10.14639/0392-100X-N2127
Objectives. The aim of this study was to compare the distribution of the angiotensin-converting enzyme (ACE) I/D polymorphism between patients with laryngeal cancer (LC) and a control group and to examine the distribution of this polymorphism with clinical parameters related to LC. Methods. We enrolled 44 LC patients and 61 healthy controls. The ACE I/D polymorphism was genotyped with the PCR-RFLP method. The distribution of ACE genotypes (II, ID, and DD) and alleles (I or D) was evaluated with Pearson’s chi-square test, and logistic regression analysis was performed for statistically significant parameters.Results. There was no significant difference in ACE genotypes and alleles between LC patients and controls (p = 0.079 and p = 0.068, respectively). Among clinical parameters related to LC (extension of tumour, node metastasis, tumour stage and tumour location), only the presence of node metastasis was found to be significant in terms of ACE DD genotype (p = 0.137, p = 0.031, p = 0.147, p = 0.321 respectively). In the logistic regression analysis, the ACE DD genotype was increased 8.3 fold in nodal metastases. Conclusions. The findings of the study suggest that ACE genotypes and alleles do not affect the prevalence of LC, but the DD genotype of ACE polymorphism may increase the risk of lymph node metastasis in LC patients.
text/xml
1827-675X
2127
en
Fatma  Caner
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
gene polymorphism
Angiotensin-converting enzyme insertion/deletion gene polymorphism in patients with laryngeal cancer
Text.Serial.Journal
https://www.actaitalica.it/
43
Laryngology
2023-02-27
2022-04-17
2023-02-28
2023-02-28
26-31
1
Fatma  Caner
2023/02/27
10.14639/0392-100X-N2127
26
Department of Otorhinolaryngology and Head & Neck Surgery, Süleyman Demirel University Faculty of Medicine, Isparta, Turkey
1827-675X
gene polymorphism
en
31
Angiotensin-converting enzyme insertion/deletion gene polymorphism in patients with laryngeal cancer
https://www.actaitalica.it/article/download/2127/915
43
ACTA OTORHINOLARYNGOLOGICA ITALICA 2023;43:26-31; doi: 10.14639/0392-100X-N2127




Laryngology


Angiotensin-converting enzyme insertion/deletion
gene polymorphism in patients with laryngeal cancer
Polimorfismo del gene di inserzione/delezione dell’enzima di conversione
dell’angiotensina in pazienti con tumore della laringe
Yusuf Çağdaş Kumbul1, Kuyaş Hekimler Öztürk2, Hasan Yasan1, Vural Akın1, Mehmet Emre Sivrice1, Fatma Caner1
1
    Department of Otorhinolaryngology and Head & Neck Surgery, Süleyman Demirel University Faculty of Medicine, Isparta, Turkey;
2
    Department of Medical Genetics, Süleyman Demirel University Faculty of Medicine, Isparta, Turkey


SUMMARY
Objectives. The aim of this study was to compare the distribution of the angiotensin-con-
verting enzyme (ACE) I/D polymorphism between patients with laryngeal cancer (LC) and
a control group and to examine the distribution of this polymorphism with clinical param-
eters related to LC.
Methods. We enrolled 44 LC patients and 61 healthy controls. The ACE I/D polymorphism
was genotyped with the PCR-RFLP method. The distribution of ACE genotypes (II, ID, and
DD) and alleles (I or D) was evaluated with Pearson’s chi-square test, and logistic regres-
sion analysis was performed for statistically significant parameters.
Results. There was no significant difference in ACE genotypes and alleles between LC
patients and controls (p = 0.079 and p = 0.068, respectively). Among clinical parameters re-
lated to LC (extension of tumour, node metastasis, tumour stage and tumour location), only
the presence of node metastasis was found to be significant in terms of ACE DD genotype
(p = 0.137, p = 0.031, p = 0.147, p = 0.321 respectively). In the logistic regression analysis,
the ACE DD genotype was increased 8.3 fold in nodal metastases.
Conclusions. The findings of the study suggest that ACE genotypes and alleles do not affect
the prevalence of LC, but the DD genotype of ACE polymorphism may increase the risk of
lymph node metastasis in LC patients.
                                                                                                  Received: April 15, 2022
KEY WORDS: laryngeal cancer, lymphatic metastasis, angiotensin converting enzyme,                 Accepted: August 4, 2022
gene polymorphism
                                                                                                  Correspondence
                                                                                                  Yusuf Çağdaş Kumbul
RIASSUNTO                                                                                         Çünür, Suleyman Demirel University, East Campus,
Obiettivi. Lo scopo di questo studio è confrontare la distribuzione del polimorfismo di ACE       School of Medicine, 32260 Merkez, Isparta, Turkey
I/D tra i pazienti con cancro laringeo e un gruppo di controllo, e correlare la distribuzione     Tel. +905355495751. Fax +902462112830
del polimorfismo ai parametri clinici del tumore.                                                 E-mail: cagdas1061@hotmail.com
Metodi. Sono stati arruolati 44 pazienti con cancro laringeo e 61 soggetti sani. Il poli-
morfismo ACE I/D è stato genotipizzato con il metodo PCR-RFLP. La distribuzione dei                 How to cite this article: Kumbul YÇ, Hekimler
genotipi ACE (II, ID e DD) e degli alleli (I o D) è stata valutata con il Pearson chi-square        Öztürk K, Yasan H, et al. Angiotensin-converting
test, e analisi logistiche di regressione sono state eseguite per i parametri statisticamente       enzyme insertion/deletion gene polymorphism
significativi.                                                                                      in patients with laryngeal cancer. Acta Otorhi-
Risultati. Non è stata dimostrata alcuna differenza statistica tra i soggetti con cancro la-        nolaryngol Ital 2023;43:26-31. https://doi.
ringeo e i controlli per quanto concerne i genotipi di ACE e gli alleli (rispettivamente            org/10.14639/0392-100X-N2127
p = 0,079 e p = 0,068). Tra i parametri clinici tumorali (estensione del tumore, metastasi
linfonodali, stadio e sede del tumore), solo la presenza di metastasi linfonodali è stata cor-    © Società Italiana di Otorinolaringoiatria
                                                                                                  e Chirurgia Cervico-Facciale
relata in maniera statisticamente significativa con il genotipo ACE DD (rispettivamente,
p = 0,137, p = 0,031, p = 0,147, p = 0,321). Nelle analisi di regressione logistica, il geno-
                                                                                                                 OPEN ACCESS
tipo ACE DD è correlato ad un incremento del rischio di metastasi linfonodali di 8,3 volte.
Conclusioni. I risultati di questo studio suggeriscono che i genotipi e gli alleli di ACE non     This is an open access article distributed in accordance with
                                                                                                  the CC-BY-NC-ND (Creative Commons Attribution-Non-
influenzano la prevalenza del cancro laringeo, mentre il genotipo DD del polimorfismo             Commercial-NoDerivatives 4.0 International) license. The
ACE potrebbe aumentare il rischio di metastasi linfonodali.                                       article can be used by giving appropriate credit and mentio-
                                                                                                  ning the license, but only for non-commercial purposes and
PAROLE CHIAVE: cancro della laringe, metastasi linfonodali, enzima di conversione                 only in the original version. For further information: https://
dell’angiotensina, polimorfismo del gene                                                          creativecommons.org/licenses/by-nc-nd/4.0/deed.en




26
                                                                                        ACE I/D gene polymorphism and larynx cancer




Introduction                                                     ogy and Medical Genetics at Süleyman Demirel University
                                                                 Faculty of Medicine, Isparta, Turkey.
Squamous cell carcinoma is the most common type of la-
ryngeal cancer (LC), accounting for 85% to 95% of all ma-
                                                                 Subjects
lignant neoplasms 1. In 2021, the estimated number of new
                                                                 All clinical and pathological parameters were collected
cases diagnosed with LC in the US was 12,620, and LC
                                                                 by chart review. The criteria for inclusion in the LC group
accounted for 0.7% of all new cancer cases. Again in 2021,
                                                                 were as follows: 1. histopathological diagnosis of laryngeal
the estimated deaths due to LC were 3,770 individuals and
                                                                 squamous cell carcinoma; 2. regular smoker; and 3. data on
LC accounted for 0.6% of all cancer deaths 2. Despite the
                                                                 clinical parameters [e.g. tumour (T), node (N), metastasis
improvements in diagnosis and treatment, survival rates are
                                                                 (M) classification, and location of tumour, according to the
still low. While the 5-year survival rate for LC was 66% in
                                                                 AJCC Cancer Staging Manual, 8th Edition] 10. The criteria
the 1970s, it decreased to 60.7% in 2017 2,3. The reason for
                                                                 for inclusion in the control group were as follows: 1) no
treatment failure is mostly advanced stage disease at the
                                                                 history of malignancy; 2) regular smoker; 3) undergoing
time of diagnosis. Therefore, otolaryngologists, radiation
                                                                 septoplasty surgery at the otolaryngology clinic; 4) a nor-
oncologists and medical oncologists are constantly seeking
                                                                 mal laryngeal examination prior to surgery. In both groups,
to improve survival.
                                                                 patients with a history of use of ACE inhibitors and/or AT
Angiotensin-converting enzyme (ACE) is a zinc metallo-
                                                                 receptor blockers and patients with genetic diseases involv-
peptidase that converts angiotensin I (AT-1) to angiotensin
                                                                 ing predisposition to malignancy were excluded. Descrip-
II (AT-2) and reduces bradykinin; hence, it controls blood
                                                                 tive information was collected on the age, gender, tumour
pressure and cardiovascular homeostasis 4. An insertion/
                                                                 localisation, TNM classification and stage. The frequency
deletion (I/D) polymorphism consisting of 287 base pairs
                                                                 of II, ID and DD genotypes and I/D alleles of the ACE gene
is defined in intron 16 of the ACE gene located on chro-
                                                                 in patients diagnosed with LC were compared with the
mosome 17q23 5. The D allele is associated with higher
                                                                 control group. In addition, the clinical findings of patients
ACE plasma concentrations than the I allele 5,6. Thus, the
DD genotype correlates with AT-2 levels. Higher AT-2             diagnosed with LC were analysed in terms of genotype and
levels are also associated with neovascularisation, cell         allele frequency. For genetic analysis, 4 cc venous blood
proliferation, inflammation and possibly cancer develop-         was taken from all patients in the study group into tubes
ment 4,7.                                                        containing EDTA during routine preoperative examina-
In previous studies, the association of ACE gene polymor-        tions. Samples were stored at -20oC in the medical genetics
phism types (DD, ID, or II) with various cancers and clini-      laboratory until DNA isolation.
cal findings in cancer patients was investigated. Carriers of
the DD genotype for the ACE gene were shown to be at risk        Genotyping
for breast and squamous cell lung cancer, and for lymph          DNA from venous blood samples was obtained using the
node metastasis in those with gastric cancer 4,8,9. A rela-      High Pure Polymerase Chain Reaction (PCR) Template
tionship between ACE gene polymorphism and colorectal,           Preparation kit (Roche Applied Science, Germany) ac-
lung, and prostate cancer was not demonstrated 4,8. As a         cording to the manufacturer’s protocol. The concentration
result, data on ACE gene polymorphisms and development           and purity of the obtained DNA samples were determined
of cancer (regardless of cancer type) are inconsistent.          by measuring with a Nanodrop 2000c spectrophotometer
Finding a relationship between ACE gene polymorphisms            (Thermo Scientific, USA). The A60/280 absorbance ratio
and development of LC or parameters associated with LC           for DNA was in the range of 1.8-2.0. Polymerase chain
[e.g., tumour (T), node (N)] might help prevent and slow         reaction was performed of each DNA sample with ACE
progression of the disease, thus contributing to increased       gene I/D polymorphism genotype allele specific primers
survival. Our study was conducted to reveal the frequency        (Forward: TGGAGACCACTCCCATCCTTTCT, Reverse:
of ACE I/D polymorphism in LC and to investigate rela-           GATGTGGCCATCACATTCGTCAGAT) and FastStart
tionships with clinical findings. To the best of our knowl-      High Fidelity PCR System, dNTPack (Roche Applied Sci-
edge, this is the first study to reveal a relationship between   ence, Germany) kit. Each polymerase chain reaction con-
ACE I/D polymorphism and LC.                                     tains 10 pmol of F and R primers, 1.8 mM MgCl2, 10 mM
                                                                 dNTP mixture, 1.25 U Taq polymerase and 250 ng genomic
                                                                 DNA. The polymerase chain reaction was carried out un-
Materials and methods                                            der the following conditions; after 10 minutes of pre-in-
The study was carried out between September 2020 and             cubation at 94°C, 35 cycles at 94°C for 2 minutes, 57°C
September 2021 at the Department of Otorhinolaryngol-            for 30 seconds, 72°C for 1 minute, and finally at 72°C for



                                                                                                                               27
Y.Ç. Kumbul et al.




                                                                             Table I. Characteristics of LC patients and controls.
                                                                              Variables                  LC patients            Controls    pa
                                                                                                           (n = 44)             (n = 61)
                                                                                                             n (%)
                                                                                 Age                      62.73 ± 9.05      61.93 ± 7.48   0.625
                                                                                 Gender
                                                                                   Male                        44/44            61/61
                                                                                   Female                       -/-              -/-
                                                                                 Tumour localisation
                                                                                   Supraglottis              7 (15.90)
                                                                                   Glottis                  35 (79.54)
                                                                                   Subglottis                 2 (4.54)
                                                                                 Tumour extension
                                                                                   T1                        14 (31.8)
                                                                                   T2                        20 (45.5)
                                                                                   T3                         5(11.4)
                                                                                   T4                         5 (11.4)
Figure 1. Gel electrophoresis of PCR-RFLP products from representative           Nodes
blood samples for ACE I/D polymorphism. Lane ladder represents DNA ladder          N0                        34 (77.2)
marker (Fermentas). I/I columns show the 490 bp band. I/D columns show the         N1                         1 (2.27)
190 bp and 490 bp bands. D/D columns show the 190 bp bands.                        N2                        9 (20.45)
                                                                                   N3                             -
                                                                                 Stage
7 minutes. After the PCR, 2% agarose gel electrophoresis                           S1                        14 (31.8)
was applied to the products and stained with GelRed Nu-                            S2                        16 (36.4)
                                                                                   S3                         5 (11.4)
cleic Acid Gel Stain (Biotium, USA). The gel was visual-
                                                                                   S4                         9 (20.5)
ised with a UV Transilluminator. I and D polymorphism                        : p-value for Independent Samples T Test.
                                                                             a

genotypes of ACE gene in the samples were determined as
II genotype single band 490 bp, ID genotype double band
190 bp and 490 bp, and DD genotype single band 190 bp                        Results
(Fig. 1).
                                                                             In this study, 44 patients with LC and 61 healthy controls
Statistical analysis                                                         were enrolled. There were no female patients in either
                                                                             group. The mean age of patients with LC was 62.73 ± 9.05
Data was analysed by using SPSS Statistics for Windows,
                                                                             years, while it was 61.93 ± 7.48 years in the control group.
Version 23.0 (IBM SPSS. Armonk, NY: IBM Corp). In
                                                                             There was no significant difference between groups in terms
descriptive findings, categorical variables are presented as
                                                                             of age (p = 0.625). The characteristics of study subjects are
number and percentage, and continuous variables are pre-
                                                                             shown in Table I (none of the LC patients had distant me-
sented as mean ± standard deviation. Whether the age vari-
                                                                             tastases at initial diagnosis). Table II shows the frequency
able was normally distributed or not was evaluated with the
                                                                             distribution of ACE genotypes and alleles among patients
Kolmogorov-Smirnov test and compared between groups
                                                                             with LC and controls. There was no significant difference
with the independent samples t test. Since the study group
                                                                             between ACE genotypes and alleles between LC patients
consisted of only males, the gender variable was not in-
                                                                             and controls (p = 0.079 and p = 0.068, respectively). In
cluded in the analysis. Pearson chi-square test was used
                                                                             addition, the relationship between the distribution of ACE
when examining the relationship of ACE genotypes and
                                                                             genotypes and alleles in patients with LC and clinical pa-
alleles between patients with LC and the control group.                      rameters is presented in Table III.
Similarly, the relationship of ACE genotypes and alleles
with clinical parameters in LC patients was evaluated with
the Pearson chi-square test. Variables that were statistically               Discussion
significant in univariate analysis were evaluated with lo-                   ACE is the key molecule involved in the formation of AT-2
gistic regression analysis. The ID genotype was taken as a                   from AT-1 and the inactivation of bradykinin. The hypoth-
reference. Results are presented with odds ratio (OR) and                    esis that increased levels of AT-2 may play a role in the de-
95% confidence intervals (CI). Statistical significance was                  velopment of any cancer has been hypothesised especially
accepted as p < 0.05.                                                        for type 1 receptors of AT-2, because they can contribute



28
                                                                                                                                      ACE I/D gene polymorphism and larynx cancer




Table II. Distribution of ACE insertion/deletion polymorphism genotypes and                        polymorphism is associated with higher ACE levels 5,6,
alleles for LC patients and controls.                                                              meaning also high levels of the AT-2 and AT-2 type 1 recep-
                           LC patients            Controls          pa                             tors. In this context, the relationship between ACE I/D gene
                             (n = 44)             (n = 61)
                               n (%)                n (%)                                          polymorphism and many solid cancer types was investigated
    ACE genotypes
                                                                                                   in several studies 4,7,9,11-13 and it is worth investigating also
      DD                           10 (22.7)                10 (16.4)             0.079            for LC.
      ID                           28 (63.6)                31 (50.8)                              A relationship was found between ACE I/D gene polymor-
      II                            6 (13.6)                20 (32.8)                              phism and the development of prostate, breast, endometrial
    ACE alleles                                                                                    cancers and oral precancerous lesions. In these studies, it
      D                            48 (54.5)                51 (41.8)             0.068
      I                            40 (45.5)                71 (58.2)                              was shown that the presence of the D allele increases the
a
    : p-value for Pearson Chi-Square Test.                                                         risk of cancer. It is thought that malignant transformation
                                                                                                   is triggered after long-term exposure to the high amount of
                                                                                                   AT-2 formed in the DD genotype of the ACE gene in nor-
to cell proliferation, angiogenesis, inflammation and cell                                         mal tissues 14-17. It is known that AT-2, which has high levels
migration through various signal transduction pathways 7.                                          in the malignant transformation process, activates some in-
Previous studies showed that the D allele in the ACE gene                                          tracellular signal transduction pathways (e.g., transforming

Table III. Comparison of ACE genotypes/alleles with clinical parameters of LC patients.
                                    Clinical parameters                                 pa                                                        OR - (CI) - pc
                                         n (%) n (%)
                                                   Tumour extension
                                                     T1+T2 T3+T4
    Genotype
      DD                                            7 (20.6) 3 (30.0)
      ID                                           24 (70.6) 4 (40.0)                                     0.137
      II                                             3 (8.8) 3 (30.0)
      Allele
      D                                           38 (55.9) 10 (50.0)
      I                                           30 (44.1) 10 (50.0)                                     0.642
                                                        Nodes
                                                        N- N+
    Genotype
      DD                                            5 (14.7) 5 (50.0)                                                                     8.333 - (1.487-46.705) - 0.016
      ID                                           25 (73.5) 3 (30.0)                                     0.031                                        1.00b
      II                                            4 (11.8) 2 (20.0)                                                                     4.167 - (0.522-33.263) - 0.178
      Allele
      D                                           35 (51.5) 13 (65.0)
      I                                            33 (48.5) 7 (35.0)                                     0.285
                                                         Stage
                                                     S1+S2 S3+S4
    Genotype
      DD                                            5 (16.7) 5 (35.7)
      ID                                           22 (73.3) 6 (42.9)                                     0.147
      II                                            3 (10.0) 3 (21.4)
      Allele
      D                                          32 (53.3) 16 (57.1)
      I                                          28 (46.7) 12 (42.9)                                      0.738
                                                 Tumour localisation
                                             Supraglottis Glottis Subglottis
    Genotype
      DD                                          1 (14.3) 9 (25.7) 0 (0)
      ID                                       4 (57.1) 23 (65.7) 1 (50.0)                                0.321
      II                                         2 (28.6) 3 (8.6) 1(50.0)
      Allele
      D                                        6 (42.9) 41 (58.6) 1 (25.0)
      I                                        8 (57.1) 29 (41.4) 3 (75.0)                                0.267
a
    : p-value for Pearson Chi-Square test; b: reference category; c: p-value for binary logistic regression (according to enter model).




                                                                                                                                                                             29
Y.Ç. Kumbul et al.




growth factor B, phosphoinositide 3-kinase, mitogen acti-         protocols to prevent lymph node metastases and/or their
vated protein kinase/extracellular signal-regulated kinase,       spread. It was shown that long-term and high-dose ACE
Janus kinase-signal transducer and activator of transcription,    inhibitor use in individuals carrying the DD genotype of
protein kinase C and rat sarcoma protein). This process oc-       the ACE gene reduces the risk of some cancers (colo-
curs when AT-2 stimulates these pathways directly or via ep-      rectal, lung, breast, and prostate cancer) 4. Thus, simple
idermal growth factor. Another reason thought to be related       identification of the ACE I/D gene polymorphism can
to tumourigenesis is the high reactive oxygen species formed      identify the population to which this treatment can be ad-
by AT-2 in tissues 7. In our study, no significant difference     ministered and might significantly contribute to survival
was found between LC and the control group in terms of the        in patients with LC.
distribution of ACE I/D gene polymorphisms. Similarly, in         Finally, our study has some limitations. Firstly, our sam-
our study, no relationship was found between the distribution     ple size was relatively small. Secondly, the levels of ACE
of ACE I/D gene polymorphisms and parameters such as T            and AT-2 in tumour tissue and plasma could not be studied
stage or tumor location. We attribute this to the fact that en-   quantitatively. We suggest that these parameters be includ-
vironmental factors such as smoking are more effective than       ed in evaluations in future studies. Thirdly, we had no LC
genetic factors for the development of LC.                        patients with distant metastasis. Therefore, we could not
One of the most important factors determining the prog-
                                                                  perform statistical analysis for this parameter.
nosis in LC patients is cervical lymph node metastasis 18.
Therefore, one way to increase survival is to focus on fac-
tors that prevent lymph node metastasis or, if metastasis         Conclusions
has occurred, to stabilise the metastasis. In a study in gas-     The findings of the study suggest that ACE genotypes and al-
tric cancer patients, there was no difference when ACE I/D        leles do not affect the prevalence of LC, but the DD genotype
polymorphisms were compared with the control group, but           of ACE polymorphism may pose a risk for lymph node metas-
it was shown that the number of metastatic lymph nodes            tasis. Well-designed studies with larger sample sizes and more
was higher with the DD genotype than with the II geno-            ethnic groups are needed to further validate these results.
type 9. In patients with colorectal carcinoma, it was report-
ed that the DD genotype of the ACE gene increases the             Conflict of interest statement
risk of lymph node metastasis 19. In another study in pa-
                                                                  The authors declare no conflict of interest.
tients with endometrial cancer, no relationship was found
between lymph node metastasis and the ACE I/D gene
                                                                  Funding
polymorphism. However, the low number of patients with
                                                                  This work was supported by The Coordinatorship of Sci-
lymph node metastases in this study may have affected
                                                                  entific Research Projects Department (BAP), Suleyman
the statistical analysis 13. In most of the above-mentioned
                                                                  Demirel University (grant number: TSG-2020-8134)
studies, it was shown that the DD genotype of the ACE
gene is associated with increased lymph node metastasis.
                                                                  Author contributions
Angiogenesis is required for an invasive tumour to metas-
                                                                  YÇK: conception and design, acquisition of data, analy-
tasise and the balance between proangiogenic factors and
anti-angiogenic factors should be disturbed in favour of          sis and interpretation of data, wrote the manuscript, criti-
proangiogenic factors for angiogenesis. One of the proan-         cal revision of the article, final approveal of the version to
giogenic factors is vascular endothelial growth factor 20.        be published. KHÖ: conception and design, acquisition of
Previous studies showed that AT-2 has a stimulating effect        data, analysis and interpretation of data, wrote the manu-
on vascular endothelial growth factor 21. We think that the       script, final approveal of the version to be published. HY:
increased risk of cervical lymph node metastasis is due to        conception and design, wrote the manuscript, critical re-
the high AT-2 levels occurring in the DD genotype of the          vision of the article, final approveal of the version to be
ACE gene. In the current study, the risk of cervical lymph        published. VA: conception and design, acquisition of data,
node metastasis in patients with LC with the DD genotype          wrote the manuscript, critical revision of the article, final
of the ACE gene was also found to be 8.3 times higher than        approveal of the version to be published. MES: conception
the other genotypes.                                              and design, wrote the manuscript, critical revision of the
The pathophysiological mechanism of the susceptibility            article, final approveal of the version to be published. FC:
of the ACE gene DD genotype for lymph node metasta-               conception and design, wrote the manuscript, critical re-
sis should be elucidated further because this intervention        vision of the article, final approveal of the version to be
may enable ACE inhibitors to be included in treatment             published.



30
                                                                                                           ACE I/D gene polymorphism and larynx cancer




Ethical consideration                                                         10
                                                                                   Amin MB, Edge SB, Greene F, et al. AJCC Cancer Staging Manual.
This study was approved by the Institutional Ethics Com-                           Eighth edition. New York, NY: Springer International Publishing
                                                                                   2017.
mittee (Ethics Committee of the Suleyman Demirel Uni-                         11
                                                                                   Dastgheib SA, Asadian F, Farbod M, et al. Association of ACE I/D,
versity School of Medicine) (approval number/protocol                              -240A > T and AT1R A1166C polymorphisms with susceptibil-
number: 04/09/2020 - 239).                                                         ity to breast cancer: a systematic review and meta-analysis based
The research was conducted ethically, with all study pro-                          on 35 case-control studies. Nucleosides Nucleotides Nucleic Acids
                                                                                   2021;40:117-135. https://doi.org/10.1080/15257770.2020.1826515
cedures being performed in accordance with the require-                       12
                                                                                   Wang ZY, Li HY, Jiang ZP, et al. Relationship between angiotensin-
ments of the World Medical Association’s Declaration of                            converting enzyme insertion/deletion gene polymorphism and pros-
Helsinki.                                                                          tate cancer susceptibility. J Cancer Res Ther 2018;14:375-380. htt-
Written informed consent was obtained from each partici-                           ps://doi.org/10.4103/0973-1482.171366
pant/patient for study participation and data publication.                    13
                                                                                   Raba G, Zawlik I, Braun M, et al. Evaluation of the association be-
                                                                                   tween angiotensin converting enzyme insertion/deletion polymor-
                                                                                   phism and the risk of endometrial cancer in and characteristics of
References                                                                         Polish women. Adv Clin Exp Med 2020;29:581-585. https://doi.
                                                                                   org/10.17219/acem/118843
1
    Slootweg PJ, Grandis JR. Tumours of the hypopharynx, larynx, tra-
    chea and parapharyngeal space. In: El-Naggar AK, Chan JKC, Gran-
                                                                              14
                                                                                   Freitas-Silva M, Pereira D, Coelho C, et al. Angiotensin I-con-
    dis JR, et al., editors. WHO classification of head and neck tumours.          verting enzyme gene insertion/deletion polymorphism and endo-
    Lyon: International Agency for Research on Cancer; 2017. pp. 77-104.           metrial human cancer in normotensive and hypertensive women.
                                                                                   Cancer Genet Cytogen 2004;155:42-46. https://doi.org/10.1016/j.
2
    Cancer Stat Facts: Laryngeal Cancer. http://seer.cancer. gov/statfacts/        cancergencyto.2004.03.020
    html/laryn.html, Accessed March 1, 2020.
                                                                              15
                                                                                   Medeiros R, Vasconcelos A, Costa S, et al. Linkage of angiotensin
3
    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J
                                                                                   I-converting enzyme gene insertion/deletion polymorphism to the
    Clin 2016;66:7-30. https://doi.org/10.3322/caac.21332
                                                                                   progression of human prostate cancer. J Pathol 2004;202:330-335.
4
    van der Knaap R, Siemes C, Coebergh JW, et al. Renin-angiotensin               https://doi.org/10.1002/path.1529
    system inhibitors, angiotensin I-converting enzyme gene insertion/        16
                                                                                   Haiman C, Henderson S, Bretsky P, et al. Genetic variation in angio-
    deletion polymorphism, and cancer: the Rotterdam Study. Cancer
    2008;112:748-757. https://doi.org/10.1002/cncr.23215                           tensin I-converting enzyme (ACE) and breast cancer risk: the multi-
                                                                                   ethnic cohort. Cancer Res 2003;63:6984-6987.
5
    Rigat B, Hubert C, Alhenc-Gelas F, et al. An insertion/deletion poly-
    morphism in the angiotensin I-converting enzyme gene accounting for
                                                                              17
                                                                                   Chung FM, Yang YH, Chen CH, et al. Angiotensin-converting enzyme
    half the variance of serum enzyme levels. J Clin Invest 1990;86:1343-          gene insertion/deletion polymorphism is associated with risk of oral
    1346. https://doi.org/10.1172/jci114844                                        precancerous lesion in betel quid chewers. Br J Cancer 2005;93:602-
                                                                                   606. https://doi.org/10.1038/sj.bjc.6602746
6
    Pasha MA, Khan AP, Kumar R, et al. Variations in angiotensin-con-
    verting enzyme gene insertion/deletion polymorphism in Indian pop-
                                                                              18
                                                                                   Kürten CHL, Zioga E, Gauler T, et al. Patterns of cervical lymph
    ulations of different ethnic origins. J Biosci 2002;27:67-70. https://         node metastasis in supraglottic laryngeal cancer and therapeutic im-
    doi.org/10.1007/bf02703684                                                     plications of surgical staging of the neck. Eur Arch Otorhinolaryngol
                                                                                   2021;278:5021-5027. https://doi.org/10.1007/s00405-021-06753-1
7
    Rachow T, Schiffl H, Lang SM. Risk of lung cancer and renin-
    angiotensin blockade: a concise review. J Cancer Res Clin Oncol
                                                                              19
                                                                                   Zheng X, Liu G, Cui G, et al. Angiotensin-converting enzyme gene
    2021;147:195-204. https://doi.org/10.1007/s00432-020-03445-x                   deletion polymorphism is associated with lymph node metastasis in
                                                                                   colorectal cancer patients in a chinese population. Med Sci Monit
8
    Dević Pavlić S, Ristić S, Flego V, et al. Angiotensin-converting en-
    zyme insertion/deletion gene polymorphism in lung cancer pa-                   2017;23:4926-4931. https://doi.org/10.12659/msm.903312
    tients. Genet Test Mol Biomarkers 2012;16:722-725. https://doi.           20
                                                                                   Folkman J. Role of angiogenesis in tumor growth and metastasis. Sem-
    org/10.1089/gtmb.2011.0306                                                     in Oncol 2002;29:15-18. https://doi.org/10.1053/sonc.2002.37263
9
    Röcken C, Lendeckel U, Dierkes J, et al. The number of lymph node         21
                                                                                   Fujita M, Hayashi I, Yamashina S, et al. Blockade of angiotensin
    metastases in gastric cancer correlates with the angiotensin I-convert-        AT1a receptor signaling reduces tumor growth, angiogenesis, and me-
    ing enzyme gene insertion/deletion polymorphism. Clin Cancer Res               tastasis. Biochem Biophys Res Commun 2002;294:441-447. https://
    2005;11:2526-2530. https://doi.org/10.1158/1078-0432.ccr-04-1922               doi.org/10.1016/s0006-291x(02)00496-5




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