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(Chest. 2006;129:718-724.)
© 2006 American College of Chest Physicians

Fluorescence Detection of Pleural Malignancies Using 5-Aminolaevulinic Acid*

Paul Baas, MD, FCCP; Martijn Triesscheijn, MSc; Sjaak Burgers, MD, PhD; Renée van Pel, MD, PhD; Fiona Stewart, PhD and Maurice Aalders, PhD

* From the Departments of Thoracic Oncology, Pathology, and Experimental Therapy, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital; and the Laser Center, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

Correspondence to: Paul Baas, MD, FCCP, Department of Thoracic Oncology, Antoni van Leeuwenhoek Hospital/The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands; e-mail: p.baas{at}nki.nl

Abstract

Study objective: Although the use of video-assisted thoracoscopy has improved the diagnostic accuracy in patients presenting with pleural diseases, not all biopsies performed are conclusive and staging of the disease is not always optimal. Fluorescence diagnosis (FD) with 5-aminolaevulinic acid (5-ALA) has been used in the diagnostic workup for various malignancies. The impact of 5-ALA–mediated FD on diagnosis and staging during video-assisted thoracoscopy was examined.

Design: Prospective, single-center study.

Setting: National cancer center.

Patients: Twenty-six patients with nonconclusive pleural effusions who were scheduled for video-assisted thoracoscopy.

Intervention: Eligible patients were administered 1,500 to 2,500 mg po of 5-ALA before video-assisted thoracoscopy. After conventional inspection with white light, fluorescence inspection of the pleural cavity was performed (D-LIGHT Auto Fluorescent System; Karl Storz; Tuttlingen, Germany). Biopsy specimens of both normal and abnormal sites, as determined from white light and FD inspection, were obtained for histologic examination.

Results: One patient was ineligible, and two patients were not evaluable because of equipment failure. One postoperative death occurred due to preexisting myocardial disease. In another patient, an empyema developed; in another patient, a postoperative infection of the lung developed. Other toxicities were minimal. A definitive diagnosis was obtained in 24 of 25 cases, with malignant mesothelioma in 15 cases, other malignancies in 5 cases, one infection, and three benign diseases. Upstaging occurred in four patients (unsuspected tumor deposits) due to FD examination. In 23 patients, a total of 111 biopsy specimens could be analyzed. When correct findings of white light and FD were compared, FD had an additional value in 21 of 111 biopsies, compared to white light with 16 of 111 biopsies.

Conclusions: FD using 5-ALA in the pleural cavity is feasible with limited side effects when used in addition to white light inspection. It improved visualization of abnormal lesions and led to upstaging in 4 of 15 mesothelioma patients.

Key Words: aminolaevulinic acid • fluorescence detection • pleural malignancies • polymeric photonic display

Thoracoscopic examination has become a standard procedure for the analysis of undiagnosed pleural diseases and for staging purposes. In the majority of patients, repeated pleural fluid examination has a diagnostic yield of 70%. For the remaining patients, more invasive procedures such as thoracoscopy have to be performed. In case of malignancy, this procedure can help with proper staging and prevent patients from undergoing a futile thoracotomy. In general, 12 to 20 biopsies are required to limit the chance of false-negative results. Despite this approach, some cases still remain undiagnosed, are understaged, or patients might even have biopsy-related complications.

Several publications123 have indicated that fluorescence diagnosis (FD) using 5-aminolaevulinic acid (5-ALA) can improve the diagnostic yield in the abdomen and thoracic cavity. Most of these studies were performed in animal models, and there are only a few reports4567 on FD using 5-ALA in humans. Of the available photosensitizers, 5-ALA has the advantage of being a naturally occurring nutrient that is metabolized by the cell to haem via an intermediate production of protoporphyrin-IX (Pp-IX). In this metabolic pathway, the final step in the transformation to haem requires ferrochelatase (Fig 1 ). The expression of this enzyme is known to be decreased in malignant and inflamed cells, leading to an accumulation of Pp-IX, which has photodynamic properties. The increase in concentration of intracellular Pp-IX and differences in pharmacokinetics between abnormal cells and normal cells facilitate the use of 5-ALA for diagnostic or therapeutic purposes. 5-ALA can be administered systemically (orally, IV) or topically and has very limited side effects. After low oral dosing, skin photosensitivity lasts for 24 to 36 h and liver enzymes may show a transient rise.


Figure 1
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  		Figure 1.. Summary of the metabolic pathway of 5-ALA into haem. The rate-limiting step is the presence of ferrochelatase.

 
In order to gain more insight in the value of FD using 5-ALA during thoracoscopy, we conducted a feasibility study. In this article, we report the preliminary results of thoracoscopic FD examination and white light inspection in patients with undiagnosed pleural effusions.

Materials and Methods

Patients
Patients were eligible when they presented with a pleural effusion or thickening and had had at least one nondiagnostic procedure (eg, thoracenthesis). Patients had to be > 18 years of age and fit for general anesthesia. Other eligibility criteria were adequate lung function parameters, PO2 levels > 7.5 kPa, PCO2 levels < 6.0 kPa, no pregnancy, adequate blood cell counts and renal function, no known hereditary porphyria, no recent pneumonia, or uncontrolled bleeding tendency. All patients had a clinical workup consisting of chest radiograph, CT scan of the thorax and upper abdomen, and ECG. The study was approved by the local Medical Ethical Committee, and written informed consent was obtained from all patients.

5- ALA
5-ALA was prepared by the local pharmacist (Professor J. Beijnen, Slotervaart Hospital, Amsterdam, the Netherlands) and delivered in capsules of 250 mg. 5-ALA was administered with ample amounts of water 3 to 4 h before thoracoscopy. Three dosage groups were defined: 1,500 mg for patients with a weight < 60 kg, 2,000 mg for patients 60 to 80 kg, and 2,500 mg for patients > 80 kg. After intake of the capsules, patients were kept in subdued light for at least 24 h.

Fluorescence Equipment
Fluorescence images were recorded (D-LIGHT Auto Fluorescent System; Karl Storz; Tuttlingen, Germany), which consists of a xenon light source with an integrated filter wheel that enables selection of white light or blue light (< 500 nm for excitation of the Pp-XI). Both illumination and observation of tissues of interest were achieved via a rigid endoscope integrated with a long-pass filter (cut-off wavelength at 470 nm), which, in turn, was connected to a dedicated charged-couple device camera and a video or digital video disc recorder for subsequent evaluation of the procedure, discussion with colleagues and to extract photographs.

The Storz camera has a white light mode and a blue light mode, in which the integration time can be increased to correct for the relative low intensity of the fluorescent light. An additional low-pass filter (> 550 nm) was placed between the endoscope and the camera to further increase the contrast of the images.

Surgical Procedure
A double-lumen tube was inserted under general anesthesia. The patient was placed on the nonafflicted side, and a sterile field was prepared. One or two entrance ports were made (Endopath; Johnson and Johnson; Cincinnati, OH) for optimal inspection of the thoracic cavity. After evacuation of the pleural fluid, the lung was blocked for ventilation to allow maximal collapse. The cavity was initially inspected with white light, followed by inspection in the fluorescence mode. The procedure was recorded to allow retrospective analysis of the sampling sites. A scoring system based on previous procedures in our hospital was used to identify lesions (Table 1 ).


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  		Table 1.. Endoscopic Appearance Scoring List*

 
During fluorescence examination, biopsy specimens were obtained from both normal and abnormal sites. Biopsy specimens of abnormal sites during white light examination were obtained; when no pathology was observed during white light examination, random biopsies ("blind") were performed. The samples were collected in formalin and processed for standard histologic examination. In case of malignancy, talc powder was insufflated to prevent recurrence of the effusion. The ports were closed after the procedure, and a suction tube was inserted for at least 24 h to allow optimal expansion of the lung. Patients were discharged from the hospital 1 to 2 days after the procedure.

Results

From January 2003 to January 2005, 26 patients (17 men and 9 women) with nondiagnostic pleural effusions were enrolled in this study. One patient was excluded from the analyses because inspection of the thoracic cavity could not be performed due to multiple adhesions.

Patient characteristics and surgical aspects are listed in Tables 2, 3 . The median age was 54 years (range, 40 to 73 years). World Health Organization performance scores (Eastern Cooperative Oncology Group) were 0 in 7 patients, 1 in 16 patients, and 2 in 2 patients. A right-sided effusion was observed in 17 patients, and a left-sided effusion was observed in 8 patients. The majority of patients (n = 19) were examined within 4 h after administration of 5-ALA, with a mean interval of 3.9 h. The procedure was uncomplicated in all but two cases and lasted 30 to 90 min (median, 45 min). The time required for the additional fluorescence examination was 10 to 15 min.


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  		Table 2.. Patient Characteristics

 

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  		Table 3.. Surgical Aspects of the Endoscopic Examination*

 
In 2 of 25 patients, endoscopic inspection could not be performed correctly after administration of 5-ALA because of endoscope failure (patients 12 and 13). The filter wheel was stuck, resulting in oversaturated recordings, which allowed no discrimination between normal and abnormal tissue. These patients were excluded from the biopsy analyses but not from toxicity analysis.

A diagnosis was obtained in 24 patients. Fifteen patients had malignant mesothelioma (epithelial, n = 14; desmoplastic, n = 1), and 5 patients had metastases from other tumors (bronchoalveolar, tongue, mammary, esophagus, and one of unknown origin). There were three patients with plaques, with or without inflammatory changes, and one empyema. Mesothelioma developed after 6 months in the patient in whom no initial diagnosis was made.

In total, 111 biopsy specimens were obtained from different sites (parietal, visceral, and diaphragmatic pleura) during examination. The specimens were grouped according to fluorescence-positive, fluorescence-negative, and white light-positive or white light-negative appearances. The pathologist was blinded to the fluorescence or white light status and assessed every specimen separately. The pathology report was limited to benign, proliferation, or malignancy/infection. In case of doubt, another pathologist was consulted. Cases in which mesothelioma was diagnosed were also evaluated by a mesothelioma expert panel. In Table 4, biopsy results are presented according to scoring.


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  		Table 4.. Biopsy Diagnoses (n = 111) and Scores for Examination With White Light and FD in Relation to the Diagnosis*

 
Both FD and white light imaging results were positive in 37 biopsies (Fig 2 , top left, A, and top right, B). Seven of these results were from biopsies with normal pleura or plaque and were therefore false-positives. A further four specimens with reactive, inflamed tissue were positive under both white light and FD inspection.


Figure 2
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  		Figure 2.. Top left, A: An example of mesothelioma with tumor spots on the diaphragm during white light examination. Top right, B: The same patient as top left, A, but now during FD. The tumor spots present as cherry red lesions, clearly demarcated from the surrounding normal tissue. Bottom left, C: Slightly thickened parietal pleura without abnormal areas during white light examination. Bottom right, D: Same location as bottom left, C, but during FD there is a small but clearly demarcated spot. Pathologic examination confirmed the diagnosis of a mesothelioma at this particular site. PPD = polymeric photonic display.

 
FD and white light imaging results were negative in 37 biopsies, of which 13 were diagnosed as having tumor or proliferating nests of cells and were therefore false-negatives. In 37 biopsies, there was a discrepancy between observations with white light and fluorescence detection (Fig 2, bottom left, C, and bottom right, D). In 26 of these discrepant biopsies, fluorescence detection was positive and white light was negative, with 19 true-positive and 7 false-positive findings. FD was false-negative in eight biopsies, of which three biopsies were from one patient with desmoplastic mesothelioma. White light results were positive over FD in 11 biopsies: 8 true-positive and 3 false-negative. The 37 biopsies with discrepancies between white light and FD imaging results have been further compared in Table 5 to determine the accuracy of the imaging results. For this analysis, a positive image was considered to be correct if the biopsy pathology showed tumor, proliferative nests, or inflamed reactive tissue but was considered to be false if the biopsy showed normal pleura or plaque. Conversely, a negative image for biopsies with tumor or proliferative cells was considered false, but a negative image for normal pleura or plaque was correct. This analysis shows that no overall improvement was observed by using FD to obtain a diagnosis in this patient group. However, for staging of mesothelioma, it was clear that in four patients FD upstaged the patient due to better visualization of lesions of the visceral pleural that could not be seen by using white light alone (Fig 3 ).


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  		Table 5.. Correct Findings of White Light or Fluorescence Thoracoscopy From Biopsy Specimens With Discrepant Imaging Results*

 

Figure 3
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  		Figure 3.. Top, A: Endoscopic overview of the right pleural cavity (patient 25) during white light examination. The right lower lobe is visible without apparent tumor involvement. More distally, a pathologic-appearing parietal pleura is visible. Bottom, B: Same location as top, A, now during FD inspection. There is a positive reflection of the visceral pleura of lower lobe. Biopsy specimens from this area proved positive for malignant mesothelioma. See Figure 2 legend for expansion of abbreviation.

 
Complications
Postoperative complications occurred in three patients, one of whom died (patient 1), a 66-year-old man with a history of myocardial and valvular disease (aortic valve prosthesis). Before the procedure, he received antibiotic prophylaxis and his standard oral anticoagulation therapy was temporarily changed to IV heparin according to the advice of his cardiologist. The total procedure took 60 min because of adhesions and tuning of the equipment, but no complications occurred. Postoperatively, he had periods of hypotension and died on day 3 after surgery of congestive heart failure. A postmortem examination showed a recent myocardial infarction (< 1 day), intact aortic valve prosthesis, severe stenosis and insufficiency of the mitral valve, and a hypertrophic and dilated myocardium. No relation with the thoracoscopic procedure itself could be established.

Another patient (patient 2) had a wound infection with a Staphylococcus aureus strain that had to be treated with oral antibiotics and rinsing of the thoracic cavity. In a third patient (patient 12), a postoperative pulmonary infection occurred that was treated with antibiotics. Skin burn grade 1 was observed in three patients within 28 to 36 h after 5-ALA administration.

Discussion

Fluorescence methods have been studied for > 2 decades with the aim of enhancing the optical contrast of abnormal (malignant) tissue over normal surrounding tissue. Subtle changes in vascularization, thickening of the superficial cell layers, and the presence of specific chromophores in tumors can lead to a change in fluorescence properties.89 For the inspection of large areas like the thoracic cavity, the use of a short-lived photodynamic drug would be helpful to boost the autofluorescence of abnormal lesions. 5-ALA is potentially suited for this task, but there is limited information available on the tumor specificity of 5-ALA–mediated fluorescence in the thoracic cavity. The first report1 on fluorescence detection with local instillation of different doses of 5-ALA in rats indicated that this approach was feasible and might be more sensitive than white light examination. We decided against local instillation of 5-ALA in patients since this approach was not well investigated and problems of distribution might occur. From a practical point of view, a flat dosing of 5-ALA was used, determined by the weight of the patient, and the interval between administration and examination was based on published literature.1011

In general, we were impressed by the colorful enhancement of lesions during fluorescence examination. Small and large lesions presented as intense cherry red spots on the monitor.

Based on fluorescence positive images, 4 of 15 patients with mesothelioma were upstaged (patients 3, 8, 21, and 25). Especially small lesions (< 3 mm) on the parietal pleura and lesions on the rim of the partly collapsed lung or diffuse spots on the visceral pleura were positive for fluorescence but could not be observed during white light examination. These abnormal reflections were confirmed by histologic examination. Normally, one does not perform "blind" biopsies of the visceral pleura, but this is clearly different when lesions are well demarcated.

We did experience a "learning curve" during the first eight patients to get accustomed to the handling of the fluorescence equipment and to interpret the fluorescence images. The management of the different switches was considered an issue that could be improved in newer generation of the fluorescence equipment. However, the additional time required in our study to perform the fluorescence imaging was acceptable, with an estimated additional 10 to 15 min including biopsies.

Concerning the false-negative results we encountered with FD, closer examination with FD of the excised specimen revealed small spots of fluorescence. Histologic examination confirmed the presence of tumor. These results were not taken into account for the overall evaluation since it was only done in a few patients.

Other methods that can be used for fluorescence detection in the thoracic cavity are the use of porphyrin compounds (Photofrin; Wyeth-Ayerst Lederle; Pearl River, NY; or Foscan; Biolitic Pharma Ltd.; Dublin, Ireland). These sensitizers have been used by several groups in an attempt to increase the discrimination between tumor and normal tissue, although success has been limited.121314 Furthermore, the porphyrins had significant side effects and required the use of expensive and complex imaging devices. We feel that the use of more potent sensitizers is not warranted for detection purposes since they tend to have longer-lasting skin phototoxicity. Autofluorescence of tumor tissue has been successfully used for detection in several organs (eg, bladder), but the major limitation is the variation in the fluorescence intensity and low specificity. To date, no reports have been published on the use of other photosensitizers in the thoracic cavity, but the use of autofluorescence has been reported.15

Methylated forms of aminolaevulinic acid, which have no skin toxicity, are available but must be administered locally.16 A possible drawback for the use in the thorax or abdomen could be the inhomogeneous distribution of the drug.

In the future, the use of higher doses of 5-ALA could also be used for imaging if local photodynamic therapy is indicated for the treatment of identified tumors. To date, this option has hardly been investigated due to the limited access of the thoracoscope in the chest cavity and the diffuse growth of pleural malignancies.

So far, we have shown only a limited benefit of 5-ALA FD for the individual patient. Staging of a disease such as mesothelioma was, however, facilitated using FD. In the future, FD might prove useful in patients presenting with limited pleural disease, but this has to be confirmed in larger trials with a more diverse patient population. A direct comparison with autofluorescence should also be made. We feel that with the proper adjustments of the system, our approach can improve staging of patients with pleural malignancies and might help to choose the proper biopsy sites during thoracoscopy.

Footnotes

Abbreviations: 5-ALA = 5-aminolaevulinic acid; FD = fluorescence diagnosis; Pp-IX = protoporphyrin-IX

Received for publication June 28, 2005. Accepted for publication October 3, 2005.

References

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  2. Gahlen, J, Prosst, RL, Pietschmann, M, et al Laparoscopic fluorescence diagnosis for intraabdominal fluorescence targeting of peritoneal carcinosis experimental studies. Ann Surg 2002;235,252-260[Medline]
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