NOTES. Tetracycline Fluorescence of Pathologic Pulmonary Tissues. P. A. Thomas, Maj, MC, USA, E. H. Merrigan,

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1 NOTES Tetracycline Fluorescence of Pathologic Pulmonary Tissues P. A. Thomas, Maj, MC, USA, E. H. Merrigan, Capt, MC, USA, and S. Tignor, Capt, MC, USA T he need for a simple, reliable means of detecting pulmonary malignancy prior to or during surgery is generally recognized. Exploration of methods which promise to augment the diagnostic efficiency of time-consuming histological techniques should continue. The tetracycline drugs are known to fluoresce under ultraviolet light excitation. This observation was exploited by Bottiger who traced the body-tissue distribution of tetracycline in experimental animals [5]. This flourescence disappears from normal tissues within 24 hours, except for bones and teeth which exhibit persistence for long periods of time [ll]. In 1957 Rall and associates observed that tumor tissue obtained upon the death of a patient with breast cancer exhibited fluorescence under ultraviolet light [ 141. The fluorescence was attributed to therapeutically administered tetracycline in the final days of the patient's life. Subsequently, tetracycline-induced fluorescence of malignant tissues from stomach, colon, breast, lung, bone, and other sources has been reported [Z, 6, 9, 10, 12, 13, 17, 181. Comparative study of selected benign tuiiiefactioiis demonstrated no fluorescence [ 121. However, persistence of flourescence in some inflammatory lesions has been observed [l, 201. In 1961 Klinger and Katz reported the detection of gastric malignancy by producing tetracycline-induced fluorescence of exfoliated cells From the Ikpartment of Surgery. I'i1or;icic Siirgcry Sen ice, \'ailey E'orgc Gctieral Hospit;il, Phocnixville, Pa. Received for publication Jan. 4, m1.: ANNALS OF THORACIC SURGERY

2 NOTE: Tetracycline Fliiorescence present in gastric sediment [8]. These investigators reported a high degree of diagnostic accuracy using this technique, which was confirmed by others conducting similar examinations [3, 4, 15, 181. The simplicity of this test, which requires only the additional preparatory period of tetracycline administration to otherwise established procedures, prompted other investigators to make similar studies. Cummins and associates compared tetracycline-induced fluorescence of gastric sediment with cytology in 146 patients, of whom 25 had gastric malignancy [7]. Five false positive examinations were encountered in addition to a number of false negative results. They concluded that the combined accuracy of both fluorescence and cytology exceeded that of either test alone, but that fluorescence without cytologic confirmation was not reliable. Sandlow and Necheles extended these diagnostic efforts to include study of fluid sediments from the peritoneum, pleura, and bronchi [16]. They reported excellent results in patients with malignant pleural effusion; however, no mention was made as to the primary tumor source in their series. Sevelius et czl. directed their study to the diagnosis of primary pulmonary malignancy [ 171. They made a comparison of sputum cytology and tetracycline-induced fluorescence of sputum sediment in the diagnosis of bronchogenic carcinoma. They were unable to demonstrate diagnostic value with the addition of the fluorescence technique. Yesner also concluded that few diagnostic gains could be anticipated by application of exfoliated tumor cell fluorescence following tetracycline therapy in lung cancer [2 11. The present study was undertaken to explore further and clarify the potential diagnostic usefulness of the tetracycline-induced fluorescence in material obtained from patients harboring bronchogenic carcinoma. An attempt was made to determine the reliability of the test in malignant in comparison to nonmalignant lung lesions by examination of surgically removed specimens requiring histological study as well as material obtained directly from the bronchial tree or pleura. MATERIALS AND METHOLIS Patients with a variety of pulmonary lesions referred to the Thoracic Surgery Service for either diagnostic evaluation or surgery were selected for study. Demethylchlortetracycline was the fluorescent antibiotic administered to these patients. This was given orally for 3 days in therapeutic doses of 600 mg. daily in two equally divided portions. A planned delay of 36 to 48 hours was allowed from the administration of the last dose to excision of tissue or collection of the specimen. The material for examination was exposed to ultraviolet light (wave length 3,600 A) in a darkened room. Gross surgical specimens were immedi- VOL. I, NO. 3, MAY,

3 THOMAS, MERRIGAN, AND TICNOR ately incised into or through the lesion and the cut surface studied under ultraviolet light in the operating room. Tissues from 8 patients who did not receive demethylchlortetracycline were examined. These were specimens harboring known tuberculous residua or chronic granulomas, studied as a control measure. Thoracentesis fluids and saline bronchial washings were centrifuged and the sediment spread on filter paper to dry after decanting of the supernatant liquid. No attempt was made to grade the results; the detection of any bright yellow fluorescence in the specimen under ultraviolet light was considered positive. K ES UL TS The gross tissue specimens from 29 patients submitting to thoracotomy have been studied (Table 1). As a control measure, 8 patients with either known pulmonary tuberculosis or etiologically undeter TABLE 1. FLUORESCENCE OF EXCISED PATHOLOGIC PULMONARY TISSUES Tetracycline Pa thology Adminisirat ion Pos. Malignant Yes 4 Tuberculosis Yes 3 3. Granuloma Yes 2 4. Tuberculosis No 0 5. Granuloma No 0 Totals 9 Fluorescence N eg. Total mined granulomata did not receive demethylchlortetracycline prior to surgery. Although the semisolid cavitary content of some of these specimens had naturally yellowish discoloration, none of these specimens fluoresced under ultraviolet light. Similar nonmalignant lesions were obtained from 13 patients who did not receive the drug prior to operation. Fluorescence was demonstrated in 3 of 7 specimens from patients with culture-proved tuberculosis. From the series of 6 etiologically undetermined granulomas examined, 2 exhibited characteristic fluorescence under ultraviolet light excitation. Eight patients with primary pulmonary malignancy received demethylchlortetracycline before operation. Four gross surgical specimens from these patients fluoresced and 4 did not. The possible influence of histological tumor classification on tetracycline-induced fluorescence is presented in Table 2. Alveolar carcinoma was encountered on 3 occasions and in no instance was fluorescence demonstrated. Sixteen patients had been prepared with demethylchlortetracycline prior to bronchoscopy and the dried bronchial washings sediments examined under ultraviolet light. Four patients with proved broncho- 322 THE ANNALS OF THORACIC SURGERY

4 NOTE: Tetracycline Fluorescence TABLE 2. FLUORESCENCE OF EXCISED MALIGNANT PULMONARY TISSUES Classification of Tumor 1. Alveolar carcinoma 0 2. Epidermoid carcinoma 2 3. Adenocarcinoma 1 4. Undifferentiated carcinoma 1 Totals 4 Fluorescence Pos. N eg. Total genic carcinoma were included in this group. The specimens from 2 of these patients with far-advanced malignant disease fluoresced. The bronchial washing from one patient with bronchial-biopsy-proved epidermoid carcinoma and widespread metastatic disease did not fluoresce. The second patient with proved malignancy from whom a nonfluorescent bronchial washing sediment was obtained had received intensive radiation therapy prior to bronchoscopic collection of the specimen. Twelve bronchial-washing sediment examinations were conducted on specimens from patients who either had known tuberculosis or who had had diagnostic evaluation for nonmalignant endobronchial disease. Two of these specimens fluoresced under ultraviolet light, both having come from patients with culture-proved tuberculosis. Two patients with pleural fluid had been prepared with demethylchlortetracycline before thoracentesis. One of these patients developed pleural effusion on completion of palliative radiation therapy for proved bronchogenic carcinoma. The pleural-fluid sediment fluoresced under ultraviolet light, and malignant cells were evident on microscopic examination of the cells from the same aspirate. The other pleural-fluid sediment did not fluoresce and was obtained from a patient with known tuberculous pleuritis. DISCUSSION The demonstration of persistent demethylchlortetracycline-induced fluorescence of malignant tissue of pulmonary origin in this study is in accord with the observations of other investigators. However, in our experience this has not been consistent, nor has it been a specific finding in lung cancer. Speculation as to the many influences which may be important would be rhetorically interesting; however, the status of vascular perfusion and tetracycline concentration relative to the time of desquamation or sequestration must play some role in drug retention or persistence of fluorescence. We have observed not only that demethylchlortetracycline enters the center of some tuberculous cavitary lesions but also that a difference in fluorescence between two cavities from the same resected lung specimen may occur. The suggestion that VVI.. I, NO. 3, MAY,

5 ~~ l HOMAS, MERRIGAN, AND TIGNOR the tetracycline may be found in the histiocyte cellular lipid complex would account for the apparent differences in reported observations [20, 211. As a diagnostic test the positive fluorescence of exfoliated cells presents an attractive addition to procedures now available. We attempted to improve on the experience of those who used sputum or gastric aspirate as the source of cells from lung cancer by direct collection of bronchial washings at bronchoscopy [7, 171. In his paper on the recovery of malignant cells from the bronchial tree for cytological examination, Umiker recited some considerations which also apply to recovery of fluorescent cells from that source [19]. The proximal obstructed bronchus and the noncell-shedding peripheral lesion will continue to thwart detection efforts. Also, the specimen collected at a given moment may not contain representative material. Finally, these factors are compounded by the fact that not all bronchogenic carcinomas exhibit tetracycline-induced fluorescence, even though centrally located and accessible. We have only been able to obtain pleural fluid for examination from 2 patients during the course of this study. This meager number of cases is partly the result of an understandable desire of the physician to proceed immediately to determine the nature of a pleural effusion, and the delay for administration of tetracycline may not be appreciated. In both of these cases the diagnosis had been established prior to our examination of the pleural-fluid sediment for fluorescence. The total available reported experience with tetracycline-induced ultraviolet fluorescence of malignant pulmonary tissues is summarized in Table 3. Although the number of cases is not large, there is apparent individual tumor variation with respect to fluorescence as well as some difference in test sensitivity among the sources of material studied. The diagnostic usefulness of this technique does not seem to approach initial expectations. TABLE 3. TETRACYCLINE-INDCICE1) FLUORESCENCE OF PIJLMONARY MALIGNANCY: SUMMARY OF REI OR I EI) RESULTS Proved Malignancy/ Posi tive Fluorescence Gross Pleural-Fluid Bronchial Gastric Source Specimen Sediment Sediment Aspirate Total Sandlow et al. [161-12/12 2/2-14/14 McLeay et al. [lo] 3/ /3 Sevelius et al. [171 3/ 1 1 /0 10/2-14/3 Cummins et al. [ o/1 10/1 Thomas et al. 8/4 1/1 4/2-13/7 [present paper1 Totals 14/8 14/13 16/6 10/1 54/ THE ANNALS OF THOKACIC SUKCEKY

6 NOTE: Tetracycline Fluorescence The observation of persistent tetracycline-induced fluorescence of some chronic inflammatory lesions of the lung as well as exfoliated cellular debris from the bronchial tree lends further support to the suggestion that the histiocytes rather than the malignant cell carry the fluorescent tetracycline material. Therefore, the presence or degree of fluorescence of a given pathological tissue may be dependent on the number of these cells associated with the lesion and specimen examined. In any event, the demonstration of positive fluorescence without histological confirmation cannot be accepted as definitive evidence of pulmonary malignancy. SUMMARY We have reported the results of our investigation of demethylchlortetracycline-induced fluorescence of malignant pulmonary tissues. Specimens from 47 patients have been studied with ultraviolet light excitation. Examination of 29 gross surgical specimens, 16 sediments of bronchial washings, and 2 sediments of aspirated pleural fluids was accomplished. Fluorescence was demonstrated in 7 of 13 specimens from patients with proved lung cancer. However, fluorescence was also noted in 7 of 26 specimens from patients with nonmalignant disease, tuberculous residua, or chronic granulomas. False positive and false negative results were obtained by study of both tissue specimens and bronchial-washing sediments. These observations fail to substantiate any significant diagnostic contribution in differentiating bronchogenic carcinoma by this technique either prior to or during surgery. ACKNOWLEDGMENT The authors wish to express their gratitude to Dr. Richard N. Fallon, of Lederle Laboratories, who supplied the Declomycin used in this study. REFERENCES 1. Ackerman, N. B., and McFee, A. S. Tetracycline Huorescence in benign and malignant tissues. Surgery 53:247, Bailey, R. W., and Levin, P. D. The clinical significance of the localization of tetracycline in certain tumors of bone. J. Surg. Res. 3:136, Berk, J. E. Additional comment on the tetracycline Huorescence test in differential diagnosis of gastric disease. Gastroenterology 45:586, Berk, J. E., and Kantor, S. M. Demethylchlortetracycline induced fluorescence of gastric sediment. J.A.M.A. 179:997, Bottiger, L. E. On the distribution of tetracycline in the body. Antibiot. Chemother. (N.Y.) 5:332, Carter, R. L., Floyd, C. E., ancl Cohn, I., Jr. Tetracycline flourescence in tumors and colon washings. Szirg. Forum 13:96, Cummins, A. J., Gompertz, M. L., and Kier, J. H. An evaluation of the tetracycline-fluorescence test in the diagnosis of gastric cancer: Comparison with cytology. Ann. Intern. Med. 61:56, VOI.. 1, NO. 3, MAY,

7 THOMAS, MERRIGAN, AND TIGNOR 8. Klinger, J., and Katz, R. Tetracycline flourescence in the diagnosis of gastric carcinoma. Gastroenterology 41:29, McLeay, J. F. The use of systemic tetracyclines and ultraviolet in cancer detection. Amer. J. Szrrg. 96:415, McLeay, J. F., and Walske, H. R. Relationship of tetracycline to carcinoma. Ann. Siirg. 156:313, Milch, R. A., Rall, 0. P., and Tobie, J. E. Hone localization of the tetracyclines. J. Nat. Cancer Znst. 19:87, Phillips, J. W., Cobb, E. G., Richards, V., Rhotles, W. D., Loehrer, D. C., and Ritchie, J. L. The disposition and retention of tetracycline in cancer. Amer. J. Surg. 100:384, Prout, G. R., Clark, R. F., Denis, L. J., antl Solomon, S. Differences in tissue fluorescence of oxytetracycline in the kidney antl metastatic prostatic carcinoma in bone. Stirg. Forum 13:513, Rall, D. P., Loo, T. L., Lane, M., antl Kelly, M. G. Appearance and persistence of fluorescent material in tumor tissue after tetracycline atlministration. J. h'at. Cancer Znst. 1979, Santllow, L. J., Allen, H. A., antl Necheles, H. The use of tetracycline fluorescence in the detection of gastric malignancy. Ann. Intern. Med. 58:409, Sandlow, L. J., antl Necheles, H. Tetracycline fluorescence in detecting malignancy. J.A.M.A. 189:363, Sevelius, H., Jimmerson, G., antl Colmore, J. P. Tetracycline fluorescence in bronchogenic carcinoma and chronic pulmonary diseases. J. ORln. Med. Ass. 56:578, Sherman, H. H., Chryssanthow, C., Sukoff, M. H., Mininberg, D., Beckman, E. M., and Weingarten, M. Tetracycline fluorescence in the diagnosis of gastric carcinoma. Gastroenterology 45:84, Umiker, W. 0. False negative reports in the cytologic diagnosis of cancer of the lung. Amer.,J. Clin. Path. 28:37, Vassar, P. S., Saunders, A. M., antl Culling, C. F. A. Tetracycline fluorescence in malignant tumors antl benign ulcers. Arch. Pnth. 69:613, Yesner, R. Personal communication, THE ANNALS OF THORACIC SURGERY