Maciej D. Zatoński
1, Tomasz Kręcicki
2, Agnieszka Jabłonka-Strom
1,
Agnieszka Zatońska
3, Agata Górecka
4, Marek Bochnia
1Autofluorescence Endoscopy
in Diagnosis of Laryngeal Lesions
Endoskopia autofluorescencyjna w diagnostyce
zmian nabłonkowych krtani
1 Department of Otolaryngology, Faculty of Dentistry, Wroclaw Medical University, Poland 2 Department of Otolaryngology, Head and Neck Surgery, Wroclaw Medical University, Poland 3 Department of Otolaryngology, District Railway Hospital, Wroclaw, Poland
4 Department of Otolaryngology, University Hospital (ASK), Wroclaw, Poland
Abstract
Background. One of the most important problems in head and neck oncology is early diagnosis of laryngeal cancer. Despite the introduction of new therapeutic methods and improvement of surgical techniques, patients’ 5-year survival rate still depends primarily on the stage of the disease at the moment of introduction of appropriate treatment.
Objectives. The goal of this study was to assess the efficacy of autofluorescence endoscopy (AFE) in diagnosis of laryngeal lesions. AFE examinations were carried out on out-patient basis, without general anesthesia and without using any photosensitizing agents (topical or systemic). Sensitivity and specificity of AFE alone, as well as AFE combined with classic white-light endoscopy, were calculated.
Material and Methods. 129 patients (30 women and 99 men) at the age between 22–89 with various laryngeal pathologies entered the study. Patients did not receive any photosensitizing agents. No tissue staining was used. Occasionally, the patients received local 10% Xylocaine anesthesia before the examination. All examinations were performed using modified laryngeal endoscopic setup. All patients underwent classical white-light endoscopy fol-lowed by AFE examination. Sites of possible pathologies were noted for further biopsy. The results obtained during white-light endoscopy and AFE were then compared to histopathological findings. Sensitivity and specificity were calculated.
Results. Suspected malignant lesions were confirmed in 63 out of 72 cases when using white-light endoscopy, and in 71 out of 72 during AFE (p = 0.0087). In precancerous lesions, 9 out of 19 were identified during white-light endoscopy, while AFE allowed to properly identify 16 out of 19 precancerous lesions (p = 0.0166). In all lesions, histologically classified as benign, the authors received 18 false positive results (white-light endoscopy) and 7 false positive results with AFE (p = 0.0072). Overall sensitivity of AFE was 95.6% and specificity was 81.6%. For white-light endoscopy the results were 79.1% and 52.6% respectively. Combined specificity of both methods was 96.7%.
Conclusions. AFE is an effective, supplementary method of diagnosing pathologies of the glottic as well as supra-glottic region of the larynx, that can be used in routine out-patient care. AFE is completely safe and has a very high sensitivity in detecting invasive cancer as well as early precancerous lesions (starting from LIN II). It allows to precisely visualize the extensions of the lesion and can be used to diagnose changes in supraglottic region. AFE can also be used in monitoring patients after treatment and in early detection of possible recurrence (Adv Clin Exp Med 2010, 19, 4, 461–467).
Key words: laryngeal cancer, autofluorescence, early diagnosis.
Streszczenie
Wprowadzenie. Diagnostyka i leczenie raka krtani jest dziś jednym z największych wyzwań w onkologii głowy i szyi. Mimo rozwoju nowych metod terapeutycznych i wprowadzeniu nowoczesnych technik chirurgicznych, w dalszym ciągu 5-letnie przeżycia w przypadku raka krtani zależą prawie wyłącznie od stanu zaawansowania choroby w chwili rozpoczęcia leczenia.
Adv Clin Exp Med 2010, 19, 4, 461–467 ISSN 1230-025X
ORIGINAL PAPERS
Cancer of the larynx makes 2–4% of all ma-lignant tumors and is the 6th most common cause
of death out of all deaths caused by malignant tu-mors in men in Poland [1]. Prognoses and survival rates depend strongly on the tumors’ advancement (stage) at the introduction of appropriate therapy. In Poland only 26% of all malignant laryngeal tu-mors is at early stages of development at the time of initial diagnosis. Therefore early diagnostics and treatment of laryngeal cancer is one of the most im-portant challenges in the head and neck oncology.
Lesions that originate in the glottic region usually present with hoarseness, which is often noticed by patients, their families or their physi-cians. In other cases, often the first noticeable sign of the neoplastic process is enlargement of regional lymph nodes. Early diagnosis of laryngeal tumors allows to introduce the treatment at early stages of the disease and increases both – the chances of sur-vival and the quality of life of patients treated from laryngeal cancer.
Most of described in the literature autofluores-cence diagnostic procedures requires general an-esthesia or photosensitizing agents. The technique described below lacks the above mentioned limita-tions. It allows to use autofluorescence endoscopy (AFE) during routine endoscopic examination of the larynx.
The goal of this study was to assess: a) the effi-ciency of AFE in diagnosing laryngeal pathologies in comparison to classic white-light endoscopy; b) specificity and sensitivity of AFE as a stand-alone method and combined with classic
endo-scopic examination; c) usefulness of AFE in treat-ment monitoring. The results were compared to histopathological findings.
Material and Methods
Material included 129 patients (30 women and 99 men). The mean age of patients was 58.6 years (22 to 89 years old). Ninety one patients with suspected malignant lesions and 38 patients with benign changes (Reinke’s Oedema, papillomas, polyps, vocal nodules, granulomas, chronic hyper-trophic laryngitis). The authors did not use general anesthesia. Sporadically the patients were sprayed with 10% Xylocaine solution. Patients did not re-ceive any photosensitizing agents, neither topically nor systemic. No tissue staining was used.
The examinations were performed using modi-fied STORZ set-up for laryngeal endoscopy. It con-sisted of white-light source (xenon) with possibil-ity to switch to blue-light during the examination, optic fiber and rigid laryngeal 70o endoscope. The
endoscope was connected to high-sensitivity CCD camera (Telecam PDD Storz). The set was equipped with system of filters eliminating reflected excita-tion light (blue). The filters were able to pass the light waves around 400 nm and over 450 nm. The pictures from the camera obtained during white and blue light examinations were recorded and stored on a PC-class computer for further assessment.
The examination started with classic white light endoscopy. The landmarks suspected of neoplastic
Cel pracy. Ocena skuteczności endoskopii autofluorescencyjnej (AFE) w diagnostyce zmian nabłonkowych w krta-ni. Opisana metoda pozwala na wykonywanie badania w trybie ambulatoryjnym, bez ogólnego znieczulenia i bez konieczności stosowania środków fotouczulających (zarówno miejscowo, jak i ogólnie). Obliczono czułość i specy-ficzność badania z wykorzystaniem endoskopii autofluorescencyjnej jako samodzielnej metody diagnostycznej oraz w połączeniu z klasycznym badaniem endoskopowym w białym świetle.
Materiał i metody. Badaniem objęto 129 pacjentów (30 kobiet i 99 mężczyzn) w wieku 22–89 lat, którzy zgłosili się do pracowni endoskopowej Kliniki Otolaryngologii. Nie podawano żadnych środków fotouczulających ani barwni-ków tkankowych. Sporadycznie stosowano znieczulenie miejscowe 10% roztworem Xylocainy przed rozpoczęciem badania. AFE wykonywano z użyciem zmodyfikowanego zestawu do endoskopii krtani zaraz po klasycznym badaniu endoskopowym. Z podejrzanych obszarów krtani pobierano następnie wycinki do badania histologicznego. Wyniki otrzymane w czasie badań endoskopowych i AFE były porównywane do wyników badań histopatologicznych.
Wyniki. Podejrzenie zmian złośliwych potwierdziło się w 63 z 72 przypadków podczas klasycznej endoskopii i w 71 z 72 przypadków w badaniu AFE (p = 0,0087). W przypadku zmian przedrakowych podczas badania w świe-tle białym zidentyfikowano 9 z 19 przypadków, a z użyciem AFE 16 z 19 (p = 0,0166). W zmianach zakwalifiko-wanych histologicznie jako łagodne otrzymano 18 fałszywie dodatnich wyników (endoskopia klasyczna) oraz 7 po badaniu AFE (p = 0,0072). Czułość AFE wyniosła 95,6%, a specyficzność 79,1%. Dla klasycznej endoskopii wyniki wynosiły odpowiednio 81,6% oraz 52,6%. Łączna czułość obu metod diagnostycznych (endoskopia w świetle białym + AFE) zastosowanych łącznie wyniosła 96,7%.
Wnioski. AFE jest skuteczną uzupełniającą metodą diagnostyczną, pozwalającą wcześnie rozpoznawać zmiany w krta-ni (zarówno w głośkrta-ni, jak i nadgłośkrta-ni). Może być używana podczas rutynowego badakrta-nia w trybie ambulatoryjnym. AFE jest całkowicie bezpieczna dla pacjenta. Charakteryzuje się bardzo wysoką czułością w rozpoznawaniu zmian nowotworowych i przednowotworowych (już od LIN II). Pozwala na precyzyjną ocenę rozległości zmian i może być używana do wczesnej diagnostyki zmian w nadgłośni. AFE może być także używana w monitorowaniu pacjentów po leczeniu oraz do wczesnego wykrywania ewentualnej wznowy (Adv Clin Exp Med 2010, 19, 4, 461–467).
changes were noted for further biopsy. When nec-essary stroboscopic examination was performed. AFE was performed right after white-light exami-nation. The picture from the camera was observed in real-time and stored on a PC computer.
All the patients suspected for malignant changes underwent standard biopsy. The material was sent for histopathological examination. The spots for biopsy were identified during previously performed white light and blue light examinations. Further treatment of the patients was dependent on histopathological findings. Material obtained by removing benign lesions underwent histopatho-logical evaluation post-operatively.
Digitally stored white and blue light examina-tions were then compared to the histopathological findings. The endoscopic findings were classified as positive, negative, false positive and false nega-tive to assess sensitivity and specificity of AFE and white-light endoscopy. These results were later statistically tested with chi-square test (Statistics Calculator 3, StatPac Inc.). Level of p < 0.05 was
considered to indicate significant difference be-tween groups.
Results
The histopathological findings performed in 129 patients included 72 cases of invasive cancer, 19 pre-cancerous states and 38 benign pathologies. The detailed diagnoses are presented in Table 1.
Malignancies that were initially suspected in classic white-light videoendoscopy were histo-pathologically confirmed in 63 out of 72 cases, while in AFE in 71 out of 72. Nine false negative results in white light examination and only 1 in AFE (Table 2). This difference was statistically sig-nificant (p = 0.0087).
AFE allowed to properly identify 16 out of 19 precancerous lesions confirmed in histopatho-logical examinations, while white light videoen-doscopy allowed for proper identification of 9 out of 19 cases. The false negative results were 3 and
Table 1. Histological findings in the analyzed samples taken from 129 patients
Tabela 1. Rozpoznania histopatologiczne w materiale pobranym od 129 pacjentów
Histopathological finding
(Rozpoznanie histopatologiczne) Number of diagnoses (Liczba rozpoznań) Carcinoma planoepitheliale keratodes 52
Dysplasia (LIN I, II, III) 29 Carcinoma planoepitheliale akeratodes 14 Leucoplakia (without dysplasia) 13 Chronic hypertrophic laryngitis 9
Reinke’s oedema 8
Carcinoma keratoblasticum 5
Laryngeal polyposis 4
Pachydermia 4
Carcinoma in-situ 4
Laryngeal papilloma 3
Vocal nodules 2
Laryngeal tuberculosis 1
Total (Suma) 148
* The total number in the table exceeds 129 (the number of the patients), because in some cases more than one pathology was diagnosed in the same patient (e.g. dysplasia and invasive cancer, dysplasia and leucoplakia, leucoplakia and chronic laryngitis, etc).
10 respectively (Table 3). This difference also showed to be statistically significant (p = 0.0166, Pearson).
Out of all 91 patients with precancerous or cancerous pathologies AFE allowed to properly identify 87 cases, while videoendoscopy 72 out of 91 (Table 4). The most significant difference was observed for early pathologies (p = 0.00081).
In the cases histologically classified as benign, white light examination allowed to properly iden-tify 20 out 38 cases (18 false positive results). AFE allowed to properly identify 31 out of 38 cases (7 false positive results) (p = 0.0072). Results are presented in Table 5.
The above presented results were used to cal-culate sensitivity (Table 6) and specificity (Table 7) of AFE and white light examination in diagnosing malignant and premalignant lesions.
Table 3. Number of positive and false negative results (precancerous lesions) during white-light and AFE exami-nations
Tabela 3. Liczba wyników dodatnich i fałszywie ujemnych (stany przedrakowe) dla AFE i endoskopii w świetle białym
Laryngeal intraepithelial neoplasia (LIN) (n=19)
(Neoplazja śródnabłonkowa krtani – LIN) (n=19)
AFE white light exami-nation
Positive (+) (Dodatni) (+)
16 9
False nega-tive (f-) (Fałszywie ujemny) (f-)
3 10
* AFE – Autofluorescence Endoscopy. ** LIN – Laryngeal Intraepithelial Neoplasia. * AFE – endoskopia autofluorescencyjna. ** LIN – neoplazja śródnabłonkowa krtani.
Table 2. Number of positive and false negative results (laryngeal cancer) during white-light and AFE examina-tions
Tabela 2. Liczba dodatnich i fałszywie ujemnych wyników (rak krtani) dla AFE i klasycznej endoskopii w świetle białym
Laryngeal carcinoma (n = 72) (Rak krtani) (n = 72)
AFE white light exami-nation
Positive (+) (Dodatni) (+)
71 63
False nega-tive (f-) (Fałszywie ujemny) (f-)
1 9
* AFE – Autofluorescence Endoscopy. * AFE – endoskopia autofluorescencyjna.
Table 4. Number of positive and false negative results (LINs and laryngeal carcinomas) during white-light and AFE examinations
Tabela 4. Liczba dodatnich i fałszywie ujemnych wyników (LIN i raki krtani) dla AFE i klasycznej endoskopii w świetle białym
LIN and Laryngeal Cancer (n = 91)
(LIN i rak krtani) (n = 91) AFE white light
examinations Positive (+)
(Dodatni) (+) 87 72 False negative (f-)
(Fałszywie
ujem-ny) (f-) 4 19
* AFE – autofluorescence endoscopy. ** LIN – laryngeal intraepithelial neoplasia. * AFE – endoskopia autofluorescencyjna. ** LIN – neoplazja śródnabłonkowa krtani.
Table 5. Number of true negative and false positive results (benign changes) obtained during white-light and AFE examinations
Tabela 5. Liczba ujemnych i fałszywie dodatnich wyników (zmiany łagodne) dla AFE I klasycznej endoskopii w świetle białym
Benign changes (n = 38) (Zmiany łagodne) (n=38) AFE white light
examinations Negative (–)
(Ujemny) (–) 31 20
False positive (f+)
(Fałszywie dodatni) (f+)
7 18
Discussion
Laryngeal cancer is the most common cancer of upper respiratory tract in men [2, 3]. Prolonged exposition to external carcinogens (such as to-bacco smoke or alcohol abuse) causes damage to large areas of upper airways mucosa, resulting in development of cancerous changes. In the recent years the authors have observed intensive develop-ment of methods of treatdevelop-ment of laryngeal cancer. Beside numerous selective and preserving surgical procedures, effective methods of radiotherapy and chemoradiotherapy were introduced to offer the patients treatment options that are more efficient, more effective, less traumatizing and provide bet-ter quality of life.
Despite this progress in the treatment, 5-year survival rate in the past 30 years did not change
and approximates at about 50% for all stages of the disease [2, 4, 5]. Two major factors contribute to this fact: delayed diagnosis and relatively high percentage of developing a second primary focus of the disease after treatment [6, 7]. Many head and neck carcinomas are preceded by so-called “precancerous lesions” (histologically defined as laryngeal intraepithelial neoplasia – LIN, or pre-viously – dysplasia). Clinically, they are possible to detect only when accompanied by easy to spot pathologies (such as leucoplakia or erythroplakia). Diagnosis and treatment of laryngeal cancer pres-ents a significant health problem, not only in Po-land, but also worldwide. Non-invasive diagnostic tool that would allow to detect dysplastic changes would also allow to prevent or early diagnose la-ryngeal cancer. Early diagnosis enables to early introduce appropriate therapy and improves the chance of patients’ survival.
Many diagnostic techniques described in avail-able literature are based on using 5-aminolevulinic acid (5-ALA) to induce fluorescence of endogenic protoporphyrin IX (PpIX). 5-ALA was usually administered intravenously 1–4 hours prior the examination. The most important drawback of this method are the side effects of photosensitiz-ers. Doses as low as 30 mg/kg of ALA can cause nausea, vomiting, elevation of liver enzymes and increase skin sensitivity to sunlight. Such side ef-fects do not present a problem during photody-namic therapy (PDD) but cannot be accepted dur-ing routine examinations [8]. 5-ALA can also be administered topically, right before the blue light examination [9–12]. Cancerous tissues tend to ac-cumulate PpIX. Achieved results are characterized
Table 6. Comparison of sensitivity of AFE and white-light endoscopy as a stand-alone and combined method of diagnosing laryngeal neoplasms
Tabela 6. Porównanie czułości AFE i endoskopii w świetle białym jako samodzielnych metod diagnostycznych oraz zastoso-wanych łącznie w diagnostyce zmian nowotworowych i przednowotworowych w krtani
Sensitivity (Czułość) AFE
% white-light endoscopy % AFE + white-light endoscopy %
Cancer (n = 72)
(Rak krtani) (n = 72) 98.6 87.5 100
LIN (n = 19)
(LIN) (n = 19) 84.2 47.3 84.2
LIN + cancer (n = 91)
(LIN + raki krtani) (n = 91) 95.6 79.1 96.7
* AFE – Autofluorescence Endoscopy. ** LIN – Laryngeal Intraepithelial Neoplasia. * AFE – endoskopia autofluorescencyjna. ** LIN – neoplazja śródnabłonkowa krtani.
Table 7. Specificity of AFE and white-light endoscopy
Tabela 7. Specyficzność AFE i endoskopii w świetle białym
Specificity (Specyficzność) AFE
% white-light endoscopy %
Benign lesions (Zmiany łagodne) (n = 38)
81.6 52.6
by strong, easy to spot, red fluorescence of cancer-ous tissues. In the literature, topical administration of 5-ALA was done by inhaling 30 mg of 5-ALA in 5 ml 0.9% NaCl solution 1–2 hours before the microlaryngoscopy. Results allowed to assess sen-sitivity and specificity of this technique (95% and 80% respectively).
In the years 2000–2004 several reports on us-ing Light-induced Fluorescence (LIF) for diagnos-ing laryngeal pathologies were published. Baletic et al. used complete autofluorescence endoscopy sys-tem (designed to diagnose lower airways) to diag-nose laryngeal epithelium. Patients were examined with flexible bronchoscope in local anesthesia. The authors assessed sensitivity of SAFE system to be 92.1% (and 73.7% in white light examination) [13]. Kulapaditharom and Boonkitticharoen showed that sensitivity of LIF system in diagnosing laryn-geal carcinomas was 100% with 87.5 specificity (in a group of 25 patients) [14]. Other researchers achieved similar results [15].
The main problems with introducing above mentioned systems based on selective spectrum enhancers or spectrometric analysis to the routine examination are high costs and necessity to use general anesthesia. The investigated tissue needs to be examined from a very close distance (about 1 cm) for example by introducing an endoscope through the Kleinsasser’s set. Using flexible bron-choscopes requires at least topical anesthesia (the optics need to be close to the vocal cords) and sig-nificant patient’s cooperation.
The technique described in this paper lacks the above mentioned limitations. It does not re-quire general anesthesia. There is no need to use any photosensitizing agents. It allows to examine the larynx from much greater distance (with rigid laryngoscope). The recorded image is of far greater quality then image acquired from flexible optics. Available laryngeal endoscopic systems can be eas-ily adopted to perform AFE. The examination can be quickly performed and repeated as often as nec-essary on out-patient basis.
The main drawback is caused by the limited amount of light available for the CCD detector. The image is too dark to be viewed by a naked eye (directly through endoscope optics). Proper exposure requires thus longer exposition time. Practically it means that the pictures is refreshed no more than 4 times per second (instead of nor-mal 25/sec rate). In order to achieve a sharp, clear picture of the larynx, the endoscope has to be held firmly without any movement for this time. This requires patient’s cooperation, although not as much as during bronchoscopy. For the same rea-son a CCD camera and a computer with monitor are required to perform the examination.
Results achieved in own study rate the sensitiv-ity of AFE at 95.6% and its specificsensitiv-ity at 81.6% and are comparable to results achieved by other authors [15–18]. Technique described in this study is sim-pler to apply. The examination itself is also easier to bear for the patient and can be performed com-pletely on out-patient basis. This allows the pres-ent auhors to question the need of using 5-ALA in diagnosing laryngeal cancer.
The big advantage of AFE is its high sensitiv-ity in detecting early stages of neoplastic trans-formation. In own material false negative results were almost exclusively found in early dysplastic lesions (LIN I and II). However a strong, green fluorescence can be observed in cases of severe keratosis on the cancer’s surface, it is usually seen in advanced tumors with accompanying lack of fluorescence in the immediate area of keratosis. Such picture does not present any diagnostic difficulties. In the examined material there was not any case that would lead to improper diag-nosis, although it is (theoretically) possible that the keratosis may completely cover the neoplastic process (potential possibility of false negative re-sults).
In own study the authors have found more positive than negative results. The false-positives were found mainly in laryngeal papillo-mas and chronic hypertrophic laryngitis, one false-positive case of leucoplakia without accompanying dysplasia, one vocal cord nodule and one case or Reinke’s oedema. There was also one patient sus-pected of laryngeal cancer, with typical lack of green fluorescence which was histopathologically diagnosed as laryngeal tuberculosis.
Reassuming – in clinical practice false-positive results present lesser problem than false-negative ones, because the consequences of false-negative results are far more dangerous for patients. High sensitivity allows for precise and early diagnosis, that in case of laryngeal cancers decides of the pa-tient’s survival and quality of life after treatment. Early detection is the key to successful treatment. AFE allows to detect pathologies that are otherwise clinically silent.
Blue-light examination seems to be an excel-lent addition to the classic white-light endoscopy of the larynx. Combining these two methods al-lows to significantly increase chances of proper diagnosis of early laryngeal neoplasia. Populariza-tion of this method may contribute to increased survival and better results of treatment of patients with laryngeal cancer.
The authors concluded that autofluorescence endoscopy is an efficient, safe and easy to per-form complementary diagnostic tool, that may be regularly used in out-patient clinics during rou-tine ENT examination of glottic and supraglottic region of the larynx. AFE is characterized by very high sensitivity and high specificity in diagnosing laryngeal cancer and pre-cancerous lesions, start-ing from mid-grade dysplasia (LIN II). AFE allows for precise biopsy and accurate assessment of the spread of the lesion.
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Address for correspondence:
Maciej Dariusz Zatoński
Department of Otolaryngology, Faculty of Dentistry Wroclaw Medical University
Borowska 213 50-556 Wrocław Poland
Tel.: +48 507 082 765 E-mail: [email protected]
Conflict of interest: None declared
