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Protoporphyrin IX Occurs Naturally in Colorectal Cancers and Their Metastases¹

Robert-Roessle-Hospital at the Max-Delbrueck-Center for Molecular Medicine, Charité, Humboldt-University at Berlin, Berlin, Germany [K. T. M., T. H., C. N., W. E. H., P. M. S.]; Division of Medical Physics and Metrological Information Technology, Section of Biomedical Optics and NMR-Measuring Techniques, Physikalisch-Technische Bundesanstalt, Berlin, Germany [B. E., D. N., H. R.]; and Division of Radiation Biology, Roswell Park Cancer Institute, Buffalo, New York [R. K. P., T. J. D.]

	ABSTRACT

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Colorectal cancers exhibit a red fluorescence. The nature of the responsible fluorophore and its eventual diagnostic potential were investigated. Thirty-three consecutive colorectal resection specimen, 32 of which with histologically confirmed cancer, and a total of 1053 palpable mesenteric nodes were fluorimetrically characterized ex vivo. Furthermore, frozen material from 28 patients was analyzed, selected for the availability of primary tumor material and metastatic tissue, e.g., lymphatic and liver metastases from the same patient. Biochemical characterization was carried out through chemical extraction and reversed phase high-performance liquid chromatography. The fluorescence spectra of tissues, tissue extracts, and standard solutions of porphyrins were determined using a pulsed solid-state laser system for excitation and an imaging polychromator, together with an intensified CCD camera for time-delayed observation. Protoporphyrin IX (PpIX) was identified as the predominant fluorophore in primary tumors and their metastases. The fluorophore occurred in the absence of necrosis and in sterile locations. In untreated cases (n = 24), PpIX fluorescence discriminates metastatically involved lymph nodes from all other palpable nodes with a sensitivity of 62% at a specificity of 78% (P < 0.0001). After neoadjuvant treatment of rectal cancer, the PpIX fluorescence level of the primary tumors was reduced and a discrimination of lymph nodes based on PpIX-fluorescence was impossible. We conclude that colorectal cancer metastases accumulate diagnostic levels of endogenous PpIX as a result of a tumor-specific metabolic alteration.

	INTRODUCTION

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The detection and visualization of macroscopically indiscernible malignancy by fluorescence detection of fluorophores, preferentially accumulated in or synthesized by tumor tissue, is tempting because of the potential ease of use and the lack of biological hazard to patient and investigator (1 , 2) . Autofluorescence features of cancer were investigated long before. The original observation of a red autofluorescence emitted under blue light from experimental tumors dates back to the French scientist Policard (3) in 1924. Others gave rather little attention to the phenomenon (4) because of its seemingly restriction to larger, ulcerated tumors easily visible by eye. Ghadially and Neish (5) reported in 1960 a series of experiments relating the presence of porphyrins to the colonization of the ulcerated tumors by bacteria, a finding that was supported by Harris and Werkhaven (6) in 1987 for cancers of the oral cavity in which a strong porphyrin fluorescence could be "wiped off" from the surface.

More recently, the fluorescence from PpIX³ formed endogenously after exogenous application of 5-ALA was demonstrated to be diagnostic in many superficial tumor diseases (7, 8, 9, 10) . The success of 5-ALA-based fluorescence detection systems in superficial applications led us to develop a similar approach for macroscopically undiscernible residual disease like lymphatic micrometastases. For this purpose, the recognition of a specific fluorescence signal in considerable tissue depth (several millimeters) was necessary. Exploiting the longer fluorescence lifetimes of porphyrins, a system of unprecedented sensitivity for porphyrins, at least so far in the clinical context, was developed. However, since the regular autofluorescence from normal and diseased human tissues, which contain considerable levels of porphyrins, may interfere with this stimulated autofluorescence from PpIX, the native autofluorescence was to be investigated in the first place.

Thus, initially as a prerequisite to detect micrometastases by PpIX fluorescence after 5-ALA administration, we studied time-dependent laser-induced autofluorescence of colorectal tumors and their metastases. We then discovered and reported the detectability and the fluorimetric characteristics of an endogenous porphyrin-like fluorophore in colorectal lymph node metastases (11) . Our present investigation aims at the identification of the fluorophore and at the description of its potential diagnostic usefulness.

	PATIENTS AND METHODS

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Patients and Tissue Samples
The lymph node study comprises 33 patients who underwent primary surgical care for suspected or proven colorectal cancer at our institution, without any preselection. Five of those patients were pretreated by HRCT and three by RCT under an independent clinical study protocol (12) . Information on patients, tumor location, tumor stage, and tumor grading, as well as pretreatment by RCT or HRCT, are reported in Table 1 . Fluorescence spectra of the primary tumor and surrounding normal mucosa were investigated immediately after excision of the surgical specimen. Subsequently, all of the palpable nodules in the adjacent mesentery of the tumor were prepared and kept moist at room temperature. All of the specimens of primary cancers were carefully washed prior to fluorescence measurements. Laser-induced fluorescence of nodules was recorded before pathological evaluation. Table 1 includes the total number of nodules investigated by fluorescence spectroscopy. Care was taken that each nodule could be kept track of during the entire procedure of characterization. The time between cessation of blood supply and the last fluorescence measurements did not exceed 4 h. Sometimes, not all of the nodules resected from a particular patient could be investigated within that time. Furthermore, a nodule investigated by fluorescence spectroscopy sometimes turned out during pathological examination to consist of several distinct nodes. As additional information, given in parentheses, Table 1 specifies for each patient the number of noninvolved and involved lymph nodes, and connective tissue nodes, examined histologically.

Pathology
When communicated, tumor stages were determined according to the revised edition of the UICC’s TNM classification of malignant tumors (13) . Routine pathological evaluation of the lymph nodes was based on a single equatorial section of each palpable node, stained by H&E. Palpable nodes containing neither lymph node structures nor metastatic tissue were classified as connective tissue nodes. Their palpable density was generally explained by the presence of fibrosis or blood vessels. A subset of lymph nodes (n = 50) with high laser-induced fluorescence intensities but classified as normal by routine pathology, were reevaluated by stepwise sectioning of the entire nodes (>10 sections). One equatorial section was stained by pancytokeratin antibody MNF 116 (Boehringer, Mannheim, Germany), while the remaining sections were read after H&E staining. Table 1 gives the number of reevaluated lymph nodes together with the number of involved lymph nodes detected in this way. The total number of noninvolved and involved lymph nodes, given in Table 1 for each patient and in Table 2 for all patients, takes the results of the reevaluation into account.

Processing.
Tissue (25–50 mg) were covered with 1 ml of Solvable, and were kept for 12 h at 50°C. The resulting dilutions were filtered (45 µm), and the clear, slightly colored filtrates were stored at room temperature in the dark.

HPLC Analysis.
The HPLC analysis was performed using a Spectra Physics solvent delivery system (Mod. SP8700, San Jose, CA, USA) and pumped with a Spectraflow 757 absorbance detector (Kratos Analytical Instruments, Ramsey, NJ). The column used (Merck, Darmstadt, Germany) was a LiChrospher 100RP-8 (4 mm x 250 mm; 5-µm particle size). Porphyrines were eluted according to the scheme described previously by Bellnier et al. (14) : 100% solvent A [60% methanol, 40% 10 mM sodium phosphate buffer (pH 7.5)] for 10 min, followed by a linear gradient to 100% solvent B [90% methanol, 10% 2 mM sodium phosphate buffer (pH 7.5)] over the next 30 min. The flow rate was 1.5 ml/min; injection volume was 20 µl. Samples dissolved in Solvable were neutralized with 1 N HCl to pH 7.5 prior to analysis. Standards were established using commercially available solutions of PpIX, uroporphyrin III, dihydrochloride, and coproporphyrine III dihydrochloride.

	Fluorescence Instrumentation and Spectroscopy

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The experimental set-up used for recording time-delayed fluorescence spectra is shown in Fig. 1 . An OPO (Model A-1, GWU-Lasertechnik, Germany) pumped by the third harmonic ( = 355 nm, E_pulse 100–140 mJ) of a Q-switched Nd:YAG laser (GCR 230-50; Spectra-Physics), provided pulsed laser radiation at a rate of 50 pulses per s, tunable between 410 nm and 2.2 µm. The energy and the duration of the OPO output pulses amounted to 15 mJ and 3 ns typically. The remaining pump laser radiation ( = 355 nm) was removed from the OPO output by means of two dichroic beam splitters (HR 355, Laseroptik GmbH, Germany), the idler wave (720 nm–2.2 µm) by a corresponding short wave pass filter. The energy of the OPO output pulses was reduced to about 300 µJ using neutral density filters. The laser beam was coupled into a 600-µm hard-clad silica fiber. The fiber tip was placed at close proximity (<2 mm distance) to the surface of the intact nodule. The laser-induced fluorescence of the tissue was collected by the same fiber and guided to the entrance slit of an optical multichannel analyzer (see Fig. 1 ), consisting of an imaging polychromator (Spectra Pro-150; Acton Research Corp.) and a cooled, intensified CCD camera (Princeton Instruments Inc.). A dichroic beam splitter (550 DRLPO2; Omega Optical Inc.) served to decouple excitation and observation optical paths, and to separate laser-induced fluorescence from backscattered laser light. In addition, a long wave pass filter (_50% = 550 nm; LL 550 Corion Corp.), at the entrance slit of the polychromator, suppressed backscattered excitation light further. The intensifier of the diode array detector was gated by an electrical pulse (-180 V) of about 20 ns duration delivered by a high-voltage pulse generator (Model 6040, Berkeley Nucleonics Corp.) synchronized and delayed to the laser pulse. For this purpose, the high-voltage pulse generator was triggered by an electrical pulse provided by the power supply of the Nd:YAG laser.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Experimental setup used for prompt and delayed detection of laser-induced fluorescence. Nd:YAG 50 Hz, Q-switched Nd:YAG-laser at 50 Hz repetition rate; SHG, second harmonic generation; THG, third harmonic generation; sync pulse, TTL-synchronizing pulse; HR355, dichroic beamsplitter to remove radiation of the pump laser ( = 355 nm).

	Data analysis

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The autofluorescence spectra exhibit well-defined bands ( = 633 nm, 700 nm) denoted as specific autofluorescence bands and a broad unstructured background termed nonspecific autofluorescence. For quantification, we take the normalized fluorescence spectra I_n(, t_d) to be the sum of the normalized specific tissue autofluorescence I_n^sp(, t_d) and the nonspecific autofluorescence background I_n^nsp(, t_d)

(1)

To correct for the nonspecific autofluorescence background in delayed (t_d = 20 ns) fluorescence spectra, we assume that (a) the prompt normalized fluorescence spectrum I_n(, 0 ns) is dominated by the nonspecific fluorescence background; and (b) the decay rate of the nonspecific fluorescence background during the delay time t_d is independent of wavelength. It follows that the nonspecific fluorescence background in delayed (t_d = 20 ns) fluorescence spectra is given by

(2)

The first factor on the right hand side of Eq. (B) takes the decay of the nonspecific fluorescence background into account. The wavelength = 595 nm was chosen because it lies outside the fluorescence bands of the specific tissue autofluorescence and the absorption band ( = 570 nm) of hemoglobin (15) .

It follows from Eqs. (A) and (B) for the intensity of the specific fluorescence bands in delayed spectra, i.e., for the normalized specific tissue autofluorescence:

(3)

The assumptions made above are met if the specific tissue autofluorescence bands in the prompt spectrum are small compared with the nonspecific fluorescence background, generally observed for normal surrounding mucosa and lymph nodes, but may be questionable in the case of tumors with strong specific tissue autofluorescence bands appearing in the prompt spectrum. However, in that case, nonspecific tissue autofluorescence background is virtually absent in delayed spectra and Eq. (C) amounts to I_n^sp(, 20 ns) I_n(, 20 ns).

Besides normalized specific tissue autofluorescence I_n^sp(633 nm, 20 ns) taken at the maximum of the main specific fluorescence band, the ratio

may be used for quantification of the spectra recorded (11) . Both of the methods are strongly correlated leading essentially to the same conclusions. This is not too surprising, because the ratio I(, 20 ns):I(595 nm, 20 ns) appears within the parentheses of Eq. (C) . Therefore, when peak values rather than fluorescence spectra are to be compared, we use the ratio R of delayed fluorescence intensities rather than the normalized specific autofluorescence I_n^sp(633 nm, 20 ns) for quantification.

	RESULTS

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Fluorescence Spectra of Primary Tumors.
The normalized autofluorescence spectra of a particular primary colonic tumor recorded at zero delay and a delay of 20 ns after pulsed laser excitation at _ex = 505 nm are illustrated in Fig. 2,a and b , respectively. To facilitate comparison of spectral shapes, the delayed spectrum of the tumor was multiplied by a factor of 4.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. a), Typical prompt (td = 0 ns) and b), delayed (td = 20 ns) fluorescence spectra of a colorectal primary tumor (ex = 505 nm). A long pass filter 50% = 550 nm is used to remove reflected light at the excitation wavelength. The fluorescence intensities are normalized to the maximum intensity of the prompt spectrum. The delayed spectrum was further multiplied by a factor of 4 to facilitate comparison. c), fluorescence emission spectra of coproporphyrin III, uroporphyrin III, and PpIX in methanol, excited at ex = 505 nm.

The Nature of the Specific Autofluorescence in Colonic Primary Tumors.
The delayed fluorescence (emission) spectrum (Fig. 2b) of the colonic primary tumor is strikingly similar to the fluorescence emission spectra of porphyrins. For comparison, Fig. 2c shows fluorescence spectra of PpIX, uroporphyrin III (Uro III), and coproporphyrin (Copro III), dissolved in methanol. Fluorescence was excited at _ex = 505 nm, the emission spectra, recorded at zero delay, were normalized to the maximum of the main fluorescence band. In particular, the fluorescence emission spectrum of PpIX closely resembles the delayed emission spectrum of the tumor shown in Fig. 2 b. Our hypothesis that the specific autofluorescence observed in tumors originates from endogenous porphyrins, in particular PpIX, is further corroborated by the fluorescence excitation spectrum of a primary colonic tumor, illustrated in Fig. 3a , presented together with the excitation spectrum of one of its involved lymph nodes (Fig. 3b) . To this end, delayed (t_d = 20 ns) fluorescence emission spectra of a primary colonic tumor were recorded at several selected excitation wavelengths ranging from _ex 480 nm up to _ex 570 nm. As can be seen from Fig. 3a , two excitation bands appear in the fluorescence excitation spectrum of the primary colonic tumor centered at _ex = 510 nm and _ex = 550 nm. These bands are shifted to slightly longer wavelengths compared to the maxima (_ex = 505 nm, _ex = 540 nm) of the Q-bands of PpIX in methanol (Fig. 2c) . The fluorescence excitation and emission spectra recorded and the fluorescence decay times (_fl 14 ns) that are observed strongly support the hypothesis that the specific autofluorescence observed in primary tumors originates from endogenous porphyrins, in particular PpIX.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Fluorescence excitation bands of a primary tumor a) and one of its involved lymph nodes b) for two observation wavelengths obs = 635 nm and obs = 690 nm. Dashed line, the excitation bands of PpIX in methanol.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Normalized fluorescence intensities at 633 nm: in colorectal mucosa (), adenoma (), and cancers (), expressed as ratio R = I(633 nm, 20 ns)/I(595 nm, 20 ns). Neoadjuvant therapies are indicated.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Comparison of a normalized specific autofluorescence spectrum of a primary colonic tumor (dotted line) with the spectrum of one of its metastatically involved lymph nodes.

Specific Autofluorescence in Lymph Nodes.
The autofluorescence spectra of two particular lymph nodes of a patient without pretreatment, one involved and one noninvolved node, are shown in Fig. 5,a and b and Fig. 5, c and d , respectively. Apart from the absorption by hemoglobin at = 570 nm, no distinct spectral features appear in both prompt fluorescence spectra. However, the fluorescence spectrum of the involved lymph node taken at a delay of t_d = 20 ns exhibits two fluorescence bands centered at about = 630 nm and = 700 nm. In contrast, no such bands can be discerned in the delayed spectrum of the noninvolved lymph node. We attribute the specific autofluorescence of the involved lymph node to the same fluorophore present in primary tumors. This result is supported by Fig. 6 , which compares the normalized specific tissue autofluorescence I_n^sp(, 20 ns) of a primary tumor and one of its regional involved lymph nodes of one particular patient (no. 6). The normalized specific fluorescence I_n^sp(, 20 ns) was calculated subtracting the nonspecific background from the delayed spectrum. Apart from an artifact centered at 560 nm, the normalized specific fluorescence spectra of the involved lymph node and of the corresponding primary tumor are strikingly similar. Furthermore, the fluorescence excitation spectra observed for both fluorescence bands (_obs = 635 nm, 690 nm), illustrated in Fig. 3b for an involved lymph node further support this result. The fluorescence excitation spectra are similar to the excitation spectra of the corresponding primary tumor (see Fig. 3a ) and of the excitation spectrum (_obs = 635 nm) of PpIX in methanol. It follows that specific tissue autofluorescence, most likely originating from PpIX, occurs not only in primary tumors but also in involved regional lymph nodes, although at a much reduced intensity. However, specific tissue autofluorescence may be observed in pathologically noninvolved lymph nodes as well.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Typical prompt (td = 0 ns) and delayed (td = 20 ns) fluorescence spectra of regional colorectal lymph nodes (ex = 505 nm). All of the spectra are normalized to the maximum intensity of the undelayed spectrum; a) and b) metastatic involved lymph node; c) and d) uninvolved lymph node.

A total of 796 nodules from patients without pretreatment were investigated by fluorescence spectroscopy consisting of 434 noninvolved lymph nodes, 90 involved lymph nodes, and 272 connective tissue nodes. In Fig. 7 , we have plotted corresponding normalized cumulative frequencies versus the ratio R of fluorescence intensities in delayed fluorescence spectra of all involved, all noninvolved lymph nodes, and all connective tissue nodes investigated. The involved nodes show a broader distribution of R values, shifted toward higher fluorescence ratios (R_median = 1.11) as compared with the noninvolved lymph nodes (R_median = 0.82) and connecting tissue nodes (R_median = 0.73). All of the distributions overlap considerably, the overlap being strongest between connective tissue nodes and noninvolved lymph nodes.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Normalized cumulative frequencies of R values for uninvolved lymph nodes (; n = 434); connective tissue nodes (; n = 272); and metastatic lymph nodes (•; n = 90) from 25 patients (Table 1) without pretreatment.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Normalized cumulative frequencies of volume of involved lymph nodes (•; n = 90) and uninvolved lymph nodes (; n = 434) from 25 patients (Table 1) with colorectal cancer and one adenoma that were not pretreated.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 9. Distribution of R values measured for one particular patient (), normal lymph nodes (n = 17); , connective tissue nodes (n = 10); •, metastatic lymph nodes (n = 21). Node number, the distance from the primary tumor (pt). For reference, the value of normal mucosa (nm) is also given.

Influence of Pretreatment on Specific Tissue Autofluorescence.
Primary tumors pretreated by HRCT or RCT presented reduced R-values. Without pretreatment, pT₂ cancers showed higher fluorescence values than pT₃ or pT₄ cancers (R_median = 18.0, 10.5, and 5.9, respectively). After pretreatment, the responders (ypT₂) did show lower R values (R_median = 1.3) than nonresponders (ypT₃; R_median = 4.8; see Table 1 and Figure 4 ). For patients after pretreatment by RCT or HRCT, the fluorescence characteristics of connective tissue nodes and of uninvolved and metastatically involved lymph nodes did not present any statistically significant differences (Fig. 10) .

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 10. Normalized cumulative frequencies of R values for uninvolved lymph nodes (, n = 94); connective tissue nodes (; n = 127), and involved lymph nodes (•; n = 36) from eight patients (Table 1) after pretreatment (HRCT, RCT).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 11. High-performance liquid chromatograms taken by reversed phase HPLC of tumor, involved lymph node and normal tissue from a particular patient of the tissue sample collection. The absorption bands ( = 405 nm) for uroporphyrin III and coprporphyrin III as well as for PpIX are indicated versus eluation time.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 12. Normalized fluorescence emission spectra of reversed phase HPLC eluates (Fig. 11) for different tissue types.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 13. Medians of R values of colorectal primary cancer (tumor), surrounding normal mucosa (normal), regional lymphatic metastases (lymph node), and liver metastases (liver). Error bars, the 68% intervals.

	DISCUSSION

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The growing knowledge about the mechanisms of 5-ALA stimulated PpIX accumulation further supports this assumption. The exact mechanisms are, however, unclear. One theory suggests an unchanged enzymatic conversion from the precursor 5-ALA to PpIX and a reduced incorporation of iron into the PpIX to heme. The latter might be a consequence of a reduced activity of the ferrochelatase, which is discussed for several malignancies (17, 18, 19, 20, 21) and was reported to be reduced by factors ranging from 50 in hepatomas as compared with normal liver (22) to a factor of 3 in colonic cancer with respect to liver (17) . This lower metabolic activity may be due to the prevalence of glycolysis over the oxidative phosphorylation (23) . It may also be indicative of a genetic alteration because a number of mutations of the ferrochelatase gene are known to cause protoporphyria.⁴ An alternative hypothesis suggests that an insufficient intracellular availability of Fe²⁺, possibly caused by an increased cell division frequency (24) . Others found that the cellular PpIX content after 5-ALA supplementation did not correlate well with the cellular proliferation rate and suggested differences in PpIX efflux from the cell (25) . Another hypothesis has suggested an increased porphobilinogen deaminase activity at a normal or marginally increased ferrochelatase activity (26 , 27) . So far, using conventional fluorescence detection systems for diagnostic purpose, it was necessary to enhance the PpIX autofluorescence by supplementation of 5-ALA. On the other hand, employing more sensitive time-resolved fluorescence instrumentation (28) , we are able to detect the same PpIX, based only on the endogenous availability of 5-ALA. To detect malignancies by PpIX fluorescence without the administration of 5-ALA certainly poses advantages. However, when PpIX autofluorescence is rather weak, as, for example, as has been observed in lymph nodes, administration of 5-ALA might improve the sensitivity of the method considerably.

Another question that arises from the data presented in this paper relates to their clinical significance. Although there is no medical demand in diagnosing by fluorescence large tumors that are easily visible by eye, the detection of macroscopically occult lymph node metastases during or after surgery may well have an impact on treatment. The concept of regional lymphatic resection dates back to Moynihan (29) and Miles (30) at the beginning of the century. However, the extent of lymphatic dissection is still controversial as well with regard to the central extension (31) as to the lateral extension for rectal cancers (32, 33, 34, 35) . Although in a number of studies in retrospective subgroup analysis, survival advantages seem to result from more extended lymphadenectomies (31 , 33 , 36 , 37) , they never reach significance for all of the patients treated and may induce considerable morbidity (33 , 34) . A more precise pre- or intraoperative selection of patients, based on proven lymphatic metastases, may allow for an individualized surgical concept. An attempt in this direction is made in what is called radioimmunoguided surgery, where radiolabelled monoclonal antibodies against various more-or-less tumor-specific antigens are used to identify occult tumor deposits intraoperatively (38) . So far, a benefit could be demonstrated for patients with recurrent disease, especially to the liver, sparing aggressive surgery to patients who would not profit from it (39 , 40) . However, this technique still suffers a number of problems that range from the development of human to mouse antibodies (41) over the restrictions associated with the use of radioactive material to the lack of an ideal tumor-associated antigen (38) . In our series, a sensitivity of 62% at a specificity of 78% may seem rather low; however, it is still far better than any conventional method of intraoperative staging, because it is mainly done by palpation. Our finding that the size of a lymph node is a bad predictor of metastatic status is well in accordance with the literature (42, 43, 44) . If one further assumes that the simulation of a fluorescence-guided biopsy protocol, which would correctly predict the lymph node status in 30 of 32 patients in this series, is transposable from the ex-vivo to the in-vivo situation, such procedure may have immediate clinical significance. However, in vivo fluorescence measurements are rather limited in depth (probably 5–8 mm).⁵ An in vivo clinical study is, therefore, necessary to describe the feasibility of the approach and the reproducibility of the results in an intraoperative situation.

Furthermore, the refinement of pathological lymph node staging by immunohistochemical (45, 46, 47) and molecular techniques (48 , 49) offers a substantial improvement in sensitivity, eventually delivering the key for an understanding of the prognostic variability in Dukes B patients. However, to carry out such extensive evaluation on any of the median 32 lymph nodes sampled in this study from each patients would be unjustifiable economically. Therefore, a fluorescence-guided and budget-priced technique to preselect regional lymph nodes that are at high risk of harboring metastatic tumor cells for in-depth pathological evaluation may represent the key for a more general use of such extended pathological lymph node staging.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Grant Schl #391-1 from the Deutsche Forschungsgemeinschaft.

² To whom requests for reprints should be addressed, at Robert-Roessle-Hospital, Lindenberger Weg 80, D-13125 Berlin, Germany. Phone: 30-9417-1400; Fax: 49-30-9417-1499; E-mail: moesta{at}rrk-berlin.de

³ The abbreviations used are: PpIX, protoporphyrin; 5-ALA, 5-aminolevulinic acid; RCT, radiochemotherapy; HRCT, hyperthermic RCT; HPLC, high-performance liquid chromatography; OPO, optical parametric oscillator.

⁴ Online Mendelian inheritance in Man, Johns Hopkins University, Baltimore, MD, World Wide Web URL: http://www.ncbi.nlm.nhi.gov/omim/

⁵ Manuscript in preparation.

Received 3/17/00. Accepted 11/20/00.

	REFERENCES

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