Abstract
The aim of this systematic review was to determine the prognostic value of Bcl-2 immunostaining in patients affected by laryngeal squamous cell carcinoma. An appropriate search was conducted on PubMed to retrieve articles dealing with this topic. A double cross-check was performed on citations and full-text articles by 2 investigators independently to review all manuscripts and perform a comprehensive quality assessment. Of 115 abstracts identified, 15 articles were included. These studies reported on 1,150 patients with histologically confirmed diagnosis of laryngeal squamous cell carcinoma. Only a few studies showed a statistical correlation between Bcl-2 immunohistochemical expression and at least 1 of the clinical and histopathological parameters considered by the authors. Moreover, these findings were also discordant between them. Overall the studies analyzed suggested that Bcl-2 expression was statistically connected with N stage (2/14), grading (2/14), disease-free survival (3/14) and overall survival (5/14). Interestingly, all of the 3 studies investigating the relation between Bcl-2 and radioresistance showed significant results in terms of recurrence-free survival and overall survival. Our review strongly suggests that the immunohistochemical staining of Bcl-2 does not correlate with tumoral aggressiveness and prognosis of patients affected by laryngeal squamous cell carcinoma and treated with primary surgery. However, an interesting connection of this protein could be demonstrated with tumoral radioresistance. Further, high-quality prospective studies should be carried out to confirm this hypothesis.
Introduction
Laryngeal squamous cell carcinoma (SCC) represents the second most common malignant neoplasm of the respiratory tract after lung cancer (1), and despite refinement of multimodal therapies over the last 20 years, 5-year survival figures remain poor (2).
The prognosis of laryngeal SCCs is defined on the basis of clinical stage, site and size of the tumor, histological grading, depth of invasion, positive surgical margins and host reaction, although, in absolute terms, the most significant prognostic indicator of survival is the presence or absence of metastatic cervical nodes (3).
For this reason, clinical staging should be supplemented by other factors which would increase our knowledge of the biologic behavior of these tumors, and biological markers capable of distinguishing patients with a good prognosis from those with a worse prognosis are thus required.
Apoptosis, or programmed cell death, is the mechanism by which fetal development and normal tissue homeostasis in adults are maintained. Dysregulation of the genes controlling apoptosis may contribute to the process of tumorigenesis by reducing the rate of cell death and allowing the accumulation of other genetic defects (4). Moreover, neoplastic cells exhibit disturbances of the process of programmed cell death and are likely to be less responsive to chemotherapeutic agents and ionizing irradiation. Identification of the gene alterations regulating apoptosis might therefore make it possible to establish a powerful therapeutic approach in patients with cancer (5).
Bcl-2 is an intracellular membrane protein capable of blocking the programmed cell death induced by several stimuli (6). At the present time the clinical relevance of Bcl-2 expression in SCC of the head and neck region is not fully understood. The aim of our review was to assess the prognostic role of the antiapoptotic protein Bcl-2 in patients affected by SCC of the larynx.
Materials and Methods
PRISMA 2009 guidelines were considered and applied whenever possible in this systematic review. In February 2014, a computerized Medline search was performed using the PubMed service of the US National Library of Medicine and the following search string was run:
(“Genes, bcl-2”[Mesh] OR “Proto-Oncogene Proteins c-bcl-2”[Mesh]) AND “Laryngeal Neoplasms”[Mesh]
The initial search returned a total of 115 results. Abstracts and titles obtained were screened independently by 2 of the authors (F.M.G. and M.R.), who subsequently met to discuss disagreements on citation inclusion.
Inclusion criteria for citations were
Cohorts of patients with histologically confirmed diagnosis of larynx SCC;
English language.
Exclusion criteria for citations were
Analysis including samples of patients with histologically confirmed diagnosis of larynx basaloid SCC;
Analysis including samples of patients affected by SCC of others organs than larynx;
Articles concerning different markers from Bcl-2.
Between the 115 articles, 16 met the initial inclusion criteria according to both authors (F.M.G. and M.R.), so they were obtained and reviewed in detail by the same 2 authors, who met and discussed disagreements on article inclusion. Inclusion criteria for full text articles and single patients identified were
Sufficient and accurate description of immunohistochemical staining;
Sufficient and accurate description of statistical analysis.
Exclusion criteria were
Analysis performed on specimens of patients with history of previous head and neck radiotherapy;
Inclusion of patients with history of previous head and neck cancers.
A total of 6 studies were excluded because of insufficient data about immunohistochemical staining or statistical analysis (4 studies), while 2 studies were ruled out because some patients had a previous history of other head and neck cancer. A further manual check was performed on the references included in the articles, and 4 additional studies were identified that met the inclusion criteria. The final number of articles included in the present review was identified, and the main information was extracted and summarized.
Results
After an initial check, full-text retrieval and manual cross-checking of references included in the articles, 14 studies, comprising a total of 1,150 subjects, clearly met the inclusion criteria and were chosen for analysis (Fig. 1). The main characteristics of these selected studies are included in Tables I and II.
Flow chart of inclusion or exclusion criteria for articles included in the review. SCC = squamous cell carcinoma.
Study design and main characteristics of included patients
R = retrospective; P = prospective; S = surgery; S-R = surgery and adjuvant radiotherapy; R = radiotherapy; n/e = not evaluated; n/a = not available.
20 tumors classified as piriform sinus carcinomas.
Bcl-2 thresholds of positivity and correlations between clinical-histopathological parameters
IHC = immunohistochemistry; DFS = disease-free survival; OS = overall survival; YES = significant correlation; NO = no significant correlation; n/e = not evaluated.
All of the studies had been performed with a retrospective cohort design. The average length of follow-up reported in 9 studies was 55.1 months, ranging from 40 to 72 months. Overall, the number of patients in each study included in this analysis varied from 21 to 172. The vast majority of patients (n = 502) had a glottic cancer, while supraglottic localization was described in 358 subjects. A transglottic tumor was described in 66 cases, and only 26 patients presented with a subglottic localization. Concerning the tumoral stage, 193 subjects were reported as pT1, 240 as pT2, 136 as pT3 and 67 as pT4. Overall 576 patients were N0, while 169 presented positive nodes.
On the basis of our review, among the articles analyzed, 7 reported at least 1 significant correlation between Bcl-2 expression and the clinical or histopathological parameters evaluated. A statistical correlation was found for N stage (2/14), grading (2/14), disease-free survival (3/14) and overall survival (5/14). Seven studies totally failed to find any statistical correlations.
Discussion
Although the classic prognostic factors continue to be of great utility in predicting the clinical behavior of a laryngeal SCC, it is still not clear why some patients affected by this tumor do better than others with the same type of lesions in terms of histological characteristics and clinical stage (20).
Identification of a malignancy's aggressive biological behavior and/or the tendency to recur or metastasize is of paramount importance to the physician where choice of treatment modality is concerned. Thus many tumor markers, cellular oncogenes and proliferation markers are currently under extensive study as potential prognostic indicators of larynx SCC (18).
The Bcl-2 family of proteins is a critical death regulator of mitochondrial integrity and of mitochondria-initiated apoptosis. The family possesses both antiapoptotic and proapoptotic members. Apoptosis-inhibiting proteins include Bcl-2 and Bcl-xL, while Bax, Bid, Bak and Bcl-xs are apoptosis-promoting proteins (21).
The antiapoptotic members of the Bcl-2 family are also attractive tumor-associated antigens for the purposes of targeted therapy, and orally bioavailable inhibitors of the Bcl-2 family capable of specifically inhibiting protein–protein interactions between BH3 and Bcl-2 at low nanomolar concentrations have the potential to mark a significant development in cancer therapy (22).
The Bcl-2 gene is an antiapoptotic-membrane-associated molecule that resides in the nuclear envelope and mitochondria. It exerts its antiapoptotic functions by modulating the mitochondrial release of cytochrome c and the interaction of apoptosis activating factors (Apaf-1) with caspase 9, Bax and c-Myc (23).
Several different strategies have been used in an attempt to overcome the cytoprotective effects of Bcl-2 in cancer, including shutting off gene transcription, inducing mRNA degradation with antisense oligonucleotides and attacking Bcl-2 directly with small-molecule drugs (24).
Numerous studies have demonstrated Bcl-2 overexpression in solid tumors, including melanoma, breast, colorectal, prostate and small cell lung cancer, as well as cancers of hematological origin (24), and it was shown that overexpression of Bcl-2 is related to poor outcomes in early-stage head and neck cancers (8). Klatka et al found that the expression of Bcl-2 protein in T lymphocytes from patients with laryngeal SCC was significantly higher than in controls (25).
Concerning laryngeal cancer, Hirvikoski et al (16) in their cohort of 172 patients with laryngeal cancer could not find any significant relation between Bcl-2 immunohistochemical expression and the clinical parameters considered. Likewise, Whisler et al (15) in a cohort comprising 40 patients did not find any significant correlation between the Bcl-2 immunohistochemical index and the patients’ prognosis. Ozdek et al (18) evaluated the expression of Bcl-2 in a cohort study of 61 patients who underwent surgery for SCC of the supraglottic larynx. No correlations were observed between Bcl-2 expression and the patients’ clinical and histopathological features. Yildirim et al (17) analyzed Bcl-2 immunoexpression on 84 subjects treated for laryngeal SCC at different pathological stages and locations. Transglottic tumors expressed significantly high levels of Bcl-2, but no connection was found with postoperative survival. In their cohort of 50 patients affected by laryngeal SCC, Klatka et al (19) did not find any significant correlations between Bcl-2 expression and patients’ T- or N-stage, histological grading or overall survival. Krecicki et al (26) analyzed the immunostaining of Bcl-2 in a total of 57 biopsies from patients with dysplastic lesion of the larynx. The immunohistochemical results did not correlate with the clinical course of dysplastic lesions of the larynx. Izawa et al (7) analyzing 67 cases of laryngeal SCC did not find any significant difference in the percentage of Bcl-2-positive cells among groups in terms of differentiation or stage. Friedman et al (8) investigated the role of Bcl-2 in predicting outcomes for advanced SCC of the larynx. They analyzed 69 patients with stage III or IV tumors, all but 6 of whom were treated with surgery plus postoperative irradiation. Expression of Bcl-2 was not associated with outcome, overall survival or disease-free survival. The authors concluded that assessing expression of Bcl-2 is unlikely to be prognostically useful for surgically treated advanced laryngeal SCC.
In certain types of cancer such as thyroid (27), breast (28) and colon cancers (29), Bcl-2 protein expression has been associated with better tumor differentiation, and with a favorable clinical outcome. Similarly, Redondo et al (9) in their study of 154 patients with laryngeal SCC reported that normal squamous epithelium was negative on staining for Bcl-2. On the other hand, Bcl-2 was up-regulated in SCC, and 25% of patients (39 of 154) showed positive Bcl-2 staining. Moreover, a higher incidence of Bcl-2 expression was associated with nodal metastasis (p = 0.01) and with supraglottic tumors (p = 0.02). Nevertheless, on multivariate analysis, Bcl-2 was an independent predictor of good prognosis. For these reasons, the authors pointed to the usefulness of this marker in distinguishing which tumors with pathologically aggressive characteristics might present a better outcome.
Also, Georgiou et al (10), analyzing the immunohistochemical expression of Bcl-2 in the tissue specimens of 38 patients with laryngeal SCC, noted that an increased level of Bcl-2 protein expression correlated with a better 5-year survival (p = 0.04). However, these findings are not in line with previous reports on carcinomas of the urinary bladder (30), prostate (31), gallbladder (32) and head and neck (33, 34), which suggested that the overexpression of Bcl-2 is linked to high histological grade, tumor progression and poor prognosis. These studies suggested that Bcl-2-positive tumors present with enhanced disease progression and are associated with an unfavorable clinical outcome.
Regarding laryngeal SCC, 2 studies appeared in accordance with those mentioned above. In fact Pruneri et al (5), in a homogeneous series of 149 patients with laryngeal SCC, reported that Bcl-2 expression was significantly related to tumor aggressiveness, and was also associated with the presence of lymph-node metastases (p = 0.001), advanced clinical stage (p = 0.001) and poor histological differentiation (p = 0.003). However, no significant association was found with overall survival. Furthermore, Jäckel et al (14), in their cohort of 88 patients with laryngeal SCC, reported that the coexpression of p53/bcl-2 was an independent predictor of patients’ outcomes and had a prognostic value superior to both parameters considered separately. In their univariate analysis, age at diagnosis, advanced clinical stage, high mitotic activity, high level of p53 positivity and positive Bcl-2 expression were significantly associated with shortened overall survival.
Regarding the relation between Bcl-2 and radioresistance, a reduction in apoptotic thymic cell death has been demonstrated in transgenic mice overexpressing this protein (35). Moreover Gallo et al (36) found a significant correlation between Bcl-2 overexpression and radioresistance in a cohort of 71 patients with head and neck SCCs. Therefore Bcl-2-related radioresistance has been hypothesized also in laryngeal SCC and investigated in series of patients who received radiotherapy. Condon et al (11) compared Bcl-2 immunohistochemical expression in preradiotherapy biopsies of radioresistant (8 cases) and radiosensitive (13 cases) laryngeal SCCs. A significant correlation between Bcl-2 overexpression and radioresistance was found (p = 0.003) suggesting that a Bcl-2 high index enabled carcinoma cells with radiotherapy-induced DNA damage to continue proliferating. Nix et al (12) analyzed the Bcl-2 expression in the biopsies of 124 patients (62 radioresistant and 62 radiosensitive) who had curative radiotherapy alone for early stage (T1-T2, N0) laryngeal SCC. In total, 53% of radioresistant tumors expressed Bcl-2 compared with 11% in the radiosensitive group (p = 0.001). In their cohort of 33 patients who underwent primary surgery followed by radiotherapy, Marioni et al (13) also investigated the relationship between Bcl-2 and response to treatment. The authors observed that disease-free survival was shorter in those cases with Bcl-2 expression >2.0% than in those with Bcl-2 expression <2.0% (p = 0.03).
On the basis of the data collected in this review, it can be noted that overall, no certainty can be obtained about the role of Bcl-2 in laryngeal SCC, and many discordant results are present. Nevertheless, in our opinion the lack of any concordance between the results presented in these 14 articles may be explained by the wide discordance between the different studies’ designs. In fact, firstly it should be noted that there was no accordance in the choice of a standard cutoff level to define as positive or negative the Bcl-2 immunoexpression. Secondly, observing the cohorts of subjects grouped in this review, it is evident that a wide discordance exists regarding the rate of tumoral stages (T and N) between different articles. Finally, the differences regarding the subsites involved, the treatment modalities and follow-up time contribute to increase the inconsistencies between the various studies.
All of the issues mentioned above represent important confounding elements for every analysis focusing on this type of cancer, most of all when clinical parameters such as recurrence-free survival and overall survival are taken into account. For these reasons, it would be totally unworkable to try to perform a statistical analysis of the data shown in the different articles analyzed.
The only indication of a hypothesis from our review, as demonstrated by the 3 studies dealing with the relations between Bcl-2 expression and radioresistance, is that Bcl-2 overexpression could represent a marker of radiotherapy's failure in patients affected by larynx SCC. However, others studies are needed to confirm this observation.
Conclusion
In conclusion, on the basis of the data reported in this review, it can be affirmed that there are still no certainties about the utility of Bcl-2 as a prognostic marker for subjects affected by laryngeal malignancies and treated with primary surgery. However, due to the wide discordance in the various studies’ design, our analysis can not categorically exclude the existence of a possible role played by this protein in the prognosis of laryngeal SCC.
On the other hand, although supported only by a small number of studies, the hypothesis that Bcl-2 could have a role in predicting tumoral radioresistance appears interesting and deserves further investigation.
Footnotes
Financial support: No grants or funding have been received for this study.
Conflict of interest: None of the authors has any financial interest related to this study to disclose.