Detection of squamous cell carcinomas and pre-cancerous lesions in the oral cavity by quantification of 5-aminolevulinic acid induced fluorescence endoscopic images

BACKGROUND AND OBJECTIVES: Studies of 5-aminolevulinic acid-induced protoporphyrin IX fluorescence have shown a sensitivity of 95-100% for oral cancer diagnosis, but the specificity is only about 50-60%. Here, we explore the applicability of quantifying PPIX fluorescence images to improve the diagnostic specificity and detect early oral lesions. STUDY DESIGN/MATERIALS AND METHODS: PPIX Fluorescence endoscopy and imaging were performed on 28 patients with a known or suspected premalignant or malignant oral cavity lesion. A total of 70 biopsies were taken from the tissue sites imaged for histological analysis. The red-to-blue and red-to-green intensity ratios were calculated from the fluorescence images to correlate with histology. RESULTS: Suspicious lesions display bright reddish fluorescence, while normal mucosas exhibit blue color background in the fluorescence images. The red-to-blue and red-to-green intensity ratios of malignant tissues are larger than those of benign tissues. Combining the two ratio diagnostic algorithms yields a sensitivity and specificity of 95% and 97%, respectively, exceeding each diagnostic algorithm alone for discriminating malignant tissue from benign tissue. CONCLUSIONS: Quantifying PPIX fluorescence endoscopic images combined with the ratio diagnostic algorithms developed in this study has the potential to significantly improve the noninvasive diagnosis of oral cavity lesions in vivo.     

Photodynamic Therapy of oral dysplasia with topical 5-aminolevulinic acid and light-emitting diode array

BACKGROUND AND OBJECTIVES: In Taiwan, more than two million people have the betel quid (BQ) chewing habit which is a risk factor related to premalignant lesion and squamous cell carcinoma of oral cavity. We developed a light-emitting diode (LED) array combined with topical 5-aminolevulinic acid (ALA) for photodynamic therapy (PDT) and evaluated its effectiveness for the treatment of oral lesions. STUDY DESIGN/MATERIALS AND METHODS: We compared the ALA-PDT effect of the homemade LED array to that of a commercial light source on cultured Ca9-22 human gingival carcinoma cells and the DMBA-induced hamster buccal pouch carcinoma model. Furthermore, we treated several patients having an oral lesion using a topical ALA delivery system and the LED array. RESULTS: The LED array light source was as effective as the commercial light source for ALA-PDT in cultured Ca9-22 cells with LD(50) of 4.5 and 4.3 J/cm(2), respectively, using an MTT assay. This light source was also effective in the DMBA-induced hamster buccal pouch carcinoma model, and in the patients of oral leukoplakia. CONCLUSIONS: ALA-PDT is effective for premalignant lesions such as mucosal dysplasia and carcinoma in situ of oral cavity. Good results could be obtained by using the homemade LED array as light source. The LED array has the advantages of low cost, high reliability, and portability. It is safe, convenient and easy to use for the treatment of oral dysplasia.     

Successful treatment of oral verrucous hyperplasia and oral leukoplakia with topical 5-aminolevulinic acid-mediated photodynamic therapy

BACKGROUND AND OBJECTIVES: Our recent studies showed that a new topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) protocol using a light-emitting diode (LED) light source is an effective and successful treatment modality for five cases of oral verrucous hyperplasia (OVH) and one case of verrucous carcinoma. In this study, we treated eight OVH lesions with the same topical ALA-PDT protocol to further confirm the efficacy of this protocol on OVH lesions. In addition, our recent study demonstrated an unsatisfactory clinical outcome for 24 oral leukoplakia (OL) lesions treated once a week by the same topical ALA-PDT protocol. Therefore, in this study 24 OL lesions were treated twice a week by the same protocol to compare whether the twice-a-week treatment modality could result in a better clinical outcome than the once-a-week treatment modality. STUDY DESIGN/MATERIALS AND METHODS: In this study, 8 OVH and 24 OL lesions were treated by the same topical ALA-PDT once a week and twice a week, respectively. RESULTS: All the former eight OVH lesions treated once a week showed complete response (CR) after 2-5 (mean, 3.8) treatments of ALA-PDT. The latter 24 OL lesions treated twice a week demonstrated CR in 8, partial response (PR) in 16, and no response in none. The present 24 OL lesions treated twice a week had a significantly better clinical outcome than the previous 24 OL lesions treated once a week (P = 0.000, chi-square test). CONCLUSIONS: We conclude that complete regression of OVH lesions can be achieved by less than six treatments of topical ALA-PDT once a week. Although the response of OL lesions to the topical ALA-PDT is not as good as the response of OVH lesions to the same therapy, all OL lesions can have at least PR after eight treatments with the topical ALA-PDT twice a week. In addition, OL lesions treated twice a week have a significantly better clinical outcome than OL lesions treated once a week.     

Recent advances in optical diagnosis of oral cancers: Review and future perspectives

Optical diagnosis techniques offer several advantages over traditional approaches, including objectivity, speed, and cost, and these label‐free, noninvasive methods have the potential to change the future workflow of cancer management. The oral cavity is particularly accessible and, thus, such methods may serve as alternate/adjunct tools to traditional methods. Recently, in vivo human clinical studies have been initiated with a view to clinical translation of such technologies. A comprehensive review of optical methods in oral cancer diagnosis is presented. After an introduction to the epidemiology and etiological factors associated with oral cancers currently used, diagnostic methods and their limitations are presented. A thorough review of fluorescence, infrared absorption, and Raman spectroscopic methods in oral cancer diagnosis is presented. The applicability of minimally invasive methods based on serum/saliva is also discussed. The review concludes with a discussion on future demands and scope of developments from a clinical point of view. © 2015 Wiley Periodicals, Inc. Head Neck 38 : E2403–E2411, 2016     

Lab-on-a-chip for oral cancer screening and diagnosis

Oral squamous cell carcinoma (OSCC) is a disfiguring and deadly cancer. Despite advances in therapy, many patients continue to face a poor prognosis. Early detection is an important factor in determining the survival of patients with OSCC. No accurate, cost-efficient, and reproducible method exists to screen patients for OSCC. As a result, many patients are diagnosed at advanced stages of the disease. Early detection would identify patients, facilitating timely treatment and close monitoring. Mass screening requires a rapid oral cancer diagnostic test that can be used in a clinical setting. Current diagnostic techniques for OSCC require modern laboratory facilities, sophisticated equipment, and elaborate and lengthy processing by skilled personnel. The lab-on-chip technology holds the promise of replacing these techniques with miniaturized, integrated, automated, inexpensive diagnostic devices. This article describes lab-on-chip devices for biomarker-based identification of oral cancer. Similar methods can be employed for the screening of other types of cancers.     

Acceleration of ALA-induced PpIX fluorescence development in the oral mucosa

BACKGROUND AND OBJECTIVES: The development of 5-aminolevulinic acid (ALA)-induced tissue fluorescence is optimal 2-4 hours after ALA application. Goal of this work was to develop a means of accelerating oral topical ALA-induced tissue fluorescence. STUDY DESIGN/MATERIALS AND METHODS: In 300 hamsters, DMBA (9,10 dimethyl-1,2-benzanthracene) cheek pouch carcinogenesis produced dysplasia in 3-5 weeks. Topical application of 20% ALA in Eucerin was followed by localized ultrasound treatment (1, 3.3 MHz) in 150 animals. In 75 animals, ALA was applied in an Oral Pluronic Lecithin Organogel (OPLO-an absorption enhancer) vehicle. Seventy-five animals received only topical ALA in Eucerin. Hamsters were sacrificed and cryosections underwent fluorescence measurements, histological evaluation, 20-180 minutes after ALA application. One-way ANOVA detected independent effects of pathology on laser-induced fluorescence (LIF). Two-way ANOVA tested for independent effect of pathology and of OPLO, ultrasound, and interaction effects. RESULTS: Ultrasound significantly (P < 0.05) accelerated tissue fluorescence development. CONCLUSIONS: Low-frequency ultrasound can accelerate ALA-induced fluorescence development.     

Comparative in vitro percutaneous penetration of 5-aminolevulinic acid and two of its esters through excised hairless mouse skin

BACKGROUND AND OBJECTIVES: ALA esters have been developed to improve PpIX production in ALA-PDT, but they do not perform as well in skin as they do in cells and the bladder. STUDY DESIGN/MATERIALS AND METHODS: The in vitro penetration across normal mouse skin of ALA and its methyl and hexyl ester was determined for different application concentrations. ALA and the esters were also applied to tape stripped skin to determine the effect of the stratum corneum. RESULTS: The penetration of ALA and the esters was higher through tape stripped skin than through normal skin (P < 0.01), showing that the stratum corneum is an important barrier. The experiments with different application concentrations indicated that the skin penetration through normal skin and tape stripped skin is highest for ALA and lowest for the hexyl ester. CONCLUSIONS: The differences in skin penetration properties could be (co-)responsible for the finding that ALA esters do not induce substantially higher PpIX levels in in vivo skin.     

Increased brain tumor resection using fluorescence image guidance in a preclinical model

BACKGROUND AND OBJECTIVES: Fluorescence image-guided brain tumor resection is thought to assist neurosurgeons by visualizing those tumor margins that merge imperceptibly into normal brain tissue and, hence, are difficult to identify. We compared resection completeness and residual tumor, determined by histopathology, after white light resection (WLR) using an operating microscope versus additional fluorescence guided resection (FGR). STUDY DESIGN/MATERIALS AND METHODS: We employed an intracranial VX2 tumor in a preclinical rabbit model and a fluorescence imaging/spectroscopy system, exciting and detecting the fluorescence of protoporphyrin IX (PpIX) induced endogenously by administering 5-aminolevulinic acid (ALA) at 4 hours before surgery. RESULTS: Using FGR in addition to WLR significantly increased resection completeness by a factor 1.4 from 68+/-38 to 98+/-3.5%, and decreased the amount of residual tumor post-resection by a factor 16 from 32+/-38 to 2.0+/-3.5% of the initial tumor volume. CONCLUSIONS: Additional FGR increased completeness of resection and enabled more consistent resections between cases.     

Clinically relevant improvement of recurrence-free survival with 5-aminolevulinic acid induced fluorescence diagnosis in patients with superficial bladder tumors

PURPOSES: Fluorescence diagnosis induced by 5-aminolevulinic acid enables more thorough transurethral resection of superficial bladder carcinoma compared with conventional white light. We performed a prospective, single institution, randomized trial to investigate whether the residual tumor rate and long-term tumor recurrence can be decreased by fluorescence diagnosis. MATERIALS AND METHODS: A total of 301 patients underwent transurethral resection of bladder tumors with white light or fluorescence diagnosis. Transurethral resection was repeated 5 to 6 weeks later to evaluate the residual tumor rate. To determine recurrence-free survival patient followup was performed every 3 months by white light cystoscopy and urine cytology. Recurrence-free survival was analyzed via Kaplan-Meier methods and multivariable Cox regression analysis. RESULTS: A total of 191 patients with superficial bladder carcinoma were available for efficacy analysis. The residual tumor rate was 25.2% in the white light arm versus 4.5% in the fluorescence diagnosis arm (p <0.0001). Median followup in the white light arm in 103 cases was 21.2 months (range 4 to 40) compared with 20.5 (range 3 to 40) in the 88 in the fluorescence diagnosis arm. Recurrence-free survival in the fluorescence diagnosis group was 89.6% after 12 and 24 months compared with 73.8% and 65.9%, respectively, in the white light group (p = 0.004). This superiority proved to be independent of risk group. The adjusted hazard ratio of fluorescence diagnosis versus white light transurethral resection was 0.33 (95% confidence interval 0.16 to 0.67). CONCLUSIONS: Fluorescence diagnosis is significantly superior to conventional white light transurethral resection with respect to the residual tumor rate and recurrence-free survival. The differences in recurrence-free survival imply that fluorescence diagnosis is a clinically relevant procedure for decreasing the number of tumor recurrences.     

Photodynamic therapy of high-grade cervical intraepithelial neoplasia with 5-aminolevulinic acid

BACKGROUND AND OBJECTIVES: To determine the safety and efficacy of 5-aminolevulinic acid (ALA) as a topically applied photosensitizer for photodynamic therapy (PDT) of cervical intraepithelial neoplasia (CIN). STUDY DESIGNS/MATERIALS AND METHODS: Forty women, who were at least 18 years old with persistent biopsy-proven CIN 2 and CIN 3 within the previous 3 months of enrollment, underwent PDT in a phase I and II design. Five escalating radiant energies (increments of 25 J/cm(2), beginning at 50-150 J/cm(2)) using a Coherent Dye Model 920 argon pumped dye laser providing light at 630 nm (maximum output 0.8 W) were used to perform PDT with a fixed dose of ALA (200 mg/ml). ALA was placed in a cervical cap fitted to the cervix. After 90 minutes, the cap was removed and the ectocervix was illuminated for 5-16 minutes, depending on the irradiance. Success was defined as the absence of CIN on Pap smear or colposcopic examination at 12-months. Patients were monitored for toxicity. RESULTS: Thirty-two women (80%) completed the study with 1 year of follow-up. Sixty percent had CIN 3 and 40% CIN 2. Success rates at 4, 8, and 12 months were 51, 46, and 31%, respectively, and were not light-dose dependent. Three patients progressed from CIN 2 to CIN 3. Toxicity was tolerable and only consisted of spotting, vaginal discharge, mild cramping, and vaginal warmth. There was no apparent dose relationship to toxicity. CONCLUSIONS: PDT at this light and ALA dose is well tolerated but has minimal activity in the treatment of CIN 2 and CIN 3. Other doses and schedules of light and ALA or novel photosensitizers may improve efficacy.     

Quantification of 5-aminolevulinic acid induced fluorescence improves the specificity of bladder cancer detection

PURPOSE: 5-Aminolevulinic acid induced fluorescence endoscopy has outstanding sensitivity for detecting early stage bladder cancer. Nevertheless, a third of the lesions that show specific fluorescence are histologically benign. We decreased the false-positive rate of 5-aminolevulinic acid induced fluorescence endoscopy by incorporating protoporphyrin IX fluorescence quantification into the standard cystoscopy procedure. MATERIALS AND METHODS: In 25 cases (53 biopsies) of a history of or suspicion for bladder cancer 5-aminolevulinic acid induced fluorescence endoscopy and fluorescence image quantification were performed. For fluorescence image quantification images obtained with a target integrating color charge-coupled device camera were digitized and stored in a personal computer. Red-to-blue ratios were calculated from fluorescence positive lesions and results were correlated with hematoxylin and eosin histology. RESULTS: Malignant fluorescence positive lesions showed significantly stronger fluorescence intensity than fluorescing lesions with benign histology. A threshold was established that decreased the false-positive rate by 30% without affecting sensitivity. CONCLUSIONS: Fluorescence image quantification is a new endoscopic method for objectively selecting multicolor fluorescence bladder lesion images for biopsy. It has the potential of eliminating human error by different surgeons with variable experience in fluorescence endoscopy.     

Oral 5‐aminolevulinic acid‐mediated photodynamic diagnosis using fluorescence cystoscopy for non‐muscle‐invasive bladder cancer: A multicenter phase III study

Objective

To confirm the reproducibility of the effectiveness and safety in photodynamic diagnosis of non‐muscle‐invasive bladder cancer using 5‐aminolevulinic acid in a prospective multicenter non‐randomized phase III trial.

Methods

A total of 61 patients with primary or recurrent non‐muscle‐invasive bladder cancer were prospectively enrolled from five hospitals between May 2015 and March 2016. 5‐Aminolevulinic acid (20 mg/kg) was orally administered 3 h before transurethral resection of bladder tumors using white light or fluorescent light. Of 60 evaluable patients, 511 specimens were obtained from tumor‐suspicious lesions and normal‐looking mucosa. The primary end‐point was sensitivity. The secondary end‐points were specificity, positive and negative predictive values, and safety.

Results

The sensitivity of the fluorescent light source (79.6%) was significantly higher (P < 0.001) than that of the white light source (54.1%). In total, 25.4% (46/181) of tumor specimens were diagnosed as positive with only the fluorescent light source. In nine (15%) of 60 patients, the risk classification and recommended treatment after transurethral resection of bladder tumors were changed depending on the additional types of tumor diagnosed by the fluorescent light source. The specificity of the fluorescent light versus white light source was 80.6% versus 95.5%. No grade 4–5 adverse event was noted. Hypotension and urticaria were severe adverse events whose relationship to oral 5‐aminolevulinic acid could not be excluded.

Conclusions

These findings confirm the diagnostic efficacy and safety of photodynamic diagnosis with 20 mg/kg of oral 5‐aminolevulinic acid, and show that transurethral resection of bladder tumors with a fluorescent light source using oral 5‐aminolevulinic acid is well tolerated.     

Long-term benefit of 5-aminolevulinic acid fluorescence assisted transurethral resection of superficial bladder cancer: 5-year results of a prospective randomized study

PURPOSE: As shown in various studies 5-aminolevulinic acid (ALA) induces fluorescence of malignant and dysplastic bladder tissue and increases tumor detection rates by about 20%. However, data on the long-term benefits are sparse. Thus, the 5-year outcome data of a prospective randomized trial comparing patients who initially underwent bladder tumor resection (TUR) under standard white light or with ALA induced fluorescence were evaluated. MATERIALS AND METHODS: A total of 115 patients with suspected superficial bladder cancer were randomized to undergo standard or ALA assisted TUR. After the second look TUR at 6 weeks patients were followed for a median of 39 (standard) and 42 (ALA) months. RESULTS: Median time to first recurrence was 5 months in the standard and 12 months in the ALA group. Recurrence-free survival was 25% in the standard and 41% in the ALA group. The recurrence rate at 2, 12, 36 and 60 months after initial TUR was 41%, 61%, 73% and 75%, and 16%, 43%, 59% and 59% in the white light and ALA groups, respectively. The total number of recurrences was 82 in the standard and 61 in the ALA group. Tumor progression occurred in 9 patients in the standard and 4 in the ALA group. Cost analysis suggests a considerable economical advantage of ALA fluorescence assisted TUR compared to the standard procedure. CONCLUSIONS: The initial advantage of improved tumor detection and decreased recurrence rates by ALA fluorescence assisted TUR is maintained for years, and effectively reduces morbidity and costs in patients with superficial bladder tumors.     

In vivo autofluorescence spectroscopy of oral premalignant and malignant lesions: distortion of fluorescence intensity by submucous fibrosis

BACKGROUND AND OBJECTIVES: To test whether autofluorescence spectroscopy can be used for the diagnosis of oral neoplasia in a high-risk population, we characterized the in vivo autofluorescence spectra from oral submucous fibrosis (OSF) lesions and oral premalignant and malignant lesions in both OSF and non-OSF patients. STUDY DESIGN/MATERIALS AND METHODS: Autofluorescence emission spectra were measured under the excitation wavelength of 330 nm, using a Xenon lamp-based fluorospectrometer coupled to a handheld optical fiber probe. Autofluorescence spectroscopies were analyzed among patients with OSF lesions, and oral lesions of epithelial hyperkeratosis (EH), epithelial dysplasia (ED), and squamous cell carcinomas (SCC) and normal oral mucosa (NOM) of healthy volunteers. RESULTS: We found that the most intensely autofluorescence emission peaks occurred at 380 nm and 460 nm. For comparing the spectral patterns among different groups of oral lesions and NOM, ratios of the area under the spectrum of 460+/-10 nm to that under the spectrum of 380+/-10 nm (denoted as A(460+/-10nm)/A(380+/-10nm)) were calculated. The mean ratio values increased gradually from OSF to NOM, to EH and ED, and to SCC. The ANOVA test showed significant differences in the ratio value among all categories of samples (P<0.01). On the other hand, we found that EH, ED, and SCC lesions on OSF patients had distorted autofluorescence intensity. The mean ratio values of EH, ED, and SCC between non-OSF and OSF patients show significant differences. Furthermore, an ANOVA test showed NOM is not distinguishable from EH and ED lesions on oral fibrotic mucosa (P>0.05). CONCLUSIONS: Autofluorescence spectroscopy can be used to diagnose EH, ED, and SCC lesions in non-OSF patients but not in OSF patients.