WO1997006724A1

WO1997006724A1 - Endoscopic imaging for the detection of cancer lesions

Info

Publication number: WO1997006724A1
Application number: PCT/FR1996/001288
Authority: WO
Inventors: Sigrid Avrillier; Dominique Ettori; Maurice Anidjar
Original assignee: Centre National De La Recherche Scientifique (Cnrs)
Priority date: 1995-08-16
Filing date: 1996-08-14
Publication date: 1997-02-27
Also published as: JPH11511048A; EP0850012A1; FR2737845A1; FR2737845B1

Abstract

The present invention relates to an endocospic imaging device for the early detection of superficial cancer ou precancer lesions, characterized in that it comprises means (10) for applying by endoscopy, to the tissues (T) to be imaged, an excitation signal having a wavelength between 300 and 320 nm, means (20) for detecting the self-fluorescence signal generated by the tissues (T) in response to the excitation, analysis means (30) for isolating the autofluorescence responses generated on the one hand by the tryptophane at about 370 nm and, on the other hand, by NADH at about 450 nm, both responses being processed in order to define an image of the tissues analyzed.

Description

ENDOSCOPIC IMAGING FOR DETECTION OF CANCER LESIONS

The present invention relates to the field of endoscopic imaging devices based on the analysis of tissue autofluorescence induced by light excitation, for example by laser excitation.

Since the mid-1970s, lasers have been used mainly in medicine for therapeutic actions consisting in destroying or coagulating tissue. In particular, it has been proposed to use lasers for therapeutic purposes in the field of ophthalmology, cardiology, orthopedics, neurosurgery, or even dentistry. However, in recent years, research efforts have tended to focus on the application of lasers to medical diagnosis. In this type of application, the useful light energy is low and does not cause any structural or functional modification of the tissues explored. Research in the field of medical diagnosis based on light excitation, for example on laser, in particular based on the analysis of excitation-induced autofluorescence, has given rise to a very abundant literature.

Numerous documents describe techniques for diagnosing atherosclerotic plaques by fluorescence analysis.

The document [1] IEEE JOURNAL OF QUANTUM ELECTRONICS, vol. QE-23, n ° 10, October 1987, "Autofluorescence Maps of Atherosclerotic Human Arteries-A New Technique in Médical Imaging", M. Sartori et al., Offers laser excitation of the tissues analyzed at a wavelength of 458nm and concludes that calcified tissues give more intensity of autofluorescence, than normal tissues, between 480 and 630nm.

The document [2] LASERS IN MEDICAL SCIENCE, vol. 4: 171, 1989, "Laser-Induced Fluorescence used in Localizing Atherosclerotic Lésions", S. Andersson-Engels et al., Offers an excitation at 337nm. The document [3] LASERS IN THE LI FE SCIENCES 3 (4), 1990,

"Excimer Laser Induced Autofluorescence from Atherosclerotic Human Arteries", GH Pettit et al., Proposes an excitation at 1 nm and concludes that the calcified tissues deliver a broader spectrum of response than healthy tissues. The document [4] IEEE JOURNAL OF QUANTUM ELECTRONICS, vol.

26, n ° 12, December 1990, "Malignant Tumor and Atherosclerotic Plaque

Diagnosis Using Laser-Induced Fluorescence ", S. Andersson-Engels et al., Offers excitation at 337nm and imagery by image overlay.

The document [5] SPIE, vol. 1201, Optical Fibers in Medicine V (1990), "Laser Induced Tissue Autofluorescence versus Exogenous Chemical Probe Induced Fluorescence as an Arterial Layer Detection Method. A Comparative Study", TG Papazoglou et al., Proposes an excitation at 308nm in the presence of a exogenous marker.

The document [6] LASERS IN SURGERY AND MEDICINE 10: 245-261 (1990), "Laser Induced Fluorescence Spectroscopy of Normal and Atherosclerotic Human Aorta Using 306-3 lOnm Excitation", JJ. Baraga et al., Analyzes the fluorescence rate of tryptophan, and collagen and elastin. He concludes that certain excitation wavelengths, notably at 325nm and 337nm do not give usable results.

The document [7] SPIE, vol. 1425, Diagnostic and Therapeutic Cardiovascular Interventions (1991), "Fluorescence Spectroscopy of Normal and Atheromatous Human Aorta: Optimum Illumination Wavelength", AL Alexander et al., Studies the excitation-induced autofluorescence between 270 and 470nm and concludes that it There are no significant differences between the autofluorescence spectrum of normal tissues and the autofluorescence spectrum of atheromatous tissues for an illumination of 270nm, 300nm or 304nm. Numerous attempts at very early diagnosis of cancer by analysis of the fluorescence induced by the tissues following a light excitation have also been proposed. More precisely, the majority of this research has focused on the diagnosis of superficial cancerous or precancerous lesions, not observable in visible light. The first tests in this area were carried out using injected markers. However, it turns out that these markers generate harmful side effects.

On this point, one can refer for example to the document [8] SPIE, vol. 1201, Optical Fibers in Medicine V (1990), "Detection of lung cancer by ratio fluorometry with and without Photofrin II", S. Lam et al., which proposes an excitation at 405nm and an analysis of the fluorescence response on the basis of the response ratio at 690nm and 560nm.

To avoid the harmful effects of these markers, we then looked at the analysis of tissue autofluorescence. Initially, research was mainly carried out on brain tissue or the lungs.

The document [9] JOURNAL OF LASER APPLICATIONS, Winter 1991, pages 46-47, R. Alfano, "Applying Optical Spectroscopy", offers an excitation at 300nm and an analysis of the autofluorescence response compared to the autofluorescence intensities at 340 and 440nm.

The document [10] ANNALES DE PHYSIQUE, Colloque n ° 1, supplement to n ° 3, vol. 17, June 1992, "Current state of the use of excimer lasers in medicine", S. Avrillier et al., Indicates that the cancerous state in cerebral white matter can result in the relative intensity of peaks at 362 and 383.6nm following an excitation at 308nm. However, this document adds that this technique is in no way usable in general in terms of diagnosis. In particular, this technique does not allow significant detection of the cancerous state of gray matter. The document [1 1] BIOMEDICAL OPTICS '93, "Excimer User

Induced Autofluorescence for Cerebral Tumors Diagnosis. Preliminary Study ", S. Avrillier et al., Proposes an illumination at 308nm and an analysis of the response of the fluorescence spectrum in a range of 250nm going from 350 to 600nm. This document concludes that the analysis of the fluorescence response requires a complex processing comprising in particular calculations on the maxima, the minima, ratios between maximum intensities and calculations on the slopes at different wavelengths of the response obtained.

The document [12] "Monte Carlo evaluation of laser induced fluorescence spectra modifications due to optical properties of the medium: Application to real spectra correction", E. Tinet et al., Indicates that numerous diagnostic researches by autofluorescence analyzes have have been proposed, but that however this technique is very complex. According to this document, the autofluorescence response depends on the excitation distribution, the geometry of the medium, the absorption coefficient of the medium, the diffraction coefficient of the medium, the distance of the observed tissue from the excitation. The document adds that this technique is particularly difficult to implement in vivo since it is difficult to obtain the optical coefficients governing the autofluorescence response, and their dependence as a function of the excitation wavelength, in vivo.

The document [13] "XeCl excimer laser-induced autofluorescence spectroscopy for human cerebral tumors diagnosis: preliminary study", S. Avrillier et al., Reports research work mainly in the field of the bronchi and gastrointestinal tract. This document mentions that the detection of autofluorescence thanks to endogenous fluorophores leads to a complex spectrum, and that the use of exogenous markers, although likely to generate side effects, makes it possible to obtain a spectrum in response that is easier to operate. . The aforementioned document [4], concludes in its chapter relating to the diagnosis on the lungs, to the need to use an external marker because of the complexity of the spectrum resulting from autofluorescence.

The document [14] LASERS IN SURGERY AND MEDICINE 11: 99-105 (1991), "Autofluorescence of Normal and Malignant Bronchial Tissue", J. Hung et al., Proposes to use excitation wavelengths of 405nm , 442nm or 488nm. This document indicates that the detected autofluorescence spectrum exhibits a characteristic decrease in intensity for carcinomas in situ.

Document [15] EP-A-512965, proposes an excitation at 442nm or 405nm. This document criticizes the previously proposed methods operating with respect to responses obtained at two or more wavelengths and proposes an exploitation by visual or mathematical combination of filtered images in bands of different wavelengths to distinguish normal tissues from tumors. . This same document EP-A-512965 indicates that the in vivo responses differ from the in vitro responses and that, moreover, this analytical technique exhibits great sensitivity as a function of the operating conditions.

The document [16] APPLIED OPTICS, vol. 28, n, n ° 12, June 15, 1989, "Pulsed and cw laser fluorescence spectra from cancerous, normal, and chemically treated normal human breast and lung tissues", GC Tang et al., proposes two excitations at 351nm and 527nm and mentions that normal tissues respond by autofluorescence to excitations at short wavelength while precancerous tissues respond by autofluorescence to excitation at higher wavelength. Attempts at diagnosis by autofluorescence analyzes, in the field of urology, have also been carried out.

The document [17] IEEE JOURNAL OF QUANTUM ELECTRONICS, vol. QE-20, n ° 12, December 1984, "Laser Induced Fluorescence Spectroscopy from Native Cancerous and Normal Tissue", RR Alfano et al., Relates tests carried out using excitation at 488nm and mentions the detection of significant peaks, in particular at 521nm, on rat bladder tumors, stressing that such peaks are not generated by healthy tissue.

The document [18] US-A-5201318 proposes a method of analysis by excitation of tissues in a wide range of 250 to 500nm, in steps of lOnm, collecting autofluorescence emissions in a range from the wavelength excitation plus lOnm at 2 times the excitation wavelength minus lOnm, in 5nm steps, establishment of an average of the emission spectra thus detected, then processing. This document concludes that the analysis technique by measuring the autofluorescence is very complex since the emission spectrum depends very much on the excitation wavelength. US-A-5201318 adds that it is necessary to register any possible pair of excitation wavelength and resulting emission wavelength to arrive at a diagnosis. The teaching of document US-A-5201318 leads to a complex visualization of the analysis results in three dimensions.

It appears from the state of the art cited above that specialists have been convinced for many years that it would be very advantageous to be able to use the fluorescence induced by a very low energy light pulse to allow at least one helps in the early diagnosis of precancerous lesions.

However today, the various research avenues explored in the field of autofluorescence analysis have led to very complex laboratory solutions that cannot be transposed into an industrial device intended for the hospital sector. The present invention now aims to improve the state of the art in order to allow the production of a simple device allowing the early detection of cancerous or precancerous surface lesions easily and reliably. This object is achieved according to the present invention thanks to an endoscopic imaging device characterized in that it comprises:

- means suitable for applying, endoscopically, to tissue to be imaged, an excitation signal of wavelength between 300 and 320 nm, preferably between 303 and 313 nm, - means capable of detecting a signal d autofluorescence generated by the tissues, following excitation, and

- analytical means able to isolate the autofluorescence responses generated, on the one hand by tryptophan around 370nm and, on the other hand by nicotine amide adenine dinucleotide (NADH) around 450nm, and process these two responses to define an image of the tissues analyzed.

As will be understood on reading the description which follows, the endoscopic imaging device in accordance with the present invention offers valuable assistance in the early diagnosis of superficial cancerous or precancerous lesions. Indeed, a ratio of the order of 1 between the autofluorescence response of NADH and the autofluorescence response of tryptophan is significant for healthy tissue, while a ratio much less than 1 is significant for tumors.

Other characteristics, aims and advantages of the present invention will appear on reading the detailed description which follows, and with reference to the appended drawings given by way of nonlimiting examples and in which:

- Figure 1 shows a schematic view of an endoscopic imaging device according to the present invention for point-by-point observation, - Figure 2 shows the autofluorescence curves obtained using an imaging device endoscopy according to the present invention, respectively on a healthy wall and on a tumor, and

- Figure 3 shows a schematic view of an imaging device according to an alternative embodiment of the present invention, for imaging an area of the tissues observed. We will first describe the structure of a point-to-point endoscopic imaging device according to the present invention illustrated in Figure 1 attached.

In this figure 1, the tissues analyzed are shown diagrammatically under the reference T.

As indicated above, the endoscopic imaging device according to the present invention essentially comprises:

- excitation means 10, - detection means 20, and

- analysis means 30.

The excitation means 10 are suitable for applying endoscopically, to the tissues T to be imaged, an excitation signal of wavelength between 300 and 320 nm, very preferably between 303 and 313 nm.

These excitation means 10 comprise a source 12 associated with a flexible optical conduit 14.

The excitation light source 12 can be the subject of numerous variants. It may, for example, be a pulsed UV laser with an XeCl excimer, or a xenon lamp with a quartz envelope followed by a bandpass filter, or alternatively any equivalent means consisting of a source emitting sufficient of light between 300 and 320 nm.

The conduit 14 is preferably formed of silica fibers. Such fibers are well suited for the conveyance of ultraviolet wavelengths. It should also be noted that optical fibers adapt well to medical techniques, in particular to endoscopy techniques because of their flexibility.

Of course, in the context of the present invention, it is advisable to use not only optical fibers suitable for carrying ultraviolet light, but also optical means associated with these fibers suitable for this purpose.

As shown diagrammatically in detail A in FIG. 1, the optical guide 14 can be formed, for example, from six emitting fibers 14 with a core diameter of 200 μm, arranged at the entrance to the conduit 14, adjacent to the source 12, in the form of a central fiber 14a surrounded by five fibers 14b.

The detection means 20 essentially comprise, according to the particular assembly of FIG. 1, a flexible optical conduit 22 associated with a spectrometer 24 followed by a detector.

The optical conduit 22 is advantageously formed of a multifiber catheter composed for example of thirteen receiving fibers. As can be seen in detail B in FIG. 1, at the distal end the thirteen receiving fibers 22 are arranged in the form of a central fiber 22a surrounded by the six emitting fibers 14, themselves surrounded by the twelve receiving fibers additional 22b.

The invention is not however limited to these particular arrangements of optical fibers, both for excitation and for detection of the emission. In particular, the invention is not limited to a concentric arrangement of the fibers used for excitation and of the fibers used for detection. A different arrangement of fibers can be used. In addition, the same fibers can be used to ensure both excitation and detection, for example by separating the excitation beam from the emission beam by a dichroic mirror or an equivalent means.

At the proximal end, adjacent to the spectrometer 24, the thirteen fibers 22 are arranged in the form of a rectilinear row compatible with the entry slit of the spectrometer, as seen in detail C in FIG. 1. A such a catheter allows optimum coupling with the spectrometer 24 and a significant reduction in the number of optical elements used compared to certain devices of the prior art. In addition, this configuration combined with the excellent qualities of the detector gives the whole apparatus very high sensitivity. A filter 26 may be disposed between the proximal end of the optical guide 22 and the spectrometer 24. The detector located downstream of the spectrometer 24 may be formed of a strip of photodiodes.

The spectrometer 24 and the associated detector make it possible to detect the autofluorescence signal generated by the tissues T following the excitation of 300 to 320 nm. The analysis and processing means 30 are suitable for isolating the autofluorescence responses generated on the one hand by tryptophan around 370 nm and, on the other hand by nicotine amide adenine dinucleotide (NADH) around 450 nm and treating these two answers to define an image of the tissue analyzed.

More precisely, the means 30 are preferably adapted to detect the autofluorescence response in the range from 360 to 380 nm (tryptophan) and in the range from 440 to 460 nm (NADH).

For this, the processing means 30 comprise for example a multichannel analyzer making it possible to take up a complete fluorescence spectrum in a single laser shot of 20 μJ and of a few nanoseconds.

The data thus obtained around 370nm and 450nm can be the subject of various treatments, in particular by computer processing. A preferential treatment in the context of the present invention consists in making the ratio of the intensities 450nm / 370nm and in selecting the ranges where this ratio corresponds to the tumor tissues. Such a treatment with respect to intensities makes it possible to eliminate numerous parasitic parameters. The inventors have in fact determined that for an excitation between 300 and 320 nm, preferably at 308 nm, there is obtained both a fluorescence signal corresponding to tryptophan, for wavelengths less than 400 nm, around 370 nm, and a NADH fluorescence band centered at around 450nm. The existence of these two signals allows an analysis of the spectra in relative intensity. We can see in Figure 2 that the ratio of fluorescence intensities at 450nm and 370nm is close to 1 for healthy tissue and less than 0.5 for tumor tissue.

Furthermore, the inventors have determined that only these range selections which are very specific to excitation and emission allow a reliable analysis.

Tests carried out by the inventors outside of these wavelength ranges at excitation and emission have in fact led to unusable results. We can quote for example, that for an excitation at 337nm only NADH is expressed by a broad band of fluorescence centered at approximately 450nm. And this method of excitation at 337nm gives many false positives (red inflammatory zone, traces of blood ...). We can also cite that for an excitation at 483nm, a similar phenomenon occurs with the fluorescence of the flavins having a single maximum around 575nm.

According to an alternative embodiment, the spectrometer 24 can be replaced by optical bandpass filters specific to the ranges 360- 380nm and 440-460nm. It is thus possible to provide two optical band pass filters placed opposite respective photodetectors or even a single photodetector placed behind an assembly comprising two band pass filters movable alternately opposite said photodetector.

We will now describe the alternative embodiment according to the present invention illustrated in Figure 3, which variant is suitable for area imaging.

This FIG. 3 shows a UV lamp 10 placed on the proximal end of a fiber 14. The fiber 14 makes it possible to excite an area of the tissues T to be observed. To this end, the fiber 14 is placed in the lighting channel of a conventional endoscope 16. The imaging path of the endoscope, formed of optical fibers 22 and adapted to observe the response of said zone to the aforementioned excitation, is as for it directed towards a detection device 20. This device 20 preferably comprises a set of filters 27, 28 specific to the bands 360-380nm and 440-460nm (for example filters 27, 28 mounted on a rotary assembly) placed in gaze from a photodetector 29, preferably of the CCD camera type. The output of the CCD camera is directed to the processing means 30.

According to another variant, the CCD camera 29 can be placed at the end of the distal end of the endoscope, that is to say inside the body observed.

The present invention thus provides valuable diagnostic assistance by superimposing contours of the tumor area (s) on a conventional video image observed by the doctor. The present invention leads to a portable device and to implementation compatible with the uses in hospital environments.

In addition, the use of the device described above can be carried out by any authorized person without requiring any particular knowledge in the medical field.

The endoscopic imaging device in accordance with the present invention finds particular application in the field of urology, but also in other fields such as ENT, gynecology (precancer of the cervix), in gastro -enterology, etc. The present invention allows early endoscopic detection of surface cancerous or precancerous lesions (severe dysplasias, carcinomas in stitu) which are flat lesions often not detectable by conventional endoscopy, and whose prognosis is severe. These lesions, which it is essential to be able to detect at the earliest, can be asymptomatic for a long time and the clinical signs which accompany them are often not specific.

The present invention which proceeds by tissue autofluorescence analysis makes it possible to avoid the use of exogenous fluorophore which is often not very selective and dangerous for the patient. The device according to the present invention uses a non-ionizing and non-invasive excitation light. It thus offers the possibility of long-term follow-up that is safe for the patient. In addition, compared to the usual diagnostic apparatuses (radiography, NMR, ecography, histological analysis) the device according to the present invention is relatively inexpensive and compact.

Of course, the present invention is not limited to the specific provisions which have just been described, but extends to any variant in accordance with its spirit.

The imaging device according to the present invention can be used by natural or artificial routes.

Claims

1. Endoscopic imaging device for the early detection of superficial cancerous or precancerous lesions, characterized in that it comprises:

means (10) capable of applying, endoscopically, on tissues (T) to be imaged, an excitation signal of wavelength between 300 and 320 nm,

- means (20) capable of detecting the autofluorescence signal generated by the tissues (T) following the excitation,

- analytical means (30) able to isolate the autofluorescence responses generated on the one hand by tryptophan around 370nm and on the other hand by NADH around 450nm and process these two responses to define an image of the tissues analyzed.

2. Device according to claim 1, characterized in that the excitation means (10) are adapted to generate an excitation signal of wavelength between 303 and 313nm.

3. Device according to one of claims 1 or 2, characterized in that the analysis means are sensitive to the autofluorescence responses in the ranges from 360 to 380nm and 440 to 460nm.

4. Device according to one of claims 1 to 3, characterized in that the analysis means (30) are adapted to determine the relationship between the autofluorescence response of NADH and the autofluorescence response of tryptophan. 5. Device according to claim 4, characterized in that the analysis means (30) are adapted to compare the ratio between the autofluorescence responses of NADH and tryptophan at a threshold value of the order of 0,

5.

6. Device according to one of claims 1 to 5, characterized in that the excitation means (10) comprise a source (12) and a flexible optical conduit (14).

7. Device according to claim 6, characterized in that the source (12) is chosen from the group comprising a pulsed UV laser with XeCl excimer, or a xenon lamp with quartz envelope possibly associated with a bandpass filter.

8. Device according to one of claims 1 to 7, characterized in that the detection means (20) comprise a flexible optical conduit (22) placed opposite a detector.

9. Device according to one of claims 6 to 8, characterized in that the optical conduit comprises UV specific silica fibers

(14, 22).

10. Device according to one of claims 1 to 9, characterized in that the detection means (20) comprise a spectrometer (24).

11. Device according to one of claims 1 to 9, characterized in that the detection means (20) comprise optical band pass filters placed opposite at least one photodetector.

12. Device according to claim 11 characterized in that the detection means (20) comprise two specific optical band pass filters of the ranges 360-380nm and 440-460nm.

13. Device according to one of claims 11 and 12, characterized in that the detection means (20) comprise two optical band pass filters placed opposite respective photodetectors.

14. Device according to one of claims 11 and 12, characterized in that the detection means (20) comprise two optical band pass filters movable alternately opposite a photodetector.

15. Device according to one of claims 1 to 14, characterized in that the detection means comprise at least one CCD camera (29) for area imaging.