Posted December 20, 2010
Pathologists will soon share examination of tissue morphology at the architectural, cellular and molecular level with other physicians. In vivo microscopy technologies enable physicians to detect pathology by visualizing tissue through innovative tomographic methods. In vivo microscopy techniques provide additional data beyond that obtainable by histological methods, including volumetric data and time/flow data. Improvements in these methods have led non-pathologists to make tissue-based diagnoses1 and perform tasks traditionally undertaken by pathologists, such as assessing tumor margins and grade.2
In some fields like ophthalmology, in vivo microscopic imaging is clinically adopted.3 For cardiology and gastroenterology, in vivo microscopic imaging is in a phase of exponential growth, and many in the field anticipate worldwide adoption within 5 years. As such, there is real potential for some in vivo microscopy technologies to reduce or even replace traditional histopathologic examination in selected settings.
In an effort to get out in front of some of these technologies near adoption, the College’s Technology Assessment Committee (TAC) has identified four technology trends that contribute to the emergence of in vivo microscopy as a competitor to histology-based diagnosis.
- Advances in optical coherence tomography (OCT)
- Advances in confocal microscopy
- Molecular imaging
- Lesional indexing
The resolution, or detail, of optical coherence tomography is approaching and may soon surpass the equivalence of high magnification optical microscopy. OCT is an interferometric technique, which uses longer (near infrared) wavelengths to gather reflectance data. When tissue reflectance is compared to reference light, a cross-sectional image can be created. Current generation OCT systems can have micron scale resolutions and can image to a depth of 2-3 mm within tissue. This resolution allows, for example, the identification of lipid pools, thin fibrous caps, and macrophages within OCT images of coronary plaques.4 Current OCT techniques generate volumetric data at microscopic resolution; a powerful advantage. One frequently cited example is the use of optical frequency domain imaging (OFDI), a type of OCT, to generate volumetric data of a 6 cm segment of gastro-esophageal junction in vivo. With these images, researchers were able to identify foci of dysplasia.5
OCT was initially described in 1991,6 and in its early generations was used predominantly in ophthalmology to image the layers of the retina in vivo to identify patients with macular pathology. No less than nine commercially available or soon to be available, spectral domain OCT (SD-OCT) retinal imaging devices exist, some of which can image at a resolution of approximately five microns.7 In the past decade, OCT has grown to other fields, most notably cardiology and gastroenterology. OCT has also spawned several variations, each with its own strengths.
OCT images have some advantages over hematoxylin and eosin stained slides. While it is true that images are typically presented in black and white, refractive substances enhance reflectance. Hence, cytoplasmic granules as found in some white blood cells are easily highlighted. Presenting volumetric data is also powerful; morphology in pathology has traditionally been limited to mental recreations of 3-dimensional volumes from two-dimensional slices of tissue. With OCT volumetric data, there will be confirmation of known histomorphology, but there will also be new insights into the microscopic architecture of disease processes. OCT can also capture live motion images, or serial images over time to document the progression or regression of a lesion.
One limitation of OCT is the depth of tissue penetration, which is not expected to reach beyond 2-3 mm of tissue, even in later generations. Another limitation is contrast, which directly affects detail and resolution. Innovations using artificial contrast media are likely to occur in the coming years, further improving the detail of the images.
Devices equipped with OCT technology give microscopic access to areas not amenable to biopsy such as the coronary arteries and retinas, and have the potential to help identify areas of tissue for biopsy. They also can replace certain biopsy procedures by offering real time microscopic, morphologic examination. When OCT diagnoses can be made with confidence, treatment might be initiated immediately, altogether bypassing the frozen section or biopsy.
Confocal microscopy is another maturing technology finding its way into the marketplace. Confocal laser microscopy (CLM) enables capture of images that are in focus across an entire plane. Commercially available systems use wavelengths of ~400-1000 nm, which allow for submicron resolution, but at the cost of a depth of penetration less than that of OCT (~300 µm). In general, acquired CLM images provide horizontal optical sections to the reviewer, rather than the traditional vertical sections seen in histology. For example, when used in skin, the devices acquire images one layer at a time (stratum corneum, stratum spinosum, basal layer, etc.) rather than a cross section of the entire skin when cut from a paraffin-embedded section. Confocal microscopy produces high-resolution images comparable to histology in gastrointestinal studies with good sensitivity and specificity for both neoplastic and inflammatory conditions of the esophagus and colon.7 CLM has been studied for use in opthalmological, gastrointestinal, cutaneous, pulmonary, and other applications.22.214.171.124 Again, images are presented in black and white, and nuclear detail, a staple of histology, is difficult to appreciate. However, this limitation may change with the refinement of contrast media. 3D reconstruction and molecular imaging are also possible with CLM.12
Pathologists are at the forefront of biomarker discovery and analysis because they are the stewards of tissue and fluids. However, in vivo microscopy technologies are enabling targeted visualization without the need to remove tissue. When you couple metabolic imaging with in vivo microscopy technologies, it is conceptually similar to applying immunohistochemical stains to live tissue. Imagine a futuristic breast scanner that can show a radiologist, oncologist, or surgeon the spiculated architecture, the glandular differentiation, and pleomorphism of a breast cancer. Now, imagine that it can also determine ER/PR and HER2 status of the tumor without the added cost of a breast biopsy (subject to sampling error) in addition to definitive excision.
There is no shortage of research pursuing in vivo molecular imaging. Biomarkers have been paired with many devices, including MRI, PET, CT, and others to help track cells,13 detect disease, to determine likely disease course, and to monitor effectiveness of therapy, among other applications.14 It is natural that new imaging techniques also seek this advantage, and in fact near infrared fluorescent dyes have been successfully coupled with OCT techniques.15 If these dyes can be linked to biomarkers, in vivo molecular imaging could be brought to the microscopic level. However, regulatory barriers are likely to exist as each new biomarker-dye combination will likely require individual FDA approval.
Lesional indexing is the stratification of a patient (or lesion) into bins based on a known cohort. This well-used statistical technique is based on analysis of characteristics of a population of patients, or samples, coupled with outcomes of those patients, in order to help predict how a patient will behave or respond in the future. To simplify the conclusions of lesional indexing, some algorithms deliver output as a likelihood of disease, or a score. Some authors have published the means by which these algorithms were derived and calculated, but many algorithms are often proprietary and therefore not disclosed. For example, Oncotype DX® is a “black box” gene assay that produces a score indicating the likelihood of response to chemotherapy, but the algorithm producing this score has not been published. However, the “score” does not provide information on the genes or algorithm for which the score is based. It is important to note that several in vivo microscopy technologies employ lesional indexing.
MELA Sciences, based in New York, is developing a point-of-care, in vivo imaging instrument called MelaFind®, which scans pigmented skin lesions with different wavelengths, compares the multispectral signature with signatures of known lesions, and advises the user on whether or not to biopsy the lesion 16. If MelaFind’s® performance is superior to that of a general practitioner, the device could decrease the percentage of lesions biopsied. On the other hand, MelaFind® may simultaneously increase the number of lesions screened. In June 2009, MELA Sciences filed a Pre-Market Approval (PMA) application with the US Food and Drug Administration (FDA) 17. In November 2010, an FDA advisory panel voted 8-7, with one member abstaining, to recommend approval for MelaFind®18.
The use of magnetic resonance spectroscopy to characterize prostate cancer is another example of lesional indexing. While this experimental technique has only been performed on ex vivo specimens, it has successfully differentiated malignant from benign prostate tissues, and has even identified a less aggressive subset, as well as a subset more likely to present with perineural invasion.19 This technology has recently been extended by co-registering spectroscopy signatures with high-resolution MRI scans.20
Pathologists may feel that morphology paired with supplemental data enhances the understanding of the disease process and is superior to lesional indexing alone. However, the popularity of lesional indexing by itself should not be underestimated, as shown by the interest in the previously mentioned gene assays.
Several other in vivo microscopy technologies examined by the TAC are not ready to challenge histopathological diagnosis. Some of these are unproven, and others seek to improve on the quality of endoscopy, rather than supplant the biopsy.21
The pathologist community has heard of the demise of the biopsy to the “virtual biopsy”, and the theories behind many of the discussed technologies are not new. Yet even though the majority of in vivo microscopy technologies are in nascent stages and some years away from adoption, several are FDA-cleared and are commercially available.22
For the in vivo technologies still in development, the TAC believes that several are building momentum and are transitioning from research to clinical practice, with interventional cardiologists, interventional radiologists, gastroenterologists, and ophthalmologists leading the charge as early adopters.
Regulatory and market forces will clearly drive adoption of technologies; as will standardization and maturation of technology, all of which are happening in parallel. Dr. Guillermo Tearney, M.D., Ph.D., Associate Professor at the Wellman Center for Photomedicine at Massachusetts General Hospital and a pioneer in OCT, has seen in vivo microscopy-related conference attendance rise from tens of researchers to thousands. The number of articles in non-ophthalmology OCT research has exploded from less than 10 articles in 1998 to 150 articles in 2008, and there are currently greater than 400 patents containing the phrase “optical coherence tomography”. The number of companies exhibiting commercial activity in OCT has risen from 5 to 25 from 1998 to 2008.23
Many translational medical technologies seek to replace invasive procedures with non-invasive or minimally invasive ones. In vivo microscopy technologies make a strong case to fill that role. If successful, these technologies may not eliminate traditional biopsies, but may allow for more accurate targeting of biopsies or decrease the need for frozen section confirmation that a lesion has been properly sampled.
The emergence of in vivo microscopy technologies both within and external to pathology and the laboratory is clearly accelerating. Some technologies will be adopted, and others will be quickly replaced by a more functional or advanced innovation. The impact of these changes on pathologists and the College of American Pathologists is unclear. However, it is evident that some technologies have the potential to cause disruption (positive or negative) of current pathology practice. On the positive side, in vivo technologies may provide a new or expanded pathway for pathologists to bring incremental value to their clinical colleagues and to improve patient care.
Pathologists should not fear these technologies; rather they should become more familiar with those nearing adoption through peer-reviewed literature and related conference curricula. Standards are in development in many of these fields utilizing in vivo microscopy. If pathologists want to be part of the ‘future story’, it is critical that they become educated about these technologies and are at the table when the utilization cases for these technologies are being determined.
The TAC would like to thank Guillermo Tearney, MD, PhD, FCAP, and Patrick FitzGibbons, MD, FCAP, for critical review and suggestions for the paper.
- Wortsman X, Wortsman J. Clinical usefulness of variable-frequency ultrasound in localized lesions of the skin. J Am Acad Dermatol. Feb;62(2):247-256.
- Chun YS, Vauthey JN, Boonsirikamchai P, et al. Association of computed tomography morphologic criteria with pathologic response and survival in patients treated with bevacizumab for colorectal liver metastases. JAMA. Dec 2 2009;302(21):2338-2344.
- Sakata, Lisandro M, DeLeon-Ortega, Julio, Sakata, Viviane, Girkin, Christopher A. Optical coherence tomography of the retina and optic nerve - a review. Clinical and Experimental Ophthalmology 2009; 37: 90-99.
- Tearney GJ, Waxman S, Shishkov M, et al. Three-dimensional coronary artery microscopy by intracoronary optical frequency domain imaging. JACC Cardiovasc Imaging. Nov 2008;1(6):752-761.
- Suter MJ, Vakoc BJ, Yachimski PS, et al. Comprehensive microscopy of the esophagus in human patients with optical frequency domain imaging. Gastrointest Endosc. Oct 2008;68(4):745-753.
- Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science. Nov 22 1991;254(5035):1178-1181.
- Kiernan DF, Mieler WF, Hariprasad SM. Spectral-domain optical coherence tomography: a comparison of modern high-resolution retinal imaging systems. Am J Ophthalmol. Jan;149(1):18-31.
- Hoffman A, Goetz M, Vieth M, Galle PR, Neurath MF, Kiesslich R. Confocal laser endomicroscopy: technical status and current indications. Endoscopy. Dec 2006;38(12):1275-1283.
- Calzavara-Pinton P, Longo C, Venturini M, Sala R, Pellacani G. Reflectance confocal microscopy for in vivo skin imaging. Photochem Photobiol. Nov-Dec 2008;84(6):1421-1430.
- Niederer RL, McGhee CN. Clinical in vivo confocal microscopy of the human cornea in health and disease. Prog Retin Eye Res. Jan;29(1):30-58.
- Thiberville L, Salaun M, Lachkar S, et al. Confocal fluorescence endomicroscopy of the human airways. Proc Am Thorac Soc. Aug 15 2009;6(5):444-449.
- Polglase AL, McLaren WJ, Skinner SA, Kiesslich R, Neurath MF, Delaney PM. A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract. Gastrointest Endosc. Nov 2005;62(5):686-695.
- Srinivas M, Heerschap A, Ahrens ET, Figdor CG, Vries IJ. (19)F MRI for quantitative in vivo cell tracking. Trends Biotechnol. Apr 26.
- Martin Goetz and Thomas D. Wang, Molecular imaging in gastrointestinal endoscopy. Gastroenterology. Mar 2010;138(3):828-833.e1.
- Xu C, Ye J, Marks DL, Boppart SA. Use of near-infrared fluorescent dyes in depth resolved spectroscopic optical coherence tomography. Conf Proc IEEE Eng Med Biol Soc. 2004;2:1214-1217.
- Cheng LL, Burns MA, Taylor JL, et al. Metabolic characterization of human prostate cancer with tissue magnetic resonance spectroscopy. Cancer Res. Apr 15 2005;65(8):3030-3034.
- Wu CL, Jordan KW, Ratai EM, et al. Metabolomic imaging for human prostate cancer detection. Sci Transl Med. Jan 27;2(16):16ra18.
- Arber N, Grinshpon R, Pfeffer J, Maor L, Bar-Meir S, Rex D. Proof-of-concept study of the Aer-O-Scope omnidirectional colonoscopic viewing system in ex vivo and in vivo porcine models. Endoscopy. May 2007;39(5):412-417.
- Holmes J. OCT technology development: where are we now? A commercial perspective. J Biophotonics. Jul 2009;2(6-7):347-352.
Technology Assessment Committee (TAC) Members at the time of original POET publication:
John W. Turner, MD, FCAP, Chair
Frederick L. Baehner, MD, FCAP
Kenneth J. Bloom, MD, FCAP
Samuel K. Caughron, MD, FCAP
Thomas J. Cooper, MD, FCAP
Richard C. Friedberg, MD, PhD, FCAP
Jonhan Ho, MD, FCAP
Federico A. Monzon, MD, FCAP
David C. Wilbur, MD, FCAP
Crystal Palmatier Jenkins, MD, Junior Member
This white paper was developed by the Technology Assessment Committee (TAC) with input from the Council on Scientific Affairs. Opinions expressed herein are solely those of the authors and do not represent those of the College of American Pathologists (CAP). No endorsement of any proprietary technology or product referenced is implied by the TAC or CAP. This report is provided for educational purposes only. None of the contents of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission of the publisher.