Laryngopharyngeal Reflux and Laryngeal Squamous Cell Carcinoma

Abstract

Background: Patients with squamous cell carcinoma (SCC) of the larynx are usually heavy smokers, and tobacco exposure has been clearly correlated with p53 tumor suppressor gene damages. In this prospective study we investigated the possible independent carcinogenic role of laryngopharyngeal reflux (LPR) by correlating the development of laryngeal SCC, with the presence of LPR and p53 alterations.

Materials and Methods: Eighty-eight consecutive patients with laryngeal SCC treated at the ENT University Clinic of Florence preoperatively underwent a 24h dual-probe-pH-monitoring and laryngeal endoscopy. Then, all patients underwent a direct microlaryngoscopy with tumor biopsy and assessment of p53 gene mutations. Statistical analysis was performed correlating the presence of LPR, smoking status and mutations of p53, by chi-squared test and logistic regression analysis.

Results: In 40% of the cases TP53 mutations were encountered. Sixteen transition mutations, 10 transversions, and 9 insertion-deletions were found. Furthermore, 22 missense, 4 frame deletions, 7 frame-shifts and 3 non-sense mutations were identified. In 34% of the patients pathologic LPR was recorded at 24h dual-probe-pH-monitoring and laryngeal endoscopy. A positive correlation between smoke and p53 mutations (p=.024, ?2 test) was found, while no correlations was recorded for LPR and p53 mutations (p>.05, ?2 test). LPR patients were at highest risk for tumors at the posterior glottic region (p=0.037, Fisher test).

Conclusion: Our study highlighted a potential key role of LPR in the pathogenesis of laryngeal SSC in non smokers and non drinkers, especially for glottic cancer of the posterior region. Moreover, our findings seems to suggest the presence of different genetic mechanisms of biomolecular damage beside those due to smoking exposure, according to the low rate of the p53 tumor suppressor gene mutations in laryngeal SCC from patients with LPR.

Keywords: Laryngopharyngeal reflux; Laryngeal squamous cell carcinoma; p53 mutation; Larynx; Cancer immunohistochemistry; Lpr gerd

Abbreviations

SCC: Squamous Cell Carcinoma; LPR: Laryngopharyngeal Reflux; IHC: Immunohistochemistry; SSCP: Single-Strand Conformational Polymorphisms; GER: Gastroesophageal Reflux; UAD: Upper Aero-Digestive; LES: Lower Oesophageal Sphincter; UES: Upper Esophageal Sphincter; PCR: Polymerase Chain Reaction

Introduction

Smoking, alcohol consumption and exposure to viral and toxic agents are well recognized risk factors for laryngeal squamous cell carcinoma (SCC). However, none of the above mentioned risk factors are present in the medical history of 5% laryngeal SCC patients [1]. The gastroesophageal reflux (GER) has been pointed out as a carcinogen for upper aero-digestive (UAD) tract cancers [2-6]. Koufman codified the term laryngopharyngeal reflux (LPR), describing a reflux that affects roughly 40–60% of patients with various voice disorders [7]. The majority of LPR-suffering patients, in fact, usually do not report classical GER symptoms like heartburn and regurgitation; instead they suffer of dry or sore throat, globus sensation, hoarsness, chronic cough, dysphagia, or buccal burning. Furthermore LPR has been correlated with posterior laryngitis, laryngeal granuloma, chronic hoarseness, pharyngitis, asthma, pneumonia, nocturnal choking, and dental diseases [8,9].

Many studies have reported a high prevalence of reflux in patients with laryngeal cancer [4-6], however smoking and alcohol consumption are not only risk factors for laryngeal SCC but usually increase reflux episodes. In fact, tobacco smoke causes a significant reduction of basal tonicity of the lower oesophageal sphincter (LES) and increases gastric acid secretion, while alcohol consumption may reduce LES tonicity and alter oesophageal motility [10,11].

Acting as carcinogens tobacco smoke and alcohol alter the repairing mechanisms of the DNA by damaging cell cycle proteins (i.e.: p53) as revealed by biomolecular studies [12-14]. The aim of our study was to evaluate the role of LPR in the carcinogenesis of laryngeal SCC, the association of reflux with tobacco and alcohol makes it difficult to separate the relative contribution of reflux in cancer development. In order to discriminate the role of LPR we assessed its prevalence in laryngeal cancer patients using 24-hour double probe pH measurements and we analyzed TP53 mutations in smoking and in not smoking LPR patients.

Materials and Methods

Patient selection

We prospectively examined 94 consecutive patients with suspect laryngeal cancer at the Otolaryngology and Head-Neck surgery department of University of Florence from January 2010 to December 2011. In all patients we recorded smoking and alcohol habits. Patients were requested to sign an informed consent and institutional board approval was obtained. All patients underwent a 24h dual-probepH- monitoring, laryngeal videoendoscopy under local anaesthesia and direct laryngoscopy under general anaesthesia to collect biopsies for diagnosis and biomolecular studies. From the initial cohort of 94 enrolled suspect lesions, the study cohort resulted in 88 consecutive patients with biopsy proven squamous cell carcinoma. Specifically, we correlated immunostaining with p53 gene mutations as assessed by means of single-strand conformational polymorphisms (SSCP). This simple method for mutation screening recognizes sequences variations through differences in the migration properties of single stranded DNA in non-denaturing polyacrylamide gels [15]. The efficacy of mutations detection with this technique has been reported to be as high as 90% [16].

24h-dual probe-pH-monitoring

24 hours dual-probe (pharyngeal and esophageal) pH monitoring was performed in all cases for diagnosing LPR. Two monocrystalline antimony pH sensors were positioned along a single catheter (diameter, 2.1 mm) with sensors 15–20 cm apart and a silver-silver chloride cutaneous reference electrode (Digitrapper Mark III, Medtronic Synectics, Maastricht, The Netherlands). Both probes (pH sensors) were calibrated simultaneously in buffer solutions pH 7 and pH 1 before monitoring. Under fibroscopic control, the proximal probe was placed in upper esophageal sphincter (UES). The second probe (distal pH sensor) was positioned at the lower esophageal sphincter (LES) using pH transition point (withdrawal technique). The distal probe was useful to detect true pharyngolaryngeal reflux episodes (pH drop at the distal probe preceding pH drop at the UES). Patients were encouraged to eat their regular meals without restrictions and to continue their usual daily routines. Pathologic laryngo-pharyngeal reflux event was defined by an abrupt decrease in pH to below 4 in the upper probe, with an accompanying or preceding decrease in pH to below 4 in the lower probe, except while eating food [17]. Pathologic LPR was defined when more than three episodes of LPR occurred. At distal probe, levels of pH <4 were considered pathologic if they were registered in more than 5.5% of total recording time, in more than 8.2% of time in upright position, and in more than 3% of time in supine position. At proximal probe, any reflux rate equal or more than 0.2% of the total time in upright position was considered pathologic as well as any reflux rate equal or more than 0.1% of the total time in supine position.

Laryngeal endoscopy

All patients underwent high definition videolaryngoscopy under local anesthesia. The findings were scored according to the reflux finding score (RFS) as described by Belafsky et al. [18]. The scale ranges from 0 (no abnormal findings) to a maximum of 26 (worst score possible). According to these authors, we considered an overall score ≥ 7 suggestive of the LPR.

Sample preparation

Formalin-fixed, paraffin-embedded tumor specimens were obtained from all of 88 laryngeal SSC patients. Tissue sections (7 to 8 μm in thickness) were placed on standard microscope slides. Specimens were deparaffinazed with xylene, rehydrated in serial graded (100%, 90%, 70%, 50%) water-ethanol solution, rinsed in deionized water, and stained with hematoxylin and eosin. The samples were dehydrated again, but slides were not fitted with a coverslip. The stained, unmounted sections were examined by microscopy. The portion of normal tissue from each specimen was identified and scraped away with sterilized blade, leaving only tumor cells for DNA extraction. If discordant results between SSCP and IHC were obtained, different areas of tumor were sampled to confirm original data.

DNA isolation

DNA from paraffin-embedded tumor sections was extracted by means of overnight incubation at 55°C in an extraction buffer: 50 mmol/L potassium chloride, 10 mmol/L tromethamine (tris) at pH 8.3, 2.5 mmol/L magnesium chloride, 0.1 mg/mL gelatin, 0.45% NP40, 0.45% polysorbate 20 (Tween 20), and 0.5 mg/mL proteinase K. The samples were boiled for 8 minutes, and centrifugated for 15 minutes at 14,000 rpm. One to 5 μL of the supernatant was used in polymerase chain reaction (PCR) mixture.

Polymerase chain reaction and single-strand conformation polymorphism analysis

Exons 5 to 8 of p53 gene were subjected to PCR with intron primers. Exon 5 was amplified with two pairs of primers, which resulted in partially overlapping fragments. Amplification consisting of 32 cycles was carried out in 25 μL total volume with 1.5 mmol/L MgCl buffer (Perkin-Elmer, Branchburg, NJ), 1 μmol/L of exon flanking primer set, 50 μmol/L each of deoxyribonucleoside triphosphate, and 0.5 units of AmplitTaq (Perkin-Elmer). Temperature and time during reaction cycles were 95°C for 1 minute, 62°C for 30 seconds, and 72°C for 30 seconds. PCR products were heat denatured and subjected to SSCP analysis by electroforesis on 6% polyacrylamide gel with 5% to 10% glycerol. Electrophoresis was carried out at 40 W for 4 hours at 4°C using the Pharmacia (Piscataway, NJ) apparatus. The gels were silver stained and dried on filter paper. All cases were amplified and run independently at least twice, with consistent results. To exclude silent CGA/CGG dimorphism in codon 213, PCR products from samples showing SSCP abnormalities in exon 6 were subjected to restriction analysis with Taq1.

Sequence analysis

PCR-amplified DNA strands were tested on 3% agarose gels (NuSieve; FMC Bioproducts, Rockland, Me), eluted from gel by phenol extraction, and concentrated by ethanol precipitation. Purified products were sequenced on an automatic sequencer (Model 373A; Applied Biosystems, Foster City, Calif) using dideoxy dye terminator method with Taq polymerase and PCR primers. Mutations were assessed on both strands.

DO-7 Immunohistochemistry

Five-micrometer paraffin-embedded sections were mounted on silane-coated slides and dried at 56°C for 30 minutes. After dewaxing and blocking endogenous peroxidase, sections were rinsed in water and then placed in 10 mmol/L of citrate acid at pH 6.0. The sections were microwaved at 750 W for 20 minutes while covered with liquid. After microwave heating, the sections were transferred to phosphate-buffered saline solution. Specimens were stained with mouse anti-p53 protein antibody DO-7 (Dako, Carpinteria, Calif) at 1:40 in phosphate-buffered saline solution. This antibody recognizes both mutant and wild type p53 protein. Primary antibody bound to antigen was detected with standard streptavidin-biotin technique (LSAB kit; Dako) and visualized with diaminobenzidine. A light hematoxylin nuclear counterstain was used. Control slides without primary antibody were normal in all cases. Immunostaining was graded as follows: +, less than 10%; ++, 10% to 50%; +++, more than 50% stained cells.

Statistical analysis

The statistical analysis was carried out with the Fisher’s exact, Χ² tests and multivariate regression analysis using STATA software (College Station, TX). Statistical significance was set at p<.05.

Results

Clinical characteristics

Of 88 patients enrolled, 79 (89.8%) were males and 9 (10.2%) females. The mean age was 60.1 years old (range 45-78). According to 7th edition of American Joint Committee on Cancer (AJCC) [19], 59(67%) T1, 22(25%) T2, 4 (4.5%) T3, 3 (3,5%) T4a with 77 (87.5%) N0 and 11 (12.5%) N1-3 were found. The most involved subsite was the glottis (46 cases, 52.2%), followed by the supraglottis in 35(39.8%) cases. The posterior supraglottic and glottic larynx was involved in 3 (3.4%) patients and 4 (4.5%) transglottic tumors were found. Tobacco smoke exposure was recorded in 75 (85.2%) patients. Most patients (n=67, 76.1%) daily consumed 2-3 drinks, whilst 17(19.3%) patients had positive medical history of an alcohol-abuse (more than 4 drinks) and only 4(4.5%) patients were abstemious. All heavy drinkers were also heavy smokers (more than 30 packs per year).

24h-dual probe-pH-monitoring outcomes

Thirty (34%) patients showed positive 24h-dual probe-pH monitoring for LPR. In these patients, values of pH <4 at proximal probe were registered as a mean rate of 0.6% (range 0.35% - 0.8%) of time in upright position; while a mean rate of 0.93% (range 0.4% - 1.5%) of time in supine position was recorded. The average number of reflux episodes was 9 (range 5 – 13) and the mean duration of reflux episodes was 21 min (range 19 – 25).

Laryngeal endoscopy outcomes

Twenty-four out of 30 LPR-suffering patients (80%) documented also a RFS more than 7 with a mean of 12.3 ± 4.7. The most common endoscopic finding was posterior commissure hypertrophy (n=23); only in 1 case a concomitant small granuloma of the posterior commissure was encountered. No patients with negative LPR at 24h-dual probe pH-monitoring scored more than 7 at RFS.

p53 Immunohistochemistry and gene status

Among 88 laryngeal SCC, IHC-positive moderate (++) and abundant (+++) staining was found in 35 cases (40%). Thirty-six (41%) cases demonstrated mutations of TP53 by SSCP analysis in exon 5,6,7,8. One sample was altered in both exon 5 and 8. As shown in Table 1, the 88 tumors in this series were stratified into distinct categories according to IHC and SSCP results. Twenty-three (26%) lesions were positive in both techniques. Twenty-seven (31%) cases neither contained mutations detectable at SSCP nor stained for p53 protein and additional 13 (15%) SSCP-negative specimens demonstrated fewer than 10% stained tumor cells. These 13 lesions were tentatively scored as IHC negative, because multiple tumor sampling failed to produce a positive SSCP assay. Thus, a total correlation between IHC and SSCP methods was attained in 63 (72%) (p=.0013, Fisher exact test). Twelve (14%) lesions displayed moderate (++) or abundant (+++) p53 staining despite negative SSCP patterns. In contrast, 11 lesions without nuclear reactivity and 2 with focal (+) staining (15%) showed SSCP shifts. These latter cases were subjected to sequence analysis, and mutation was always found, confirming the good analytical power of SSCP [Table 2]. Ten mutations were either nonsense or frameshift alterations, thus generating a truncated protein. Two were base substitutions, leading to conservative amino acid changes (Arg to His, and Thr to Asn), whereas remaining base substitution was of non conservative type (Arg to Gly) and involved codon 280. Sixteen (45.7%) transition mutations, 10 (28.57%) transversions (from G:C to A:T in 8 of 10), and 9 (25.71%) insertiondeletion were found. Further 22 missense, 7 frame shift and 3 nonsense mutations and 4 frame deletions were identified. In this series, 13 mutations only involved DNA binding portion of p53 whilst the remnants 24 mutations determined conformational alterations resulting in an abnormal structure of this protein.