# 5-ALA Mediated Fluorescence Detection of  Gastrointestinal Tumors

Authors: Ekaterina Borisova; Borislav Vladimirov; Latchezar Avramov
Journal: Advances in Optical Technologies
 (2008)

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## Abstract

Delta-aminolevulinic acid/protoporphyrin IX is applied for fluorescent tumor detection in the upper part of gastrointestinal tract. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. In such way, 1D detection and 2D visualization of the lesions' fluorescence are received, and both advantages and limitations of these methodologies are discussed in relation to their clinical applicability. Comparison of the spectra received from normal mucosa, inflammatory, and tumor areas is applied to evaluate the feasibility for development of simple but effective algorithm based on dimensionless ratio of the fluorescence signals at 560 and 635 nm, for differentiation of normal/abnormal gastrointestinal tissues.


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## Body

## 1. Introduction

The limitations of standard endoscopy for detection and evaluation of cancerous changes in
gastrointestinal tract are significant challenge and initiative development of
new diagnostic modalities, including optical detection of tissues alterations.
One of the most widely examined spectroscopic techniques is laser- or light-induced
fluorescence spectroscopy (LIFS), because of its rapid and highly sensitive
response to early biochemical and morphological changes in the tissues. Fluorescent
diagnosis of tumor tissues becomes a valuable tool in the clinical practice.
This technique could be applied for detection and evaluation of tumors in
different localizations using endoscopic equipment. Such combined white-light
and fluorescent mode endoscopic systems are already developed and introduced in
the clinic for the needs of bronchoscopy and lung cancer diagnosis, like
D-Light system of Karl Storz GmbH, Tuttlingen, Germany, diagnostic
autofluorescence endoscope (DAFE) system of Richard Wolf GmbH, Knittlingen, Germany, lung
fluorescence endoscopy (LIFE) system of Xillix Technologies Corp., Richmond, Canada [1–4].However, fluorescent gastroscopes are still on its research and development phases and
from the best we know that the few existing systems, such as Olympus Evis Lucera, have not received yet approvals from FDA for access
to the broad clinical market [5]. This system is a digestive tract videoscope
used for observing blood vessels in mucous membranes under infrared light in
the regions 790–820 nm and 905–970 nm. Variation of Xillix fluorescent endoscopic system is Xillix-LIFE-GI which is
applied for autofluorescence detection of stomach neoplasia and has approval for Japan and European countries.Several fluorescent endoscopy systems are developed and proposed also for practical applications
by different research teams, demonstrating very good clinical results [4, 6–8], using autofluorescence or exogenous fluorescence detection of
gastrointestinal neoplasia.Despite of the fluorescent endoscopic systems developed mentioned above, the fluorescent
diagnosis of tumors of the upper part of gastrointestinal tract still is very
interesting and extensive research and development task worldwide. Spectral diagnosis can provide imaging and
point spectroscopic information in both morphological and biochemical data modes [1, 4, 9]. With the optimization of the
procedures and evaluation of this technique diagnostic added value, through
development of appropriate algorithms based on fluorescence properties of the
investigated sites, a novel high-sensitive diagnostic tool could be successfully
applied as complementary to the standard white light endoscopy [10–13].In the case of autofluorescence detection, high sensitivity and specificity could be achieved
if complex algorithms are applied for differentiation of the spectra [14]. However,
on the current moment detecting the difference in
autofluorescence as a gastroendoscopic image still has been relatively
difficult task because of its faintness. Only combination of powerful light
sources and highly sensitive detectors will lead to the development of
autofluorescence gastroscopy clinical systems [1, 13]. Therefore, recent real-time
gastrointestinal fluorescence endoscopy is all based on the use of exogenous
fluorophores [1, 8, 12], as
the addition of exogenous fluorescent compounds increases the contrast, improves endoscopic resolution and sampling,
and could be used to receive better 2D visualization for the needs of
clinicians.Unfortunately, one typical problem of exogenous fluorophores’ application, for example 5-ALA/PpIX
for detection of gastrointestinal tumors, is the moderate specificity achieved.
High false-positive values are obtained, mainly due to inflammation areas in
the organs under interest. Sensitivity and specificity reported in different
studies related to detection of esophageal neoplasia are 76% and 63% [15], 77%
and 71% [16], 80% and 56% [17], respectively. Moreover,
these values undergo significant changes in dependence of the amount of the photosensitizer applied—5, 10, 20, or 30 mg/kg 5-ALA and the way of sensitization-orally, using
enema, or spray catheter [17, 18]. These differences are quite large—as the sensitivity and specificity for the
cases of 10 mg/kg and 30 mg/kg oral application are 80% and 56%, as well as 100% and 27%, respectively. Local application of 5-ALA using spray catheter also reveals
better values for sensitivity of the dysplastic lesions evaluation in colon
than oral application of the drug, but specificity is lower—62% versus 73%
(in the case of 20 mg/kg oral application) [18]. With increasing of the 5-ALA
concentration orally applied, the specificity decreases very rapidly.
Therefore, to achieve some optimization of the procedures and proper diagnosis,
it is proposed to work with orally applied aminolevulinic acid, with
concentration in the frames of 15–20 mg/kg for the
gastrointestinal tract tumor detection [17, 18].In the recent study, delta-aminolevulinic acid/protoporphyrin IX (5-ALA/PpIX) is also
applied as fluorescent marker for tumor detection in esophagus and stomach. Normal
and cancerous mucosas of esophagus and stomach as well as inflammatory areas fluorescence spectra are
detected and compared in this study. Rapid lesions border determination using
exogenous fluorescence signal is obtained in 1D scanning spectroscopic mode.
Our results from in vivo
detection show very good differentiation between normal and abnormal tissues in
1D spectroscopic regime, but moderate discrimination in 2D imaging. In the case
of 2D video visualization, the problem of high autofluorescence signal in the
red spectral region gives low contrast between normal and abnormal mucosas when standard CCD
camera of the endoscope is applied. This problem is in a process of resolve in
our further investigations.


## 2. Methods and Materials

Two variants of the fluorescent complimentary
equipment to the standard gastroscope were applied for the needs of fluorescent
diagnosis of the neoplasia in upper part of gastrointestinal tract. In the
first one, we built light source that replace standard white light illuminator
of the endoscope, and through instrumental channel a fluorescence collecting
fiber (quartz-polymer single fiber,d=600 μm, NA=0.22) was
applied. In the second variant, a fiber bundle was applied though instrumental
channel of the endoscope, as the excitation light was delivered through central
1 fiber, and fluorescence response of the tissue was collected by surrounding 7
fibers and delivered to microspectrometer. The fiber bundle is specially
developed for endoscopic applications in gastrointestinal tract (Polironik
Ltd., Moscow, Russia), as the bundle used in this case consists of 8 quartz fibers, central one with
diameter 200 μm, and surrounding 7 fibers with diameter 100 μm, all of them
with numerical aperture 0.22. The central fiber is detached by thin aluminum
folio from the surrounding fibers to avoid cross-signal between the fibers.
Special resin is used on the end tip of the fiber bundle applied, due to its
application in gastrointestinal tract—for patient
safety and for enlarged resistance on the severe conditions in stomach
environment. Both modalities have their advantages and disadvantages that will
be described below.In the first case, a special light source was developed for the needs of our experimental work,
based on 405 nm high-power light-emitting diode OTLH-0360-UV-UV HIGH FLUX LED
ILLUMINATOR (25 mW, Rhopoint Components Ltd, Oxted-Surrey, UK). This
opto-mechanical device could replace common white light source of the standard
endoscopic equipment (Olympus Corporation, Hertfordshire, UK)
that is in use in the University Hospital “Queen Giovanna,” Sofia, Bulgaria.
Through endoscopic instrumental channel, a quartz-polymer fiber was applied to
return information about fluorescence to microspectrometer USB4000 (spectral
range—350–1000 nm, FWHM∼2 nm, Ocean Optics Inc., Fla, USA).
A computer was used to control the spectrometric system and to store and
display the data measured using specialized software Spectra Suite (Ocean Optics
Inc.). Usage of the standard light guide of the endoscope for delivery of
excitation light is beneficial due to the same geometry of illumination—view angle and illuminated area are preserved
as for the white-light illumination mode. However, the optics of the light
guide was not optimized for the short wavelength range and strong losses
appeared in this case, therefore 2D visualization of the lesions obtained was
not optimal for clinical observation needs [13].In the second variant, excitation source was light emitting diode illuminator—AFS-405
(Polironik Ltd., Moscow, Russia) on 405 nm with 25 mW output
power on the end of the fiber tip. Numerical aperture of the fiber was lower
than that one of light guide of the endoscope itself, and about 60% of the view
area was illuminated in this case, reducing the field of view for the
clinicians. However, in this case excitation light intensity was not faded by
delivery optics, and bright 2D video visualization was achieved.In the both cases, spectroscopic 1D measurements were with very good quality (high value of
the signal-to-noise ratio), due to the significant sensitivity of the microspectrometer
applied.In the study, delta-aminolevulinic acid/protoporphyrin IX (“ALASENS,” NIOPIK JSCo, Russia) was
used as fluorescent marker for dysplasia and tumor detection in esophagus and
stomach. Theδ-ALA is administered per os six hours before measurements at dose
20 mg/kg weight, according to clinical experience in gastrointestinal diagnostic
applications of δ-ALA/PpIX fluorescence [4, 17].Fluorescence
diagnosis of esophageal and stomach lesions was made during standard endoscopic
examinations of the patients in Gastroenterology Department of University
Hospital “Queen Giovanna.” Twelve esophageal tumors and fifteen stomach tumors
were detected using fluorescence endoscopy. The spectral measurements are made
on several anatomic points during endoscopic procedures—on normal mucosa and cancerous sites. In the
case of stomach endoscopic observations, spectral data from inflammatory areas
were also detected and stored for subsequent analysis. All procedures are
developed after local ethical committee approval received for the protocol of
exogenous fluorescence diagnostic modality verification.Five to seven points
were measured from every section and averaged spectrum was used for evaluation
of their state. The resultant spectrum was smoothed using Savitzky-Golay
algorithm to reduce instrumental noise of the spectrometric system used. All
results presented from spectral measurements in this paper are normalized with
respect to back-scattered excitation signal at 405 nm from the mucosa surface
for appropriate comparison of the signals obtained.Measurements of
the fluorescence spectra were made in 1D using spectrometer fibers and 2D
visualization, and record of protoporphyrin IX distribution in the mucosa was
made using video system of the endoscopic equipment. Up to three biopsy samples
are collected from every suspicious area observed during observations of the
patients using standard endoscopic biopsy clips and send for histological
evaluation. Standard histology of all suspicious areas was used as “gold”
standard, for comparison with the results obtained from spectral measurements.


## 3. Results

The spectral measurements in vivo were made on several anatomic points during endoscopic
procedures—on normal mucosa
and suspicious sites, known from the previous diagnostic evaluation of the
exact patient or where some red fluorescence signal was observed during current
endoscopic observation. Standard histology applied as a “gold” standard for
comparison with the results obtained from spectral measurements, revealed very
good correlation between the fluorescence signals detected and histology
examination results.The fluorescence detected from tumor sites has very complex spectral origins. It consists of
autofluorescence, fluorescence from exogenous fluorophores, and reabsorption
from the chromophores accumulated in the tissue under investigation [1, 7–15, 19].Spectral features observed during endoscopic investigations could be distinct as the
next regions, according to their origin and spectral region appearance, after
excitation at 405 nm:(i)
450–650 nm
region, where tissue autofluorescence is observed;(ii)
630–710 nm
region, where fluorescence of PpIX is clearly pronounced;(iii)
530–580 nm
region, where minima in the autofluorescence signal are observed, related to
reabsorption of oxy-hemoglobin in this spectral area.Normal mucosa has bright autofluorescence, related mainly to the emission of coenzymes and
protein cross-links [19]. The intensity of autofluorescence in the case of
neoplasia rapidly decreases, which could be used as additional indicator of
pathology evaluation; see Figures 1(a) and 1(b).Fluorescence spectra of normal mucosa
and tumor of patient with (a) esophageal carcinoma and patient with (b) stomach
carcinoma at 405 nm excitation. Spectra are presented with their standard
deviation, resulting of calculation of the mean value from the spectra detected
from one section.On Figure1 are presented spectra of normal
mucosa and tumor sites for esophagus and stomach for two patients. Standard
deviation presented is a result of averaging of the spectra detected from
different points of the pathological and normal tissues surface, respectively. In the case of
stomach, autofluorescence of normal mucosa was significantly higher than that
of tumor, and the contrast observed in green spectral area is usually higher
than 3,5:1. For the esophagus fluorescence, this difference is not so strongly
pronounced, but the autofluorescence of normal mucosa was also higher for all
cases observed.Such high autofluorescence leads to problems in
2D video-observation of the stomach tumor fluorescence, as the values of the area
of the autofluorescence spectrum of normal mucosa for the region >600 nm in
comparison with the same region for tumor fluorescence are comparable, and the
ratio values between total areas of normal versus tumor spectra in the region 600–800 nm vary from
0.7 to 1.1 for different patients. This effect could not be avoided by
application of filter before CCD camera, as the long-pass filter (>600 nm)
passed both signals—from normal
mucosa autofluorescence and from exogenous PpIX tumor fluorescence.For both anatomic areas, esophagus and stomach,
the same procedure was applied, 20 mg/kg 5-ALA oral application 6 hours before
endoscopic observations. However, we received relatively big deviations of the
fluorescence intensity of PpIX emission for both localizations for different
patients, as in esophagus neoplastic lesions the fluorescence intensity of
exogenous fluorophore was higher as a general than that for the stomach cancer
sites; see Figures2(a) and 2(b). On Figure 2 schematically are presented two endmost
cases—of the highest signal detected from tumor—“Tumor 1” and the
lowest signal detected from tumor—“Tumor 2” for both localizations—esophagus and
stomach. All spectra are normalized with respect to the back-scattered
excitation signal at 405 nm. In general, stomach tumor PpIX fluorescence is
about two times lower than that from esophagus neoplastic areas. It could be
related to differences in the time of accumulation or the specific accumulation
in both anatomic areas.Fluorescence spectra of the lesions
of two different patients with (a) esophageal tumors and two patients with (b) stomach
tumors at 405 nm excitation. Spectra presented outlying cases of the highest
(“Tumor 1”) and the lowest (“Tumor 2”) signals received from tumors in both
localizations in different patients.
(a)(b)When inflammatory areas occurred in the organ
under investigation, red fluorescence is also observed, which could give false-positive
results for tumor determination, during observation in video channel of the
endoscope. This fluorescence is observed due to the accumulation of PpIX in the
both tumor and inflammatory areas. These signals could be distinguished when 1D
measurements of the inflammation are carried out; see Figure3. The
contrast between the fluorescent signals at 635 nm between tumor regions and
inflammations observed in all patients, where such comparison was possible,
usually is higher than two. In such way, we could be sure in general that by using
this detection approach one could distinguish inflammation from tumor site, and
moreover, could distinguish inflammatory areas from normal mucosa.Figure 3
Fluorescence spectra of stomach
inflammation and tumor of one patient, using excitation at 405 nm. Spectra are
presented with their standard deviation, received after averaging of several
point measurements in the respective abnormal mucosa areas.However, the fluorescent intensities of the
maximum at 635 nm of inflammatory area detected from stomach wall are close to
the lowest signals received from tumors (see Figure2(b)). Therefore, an
additional criterion could be applied for better differentiation of inflammation
from tumor. We used for these goals a dimensionless ratio R=I635/I560.
In such way, we received excellent differentiation of malignancies from benign
and normal tissues of the stomach with significant gap between values corresponded;
see Figure 4.Figure 4
Dimensionless ratio (•) R=I635/I560 calculated for all cases detected from normal mucosa, inflammation, and tumor
of stomach. Lines represent the mean values of this ratio calculated.On Figure4 are presented values of the
dimensionless ratio of the all cases detected with tumors and inflammations in
stomach wall, compared with normal mucosa values. Using this simple algorithm,
very good differentiation tumor/inflammation is obtained that could be applied
for clinical practice needs.Moreover, similar approaches are proposed by
other research groups for differentiation of normal and cancerous sites in
bronchi and lungs. Using green and red band-pass filters, two complimentary
images are received and ratio between them is calculated to receive more
contrast image and to improve sensitivity and specificity of the fluorescent
endoscopy approach [8, 20, 21].


## 4. Discussion

Gastrointestinal
tumors have major place in the statistics of newly developed cancers every
year, and usually the tumors are detected on advanced III and IV stages, where
perspectives for the patients are not very optimistic. Up to now, white light
endoscopy is the main method in detection of gastrointestinal tumors.
White-light endoscopy is well-established and wide used modality. However,
despite the many technological advances that have been occurred, conventional
white-light endoscopy is suboptimal and usually detects lesions, which already
have symptoms of obstruction, bleeding and pain, related to tumor growth.
Misdiagnoses, related to difficulties in differentiation of inflammatory from
initial stage adenocarcinoma, also have negative effect on the diagnostic
accuracy [1]. Only experienced gastroenterologists with long practice in
endoscopy observations could find slight initial changes to dysplastic and
neoplastic stages of esophageal, stomach or colon mucosa.In gastroenterology, several optical methods are applied recently, such as optical
coherent tomography [7], chromo-endoscopy, confocal fluorescent microscopy [10],
Raman spectroscopy [22], reflectance spectroscopy [11], and laser- and
light-induced fluorescence spectroscopy [4, 15–17]. Combination
of optical techniques (laser autofluorescence and diffuse reflectance) is also
applied to increase the values of sensitivity and specificity of diagnostic
procedure up to 93% and 100%, respectively [11].Exogenous fluorescence
spectroscopy is suggested to be very promising modality for early diagnosis of
gastrointestinal tumors, and one of the most widely applied compound is
delta-aminolevulinic acid/protoporphyrin IX [9, 16, 17]. 5-ALA is a natural
precursor of heme, which induces the formation of endogenous PpIX. The
administration of exogenous 5-ALA results in the accumulation of PpIX in tissue
due to feedback inhibition of the final step of the heme biosynthetic cycle.
Enzymatic differences in dysplastic tissue (e.g., decreased ferrochelatase
activity) can lead to an increase in PpIX fluorescence in the tumor cells. Fluorescence spectroscopy of gastrointestinal
tract is not yet in routine clinical use, but increasingly compelling data in
patients will likely to lead to its introduction to the diagnostic practice in
near future.In the current study are presented data from
exogenous fluorescence spectroscopy of esophageal and stomach tumors in vivo after oral application of
20 mg/kg 5-ALA six hours before spectroscopic measurements and video
visualization of the patients using excitation at 405 nm. The spectra received
in 1D point spectroscopic measurements consist from three fundamental compounds—autofluorescence
of endogenous fluorophores, fluorescence of exogenous protoporphyrin IX, and
reabsorption of their emission from the hemoglobin. Video 2D observation which is
the most user—friendly tool for the needs of anatomically connected
visualization of the pathology from the clinicians is not presented, as due to
high level of autofluorescence of the gastrointestinal wall in the red spectral
region, it is not representative enough. Similar observations were found from
other investigators for detection of esophageal carcinoma lesions [23]. This
problem could be solved by change of excitation wavelength applied and this
task is in a process of solving in our further investigations using
longer wavelengths for excitation of PpIX (e.g., its peaks of absorption at 509 nm, 544 nm, or 584 nm), where autofluorescence is not so strong factor, as well
as back scattered excitation light from the mucosal surface does not lie in the
spectral region of PpIX fluorescence itself.In the case of
normal mucosa, the hemoglobin reabsorption is not strongly pronounced. The
mucosal autofluorescence is observed in the region of 450–700 nm and could
be related to signals from protein cross-links, coenzymes, and phospholipids,
using excitation at 405 nm [4, 19, 24–26]. The lack of fluorescence peaks
at 636 nm and 704 nm for normal mucosa is an indication for highly selective
accumulation of 5-ALA/PpIX only in abnormal sites and gives high contrast when
lesion borders are determined. In the cases of advanced tumor progress, where necrosis
appears, a lack of autofluorescence or exogenous signal is noticed, which visually is observed as a
dark spot in the center of tumor lesion.In the case of
inflammation and tumor very strong minima are presented in the spectra at 545
and 573 nm, related to oxy-hemoglobin reabsorption of the autofluorescence
signals from these areas. These strongly pronounced minima in the green
spectral region could be used as additional indication of abnormality of the
tissue investigated.Dimensionless
ratio at two wavelengths 560 nm and 635 nm is proposed to be used for better
differentiation of tumors from inflammatory areas. This ratio allows receiving
of 100% selective discrimination of tumors versus mucosal inflammation.
However, additional comparison must be applied when dysplasia is evaluated. On
this stage, only three cases of dysplasia are evaluated during clinical
observations and their ratio' values lie between values of inflammations’ and
tumors’ spectral ratios, namely, values ofR for detected spectra of inflammation
are between 1<R<1,6, of dysplasia—1,5<R<2,3, and 2,5<R<4,4 for
the tumors' fluorescence spectra measured.


## 5. Conclusions

Advances in spectroscopic instruments will
improve imaging’s role as a facilitator of research translation. Results
received in our study could serve for development of novel tools for
quantifying in vivo tumor
growth and origin and for accelerating the transition from preclinical studies
to early clinical trials and to routine diagnostic practice. Fluorescent
spectroscopy and imaging will help for further understanding of
gastrointestinal tract tumors and to improve cancer patients’ lives.



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