 mg/m ${}^{2}$ ) bolus on day 1, then 5-FU (1200 mg/m ${}^{2}$ /day) continuous infusion in 22 h on days 1–2, to be repeated every 2 weeks. Tumor response was assessed according to the revised Response Evaluation Criteria in Solid Tumors (RECIST) criteria (version 1.1) [20].

The follow-up to detect early recurrence was completed according to a standard protocol that included colonoscopy, abdominal ultrasound, and chest radiography every 6 months/2 years then every year later on. Assessment of CEA level was done every 3 months/ 2 years, then every 6 months later on. The follow-up aimed to detect early treatment failure (tumor recurrence).

2.3 Immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded tissue (FFPE) blocks were serially sectioned into 3–5 $\mu$ m sections followed by deparaffinization in xylene and rehydration in descending series of alcohols. For antigen retrieval, 10 mM citrate buffer (pH 6.0) at the microwave for 20 min was used. We used 3% hydrogen peroxide for about 10 min to block the endogenous peroxidase activity. After repeat washing in PBS, the slides were incubated with ERCC1 antibody [catalog no: ab2356, 8F1 clone, dilution 1:100, (Abcam)], monoclonal PARP-1 antibody (F-2, dilution 1: 500; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal AQP1 antibody (cat. no. HPA019206; Atlas antibodies AB, Bromma, Sweden). The binding site of primary antibodies was visualized by using the polymer detection system; the Dako EnVision ${}^{\text{TM}}$ kit (Dako, Copenhagen, Denmark). Finally, the tissue sections were counterstained with Meyer’s hematoxylin, dehydrated and mounted. Negative controls were obtained by the replacement of the primary antibodies with non-immune serum. Positive and negative controls were stained in the same setting with the studied cases.

2.4 Immunohistochemistry assessment

2.4.1 ERCC1 expression

ERCC1 assessment based on the percentage of positive tumor cells/10 HPF. Score 0: complete negative staining, score 1: $<$ 50% positivity and the score of 2: positivity in $>$ 50% of the neoplastic cells. Positive expression of ERCC1 was defined as score 2 [21].

2.4.2 PARP-1 expression

Both intensity (0–3) and pattern scores (1–6) of nuclear PARP-1 expression were evaluated. Each intensity score was multiplied by its parallel pattern score (1 $=$ 0–4% of positive cells; 2 $=$ 5–19%; 3 $=$ 20–39%; 4 $=$ 40–59%; 5 $=$ 60–79%; 6 $=$ 80–100%) to obtain the final score. Nuclear PARP-1 expression was graded as low (0–9) or high (10–18) [22].

2.4.3 AQP1 expression

AQP1 membranous or cytoplasmic staining within the tumor cells was semi-quantitatively scored as 0 (0–5%), 1 (5–20%), 2 (20–50%) or 3 ( $>$ 50%). Subsequently, tumors determined to have scores of 2 and 3 were considered to have AQP1-positive expression, but tumors with scores of 0 and 1 were considered to be negative for AQP1 expression [6].

Table 1
Clinicopathological features, immunohistochemical markers and outcome of 60 patients with colorectal carcinoma

Characteristics	All patients		Characteristics	All patients
	( $N=$ 60)			( $N=$ 60)
	No.	%		No.	%
Age			Stage
Mean $\pm$ SD	46.16 $\pm$ 10.74		Stage II	22	36.7%
Median (range)	48 (20–66)		Satge III	38	63.3%
$\leqslant$ 50 years	37	61.7%	ERCC1
$>$ 50 years	23	38.3%	Negative	25	41.7%
Sex			Positive	35	58.3%
Male	42	70%	PARP1
Female	18	30%	Low	22	36.7%
Location			High	38	63.3%
Right colon	20	33.3%	AQP1
Left colon	40	66.7%	Negative	35	58.3%
Histopathology			Positive	25	41.7%
Adenocarcinoma	48	80%	Adjuvant chemotherapy
Mucinous	12	20%	Not recieved	10	16.7%
Grade			Received	50	83.3%
Grade I	6	10%	Follow-up duration (months)
Grade II	25	41.7%	Mean $\pm$ SD	38.53 $\pm$ 9.34
Grade III	29	48.3%	Median (range)	41 (20–48)
T			Relapse
T2	10	16.7%	Absent	37	61.7%
T3	33	55%	Present	23	38.3%
T4	17	28.3%	Mortality
N			Alive	49	81.7%
N0	22	36.7%	Died	11	18.3%
N1	22	36.7%
N2	16	26.7%

Categorical variables were expressed as number (percentage). Continuous variables were expressed as mean $\pm$ SD and median (range).

2.5 Statistics

Continuous variables were expressed as the mean $\pm$ SD & median (range), and the categorical variables were expressed as a number (percentage). Continuous variables were checked for normality by using a Shapiro-Wilk test. Mann Whitney U test was used to compare two groups of non-normally distributed variables. Percent of categorical variables were compared using Pearson’s Chi-square test or Fisher’s exact test when was appropriate. Trend of change in the distribution of relative frequencies between ordinal data was compared using a Chi-square test for trend. Overall Survival (OS) was calculated as the time from diagnosis to death or the most recent follow-up contact (censored). Disease-Free Survival (DFS) was calculated as the time from the start of treatment to date of relapse or the most recent follow-up contact that a patient was known as relapse free. Stratification of OS and DFS was done according to the studied biomarkers. These time-to-event distributions were estimated using the method of Kaplan-Meier plot, and compared using a two-sided exact log-rank test. All tests were two sided. A $p$ -value $<$ 0.05 was considered significant. All statistics were performed using SPSS 22.0 for Windows (SPSS Inc., Chicago, IL, USA) and MedCalc Windows (MedCalc Software bvba 13, Ostend, Belgium).

3. Results

3.1 Clinicopathological characteristics of the patients

As presented in Table 1, the mean age of the studied patients at the initial diagnosis was 46.17 $\pm$ 10.7 years (range 20–66 years) where 70% were males. Grade II tumors represented 48.3% of the cases and about 66.7% had their primary tumor in the left side. Most of the patients (63.3%) were at stage III, while 80% had adenocarcinoma type (Fig. 1). Lymph node metastasis was noted in 63.3% of the patients, while only 36.7% of patients presented without lymph node metastasis. The mean follow-up duration was 38.5 months (range 20–48 m) during which 38.3% of the cases exhibited relapse and 11 patients died.

Figure 1.

A. Represenative case of colon adenocarcinoma (H&E, $\times$ 400), B. Represenative case of colon mucinous carcinoma (H&E, $\times$ 400).

Figure 2.

Representative images of ERCC1 immunohistochemical staining A. Nuclear ERCC1 expression in grade II colon adenocarcinoma (IHC, $\times$ 200), B. Nuclear ERCC1 expression in grade II colon adenocarcinoma (IHC, $\times$ 400), C. Nuclear ERCC1 expression in grade III colon adenocarcinoma (IHC, $\times$ 100).

Figure 3.

Representative images of PARP1 immunohistochemical staining A. Nuclear PARP1 expression in grade II colon adenocarcinoma (IHC, $\times$ 400), B. Nuclear PARP1 expression in grade III colon adenocarcinoma (IHC, $\times$ 400).

Figure 4.

Representative images of AQP1 immunohistochemical staining A. Negative expression of AQP in normal mucosa (IHC, $\times$ 400), B. Membranous AQP1 expression in grade I colon adenocarcinoma (score 1) (IHC, $\times$ 400), C. Membranous AQP1 expression in grade II colon adenocarcinoma (score 3) (IHC, $\times$ 200), D. Membranous AQP1 expression in grade III colon adenocarcinoma (score 3) (IHC, $\times$ 400).

3.2 Association between ERCCI, PARP-1, and AQP1 expression and clinicopathological characteristics, Table 2

Positive nuclear ERCC1 expression was identified in 58.3% of the patients (Fig. 2). Positive ERCC1 expres-

Table 2
Relation between clinicopathological features and immunohistochemical staining ERCC1, PAPR1 and AQP1 in colorectal carcinoma patients ( $N=$ 60)

Characteristics	All		ERCC1				$p$ -value		PARP1				$p$ -value		AQP1				$p$ -value
	patients		Negative		Positive				Low		High				Negative		Positive
	( $N=$ 60)		( $N=$ 25)		( $N=$ 35)				( $N=$ 22)		( $N=$ 38)				( $N=$ 35)		( $N=$ 25)
	No. (%)		No. (%)		No. (%)				No. (%)		No. (%)				No. (%)		No. (%)
Age
Mean $\pm$ SD	46.16 $\pm$ 10.74		47.56 $\pm$ 9.44		45.17 $\pm$ 11.62			0.573 ${}^{*}$	43.59 $\pm$ 10.79		47.65 $\pm$ 10.57			0.077 ${}^{*}$	45.65 $\pm$ 10.75		46.88 $\pm$ 10.92			0.946 ${}^{*}$
Median (range)	48	(20–66)	48	(20–63)	46	(20–66)			45.50	(20–63)	50.05	(20–66)			48	(20–60)	45	(23–66)
$\leqslant$ 50 years	37	(61.7%)	17	(45.9%)	20	(54.1%)		0.394 ${}^{\ddagger}$	18	(48.6%)	19	(51.4%)		0.015 ${}^{\ddagger}$	23	(62.2%)	14	(37.8%)		0.445 ${}^{\ddagger}$
$>$ 50 years	23	(38.3%)	8	(34.8%)	15	(65.2%)			4	(17.4%)	19	(82.6%)			12	(52.2%)	11	(47.8%)
Sex
Male	42	(70%)	21	(50%)	21	(50%)		0.046 ${}^{\ddagger}$	17	(40.5%)	25	(59.5%)		0.350 ${}^{\ddagger}$	28	(66.7%)	14	(33.3%)		0.046 ${}^{\ddagger}$
Female	18	(30%)	4	(22.2%)	14	(77.8%)			5	(27.8%)	13	(72.2%)			7	(38.9%)	11	(61.1%)
Location
Right colon	20	(33.3%)	15	(75%)	5	(25%)	$<$	0.001 ${}^{\ddagger}$	11	(55%)	9	(45%)		0.037 ${}^{\ddagger}$	15	(75%)	5	(25%)		0.064 ${}^{\ddagger}$
Left colon	40	(66.7%)	10	(25%)	75	(75%)			11	(27.5%)	29	(72.5%)			20	(50%)	20	(50%)
Grade
Grade I	6	(10%)	6	(100%)	0	(0%)	$<$	0.001 ${}^{\S}$	5	(83.3%)	1	(16.7%)		0.003 ${}^{\S}$	6	(100%)	0	(0%)	$<$	0.001 ${}^{\S}$
Grade II	25	(41.7%)	18	(72%)	7	(28%)			11	(44%)	14	(56%)			19	(76%)	6	(24%)
Grade III	29	(48.3%)	1	(3.4%)	28	(96.6%)			6	(20.7%)	23	(79.3%)			10	(34.5%)	19	(65.5%)
T
T2	10	(16.7%)	5	(50%)	5	(50%)		0.252 ${}^{\S}$	4	(40%)	6	(60%)		0.151 ${}^{\S}$	5	(50%)	5	(50%)		0.053 ${}^{\S}$
T3	33	(55%)	15	(45.5%)	18	(54.5%)			15	(45.5%)	18	(54.5%)			16	(48.5%)	17	(51.5%)
T4	17	(28.3%)	5	(29.4%)	12	(70.6%)			3	(17.6%)	14	(82.4%)			14	(82.4%)	3	(17.6%)
N
N0	22	(36.7%)	19	(86.4%)	3	(13.6%)	$<$	0.001 ${}^{\S}$	12	(54.5%)	10	(45.5%)		0.003 ${}^{\S}$	21	(95.5%)	1	(4.5%)	$<$	0.001 ${}^{\S}$
N1	22	(36.7%)	5	(22.7%)	17	(77.3%)			9	(40.9%)	13	(59.1%)			10	(45.5%)	12	(54.5%)
N2	16	(26.7%)	1	(6.2%)	15	(93.8%)			1	(6.2%)	15	(93.8%)			4	(25%)	12	(75%)
Stage
Stage II	22	(36.7%)	19	(86.4%)	3	(13.6%)	$<$	0.001 ${}^{\ddagger}$	12	(54.5%)	10	(45.5%)		0.029 ${}^{\ddagger}$	21	(95.5%)	1	(4.5%)	$<$	0.001 ${}^{\ddagger}$
Satge III	38	(63.3%)	6	(15.8%)	32	(84.2%)			10	(26.3%)	28	(73.7%)			14	(36.8%)	24	(63.2%)
ERCC1
Negative	25	(41.7%)							17	(68%)	8	(32%)	$<$	0.001 ${}^{\ddagger}$	20	(80%)	5	(20%)		0.004 ${}^{\ddagger}$
Positive	35	(58.3%)							5	(14.3%)	30	(85.7%)			15	(42.9%)	20	(57.1%)
PARP1
Low	22	(36.7%)	17	(77.3%)	5	(22.7%)	$<$	0.001 ${}^{\ddagger}$							14	(63.6%)	8	(36.4%)		0.526 ${}^{\ddagger}$
High	38	(63.3%)	8	(21.1%)	30	(78.9%)									21	(55.3%)	17	(44.7%)
AQP1
Negative	35	(58.3%)	20	(57.1%)	15	(42.9%)		0.004 ${}^{\ddagger}$	14	(40%)	21	(60%)		0.526 ${}^{\ddagger}$
Positive	25	(41.7%)	5	(20%)	20	(80%)			8	(32%)	17	(68%)

Categorical variables were expressed as number (percentage); ${}^{*}$ Mann Whitney U test; ${}^{\ddagger}$ Chi-square test; ${}^{\S}$ Chi-square test for trend; $p<$ 0.05 is significant.

Table 3

Relation between immunohistochemical staining ERCC1, PAPR1 and AQP1 and outcome in colorectal carcinoma patients received adjuvant chemotherapy ( $N=$ 50)

Outcome	Received	ERCC1		$p$ -value	PARP1		$p$ -value	AQP1		$p$ -value
	chemotherapy	Negative	Positive		Low	High		Negative	Positive
	( $N=$ 50)	( $N=$ 15)	( $N=$ 35)		( $N=$ 15)	( $N=$ 35)		( $N=$ 25)	( $N=$ 25)
	No. (%)	No. (%)	No. (%)		No. (%)	No. (%)		No. (%)	No. (%)
Relapse
Absent	30 (60%)	13 (86.7%)	17 (48.6%)	0.012 ${}^{\ddagger}$	13 (86.7%)	17 (48.6%)	0.012 ${}^{\ddagger}$	8 (32%)	22 (88%)	$<$ 0.001 ${}^{\ddagger}$
Present	20 (40%)	2 (13.3%)	18 (51.4%)		2 (13.3%)	18 (51.4%)		17 (68%)	3 (12%)
DFS
Mean (months) (95% CI)	39.79 months	45.79 months	37.07 months	0.005 ${}^{\dagger}$	45.64 months	37.34 months	0.016 ${}^{\dagger}$	35.72 months	45.02 months	0.002 ${}^{\dagger}$
	(36.61–42.97)	(42.83–48.76)	(33.04–41.09)		(42.55–48.73)	(33.30–41.37)		(31.21–40.22)	(41.76–48.27)
1 year DFS	100%	100%	100%		100%	100%		100%	100%
2 year DFS	77.2%	100%	67.5%		100%	67.5%		64%	92%
3 year DFS	70%	92.9%	60%		92.9%	60.7%		52%	92%
4 year DFS	49.4%	85.1%	26%		83.6%	35.5%		29.2%	85.4%
Mortality
Alive	39 (78%)	14 (93.3%)	25 (71.4%)	0.139 ${}^{\ddagger}$	14 (93.3%)	25 (71.4%)	0.139 ${}^{\ddagger}$	18 (72%)	21 (84%)	0.306 ${}^{\ddagger}$
Died	11 (22%)	1 (6.7%)	10 (28.6%)		1 (6.7%)	10 (28.6%)		7 (28%)	4 (16%)
OS
Mean (months) (95% CI)	43.13 months	46.13 months	41.51 months	0.062 ${}^{\dagger}$	46.13 months	41.62 months	0.077 ${}^{\dagger}$	41.96 months	44.36 months	0.543 ${}^{\dagger}$
	(40.54–45.72)	(42.59–49.66)	(38.13–44.88)		(42.59–49.66)	(38.26–44.98)		(38.13–45.78)	(41.09–47.63)
1 year OS	100%	100%	100%		100%	100%		100%	100%
2 year OS	91.5%	93.3%	90.3%		93.3%	90.3%		88%	96%
3 year OS	77.9%	93.3%	70%		93.3%	70.4%		71.8%	85.1%
4 year OS	74.2%	93.3%	63%		93.3%	64.5%		71.8%	73%

Continuous variables were expressed as mean (95% CI); categorical variables were expressed as number (percentage); ${}^{\ddagger}$ Chi-square test; ${}^{\dagger}$ Log rank test; $p<$ 0.05 is significant.

sion was significantly associated with left-sided tumors ( $P<$ 0.001) besides the aggressive tumor characteristics, including high grade, lymph node metastasis and advanced tumor stage ( $P<$ 0.001 for each).

High nuclear PARP-1 expression was observed in 63.3% of the patients (Fig. 3). High PARP-1 expression was significantly associated with the tumor grade and lymph node metastasis ( $P=$ 0.003 for each) besides the tumor stage ( $P=$ 0.029).

Positive membranous AQP1 expression was identified in 41.7% of the patients (Fig. 4). Negative AQP1 expression was significantly associated with the right-sided tumors vs. left-sided tumors ( $P=$ 0.06) where 75% of right-sided tumors showed negative AQP1 expression. Positive AQP1 expression was significantly associated with the aggressive tumor characteristics, including high grade, lymph node metastasis and advanced tumor stage ( $P<$ 0.001 for each). However, AQP1 expression had no significant association with the other tumor characteristics.

Table 4

Relation between immunohistochemical staining ERCC1/PAPR1/ AQP1 and outcome in colorectal carcinoma patients ( $N=$ 29)

Outcome	ERCC1/PAPR1/AQP1		$p$ -value
	$+$ ve/High/ $-$ ve	$+$ ve/High/ $+$ ve
	( $N=$ 14)	( $N=$ 15)
	No. (%)	No. (%)
Relapse
Absent	0 (0%)	12 (80%)	$<$ 0.001 ${}^{\ddagger}$
Present	14 (100%)	3 (20%)
DFS
Mean (months)	28.57 months	43.05 months	$<$ 0.001 ${}^{\dagger}$
(95% CI)	(23.82–33.31)	(37.87–48.22)
1 year DFS	100%	100%
2 year DFS	35.7%	86.7%
3 year DFS	21.4%	86.7%
4 year DFS	0%	75.8%
Mortality
Alive	7 (50%)	12 (80%)	0.128 ${}^{\ddagger}$
Died	7 (50%)	3 (20%)
OS
Mean (months)	37.10 months	43.17 months	0.228 ${}^{\dagger}$
(95% CI)	(31.36–42.84)	(38.64–47.69)
1 year OS	100%	100%
2 year OS	78.6%	100%
3 year OS	49%	80.8%
4 year OS	49%	53.9%

3.3 Prognostic relevance of ERCCI, PARP-1, and AQP1 expression in stage II–III colon cancer; Tables 3 and 4

During the follow-up period, 23 patients (38.3%) suffered progressive disease after curative surgery (failure of therapy) and 11 patients (18.3%) died. Out of the 60 patients, 50 patients received adjuvant chemotherapy following curative surgery, while 10 patients were treated with surgery alone. Among the patients that have received adjuvant chemotherapy, relapse was detected in 20 cases (40%). Relapse had a positive association with the ERCC1 expression ( $P=$ 0.012) and PARP-1 expression ( $P=$ 0.012). In contrast, relapse was negatively associated with AQP1 expression ( $P<$ 0.001) where 68% of negative AQP1 expression had a relapse and 88% of positive cases showed no relapse.

Statistical analysis of the survival data revealed that shorter DFS was significantly associated with positive ERCC1, high PARP-1, and positive AQP1 expression ( $P=$ 0.005, 0.016, 0.002, respectively). In contrast, a non-significant association between OS with ERCC1, PARP1, AQP1 ( $P>$ 0.05) was detected. Kaplan-Meier plot curves that were stratified according to ERCC1, PARP-1, and AQP1 expression were presented in (Figs 5 and 6).

Figure 5.

Kaplan Meier plot for overall survival (OS): A. Stratified by ERCC1 IHC staining ( $P=$ 0.062), B. Stratified by PARP1 IHC staining ( $P=$ 0.077), C. Stratified by AQP1 IHC staining ( $P=$ 0.543).

Figure 6.

Kaplan Meier plot for disease-free survival (DFS): A. Stratified by ERCC1 IHC staining ( $P=$ 0.005), B. Stratified by PARP1 IHC staining ( $P=$ 0.016), C. Stratified by AQP1 IHC staining ( $P=$ 0.002).

4. Discussion

Colon cancer prognosis and therapy choice basically depend on pathology-related staging that infrequently predicts the CC clinical outcome perfectly. About 20–40% of patients with relatively low-grade, stage II rapidly progress and die [23].

Colon cancer therapy includes surgery, with or without adjuvant chemotherapy that is mainly based on 5-fluorouracil (5-FU). 5-FU results in thymidylate synthase inhibition, reduction of thymidine triphosphates available for DNA synthesis, and misincorporation of FdUTP (5-FdUrd triphosphate) into DNA [24]. Even with therapy, tumor progression is common within a few years, indicating drug resistance [25].

Oxaliplatin resistance is commonly seen in CC patients and so the recognition of sensitive predictive biomarkers is needed before the start of therapy to define patients who are prone to benefit from oxaliplatin. Understanding the mechanism of action of oxaliplatin-based chemotherapy on a molecular level combined with well-developed detection techniques of the genetic profile of the patients had led to great progress in individualizing treatment in advanced CC [7]. The genetic diversity between different CC cases may explain a superior response to one regimen than the others [26].

ERCC1 is a key enzyme in the NER pathway that is essential for repair of platinum-DNA adducts, and so associated with resistance to therapy containing platinum compounds. ERCC1 was suggested as a promising marker in the CC. ERCC1-overexpressing tumor cells are thought to be more resistant to platinum-based chemotherapy, suggesting that chemotherapy would be more effective in ERCC1-negative cancer [27].

In the present study, positive ERCC1 expression was noted in 58.3% of our cases. Its expression was significantly associated with the left-sided tumors and the aggressive tumor characteristics, including high grade, lymph node metastasis, and advanced tumor stage. Likewise in previous studies, ERCC1 positivity was observed in 45% and 55% of patients with colorectal cancer stage III, respectively [28, 21]. In a previous study, ERCC1 expression was detected in 72% of the studied cases and showed no significant association with the clinicopathological parameters apart from a tumor site [29]. On the other hand, Liang et al. reported that patients with nasopharyngeal carcinoma, the ERCC1 expression increased significantly with higher tumor stages and clinical stages ( $P<$ 0.05). Therefore, at least in that type of malignancy, ERCC1 seemed to be a valid biological indicator to predict prognosis [30]. This discrepancy may be due to numerous factors, including the antibody used, the scoring method, and the preparation of the paraffin-embedded tissue blocks.

Regarding the predictive value of ERCC1, some authors concluded that the high ERCC1 expression predicted bad response to chemotherapy and survival outcome [31], while other authors didn’t find any significant association between ERCC1 expression and clinical prognosis [32].

ERCC1 expression has been assessed by IHC in some previous clinical studies including patients who received oxaliplatin-based chemotherapy and showed a significantly longer DFS and OS in patients with low ERCC1 protein expression compared to those with high ERCC1 expression [21, 33, 34]. In the present study, we confirmed these findings where ERCC1 expression was related to tumor relapse ( $P=$ 0.012) and shorter DFS ( $P=$ 0.005) but no correlation was observed with OS. This finding confirms the recognized function of ERCC1 in DNA repair after platinum therapy. Tumor cells with high ERCC1 expression have a superior DNA repair capacity that could efficiently diminish the antitumor effect of oxaliplatin, leading to unfortunate prognosis of these patients. Moreover, ERCC1 expression often has a high DNA-repair capability, and upon exposure to oxaliplatin, will undergo relatively less apoptosis. Apoptosis is one of the central mechanisms by which platinum compounds excute their antineoplastic activity, so less apoptosis is correlated to reduced therapy efficiency and leading to treatment failure [35].

In contrast to these results, some studies couldn’t reach the same conclusion, where Kim et al. found that ERCC1 expression was not significantly correlated with DFS in patients with stage II–III colon cancer treated with FOLFOX adjuvant chemotherapy [36]. Moreover, Lenz et al. didn’t find a significant predictive value of ERCC1 in cases received oxaliplatin-based regimens, but he remarked a suggestion that low ERCC1 expression was associated with longer PFS compared to those with high expression [37]. The contradictory results reported in these studies may be explained by differences in their design, heterogeneity in the target patients, the methods used to detect ERCC1 (IHC, RT-qPCR or SNP), different cutoff to stratify low vs. high besides differences in their endpoints. Furthermore, in some studies, due to the short follow-up period of 15 months, no correlation of DFS and OS with ERCC1 expression was observed [29].

Furthermore, we evaluated PARP-1 expression in the studied cases to address detailed information and expand our shortened knowledge about its expression level in this prevalent cancer. Despite considerable progress in the estimation of the PARP-1 role in DNA repair and various biological processes, the level of PARP-1 expression in different tumors, including CC is largely unknown but intensive research is ongoing. This knowledge is actually crucial to properly evaluate the outcome of clinical trials concerning targeted treatment with the PARP-1 inhibitors [38].

PARP-1 has a fundamental task in the maintenance of genome stability and the chromatin structure. PARP-1 has a role in different DNA repair pathways, as base excision repair (BER) and DNA single-strand break (SSB) repair [39]. Therefore, PARP-1-deficient mice show hypersensitivity to DNA damaging agents, with an enhanced genomic instability [40].

In our study, high PARP-1 expression was found in 63.3% of the studied cases. However, in other studies the expression was 97.7% [41], and 82.1% [42]. These findings may have a clinical implication because the estimation of PARP-1 expression in the tumor may improve the selection of patients with colon cancer for PARP inhibitor therapy. In a previous study [22], it was noted the high PARP-1 expression in 68.2% of the studied CC cases and there was a significant association of PARP-1 expression with the site of CC ( $P=$ 0.001) and tumor stage ( $P=$ 0.018). In contrast to these findings, our study revealed no relation to the site, but strongly related to the grade stage, and lymph node metastasis. In addition, we noted a significant relationship between high PARP-1 expression and relapse rate ( $P=$ 0.012) and shorter DFS ( $P=$ 0.016) and the mortality was higher in those patients. Our results confirmed the previous preclinical studies which verified the improvement of radiosensitivity and chemotherapy response in experimental models of CC by PARP-1 inhibitors [15].

PARP-1 inhibition emerged as a therapeutic target, and recent studies verified dramatic enhancement of chemotherapeutic agents in the presence of PARP inhibition [43]. New data indicate that besides the catalytic inhibition of PARP activity, PARP inhibitors (PARPi) provoke cytotoxic PARP-DNA complexes through PARP “trapping” that augment the cytotoxicity of alkylating agents. It is therefore of extreme significance to spot the molecular character that acts not only as biomarkers for the patient stratification, but also proposes insights into the mechanisms of chemotherapy resistance [44].

Although numerous studies have analyzed the prognostic significance of AQP1 in human cancer, only few clinical studies have assessed the association between AQP1 expression and the response to chemotherapy. Therefore, the present study focused on evaluating the potential contribution of AQP1 expression to chemotherapeutic outcomes in patients with stage II–III colon cancer who received 5-FU-based adjuvant chemotherapy. In the current study, we noted positive expression of AQP1 in 41.7% of the cases with a significant relation to aggressive tumor features including high grade, lymph node metastasis, and tumor stage. In accordance with this, previous studies [45, 46] have revealed AQP1 expression in 41.8% and 35.8% of patients, respectively. This was significantly associated with tumor grade, advanced tumor stage, and lymph node metastasis. These findings suggest that a tumor-promoting role of AQP1 that particularly affect lymph node metastasis and vascular invasion. In vitro studies have also reported that AQP1 increases the potential of invasion and migration in CC cell lines [47, 48]. This may suggest that AQP1 has the potential to contribute to tumor progression, particularly in left-sided colorectal tumors. However, Kang et al. reported that marked positive AQP1 expression was negatively associated with lymph node metastasis in stage I–III colon cancer. Therefore, further studies are required to specifically clarify the clinical and biological significance of AQP1 in colon cancer [49].

Regarding the prognostic significance of AQP1 expression, Yoshida et al. concluded that AQP1 is a poor prognostic factor for overall survival (OS) in patients with stage II–III CC using multivariate analysis [46], whereas Kang et al. identified that AQP1 expression had no significant impact on OS or DFS in patients with stage I–III CC [49]. In contrast to these findings, in the present study, we identified a significant survival impact of AQP1 expression on DFS. Furthermore, our results revealed that patients with AQP1 positive cancer had improved therapeutic outcomes. Similar to our findings, Imaizumi et al. reported that patients with AQP1-negative CC exhibited an inferior response to chemotherapy and may even be harmed by 5-FU-based treatment as demonstrated by the reduction in DFS in the AQP1-negative CC chemotherapy-treated group [45]. These findings suggest that AQP1-positive colon cancer have a superior sensitivity to 5-FU-based adjuvant chemotherapy compared with AQP1-negative cases. Furthermore, there is currently no sufficient explanation as to why AQP1-negative tumors have a poorer prognosis following treatment with adjuvant chemotherapy, suggesting the requirement for underlying mechanistic investigation in future studies [45].

On the other hand, in ovarian cancer cell lines, AQP1 expression was associated with sensitivity to cisplatin [50]. Liu and Zhu indicated that AQP1 expression was downregulated by combination therapy of celecoxib and afatinib in a lung cancer cell line. In view of these results, it is hypothesized that AQP1 may potentially modulate the sensitivity to anticancer drugs. A potential explanation for this discrepancy in the clinical impact of AQP1 between cancer types may be due to the multifaceted roles of AQP1 across various organs and tumor cells [51].

In conclusion, ERCC1, PARP1, and AQP1 are adverse prognostic biomarkers of aggressive colon cancer. Moreover, oxaliplatin usage in adjuvant chemo- therapy may not be beneficial for patients with positive ERCC1, high PARP1, and AQP1-negative tumors. Therefore, selecting alternative chemotherapeutic regimens or even adding targeted agents as PARP inhibitors may present a novel strategy. We hope that subsequent investigations may confirm our findings, paving the way to the implementation of these biomarkers in routine clinical practice.

Footnotes

Conflict of interest

The authors declare that they have no conflict of interest.

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ERCC1,PARP-1,and AQP1 as predictive biomarkers in colon cancer patients receiving adjuvant chemotherapy

Abstract

BACKGROUND:

OBJECTIVES:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients’ selection

2.2 Treatment schedule and follow-up

2.3 Immunohistochemistry (IHC)

2.4 Immunohistochemistry assessment

2.4.1 ERCC1 expression

2.4.2 PARP-1 expression

2.4.3 AQP1 expression

Table 1 Clinicopathological features, immunohistochemical markers and outcome of 60 patients with colorectal carcinoma

3. Results

3.1 Clinicopathological characteristics of the patients

Table 2 Relation between clinicopathological features and immunohistochemical staining ERCC1, PAPR1 and AQP1 in colorectal carcinoma patients ( N = 60)

Footnotes

Conflict of interest

References

Table 1
Clinicopathological features, immunohistochemical markers and outcome of 60 patients with colorectal carcinoma

Table 2
Relation between clinicopathological features and immunohistochemical staining ERCC1, PAPR1 and AQP1 in colorectal carcinoma patients ( $N=$ 60)