Abstract
Background/Aim: Due to the high recurrence rates of squamous cell carcinoma of the head and neck (SCCHN) and de-novo local secondary carcinomas, a close monitoring of patients is mandatory. In doubtful cases, a clearance by histological biopsy is necessary. This, however, bears potential complications. We analyzed the application of combined cytology and immunocytology in cytological brush smears for diagnosing pre-malignant and malignant lesions of the oral/oropharyngeal cavity. Materials and Methods: Brush biopsies of 30 subsequently histologically-confirmed oral/oropharyngo-/laryngeal cavity cancer cases (all then in a recurrence status) and normal mucosa were obtained for routine cytology and immunocytology for cytokeratin-8 (CK-8). Additionally 20 samples with inflammatory lesions were investigated. Results: Our results showed a high rate for positive prediction of oral/oropharyngo-/laryngeal dysplasia/cancer cases. Accordingly, 82% of all subsequently confirmed cases were detected by cytology alone (sensitivity). The specificity, however, of cytology was distinctly lower since several doubtful cases contained only inflammatory lesions (specificity 85%). The addition of CK-8-immunocytology did not increase the sensitivity, since the rate of detected cases by immunocytology was comparable to routine cytology (79%); however, the addition of immunocytology significantly increased the specificity (up to 90%). Conclusion: Routine cytology is a simple, non-invasive and cost-effective method for routine control and screening of dysplastic oral/oropharyngo-/laryngeal lesions. In doubtful cases, the addition of CK-8-immunocytology is very helpful for the distinction of reactive from neoplastic cases.
The diagnosis of malignant squamous cell carcinomas of the oral/oropharyngo-/ laryngeal region is usually established through histology. While this initial confirmation by biopsy is mandatory, it is difficult to monitor the post-therapeutic status by recurrent biopsies, due to the risks and side-effects of repeated traumatization of the tissue, enhanced bleeding risk, infection etc. Furthermore, it is well-known that both the recurrence of squamous cell carcinoma of the head and neck (SCCHN) and the incidence of locoregional secondary carcinomas are high (1). This is due to the fact that field carcinogenesis - mainly caused by tobacco and alcohol - affect the complete oro-pharyngo-/laryngeal area (2). It is, therefore, of utmost importance to early identify cancer cases particularly during the follow-up of these patients. In order to reduce the risks of complications other procedures than histology may be sought. Other medical specialities, such as gynecology or pneumology, already use brush cytology for monitoring of cervical and pulmonary cancer.
The aim of the present study was to investigate the efficiency of cytology on the identification of pre-malignant and malignant lesions of the oral/oropharyngo-/larygeal cavity during the monitoring process of curatively treated tumor patients and to test the application of special tests by immunocytology in order to enhance the screening results.
Material and Methods
Materials. Oral brushes were obtained from 30 patients with histologically-confirmed squamous cell carcinoma of the oral cavity, the pharynx or the larynx (SCCHN) during post-treatment periods with the clinical features of tumor recurrence or doubtful cases of recurrent tumor. The smears were obtained from normal and pathological mucosa by using a lamellar brush as routinely done in gynecological testings. Additionally 20 cases with histologically confirmed inflammatory mucosa lesions were additionally investigated for comparison. In all cases, subsequent tissue biopsies were removed for histological investigation.
Methods. Routine staining of the smears was performed by May-Grünwald-Giemsa stains (MGG). Additionally, a parallel preparation of each smear was used for immunocytochemical staining for cytokeratin-8 using a monospecific antibody (Dako GmbH, Hamburg, Germany). The antibody reaction was visualized by a secondary antibody system (detection system APAAP) as also routinely done (Zytomed Systems GmbH, Berlin, Germany). Parallel histological biopsies of the same region were histologically investigated following routine embedding into paraffin wax. All samples were subsequently evaluated by light microscopy.
Results
In 6/50 cases (12%) the immunocytochemical smear could not be evaluated due to insufficient quality (drying-up of the sample, staining artifact), while all routine smears could be assessed.
By routine cytology, 82% of subsequently confirmed cancer cases (Figure 1G) could be identified as dysplastic/probably dysplastic. In the inflammation group (Figure 1D) there were 14% false-positive/doubtful cases; 4% of normal mucosa (Figure 1A) smears were also ranked as doubtful (Figure 1B, 1E and 1H).
By immunocytochemistry, using cytokeratin-8 antibodies (CK-8), it was seen that 79% of cancer cases were positively identified as dysplastic/possibly dysplastic (Figure 1I) that is not significantly different from routine cytology. Inflammatory cases were false-positive/doubtful in 9% of cases (Figure 1F), but none of the normal mucosa cases reacted positive (0%, Figure 1C).
Discussion
Generally, the prognosis of SCCHN is still very poor. The five-year survival of advanced-stage cancer cases ranges to only 22%-50%. A major problem is local recurrence and the high incidence of a secondary carcinoma in the head and neck region (1). This fact is mainly due to field cancerization caused by alcohol and tobacco (3). Routinely, the diagnosis of tumor recurrence/de novo-carcinoma is assessed by biopsy that is an invasive procedure with possible complications. Therefore, other potential diagnostic procedures may be evaluated for monitoring cancer patients. The aim of our study was therefore to analyze the results of a combination of cytology and immunocytochemistry for diagnosing pre-malignant and malignant lesions of the oral cavity/oropharynx or larynx.
Previously, Shroff (4) already proved that bronchial brush cytology provides good values for sensitivity and accuracy in the diagnosis of bronchopulmonary carcinomas. Similarly, Choudhury et al. (5) confirmed those observations in bronchial brush cytology and bronchial washings for the diagnosis of non-neoplastic versus neoplastic bronchopulmonary lesions. They could demonstrate that both, sensitivity (81%) and specificity (86%) were sufficiently high to identify cancer cases. Therefore, bronchial brush cytology has been established as a good diagnostic tool in certain cases, particularly in those cases where tissue biopsies provide enhanced risks of side-effects or complications.
Very similarly and established since many decades, the screening for cervical cancer smear preparates is routinely used including different forms and extent of cervical cancer (6, 7, 8, 9). These studies demonstrated a very high accuracy of cytology in various conditions.
To date, head and neck cancer is routinely diagnosed by biopsy. We could show a high sensitivity and high specificity of oropharyngeal brush biopsies. Up until now, no similar study has been performed on post-treatment monitoring samples. However, existing studies on the value of smear cytology compared to histology in pre-treatment cases revealed a very high conformity of the tests with 89% of identical results between both techniques (10). Similar results have also been reported by others (11, 12, 13) with specificity ranges between 80 and 100% which is at a comparable range as in our study.
Cytokeratin-8 is an intermediate filament protein with aberrant expression on the cell surface of SCCHN (14). Histological findings demonstrate that with increasing de-differentiation the amount of CK-8 expression increases (15). In our study, we combined routine cytology with immunocytology for CK-8 which increased the specificity for the detection of malignant/pre-malignant conditions.
Oropharyngeal cancer is in 25% of cases associated with HPV-infection (16). Holmes et al. (17) showed that brushes of oropharyngeal cancer are also suitable for HPV analysis. Using the hybrid capture 2 assay, already widely used for high-risk HPV in cervical brushes to squamous cell carcinoma of the head and neck, all cytologic preparations were classified correctly (18).
In summary, our data demonstrate that routine cytology is a simple, non-invasive and cost-effective method for routine control and screening of dysplastic oropharyngeal lesions. Using additional immuncytochemical methods the specificity of detection of dysplastic lesions is increased.
Histology, cytology and immunocytochemistry of normal oral mucosa, inflammatory oral lesion and invasive oral squamous cell cancer in comparison. Upper row: Normal oral mucosa: (from left to right) (A) H&E (magnification ×100), (B) cytology (MGG, ×400), (C) immunocytochemistry (CK-8, ×400). Middle row: Inflammatory lesion of oral mucosa: (from left to right) (D) H&E (×100), (E) cytology (MGG, ×400), (F) immunocytochemistry (CK-8, ×400). Lower row: (G) invasive squamous cell carcinoma of the oral mucosa: (from left to right) (G) H&E (×100), (H) cytology (MGG, ×400), (I) immunocytochemistry (CK8, ×600).
- Received August 29, 2015.
- Revision received September 1, 2015.
- Accepted September 5, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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