Oral Oncology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Review
Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: A comprehensive review Muriel Abbaci a,b,⇑, Ingrid Breuskin c, Odile Casiraghi d, Frederic De Leeuw a,b, Malek Ferchiou d, Stephane Temam c, Corinne Laplace-Builhé a,b,⇑ a
Gustave Roussy, Imaging and Cytometry Platform, IRCIV, 114, rue Edouard Vaillant, Villejuif 94805, France Univ Paris-Sud, UMR CNRS 8081-IR4M, Orsay F-91401, France Gustave Roussy, Department of Otorhinolaryngology and Head and Neck Surgery, France d Gustave Roussy, Department of Pathology, France b c
a r t i c l e
i n f o
Article history: Received 14 February 2014 Received in revised form 9 May 2014 Accepted 11 May 2014 Available online xxxx Keywords: Confocal laser endomicroscopy Optical biopsy Fluorescence Reflectance Molecular imaging Head and neck cancer
s u m m a r y Histological assessment is an essential tool in the diagnosis and guidance of the treatment of various diseases, in particular cancer, of the head and neck. Recent major advances in optical imaging techniques have made it possible to acquire high-resolution in vivo images at the cellular scale. Confocal endomicroscopy is a non-invasive technique, which can be highly useful whenever meaningful in situ histological information is required. The technical aspects of confocal endomicroscopy are introduced, followed by an overview of major clinical studies in the field of head and neck cancer. Ongoing technical developments, contributing to improvements in imaging of the upper aero-digestive tract, are also discussed. Finally, the potential complementarities of functional and molecular imaging, as compared to morphological endomicroscopy, are highlighted. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Head and neck cancer represents 10% of all types of cancer, and is responsible for 11% of cancer-specific deaths [1]. According to the European Cancer Observatory, in 2012: lip, oral cavity, and pharynx cancer had the sixth highest cancer incidence rate in Europe. Despite current progress in their treatment, these cancers have a poor prognosis [2]. Histopathological assessment is a multi-step process [3] involving a macroscopic evaluation, tissue coloration, and a microscopic examination, requiring at least several minutes. Both of these are usually performed outside the operating room. To address the limitations of a white light examination, which provides a macroscopic view of superficial mucosa only, new optical methods based on light-tissue interactions have recently been proposed. They provide additional information on pathological tissues, and can be used to provide a true ‘‘optical biopsy’’ [4]. Among these techniques, confocal endomicroscopy (CEM) has been described as one of the most promising tools for the assessment ⇑ Corresponding authors at: Gustave Roussy, Imaging and Cytometry Platform, IRCIV, 114, rue Edouard Vaillant, Villejuif 94805, France. Tel.: +33 142116015/ 142116672. E-mail addresses: [email protected] (M. Abbaci), corinne. [email protected] (C. Laplace-Builhé).
of non-invasive morphological examinations in real time, and at high magnifications, using reflected or fluorescent light. This technique has been cited for diagnosis and surveillance in many applications such as dermatology [5], gastroenterology [6] and, more recently, oral pathologies [7]. The present review discusses the major developments and clinical results obtained for imaging of the head and neck by endomicroscopy. A number of current technical and scientific developments, together with their potential impact on the future implementation of CEM as a new tool for the ‘‘real time histology in situ’’ of head and neck cancer, are also considered.
Principle of confocal endomicroscopy Confocal microscopy is an adaptation of conventional optical microscopy, in which the illumination from a laser source is associated with a pinhole in such a way as to geometrically eliminate information lying outside the focal plane [8]. The resulting image is an optical section taken at a specific depth from the surface, the width of which depends on the confocal technology. The light returned to the detector by consecutively scanned points in the x-y plane is digitized so as to produce a high-resolution image of the region of interest. Each image provides an optical section, taken
http://dx.doi.org/10.1016/j.oraloncology.2014.05.002 1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Abbaci M et al. Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: A comprehensive review. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.05.002
2
M. Abbaci et al. / Oral Oncology xxx (2014) xxx–xxx
with a field of view corresponding to approximately one focal plane within the specimen [9]. Fibered confocal endomicroscopy (CEM), derived from confocal microscopy, is an in vivo non-invasive imaging tool enabling imaging of tissues at cell level. Since tissue removal is unnecessary, the real time on-screen view is an ‘‘optical biopsy’’. As opposed to conventional histopathology, the images obtained with CEM are parallel, rather than perpendicular, to the tissue surface sections. Whereas histopathology provides only a static picture of small biopsies from an organ, CEM can provide a direct and dynamic in situ visualization of selected areas, with various flow rates according to the instrument’s performance (Table 1). In vivo confocal reflectance endomicroscopy [10] is based on the backscattered light from cellular structures, which provide the image signal. In this mode, the image contrast results from variations in the refractive index of the cellular components highlighting the cellular morphology [11]. In contrast, in vivo confocal fluorescence endomicroscopy permits the characterization of microstructures and cellular details. It is based either on auto-fluorescence from the tissue, or on the administration of exogenous dyes. Fluorescent dyes already assessed for head and neck clinical microimaging In the context of in vivo confocal endomicroscopy, five major fluorescent dyes are referred to clinical head and neck studies. Table 2 presents a descriptive overview of these fluorescent dyes, which can be either topically applied or intravenously (IV) injected. Fluorescein, which is widely used as contrast agent in CEM imaging of the gastrointestinal tract [12], has also been evaluated in oral endomicroscopy, after IV injection, and allows to distinguish between normal and neoplastic mucosa [13]. After its rapid diffusion out of blood vessels, the fluorophore stains the interstitial space. The systemic clearance of the dye is essentially complete by 48–72 h. The topical application of Acriflavine [14] or Proflavine [15] (principal component of acriflavine), a fluorescent dye from the acridine family, results in high nuclear loading and diffuse cytoplasmic staining of tissues on CEM images. When combined with multimodal imaging (autofluorescence imaging and CEM) of the upper aerodigestive tract, the dye helped to accurately identify neoplastic tissue and pre-malignant lesions [15]. Hypericin [7] and 5-aminolevulinic (5-ALA) acid [16] are known for tumor selectivity in macroscopic fluorescence endoscopy [17,18]. They have been successfully reported in CEM imaging of the oral cavity after topical administration. Recently, our team developed a new in vivo technique for larynx imaging, following staining with a patent blue V solution (Fig. 1), and a pilot study is currently underway to clinically assess CEM imagery with this dye. We must note that topical application of any contrast agent usually limits its penetration to the upper cell layers (about 60– 100 lm in depth, according to our measurements - unpublished data) and can clearly limit the detection of lesions such as submucosal tumors, regardless of the imaging tool capabilities. Finally, no information has been provided to date on the tissue clearance during CEM imaging protocols after topical application of the abovecited fluorescent dyes for upper aerodigestive tract. Clinical reflectance micro-imaging of the head and neck Approximately ten years ago, ex vivo studies revealed the promising potential of confocal reflectance microscopy for the analysis of the human oral cavity, through the use of laryngeal biopsies, and for the discrimination between normal and cancerous tissues [19,20]. Clark et al. described a higher nuclear density and the
presence of keratin pearls in squamous cell carcinoma [20]. Reflectance imaging also provided in vivo information on tissue architecture and cellular morphology, through the use of prototypes dedicated to the oral cavity, based on fiber optic technology [21] or on the use of a bench type of microscope [22] (Table 1). The epithelium and the lamina propria in lip mucosa were imaged by White et al., in six healthy subjects at a depth of 490 lm. The papillae structures and extracellular matrix of the tongue were revealed down to a depth of 250 lm, using a specific excitation wavelength at 1064 nm [22]. According to these authors, the limited penetration at this wavelength could be attributed to scattering in the superficial epithelium and deeper connective tissues. Although irregular and keratinized areas perturbed the image acquisition and quality, a good qualitative and quantitative correlation was reported overall, between reflectance imaging and conventional histology. Maitland et al. obtained reflectance images from 20 oral sites, in eight patients with squamous cell carcinoma [11]. Examination of the oral mucosa was achieved using an acid acetic wash, to increase the contrast between the cytoplasm and the nuclei. Investigated sites, such as those with hyperkeratosis, hyperplasia, inflammation, dysplasia and invasive squamous cell carcinoma, were then correlated by means of histopathological analyses. The reflectance images were evaluated mainly on the basis of the distribution and morphology of the nuclei, and structural and cellular modifications were identified according to tumor area. Finally, Contaldo et al. successfully evaluated the lucid vivascopeÒ system (widely used in dermatology) to acquire in vivo images from 50 healthy oral cavities (lip, cheek, gingiva and tongue) [23].
Clinical fluorescence micro-imaging of the head and neck The application of fluorescence confocal microscopy to the diagnosis of lesions in head and neck medicine was also evaluated. Thong et al. achieved in vivo fluorescence imaging with a rigid endomicroscope from Optiscan Ltd [24,25]. Using a 488 nm excitation source, they investigated normal epithelium and squamous cell carcinoma of the tongue, following the topical application of 5-ALA. Images of normal tissue and tumors could be distinguished 45 min after staining [24]. Our research group conducted an ex vivo study based on the topical application of acriflavine and fluorescein, in order to evaluate the cellvizioÒ probe-based confocal laser endomicroscope (Table 1). Our preliminary studies [26] showed promising imaging capabilities, in distinguishing squamous cell carcinoma from squamous epithelium. Haxel et al. described a pilot clinical study on 5 patients, following the intravenous administration of fluorescein in order to image 5 different oral or oropharyngeal regions [27]. Although the dimensions and flexibility of the GI-endomicroscope (Optiscan/Pentax) used in this study were not suitable for the examination of the entire oropharyngeal area, various architectural and cellular details could be revealed. A rigid endomicroscope dedicated to head and neck examinations was tested in vivo by Pogorzelski et al. on 15 patients with HNSCC, for the purposes of imaging tumors and tumor margins [28]. Their data mainly describes the architecture, cellular and capillary morphology of these tumors. Ex vivo non confocal images were obtained by Vila et al., from 38 patients after proflavine [29] staining with a prototype high resolution micro-endoscope (HMRE) [30]. A blind review, carried out by seven head and neck specialists to discriminate between cancerous and benign lesions, revealed a sensitivity of 98% and a specificity of 92%, based on the interpretation of 36 images (11 benign and 25 cancerous) [29]. A recent study [14] proposed an atlas of CEM images (lip, ventral tongue, dorsal tongue and gingiva) from six healthy nonsmokers after topical application of acriflavine.
Please cite this article in press as: Abbaci M et al. Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: A comprehensive review. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.05.002
3
M. Abbaci et al. / Oral Oncology xxx (2014) xxx–xxx Table 1 Imaging parameters of commercial and prototype confocal endomicroscopes used for in vivo head and neck imaging.
a
Fiberoptic confocal reflectance microscope [11]
Lucid vivascope3000 [23]
OptiscanÒ RCE [24]
Optiscan Endomicroscope [28]
CellvizioÒ with gastroflex UHD
OptiscanÒ/PentaxÒ Endomicroscope [27]
Mode Type
Reflectance Fibered rigid probe
Reflectance Not fibered
Fluorescence Rigid probe
Excitation wavelength Penetration depth Frame rate
1064 nm
830 nm
488 nm
Fluorescence Rigid probe (32 mm) 488 nm
Fluorescence Fibered flexible probe 660 nm
Fluorescence Fibered probe integrated into a colonoscope 488 nm
NSa 15 frames/s NS
0–450 lm 6 frame/s for 1000 1000 pixels
0–300 lm 1–2 frames/s for 512 512 pixels
0–250 lm 0.8 images/s for 1024 1024 pixels
Field of view
230 230 lm
500 500 lm
390 390 lm
475 475 lm
0–250 lm 0.8 frame/s for 1024 1024 pixels 1.6 frame/s For 512 1024 pixels 475 475 lm
Lateral resolution Axial resolution (optical slice thickness) Distal tip diameter
2 lm
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Review
Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: A comprehensive review Muriel Abbaci a,b,⇑, Ingrid Breuskin c, Odile Casiraghi d, Frederic De Leeuw a,b, Malek Ferchiou d, Stephane Temam c, Corinne Laplace-Builhé a,b,⇑ a
Gustave Roussy, Imaging and Cytometry Platform, IRCIV, 114, rue Edouard Vaillant, Villejuif 94805, France Univ Paris-Sud, UMR CNRS 8081-IR4M, Orsay F-91401, France Gustave Roussy, Department of Otorhinolaryngology and Head and Neck Surgery, France d Gustave Roussy, Department of Pathology, France b c
a r t i c l e
i n f o
Article history: Received 14 February 2014 Received in revised form 9 May 2014 Accepted 11 May 2014 Available online xxxx Keywords: Confocal laser endomicroscopy Optical biopsy Fluorescence Reflectance Molecular imaging Head and neck cancer
s u m m a r y Histological assessment is an essential tool in the diagnosis and guidance of the treatment of various diseases, in particular cancer, of the head and neck. Recent major advances in optical imaging techniques have made it possible to acquire high-resolution in vivo images at the cellular scale. Confocal endomicroscopy is a non-invasive technique, which can be highly useful whenever meaningful in situ histological information is required. The technical aspects of confocal endomicroscopy are introduced, followed by an overview of major clinical studies in the field of head and neck cancer. Ongoing technical developments, contributing to improvements in imaging of the upper aero-digestive tract, are also discussed. Finally, the potential complementarities of functional and molecular imaging, as compared to morphological endomicroscopy, are highlighted. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Head and neck cancer represents 10% of all types of cancer, and is responsible for 11% of cancer-specific deaths [1]. According to the European Cancer Observatory, in 2012: lip, oral cavity, and pharynx cancer had the sixth highest cancer incidence rate in Europe. Despite current progress in their treatment, these cancers have a poor prognosis [2]. Histopathological assessment is a multi-step process [3] involving a macroscopic evaluation, tissue coloration, and a microscopic examination, requiring at least several minutes. Both of these are usually performed outside the operating room. To address the limitations of a white light examination, which provides a macroscopic view of superficial mucosa only, new optical methods based on light-tissue interactions have recently been proposed. They provide additional information on pathological tissues, and can be used to provide a true ‘‘optical biopsy’’ [4]. Among these techniques, confocal endomicroscopy (CEM) has been described as one of the most promising tools for the assessment ⇑ Corresponding authors at: Gustave Roussy, Imaging and Cytometry Platform, IRCIV, 114, rue Edouard Vaillant, Villejuif 94805, France. Tel.: +33 142116015/ 142116672. E-mail addresses: [email protected] (M. Abbaci), corinne. [email protected] (C. Laplace-Builhé).
of non-invasive morphological examinations in real time, and at high magnifications, using reflected or fluorescent light. This technique has been cited for diagnosis and surveillance in many applications such as dermatology [5], gastroenterology [6] and, more recently, oral pathologies [7]. The present review discusses the major developments and clinical results obtained for imaging of the head and neck by endomicroscopy. A number of current technical and scientific developments, together with their potential impact on the future implementation of CEM as a new tool for the ‘‘real time histology in situ’’ of head and neck cancer, are also considered.
Principle of confocal endomicroscopy Confocal microscopy is an adaptation of conventional optical microscopy, in which the illumination from a laser source is associated with a pinhole in such a way as to geometrically eliminate information lying outside the focal plane [8]. The resulting image is an optical section taken at a specific depth from the surface, the width of which depends on the confocal technology. The light returned to the detector by consecutively scanned points in the x-y plane is digitized so as to produce a high-resolution image of the region of interest. Each image provides an optical section, taken
http://dx.doi.org/10.1016/j.oraloncology.2014.05.002 1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Abbaci M et al. Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: A comprehensive review. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.05.002
2
M. Abbaci et al. / Oral Oncology xxx (2014) xxx–xxx
with a field of view corresponding to approximately one focal plane within the specimen [9]. Fibered confocal endomicroscopy (CEM), derived from confocal microscopy, is an in vivo non-invasive imaging tool enabling imaging of tissues at cell level. Since tissue removal is unnecessary, the real time on-screen view is an ‘‘optical biopsy’’. As opposed to conventional histopathology, the images obtained with CEM are parallel, rather than perpendicular, to the tissue surface sections. Whereas histopathology provides only a static picture of small biopsies from an organ, CEM can provide a direct and dynamic in situ visualization of selected areas, with various flow rates according to the instrument’s performance (Table 1). In vivo confocal reflectance endomicroscopy [10] is based on the backscattered light from cellular structures, which provide the image signal. In this mode, the image contrast results from variations in the refractive index of the cellular components highlighting the cellular morphology [11]. In contrast, in vivo confocal fluorescence endomicroscopy permits the characterization of microstructures and cellular details. It is based either on auto-fluorescence from the tissue, or on the administration of exogenous dyes. Fluorescent dyes already assessed for head and neck clinical microimaging In the context of in vivo confocal endomicroscopy, five major fluorescent dyes are referred to clinical head and neck studies. Table 2 presents a descriptive overview of these fluorescent dyes, which can be either topically applied or intravenously (IV) injected. Fluorescein, which is widely used as contrast agent in CEM imaging of the gastrointestinal tract [12], has also been evaluated in oral endomicroscopy, after IV injection, and allows to distinguish between normal and neoplastic mucosa [13]. After its rapid diffusion out of blood vessels, the fluorophore stains the interstitial space. The systemic clearance of the dye is essentially complete by 48–72 h. The topical application of Acriflavine [14] or Proflavine [15] (principal component of acriflavine), a fluorescent dye from the acridine family, results in high nuclear loading and diffuse cytoplasmic staining of tissues on CEM images. When combined with multimodal imaging (autofluorescence imaging and CEM) of the upper aerodigestive tract, the dye helped to accurately identify neoplastic tissue and pre-malignant lesions [15]. Hypericin [7] and 5-aminolevulinic (5-ALA) acid [16] are known for tumor selectivity in macroscopic fluorescence endoscopy [17,18]. They have been successfully reported in CEM imaging of the oral cavity after topical administration. Recently, our team developed a new in vivo technique for larynx imaging, following staining with a patent blue V solution (Fig. 1), and a pilot study is currently underway to clinically assess CEM imagery with this dye. We must note that topical application of any contrast agent usually limits its penetration to the upper cell layers (about 60– 100 lm in depth, according to our measurements - unpublished data) and can clearly limit the detection of lesions such as submucosal tumors, regardless of the imaging tool capabilities. Finally, no information has been provided to date on the tissue clearance during CEM imaging protocols after topical application of the abovecited fluorescent dyes for upper aerodigestive tract. Clinical reflectance micro-imaging of the head and neck Approximately ten years ago, ex vivo studies revealed the promising potential of confocal reflectance microscopy for the analysis of the human oral cavity, through the use of laryngeal biopsies, and for the discrimination between normal and cancerous tissues [19,20]. Clark et al. described a higher nuclear density and the
presence of keratin pearls in squamous cell carcinoma [20]. Reflectance imaging also provided in vivo information on tissue architecture and cellular morphology, through the use of prototypes dedicated to the oral cavity, based on fiber optic technology [21] or on the use of a bench type of microscope [22] (Table 1). The epithelium and the lamina propria in lip mucosa were imaged by White et al., in six healthy subjects at a depth of 490 lm. The papillae structures and extracellular matrix of the tongue were revealed down to a depth of 250 lm, using a specific excitation wavelength at 1064 nm [22]. According to these authors, the limited penetration at this wavelength could be attributed to scattering in the superficial epithelium and deeper connective tissues. Although irregular and keratinized areas perturbed the image acquisition and quality, a good qualitative and quantitative correlation was reported overall, between reflectance imaging and conventional histology. Maitland et al. obtained reflectance images from 20 oral sites, in eight patients with squamous cell carcinoma [11]. Examination of the oral mucosa was achieved using an acid acetic wash, to increase the contrast between the cytoplasm and the nuclei. Investigated sites, such as those with hyperkeratosis, hyperplasia, inflammation, dysplasia and invasive squamous cell carcinoma, were then correlated by means of histopathological analyses. The reflectance images were evaluated mainly on the basis of the distribution and morphology of the nuclei, and structural and cellular modifications were identified according to tumor area. Finally, Contaldo et al. successfully evaluated the lucid vivascopeÒ system (widely used in dermatology) to acquire in vivo images from 50 healthy oral cavities (lip, cheek, gingiva and tongue) [23].
Clinical fluorescence micro-imaging of the head and neck The application of fluorescence confocal microscopy to the diagnosis of lesions in head and neck medicine was also evaluated. Thong et al. achieved in vivo fluorescence imaging with a rigid endomicroscope from Optiscan Ltd [24,25]. Using a 488 nm excitation source, they investigated normal epithelium and squamous cell carcinoma of the tongue, following the topical application of 5-ALA. Images of normal tissue and tumors could be distinguished 45 min after staining [24]. Our research group conducted an ex vivo study based on the topical application of acriflavine and fluorescein, in order to evaluate the cellvizioÒ probe-based confocal laser endomicroscope (Table 1). Our preliminary studies [26] showed promising imaging capabilities, in distinguishing squamous cell carcinoma from squamous epithelium. Haxel et al. described a pilot clinical study on 5 patients, following the intravenous administration of fluorescein in order to image 5 different oral or oropharyngeal regions [27]. Although the dimensions and flexibility of the GI-endomicroscope (Optiscan/Pentax) used in this study were not suitable for the examination of the entire oropharyngeal area, various architectural and cellular details could be revealed. A rigid endomicroscope dedicated to head and neck examinations was tested in vivo by Pogorzelski et al. on 15 patients with HNSCC, for the purposes of imaging tumors and tumor margins [28]. Their data mainly describes the architecture, cellular and capillary morphology of these tumors. Ex vivo non confocal images were obtained by Vila et al., from 38 patients after proflavine [29] staining with a prototype high resolution micro-endoscope (HMRE) [30]. A blind review, carried out by seven head and neck specialists to discriminate between cancerous and benign lesions, revealed a sensitivity of 98% and a specificity of 92%, based on the interpretation of 36 images (11 benign and 25 cancerous) [29]. A recent study [14] proposed an atlas of CEM images (lip, ventral tongue, dorsal tongue and gingiva) from six healthy nonsmokers after topical application of acriflavine.
Please cite this article in press as: Abbaci M et al. Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: A comprehensive review. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.05.002
3
M. Abbaci et al. / Oral Oncology xxx (2014) xxx–xxx Table 1 Imaging parameters of commercial and prototype confocal endomicroscopes used for in vivo head and neck imaging.
a
Fiberoptic confocal reflectance microscope [11]
Lucid vivascope3000 [23]
OptiscanÒ RCE [24]
Optiscan Endomicroscope [28]
CellvizioÒ with gastroflex UHD
OptiscanÒ/PentaxÒ Endomicroscope [27]
Mode Type
Reflectance Fibered rigid probe
Reflectance Not fibered
Fluorescence Rigid probe
Excitation wavelength Penetration depth Frame rate
1064 nm
830 nm
488 nm
Fluorescence Rigid probe (32 mm) 488 nm
Fluorescence Fibered flexible probe 660 nm
Fluorescence Fibered probe integrated into a colonoscope 488 nm
NSa 15 frames/s NS
0–450 lm 6 frame/s for 1000 1000 pixels
0–300 lm 1–2 frames/s for 512 512 pixels
0–250 lm 0.8 images/s for 1024 1024 pixels
Field of view
230 230 lm
500 500 lm
390 390 lm
475 475 lm
0–250 lm 0.8 frame/s for 1024 1024 pixels 1.6 frame/s For 512 1024 pixels 475 475 lm
Lateral resolution Axial resolution (optical slice thickness) Distal tip diameter
2 lm
