Multispectral Imaging Autofluorescence Microscopy in Colonic and Gastric Cancer Metastatic Lymph Nodes
2007; Elsevier BV; Volume: 5; Issue: 2 Linguagem: Inglês
10.1016/j.cgh.2006.11.013
ISSN1542-7714
AutoresDesirée Pantalone, F Andréoli, Franco Fusi, Venere Basile, G. Romano, Gianmario Giustozzi, Luigi Rigacci, Renato Alterini, Monica Monici,
Tópico(s)Infectious Diseases and Mycology
ResumoBackground & Aims: The lymphadenectomy and extended lymphadenectomy procedures have been points of controversy in surgical oncology. The methods available for the detection of metastatic lymph nodes are numerous. These include lymphoscintigraphy and radiolabeled antibody detection, but in most cancers the currently used technique is sentinel lymph node identification, performed primarily through the use of immunohistochemistry. We propose the application of autofluorescence (AF)-based techniques for lymph node evaluation in colorectal and gastric tumors. Methods: We studied 30 clinical cases: 15 colorectal cancers and 15 gastric cancers. All of the patients were in the advanced stages of the disease and were candidates for adjuvant therapy. Autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy have been used to analyze the AF emission of metastatic lymph node sections, excited with 365-nm wavelength radiation. The AF spectra were recorded in the range of 400–700 nm. Monochrome AF images were acquired sequentially through interference filters peaked at 450, 550, and 650 nm, and then combined together in a single red-green-blue image. The AF pattern and the emission spectrum of metastatic lymph nodes have unique characteristics that can be used to distinguish them from the normal ones. Results: The results, compared with standard histopathologic procedures and with specific staining methods, supplied a satisfactory validation of the proposed technique, revealing the possibility of improving the actual diagnostic procedures for malignant lymph node alterations. Conclusions: With the development of appropriate instrumentation, the proposed technique could be particularly suitable in intrasurgical diagnosis of metastatic lymph nodes. Background & Aims: The lymphadenectomy and extended lymphadenectomy procedures have been points of controversy in surgical oncology. The methods available for the detection of metastatic lymph nodes are numerous. These include lymphoscintigraphy and radiolabeled antibody detection, but in most cancers the currently used technique is sentinel lymph node identification, performed primarily through the use of immunohistochemistry. We propose the application of autofluorescence (AF)-based techniques for lymph node evaluation in colorectal and gastric tumors. Methods: We studied 30 clinical cases: 15 colorectal cancers and 15 gastric cancers. All of the patients were in the advanced stages of the disease and were candidates for adjuvant therapy. Autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy have been used to analyze the AF emission of metastatic lymph node sections, excited with 365-nm wavelength radiation. The AF spectra were recorded in the range of 400–700 nm. Monochrome AF images were acquired sequentially through interference filters peaked at 450, 550, and 650 nm, and then combined together in a single red-green-blue image. The AF pattern and the emission spectrum of metastatic lymph nodes have unique characteristics that can be used to distinguish them from the normal ones. Results: The results, compared with standard histopathologic procedures and with specific staining methods, supplied a satisfactory validation of the proposed technique, revealing the possibility of improving the actual diagnostic procedures for malignant lymph node alterations. Conclusions: With the development of appropriate instrumentation, the proposed technique could be particularly suitable in intrasurgical diagnosis of metastatic lymph nodes. Lymph node metastases are one of the most important prognostic factors in cancer. 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Monici M. et al.Multicolor imaging autofluorescence microscopy: a new technique for the discrimination of normal and neoplastic tissues and cells.Recent Res Dev Photochem Photobiol. 2002; 6: 79-93Google Scholar The potential use of cell and tissue AF for diagnostic purposes already was recognized by Stübel42Stübel H. Die Fluoreszenz tierischer Gewebe im ultravioletten Licht.Pflugers Arch. 1911; 142: 1Crossref Scopus (72) Google Scholar 90 years ago. In 1924, Policard43Policard A. Etudes sur les aspects offerts par des tumours expérimentales examinees à la lumière de Woods.C R Soc Biol. 1924; 91: 1423-1425Google Scholar reported a study concerning the AF of tumors, attributed to endogenous porphyrins. Nevertheless, because of difficulties in detecting and interpreting the AF signals, owing to low intensity and spectral complexity, respectively, fluorescence microscopy was, in the past, mostly oriented to the use of exogenous markers. 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Bernabei P.A. et al.Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues.Photochem Photobiol. 2000; 71: 737-742Crossref PubMed Scopus (36) Google Scholar we showed that AF imaging allows discrimination between hyperplastic and primary neoplastic lymph nodes. Here, we report the results obtained by applying both autofluorescence microspectroscopy (AMS) and multispectral imaging autofluorescence microscopy (MIAM) to the diagnosis of secondary neoplastic lymph nodes in gastric and colorectal cancer. The AF pattern and the emission spectrum of metastatic lymph nodes have unique characteristics that can be used to distinguish them from the normal ones. For this study, we enrolled a total of 30 patients: 15 affected by colorectal cancer and 15 affected by gastric cancer. Two control lymph nodes were obtained from informed nonneoplastic patients. We expressly chose neoplastic patients who were in the advanced stages of the disease and who were candidates for adjuvant therapy. In these patients, the removal of suspected metastatic lymph nodes, independent of location, did not affect the individual patient's scheduled procedure. None of the patients had received neoadjuvant therapy. Each patient signed a written consent form before enrollment in the present study. A database was opened to register patient information, preoperative and histopathologic staging, adjuvant therapy, and follow-up data. The lymph node biopsy specimens were collected during surgical resection of the tumor. Fresh lymph node biopsy specimens were divided into 2 parts: the former, formalin-fixed and paraffin-embedded, were processed using standardized histochemical and immunohistochemical techniques for diagnostic purposes; the latter were frozen in liquid nitrogen and then stored at −80°C until the MIAM and AMS analyses. The results obtained with the different techniques then were compared. The specimens, obtained as described earlier, were fixed in 10% formalin and then processed in paraffin. H&E-stained sections from each patient were assessed by the pathologists. A representative section of 4 μm from each lymph node was selected for the immunohistochemical analysis. All sections were dewaxed in Bio-Clear (Bio-Optica, Milano, Italy) and hydrated with grade ethanol concentration progressively diluted with distilled water until 0% ethanol. The streptavidin-biotin peroxidase method was used for immunostaining. As the primary antibody, we used a cocktail of broad-spectrum anticytokeratin, clone AE1/AE3 (BioGenex, San Ramon, CA). Antigen retrieval was performed routinely by microwave pretreatment (Microwave MicroMed T/T Mega; Milestone, Bergamo, Italy) in 10 mmol/L sodium citrate buffer (pH 6.0) for 30 minutes. All tissue sections were placed in the Ventana Nexes automated stainer (Ventana Medical Systems Inc., Tucson, AZ) with the iVIEW DAB Detection Kit (Ventana) as the revelation system. The pancytokeratin monoclonal antibody AE1/AE3 then was incubated for 20 minutes. After the staining run was completed, the tissue sections were removed from the stainer, counterstained with Mayer's hematoxylin, dehydrated, and mounted with Permount (Biomeda, Foster City, CA). Formalin-fixed, paraffin-embedded sections of gastrointestinal (GI) carcinoma were used as positive controls. Negative control sections for immunostaining were stained with the omitted primary antibody. The same procedure was applied on sections contiguous to the ones analyzed by AMS and MIAM. In this case, antigen retrieval was not necessary because the tissue was frozen and not fixed. Lymph node sections, 5-μm thick, were obtained from frozen biopsy specimens, mounted on slides, and immediately were analyzed by AMS and MIAM. Tissue AF was analyzed by an inverted epifluorescence microscope (Nikon Eclipse TE 2000 E; Nikon, Firenze, Italy). Oil-immersion CFI (Chrome-Free Infinity) S fluor 40× (numerical aperture = 1.3) and CFI S fluor 20× (numerical aperture = 0.75) objectives were used. The excitation light source was a high-pressure mercury lamp (HBO 100 W, Osram Milano, Italy). The 365-nm excitation wavelength was obtained by filtering the mercury lamp emission by an interference filter (10-nm full width at half maximum, 365FS10-25; Andover Corp., Salem, NH), which excluded light coming from nearby mercury lamp emission lines other than the one selected. The fluorescence signal was transmitted through a dichroic mirror at 400 nm (DM400; Nikon) and detected by a spectrophotometer, for spectral measurements, or by a Hires IV cooled digital CCD camera (DTA, Italy), equipped with a Kodak KAF261E detector (20 μm, 512 × 512 pixels), for image acquisition. Therefore, the whole system allowed sequential measurements of spectra and images on the same sample. The spectrophotometer was based on a polychromator (SpectraPro 500i Action Research Corporation, Action, MA; 500-mm focal length), connected to the microscope through an optical fiber bundle (1-mm diameter) and comprising a 16-bit Hires III cooled digital CCD device (DTA) with a back-illuminated SITe sensor (24 μm, 330 × 1100 pixels Scientific Imaging Technology, Tigard, OR); the overall wavelength resolution is about 0.34 nm in the selected range of 375–750 nm. The SpectraPro was equipped with grating (150 lines/mm) blazed at 380 nm. The spectrum analyzer was calibrated in wavelength using spectral lamps (Hg, Cd, Kr, Xe). Intensity calibration was performed by use of a calibrated halogen lamp (EG&G, Princeton, NJ); the maximum error in wavelength measurement was ±1 nm over the whole considered bandwidth. Each spectrum was calibrated for modifications induced by the detection optics. The correction function for the microscope optics was obtained by measuring the light spectrum of a halogen lamp directly through the fiber optic bundle of the spectrum analyzer and dividing it by the spectrum of the same lamp passing also through the microscope optics. Peak identification was performed on mean curves (15 acquisitions on different tissue areas, showing similar histologic conditions as judged from AF images). Peak positions were identified by means of polynomial fits applied locally to mediated spectrum curves; maximum differences in fluorescence peaks were calculated by subtracting peak positions. All the calibration procedures were performed one time only. Before every measurement section the efficacy of the calibration was checked by fluorescence standards. AF imaging was accomplished by using a motorized filter wheel, containing up to 8 different interference filters, placed in front of the CCD detector. This allowed for multispectral sequential acquisition in different emission bands. The choice of the filter combination was made on the basis of the main spectral bands determined by preliminary analysis of the AF spectra. Both the CCD camera and the filter wheel were controlled by a modified routine running under Visa software (DTA). AF images were digitized directly in the CCD controller with 16-bit dynamics and transferred to the storage computer on a digital interface. To determine the observation field dimensions we measured the spatial calibration factor (μm/pixels) by using 6-μm diameter fluorescent microspheres (Molecular Probes, Leiden, The Netherlands) observed by a 100× objective. The corresponding calibration factor was 0.13 μm/pixel−1. Then, the size of the field with 40× and 20× objectives was of about 172 × 172 μm and 345 × 345 μm, respectively. To evaluate the potential of the proposed AF-based techniques for application in diagnostics, the concordance of different raters in interpreting images was tested by applying κ statistics on a group of 35 AF images (30 from neoplastic lymph nodes and 5 from controls). Unweighted κ values (degree of agreement)50Altman D.G. Practical statistics for medical research. Chapman and Hall, London1991Google Scholar were evaluated considering the 3 possible pairings among 3 different raters, who interpreted the images in a blinded fashion according to the 3 categories: metastatic, probably metastatic, and hyperplastic (normal) lymph node sections. Both multicolored imaging and spectroscopic techniques were used to characterize the AF properties of metastatic lymph nodes and to distinguish them from normal lymph nodes. Biopsy sections of the lymph nodes, prepared as described in the Materials and Methods section, were processed by AMS and MIAM, sequentially. Comparison between the AF spectra of metastatic lymph nodes and the control lymph nodes (Figure 1) revealed meaningful differences: the maximum had shifted toward red by more than 20 nm, being 437 nm for normal and 459 nm for metastatic lymph nodes, respectively. Moreover, the spectra of the latter showed a wide broadening toward the green-red region of the electromagnetic spectrum. At times, a shoulder or even a second peak was observed in the same region (Figure 2). The difference between the AF spectra of normal and metastatic tissue sections was calculated, giving maximum results at 504 nm (Figure 1).Figure 2AF spectrum of a bioptic section from a metastatic lymph node. The primary tumor was a gastric carcinoma. Besides the principal peak at about 460 nm, a second peak at 602 nm can be observed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The multicolored imaging technique allowed us to attain monochrome images of the specimens, related to the fluorescence of one or more endogenous chromophores emitting in a narrow band of wavelengths, which then were recombined in a single multicolored image. In lymph node tissues, considering the spectral characteristics observed at 365-nm excitation, we expected contributions from collagen, elastin, and nicotinamide adenine dinucleotide (phosphate) in the blue band, as well as from flavins in the green band. A possible contribution in the red, as a result of fluorophores, such as lipopigments and porphyrins, also was evaluated. The spectral bands for monochrome image acquisition (see Materials and Methods section) were chosen considering the emission peaks of these fluorophores. Significant examples of AF images from nonneoplastic and neoplastic lymph node sections are shown in Figure 3. As we already reported in a previous study,49Rigacci L. Alterini R. Bernabei P.A. et al.Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues.Photochem Photobiol. 2000; 71: 737-742Crossref PubMed Scopus (36) Google Scholar the AF pattern of nonneoplastic lymph nodes (Figure 3A) allowed for easy recognition of the usual structure of the organ, with the cortical follicles separated by connective trabeculae arising from the capsule and penetrating into the medullar part of the organ. Connective trabeculae were strongly fluorescent, being composed mainly of collagen and elastin. However, as expected, a very faint fluorescence characterized the follicles, because of the low AF of the lymphocytes,51Wyszecki G. Stiles W.S. Color science-concepts and methods, quantitative data and formulae. John Wiley & Sons, New York1982Google Scholar the most represented cell population in lymph nodes. When excited at 365 nm, a wavelength not suitable to induce nuclear AF, the lymphocytes showed a fluorescence emission lower than other cell types becau
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