Positive BCL2L12 expression predicts favorable prognosis in patients with laryngeal squamous cell carcinoma

Abstract

BACKGROUND:

Laryngeal squamous cell carcinoma (LSCC) constitutes the third most frequent head and neck cancer. Several tissue biomarkers have been studied for their prognostic significance in LSCC.

OBJECTIVE:

To investigate the prognostic significance of BCL2L12, a new member of the BCL2 family, in primary LSCC along with well-examined biomarkers such as BCL2 and BAX.

METHODS:

Cancerous tissue specimens of patients with primary LSCC were collected during 2005 and 2012 as pretreatment tissue biopsy. The specimens were immunohistochemically evaluated for the protein expression of BCL2L12, BCL2 and BAX. Kaplan-Meier survival curves and Cox proportional hazard regression models were performed to evaluate prognosis.

RESULTS:

In the study cohort of 78 patients with primary LSCC, Kaplan-Meier survival curves demonstrated that advanced-stage LSCC patients with BCL2L12-positive tumors had significantly higher OS time in comparison with advanced-stage LSCC patients with BCL2L12-negative tumors ( $p=$ 0.014). Also, advanced-stage LSCC patients with BCL2L12-positive tumors had significantly lower risk of death from LSCC compared to advanced-stage LSCC patients with BCL2L12-negative tumors (HR $=$ 0.228, 95%CI $=$ 0.063–0.833, $p=$ 0.025).

CONCLUSIONS:

BCL2L12 protein expression could be used as a favorable prognostic tissue biomarker in patients with primary advanced-stage LSCC. On the contrary, BCL2 and BAX did not correlate with prognosis in patients with primary LSCC.

Keywords

Laryngeal neoplasms BCL2L12 biomarkers prognosis survival

1. Introduction

Head and neck squamous cell carcinoma (HNSCC) constitutes the eighth most commonly diagnosed cancer, accounting for approximately 6% of all cancer cases worldwide [1]. Laryngeal squamous cell carcinoma (LSCC) was the most frequent among subtypes of HNSCC until the beginning of the 21st century [2].

Several tissue and serum biomarkers have been studied for their prognostic significance in LSCC using distinct techniques, including immunohistochemistry [3], polymerase chain reaction (PCR) and real-time PCR [4]. For instance, Foteinou et al. observed that low mRNA expression levels of kallikrein-related peptidase 4 (KLK4) predicted shorter disease-free survival (DFS) in LSCC [4]. Additionally, Kumar et al concluded that tumor expression of P53 and BCL-xL might be correlated with successful laryngeal preservation in patients with laryngeal cancer treated with chemotherapy (CTH) and radiotherapy (RT) [5]. Also, Zhang et al reported that LSCC patients with positive cyclin D1 protein expression had significant higher median survival time than LSCC patients with negative cyclin D1 protein expression [6].

Data are controversial about correlation of BCL2 family genes expression and prognosis in LSCC. Jackel et al. reported that positive BCL2 protein expression predicted shorter overall survival (OS) [7], while others supported that positive [8] or high [9]BCL2 protein expression were associated with better OS. In other studies, BCL2 protein expression was not correlated with prognosis in early-stage LSCC [10] or LSCC of any stage [11]. Interestingly, high BCL2 protein expression was associated with resistance to radiotherapy in LSCC [3, 12]. On the contrary, data are consistent that BAX protein expression does not predict DFS or OS in LSCC [8, 7].

The BCL2L12 gene, a member of the BCL2 family, was discovered and cloned in 2001 [13]. The prognostic significance of BCL2L12 mRNA expression has already been assessed in several cancer types [14, 15, 16]. Fendri et al. observed that BCL2L12 mRNA expression was a significant predictor of unfavorable prognosis in patients with nasopharyngeal carcinoma (NPC) [17]. Moreover, Geomela et al. observed that BCL2L12 mRNA expression was lower in advanced-stage LSCC. The authors suggested that BCL2L12 mRNA expression might serve as potential prognostic biomarkers in LSCC [18].

To enhance the knowledge of this subject, we investigated the prognostic value of BCL2L12 expression in patients with primary LSCC. We also examined the association of BCL2 and BAX protein expression (as well-examined biomarkers) with prognosis in the same cohort of primary LSCC.

2. Methods

2.1 Collection of tissue specimens

Prospectively collected cancerous laryngeal tissue specimens were obtained from patients with LSCC as pretreatment tissue biopsy, at the first Department of Otorhinolaryngology, Hippocration Hospital, Medical University of Athens, National and Kapodistrian University of Athens. All tissue specimens were examined by a pathologist of the Hippocration Hospital, who confirmed the malignancy. The specimens were collected between 2005 and 2012. The study was approved by the Institutional Ethics Committee of the National and Kapodistrian University of Athens, in accordance with the ethic standards of the 1964 Declaration of Helsinki and its latter amendments. Inclusion criteria included patients with a primary LSCC. Exclusion criteria included patients with recurrence, patients with metastatic disease at time of diagnosis and patients who refused treatment.

All tissue specimens were accompanied by detailed data such as age at diagnosis, TNM stage (VII Edition), primary tumor site (T-status), nodal status (N), metastasis (M), alcohol consumption and smoking (pack-years index). Laryngeal cancer site (glottic, supraglottic or subglottic area), multiple primary cancer sites, type of treatment, radiotherapy, chemotherapy and type of surgery were also recorded. Follow-up information was available for all patients and included disease-free survival (DFS) status, overall survival (OS) status (deceased or alive) along with the dates of the events, region of metastatic disease and cause of death. TNM stage, T-status and nodal status were analyzed independently to detect possible subgroups, as previously observed [19].

2.2 Immunohistochemistry

Paraffin sections 5 $\mu$ m thick, mounted on superfrost glass slides, were used for the immunohistochemical detection of apoptosis-related proteins. Sections were deparaffinized in xylol, rehydrated in a graded ethanol series, and then subjected to microwave antigen retrieval (4 $\times$ 10 minutes, 800 w, in 0.1 M citrate acid buffer solution, pH 6). Endogenous peroxidase activity was blocked using 0.1% hydrogen peroxide. Sections were then incubated at 4 ${}^{\circ}$ C overnight with one of the following primary antibodies at the specified dilution: Bcl-2-like protein 12 (Thermo Fisher Scientific Pierce PA5-22209, dilution 1:150), Anti-BCL-2, (Thermo Fisher Scientific Pierce MA5-15668, dilution 1:500) and bax (SantaCruz 7480, dilution 1:80). Immunohistochemical staining was performed using the ChemMateTM EnVisionTM system (DAKO K5007) according to instructions provided by the manufacturer. Sections were then counterstained with Mayer hematoxylin, dehydrated, cleared and mounted. Appropriate positive controls were used according to the manufacturer’s guide for each antibody.

2.3 Immunohistochemical evaluation

Sections were evaluated under a light microscope. Ten fields at x200 were randomly selected and examined by two other experienced pathologists of the Department of Pathology, independently. In case of disagreement, the final score was determined by general agreement. All markers were separately evaluated in the cancerous cell population, the tumor-adjacent dysplastic epithelium and the stroma. The percentage of immunopositive cells (below 30% was characterized as weakly stained, between 31% and 69% as moderately stained, and above 70% as strongly stained) and a three-scale intensity scheme (staining intensity: negative $=$ 0, weak $=$ 1, moderate $=$ 2 and high $=$ 3) were combined and a semi-quantitative score was calculated, as discussed elsewhere [20]. For the statistical analysis, BCL2L12 final score 0 was classified as negative and 1–3 as positive. BCL2 final score of 0 and 1 were classified as low, while 2 and 3 were classified as high. Similarly, BAX final score of 0 and 1 were classified as low, while 2 and 3 were classified as high.

2.4 Statistical analysis

Descriptive statistics analysis was initially per- formed. Associations between staining positivity of BCL2L12, BCL2 and BAX and clinicopathological variables of LSCC patients were analyzed using the chi-square ( $\chi^{2}$ ) test (or the Fisher’s exact test, where appropriate). Kaplan-Meier DFS and OS analysis were performed and any differences between survival curves were evaluated by the log-rank (Mantel-Cox) test. Cox proportional hazard regression models were also built to assess the relationship between the established or potential prognostic biomarkers and the relative risk for patient recurrence or death. The level of significance for all statistical tests was defined at a probability value of less than 0.05 ( $p<$ 0.05).

Table 1
Clinicopathological characteristics of 78 LSCC patients

Variable		Number of
		patients (%)
Gender	Men	73/78 (93.6%)
	Women	5/78 (6.4%)
Laryngeal site	Supraglottic	29/78 (37.2%)
	Glottic	48/78 (61.5%)
	Subglottic	1/78 (1.3%)
Smoking ${}^{\rm a}$	Below 10 cigarettes daily	3/78 (3.9%)
	10–20 cigarettes daily	5/78 (6.4%)
	More than 20 cigarettes daily	70/78 (89.7%)
Alcohol	None or normal	21/78 (26.9%)
consume	Mild ${}^{\rm b}$ or heavy ${}^{\rm b}$	57/78 (73.1%)
Other cancer	Yes	7/78 (8.97%)
types	No	71/78 (91.03%)

${}^{\rm a}$ Since adulthood. ${}^{\rm b}$ More than 1 glass alcohol daily.

Table 2

Primary treatment of 78 LSCC patients

Variable (number of patients)	Primary treatment (number of patients)
Early TNM stage (26/78)	Primary RT ${}^{\rm{a}}$	Primary transoral surgery with CO ${}_{2}$ -laser ${}^{\rm{b}}$
I (15/78)	4	11
II (11/78)	5	6
Advanced TNM stage (52/78)	Primary CRTH ${}^{\rm{c}}$	Primary surgery with or without adjuvant RT/CRTH ${}^{\rm{c}}$
		Transoral CO ${}_{2}$ -laser ${}^{\rm b}$	Total laryngectomy ${}^{\rm b}$
III (25/78)	6	3	16
IV (27/78)	0	0	27

${}^{\rm a}$ Radiotherapy. ${}^{\rm b}$ With or without neck dissection. ${}^{\rm c}$ Concurrent chemoradiotherapy.

Table 3

Associations between BCL2L12 protein status and categorical clinicopathological variables of 78 LSCC patients

T (primary tumor site)
Variable	Total (%)	Number of patients (%)
		BCL2L12-positive	BCL2L12-negative	$p$ -value
T1	15 (19.2%)	7/15 (46.7%)	8/15 (53.3%)	0.013 ${}^{\rm a}$
T2	12 (15.4%)	11/12 (91.7%)	1/12 (8.3%)
T3	30 (38.5%)	12/30 (40.0%)	18/30 (60.0%)
T4	21 (26.9%)	14/21 (66.7%)	7/21 (33.3%)
N (nodal status)
N0	64 (82.1%)	37/64 (57.8%)	27/64 (42.2%)	$>$ 0.2 ${}^{\rm a}$
N1	8 (10.3%)	5/8 (62.5%)	3/8 (37.5%)
N2	6 (7.7%)	2/6 (33.3%)	4/6 (66.7%)
TNM stage
I/II	26 (33.3%)	17/26 (65.4%)	9/26 (34.6%)	0.188 ${}^{\rm b}$
III/IV	52 (66.7%)	27/52 (51.9%)	25/52 (48.1%)
Histological grade
Low	12 (15.4%)	7/12 (58.3%)	5/12 (41.7%)	$>$ 0.2 ${}^{\rm a}$
Intermediate	42 (53.8%)	26/42 (61.9%)	16/42 (38.1%)
High	24 (30.8%)	11/24 (45.8%)	13/24 (54.2%)

${}^{\rm a}$ Calculated by chi-square ( $\chi^{2})$ test. ${}^{\rm b}$ Calculated by Fisher’s exact test.

3. Results

3.1 Clinicopathological characteristics of the study cohort

The study included 78 cancerous laryngeal tissue specimens. The mean age of the patients was 61.76 $\pm$ 1.118 years (range from 36 to 90 years). The 25 ${}^{\rm th}$ , 50 ${}^{\rm th}$ (median age) and 75 ${}^{\rm th}$ percentile were 55.75, 61.50 and 68.25 years respectively. Other clinicopathological characteristics are presented in Table 1.

Patients’ primary treatment is presented in Table 2. All tumors were resected with clear margins either with transoral surgery with CO ${}_{2}$ laser or with total laryngectomy. In general, the authors preferred single modality treatment by early-stage LSCC patients and multimodal treatment by advanced-stage LSCC patients. The authors performed no neck dissection (ND) in patients with early-stage LSCC. Ipsilateral or bilateral selective ND of levels II-IV was performed to detect potential micro-metastastic disease in cN0 patients with advanced-stage glottic or supraglottic LSCC respectively. Ipsilateral modified radical ND was performed by clinical or radiological suspicion (cN $+$ ) of pathological cervical lymph nodes.

During the follow-up period, the mean DFS time was 71.37 $\pm$ 4.754 months (minimum 0.5 and maximum 120 months). The 25 ${}^{\rm th}$ , 50 ${}^{\rm th}$ (median DFS) and 75 ${}^{\rm th}$ percentile were 25.50, 77.50 and 119.25 months respectively. The mean OS time was 80.91 $\pm$ 4.150 months (minimum 0.5 and maximum 120 mon- ths). The 25 ${}^{\rm th}$ , 50 ${}^{\rm th}$ (median OS) and 75 ${}^{\rm th}$ percentile were 63.00, 85.00 and 120.00 months respectively. During the follow-up period, 38/78 patients (48.7%) presented with recurrence and 34/78 patients (43.6%) died; fourteen of them (14/78 – 17.9%) due to the disease. Also, 15/78 patients (19.2%) developed distant metastatic disease; the majority of them in the lungs (12/15 – 80.0%).

3.2 BCL2L12 protein expression in LSCC tissue specimens and its association with patients’ clinicopathological variants

BCL2L12 protein expression was categorized as positive or negative, as described in Material and methods. Thus, 44/78 (56.4%) LSCCs were classified as BCL2L12-positive and 34/78 (43.6%) as BCL2L12-negative (Table 3). Primary tumor site was found to be significant correlated to BCL2L12 protein expression status. LSCCs of high T-status were more frequently BCL2L12-negative than LSCCs of low T-status ( $p=$ 0.013). No significant associations were observed between BCL2L12 protein expression and nodal status ( $p>$ 0.2), TNM stage ( $p=$ 0.188) and histological grade ( $p>$ 0.2) (Table 3).

Table 4
Associations between BCL2 and BAX protein status and categorical clinicopathological variables of 78 LSCC patients

T (primary tumor site)
Variable	Total (%)	Number of patients (%)
		BCL2-high ${}^{\rm a}$	BCL2-low ${}^{\rm a}$	$p$ -value	BAX-high ${}^{\rm a}$	BAX-low ${}^{\rm a}$	$p$ -value
T1	15 (19.2%)	4 (26.7%)	11 (73.3%)	$>$ 0.2 ${}^{\rm b}$	7 (46.7%)	8 (53.3%)	0.036 ${}^{\rm b}$
T2	12 (15.4%)	7 (58.3%)	5 (41.7%)		3 (25.0%)	9 (75.0%)
T3	30 (38.5%)	12 (40.0%)	18 (60.0%)		8 (26.7%)	22 (73.3%)
T4	21 (26.9%)	7 (33.3%)	14 (66.7%)		1 (4.8%)	20 (95.2%)
N (nodal status)
N0	64 (82.1%)	27 (42.2%)	37 (57.8%)	0.127 ${}^{\rm b}$	16 (25.0%)	48 (75.0%)	$>$ 0.2 ${}^{\rm b}$
N1	8 (10.3%)	3 (37.5%)	5 (62.5%)		2 (25.0%)	6 (75.0%)
N2	6 (7.7%)	0 (0.0%)	6 (100.0%)		1 (16.7%)	5 (83.3%)
TNM stage
I/II	26 (33.3%)	11 (42.3%)	15 (57.7%)	$>$ 0.2 ${}^{\rm c}$	10 (38.5%)	16 (61.5%)	0.040 ${}^{\rm c}$
III/IV	52 (66.7%)	19 (36.5%)	33 (63.5%)		9 (17.3%)	43 (82.7%)
Histological stage
Low	12 (15.4%)	3 (25.0%)	9 (75.0%)	0.194 ${}^{\rm b}$	3 (25.0%)	9 (75.0%)	$>$ 0.2 ${}^{\rm c}$
Intermediate	42 (53.8%)	20 (47.6%)	22 (52.4%)		13 (31.0%)	29 (69.0%)
High	24 (30.8%)	7 (29.2%)	17 (70.8%)		3 (12.5%)	21 (87.5%)

${}^{\rm a}$ Quotients similar to Table 3. ${}^{\rm b}$ Calculated by chi-square ( $\chi^{2})$ test. ${}^{\rm c}$ Calculated by Fisher’s exact test.

3.3 BCL2 and BAX protein expression in LSCC tissue specimens and their association with patients’ clinicopathological variants

BCL2 protein expression was categorized as high or low, as described in Material and methods. Thus, 30/78 (38.5%) LSCCs were classified as BCL2-high and 48/78 (61.5%) as BCL2-low (Table 4). No significant associations were observed between BCL2 protein expression and T-status ( $p>$ 0.2), nodal status ( $p=$ 0.127), TNM stage ( $p>$ 0.2) and histological grade ( $p=$ 0.194).

BAX protein expression was also categorized as high or low, as described in Material and methods (2.3). Thus, 19/78 (24.4%) LSCCs were classified as BAX-high and 59/78 (75.6%) as BAX-low (Table 4). T-status and TNM stage were found to be significantly correlated to BAX protein expression status. LSCCs of high T-status and high TNM stage were more frequently BAX-low than LSCCs of low T-status ( $p=$ 0.036) and low TNM stage ( $p=$ 0.040). No significant associations were observed between BAX protein expression and nodal status and histological grade ( $p>$ 0.2).

3.4 The role of BCL2L12 protein as a prognostic factor of LSCC patients’ survival

In order to evaluate BCL2L12 protein expression in terms of predicting survival outcome, we performed Kaplan-Meier survival analysis. In Kaplan-Meier survival curves with regard to DFS time, no significant difference was observed between LSCC patients with BCL2L12-positive tumors and LSCC patients with BCL2L12-negative tumors ( $p>$ 0.2). On the contrary, LSCC patients with BCL2L12-positive tumors had significantly higher OS time in comparison with LSCC patients with BCL2L12-negative tumors ( $p=$ 0.018) (Fig. 1).

Figure 1.

Kaplan-Meier survival curves for overall survival (OS) of LSCC patients with BCL2L12-positive tumors and LSCC patients with BCL2L12-negative tumors. $Y$ -axis: Survival probability (%); $x$ -axis: Overall survival in months; LSCC patients with BCL2L12-positive tumors had significantly higher OS time in comparison with LSCC patients with BCL2L12-negative tumors ( $p=$ 0.018).

Figure 2.

Kaplan-Meier survival curves for overall survival (OS) of advanced-stage LSCC patients with BCL2L12-positive tumors and advanced-stage LSCC patients with BCL2L12-negative tumors. $Y$ -axis: Survival probability (%); $x$ -axis: Overall survival in months; Advanced-stage LSCC patients with BCL2L12-positive tumors had significantly higher OS time in comparison with advanced-stage LSCC patients with BCL2L12-negative tumors ( $p=$ 0.014).

Table 5

BCL2L12 protein expression, BCL2, BAX protein expression and survival of 78 LSCC patients

Variable	Overall survival
	HR ${}^{\rm a}$	95% CI ${}^{\rm b}$	$p$ -value
Univariate analysis
BCL2L12 protein expression of 78 LSCC patients (regardless
TNM stage)
Negative	1.00		0.028
Positive	0.272	0.085–0.869
BCL2L12 protein expression of 26 early-stage LSCC patients
Negative	1.00		$>$ 0.2
Positive	42.189	0.000–920960403.876
BCL2L12 protein expression of 52 advanced-stage LSCC
patients
Negative	1.00		0.025
Positive	0.228	0.063–0.833
BCL2 protein expression of 78 LSCC patients
Negative	1.00		$>$ 0.2
Positive	1.312	0.455–3.784
BAX protein expression of 78 LSCC patients
Negative	1.00		$>$ 0.2
Positive	0.490	0.109–2.195

${}^{\rm a}$ Hazard ratio, estimated from Cox proportional hazard regression model. ${}^{\rm b}$ Confidence interval of the estimated hazard ratio.

We also used Kaplan-Meier survival curves to further evaluate BCL2L12 protein expression in terms of predicting OS in early-stage (TNM stage I/II) and advanced-stage (TNM stage III/IV) LSCC patients. With regard to OS time, no significant difference was observed between early-stage LSCC patients with BCL2L12-positive tumors and early-stage LSCC patients with BCL2L12-negative tumors ( $p>$ 0.2). On the contrary, advanced-stage LSCC patients with BCL2L12-positive tumors had significantly higher OS time in comparison with advanced-stage LSCC patients with BCL2L12-negative tumors ( $p=$ 0.014) (Fig. 2). As expected, early-stage LSCC patients had significantly higher OS time in comparison with ad- vanced-stage LSCC patients ( $p=$ 0.015).

In Cox univariate survival analysis (Table 5), LSCC patients with BCL2L12-positive tumors had significantly lower risk of death compared to LSCC patients with BCL2L12-negative tumors (hazard ratio [HR] $=$ 0.272, 95% confidence interval [95%CI] $=$ 0.085–0.869, $p=$ 0.028). Also, no significant difference was observed between early-stage LSCC patients with BCL2L12-positive tumors and early-stage LSCC patients with BCL2L12-negative tumors. On the contrary, advanced-stage LSCC patients with BCL2L12-positive tumors had significantly lower risk of death compared to advanced-stage LSCC patients with BCL2L12-negative tumors (HR $=$ 0.228, 95%CI $=$ 0.063–0.833, $p=$ 0.025).

3.5 BCL2 and BAX protein expression is not associated with prognosis in patients with LSCC

In order to evaluate BCL2 and BAX protein expression in terms of predicting survival outcomes, we also performed Kaplan-Meier survival analysis. In Kaplan-Meier survival curves with regard to DFS and OS time, no significant difference was observed between LSCC patients with BCL2-high tumors (or BAX-high tumors) and LSCC patients with BCL2-low tumors (or BAX-low tumors) ( $p>$ 0.2).

In Cox univariate survival analysis (Table 5) with regard to survival probabilities, no significant difference was observed between LSCC patients with BCL2-high tumors (or BAX-high tumors) and LSCC patients with BCL2-low tumors (or BAX-low tumors) ( $p>$ 0.2).

4. Discussion

Recently, Geomela et al. observed that BCL2L12 mRNA expression was lower in advanced-stage LSCC in a cohort of patients with HNSCC. The authors suggested that BCL2L12 mRNA expression might represent a potential prognostic biomarker in LSCC [18]. Thus, the authors of the current study performed a review of the literature in Pubmed Database using combinations of the following keywords: ‘BCL2L12’, ‘larynx’, ‘laryngeal’, ‘cancer’, ‘survival’ and ‘prognosis’. Only Geomela’s study [18] matched our search terms. To enhance the knowledge of this subject, we examined the prognostic value of BCL2L12 expression in patients with primary LSCC. To further investigate the prognostic significance of BCL2 and BAX, we also examined the association of BCL2 and BAX protein expression with prognosis in primary LSCC.

The evaluation was performed with immunohistochemical analysis. Immunohistochemistry is an optimal technique to assess protein expression and it is easily and routinely used in clinical practice. The use of a semi-quantitative score for the evaluation has been described in several studies [21, 20]. Alternatively, a quantitative real-time PCR methodology, aiming at the determination of mRNA expression levels, could be used for the same purpose, as recommended by multiple studies [17, 18, 22, 23].

Our data demonstrated that BCL2L12 protein was significantly more frequently expressed in primary LSCC of low T-status than in primary LSCC of high T-status. These data indicated that positive BCL2L12 protein expression in primary LSCC could be correlated with better prognosis. In accordance with the aforementioned results, we observed in the COX univariate regression analysis that positive BCL2L12 protein expression in primary advanced-stage LSCC predicted a lower risk of death. This implied that positive BCL2L12 protein expression could be used as a favorable prognostic tissue biomarker of higher OS in advanced-stage primary LSCC. Accordingly, Kaplan-Meier survival analysis demonstrated that advanced-stage LSCC patients with BCL2L12-positive tumors had significantly higher OS time in comparison with advanced-stage LSCC patients with BCL2L12-negative tumors. As expected, early-stage LSCC patients had significantly higher OS time in comparison with ad- vanced-stage LSCC patients. Interestingly, positive BCL2L12 protein expression did not predict lower risk of recurrence. Similarly, in Kaplan-Meier survival curves, LSCC patients with BCL2L12-positive tumors had not significantly lower DFS-time in comparison with LSCC patients with BCL2L12-negative tumors.

BCL2L12, a novel member of the BCL2 apoptosis-related family, is involved in apoptosis and several malignancies. Data imply that BCL2L12 mRNA expression may be considered as favorable biomarker in gastric [14], colon [15] and breast cancer [24]. Our results suggest that BCL2L12 protein expression could be also used as a favorable prognostic tissue biomarker in patients with primary advanced-stage LSCC. Undoubtedly, further studies are needed to examine the potential application of BCL2L12 protein expression in clinical practice as a favorable prognostic tissue biomarker in primary advanced-stage LSCC and to elucidate the biochemical pathways in which BCL2L12 is involved.

Furthermore, our data demonstrated that high or low BCL2 and BAX protein expression did not predict recurrence or death. Also, high or low BCL2 and BAX protein expression did not significantly correlate with DFS and OS time. These results are in accordance with previously published studies investigating BCL2 [25] and BAX [8] expression in LSCC. The difference between BCL2L12 and BCL2 prognostic role in LSCC could be attributed to the fact that BCL2 is merely antiapoptotic, whereas BCL2L12 has been shown to act as a double-edge sword, either facilitating or impeding apoptosis, depending on the cell type and the expressed BCL2L12 splice variants and protein isoforms [26, 27].

Our study group consisted of 78 LSCC patients. The clinicopathological characteristics of our study group resembled characteristics of study groups described in larger studies with patients with laryngeal cancer [28, 29]. Thus, our smaller study group could be considered representative of laryngeal cancer patients. Additionally, treatment strategies correlated with international standards [30, 31, 32, 33, 34, 35]. Nevertheless, our preference for total laryngectomy in the advanced-stage LSCC could be criticized, since Jenckel et al. implied that aim of every treatment decision should be larynx preservation and preservation of laryngeal function [36]. However, the following studies supported our preference. Richard et al. noticed a significant better 2-year survival in T3-LSCC patients treated with surgery and adjuvant RT in comparison with T3-LSCC patients treated with concurrent CRTH alone ( $p<$ 0.02) [34]. Similarly, in a study including 221 T4-LSCC patients and a 3-decade survey, Rosenthal et al. observed that T4-LSCC patients treated with total laryngectomy and adjuvant RT had significantly better local control in comparison with T4-LSCC patients treated with larynx preservation protocols [35]. Furthermore, the prospective collection of tissue samples and accompanying data also belonged to the strengths of the study. Lastly, the authors used immunohistochemistry, a simple, easily performed technique to minimize the effort and identify potential prognostic significance of BCL2L12 in LSCC. The lack of more accurate techniques like real-time PCR clearly belongs to the limitations of this work and should be applied in the forthcoming studies.

In conclusion, positive protein expression of BCL2 L12 gene, a member of the BCL2-family, predicted significantly higher OS time and lower risk of death in patients with primary advanced-stage LSCC. Our results imply that BCL2L12 protein expression could be used as a favorable prognostic tissue biomarker in patients with primary advanced-stage LSCC. Further studies are needed to examine its use in the clinical practice. On the contrary, BCL2 and BAX protein expression did not correlate with prognosis in primary LSCC. Our data suggest that BCL2 and BAX cannot be used as potential prognostic tissue biomarkers in patients with primary LSCC.

Footnotes

Conflict of interest

The authors declare that they have no competing interests.

References

Siegel

R.L.

Miller

K.D.

and Jemal

, Cancer statistics, 2017, CA Cancer J Clin 67(1) (2017), 7–30. doi: 10.3322/caac.21387.

Kokoska

M.S.

Piccirillo

J.F.

El-Mofty

S.K.

Emami

Haughey

B.H.

and Schoinick

S.B.

, Prognostic significance of clinical factors and p53 expression in patients with glottic carcinoma treated with radiation therapy, Cancer 78(8) (1996), 1693–1700.

Condon

L.T.

Ashman

J.N.

Ell

S.R.

Stafford

N.D.

Greenman

and Cawkwell

, Overexpression of Bcl-2 in squamous cell carcinoma of the larynx: a marker of radioresistance, International Journal of Cancer Journal International Du Cancer 100(4) (2002), 472–475. doi: 10.1002/ijc.10503.

Foteinou

Kontos

C.K.

Giotakis

A.I.

and Scorilas

, Low mRNA expression levels of kallikrein-related peptidase 4 (KLK4) predict short-term relapse in patients with laryngeal squamous cell carcinoma, Biol Chem 395(9) (2014), 1051–1062. doi: 10.1515/hsz-2014-0139.

Kumar

Cordell

K.G.

D’Silva

Prince

M.E.

Adams

M.E.

Fisher

S.G.

et al., Expression of p53 and Bcl-xL as predictive markers for larynx preservation in advanced laryngeal cancer, Archives of Otolaryngology – Head & Neck Surgery 134(4) (2008), 363–369. doi: 10.1001/archotol.134.4.363.

Zhang

Liu

Sun

et al., KAI1/CD82 and cyclin D1 as biomarkers of invasion, metastasis and prognosis of laryngeal squamous cell carcinoma, Int J Clin Exp Pathol 6(6) (2013), 1060–1067.

Jackel

M.C.

Sellmann

Youssef

Dorudian

M.A.

and Fuzesi

, Prognostic significance of expression of p53, bcl-2 and bax in squamous epithelial carcinoma of the larynx–a multivariate analysis, HNO 49(3) (2001), 204–211.

Boonyaphiphat

Pruegsanusak

and Thongsuksai

, The prognostic value of p53, Bcl-2 and Bax expression in laryngeal cancer, J Med Assoc Thai 95(10) (2012), 1317–1320.

Georgiou

Gomatos

I.P.

Ferekidis

Syrigos

Bistola

Giotakis

et al., Prognostic significance of p53, bax and bcl-2 gene expression in patients with laryngeal carcinoma, European Journal of Surgical Oncology: The Journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 27(6) (2001), 574–580. doi: 10.1053/ejso.2001.1131.

10.

Gurlek

Abaka

C.D.

Ozkan

Saraydaroglu

Kurt

and Cetintas

S.K.

, The evaluation of bcl-2 expression as a prognostic marker in early stage laryngeal cancer, Tumori 99(6) (2013), 682–688. doi: 10.1700/1390.15456.

11.

Spafford

M.F.

Koeppe

Pan

Archer

P.G.

Meyers

A.D.

and Franklin

W.A.

, Correlation of tumor markers p53, bcl-2, CD34, CD44H, CD44v6, and Ki-67 with survival and metastasis in laryngeal squamous cell carcinoma, Archives of Otolaryngology – Head & Neck Surgery 122(6) (1996), 627–632.

12.

Nix

Cawkwell

Patmore

Greenman

and Stafford

, Bcl-2 expression predicts radiotherapy failure in laryngeal cancer, British Journal of Cancer 92(12) (2005), 2185–2189. doi: 10.1038/sj.bjc.6602647.

13.

Scorilas

Kyriakopoulou

Yousef

G.M.

Ashworth

L.K.

Kwamie

and Diamandis

E.P.

, Molecular cloning, physical mapping, and expression analysis of a novel gene, BCL2L12, encoding a proline-rich protein with a highly conserved BH2 domain of the Bcl-2 family, Genomics 72(2) (2001), 217–221. doi: 10.1006/geno.2000.6455S0888-7543(00)96455-3 [pii].

14.

Florou

Papadopoulos

I.N.

and Scorilas

, Molecular analysis and prognostic impact of the novel apoptotic gene BCL2L12 in gastric cancer, Biochem Biophys Res Commun 391(1) (2010), 214–218. doi: S0006-291X(09)02194-9 [pii] 10.1016/j.bbrc.2009.11.034.

15.

Kontos

C.K.

Papadopoulos

I.N.

and Scorilas

, Quantitative expression analysis and prognostic significance of the novel apoptosis-related gene BCL2L12 in colon cancer, Biol Chem 389(12) (2008), 1467–1475. doi: 10.1515/BC.2008.17310.1515/BC.2008.173 [pii].

16.

Talieri

Diamandis

E.P.

Katsaros

Gourgiotis

and Scorilas

, Expression of BCL2L12, a new member of apoptosis-related genes, in breast tumors, Thromb Haemost 89(6) (2003), 1081–1088. doi: 10.1267/THRO0306108103061081[pii].

17.

Fendri

Kontos

C.K.

Khabir

Mokdad-Gargouri

and Scorilas

, BCL2L12 is a novel biomarker for the prediction of short-term relapse in nasopharyngeal carcinoma, Mol Med 17(3–4) (2011), 163–171. doi: 10.2119/molmed.2010.00056.

18.

Geomela

P.A.

Kontos

C.K.

Yiotakis

and Scorilas

, Quantitative expression analysis of the apoptosis-related gene, BCL2L12, in head and neck squamous cell carcinoma, Journal of Oral Pathology & Medicine: Official Publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 42(2) (2013), 154–161. doi: 10.1111/j.1600-0714.2012.01190.x.

19.

Giotakis

A.I.

Kontos

C.K.

Manolopoulos

L.D.

Sismanis

Konstadoulakis

M.M.

and Scorilas

, High BAX/BCL2 mRNA ratio predicts favorable prognosis in laryngeal squamous cell carcinoma, particularly in patients with negative lymph nodes at the time of diagnosis, Clin Biochem 49(12) (2016), 890–896. doi: 10.1016/j.clinbiochem.2016.04.010.

20.

Micozkadioglu

Unal

Pata

Y.S.

Basturk

and Cinel

, Prognostic value of expression of p53, proliferating cell nuclear antigen, and c-erbB-2 in laryngeal carcinoma, Medical Science Monitor: International Medical Journal of Experimental and Clinical Research 14(6) (2008), Cr299–304.

21.

Jackel

M.C.

Sellmann

Dorudian

M.A.

Youssef

and Fuzesi

, Prognostic significance of p53/bcl-2 co-expression in patients with laryngeal squamous cell carcinoma, The Laryngoscope 110(8) (2000), 1339–1345. doi: 10.1097/00005537-200008000-00022.

22.

Friedman

Grey

Venkatesan

T.K.

Bloch

Chawla

Caldarelli

D.D.

et al., Prognostic significance of Bcl-2 expression in localized squamous cell carcinoma of the head and neck, Ann Otol Rhinol Laryngol 106(6) (1997), 445–450.

23.

Gallo

Bianchi

and Porfirio

, Bcl-2 overexpression and smoking history in head and neck cancer, J Natl Cancer Inst 87(13) (1995), 1024–1025.

24.

Thomadaki

Talieri

and Scorilas

, Prognostic value of the apoptosis related genes BCL2 and BCL2L12 in breast cancer, Cancer Lett 247(1) (2007), 48–55. doi: S0304-3835(06)00174-1[pii] 10.1016/j.canlet.2006.03.016.

25.

Gioacchini

F.M.

Alicandri-Ciufelli

Rubini

Magliulo

and Re

, Prognostic value of Bcl-2 expression in squamous cell carcinoma of the larynx: A systematic review, Int J Biol Markers 30(2) (2015), e155–160. doi: 10.5301/jbm.5000116.

26.

Adamopoulos

P.G.

Kontos

C.K.

Tsiakanikas

and Scorilas

, Identification of novel alternative splice variants of the BCL2L12 gene in human cancer cells using next-generation sequencing methodology, Cancer Lett 373(1) (2016), 119–129. doi: 10.1016/j.canlet.2016.01.019.

27.

Kontos

C.K.

and Scorilas

, Molecular cloning of novel alternatively spliced variants of BCL2L12, a new member of the BCL2 gene family, and their expression analysis in cancer cells, Gene 505(1) (2012), 153–166. doi: 10.1016/j.gene.2012.04.084.

28.

Dietz

, Epidemiology of laryngeal cancer, Laryngorhinootologie 83(11) (2004), 771–772. doi: 10.1055/s-2004-826025.

29.

MacNeil

S.D.

Liu

Shariff

S.Z.

Thind

Winquist

Yoo

et al., Secular trends in the survival of patients with laryngeal carcinoma, 1995–2007, Curr Oncol 22(2) (2015), e85–99. doi: 10.3747/co.22.2361.

30.

Johnson

J.T.

, Carcinoma of the larynx: Selective approach to the management of cervical lymphatics, Ear Nose Throat J 73(5) (1994), 303–305.

31.

Rodrigo

J.P.

Cabanillas

Franco

and Suarez

, Efficacy of routine bilateral neck dissection in the management of the N0 neck in T1-T2 unilateral supraglottic cancer, Head Neck 28(6) (2006), 534–539. doi: 10.1002/hed.20359.

32.

Silver

C.E.

Beitler

J.J.

Shaha

A.R.

Rinaldo

and Fesrlito

, Current trends in initial management of laryngeal cancer: the declining use of open surgery, Eur Arch Otorhinolaryngol 266(9) (2009), 1333–1352. doi: 10.1007/s00405-009-1028-2.

33.

Wiegand

, Evidence and evidence gaps of laryngeal cancer surgery, GMS Curr Top Otorhinolaryngol Head Neck Surg 15 (2016), Doc03. doi: 10.3205/cto000130.

34.

Richard

J.M.

Sancho-Garnier

Pessey

J.J.

Luboinski

Lefebvre

J.L.

Dehesdin

et al., Randomized trial of induction chemotherapy in larynx carcinoma, Oral Oncol 34(3) (1998), 224–228.

35.

Rosenthal

D.I.

Mohamed

A.S.

Weber

R.S.

Garden

A.S.

Sevak

P.R.

Kies

M.S.

et al., Long-term outcomes after surgical or nonsurgical initial therapy for patients with T4 squamous cell carcinoma of the larynx: A 3-decade survey, Cancer 121(10) (2015), 1608–1619. doi: 10.1002/cncr.29241.

36.

Jenckel

and Knecht

, State of the art in the treatment of laryngeal cancer, Anticancer Res 33(11) (2013), 4701–4710.

Positive BCL2L12 expression predicts favorable prognosis in patients with laryngeal squamous cell carcinoma

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Methods

2.1 Collection of tissue specimens

2.2 Immunohistochemistry

2.3 Immunohistochemical evaluation

2.4 Statistical analysis

Table 1 Clinicopathological characteristics of 78 LSCC patients

3.1 Clinicopathological characteristics of the study cohort

3.2 BCL2L12 protein expression in LSCC tissue specimens and its association with patients’ clinicopathological variants

Table 4 Associations between BCL2 and BAX protein status and categorical clinicopathological variables of 78 LSCC patients

3.4 The role of BCL2L12 protein as a prognostic factor of LSCC patients’ survival

4. Discussion

Footnotes

Conflict of interest

References

Table 1
Clinicopathological characteristics of 78 LSCC patients

Table 4
Associations between BCL2 and BAX protein status and categorical clinicopathological variables of 78 LSCC patients