- Email: [email protected]
Apoptosis in Vocal Fold Polyps
ARTICLE IN PRESS Apoptosis in Vocal Fold Polyps *Abdul-Latif Hamdan, *Georges Tabet, †Zein Saadeddin, *Rachel Btaiche, and ‡Ibrahim Khalifeh, *†‡Beirut, Lebanon Summary: Objective. To examine the degree of apoptosis and its role in the pathogenesis of polyps of the vocal folds. Material and Method. A review of the pathology department’s archives for patients diagnosed with vocal fold polyps between 2010 and 2016 has been conducted. As a control group, gross and microscopically intact vocal fold from laryngectomy specimens was collected. A total of 61 vocal fold polyps from 51 patients and 41 unremarkable vocal folds from the control group were identified. Microscopically, the parameters studied were as follows: apoptosis, mitosis, inflammation, and exocytosis. Apoptotic index (number of apoptotic cells) was determined by the number of apoptotic cells per millimeter square in the epithelium. Apoptotic cells were readily identified by deeply shrunken eosinophilic cells detached from the surrounding environment with pyknotic-degenerated nuclei. Results. In polyps, the apoptotic index was statistically higher than the control group (ρ = 0.000). In addition, the increased apoptotic index in polyps showed a statistically proportional increase in mitotic index, inflammation, and exocytosis, which were significantly higher compared to control group. Conclusion. As a key for several therapeutic modalities, manipulation of apoptosis can be a future route for approaching vocal fold polyps by deciphering the complex signal pathways that allow the specified apoptotic cell to be targeted without damaging its surrounding counterpart. Key Words: Polyps–Vocal folds–Apoptosis–Mitosis–Epithelium.
INTRODUCTION Vocal fold polyps are among the most common exudative lesions of the vocal folds arising from the superficial layer of the lamina propria. These lesions are usually unilateral located at the free edge of the vocal fold with either a pale translucent or hemorrhagic appearance. The base can be sessile or pedunculated leading to aperiodic vibration of the vocal folds which in turn leads to a change in voice quality.1 This latter is perceived as dysphonia secondary to mass imbalance in the vocal folds often associated with compensatory hyperactivity of the supraglottic laryngeal structures. Acoustically there is a drop in the fundamental frequency and an increase in the perturbation parameters and noise to harmonic ratio.2 The most accepted therapeutic modality is phonomicrosurgery for removal of the lesions coupled with voice therapy and rehabilitation that result in improvement in the self-reported, perceptual, acoustic, and aerodynamic measures.3 The change in voice quality in patients with polyps both perceptually and acoustically has been attributed to structural and histological changes in the submucosal plane. Remacle et al has previously reported histological changes in patients with benign vocal fold lesions among which are vocal fold polyps. Pathological examination of the removed specimens revealed the presence of vascular proliferation and fibrosis.4,5 These results were further corroborated by numerous authors, confirming the preponderance of fibrosis, neovascularization, and bleeding in Accepted for publication March 31, 2017. From the *Department of Otolaryngology—Head & Neck Surgery, American University of Beirut Medical Center, Beirut, Lebanon; †Faculty of Medicine, American University of Beirut, Beirut, Lebanon; and the ‡Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon. Address correspondence and reprint requests to Ibrahim Khalifeh, Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, P.O. Box 11-0236 Beirut, Lebanon. E-mail: [email protected] Journal of Voice, Vol. ■■, No. ■■, pp. ■■-■■ 0892-1997 © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvoice.2017.03.023
patients with basal membrane thickening and epithelial hyperplasia nodules exhibited.6,7 The etiology behind the microscopic and structural changes as well as the alterations in the immune expression of many of the extracellular matrix constituents in patients with polyps has been attributed mainly to exogenous and endogenous factors such as smoking, reflux, and phonotraumatic behavior with little discussion at all on the role of apoptosis in the pathogenesis of these lesions. Apoptosis plays a very important protective role in the remodeling of tissues and their proliferation and carries a pivotal role in various processes, including cell death, development and aging, and defense mechanism during immune reactions and in response to disease or noxious agents.8,9 To that end, apoptosis is a key indicator of the proliferative activity of any cell group and provides the balance or homeostasis in any tissue proliferation activity. In addition, a process of selective apoptosis might be involved in the development of the lamina propria within the human vocal fold which bears important consequences on the human’s ability to phonate and engage in complex singing and speaking.10 Given the important role of apoptosis in tissue remodeling, the authors of this manuscript hypothesize that dysregulation of apoptosis may be a contributing factor to the onset or perpetuation of diseases of the lamina propria. No previous report has investigated the apoptotic activity in the vocal fold epithelial lining in patients with vocal fold polyps. The hypothesis is that there is elevated index of apoptosis in patients with vocal fold polyps compared to a control group with normal vocal folds. The purpose of this investigation is to examine the degree of apoptosis and its role in the pathogenesis of polyps of the vocal folds. MATERIALS AND METHODS After having obtained an approval from the institution research board, a review of the pathology department’s archives for patients diagnosed with vocal fold polyps between 2010 and 2016 has been conducted. As a control group, gross and
ARTICLE IN PRESS 2
FIGURE 1. A. A vocal fold polyp with hyperplastic lining epithelium and a fibrous core with intervening vascular proliferation. B. Apoptotic cell in the epithelium with deeply shrunken eosinophilic cytoplasm detached from the surrounding cells and pyknotic-degenerated nuclei. C. Mitotic figures in the lining epithelium of the vocal fold polyp. microscopically intact vocal fold from laryngectomy specimens was collected. Only patients with full clinical history and available pathology material were included in the study. Clinical data included age, gender, and history of smoking. Diagnosis of polyp was established microscopically by the pres-
Journal of Voice, Vol. ■■, No. ■■, 2017
ence of polypoid protrusion in the vocal fold with intense fibrous core and intervening vascular proliferation with acute and chronic hemorrhage11,12 (Figure 1A). Microscopically, the parameters studied were as follows: apoptosis, mitosis, inflammation, and exocytosis. Apoptotic index (number of apoptotic cells) was determined by the number of apoptotic cells per millimeter square in the epithelium.13 Apoptotic cells were readily identified by deeply shrunken eosinophilic cells detached from the surrounding environment with pyknotic-degenerated nuclei, which is a well-established criterion for identification of apoptotic cells as an alternative to the Terminal deoxynucleotidyl transferasemediated d-UTP Nick End Labeling (TUNEL) method of quantification14 (Figure 1B). Mitotic index (number of mitotic cells) was determined by the number of mitotic cells per millimeter square in the epithelium (Figure 1C). Inflammation was quantified in the subepithelium and divided into nonbrisk and brisk. Nonbrisk describes absent to patchy presence of inflammatory cells. Brisk is defined as inflammatory cells completely cuffing the epithelium (Figure 2A). Exocytosis is established by inflammatory cells migrating into the overlying epithelium and was divided into nonbrisk and brisk. Nonbrisk describes absent to focal presence of inflammatory cells in the overlying epithelium. Brisk is defined as numerous and diffuse inflammatory cells in the epithelium (Figure 2B). The microscopic features were evaluated and graded on multiple hematoxylin and eosin stained slides by two of the authors in a blinded fashion (IK and GT). The same variables (apoptosis, mitosis, inflammation, and exocytosis) were entertained in the control specimens. Statistical analysis Means and standard deviations were reported for continuous variables, whereas frequencies and percentages were reported for
FIGURE 2. A. A vocal fold polyp with brisk inflammation cuffing the overlying epithelium. B. A vocal fold polyp with brisk exocytosis showing inflammatory cells migrating to the squamous epithelium. C. Apoptotic cell cuffed by inflammatory cells. D. Clustering of apoptotic cells in vocal fold polyp.
ARTICLE IN PRESS Abdul-Latif Hamdan, et al
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Apoptosis in Vocal Fold Polyps
TABLE 1. Summary of the Clinical Variables Between Vocal Fold Polyps and the Control Group
Variables Age Gender
Female Male Nonsmokers Smokers
Smoking
Polyps (n = 61) Mean ± SD* Frequency (%)†
Controls (n = 41) Mean ± SD* Frequency (%)†
44.06 ± 12.483 * 18 (35.3%)† 33 (64.7%)† 17 (33.3%)† 34 (66.7%)†
64.31 ± 9.216* 5 (19.2%)† 21 (80.8%)† 8 (30.8%)† 18 (69.2%)†
* Mean ± SD. † Frequency (%). Abbreviation: SD, standard deviation.
categorical variables. The nonparametric alternative of independent t test, the Mann-Whitney U test, was used since the sample size is relatively small and the data are not normally distributed. Odds ratio and 95% confidence interval were computed using bivariate logistic regression. Results were considered significant at P value <0.05. Data were analyzed using SPSS version 22.0 (SPSS Inc, Chicago, IL). Inter-rater reliability Cohen’s kappa test statistic was computed to determine the proportion of agreement between rater 1 and rater 2 in the examination of 15% of the total sample for each of the outcomes: apoptosis, mitosis, exocytosis, and inflammation. A value of Κ < 0.20 was considered poor agreement. Rater 1 is the last author of this manuscript and rater 2 is a Board Certified Pathologist. RESULTS Demographic data In the search preformed, 61 vocal fold polyps from 51 patients and 41 unremarkable vocal folds from the control group were identified. The mean age for the patients with polyps was 44.1
versus 64.3 for the control group. Male gender and smokers were dominant in both groups. See Table 1.
Microscopic findings Microscopically, in polyps the apoptotic index was statistically higher than the control group (ρ = 0.000). In addition, the increased apoptotic index in polyps showed statistically proportional increase in mitotic index, inflammation, and exocytosis. When comparing mitotic index, inflammation, and exocytosis between polyps and the control group, polyps show significantly higher values among all three variables. See Table 2. Such findings confirm the close interaction between the four microscopic variables included in this study. Such interaction is also highlighted by the inflammatory cells cuffing the apoptotic cells (Figure 2C) and increased exocytosis in areas with high apoptotic and mitotic indexes (Figure 2D).
Inter-rater reliability Cohen’s kappa test statistic on 15% of the observations independently coded by both assessors for apoptosis, mitosis, exocytosis, and inflammation was 0.53, 0.26, 1.0, and 0.83,
TABLE 2. Summary of the Microscopic Variables Between Vocal Fold Polyps and the Control Group
Variables
Polyps (n = 61) Mean ± SD* Frequency (%)†
Controls (n = 41) Mean ± SD* Frequency (%)†
P value
OR (95% CI)
Apoptosis
7.84 ± 5.817*
0.59 ± 0.774*
0.000 < 0.05
Mitosis
6.02 ± 3.905*
0.83 ± 1.070*
0.000 < 0.05
17 (27.9%)† 44 (72.1%)† 15 (24.6%)† 46 (75.4%)†
39 (95.2%)† 2 (4.8%)† 40 (97.5%)† 1 (2.5%)†
0.000 < 0.05
5.65 (2.62–12.16) 5.79 (2.68–12.48) 50 (11–232) 122 (15–970)
Inflammation: Exocytosis:
Nonbrisk Brisk Nonbrisk Brisk
Results were significant at P value < 0.05. * Mean ± SD. † Frequency (%). Abbreviations: CI, confidence interval; OR, odds ratio; SD, standard deviation.
0.000 < 0.05
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TABLE 3. Inter-rater Reliability Apoptosis Rater 1 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Κ
Mitosis
Rater 2
2 1 1 0 3 9 3 4 6 4
2 2 1 0 3 9 3 3 5 3 0.53
Rater 1
Exocytosis
Rater 2
5 3 5 4 5 5 7 6 5 7
4 3 3 2 5 5 9 4 5 8 0.26
respectively. Therefore, the hypothesis of random determinations was rejected. See Table 3. DISCUSSION Apoptosis, derived from the Greek word “απο´ πτωσις” or the falling of leaves or petals, is a form of noninflammatory programmed cell death characterized by cellular condensation which is distinct from the process of necrosis that involves an accidental form of cell death accompanied by cellular swelling and inflammation. The apoptosis phenomena were first described by German scientist Carl Vogt in 1842, but the term apoptosis was not used until year 1972 by John Foxton Ross Kerr group.15 Apoptosis has since been recognized as a distinctive mode of “programmed” cell death involving the genetically determined elimination of cells.8 Apoptosis has been described not only in physiological processes but also in pathological processes including cancer, inflammatory diseases, neurodegenerative disorders, and infections. It is characterized by the presence of membrane blebbing and the appearance of highly condensed chromatin with activation of an endonucleolytic process leading to the sequential cleavage of chromosomal DNA.16 Although there are a wide variety of stimuli and conditions triggering apoptosis, not all cells will necessarily die in response to the same stimulus.8 Tumor necrosis factor alpha (TNF-α) is an example of a substance that can push a cell to apoptosis via TNF receptorinduced assembly as well as activation of the transcription factor nuclear factor κB which can override the apoptotic signal by producing survival proteins.17 Programmed cell death can be realized mainly through two routes: intrinsic and extrinsic pathways. The intrinsic pathway is represented by intrinsic apoptotic proteins affecting the mitochondria. Recent evidence shows that nitric oxide is one of those proteins.18 These proteins create pores in the mitochondrial membranes or increase the permeability of the organelle in order to release SMAC (second mitochondria-derived activator of caspases), which in turn attaches to inhibitors of apoptosis and disables them. This cascade of events leads to the activation of caspases which propagate the cell into apoptosis.19 The extrinsic pathway is mainly governed by two mechanisms: the
Rater 1
Inflammation
Rater 2
2 2 1 1 2 2 1 2 2 2
2 2 1 1 2 2 1 2 2 2 1.0
Rater 1
Rater 2
1 2 0 1 1 2 1 2 2 2
1 2 0 1 1 2 1 1 2 2 0.83
TNF model (TNF induced) and the Fas-Fas ligand mediated model. TNF-α is produced by many inflammatory cells but mainly by macrophages. When TNF-α binds to TNFR1 (TNF-α receptor 1), a cascade of reactions occurs including the activation of an intermediary protein TRADD (TNF receptor-associated death domain) that leads to the activation of caspases and eventually completes the process of programmed cell death.20 The FasFas pathway is governed by FasL (Fas ligand) binding to Fas receptor which activates FADD (Fas-associated death domain) protein that triggers caspases activities resulting in apoptosis.21 Apoptosis and mitosis are in constant interplay in order to create cellular homeostasis. As mitosis is increased, apoptosis will increase in order to keep the tissue in balance. Hence apoptosis is a sign of cellular proliferation. No previous study has investigated the degree of apoptosis in patients with vocal fold polyps. There has been only one recent publication on apoptosis in patients with chronic adenoiditis and adenoid hypertrophy.22 Onal et al showed that patients who had a high number of apoptotic cells in their adenoid tissue had a higher degree of choanal occlusion when compared with patients who had lower count of apoptotic cells in their adenoids.22 Our investigation is the first to cast information on apoptotic activity in patients with benign vocal fold masses, in particular polyps. The results not only highlighted the significantly higher apoptotic index in vocal fold polyps but also accentuated the mutual relation among apoptosis, mitosis, and inflammation as in other processes. Such findings emphasize the high turnover of the epithelium lining of polyps and the close interaction between the inflammatory cascade and apoptosis. The increased apoptosis and mitosis in patients with vocal fold polyps can be attributed to multiple factors including phonotraumatic behavior, laryngopharyngeal reflux, and history of smoking in addition to other environmental factors. As a key for several therapeutic modalities, manipulation of apoptosis can be a future route for approaching vocal fold polyps. This could lead to several therapeutic benefits by deciphering the complex signal pathways that allow the specified apoptotic cell to be targeted without damaging its surrounding counterpart. There are two main limitations to this study. One is the high prevalence of smoking in both groups, polyps and controls that
ARTICLE IN PRESS Abdul-Latif Hamdan, et al
Apoptosis in Vocal Fold Polyps
could have masked or accentuated the true apoptotic activity. Second is the age difference between the two groups. Aging acts in a similar way to cancer when it comes to apoptosis as both these entities accumulate dysfunction in apoptosis and cellular senescence. However, unlike cancer, aging can be related to an increase or a decrease in apoptosis depending on different tissues.23,24 CONCLUSION This study is the first to investigate apoptosis activity in patients with vocal cord polyps. A larger prospective study with the opposite vocal fold as control may substantiate the results of this investigation, namely the significantly higher apoptotic index. REFERENCES 1. Hirano M. Clinical Examination of Voice. Springer; 1981. 2. Petrovic-Lazic M, Babac S, Vukovic M, et al. Acoustic voice analysis of patients with vocal fold polyp. J Voice. 2011;25:94–97. 3. Johns MM, Garrett CG, Hwang J, et al. Quality-of-life outcomes following laryngeal endoscopic surgery for nonneoplastic vocal fold lesions. Ann Otol Rhinol Laryngol. 2004;113:597–601. 4. Remacle M, Lagneau G, Marbaix E, et al. Exudative laryngeal diseases of Reinke’s space. Ann Otolaryngol Chir Cervicofac. 1991;109:33–38. 5. Remacle M, Degols JC, Delos M. Exudative lesions of Reinke’s space. An anatomopathological correlation. Acta Otorhinolaryngol Belg. 1995;50:253– 264. 6. Dikkers FG, Nikkels PG. Lamina propria of the mucosa of benign lesions of the vocal folds. Laryngoscope. 1999;109:1684–1689. 7. Volic´ SV, Klapan I, Seiwerth S, et al. Extracellular matrix of Reinke’s space in some pathological conditions. Acta Otolaryngol. 2004;124:505–508. 8. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.
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9. Vaux DL, Korsmeyer SJ. Cell death in development. Cell. 1999;96:245– 254. 10. Hartnick CJ, Rehbar R, Prasad V. Development and maturation of the pediatric human vocal fold lamina propria. Laryngoscope. 2005;115:4– 15. 11. Martins RH, Defaveri J, Domingues MA, et al. Vocal polyps: clinical, morphological, and immunohistochemical aspects. J Voice. 2011;25:98–106. 12. Nunes RB, Behlau M, Nunes MB, et al. Clinical diagnosis and histological analysis of vocal nodules and polyps. Braz J Otorhinolaryngol. 2013;79:434–440. 13. Garrity MM, Burgart LJ, Riehle DL, et al. Identifying and quantifying apoptosis: navigating technical pitfalls. Mod Pathol. 2003;16:389–394. 14. Jerome KR, Vallan C, Jaggi R. The TUNEL assay in the diagnosis of graft-versus-host disease: caveats for interpretation. Pathology. 2000;32:186– 190. 15. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239– 257. 16. Wu M, Ding H-F, Fisher DE. Apoptosis: molecular mechanisms. In: Encyclopedia of Life Sciences. 2001 doi:10.1038/npg.els.0001150. 17. Ward C, Rossi AG, Haslett C, et al. Apoptosis: future perspectives. Essays Biochem. 2003;39:155–162. 18. Brune B. Nitric oxide: no apoptosis or turning it on? Cell Death Differ. 2003;10:864–869. 19. Fesik SW, Shi Y. Structural biology. Controlling the caspases. Science. 2001;294:1477–1478. 20. Chen G, Goeddel DV. Tnf-R1 signaling: a beautiful pathway. Science. 2002;296:1634–1635. 21. Wajant H. The Fas signaling pathway: more than a paradigm. Science. 2002;296:1635–1636. 22. Onal M, Yilmaz T, Bilgic E, et al. Possible role of apoptosis in pathogenesis of adenoid hypertrophy and chronic adenoiditis: prospective case-control study. Auris Nasus Larynx. 2015;42:449–452. 23. Cerella C, Grandjenette C, Dicato M, et al. Roles of apoptosis and cellular senescence in cancer and aging. Curr Drug Targets. 2016;17:405–415. 24. Warner HR. Aging and regulation of apoptosis. Curr Top Cell Regul. 1997;35:107–121.
INTRODUCTION Vocal fold polyps are among the most common exudative lesions of the vocal folds arising from the superficial layer of the lamina propria. These lesions are usually unilateral located at the free edge of the vocal fold with either a pale translucent or hemorrhagic appearance. The base can be sessile or pedunculated leading to aperiodic vibration of the vocal folds which in turn leads to a change in voice quality.1 This latter is perceived as dysphonia secondary to mass imbalance in the vocal folds often associated with compensatory hyperactivity of the supraglottic laryngeal structures. Acoustically there is a drop in the fundamental frequency and an increase in the perturbation parameters and noise to harmonic ratio.2 The most accepted therapeutic modality is phonomicrosurgery for removal of the lesions coupled with voice therapy and rehabilitation that result in improvement in the self-reported, perceptual, acoustic, and aerodynamic measures.3 The change in voice quality in patients with polyps both perceptually and acoustically has been attributed to structural and histological changes in the submucosal plane. Remacle et al has previously reported histological changes in patients with benign vocal fold lesions among which are vocal fold polyps. Pathological examination of the removed specimens revealed the presence of vascular proliferation and fibrosis.4,5 These results were further corroborated by numerous authors, confirming the preponderance of fibrosis, neovascularization, and bleeding in Accepted for publication March 31, 2017. From the *Department of Otolaryngology—Head & Neck Surgery, American University of Beirut Medical Center, Beirut, Lebanon; †Faculty of Medicine, American University of Beirut, Beirut, Lebanon; and the ‡Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon. Address correspondence and reprint requests to Ibrahim Khalifeh, Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, P.O. Box 11-0236 Beirut, Lebanon. E-mail: [email protected] Journal of Voice, Vol. ■■, No. ■■, pp. ■■-■■ 0892-1997 © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvoice.2017.03.023
patients with basal membrane thickening and epithelial hyperplasia nodules exhibited.6,7 The etiology behind the microscopic and structural changes as well as the alterations in the immune expression of many of the extracellular matrix constituents in patients with polyps has been attributed mainly to exogenous and endogenous factors such as smoking, reflux, and phonotraumatic behavior with little discussion at all on the role of apoptosis in the pathogenesis of these lesions. Apoptosis plays a very important protective role in the remodeling of tissues and their proliferation and carries a pivotal role in various processes, including cell death, development and aging, and defense mechanism during immune reactions and in response to disease or noxious agents.8,9 To that end, apoptosis is a key indicator of the proliferative activity of any cell group and provides the balance or homeostasis in any tissue proliferation activity. In addition, a process of selective apoptosis might be involved in the development of the lamina propria within the human vocal fold which bears important consequences on the human’s ability to phonate and engage in complex singing and speaking.10 Given the important role of apoptosis in tissue remodeling, the authors of this manuscript hypothesize that dysregulation of apoptosis may be a contributing factor to the onset or perpetuation of diseases of the lamina propria. No previous report has investigated the apoptotic activity in the vocal fold epithelial lining in patients with vocal fold polyps. The hypothesis is that there is elevated index of apoptosis in patients with vocal fold polyps compared to a control group with normal vocal folds. The purpose of this investigation is to examine the degree of apoptosis and its role in the pathogenesis of polyps of the vocal folds. MATERIALS AND METHODS After having obtained an approval from the institution research board, a review of the pathology department’s archives for patients diagnosed with vocal fold polyps between 2010 and 2016 has been conducted. As a control group, gross and
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FIGURE 1. A. A vocal fold polyp with hyperplastic lining epithelium and a fibrous core with intervening vascular proliferation. B. Apoptotic cell in the epithelium with deeply shrunken eosinophilic cytoplasm detached from the surrounding cells and pyknotic-degenerated nuclei. C. Mitotic figures in the lining epithelium of the vocal fold polyp. microscopically intact vocal fold from laryngectomy specimens was collected. Only patients with full clinical history and available pathology material were included in the study. Clinical data included age, gender, and history of smoking. Diagnosis of polyp was established microscopically by the pres-
Journal of Voice, Vol. ■■, No. ■■, 2017
ence of polypoid protrusion in the vocal fold with intense fibrous core and intervening vascular proliferation with acute and chronic hemorrhage11,12 (Figure 1A). Microscopically, the parameters studied were as follows: apoptosis, mitosis, inflammation, and exocytosis. Apoptotic index (number of apoptotic cells) was determined by the number of apoptotic cells per millimeter square in the epithelium.13 Apoptotic cells were readily identified by deeply shrunken eosinophilic cells detached from the surrounding environment with pyknotic-degenerated nuclei, which is a well-established criterion for identification of apoptotic cells as an alternative to the Terminal deoxynucleotidyl transferasemediated d-UTP Nick End Labeling (TUNEL) method of quantification14 (Figure 1B). Mitotic index (number of mitotic cells) was determined by the number of mitotic cells per millimeter square in the epithelium (Figure 1C). Inflammation was quantified in the subepithelium and divided into nonbrisk and brisk. Nonbrisk describes absent to patchy presence of inflammatory cells. Brisk is defined as inflammatory cells completely cuffing the epithelium (Figure 2A). Exocytosis is established by inflammatory cells migrating into the overlying epithelium and was divided into nonbrisk and brisk. Nonbrisk describes absent to focal presence of inflammatory cells in the overlying epithelium. Brisk is defined as numerous and diffuse inflammatory cells in the epithelium (Figure 2B). The microscopic features were evaluated and graded on multiple hematoxylin and eosin stained slides by two of the authors in a blinded fashion (IK and GT). The same variables (apoptosis, mitosis, inflammation, and exocytosis) were entertained in the control specimens. Statistical analysis Means and standard deviations were reported for continuous variables, whereas frequencies and percentages were reported for
FIGURE 2. A. A vocal fold polyp with brisk inflammation cuffing the overlying epithelium. B. A vocal fold polyp with brisk exocytosis showing inflammatory cells migrating to the squamous epithelium. C. Apoptotic cell cuffed by inflammatory cells. D. Clustering of apoptotic cells in vocal fold polyp.
ARTICLE IN PRESS Abdul-Latif Hamdan, et al
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Apoptosis in Vocal Fold Polyps
TABLE 1. Summary of the Clinical Variables Between Vocal Fold Polyps and the Control Group
Variables Age Gender
Female Male Nonsmokers Smokers
Smoking
Polyps (n = 61) Mean ± SD* Frequency (%)†
Controls (n = 41) Mean ± SD* Frequency (%)†
44.06 ± 12.483 * 18 (35.3%)† 33 (64.7%)† 17 (33.3%)† 34 (66.7%)†
64.31 ± 9.216* 5 (19.2%)† 21 (80.8%)† 8 (30.8%)† 18 (69.2%)†
* Mean ± SD. † Frequency (%). Abbreviation: SD, standard deviation.
categorical variables. The nonparametric alternative of independent t test, the Mann-Whitney U test, was used since the sample size is relatively small and the data are not normally distributed. Odds ratio and 95% confidence interval were computed using bivariate logistic regression. Results were considered significant at P value <0.05. Data were analyzed using SPSS version 22.0 (SPSS Inc, Chicago, IL). Inter-rater reliability Cohen’s kappa test statistic was computed to determine the proportion of agreement between rater 1 and rater 2 in the examination of 15% of the total sample for each of the outcomes: apoptosis, mitosis, exocytosis, and inflammation. A value of Κ < 0.20 was considered poor agreement. Rater 1 is the last author of this manuscript and rater 2 is a Board Certified Pathologist. RESULTS Demographic data In the search preformed, 61 vocal fold polyps from 51 patients and 41 unremarkable vocal folds from the control group were identified. The mean age for the patients with polyps was 44.1
versus 64.3 for the control group. Male gender and smokers were dominant in both groups. See Table 1.
Microscopic findings Microscopically, in polyps the apoptotic index was statistically higher than the control group (ρ = 0.000). In addition, the increased apoptotic index in polyps showed statistically proportional increase in mitotic index, inflammation, and exocytosis. When comparing mitotic index, inflammation, and exocytosis between polyps and the control group, polyps show significantly higher values among all three variables. See Table 2. Such findings confirm the close interaction between the four microscopic variables included in this study. Such interaction is also highlighted by the inflammatory cells cuffing the apoptotic cells (Figure 2C) and increased exocytosis in areas with high apoptotic and mitotic indexes (Figure 2D).
Inter-rater reliability Cohen’s kappa test statistic on 15% of the observations independently coded by both assessors for apoptosis, mitosis, exocytosis, and inflammation was 0.53, 0.26, 1.0, and 0.83,
TABLE 2. Summary of the Microscopic Variables Between Vocal Fold Polyps and the Control Group
Variables
Polyps (n = 61) Mean ± SD* Frequency (%)†
Controls (n = 41) Mean ± SD* Frequency (%)†
P value
OR (95% CI)
Apoptosis
7.84 ± 5.817*
0.59 ± 0.774*
0.000 < 0.05
Mitosis
6.02 ± 3.905*
0.83 ± 1.070*
0.000 < 0.05
17 (27.9%)† 44 (72.1%)† 15 (24.6%)† 46 (75.4%)†
39 (95.2%)† 2 (4.8%)† 40 (97.5%)† 1 (2.5%)†
0.000 < 0.05
5.65 (2.62–12.16) 5.79 (2.68–12.48) 50 (11–232) 122 (15–970)
Inflammation: Exocytosis:
Nonbrisk Brisk Nonbrisk Brisk
Results were significant at P value < 0.05. * Mean ± SD. † Frequency (%). Abbreviations: CI, confidence interval; OR, odds ratio; SD, standard deviation.
0.000 < 0.05
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TABLE 3. Inter-rater Reliability Apoptosis Rater 1 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Κ
Mitosis
Rater 2
2 1 1 0 3 9 3 4 6 4
2 2 1 0 3 9 3 3 5 3 0.53
Rater 1
Exocytosis
Rater 2
5 3 5 4 5 5 7 6 5 7
4 3 3 2 5 5 9 4 5 8 0.26
respectively. Therefore, the hypothesis of random determinations was rejected. See Table 3. DISCUSSION Apoptosis, derived from the Greek word “απο´ πτωσις” or the falling of leaves or petals, is a form of noninflammatory programmed cell death characterized by cellular condensation which is distinct from the process of necrosis that involves an accidental form of cell death accompanied by cellular swelling and inflammation. The apoptosis phenomena were first described by German scientist Carl Vogt in 1842, but the term apoptosis was not used until year 1972 by John Foxton Ross Kerr group.15 Apoptosis has since been recognized as a distinctive mode of “programmed” cell death involving the genetically determined elimination of cells.8 Apoptosis has been described not only in physiological processes but also in pathological processes including cancer, inflammatory diseases, neurodegenerative disorders, and infections. It is characterized by the presence of membrane blebbing and the appearance of highly condensed chromatin with activation of an endonucleolytic process leading to the sequential cleavage of chromosomal DNA.16 Although there are a wide variety of stimuli and conditions triggering apoptosis, not all cells will necessarily die in response to the same stimulus.8 Tumor necrosis factor alpha (TNF-α) is an example of a substance that can push a cell to apoptosis via TNF receptorinduced assembly as well as activation of the transcription factor nuclear factor κB which can override the apoptotic signal by producing survival proteins.17 Programmed cell death can be realized mainly through two routes: intrinsic and extrinsic pathways. The intrinsic pathway is represented by intrinsic apoptotic proteins affecting the mitochondria. Recent evidence shows that nitric oxide is one of those proteins.18 These proteins create pores in the mitochondrial membranes or increase the permeability of the organelle in order to release SMAC (second mitochondria-derived activator of caspases), which in turn attaches to inhibitors of apoptosis and disables them. This cascade of events leads to the activation of caspases which propagate the cell into apoptosis.19 The extrinsic pathway is mainly governed by two mechanisms: the
Rater 1
Inflammation
Rater 2
2 2 1 1 2 2 1 2 2 2
2 2 1 1 2 2 1 2 2 2 1.0
Rater 1
Rater 2
1 2 0 1 1 2 1 2 2 2
1 2 0 1 1 2 1 1 2 2 0.83
TNF model (TNF induced) and the Fas-Fas ligand mediated model. TNF-α is produced by many inflammatory cells but mainly by macrophages. When TNF-α binds to TNFR1 (TNF-α receptor 1), a cascade of reactions occurs including the activation of an intermediary protein TRADD (TNF receptor-associated death domain) that leads to the activation of caspases and eventually completes the process of programmed cell death.20 The FasFas pathway is governed by FasL (Fas ligand) binding to Fas receptor which activates FADD (Fas-associated death domain) protein that triggers caspases activities resulting in apoptosis.21 Apoptosis and mitosis are in constant interplay in order to create cellular homeostasis. As mitosis is increased, apoptosis will increase in order to keep the tissue in balance. Hence apoptosis is a sign of cellular proliferation. No previous study has investigated the degree of apoptosis in patients with vocal fold polyps. There has been only one recent publication on apoptosis in patients with chronic adenoiditis and adenoid hypertrophy.22 Onal et al showed that patients who had a high number of apoptotic cells in their adenoid tissue had a higher degree of choanal occlusion when compared with patients who had lower count of apoptotic cells in their adenoids.22 Our investigation is the first to cast information on apoptotic activity in patients with benign vocal fold masses, in particular polyps. The results not only highlighted the significantly higher apoptotic index in vocal fold polyps but also accentuated the mutual relation among apoptosis, mitosis, and inflammation as in other processes. Such findings emphasize the high turnover of the epithelium lining of polyps and the close interaction between the inflammatory cascade and apoptosis. The increased apoptosis and mitosis in patients with vocal fold polyps can be attributed to multiple factors including phonotraumatic behavior, laryngopharyngeal reflux, and history of smoking in addition to other environmental factors. As a key for several therapeutic modalities, manipulation of apoptosis can be a future route for approaching vocal fold polyps. This could lead to several therapeutic benefits by deciphering the complex signal pathways that allow the specified apoptotic cell to be targeted without damaging its surrounding counterpart. There are two main limitations to this study. One is the high prevalence of smoking in both groups, polyps and controls that
ARTICLE IN PRESS Abdul-Latif Hamdan, et al
Apoptosis in Vocal Fold Polyps
could have masked or accentuated the true apoptotic activity. Second is the age difference between the two groups. Aging acts in a similar way to cancer when it comes to apoptosis as both these entities accumulate dysfunction in apoptosis and cellular senescence. However, unlike cancer, aging can be related to an increase or a decrease in apoptosis depending on different tissues.23,24 CONCLUSION This study is the first to investigate apoptosis activity in patients with vocal cord polyps. A larger prospective study with the opposite vocal fold as control may substantiate the results of this investigation, namely the significantly higher apoptotic index. REFERENCES 1. Hirano M. Clinical Examination of Voice. Springer; 1981. 2. Petrovic-Lazic M, Babac S, Vukovic M, et al. Acoustic voice analysis of patients with vocal fold polyp. J Voice. 2011;25:94–97. 3. Johns MM, Garrett CG, Hwang J, et al. Quality-of-life outcomes following laryngeal endoscopic surgery for nonneoplastic vocal fold lesions. Ann Otol Rhinol Laryngol. 2004;113:597–601. 4. Remacle M, Lagneau G, Marbaix E, et al. Exudative laryngeal diseases of Reinke’s space. Ann Otolaryngol Chir Cervicofac. 1991;109:33–38. 5. Remacle M, Degols JC, Delos M. Exudative lesions of Reinke’s space. An anatomopathological correlation. Acta Otorhinolaryngol Belg. 1995;50:253– 264. 6. Dikkers FG, Nikkels PG. Lamina propria of the mucosa of benign lesions of the vocal folds. Laryngoscope. 1999;109:1684–1689. 7. Volic´ SV, Klapan I, Seiwerth S, et al. Extracellular matrix of Reinke’s space in some pathological conditions. Acta Otolaryngol. 2004;124:505–508. 8. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.
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