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Maspin expression and anti-apoptotic pathway regulation by bcl2 in laryngeal cancer
Annals of Diagnostic Pathology 45 (2020) 151471
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Annals of Diagnostic Pathology journal homepage: www.elsevier.com/locate/anndiagpath
Original Contribution
Maspin expression and anti-apoptotic pathway regulation by bcl2 in laryngeal cancer
T
Andrea Lovatoa, Leonardo Franzb, Valentina Carraroc, Luigia Bandolinb, Giacomo Controb, Giancarlo Ottavianob, Cosimo de Filippisa, Stella Blandamurac, Lara Alessandrinic, ⁎ Gino Marionib, a
Department of Neuroscience DNS, Audiology Unit, University of Padova, Treviso, Italy Department of Neuroscience DNS, Otolaryngology Section, University of Padova, Padova, Italy c Department of Medicine DIMED, University of Padova, Padova, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Laryngeal squamous cell carcinoma Maspin pattern Bcl-2 Apoptosis Prognosis
Objectives: Comprehension of the interplay of pro-apoptotic and anti-apoptotic stimuli in laryngeal squamous cell carcinoma (LSCC) is crucial to understand tumor development, biological behavior and treatment response. Bcl-2 family proteins mainly regulate the apoptotic signal cascade. In some cancers, maspin seems to influence the balance between pro-apoptosis and anti-apoptosis bcl-2 family proteins. The aim of this study was to investigate the potential relationship between bcl-2 anti-apoptotic factor and the tumor suppressor maspin in LSCC. Materials and methods: 31 consecutive patients who underwent primary surgery and post-operative radiotherapy for LSCC were evaluated retrospectively. For each case, immunohistochemistry assays for bcl-2 and maspin were performed. Data were also collected on N-status, pT stage, grading, recurrence and disease-free survival (DFS). Results: Patients with nuclear maspin pattern of expression showed a significantly lower recurrence rate (p = 0.04) and longer DFS (p = 0.0018). The expression of bcl-2 was not associated with recurrence rate or DFS either in the whole cohort or in cases with nuclear maspin pattern, while in patients with non-nuclear maspin pattern, a statistical trend was found toward a shorter DFS for bcl-2 positive cases (p = 0.062). In the multivariate model, only maspin expression pattern retained its independent prognostic significance (p = 0.006). Conclusions: Nuclear maspin pattern seemed to be an independent positive prognostic factor, while bcl-2 prognostic value was related to maspin expression pattern. Further investigations are needed to support the use of bcl-2 inhibitors in multimodality or multitarget strategies against advanced LSCCs, also considering the role and expression of tumor suppressor genes.
1. Introduction Laryngeal squamous cell carcinoma (LSCC) represents the 12th most common human malignancy in the adult age, according to the Surveillance Epidemiology and End Results (SEER) program of the United States [1]. Prognosis in early LSCC was generally considered quite good [2]. On the contrary, in advanced LSCC survival rates have not improved despite novelties in the therapeutic techniques (including CO2 laser-assisted transoral surgery, open reconstructive techniques, radiotherapy and chemotherapy) [3]. LSCCs have a 5-year survival rate of 80% to 90% for stage I/II disease, but up to 50% of advanced patients with LSCC experience recurrence following frontline therapy [4]. Consequently, in recent years oncological research on LSCC has focused
⁎
on tumor biology in order to identify prognostic markers and select potential treatment targets [5]. A deeper knowledge of the roles and relationships between oncogenes and tumor suppressor genes is crucial to understand development, biological behavior and treatment response [6]. Accordingly, the comprehension of balance regulation between pro-apoptotic and anti-apoptotic stimuli is important [7]. The activation of apoptotic signal cascade depends mainly on the Bcell lymphoma-2 (bcl-2) family of mitochondrial proteins. Bcl-2 is one of the most important anti-apoptotic factors. It acts as an oncogene and was found overexpressed in solid tumors, including breast, colorectal, prostate and small cell lung cancer, as well as in malignancies of hematological origin [8]. In LSCC, bcl-2 expression was preliminarily associated with a negative prognosis [9].
Corresponding author at: Department of Neuroscience DNS, Otolaryngology Section, University of Padova, Padova, Italy. E-mail address: [email protected] (G. Marioni).
https://doi.org/10.1016/j.anndiagpath.2020.151471
1092-9134/ © 2020 Published by Elsevier Inc.
Annals of Diagnostic Pathology 45 (2020) 151471
A. Lovato, et al.
scheduled as follows: (i) once a month for the 1st year; (ii) every 2 months in the 2nd year; (iii) every 3 months in the 3rd year; (iv) every 4 months in the 4th year; (v) every 6 months in the 5th year; and (vi) every 12 months thereafter. Patients underwent neck ultrasonography and chest X-ray at least yearly. Contrast-enhanced CT of the neck or total body positron emission tomography (PET-CT) were performed when necessary. Mean follow-up time was 58.3 ± 45.7 months (median, 43 months).
Maspin (mammary serine protease inhibitor) is a tumor suppressor gene that plays a role in various tumor related processes such as the inhibition of cell migration, cell invasion, angiogenesis, as well as improvement in cell adhesion and induction of programmed cell death [10]. Maspin nuclear localization in cancer cells has been reported as necessary for its tumor suppressor activity [11]. As a tumor suppressor, maspin interacts with the cellular signaling pathways of different oncogenes. Maspin overexpression was found modulating tumor cell apoptosis through the regulation of bcl-2 family proteins in mouse mammary tumor TM40D cells [12]. Also in prostate cancer, maspin seemed to influence the balance between apoptosis regulation proteins [13]. In LSCC, maspin nuclear expression was associated with the expression of M30, a protein expressed by epithelial cells during early apoptosis [14]. However, there is no evidence regarding a possible association between maspin expression and apoptosis control by bcl-2 family members in LSCC. The main aim of this exploratory study was to conduct a clinicopathological investigation into the potential relationship of bcl-2 anti-apoptotic factor and the tumor suppressor maspin in LSCC. In addition, the prognostic value of bcl-2 was evaluated in LSCC sub-cohorts characterized by different patterns of maspin expression.
2.2. Radiotherapy All 31 patients received external beam radiation therapy postoperatively using the same 6 MV photon beam delivered by a linear accelerator, at the Radiotherapy and Nuclear Medicine Unit, Istituto Oncologico Veneto, Padova. Patients were first immobilized with a thermoplastic mask, and CT images were acquired in the treatment position to allow for 3D treatment planning. The method used to administer the PORT depended on the target volume. In most cases, the dose was delivered using 2 parallel opposite fields up to 40 Gy, then the fields were shielded to cover the spine and matched with electron beams (8–10 MeV) on the spinal chains. In selected cases, the dose was delivered with multiple beams to cover the whole planned target volume. Conventional fractionation was used, i.e. 1.8–2 Gy/fraction daily for a total dose ranging from 50 to 70 Gy.
2. Materials and methods 2.1. Patients
2.3. Immunohistochemistry
The study was conducted in accordance with the principles of the Helsinki Declaration. Data were examined in agreement with the Italian privacy and sensible data laws and the Otolaryngology Section's University of Padova internal rules. Before undergoing surgery, all patients included in the study signed a detailed informed consent form. The investigation involved 31 consecutive patients (25 males, 6 females; mean age 67.5 ± 9.8 years) who underwent primary surgery followed by post-operative RT (PORT) for LSCC according to the currently-accepted indications for post-operative radiotherapy [3]. The laryngeal surgery was performed by the same surgical team for all the patients with unilateral or bilateral neck lymph node dissection in 29 cases. Before the surgery, all patients were investigated with microlaryngoscopy with laryngeal biopsies, upper aerodigestive tract endoscopy, neck ultrasonography (with or without fine needle aspiration cytology), head and neck contrast-enhanced computed tomography (CT) and/or magnetic resonance imaging (MRI), chest X-ray and liver ultrasonography. According to the 2017 TNM classification (8th edition) [15], the pathological stage of the primary lesion (pT) was T1 in 1 case, T2 in 10, T3 in 11, T4 in 9. For the N stage (pN), 14 patients were pN0, 14 were pN2 (3 pN2a, 6 pN2b, 5 pN2c), 1 was pN3. No distant metastasis (M) were found at diagnosis. Considering stage grouping, one of the 31 LSCC patients (3.2%) was stage I, 7 (22.6%) stage II, 4 (12.9%) stage III and 19 (61,3%) stage IV. As for the pathological grading, 3 patients were G1, 18 patients G2 and 10 G3 (Table 1). As previously reported [16,17], in our institution the clinical followup after treatment (adjusted to patients' individual characteristics) was
All tissues were fixed in 4% para-formaldehyde, embedded in paraffin and then stained with a fully automated system (Bond-maX; Vision BioSystems, UK). From each of the 31 paraffin-embedded blocks, 4 μm thick sections were cut. The sections were then deparaffinised in Bond Dewax Solution (Vision BioSystems, UK) at 72 °C, rinsed in ethanol and rehydrated in distilled water. Antigens were retrieved by heating sections for 30 min at 99 °C in Bond Epitope Retrieval Solution 1 (Vision BioSystems, UK). Endogenous peroxidase was blocked with 3% hydrogen peroxide before 30 min of incubation with anti-human maspin (mouse monoclonal antibody, clone EAW24, Novocastra Laboratories, Newcastle upon Tyne, UK; diluted 1:100) and anti-human bcl-2 (mouse monoclonal antibody, clone 124, Dako, Glostrup, Denmark; diluted 1:200). Specimens were then washed with phosphate-buffered saline (pH 7.0) and incubated with a Bond Polymer Refine Detection Kit (Vision BioSystems, UK) according to the manufacturer's protocols. The sections were then dehydrated, cleared, mounted and counterstained with Meyer's hematoxylin. Normal breast tissue was used as a positive maspin control, while samples from formalin-fixed, paraffin-embedded human tonsil were used as a positive bcl-2 control. Serum without the primary antibody was used as a negative control. 2.3.1. Maspin subcellular localization and bcl-2 expression The pathologists interpreting the sections were unaware of the patients' clinical outcomes. For each case, 40 non-overlapping fields of the less-differentiated areas of SCC, with no evidence of necrosis or
Table 1 Maspin expression pattern and bcl-2 expression in relation to classical clinicopathological and prognostic variables in LSCC: whole series. Clinicopathological variables
No. of cases
Maspin pattern of expression ( nuclear/non-nuclear)
Mean bcl-2 expression % ± SD
Mean disease-free survival in months ± SD
Recurrence rate (%)
pT1 - pT2 pT3 - pT4 G1 - G2 G3 pN+ N0 (cN0 + pN0) Without cancer recurrence With cancer recurrence
11 20 21 10 15 16 14 17
3/8 6/14 7/14 2/8 2/13 7/9 7/7 2/15
9.6 8.4 8.7 9.0 8.7 8.7 9.0 8.7
35.5 43.0 44.3 39.3 41.7 44.3 44.7 41.7
7 (63.6%) 10 (50.0%) 11 (52.4%) 6 (60.0%) 10 (66.7%) 7 (43.8%)
± ± ± ± ± ± ± ±
16.0 15.4 15.6 15.8 15.6 15.6 15.8 15.6
2
± ± ± ± ± ± ± ±
45.1 46.7 47.0 45.8 47.0 46.9 47.7 47.0
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A. Lovato, et al.
(p = 0.68), or grading (p = 0.68). The recurrence rate was significantly lower among LSCC patients with a nuclear vs non-nuclear maspin pattern of expression (Fisher's exact test, p = 0.04). DFS was significantly longer comparing the patients with a maspin nuclear pattern of expression and those with a non-nuclear one (log-rank test, p = 0.0018) (Fig. 2A).
hemorrhage, were assessed at ×400 magnification. Considering a minimum of 600 carcinoma cells, the pathologists visually assessed maspin expression and classified its subcellular distribution pattern as nuclear (almost exclusively nuclear or nuclear and cytoplasmic) or nonnuclear (showing only cytoplasmic or no reactivity). According to our previous report [6], only the subcellular maspin distribution pattern is associated with prognosis in LSCC, so in this investigation the pathologist did not quantify maspin expression. Bcl-2 granular cytoplasmic expression was evaluated in carcinoma cells from the same areas, estimating the percentage of bcl-2 stained cells.
3.2.3. Bcl-2 expression in LSCC, its clinicopathological features and prognosis LSCC specimens were considered as bcl-2-negative in the case of no immunohistochemical staining (expression of bcl-2: 0%) and bcl-2-positive in the case of any detectable immunohistochemical reaction (range of bcl-2 expression: 2–60%, Fig. 1E, F). Mann-Whitney U test identified a statistical trend toward higher bcl2 expression in T3-T4 vs T1-T2 patients (p = 0.063), while no significant differences for N status (p = 0.49), or pathological grade (p = 0.61). The expression of bcl-2 in LSCC was not associated with recurrence rate (Fisher's exact test, p = 0.093), or DFS (log-rank test, p = 0.072) (Fig. 2B).
2.4. Statistical analysis The statistical tests applied were Fisher's exact test, and the MannWhitney U test, as appropriate. The log-rank test was used to compare disease-free survival (DFS) (in months), stratified by the variables analyzed. As a cut-off for the immunohistochemical expression of bcl-2 in the LSCC, we chose the median value (0%) for binarizing the continuous variable (bcl-2 expression > 0% as bcl-2 positive and bcl-2 expression =0% as bcl-2 negative). The median value for the cohort of patients considered was the analytically best-fitting value and did not suffer from any subjectivity. In the multivariate analysis, recurrence rate estimates were based on the assumption of no collinearity or interactions between significant variables in the final model. Variables considered for multivariate analysis were those that showed a p-value < 0.1 in the univariate analysis. Cox proportional hazards regression identified the significant predictors of carcinoma recurrence. A p-value < 0.05 was considered significant, while values in the range between 0.05 and 0.07 were assumed to indicate a statistical trend. STATA 15.1 IC statistical package (Stata Corp LP, College Station, TX, USA) was used for all analyses.
3.2.4. Bcl-2 clinicopathological role in LSCCs according to maspin pattern Table 2 reports bcl-2 expression according to clinicopathological variables and maspin pattern in LSCCs. In LSCC with nuclear maspin pattern (9 cases), Mann–Whitney U test found significantly higher bcl-2 expression in N+ vs. N0 patients (p = 0.037), while there were no significant differences according to pT (p = 0.082) or grade (p = 0.87). No significant association was found between bcl-2 expression and the clinical outcome (recurrence rate, p = 0.86; DFS, log-rank test p = 0.78). In LSCCs with non-nuclear maspin pattern (22 cases), a statistical trend was found toward a shorter DFS in bcl-2 positive LSCCs (log-rank test, p = 0.062), but no significant difference in recurrence rate (p = 0.34). Mann–Whitney U test found no significant difference in bcl2 expression when the patients were distributed by pT (p = 0.24), N status (p = 0.61), or pathological grade (p = 0.46).
3. Results 3.1. Clinical outcome
3.3. Multivariate analysis Fourteen patients (45.2%) experienced no disease recurrence after surgery plus adjuvant RT, while 17 (54.8%) relapsed after a mean of 21.8 months (SD 26.3 months).
Multivariate recurrence rate estimates were based on Cox's proportional hazards model and on the assumption that there were no interactions between significant variables in the final model. No multicollinearity was found (the program identifies the collinear variables by default). The variable selection modality for backward elimination was set at p = 0.10 for univariate analysis according to disease recurrence (0/1). The multivariate model showed that, in the whole population, only maspin expression pattern (nuclear vs non-nuclear) retained its independent significance (hazard ratio 0.11, 95% CI 0.02–0.54, p = 0.006), while for bcl-2 expression (bcl-2 positive vs bcl-2 negative) no significant association was found with cancer recurrence risk (hazard ratio 2.49, 95% CI 0.91–6.84, p = 0.08). In the two sub-cohorts of LSCCs – nuclear (9 cases) and non-nuclear maspin pattern (22 cases)-, using the same variable selection modality for backward elimination, no variables were found to be suitable for entering the multivariate analysis model. Multivariate analysis was therefore not applicable in these subgroups.
3.2. Univariate analysis 3.2.1. Conventional clinicopathological variables In terms of disease recurrence rate, Fisher's exact test identified no significant differences in patients' distribution for lymph node status (N0 vs N+) (p = 0.29), pT (pT1–T2 vs pT3–T4) (p = 1.00), or pathological grade (G1–G2 vs G3) (p = 1.00). The log-rank test showed no significant differences in DFS (in months) when patients were stratified by N status (p = 0.09), pT (p = 0.82), or pathological grade (p = 0.27). 3.2.2. Maspin sub-cellular pattern of expression in LSCC, its clinicopathological features and prognosis One of the considered specimens was maspin-negative. Twenty-one out of 31 cases were characterized by a maspin cytoplasmic pattern of sub-cellular localization (Fig. 1A, B), 4 cases by a nuclear-cytoplasmic pattern and 5 by a nuclear pattern (Fig. 1C, D, respectively). Considering the evidence that maspin tumor-suppressor activity in LSCC is due to maspin localized in cellular nuclei [14], cases with nuclear or nuclear-cytoplasmic patterns were combined for statistical analysis. Table 1 summarizes the main clinicopathological and prognostic variables considering the whole series. Fisher's exact test showed no significant difference in the distribution of the maspin expression pattern (nuclear vs non-nuclear) vis-à-vis N-status (p = 0.11), pT
4. Discussion In this investigation on 31 LSCCs treated with surgery followed by RT, 23 patients were at stage III or IV on presentation. The recurrence rate was almost 55% despite radical surgery (without positive margins at final histological examinations) and adjuvant RT performed in accordance with currently-accepted protocols [3]. This confirmed that the prognosis of advanced cases of LSCC still remains poor despite 3
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Fig. 1. LSCC showing cytoplasmic maspin expression (A, B); LSCC featuring diffuse nuclear maspin expression (C, D); LSCC (Hematoxylin & Eosin) (E) with positive immunostaining for bcl-2 (F) (original magnification: A, B, C = 200x; D, E, F = 400x).
[8]. Bcl-2 is one of the main anti-apoptotic factors. In LSCCs, a decreased bcl-2 expression was associated with longer disease-free and overall survival of patients with LSCCs [20]. On the contrary, [21] found that bcl-2 over-expression was associated with increased grade and transglottic extension of LSCC. The aim of this exploratory clinicopathological investigation was to explore the relationship between maspin pattern of expression and bcl2 in LSCCs that underwent primary surgery followed by PORT. The main strength of this investigation lies in the homogeneity of the series of patients considered because: (i) they all underwent primary laryngeal surgery; (ii) their surgical treatment was performed consecutively by the same team; (iii) only surgical specimens (not biopsies) of LSCC were assessed; (iv) only squamous cell carcinomas located in a single head and neck structure (the larynx) were considered – this is because the role and subcellular localization of maspin differ in cancers developing at different head and neck sites [14], (v) PORT was appropriately performed in all cases; (vi) oncological follow-up criteria were defined, (vi) both univariate and multivariate statistical data analyses were applied. Instead, the main weaknesses of the study concern the retrospective setting of the investigation and the limited
multimodal treatment. The identification of molecular markers that may support diagnosis and predict prognosis and treatment response is therefore necessary. The tumor suppressor protein maspin could be considered as a promising molecular marker for prognostic evaluation in LSCC. Maspin showed different subcellular localizations, with different biological and prognostic significance. The presence of maspin in the nucleus of cancer cells has been reported as fundamental for a tumor suppressor activity [18,11]. Using immunohistochemistry, we had previously studied maspin expression in a large series of consecutive cases of operable LSCC [19]: the rates of lymph node metastases occurrence and recurrent disease were lower in patients with a nuclear pattern of maspin expression. The role of maspin was investigated in the LSCC apoptotic mechanism by M30, a protein expressed by epithelial cells during early apoptosis: M30 expression was significantly higher in the group of malignancies with maspin nuclear localization [14]. The activation of apoptotic signal cascade depends mainly on the release of mitochondrial proteins; they regulate the efflux from the mitochondrial inter-membrane space of cytochrome c that activates caspases 3, 8 and 9 in the cytoplasm, prompting the apoptotic process 4
Annals of Diagnostic Pathology 45 (2020) 151471
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prognostic factor (p = 0.006) in terms of disease recurrence. These results confirmed what was found in our previous investigation on LSCCs [19] and the importance of maspin nuclear localization in order to produce its tumor suppressive role. Interestingly, bcl-2 prognostic value seems to be associated to maspin expression pattern: in fact, in the sub-cohort of patients with non-nuclear maspin expression, bcl-2 positive LSCCs showed shorter DFS (statistical trend, p = 0.06). On the contrary, in the whole series and in the sub-cohort with nuclear maspin expression, bcl-2 was not significantly associated with laryngeal carcinoma prognosis. Considering this preliminary evidence, the hypothesis that maspin nuclear expression in LSCC could influence the apoptotic process by regulating blc-2 functions seemed to be supported. In prostate cancer, maspin was found to drive overexpression of pro-apoptotic bcl-2 family members, such as Bax, thus modifying the balance between mitochondrial apoptosis regulation proteins [13]. Moreover, maspin overexpression modulated tumor cell apoptosis through the regulation of bcl-2 family proteins in mouse mammary tumor TM40D cells [12]. In LSCC, this is the first report that analyzed the possible relationship between maspin and bcl-2 expressions. Given that bcl-2 has s critical role in the regulation of apoptosis and tumor progression, bcl-2 inhibitors hold promise in cancer therapy. Venetoclax is a first in-class bcl-2 selective inhibitor, recently FDA-approved for chronic lymphocytic leukemia (CLL) patients with 17p deletion who have received at least one prior treatment [22]. In small-cell lung cancers with high bcl-2 expression, venetoclax reduces the binding of pro-apoptotic ‘bcl-2-like protein 11’ (BIM) to BCL-2, destabilizing the bcl-2/BIM complex and ultimately, inducing apoptosis [23]. Further investigations are needed to establish the future prospects for incorporating modern bcl-2 inhibitors in multimodality or multitarget strategies against advanced LSCCs. According to our preliminary results, it could be rational to consider such strategies especially in patients with non-nuclear maspin pattern of expression in which bcl-2 gene seemed to exert its oncogenic effect.
Fig. 2. Disease-free survival in the whole series of LSCC patients, estimated on the basis of maspin pattern (nuclear vs non-nuclear) (A), and bcl-2 expression (B); time interval (abscissa).
Acknowledgments Table 2 LSCCs with and without maspin nuclear expression: bcl-2 expression in relation to classical clinicopathological and prognostic variables. Clinicopathological variables
No. of cases
Mean bcl-2 expression % ± SD
With maspin nuclear expression pT1 - pT2 3 11.3 ± 14.9 pT3 - pT4 6 9.0 ± 15.8 G1 - G2 7 9.0 ± 15.8 G3 2 1.0 ± 1.4 pN+ 2 8.0 ± 9.1 N0 (cN0 + pN0) 7 9.0 ± 15.8 Without cancer 7 9.0 ± 15.8 recurrence With cancer 2 11.3 ± 14.9 recurrence Without maspin nuclear expression pT1 - pT2 8 9.9 pT3 - pT4 14 8.7 G1 - G2 14 9.0 G3 8 9.0 pN+ 13 8.7 N0 (cN0 + pN0) 9 8.8 Without cancer 7 9.6 recurrence With cancer 15 8.7 recurrence
± ± ± ± ± ± ±
17.2 15.6 15.8 15.8 15.6 15.5 16.0
± 15.6
Mean diseasefree survival in months ± SD
Recurrence rate (%)
34.2 44.7 44.7 76.0 62.8 44.7 44.7
2 0 1 1 1 1
The authors thank Vincenza Guzzardo, BD, Department of Medicine DIMED, University of Padova, Italy for her pathological technical contribution, and Alison Garside for correcting the English version of this paper. Funding
± ± ± ± ± ± ±
21.0 47.7 47.7 53.7 51.9 47.7 47.7
(66.7%) (0.0%) (14.3%) (50.0%) (50.0%) (14.3%)
This study was supported in part by grant No. DOR1951845/19 (G. Marioni) from the University of Padova. Declaration of competing interest The authors declare that they have no conflict of interest.
32.2 ± 37.2
References 29.7 41.7 43.1 39.3 41.7 39.5 33.8
± ± ± ± ± ± ±
42.8 47.0 47.3 45.8 47.0 46.1 37.7
5 (62.5%) 10 (71.4%) 10 (71.4%) 5 (62.5%) 9 (69.2%) 6 (66.6%)
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41.7 ± 47.0
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Contents lists available at ScienceDirect
Annals of Diagnostic Pathology journal homepage: www.elsevier.com/locate/anndiagpath
Original Contribution
Maspin expression and anti-apoptotic pathway regulation by bcl2 in laryngeal cancer
T
Andrea Lovatoa, Leonardo Franzb, Valentina Carraroc, Luigia Bandolinb, Giacomo Controb, Giancarlo Ottavianob, Cosimo de Filippisa, Stella Blandamurac, Lara Alessandrinic, ⁎ Gino Marionib, a
Department of Neuroscience DNS, Audiology Unit, University of Padova, Treviso, Italy Department of Neuroscience DNS, Otolaryngology Section, University of Padova, Padova, Italy c Department of Medicine DIMED, University of Padova, Padova, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Laryngeal squamous cell carcinoma Maspin pattern Bcl-2 Apoptosis Prognosis
Objectives: Comprehension of the interplay of pro-apoptotic and anti-apoptotic stimuli in laryngeal squamous cell carcinoma (LSCC) is crucial to understand tumor development, biological behavior and treatment response. Bcl-2 family proteins mainly regulate the apoptotic signal cascade. In some cancers, maspin seems to influence the balance between pro-apoptosis and anti-apoptosis bcl-2 family proteins. The aim of this study was to investigate the potential relationship between bcl-2 anti-apoptotic factor and the tumor suppressor maspin in LSCC. Materials and methods: 31 consecutive patients who underwent primary surgery and post-operative radiotherapy for LSCC were evaluated retrospectively. For each case, immunohistochemistry assays for bcl-2 and maspin were performed. Data were also collected on N-status, pT stage, grading, recurrence and disease-free survival (DFS). Results: Patients with nuclear maspin pattern of expression showed a significantly lower recurrence rate (p = 0.04) and longer DFS (p = 0.0018). The expression of bcl-2 was not associated with recurrence rate or DFS either in the whole cohort or in cases with nuclear maspin pattern, while in patients with non-nuclear maspin pattern, a statistical trend was found toward a shorter DFS for bcl-2 positive cases (p = 0.062). In the multivariate model, only maspin expression pattern retained its independent prognostic significance (p = 0.006). Conclusions: Nuclear maspin pattern seemed to be an independent positive prognostic factor, while bcl-2 prognostic value was related to maspin expression pattern. Further investigations are needed to support the use of bcl-2 inhibitors in multimodality or multitarget strategies against advanced LSCCs, also considering the role and expression of tumor suppressor genes.
1. Introduction Laryngeal squamous cell carcinoma (LSCC) represents the 12th most common human malignancy in the adult age, according to the Surveillance Epidemiology and End Results (SEER) program of the United States [1]. Prognosis in early LSCC was generally considered quite good [2]. On the contrary, in advanced LSCC survival rates have not improved despite novelties in the therapeutic techniques (including CO2 laser-assisted transoral surgery, open reconstructive techniques, radiotherapy and chemotherapy) [3]. LSCCs have a 5-year survival rate of 80% to 90% for stage I/II disease, but up to 50% of advanced patients with LSCC experience recurrence following frontline therapy [4]. Consequently, in recent years oncological research on LSCC has focused
⁎
on tumor biology in order to identify prognostic markers and select potential treatment targets [5]. A deeper knowledge of the roles and relationships between oncogenes and tumor suppressor genes is crucial to understand development, biological behavior and treatment response [6]. Accordingly, the comprehension of balance regulation between pro-apoptotic and anti-apoptotic stimuli is important [7]. The activation of apoptotic signal cascade depends mainly on the Bcell lymphoma-2 (bcl-2) family of mitochondrial proteins. Bcl-2 is one of the most important anti-apoptotic factors. It acts as an oncogene and was found overexpressed in solid tumors, including breast, colorectal, prostate and small cell lung cancer, as well as in malignancies of hematological origin [8]. In LSCC, bcl-2 expression was preliminarily associated with a negative prognosis [9].
Corresponding author at: Department of Neuroscience DNS, Otolaryngology Section, University of Padova, Padova, Italy. E-mail address: [email protected] (G. Marioni).
https://doi.org/10.1016/j.anndiagpath.2020.151471
1092-9134/ © 2020 Published by Elsevier Inc.
Annals of Diagnostic Pathology 45 (2020) 151471
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scheduled as follows: (i) once a month for the 1st year; (ii) every 2 months in the 2nd year; (iii) every 3 months in the 3rd year; (iv) every 4 months in the 4th year; (v) every 6 months in the 5th year; and (vi) every 12 months thereafter. Patients underwent neck ultrasonography and chest X-ray at least yearly. Contrast-enhanced CT of the neck or total body positron emission tomography (PET-CT) were performed when necessary. Mean follow-up time was 58.3 ± 45.7 months (median, 43 months).
Maspin (mammary serine protease inhibitor) is a tumor suppressor gene that plays a role in various tumor related processes such as the inhibition of cell migration, cell invasion, angiogenesis, as well as improvement in cell adhesion and induction of programmed cell death [10]. Maspin nuclear localization in cancer cells has been reported as necessary for its tumor suppressor activity [11]. As a tumor suppressor, maspin interacts with the cellular signaling pathways of different oncogenes. Maspin overexpression was found modulating tumor cell apoptosis through the regulation of bcl-2 family proteins in mouse mammary tumor TM40D cells [12]. Also in prostate cancer, maspin seemed to influence the balance between apoptosis regulation proteins [13]. In LSCC, maspin nuclear expression was associated with the expression of M30, a protein expressed by epithelial cells during early apoptosis [14]. However, there is no evidence regarding a possible association between maspin expression and apoptosis control by bcl-2 family members in LSCC. The main aim of this exploratory study was to conduct a clinicopathological investigation into the potential relationship of bcl-2 anti-apoptotic factor and the tumor suppressor maspin in LSCC. In addition, the prognostic value of bcl-2 was evaluated in LSCC sub-cohorts characterized by different patterns of maspin expression.
2.2. Radiotherapy All 31 patients received external beam radiation therapy postoperatively using the same 6 MV photon beam delivered by a linear accelerator, at the Radiotherapy and Nuclear Medicine Unit, Istituto Oncologico Veneto, Padova. Patients were first immobilized with a thermoplastic mask, and CT images were acquired in the treatment position to allow for 3D treatment planning. The method used to administer the PORT depended on the target volume. In most cases, the dose was delivered using 2 parallel opposite fields up to 40 Gy, then the fields were shielded to cover the spine and matched with electron beams (8–10 MeV) on the spinal chains. In selected cases, the dose was delivered with multiple beams to cover the whole planned target volume. Conventional fractionation was used, i.e. 1.8–2 Gy/fraction daily for a total dose ranging from 50 to 70 Gy.
2. Materials and methods 2.1. Patients
2.3. Immunohistochemistry
The study was conducted in accordance with the principles of the Helsinki Declaration. Data were examined in agreement with the Italian privacy and sensible data laws and the Otolaryngology Section's University of Padova internal rules. Before undergoing surgery, all patients included in the study signed a detailed informed consent form. The investigation involved 31 consecutive patients (25 males, 6 females; mean age 67.5 ± 9.8 years) who underwent primary surgery followed by post-operative RT (PORT) for LSCC according to the currently-accepted indications for post-operative radiotherapy [3]. The laryngeal surgery was performed by the same surgical team for all the patients with unilateral or bilateral neck lymph node dissection in 29 cases. Before the surgery, all patients were investigated with microlaryngoscopy with laryngeal biopsies, upper aerodigestive tract endoscopy, neck ultrasonography (with or without fine needle aspiration cytology), head and neck contrast-enhanced computed tomography (CT) and/or magnetic resonance imaging (MRI), chest X-ray and liver ultrasonography. According to the 2017 TNM classification (8th edition) [15], the pathological stage of the primary lesion (pT) was T1 in 1 case, T2 in 10, T3 in 11, T4 in 9. For the N stage (pN), 14 patients were pN0, 14 were pN2 (3 pN2a, 6 pN2b, 5 pN2c), 1 was pN3. No distant metastasis (M) were found at diagnosis. Considering stage grouping, one of the 31 LSCC patients (3.2%) was stage I, 7 (22.6%) stage II, 4 (12.9%) stage III and 19 (61,3%) stage IV. As for the pathological grading, 3 patients were G1, 18 patients G2 and 10 G3 (Table 1). As previously reported [16,17], in our institution the clinical followup after treatment (adjusted to patients' individual characteristics) was
All tissues were fixed in 4% para-formaldehyde, embedded in paraffin and then stained with a fully automated system (Bond-maX; Vision BioSystems, UK). From each of the 31 paraffin-embedded blocks, 4 μm thick sections were cut. The sections were then deparaffinised in Bond Dewax Solution (Vision BioSystems, UK) at 72 °C, rinsed in ethanol and rehydrated in distilled water. Antigens were retrieved by heating sections for 30 min at 99 °C in Bond Epitope Retrieval Solution 1 (Vision BioSystems, UK). Endogenous peroxidase was blocked with 3% hydrogen peroxide before 30 min of incubation with anti-human maspin (mouse monoclonal antibody, clone EAW24, Novocastra Laboratories, Newcastle upon Tyne, UK; diluted 1:100) and anti-human bcl-2 (mouse monoclonal antibody, clone 124, Dako, Glostrup, Denmark; diluted 1:200). Specimens were then washed with phosphate-buffered saline (pH 7.0) and incubated with a Bond Polymer Refine Detection Kit (Vision BioSystems, UK) according to the manufacturer's protocols. The sections were then dehydrated, cleared, mounted and counterstained with Meyer's hematoxylin. Normal breast tissue was used as a positive maspin control, while samples from formalin-fixed, paraffin-embedded human tonsil were used as a positive bcl-2 control. Serum without the primary antibody was used as a negative control. 2.3.1. Maspin subcellular localization and bcl-2 expression The pathologists interpreting the sections were unaware of the patients' clinical outcomes. For each case, 40 non-overlapping fields of the less-differentiated areas of SCC, with no evidence of necrosis or
Table 1 Maspin expression pattern and bcl-2 expression in relation to classical clinicopathological and prognostic variables in LSCC: whole series. Clinicopathological variables
No. of cases
Maspin pattern of expression ( nuclear/non-nuclear)
Mean bcl-2 expression % ± SD
Mean disease-free survival in months ± SD
Recurrence rate (%)
pT1 - pT2 pT3 - pT4 G1 - G2 G3 pN+ N0 (cN0 + pN0) Without cancer recurrence With cancer recurrence
11 20 21 10 15 16 14 17
3/8 6/14 7/14 2/8 2/13 7/9 7/7 2/15
9.6 8.4 8.7 9.0 8.7 8.7 9.0 8.7
35.5 43.0 44.3 39.3 41.7 44.3 44.7 41.7
7 (63.6%) 10 (50.0%) 11 (52.4%) 6 (60.0%) 10 (66.7%) 7 (43.8%)
± ± ± ± ± ± ± ±
16.0 15.4 15.6 15.8 15.6 15.6 15.8 15.6
2
± ± ± ± ± ± ± ±
45.1 46.7 47.0 45.8 47.0 46.9 47.7 47.0
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(p = 0.68), or grading (p = 0.68). The recurrence rate was significantly lower among LSCC patients with a nuclear vs non-nuclear maspin pattern of expression (Fisher's exact test, p = 0.04). DFS was significantly longer comparing the patients with a maspin nuclear pattern of expression and those with a non-nuclear one (log-rank test, p = 0.0018) (Fig. 2A).
hemorrhage, were assessed at ×400 magnification. Considering a minimum of 600 carcinoma cells, the pathologists visually assessed maspin expression and classified its subcellular distribution pattern as nuclear (almost exclusively nuclear or nuclear and cytoplasmic) or nonnuclear (showing only cytoplasmic or no reactivity). According to our previous report [6], only the subcellular maspin distribution pattern is associated with prognosis in LSCC, so in this investigation the pathologist did not quantify maspin expression. Bcl-2 granular cytoplasmic expression was evaluated in carcinoma cells from the same areas, estimating the percentage of bcl-2 stained cells.
3.2.3. Bcl-2 expression in LSCC, its clinicopathological features and prognosis LSCC specimens were considered as bcl-2-negative in the case of no immunohistochemical staining (expression of bcl-2: 0%) and bcl-2-positive in the case of any detectable immunohistochemical reaction (range of bcl-2 expression: 2–60%, Fig. 1E, F). Mann-Whitney U test identified a statistical trend toward higher bcl2 expression in T3-T4 vs T1-T2 patients (p = 0.063), while no significant differences for N status (p = 0.49), or pathological grade (p = 0.61). The expression of bcl-2 in LSCC was not associated with recurrence rate (Fisher's exact test, p = 0.093), or DFS (log-rank test, p = 0.072) (Fig. 2B).
2.4. Statistical analysis The statistical tests applied were Fisher's exact test, and the MannWhitney U test, as appropriate. The log-rank test was used to compare disease-free survival (DFS) (in months), stratified by the variables analyzed. As a cut-off for the immunohistochemical expression of bcl-2 in the LSCC, we chose the median value (0%) for binarizing the continuous variable (bcl-2 expression > 0% as bcl-2 positive and bcl-2 expression =0% as bcl-2 negative). The median value for the cohort of patients considered was the analytically best-fitting value and did not suffer from any subjectivity. In the multivariate analysis, recurrence rate estimates were based on the assumption of no collinearity or interactions between significant variables in the final model. Variables considered for multivariate analysis were those that showed a p-value < 0.1 in the univariate analysis. Cox proportional hazards regression identified the significant predictors of carcinoma recurrence. A p-value < 0.05 was considered significant, while values in the range between 0.05 and 0.07 were assumed to indicate a statistical trend. STATA 15.1 IC statistical package (Stata Corp LP, College Station, TX, USA) was used for all analyses.
3.2.4. Bcl-2 clinicopathological role in LSCCs according to maspin pattern Table 2 reports bcl-2 expression according to clinicopathological variables and maspin pattern in LSCCs. In LSCC with nuclear maspin pattern (9 cases), Mann–Whitney U test found significantly higher bcl-2 expression in N+ vs. N0 patients (p = 0.037), while there were no significant differences according to pT (p = 0.082) or grade (p = 0.87). No significant association was found between bcl-2 expression and the clinical outcome (recurrence rate, p = 0.86; DFS, log-rank test p = 0.78). In LSCCs with non-nuclear maspin pattern (22 cases), a statistical trend was found toward a shorter DFS in bcl-2 positive LSCCs (log-rank test, p = 0.062), but no significant difference in recurrence rate (p = 0.34). Mann–Whitney U test found no significant difference in bcl2 expression when the patients were distributed by pT (p = 0.24), N status (p = 0.61), or pathological grade (p = 0.46).
3. Results 3.1. Clinical outcome
3.3. Multivariate analysis Fourteen patients (45.2%) experienced no disease recurrence after surgery plus adjuvant RT, while 17 (54.8%) relapsed after a mean of 21.8 months (SD 26.3 months).
Multivariate recurrence rate estimates were based on Cox's proportional hazards model and on the assumption that there were no interactions between significant variables in the final model. No multicollinearity was found (the program identifies the collinear variables by default). The variable selection modality for backward elimination was set at p = 0.10 for univariate analysis according to disease recurrence (0/1). The multivariate model showed that, in the whole population, only maspin expression pattern (nuclear vs non-nuclear) retained its independent significance (hazard ratio 0.11, 95% CI 0.02–0.54, p = 0.006), while for bcl-2 expression (bcl-2 positive vs bcl-2 negative) no significant association was found with cancer recurrence risk (hazard ratio 2.49, 95% CI 0.91–6.84, p = 0.08). In the two sub-cohorts of LSCCs – nuclear (9 cases) and non-nuclear maspin pattern (22 cases)-, using the same variable selection modality for backward elimination, no variables were found to be suitable for entering the multivariate analysis model. Multivariate analysis was therefore not applicable in these subgroups.
3.2. Univariate analysis 3.2.1. Conventional clinicopathological variables In terms of disease recurrence rate, Fisher's exact test identified no significant differences in patients' distribution for lymph node status (N0 vs N+) (p = 0.29), pT (pT1–T2 vs pT3–T4) (p = 1.00), or pathological grade (G1–G2 vs G3) (p = 1.00). The log-rank test showed no significant differences in DFS (in months) when patients were stratified by N status (p = 0.09), pT (p = 0.82), or pathological grade (p = 0.27). 3.2.2. Maspin sub-cellular pattern of expression in LSCC, its clinicopathological features and prognosis One of the considered specimens was maspin-negative. Twenty-one out of 31 cases were characterized by a maspin cytoplasmic pattern of sub-cellular localization (Fig. 1A, B), 4 cases by a nuclear-cytoplasmic pattern and 5 by a nuclear pattern (Fig. 1C, D, respectively). Considering the evidence that maspin tumor-suppressor activity in LSCC is due to maspin localized in cellular nuclei [14], cases with nuclear or nuclear-cytoplasmic patterns were combined for statistical analysis. Table 1 summarizes the main clinicopathological and prognostic variables considering the whole series. Fisher's exact test showed no significant difference in the distribution of the maspin expression pattern (nuclear vs non-nuclear) vis-à-vis N-status (p = 0.11), pT
4. Discussion In this investigation on 31 LSCCs treated with surgery followed by RT, 23 patients were at stage III or IV on presentation. The recurrence rate was almost 55% despite radical surgery (without positive margins at final histological examinations) and adjuvant RT performed in accordance with currently-accepted protocols [3]. This confirmed that the prognosis of advanced cases of LSCC still remains poor despite 3
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Fig. 1. LSCC showing cytoplasmic maspin expression (A, B); LSCC featuring diffuse nuclear maspin expression (C, D); LSCC (Hematoxylin & Eosin) (E) with positive immunostaining for bcl-2 (F) (original magnification: A, B, C = 200x; D, E, F = 400x).
[8]. Bcl-2 is one of the main anti-apoptotic factors. In LSCCs, a decreased bcl-2 expression was associated with longer disease-free and overall survival of patients with LSCCs [20]. On the contrary, [21] found that bcl-2 over-expression was associated with increased grade and transglottic extension of LSCC. The aim of this exploratory clinicopathological investigation was to explore the relationship between maspin pattern of expression and bcl2 in LSCCs that underwent primary surgery followed by PORT. The main strength of this investigation lies in the homogeneity of the series of patients considered because: (i) they all underwent primary laryngeal surgery; (ii) their surgical treatment was performed consecutively by the same team; (iii) only surgical specimens (not biopsies) of LSCC were assessed; (iv) only squamous cell carcinomas located in a single head and neck structure (the larynx) were considered – this is because the role and subcellular localization of maspin differ in cancers developing at different head and neck sites [14], (v) PORT was appropriately performed in all cases; (vi) oncological follow-up criteria were defined, (vi) both univariate and multivariate statistical data analyses were applied. Instead, the main weaknesses of the study concern the retrospective setting of the investigation and the limited
multimodal treatment. The identification of molecular markers that may support diagnosis and predict prognosis and treatment response is therefore necessary. The tumor suppressor protein maspin could be considered as a promising molecular marker for prognostic evaluation in LSCC. Maspin showed different subcellular localizations, with different biological and prognostic significance. The presence of maspin in the nucleus of cancer cells has been reported as fundamental for a tumor suppressor activity [18,11]. Using immunohistochemistry, we had previously studied maspin expression in a large series of consecutive cases of operable LSCC [19]: the rates of lymph node metastases occurrence and recurrent disease were lower in patients with a nuclear pattern of maspin expression. The role of maspin was investigated in the LSCC apoptotic mechanism by M30, a protein expressed by epithelial cells during early apoptosis: M30 expression was significantly higher in the group of malignancies with maspin nuclear localization [14]. The activation of apoptotic signal cascade depends mainly on the release of mitochondrial proteins; they regulate the efflux from the mitochondrial inter-membrane space of cytochrome c that activates caspases 3, 8 and 9 in the cytoplasm, prompting the apoptotic process 4
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prognostic factor (p = 0.006) in terms of disease recurrence. These results confirmed what was found in our previous investigation on LSCCs [19] and the importance of maspin nuclear localization in order to produce its tumor suppressive role. Interestingly, bcl-2 prognostic value seems to be associated to maspin expression pattern: in fact, in the sub-cohort of patients with non-nuclear maspin expression, bcl-2 positive LSCCs showed shorter DFS (statistical trend, p = 0.06). On the contrary, in the whole series and in the sub-cohort with nuclear maspin expression, bcl-2 was not significantly associated with laryngeal carcinoma prognosis. Considering this preliminary evidence, the hypothesis that maspin nuclear expression in LSCC could influence the apoptotic process by regulating blc-2 functions seemed to be supported. In prostate cancer, maspin was found to drive overexpression of pro-apoptotic bcl-2 family members, such as Bax, thus modifying the balance between mitochondrial apoptosis regulation proteins [13]. Moreover, maspin overexpression modulated tumor cell apoptosis through the regulation of bcl-2 family proteins in mouse mammary tumor TM40D cells [12]. In LSCC, this is the first report that analyzed the possible relationship between maspin and bcl-2 expressions. Given that bcl-2 has s critical role in the regulation of apoptosis and tumor progression, bcl-2 inhibitors hold promise in cancer therapy. Venetoclax is a first in-class bcl-2 selective inhibitor, recently FDA-approved for chronic lymphocytic leukemia (CLL) patients with 17p deletion who have received at least one prior treatment [22]. In small-cell lung cancers with high bcl-2 expression, venetoclax reduces the binding of pro-apoptotic ‘bcl-2-like protein 11’ (BIM) to BCL-2, destabilizing the bcl-2/BIM complex and ultimately, inducing apoptosis [23]. Further investigations are needed to establish the future prospects for incorporating modern bcl-2 inhibitors in multimodality or multitarget strategies against advanced LSCCs. According to our preliminary results, it could be rational to consider such strategies especially in patients with non-nuclear maspin pattern of expression in which bcl-2 gene seemed to exert its oncogenic effect.
Fig. 2. Disease-free survival in the whole series of LSCC patients, estimated on the basis of maspin pattern (nuclear vs non-nuclear) (A), and bcl-2 expression (B); time interval (abscissa).
Acknowledgments Table 2 LSCCs with and without maspin nuclear expression: bcl-2 expression in relation to classical clinicopathological and prognostic variables. Clinicopathological variables
No. of cases
Mean bcl-2 expression % ± SD
With maspin nuclear expression pT1 - pT2 3 11.3 ± 14.9 pT3 - pT4 6 9.0 ± 15.8 G1 - G2 7 9.0 ± 15.8 G3 2 1.0 ± 1.4 pN+ 2 8.0 ± 9.1 N0 (cN0 + pN0) 7 9.0 ± 15.8 Without cancer 7 9.0 ± 15.8 recurrence With cancer 2 11.3 ± 14.9 recurrence Without maspin nuclear expression pT1 - pT2 8 9.9 pT3 - pT4 14 8.7 G1 - G2 14 9.0 G3 8 9.0 pN+ 13 8.7 N0 (cN0 + pN0) 9 8.8 Without cancer 7 9.6 recurrence With cancer 15 8.7 recurrence
± ± ± ± ± ± ±
17.2 15.6 15.8 15.8 15.6 15.5 16.0
± 15.6
Mean diseasefree survival in months ± SD
Recurrence rate (%)
34.2 44.7 44.7 76.0 62.8 44.7 44.7
2 0 1 1 1 1
The authors thank Vincenza Guzzardo, BD, Department of Medicine DIMED, University of Padova, Italy for her pathological technical contribution, and Alison Garside for correcting the English version of this paper. Funding
± ± ± ± ± ± ±
21.0 47.7 47.7 53.7 51.9 47.7 47.7
(66.7%) (0.0%) (14.3%) (50.0%) (50.0%) (14.3%)
This study was supported in part by grant No. DOR1951845/19 (G. Marioni) from the University of Padova. Declaration of competing interest The authors declare that they have no conflict of interest.
32.2 ± 37.2
References 29.7 41.7 43.1 39.3 41.7 39.5 33.8
± ± ± ± ± ± ±
42.8 47.0 47.3 45.8 47.0 46.1 37.7
5 (62.5%) 10 (71.4%) 10 (71.4%) 5 (62.5%) 9 (69.2%) 6 (66.6%)
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41.7 ± 47.0
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