J. Photochem. Photobiol. B: Biol., 14 (1992) 219-230 219

Light-induced fluorescence spectroscopy of adenomas, adenocarcinomas and non-neoplastic mucosa in human colon I. In vitro measurements

Renato Marchesini’, Marco Brambilla, Emanuele Pignoli

Division of Health Physics, Zstituto Nazionale Tumori, Via Venezian 1, Z-20133 Milan (Ztaly)

Giovanni Bottiroli, Anna Cleta Croce

Centro Zstochimica CNR, Pavia (Italy)

Marco Dal Fante, Pasquale Spinelli

Division of Endoscopy, Zstituto Nazionale Tutnori, Ka Venezian I, Z-20133 Milan (Italy)

Silvana di Palma

Division of Pathology, Zstituto Nazionale Tumori, Via Venezian I, Z-20133 Milan (Italy)

(Received October 14, 1991; accepted January 21, 1992)

Abstract

In an attempt to evaluate whether induced fluorescence could be exploited to discriminate neoplastic from non-neoplastic tissue, fluorescence spectroscopy was performed at 450-800 nm on 83 biopsy specimens of colonic mucosa. Measurements showed that fluorescence spectra of adenoma, adenocarcinoma and non-neoplastic mucosa manifest dissimilar patterns. Nine variables, whose photophysical and/or biological bases need further investigation, were derived from the spectra. Discriminant functions between the groups of lesions were determined by using a stepwise discriminant analysis. The diagnostic test had a sensitivity of 80.6% and 88.2%, and a specificity of 90.5% and 95.2% in discriminating neoplastic from non-neoplastic mucosa and adenoma from non-neoplastic mucosa respectively. These results suggest that fluorescence spectroscopy has the potential to improve endoscopic diagnosis of premalignant and malignant lesions of colonic mucosa.

Keywords: Native fluorescence, human colon, adenocarcinoma, adenoma, spectrofluorimeter.

1. Introduction

Detection of fluorescence in human tissues is under investigation as a possible method to aid in the clinical diagnosis of diseases. For instance, the property of some fluorescent drugs, mainly porphyrin derivatives, of being preferentially accumulated in tumour tissues with respect to normal surrounding tissue, provides the basis for a diagnostic approach in oncology [l-3]. The fluorescence of endogenous chromophores, i.e. autofluorescence, has recently been exploited to discriminate normal from diseased tissue [&lo]. Under excitation at a suitable wavelength, many biological components,

+Author to whom correspondence should be addressed

Elsevier Sequoia

220

either related to structural aspects of the tissue or involved in metabolic and functional processes of the cells, are fluorescent. Thus, spectral analysis of autofluorescence can provide information on the characterist ics of a given tissue, since the propert ies of the emission are dependent on the composit ion and environment of the biological substrate [11-13].

Among the various solid tumours suitable for a fluorescence investigation, we have considered adenoma and adenocarc inoma of the colon and rectum, since the lat ter is the second most common cause of cancer-re la ted death in Western industrialized nations [14]. Adenoma is commonly believed to be the precursor of adenocarcinoma, and various stages of tumour progression can be found in the same patient [15]. Genet ic and environmental factors are believed to initiate and promote malignant progression, thereby resulting in a field defect of colonic mucosa which can produce adenomatous polyps and may evolve into carcinoma [16]. Moreover, in familial ad- enomatous polyposis (FAP), a disease caused by a genetic defect recently localized in the long arm of chromosome 5 [17, 18], hundreds of colorectal adenomas are present and, if untreated, progress toward cancer in virtually 100% of the affected individuals [19]. Endoscopic explorat ion of the large intest ine permits observation of various lesions at different stages of malignant progression.

The non-invasive fluorescent technique in diagnosis has not yet been fully in- vestigated. Kapadia et al. [8] repor ted that analysis of laser-induced fluorescence (LIF) spectra using a he l ium-cadmium laser (325 nm) on specimens removed at colonoscopy or at surgery enables the detect ion of adenomatous transformation in colonic mucosa with an overall accuracy of 92%. Cothren et al. [9], who used an excitation wavelength of 370 nm, repor ted that adenoma can be distiguished in v ivo from normal colon with a sensitivity of 100% when their fluorescence intensities at 680 nm and 460 nm are compared. Finally, Yakshe et al. [7] used LIF with an excitation at 325 or 337 nm for in vitro diagnosis of colonic adenocarc inoma and showed different spectral emissions with respect to normal mucosa, depending on the muscle tissue infiltration of the tumour. A more exhaustive a t tempt to evaluate fluorescence spectroscopy potentials has been carried out by Richard-Kor tum et al. [10], who utilized fluorescence excitat ion-emission matrices to characterize colonic dysplasia autofluorescence.

The findings support the hypothesis that fluorescence spectroscopy might provide a method of tissue differentiation or diagnosis. Unfortunately, data repor ted in the above-mentioned papers do not allow a comparison of the characterist ic emission spectra of normal tissue, premalignant and malignant lesions, since differences in spectroscopic arrangement and analytical procedures do not permit a univocal diagnostic parameter to be derived.

The purpose of the present study was to characterize the emission spectra of neoplastic and non-neoplast ic colonic mucosa and to evaluate the possibility of: (i) discriminating malignancy from premalignancy; (ii) differentiating neoplastic from non- neoplastic tissue by analysing in vitro their induced fluorescence.

2. Materials and methods

Fluorescence spectroscopy was per formed on 83 biopsy samples removed at colonoscopy from 45 patients. In 17 pat ients a sessile, recto-sigmoidal adenoma, 3-5 cm in diameter , was present . In 19 pat ients an advanced carcinoma 5-10 cm long was located in the rectum (n = 12), sigmoid (n = 1) and colorectal anastomosis (n = 6). Five patients had FAP and the remaining four pat ients had no lesions.

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Analgesic drugs (e.g. diclofenac or meperidine) taken by the patients during the week preceding colonoscopy, as well as laxatives for bowel preparation (i.e. oral electrolyte lavage solution, e.g. ColyteR, or a senna preparation, e.g. X-PrepR), were recorded. Except for the X-PrepR solution, fluorescence analysis performed on the drugs showed that they did not affect the spectral profile of the biopsy autofluorescence. Since X-PrepR solution, under excitation at 410 nm showed a broad band fluorescence emission at 512 nm, patients were advised to avoid its use before colonoscopy.

Samples were taken by means of a biopsy miniforceps from the apparently non- necrotic area of adenoma and carcinoma and from the normal appearing mucosa adjacent (2-10 cm) to the lesion. In the patients without lesions and in those bearing FAP, biopsies were taken from the normal appearing mucosa. All biopsies were of similar volume, approximately of 10 mm 3. Biopsies removed from normal appearing colon were histologically composed of mucosa and submucosa, whereas samples from adenoma and carcinoma contained mainly tumour tissue.

After tissue excision, specimens were stored at 4 “C, and spectroscopy was performed within 2 h. Following spectral acquisition, all biopsies were frxcd in formalin and stained with hematoxylin and eosin for histological examination.

The histological diagnoses were: villous adenoma (13 casts), tubular adenoma (two cases), tubulovillous adenoma (two cases), adenocarcinoma (19 cases), and non- neoplastic mucosa (47 cases, five of them from the patients bearing FAP). In fact, 42 out of the 47 cases of non-neoplastic mucosa were considered, since the five non- neoplastic samples from the patients bearing FAP were examined separately. Comparison of fluorescence spectra was performed on four groups, namely: adenocarcinoma (II = 19); adenoma (n = 17); FAP (n = 5); non-neoplastic mucosa (n = 42).

Fluorescence intensity on the 45G300 nm range was recorded with a spcctro- fluorimeter (F3000, Hitachi, Japan) at an excitation wavelength of 410 nm, which was passed through an additional interference filter. A 420 nm long pass, low fluorescence filter was used to block scattered excitation light. Excitation and emission bandpass was 5 nm. Scan speed was set at 120 nm min-‘. The fluorescence signals were stored in a personal computer to be processed.

A drop of saline solution was used to moisten the sample to be analysed, which was carefully unfolded and placed between two slides, each of them with a rcctangular- shaped well (4 mm high, 8 mm long, 0.1 mm wide). The 0.2 mm optical path cuvette thus assembled was inserted in a metal frame and oriented 315” relative to the incident beam inside the cell holder of the spectrofluorimeter. Since biopsy had a volume of about 10 mm3, tissue filled up the entire path, being squeezed within the two wells. In order to continuously monitor intensity of excitation light, whose spot was 5 mm long and 1 mm high, or light transmitted by the sample, a simple optical system composed of a lens (50 mm focal length, 30 mm diameter), a mirror and a photodiode was positioned along the incident beam path, within the sample compartment. The scheme of the optical system is reported in Fig. 1. Intensity of the excitation light was about 10 pW. Transmission of the specimens was, on the average, 10%.

Nine spectral intensity features were chosen as input data in a discriminant analysis, as follows. For each fluorescence emission spectrum, six variables were obtained as the ratio of intensity at 460, 470, 480, 510, 520 and 530 nm with respect to that at 500 nm, and three variables were obtained as the fluorescence intensity measured at 480, 500 and 635 nm. Discriminant functions among adenoma, carcinoma and normal mucosa were determined by using the stepwise discriminant analysis reported in the BMDP-P7M computer program [20]. The stepwise analysis includes in the discriminant

222

z 1 q IN Sample well .

I Exci!:ltion

I --

light

410 nm

0.2 mm Fluorescence emission

420 nm L.P.F.

uvette holder

50 mm focal lens

Mirror

Fig. 1. Experimental set up for fluorescence measurements.

function one variable at each step until no further gain in the group separation results from the insertion of other variables [21].

3. Results

The mean fluorescence spectra of the non-neoplastic, adenomatous and carci- nomatous specimens are shown in Fig. 2. The intensity and shape of the mean spectrum of the 42 cases of non-neoplastic mucosa differed from those of the tumoural tissues. The amplitude of the emission band at 460-530 nm gradually decreased from non- neoplastic to carcinomatous and then to adenomatous mucosa. The fluorescence intensity of the five biopsies from normal appearing mucosa in FAP bearing patients was consistent with that of adenomas, although, owing to the small number of cases, the

223

Fluorescence Intensity

560 010 660

Wavelength (nm)

Fig. 2. Average fluorescence intensity of: (a) 42 samples of normal colonic mucosa (. . . . .); (b) 17 samples of adenoma (-); (c) 19 samples of carcinoma (-); (d) five samples of non polypoid colonic mucosa (- - -).

difference was not significant (P=O.2) when they were compared to the other cases of non-neoplastic mucosa.

The peak position of the fluorescence emission of adenoma and carcinoma appeared slightly red-shifted compared with that of non-neoplastic mucosa. Moreover, carcinoma exhibited an emission band at 635 nm, along with shoulders in the 650-100 nm region, which was occasionally detectable in the other tissues to a lesser extent.

To better estimate the difference in shape in the blue-green region among the four fluorescence spectra, for each single measurement the fluorescence intensities between 460 and 530 nm were normalized. Figure 3 shows the spectrum obtained by averaging the fluorescence ratio of the single measurement for each of the four above- mentioned groups, after normalization at 500 nm. The spectra appear somewhat different in the shape. A comprehensive analysis indicates that the spectra consist of more than one spectral component, since differing peaks and shoulders can be recognized, centered at about 470, 485 and 505 nm respectively. Adenoma and adenocarcinoma showed a similar pattern with a maximum that was slightly red-shifted with respect to that of non-neoplastic mucosa. Colonic mucosa of FAP bearing patients showed a fluorescence shape close to that of non-neoplastic mucosa.

Table 1 reports the mean values (*mean standard error (SEM)) of the nine selected variables. Comparison among the groups showed that most of the data related to non-neoplastic mucosa were significantly different compared to those of neoplastic tissues. In contrast, no difference was found between adenoma and adenocarcinoma except for fluorescence emission at 635 nm. In view of the absence of a significant difference between adenoma and adenocarcinoma, we pooled the whole set of their correspondent data and used multivariate regression as a method of discriminating neoplasms from non-neoplastic mucosa.

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, 2 Fluorescence lntenslty (a.u.) I

J

460 470 400 490 500 510 520 530

Wavelength

Fig. 3. Average fluorescence intensity, after normalization at 500 nm of each single measurement, of the spectra reported in Fig. 2.

TABLE 1

Mean values (f SEM) of nine selected variables evaluated from the fluorescent spectrum at different wavelengths

Non-neoplastic mucosa Adenoma (n =42) (n = 17)

Adenocarcinoma (n = 19)

Z[460]/1[500] 0.95 + 0.03 Z[47O]/Z[500] 1.07 f 0.02 1[480]/1[500] 1.09 + 0.02 1[510]/1[500] 0.89 f 0.01 1[520]/1[500] 0.77 f 0.01 Z[53O]/Z[500] 0.64f0.01 Z[480] 5.67kO.81 Z[500] 4.97 + 0.60 Z[ 6351 0.48 f 0.04

0.85 + 0.05a 0.86 f 0.04” 0.92* 0.05’ 0.97 + 0.03b 0.94 f 0.03’ 0.99 f 0.02’ 0.99 f 0.01’ 0.94 * O.OIC 0.90 f 0.02’ 0.83 f 0.02” 0.80 f 0.02’ 0.71 f 0.03b 2.36 f 1.07b 4.06 f 0.85 2.34 f l.OOb 3.90 f 0.78 0.34 + 0.08 2.21 f 0.7gcsd

“P<O.O8 compared to non-neoplastic mucosa. bP < 0.05 compared to non-neoplastic mucosa. ‘P < 0.01 compared to non-neoplastic mucosa. dP<O.Ol compared to adenoma. Probability values are given for a two-tailed Student’s t-test.

The stepwise regression used a partial F-test (F to enter equal to or greater than 4; F to remove less than 4) to sequentially incorporate a subset of the nine variables into a discriminant function (DF). The DF derived by analysing the whole set of

225

tumoural tissues versus the non-neoplastic cases (DFl) resulted in the following expression: DFl = 1.95 + 10.92 x (1[460]/1[500])

- 22.18 x (Z[480]/1[500]) +41.98x (1[520]/1[500]) -33.50x (Z[53O]/Z[500]) + 0.25 x F[635]

Four variables were not considered by the computer program in DFl because they were no longer significant in discriminating between the two groups. It is worth noting that a variable that appeared to be fairly important at an early stage may become superfluous at a later stage because of the relationship between it and other variables already in the model. If that variable is forced into a discriminant function a loss in discriminatory power can even occur [21]. Figure 4 shows the frequency distribution of the canonical variable, i.e. the score calculated by DFl for neoplastic and non- neoplastic colonic mucosa. For representative purposes, cases were grouped at a step interval of 0.5. The mean value (&SD) of the canonical variable was 1.21+ 1.1 and - 1.04 _t 0.88 for neoplastic and non-neoplastic cases respectively, with a discriminating threshold set, by the computer program, at -0.06. Of the 36 cases of histologically proven neoplasia 29 were correctly classified. Of the 42 cases of non-neoplastic mucosa 38 were correctly classified. The test had a sensitivity of 80.6% and a specificity of 90.5%. The actual classification rates were usually less than expected according to the sensitivity reported above, since DFl was determined using the same samples which were themselves the object of classification. Nevertheless, the computer program provides a jack-knifed classification table (i.e. every case is classified by using a DF obtained

Freauencv dlstrlbution t%i 50

40

30

20

10

0

m Neoplastlc

m Non-woplastlc

-3.75 -2.25 -0.75 0.75 2.2:~ 3.75

Canonical variabit

Fig. 4. Frequency distribution of the canonical variable for neoplastic and non-neoplastic samples.

226

from the remaining n - 1 cases), thus allowing a better estimate of the most probable misclassification rates. This procedure resulted in a sensitivity of 77.8% and a specificity of 90.5%.

Discriminant analysis was also performed across the other groups, i.e. adenoma versus normal mucosa (DF2), adenocarcinoma versus normal mucosa (DF3), and adenoma versus adenocarcinoma (DF4). The DF2 resulted in the following expression: DF2 = 16.08 + 11.04 x (F[460]/F[500])

- 25.10 x (F[480]/F[SOO]) The mean value (&SD) of the canonical variable was 1.94* 1.1 and -0.79kO.91 for adenoma and non-neoplastic cases respectively, with a discriminating threshold set at -0.10. Of the 17 cases of adenoma 15 were correctly classified. Of the 42 cases of non-neoplastic mucosa 40 were correctly classified. The test showed a specificity of 95.2% and a sensitivity of S&2%, and the latter figure reduced by the jack-knifed classification to 82.4%.

Poor results were obtained when discriminant analysis was applied between carcinoma and non-neoplastic mucosa (i.e. DF3), and between adenoma and carcinoma (i.e. DF4). The DF3 and DF4 resulted in the following expressions: DF3 = - 29.85 + 42.00 x (F[510]/F[500])

- 13.09 x (F[530]/F[500]) + 0.34 xF[635]

DF4 = - 7.84 + 9.52 x (F[520]/F[500]) - 0.29 xF[635]

Table 2 summarizes results obtained following the various discriminant analyses.

4. Discussion

Spectrofluorimetric analysis performed in biopsy samples showed that three main features characterized the difference in the autofluorescence emission spectra. Firstly,

TABLE 2

Classification of discriminant analysis applied to various entry groups in relation to histology

Histology

Neoplastic Non-neoplastic

Sensitivity

(%I

Specificity

(“ro)

Neoplastic Non-neoplastic

Adenoma Non-neoplastic

Carcinoma Non-neoplastic

Carcinoma Adenoma

29”

7

Adenoma 15

2

Carcinoma 11

8

Carcinoma 11

8

4

38

Non-neoplastic

80.6 90.5

Non-neoplastic 3

39

Adenoma 4

13

88.2 95.2

57.9 92.9

57.9 76.5

“Number of cases.

227

the maximum of intensity, in the 486500 nm region, increased in passing from adcnoma to carcinoma and then to non-neoplastic mucosa. Secondly, the fluorescence emission seemed to be due to a superimposition of at least three main emission bands, centcrcd at about 470, 485 and 505 nm. Thirdly, a characteristic peak at 635 nm was usually well evidenced only in biopsies of carcinoma.

With regard to the first feature, our results agree reasonably with those rcportcd in previously published papers. Cothren et al. [9] . h s owed that the fluorescence emission intensity is consistently lower in adenoma than normal mucosa. Such a difference allowed a good discrimination between neoplastic and non-neoplastic mucosa. Yakshc et al. [7] reported that colonic malignancy could give high or low fluorescence intensity depending on whether or not the cancer had abundant connective tissue stroma.

Adenoma, non-neoplastic mucosa and carcinoma usually show a respectively increasing amount of the connective tissue that might correlate with our findings in intensity of autofluorescence. An adenoma is composed almost entirely of epithclial cells with a small amount of stroma; normal mucosa has a mixture of conncctivc tissue and epithelium as does carcinoma, but in the latter the stromal reaction is often very marked [22, 231. Moreover, host cells or inflammatory cells, which arc involved in the response of the host to colonic carcinoma, arc distributed in the stromal compartment of carcinomas [24]. If we assume that fluorescence intensity emitted by the connective tissue predominates over that emitted by the epithclial cell layer, it follows that fluorescence intensity somehow reflects a different balance between collagen and epithelial cell content in the biopsy samples when similar biopsy volumes are involved, as it was in our case.

The previous assumptions imply that adenoma fluoresces less than normal mucosa and adenocarcinoma. To investigate the hypothesis about the relative content of stroma and epithelium, WC performed fluorescence measurcmcnts on five biopsies removed from non-polypoid colorectal mucosa of patients with FAP, where a general increase in thickness of the epithelial component is known to occur [25]. Results showed that FAP fluorescence intensity reflects that typical of adenoma (Fig. 2). Although FAP fluorescence intensity is comparable to that of adenomas, the pattern of autofluorcscence remains that typical of non-neoplastic mucosa.

As regards the different spectral shapes among non-neoplastic, adenomatous and carcinomatous mucosa (Fig. 3), statistical evaluation showed a significant difference when adenoma and carcinoma were compared with non-ncoplastic mucosa. These findings correspond with the results reported by Kapadia et al., who showed that the mean spectrum of normal colonic mucosa differs from that of adcnomatous colonic mucosa and has a blue-shifted maximal intensity peak [8]. The difference in the fluorescence emission shape could be due to the presence of different fluorophores or to differing amounts of similar fluorophores.

Scattering and absorption processes in the optically thick samples, along with the variability from subject to subject, result in a lack or reduction of the structure of the fluorescence spectrum, which prevents the separate peaks or shoulders being distinctly recognized, as otherwise evidenced in Fig. 3. As to the chemical nature of the fluorophores, nothing can be inferred for certain at present. In fact, it is known that tissue light-induced fluorescence is generated by a variety of chromophores (i.e. NADH, flavins, riboflavins, pyridoxal 5’-phosphate, vitamins and porphyrins are the most common) whose identification and relative contribution to the overall tissue fluorescence have not yet been fully elucidated, in spite of the amount of work done [lo]. The reasons why the different fluorescence patterns pass from non-neoplastic to neoplastic tissue remain a matter for further investigation. It has been suggested that

228

the differcncc might be attributable to a decreased content of NADH and pyridoxal 5’-phosphate when cancerous states are involved [lo].

The presence of the peak at 635 nm and the broad band between 660 and 700 nm in the adenocarcinoma mean fluorescence spectrum can reasonably be ascribed to endogeneous porphyrins. Fluorescence macroscopic observation with Wood’s lamp cvidcnced the presence of red-fluorescing hot spots within the tissue structure. Their non-homogeneous distribution within the biopsy sample could account for the remarkable variability in intensity observed under the spectrofluorimetric experimental conditions. Histology did not show a relationship between the hot spots and microscopic alterations in tissue or the cellular structures responsible for the emission. The presence of an unusual amount of endogeneous porphyrins in cancerous tissue has been reported by several authors, but the rationale for such a finding remains obscure [26].

The nine variables used in the discriminant analysis were chosen after several attempts based upon empirical approaches. One of the problems related to discriminant analysis is that of finding the variables which discriminate best between groups. In our case the rationale for searching for the variables was that of selecting parameters which showed a highly significant difference when the groups were compared. Table 1 shows that almost all the selected variables were significantly different when carcinoma and adenoma were compared to non-neoplastic mucosa. Nevertheless, several of them were not included in the discriminant functions because the stepwise discriminant analysis procedure operates with variables which have the most significant F-value after adjusting for variables already included in the model.

Multivariate regression analysis discriminates adenomatous tissue from non-nco- plastic mucosa with both sensitivity (88.2%) and specificity (93.2%) better than those found when the other groups are compared with one another. As far as carcinoma is concerned, it is worth noting that emission intensity at 635 nm was always included in the discriminant functions, i.e. DFl, DF3 and DF4. Thus, the peak at 635 nm may be considered characteristic of carcinoma. Nevertheless, the remarkable variability in intensity from sample to sample did not allow improvement of carcinoma classification when compared to the other groups. Interestingly, apart from emission intensity at 635 nm, only variables related to the spectral shape were included in all the DFs. This means that tissue spectra are better differentiated when some form of normalization is applied. This finding is confirmed by the excellent results reported by Kapadia et al. who employed a stepwise regression analysis to relate the spectral intensity features to tissue type following normalization of tissue spectra to the total integrated light intensity [8].

Notwithstanding the great variability from sample to sample and from patient to patient, fluorescence characteristics of adenoma show the greatest statistical difference when compared to those of normal mucosa. It should be mentioned that one of the main sources of uncertainty affecting fluorescence emission is the variability in morphology of the biopsy samples. In fact, although all the biopsies were histologically representative, samples of neoplastic lesions were affected by the presence of variable portions of non-neoplastic tissue, particularly when carcinoma biopsies were considered. On the basis of the previous hypothesis concerning the different relative contributions of the histological components (i.e. different emission of stromal tissue compared to epithelial tissue), the fluorescence pattern may be influenced to a large extent by varying the ratio between the number of epithelial cells and amount of connective tissue. A retroprospective histological examination of the neoplastic cases misclassified by dis- criminant analysis revealed that most of them exhibited a predominance of stroma and granular tissue with respect to the epithelial layer. Furthermore, the area involved

229

with tumour cells had, in several cases of carcinoma, a very minimal extension with respect to the remaining non-neoplastic tissue.

Interpretation of the fluorescence spectra remains difficult owing to the complexity of the physical and biochemical processes involved. Moreover, the limited reported cases do not permit us to stress the validity of the hypotheses used to explain the observed differences between neoplastic and non-neoplastic colonic mucosa. Detection of early cancer or premalignant changes in the gastrointestinal tract is one of the main requisites for a high cure rate. Our results suggest that fluorescence spectroscopy has the potential to improve endoscopic diagnosis of premalignant and malignant lesions of the gastrointestinal tract. A fluorescence spectroscopic diagnostic system for in viva measurements is being developed by our group, and preliminary results seem to confirm the findings shown by the in vitro measurements [27].

Acknowledgements

The authors gratefully acknowledge the skilful technical assistance of Ambrogio Colombo. This work was supported in part by the Special Projects “Applicazioni cliniche della ricerca oncologica” and “Tecnologie Elettroottiche”, grant 90.00208.65, C.N.R., Rome, Italy.

References

1 A. E. Profio, Review of fluorescence diagnosis using porphyrins, SPZE, 905 (1988) 150-156.

2 A. Andersson-Engels, J. Johansson, K. Svanberg and S. Svanberg, Fluorescence imaging and point measurements of tissue: applications to the demarcation of malignant tumors and

atherosclerotic lesions from normal tissue, Phofochem. PhofobioZ., 53 (1991) 807-814. 3 M. Dal Fante, G. Bottiroli, P. Spinelli, Behaviour of haematoporphyrin derivative in adenomas

and adenocarcinomas of the colon: a microfluorimetric study, Lasers Med. Sci., 3 (1988)

165-171. 4 R. R. Alfano, W. Lam, H. Zarrabi, M. A. Alfano, J. Cordero, D. Tata and C. Swinberg,

Human teeth with and without caries studied by laser scattering, fluorescence and absorption spectroscopy, IEEE Q.E., 20 (1984) 1512-1516.

5 R. R. Alfano, A. Pradhan and G. C. Tang, Optical spectroscopic diagnosis of cancer and normal breast tissues, .Z. Opt. Sm. Am. B, 6 (1989) 1015-1023.

6 R. Richards-Kortum, A. Metha, G. Hayes, R. Cothren, T. Kolubayev, C. Kittrell, N. B. Ratliff, J. R. Kramer and M. S. Feld, Spectral diagnosis of atherosclerosis using an optical catheter, Am. Heart J., 118 (1989) 381-391.

7 P. N. Yakshe, R. F. Bonner, R. Patterson, M. B. Leon and D. E. Fleischer, Laser induced

fluorescence spectroscopy (LIFS): can it be used in the diagnosis and treatment of colonic malignancy?, Am. J. GastroenteroZ., 84 (1989) 1199 (abstract).

8 C. R. Kapadia, F. W. Cutruzzola, K. M. O’Brien, M. L. Stetz, R. Enriquez and L. I.

Deckelbaum, Laser-induced fluorescence spectroscopy of human colonic mucosa, Gasho- enterology, 99 (1990) X0-157.

9 R. M. Cothren, R. Richards-Kortum, M. V. Sivak, M. Fitzmaurice, R. P. Rava, G. A. Boyce,

M. Doxtader, R. Blackman, T. B. Ivanc, G. B. Hayes, M. S. Feld and R. E. Petras, Gastrointestinal tissue diagnosis by laser-induced fluorescence spectroscopy at endoscopy, Gastrointest. Endoscopy, 36 (1990) 105-l 11.

10 R. Richard-Kortum, R. P. Rava, R. E. Petras, M. Fitzmaurice, M. Sivak and M. Feld, Spectroscopic diagnosis of colonic dysplasia, Phofochem. PhotobioZ., 53 (1991) 777-786.

230

11 E. Kohen, C. Kohen, J. C. Hirschberg, A. Wonters and B. Thorell, Multisite topographic

microfluorometry of intracellular and exogeneous fluorochromes, Photochem. PhotobioZ., 27

(1978) 259-1268.

12 R. C. Benson, R. A. Meyer, M. E. Zaruba and G. M. McKhann, Cellular autofluorescence: is it due’to flavins?, J. Histochem. Cytochem., 27 (1979) 44-49.

13 G. M. Baremboim, A. N. Domanskii and K. K. Turoverov (Eds.), Luminescence of Biopoiymers and Cells, Plenum Press, New York, 1969, pp. 64-75.

14 E. Silverberg and J. A. Lubera, Cancer statistics, 1988, CA-A, 39 (1989) 3-20.

15 S. J. Winawer, A. G. Zauber, E. Stewart and M. J. O’Brien, The natural history of colorectal

cancer, Cancer, 67 (1991) 1143-1149. 16 T. Muto, H. J. R. Bussey and B. C. Morson, The evolution of cancer of the colon and

rectum, Cancer, 36 (1975) 2251-2270. 17 W. F. Bodmer, C. J. Bailay, J. Bomer, H. J. R. Bussey, A. Ellis, P. Gorman, F. C. Lucibello,

V. A. Murday, S. H. Rider, P. Scambler, D. Sheer, E. Solomon and N. K. Spurr, Localization

of the gene for familial adenomatous polyposis on chromosome 5, Nature, 328 (1987) 614-616. 18 M. Leppert, M. Dobbs, P. Scambler, P. O’Connell, Y. Nakamura, D. Staffer, S. Woodward,

R. Burt, J. Hugues, E. Gardner, M. Lathrop, J. Wasmuth, J. M. Lalouel and R. White, The

gene for familial polyposis coli maps to the long arm of chromosome 5, Science, 238 (1987)

1411-1413. 19 B. C. Morson and H. J. R. Bussey, Magnitude of risk for cancer in patients with colorectal

adenomas, Br. J. Surg., 72s (1985) 23-28. 20 L. Engelman, J. W. Frane and R. I. Jennrich, BMDP Biomedical Computer Program. P-series,

University of California Press, Los Angeles, CA, 1977, pp. 711-734.

21 D. G. Kleinbaum, L. L. Kupper and K. E. Muller, Applied Regression Analysis and other Multivariate Methods, PWS-Kent, Boston, MA, 1988.

22 G. Sauter, A. Nerlich, U. Spengler, R. Kopp and A. Pfeiffer, Low diacylglycerol values in colonic adenomas and colorectal cancer, Gut, 31 (1990) 1041-1045.

23 H. Ohtani and N. Sasano, Stromal cell changes in human colorectal adenomas and aden- ocarcinomas, Virchows Arch. Puthol. Anat. Physical, 401 (1983) 209-222.

24 M. C. McGinnis, E. L. Bradley T. P. Pretlow, R. Ortiz-Reyes, C. J. Bowden, T. A. Stellato

and T. G. Pretlow, Correlation of stromal cells by morphomeric analysis with metastatic behavior of human colonic carcinoma, Cancer Res., 49 (1989) 5989-5993.

25 B. C. Morson, I. M. P. Dawson, D. W. Day, J. R. Jass, A. B. Price and G. T. Williams, Morson & Dawson’s Gastrointestinal Pathology, Blackwell Scientific Publications, Oxford, 1990,

pp. 581-583. 26 Y. Yualong, Y. Yanming, L. Fuming, L. Yufen and M. Paozhong, Characteristic autofluorescence

for cancer and its origin, Lasers Surg. Med., 7 (1987) 528-532. 27 G. Bottiroli, A. C. Croce, R. Marchesini, E. Pignoli, M. da1 Fante, S. di Palma and P.

Spinelli, In vivo fluorescence spectroscopy of adenomas, adenocarcinomas and non-neoplastic mucosa in human colon, Lasers Surg. Med., 3S (1991) 27 (abstract).