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Keywords:

  • laryngeal cancer;
  • aneuploidy;
  • fluorescence in situ hybridization;
  • precursor lesions;
  • head and neck cancer

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

BACKGROUND:

Most cases of laryngeal cancer are preceded by precursor lesions which, if left untreated, can progress toward an invasive cancer. The objective of this study was to investigate the presence of chromosomal numerical aberrations in cells that were collected by noninvasive brush sampling from laryngeal lesions.

METHODS:

Laryngeal brush samples from 52 patients were analyzed simultaneously for morphology and fluorescence in situ hybridization (FISH) using centromeric probes for chromosome 17, chromosome 8, and a locus-specific instability (LSI) v-myc avian myelocytomatosis viral oncogene homolog (myc) proto-oncogene protein (C-MYC) probe for the MYC gene. The patients were divided according to histopathologic diagnosis. Group 1 included patients with squamous cell carcinoma, carcinoma in situ, and severe dysplasia; Group 2 included patients with moderate dysplasia, mild dysplasia, and hyperplasia; and Group 3 included patients with benign nondysplastic lesions.

RESULTS:

The proportion of cells with MYC and chromosome 8 gains demonstrated significant trends toward being the highest in Group 1 and the lowest in Group 3 (P = .001 and P = .003, respectively). No significant trend was observed for chromosome 17. Mann-Whitney Bonferroni-corrected analyses revealed that the most significant contribution was the difference between Groups 1 and 3 (P = .0195 for MYC gains and P = .036 for chromosome 8 gains). When using a cutoff point of 4% aneuploid cells (ACs), both MYC and chromosome 8 differed significantly between groups (P = .030 and P = .037, respectively).

CONCLUSIONS:

The current results suggested that FISH analysis of brush samples obtained noninvasively from suspicious laryngeal lesions can augment the clinical examination in predicting the nature of the lesions and can aid clinicians in monitoring and follow-up of high-risk patients. Cancer (Cancer Cytopathol) 2011. © 2011 American Cancer Society.

Laryngeal cancer (mainly cancer of the vocal cords) is a common cancer in men world wide, and tobacco smoking is the main cause.1-4 It is believed that squamous cell carcinoma (SCC), which comprises >90% of laryngeal malignancies, progresses through a series of well defined clinical and histopathologic stages in the form of precursor lesions with various degrees of dysplasia.5-7 Clinically, these lesions appear mainly along the true vocal cord as white exophytic or shallow patches (leukoplakia).8 The clinical appearance, however, is not reliably diagnostic of the histologic grade, and histopathologic examination is essential for the diagnosis.7 Moreover, because >50% of reported leukoplakia lesions reveal no dysplasia on biopsy, these patients are subjected to unnecessary surgical procedures.6, 7 Certain prediction of malignancy in laryngeal lesions can be achieved with the aid of new technologies, such as videostroboscopy, contact endoscopy, fluorescence endoscopy, high-frequency ultrasound, and optical coherence tomography, however; these techniques are not qualified to completely assess epithelial alterations, and biopsy is always needed.9

Exfoliative cytology, although it is an established procedure in the diagnosis of carcinoma of the cervix and bronchus, has not been practiced to the same extent in premalignant laryngeal lesions. Laryngeal cytology has been investigated in several studies with variable results, ranging from an extremely high percentage of compatibility with the histopathologic finding to <50% compatibility.10-13 Therefore, exfoliative laryngeal cytology is used only sporadically, and the majority of otorhinolaryngology departments still prefer conventional biopsy, which remains the gold standard.10 Therefore, an objective, noninvasive method is needed for the early detection of laryngeal lesions that may progress to malignancy: a method that will be sensitive and easy to perform in the daily clinical setting.

Molecular cytogenetics may provide new insights into the potentially precancerous changes in cells obtained noninvasively from the surface of suspicious laryngeal lesions. Alteration in the number of chromosomes, defined as aneuploidy, stands out as the most consistent marker of malignancy and is the earliest and most distinctive preneoplastic genotype.14-16 DNA ploidy has been analyzed by several groups, and the results indicate that aneuploidy is an early event in laryngeal carcinogenesis.17-19 Studies using cytogenetic analysis, fluorescence in situ hybridization (FISH), and comparative genomic hybridization have demonstrated that a consistent set of chromosome regions frequently is altered in laryngeal SCC (LSCC), including gains on 3q, 5p, 8q, and 11q13 and losses on 3p and 9p. However, those studies either used archival material or were conducted on a limited number of patients with laryngeal cancer.

In several recent studies, we analyzed brush samples obtained noninvasively from various oral premalignant and malignant lesions, and the results suggested that aneuploidy is an early event in oral carcinogenesis.20-22 The results also suggested that the presence of numerical aberrations in cells obtained by brush samples predicts the malignant behavior of a suspicious oral lesion.20-22

The objective of the current study was to evaluate the presence of chromosomal numerical aberrations in cells that were collected by noninvasive brush sampling from laryngeal lesions using simultaneous morphologic and FISH analyses. The results were investigated for a correlation with the accepted gold-standard histopathologic diagnosis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Study Population

The study population consisted of 52 patients, including 46 consecutive patients who were referred to the Department of Otolaryngology Head and Neck Surgery at the Chaim Sheba Medical Center for evaluation of vocal cord pathology and 6 additional patients who had suspected recurrence of a previous laryngeal cancer and were studied as a separate group. We excluded patients who had any malignant disease other than laryngeal carcinoma and patients who were receiving treatment with steroids or other immunomodulators.

Consent was obtained from all patients in accordance with a protocol approved by the Institutional Review Board for Clinical Studies at the Sheba Medical Center and by the Ministry of Health for the use of genetic material. All study participants signed an informed consent form and completed a detailed questionnaire on smoking and drinking habits. The data collected included information on age, sex, signs and symptoms, sites of involvement, and the clinical description.

Sample Collection

Patients were examined before surgery, and a clinical diagnosis was given. All samples were taken in the operating room during a direct laryngoscopy procedure. A flexible brush was guided manually to reach the larynx, and fine rotational movements allowed the harvest of cells from the mucosa. Brush samples were taken from the mucosal surface of all inspected lesions regardless of the clinical diagnosis. The brush was placed in RPMI 1640 medium containing 10% fetal calf serum (Gibco-Invitrogen, Carlsbad, Calif). Only samples that had >50 cells collected for analysis were included in the study. After the brush sample, a biopsy sample was taken for histopathologic diagnosis.

Histopathologic Grading of the Biopsy Samples

All patients underwent excisional or incisional biopsy after the brush samples were obtained, and the tissues were submitted for histologic examination. All biopsy samples that we examined were formalin-fixed, paraffin-embedded tissues. Five-micron-thick slides were cut and stained with standard hematoxylin and eosin. The histopathologic diagnosis was confirmed for each lesion by 2 pathologist (B.S. and A.H.) using criteria established by the World Health Organization23 as hyperplasia (increased cell number with regular stratification), mild dysplasia (architectural disturbances limited to the lower third of the epithelium accompanied by cytologic atypia), moderate dysplasia (architectural disturbance extending into the middle third of the epithelium), severe dysplasia (architectural disturbances in over two-thirds of the epithelium with associated cytologic atypia), carcinoma in situ (full-thickness architectural abnormalities accompanied by pronounced cytologic atypia), invasive SCC. Patients were accepted for the study only when a consensus was reached on the classification of dysplasia. The patients were divided into the following 3 groups: Group 1 included patients who were diagnosed histologically with SCC, carcinoma in situ, and severe dysplasia (10 patients); Group 2 included patients who were diagnosed histologically with moderate dysplasia, mild dysplasia, and hyperplasia (11 patients); and Group 3 included patients who were diagnosed histologically with nondysplastic lesions (25 patients).

Combined Morphology and FISH Analysis

Combined analysis of morphology and FISH was performed using the Duet system (BioView Ltd., Rehovot, Israel). The parallel analysis of morphology and FISH allows the clear identification of small populations of nondiploid cells among overlapping diploid cells, enables clear distinction between epithelial and inflammatory cells, and avoids false polyploidy caused by overlapping nuclei. The technical procedure was described in detail in our previous publications.20-22

FISH was performed using alpha satellite probes for chromosomes 17 and 8 and a locus-specific instability (LSI) v-myc avian myelocytomatosis viral oncogene homolog (myc) proto-oncogene protein (C-MYC) probe for the MYC gene. Two slides from each sample were used. On 1 slide, dual-color FISH was performed using chromosome 17 labeled with SpectrumGreen and chromosome 8 labeled with SpectrumOrange (Vysis, Downers Grove, Ill) (Fig. 1a,b). On the second slide, the LSI C-MYC probe was used (Vysis) (Fig. 1c,d).

thumbnail image

Figure 1. (a-d) These photomicrographs illustrate simultaneous morphology and fluorescence in situ hybridization (FISH) analyses of brush-collected cells using centromeric probes (a,b) for chromosome 8 (red signals) and chromosome 17 (green signals) and (c,d) for LSI C-MYC probe for the MYC gene (yellow signals). The cells in a and c were stained with Giemsa, and the cells in b and d are the same cells shown in a and c, respectively, analyzed with FISH (original magnification, ×1000). In b, 3 epithelial cells are shown, including 1 normal diploid cell and 2 cells with 2 green signals and 3 red signals (arrow) denoting trisomy of chromosome 8. In d, an epithelial cell is shown with 3 yellow signals denoting MYC gene amplification.

Download figure to PowerPoint

The percentage of cells with >2 signals of chromosome 17, chromosome 8, and the MYC gene in the entire cell population was calculated for each sample (gains were defined as >2 copies). Aneuploid cells (ACs) were defined as cells that had >2 FISH signals. The selection of chromosomal probes for the current study was based on a literature review and our previous studies. Chromosome 8 was analyzed successfully to detect ACs in premalignant and malignant lesions in the oral mucosa.20, 21 Gains of chromosomes 8 and 17 reportedly are altered in LSCC,18, 24, 25 and 8q was the most common arm gain.26, 27 These chromosomal probes can disclose numerical as well as structural aberrations; for example, 3 signals of 8q can be detected even if the centromeric 8 probe yields 2 signals (ie, gains of the MYC gene, which is located on 8q24).

Statistics

A nonparametric analysis was used mainly because of the relatively small sample size and non-normal distribution of the variable of interest (the proportion of ACs). To test the trend, we used the Cuzick test for trend. This function provides a Wilcoxon-type test for trend across a group of 3 or more independent, random samples.28 In addition, the data were presented in tables using median, interquartile range, and range values. The Mann-Whitney test was used for comparisons between 2 groups, and the Kruskal-Wallis nonparametric analysis of variance was used for comparisons between more than 2 groups. The uncorrected P values for differences between all pairs of groups and P values after Bonferroni correction for multiple comparisons are presented. We used an “overall zero test” to separate patients with cancer (Group 1: SCC, carcinoma in situ, and severe dysplasia) from patients without cancer (Group 3). The test was defined as follows: If ACs were not identified in any of the 3 probes used (C-MYC, chromosome 8, and chromosome 17), then it was not cancer. A cutoff point for the proportion of ACs was determined as the maximum significant difference between the 3 groups. All P values are 2-sided, and calculations were done using the STATA SE 10 statistical software package (Stata Corporation, College Station, Tex).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Clinical and demographic data on the study population are presented in Tables 1 and 2. The mean patient age was 51 years (range, 19-79 years), and the ratio of men to women was 3:1. Hoarseness was the main symptom, and 40 patients (87%) experienced hoarseness before the first examination. Most patients were heavy smokers (34 patients; 74%), and their mean smoking history was 32 pack-years (range, 2-120 years). The percentage of smokers differed between the groups (P = .031) and was the lowest in Group 3. Age, sex, and smoking status did not have any significant effect on the correlation of ACs and histopathology.

Table 1. Clinical and Demographic Results From the Study Group
  Sex   
GroupMean Age [Range], yNo. of MenNo. of WomenNo. of Smokers (%)No. of Pack-Years [Range]No. With Hoarseness (%)
159 [38-79]918 (80)42 [23-120]9 (90)
253 [43-76]9211 (100)61 [26-120]7 (64)
344 [19-79]16915 (60)16 [2-80]24 (96)
Total51 [19-79]341234 (74)32 [2-120]40 (87)
Table 2. Clinical, Histopathologic, and Fluorescence In Situ Hybridization Results From the Study Group
       Percentage of ACs
       MYCChromosome 8Chromosome 17
PatientaAge, ySexSmokerClinical DiagnosisHistopathologyACs, %b3 Copies4 Copies3 Copies4 Copies3 Copies4 Copies
  • Abbreviations: AC, aneuploid cells; BL, benign lesion (polyp, cyst, nodule, or and Reinke edema); CIS, carcinoma in situ; Dys, dysplasia; ND, no dysplasia; SCC, squamous cell carcinoma; VF, vocal fold.

  • a

    Group 1 included patients with squamous cell carcinoma, carcinoma in situ, and severe dysplasia; Group 2 included patients with moderate dysplasia, mild dysplasia, and hyperplasia; and Group 3 included patients with benign nondysplastic lesions.

  • b

    The proportion of ACs presented is the highest proportion observed in each sample.

  • c

    Five percent of the cells had over 6 signals of MYC gene.

  • d

    In Patient 12, chromosome 17 monosomy was observed in 15% of cells, which was an exceptional event in the current study.

Group 1            
 174ManNoLeukoplakiaSCC95905c
 260ManYesExophytic lesionSCC8060205322
 376ManYesLeukoplakiaSCC2200000
 457ManNomonocorditisCIS262062323
 561ManYesLeukoplakiaCIS181448020
 679ManYesLeukoplakiaCIS8326000
 738ManYesmonocorditisCIS220
 869ManYesLeukoplakiaSevere Dys2525000
 955WomanYesExophytic lesionSevere Dys16160150
 1079ManYesLeukoplakiaSevere Dys5.55.503.5000
Group 2            
 1176ManYesLeukoplakiaModerate Dys202006000
 1265ManYesBLModerate Dys15d000000
 1351WomanYesBLMild Dys5045050000
 1451ManYesLeukoplakiaMild Dys3536035000
 1543ManYesLeukoplakiaMild Dys5.75.7%01.40
 1654ManYesleukoplakiaMild Dys54101
 1748ManYesleukoplakiaMild Dys4.64.602.7000
 1852WomanYesBLMild Dys1.51.50
 1957ManYesLeukoplakiaHyperplasia7516000
 2031ManYesLeukoplakiaHyperplasia0000000
 2155ManYesBLHyperplasia0000000
Group 3            
 2255ManYesLeukoplakiaND3636036000
 2336ManYesBLND25020250120
 2447ManYesBLND8.38.3000
 2555ManYesVF irregularityND7703.403.40
 2653WomanYesBLND88070
 2745WomanNoSulcusND6601.4000
 2879ManYesLeukoplakiaND66000
 2943ManNoBLND5500000
 3054ManYesBLND55000
 3141WomanNoBLND404
 3220WomanNoBLND440
 3340WomanNoBLND3.53.50002.70
 3430ManNoBLND2.52.500000
 3543ManNoBLND2201.2501.250
 3658ManYesBLND1.61.60001.20
 3754ManYesBLND1001000
 3856ManYesBLND0.50.5000.300.3
 3947ManYesBLND0000000
 4059WomanYesBLND0000000
 4119ManYesBLND0000000
 4242ManYesBLND0000000
 4321WomanNoBLND0000000
 4443WomanNoBLND0000000
 4550ManYesBLND0000000
 4636WomanNoBLND0000000

Before surgery, all patients were examined received a clinical diagnosis (Table 2). In Group 1, 6 patients were diagnosed with leukoplakia, 2 were diagnosed with exophytic lesions, and 2 were diagnosed with monocorditis. In Group 2, 7 patients were diagnosed clinically with leukoplakia, 3 were diagnosed with Reinke edema, and 1 was diagnosed with a nodule. In Group 3, most patients were diagnosed with clinically benign lesions (polyp, cyst, nodule, or Reinke edema), 1 was diagnosed with a vocal fold irregularity, 1 was diagnosed with sulcus, and only 2 were diagnosed with leukoplakia.

Combined FISH and Morphologic Analysis

An average of 170 cells (range, 50-750 cells) were analyzed in each sample. Because of the limited number of cells in some samples, FISH was performed with only 1 or 2 probes. ACs were detected in 36 samples (Tables 2 and 3). The number of patients who had detectable ACs and the percentage of ACs in the samples increased from Group 3 to Group 1. ACs were identified in all samples from Group 1 (range, 2%-95%), in 9 of 11 samples from Group 2 (range, 1.5%-50%), and in 15 of 25 samples from Group 3 (in all but 2 samples, the range of ACs was 0.5%-8.3% of the examined cells). In Patients 22 and 23, 36% and 25% ACs were detected, respectively.

Table 3. Median, Interquartile Range, and Range for Proportions of Aneuploid Cells
 MYC, %Chromosome 8, %Chromosome 17, %
GroupaRangeMedian (IQR)RangeMedian (IQR)RangeMedian (IQR)
  • Abbreviation: IQR, interquartile range.

  • a

    Group 1 included patients with squamous cell carcinoma, carcinoma in situ, and severe dysplasia; Group 2 included patients with moderate dysplasia, mild dysplasia, and hyperplasia; and Group 3 included patients with benign nondysplastic lesions.

  • b

    Fifteen percent had deletion of chromosome 17.

12-955.5 (3-20)0-758 (2.75-20.7)0-50 (0-2)
20-454.8 (0-20)0-504 (1.4-6)0-15b0 (0-0)
30-360.5 (0-4)0-360 (0-5)0-120 (0-0)

The Cuzick test for trend revealed that the proportion of cells with MYC gains demonstrated a significant trend and as highest in Group 1 and lowest in Group 3 (P = .001). The trend also was significant for chromosome 8 gains (P = .003). No significant trend was observed for chromosome 17. The median, interquartile range, and range values for the proportions of ACs in the 3 groups are presented in Table 3. In Group 1, the median proportion of ACs was higher for both MYC and chromosome 8 gains compared with Groups 2 and 3.

Table 4 presents a statistical analysis of the comparison between the 3 groups regarding the proportion of ACs. Kruskal-Wallis analysis revealed a significant difference between the groups with regard to MYC and chromosome 8 gains (P = .0257 and P = .0239, respectively). This difference was not observed with regard to chromosome 17 gains (P = .3104). Mann-Whitney analysis revealed that the most significant contribution was the difference between Group 1 and Group 3 (MYC gains, P = .0065; chromosome 8 gains, P = .0120). No difference was observed between Groups 1 and 2 or between Groups 2 and 3 regarding all probes used. It is important to note that the differences between Groups 1 and 3 for MYC and chromosome 8 remained significant after Bonferroni correction for multiple comparisons (P = .019 and P = .036, respectively).

Table 4. Statistical Analysis of Comparisons Between the 3 Groups Regarding the Proportion of Aneuploid Cells
 P
ComparisonaMYCChromosome 8Chromosome 17
  • Abbreviation: ANOVA, analysis of variance.

  • a

    Group 1 included patients with squamous cell carcinoma, CIS, and severe dysplasia; Group 2 included patients with moderate dysplasia, mild dysplasia, and hyperplasia; and Group 3 included patients with benign nondysplastic lesions.

  • b

    P value after Bonferroni correction for multiple comparisons.

Kruskal-Wallis ANOVA   
 Groups 1, 2, and 3.0257.0239.3104
Mann-Whitney comparison of pairs of groups   
 Groups 1 and 2.4367.4397 
 Groups 2 and 3.1601.0766 
 Groups 1 and 3.0065 (.0195b).0120 (.036b) 

ACs were not detected in 10 samples (21.7%). Two of those samples were diagnosed as hyperplasia (Group 2), and 8 were diagnosed as nondysplastic lesions (Group 3). By using the Cuzick test for trend, we observed that the proportion of patients with no MYC and chromosome 8 gains demonstrated a significant trend toward being lowest in Group 1 and highest in Group 3 (P = .033 and P = .016, respectively). No significant trend was observed for chromosome 17. The specificity of the “overall zero test” (percentage of correct answers in cancer patients) is 100% (Wilson 95% confidence interval, 72%-100%), and its sensitivity (percentage of correct answers in patients without cancer) is 33% (Wilson 95% confidence interval, 20%-50%). An exceptional finding was Patient 12, in which chromosome 17 monosomy was detected in 15% of cells.

Cutoff Point

A search for a cutoff point revealed that, when using a 4% ACs cutoff point, both MYC and chromosome 8 differed significantly between the groups (P = .030 and P = .037, respectively). The percentage of patients with >4% ACs increased in relation to the histologic degree of dysplasia and was 36% in Group 3, 73% in Group 2, and 80% in Group 1 (P = .028). Of 24 patients who had samples with >4% ACs, 21 were smokers; whereas, of 22 patients who had samples with <4% ACs, 13 were smokers (P = .044).

Post-SCC Cases

The separate group of 6 patients who previously had laryngeal cancer was analyzed with the same probes (Table 5). All patients had results that were clinically suspicious for disease recurrence. Three patients were diagnosed histopathologically with recurrent carcinoma, 1 patient was diagnosed with hyperplasia, and 2 patients were diagnosed with nondysplastic lesions. ACs were identified in all 6 samples; however, greater proportions of ACs were detected in samples from patients who had carcinoma (range, 6.7%-80%) compared with samples from patients who had nonmalignant lesions (2.8%-4%).

Table 5. Clinical, Histopathologic, and Fluorescence In Situ Hybridization Results in Patients With Suspected Recurrence of a Laryngeal Carcinoma
    Percentage of ACs
    MYCChromosome 8Chromosome 17
PatientClinical DiagnosisHistopathologyACs, %3 Copies4 Copies3 Copies4 Copies3 Copies4 Copies
  1. Abbreviations: AC, aneuploid cells; CIS, carcinoma in-situ; ND, no dysplasia; SCC, squamous cell carcinoma.

12780LeukoplakiaCIS80800800100
14608LeukoplakiaSCC18.3171.300
17068Exophytic lesionSCC6.76.706000
15676LeukoplakiaHyperplasia2.82.802.8000
16524Exophytic lesionND4401.3000
16526Exophytic lesionND33000

Follow-Up

In most patients, follow-up was short (up to 2 years) and included direct laryngoscopic examination, which did not reveal progression in any patients. Two patients were lost to follow-up. All patients who had invasive cancer and carcinoma in situ received radiotherapy, and 2 other patients underwent surgery. No patients developed recurrent tumors.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

The results from this study emphasize the importance of noninvasive analysis using interphase FISH as a specific monitor of aneuploidy in patients with laryngeal carcinogenesis. We have demonstrated that aneuploidy involving chromosome 8 and gains in the MYC gene increase progressively alongside the well defined histologic groups.

The current standard for assessing the risk of developing cancer in suspicious laryngeal lesions depends on histologic examination. The decision whether to biopsy a suspicious lesion relies solely on clinical examination; however, physical examination with laryngoscopy has proven inadequate for the detection of dysplastic and malignant lesions, and >50% of the reported leukoplakia lesions reveal no dysplasia on biopsy.7 In addition, these patients often undergo unnecessary biopsy procedures, which may disrupt their vocal cord function.

FISH analysis can provide a reliable, objective technique that can augment the clinical and cytologic examinations. Our current results suggest that noninvasive monitoring of aneuploidy in suspicious lesions may provide useful additional information for the clinical management of these patients. In particular, such monitoring may help identify those patients who are at low risk for cancer when no gains of MYC or chromosome 8 are detected. Conversely, when aneuploidy and MYC gains are detected, noninvasive monitoring of aneuploidy may help identify those patients who are at increased risk developing recurrent disease and, thus, require closer surveillance.

Because of its relative ease of performance and high resolution, FISH is used extensively to screen for the common chromosomal abnormalities, both numerical and structural, this technique is now offered by the majority of cytogenetic laboratories. The advantages of the combined morphologic and FISH analysis used in the current study include the rapid and efficient identification of small, residual populations of ACs among overlapping diploid cells; the distinction of ACs from proliferating mitotic cells and from inflammatory cells; and avoiding false polyploidy caused by overlapping nuclei. This method has been tested successfully by us and by other authors.29, 30 Chromosome 8, chromosome 17, and MYC were chosen for the current analysis based on the literature and our previous studies.20-23, 25, 31 Chromosome 8 is the site of several genes that have crucial functions in the tumorigenesis of several solid and hematopoietic malignancies.32-38 Among the genes on chromosome 8, MYC, located at 8q24, has been studied the most, and overexpression of the MYC gene is a frequent alteration and has been described in several types of human cancer.39-41

The proportion of ACs with MYC and chromosome 8 gains demonstrated a significant trend toward being highest in Group 1 and lowest in Group 3 (Cuzick test for trend; P = .001 and P = .003, respectively), but this trend was not observed for chromosome 17. These results are in agreement with previous studies in which the most common chromosomal gains observed in LSCC were 8q gains and especially gains and amplifications of the MYC gene.7, 39, 40 Concerning chromosome 17, Gale et al42 indicated that numerical changes in chromosomes 7 and 17 may contribute to critical events in laryngeal carcinogenesis. Those results suggested that chromosome 17 gain is not a constant event in laryngeal carcinogenesis. In the current study, an interesting finding in 1 sample (Patient 12) was the detection of chromosome 17 monosomy in 15% of cells. Chromosome 17 monosomy has been described previously in patients with head and neck SCC and has been associated with p53 overexpression.43 Because p53 is a crucial gene in head and neck carcinogenesis, it is assumed that chromosome 17 monosomy is the consequence of p53 gene alteration.43 Nevertheless, because we detected only a single example of chromosome 17 monosomy, conclusions cannot be made.

Several studies have provided evidence supporting the role of genetic tests in augmenting histopathologic evaluation of the risk for developing laryngeal cancer using microsatellite analysis to determine loss of heterozygosity in chromosome regions mainly at 9p, 3p, and 17p.24, 44 Nevertheless, caution is necessary in interpreting those data and their potential clinical implications, because they were retrospective studies and contained unavoidable selection bias, which often makes it difficult to reach a final conclusion.

Several authors have associated aneuploidy with an increased risk of developing laryngeal cancer. Abou-Elhamd and Habib,45 using flow cytometry, observed abnormal DNA content in 44% of precancerous lesions from head and neck origin, including the larynx. Chi et al19 observed aneuploidy in 20 of 23 patients with leukoplakia that progressed to cancer. Gale et al42 used non-FISH analysis and observed that polysomy of chromosomes 7 and 17 was correlated with progressive histologic grades. In several studies, Veltman and colleagues17, 18, 24 reported the presence of numerical aberrations for chromosomes 1 and 7 in dysplastic laryngeal lesions, especially in those that progressed to cancer. However, most of those studies used archival material (mainly paraffin-embedded tissue), and the use of paraffin-embedded tissue sections can be accompanied by confounding problems, such as nuclear slicing, variations of hybridization efficiency, and counting error, which may be responsible for the excess of monosomic cells.

A search of the literature disclosed only 1 study that used FISH analysis on brush sampling of laryngeal lesions.17 That study indicated that most tumor brush specimens contained numeric chromosomal aberrations in at least 5% of the cells collected; however, the authors analyzed only 10 patients with laryngeal cancer.17 To our knowledge, the current study is the first to use molecular cytogenetics as an aid in augmenting the clinical evaluation of laryngeal lesions using noninvasive brush sampling. The proportion of cells with MYC and chromosome 8 gains demonstrated a significant trend toward being highest in patients with histopathologically diagnosed severe dysplasia, carcinoma in situ, and invasive cancer (Group 1) and lowest in patients with benign nondysplastic lesions (Group 3; Cuzick test for trend; P = .001 and P = .003, respectively). Mann-Whitney analysis revealed that the most significant contribution was the differences between Group 1 and Group 3, which remained significant after Bonferroni correction for multiple comparisons (P = .0195 for MYC gains and P = .036 for chromosome 8 gains). Patients in Group 2 did not differ significantly from either Group 1 or Group 3 with regard with the proportion of ACs when all samples were analyzed. This may be explained by the subjectivity of the histopathologic examination and because at least some patients in Group 2 are in a transitional state in which genetic changes already are occurring but no morphologic changes can be detected in the mucosa. When ACs are not observed in a sample, it is most probably a noncancerous lesion (the specificity of the “overall zero test” was 100%; Wilson 95% confidence interval, 72%-100%).

The search for a cutoff point revealed that the percentage of patients with >4% ACs increased in relation to the histologic degree of dysplasia (36% in Group 3, 73% in Group 2, and 80% in Group 1; P = .028); and both MYC and chromosome 8 differed significantly between the groups (P = .030 and P = .037, respectively). Most patients who had samples with >4% ACs were heavy smokers (P = .044), which correlates well with the finding that smoking is related directly to laryngeal cancer development.1-4 The 4% cutoff point also was tested on a limited number of post-SCC patients who had clinical findings that were suspicious for recurrence. Although those results were not statistically significant, patients in that group who were diagnosed histopathologically with carcinoma had >6.7% ACs compared with <4% ACs in the 3 patients who were diagnosed with nondysplastic lesions.

The severity of dysplasia on the initial biopsy of a laryngeal lesion has been associated with an increased risk of malignant conversion;7 nevertheless, a 3.8% chance of developing invasive laryngeal cancer was reported even in the absence of dysplasia.7 Nine patients with nondysplastic lesions (in Group 3) and 6 patients with mild dysplasia and hyperplasia (Group 2) had >4% ACs in the collected samples, and these patients usually are classified as low-risk and are followed only for a short period (up to 2 years).46, 47 Therefore, we suggest that these patients should be classified as high-risk and should be controlled in short intervals for long periods by direct laryngoscopic inspection; and, whenever needed, with the aid of brush sampling and FISH analysis, they should undergo a biopsy for any suspected lesion. In the current study, 2 years of follow-up did not reveal progression in any of our patients; however, the follow-up was relative short in view of the slow progression of laryngeal precursor lesions,7 mainly in smokers, because the latency between carcinogen exposure and the appearance of malignancy may be as long as 25 years.48

Because it is a recently developed method, the clinical application of simultaneous morphology and FISH analysis of laryngeal brush samples has yet to be established, and larger studies are needed. Future studies should include longer periods of follow-up and correlation of FISH analysis with cytology and histopathology results. Nevertheless, as a noninvasive technique that can augment the clinical examination in predicting the nature of lesions without the need for a biopsy, this method has potentially significant clinical benefits. In the current study and in our previous studies in oral mucosa,20-22 we observed a high correlation between the simultaneous morphology and FISH analysis method and the gold-standard histopathologic diagnosis. Thus, we believe that this method has potential as a powerful tool for clinicians to monitor and follow high-risk patients and can help avoid unnecessary surgical interventions.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

This work was supported by the Flight Attendants Medical Research Institute (Grant 42214) and in part by the Israel Cancer Association (Grant 20100092).

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

We thank Dr. Galina Yshoev for her excellent technical assistance and Dr. Ilya Novikov for the statistical analysis.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES