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In Vivo and Ex Vivo Microscopy: Moving Toward the Integration of Optical Imaging Technologies Into Pathology Practice.

Optical imaging technologies have emerged as viable modalities for clinical diagnosis and patient management. A confluence of events has facilitated this progress: (1) the advancement and validation of technologies that produce high-resolution digital images of tissues in real time without the need to remove, process, fix, or stain the tissue; (2) the widespread use and acceptance of digital images for histologic interpretation and quantitation (eg, whole slide scanning, image analysis software); (3) commercially available, US Food and Drug Administration-approved instruments for clinical use (such as optical coherence tomography and confocal microscopy); and (4) Current Procedural Terminology (CPT) billing code approval for in vivo microscopy (IVM) procedures and image analysis. However, in order for these technologies to mature to standard clinical care, an expert is required to interpret and assess imaging data. Because of the strong similarities between optical microscopic images and traditional white light microscopy, we propose that pathologists are ideally suited to take on the emerging role of expert in optical microscopy. (1) In this paper, we will examine optical imaging modalities that are at the forefront of clinical adoption, explore example clinical applications that these modalities can impact, and discuss the role that pathologists have the opportunity to fill in this endeavor.

OPTICAL IMAGING MODALITIES

Numerous optical imaging technologies exist that have the potential for clinical impact. In this paper, we focus on 2 imaging technologies (Table 1) that are commercially available for clinical applications: optical coherence tomography (OCT) and confocal microscopy.

Optical coherence tomography is a high-resolution optical imaging modality that generates depth-resolved, cross-sectional images with resolutions up to 10 pm. (2-5) Analogous to ultrasound, OCT uses near-infrared light, instead of sound, as its source. With OCT, contrast is generated endogenously based on optical index of refraction mismatches, with depth penetration of 2 to 3 mm within tissue. No exogenous contrast agents or transducing media are required. Technologic advancements occurring during the last decade have allowed for rapid image acquisition rates of more than 200 frames per second, such that large 3-dimensional scans at near-cellular resolution can be acquired in seconds. (6-13) OCT is well suited for superficial applications, such as retinal imaging. (14-17) The development of OCT catheters, which can perform either helical or longitudinal scanning, has led to many endoscopic applications. These include cardiovascular, upper and lower gastrointestinal, pulmonary, laryngeal, and genitourinary imaging. (9,18-60) Needle-based and laparoscopic OCT catheter devices have also been developed for imaging in solid organs, such as breast, pancreas, lung parenchyma, kidney, ovary, and prostate. (40,41,59,61-75) Swallowable, capsule-based OCT probes have also been developed for upper GI imaging without the need for sedation or traditional white light endoscopy. (76-80) Higher-resolution forms of OCT are also under development and have achieved resolutions of 1 [micro]m in the ex vivo setting. (81-840

Confocal microscopy is an optical imaging technique that typically uses a pinpoint beam of light scanned across a tissue to create an en face, or transverse, image. The pinpoint blocks out-of-focus light to generate very high-resolution images (<1 [micro]m). This method of obtaining subcellular resolution comes at a trade-off for depth penetration, with a maximum of 300 [micro]m. (85-88) Line-scanned confocal methods have also been developed. Confocal microscopy measures fluorescence emitted from tissue to generate contrast, which can either be from endogenous autofluorescence, from structures such as collagen, or from the use of exogenous fluorescent dyes, such as fluorescein, injected intravascularly or applied topically. (89) Confocal microscopy can be used for surface applications, such as dermatologic imaging. (90-93) Endoscope-based confocal imaging devices have been developed, termed confocal laser endomicroscopy (CLE), which allow for luminal organ imaging. Endoscope-based CLE is a device that incorporates a scanner into the endoscope to scan the pinpoint beam across the tissue to generate a 2-dimensional image. Probe-based CLE is a different device design that contains a fiber bundle to simultaneously acquire multiple pinpoint locations within tissue, allowing for 2-dimensional imaging. The field of view for these devices ranges from 200 to 600 pm, with imaging depths of 100 to 250 [micro]m. Image acquisition speeds also range from 1 to 12 frames per second, with the more rapid image acquisition allowing for video-rate imaging. Example applications of CLE include upper and lower gastrointestinal imaging, cystoscopy, and pulmonary imaging. (94-115) Needle-based confocal devices have also been developed, providing access to solid organ imaging. (116-120)

WHAT ARE IN VIVO AND EX VIVO MICROSCOPY?

In vivo microscopy defines imaging techniques, such as OCT and CLE, that allow tissue to be viewed in living patients, at high resolution, in real time, without tissue removal, fixation, freezing, or staining. Some of the most potent IVM applications have been developed in areas where conventional imaging techniques (such as white light endoscopy, computed tomography, magnetic resonance imaging, and mammography) have resolution limitations. In vivo microscopy technologies, such as OCT, enable rapid, high-resolution surveillance of large areas of tissue, such as the entire length of an esophagus, within minutes. This enables more precisely directed biopsies of image-specific anomalies, such as dysplasia in Barrett esophagus. It can also serve as a surrogate for frozen section, such as in Mohs surgery or breast resection. In vivo microscopy can also provide a method of microscopic assessment in sites that may preclude invasive biopsy procedures, such as in the eye, brain, and coronary arteries. It can also be used in scenarios where patients may be too frail to undergo an invasive biopsy, such as in fibrotic interstitial lung diseases. In vivo microscopy technologies can also provide intraprocedural biopsy guidance, such as the use of needle-based IVM for biopsy guidance in pulmonary nodules, breast lesions, and other organ systems. Optical imaging in the in vivo setting also provides unique opportunities for dynamic tissue assessment that are not attainable with traditional microscopy or EVM, such as the ability to visualize red blood cells moving within capillaries. Example applications for IVM are detailed in Table 2.

Ex vivo microscopy (EVM) offers the same capacity for high-resolution visualization of tissue without fixation, processing, and staining, but in tissue that has been removed from the patient. Ex vivo microscopy has the potential for many clinical applications that may enhance our practice as pathologists. Examples of applications include: (1) rapid assessment of biopsy adequacy without alteration or loss of biopsy tissue, which would be applicable to biopsies in many organ systems, (2) more directed sampling of specimens on the gross bench, (3) better specimen triaging and selection of tissue for genomic molecular studies and biobanking, and (4) intraoperative assessment of resections and sentinel lymph nodes as a complement to, or possibly in lieu of, frozen sections. Example applications of EVM are detailed in Table 3.

IVM AND EVM IMAGE INTERPRETATION: PARALLELS TO TRADITIONAL MICROSCOPY

In vivo microscopy and EVM provide microscopic images of tissue architecture, and thus the imaging features seen closely parallel the morphologic features pathologists assess and interpret during their routine clinical practice. For the vast majority of clinical applications, the characteristic imaging features of specific entities have been developed and validated using corresponding histologic features as the comparator. (24,25,29,54,55,58,59) In the same way that pathologists have learned to interpret immunohistochemical staining for routine diagnostic histopathology, air-dried Diff-Quik versus alcohol-fixed Papanicolaou staining in cytopathology preparations, and electron microscopy images for renal and muscle biopsies, IVM and EVM images are different but interpretable by pathologists who are already experts in microscopic diagnosis. OCT provides cross-sectional images that are very similar in orientation to traditional histology. The near-cellular resolution of OCT is analogous to low- to medium-power objectives using a standard microscope, providing predominantly microarchitectural information. Because of the rapid image acquisition speeds, large volumetric imaging stacks can be obtained with OCT that can be reconstructed in different planes, which has the advantage of being viewed in multiple planes simultaneously, similar in principle to computed tomography scans. Confocal microscopy provides transverse, or en face, views of tissue that are perpendicular to the tissue plane typically visualized with traditional microscopy. However, its high resolution allows for visualization of cellular detail analogous to high-power microscopy. Image processing software advances are also now able to convert grey value images to pseudocolor palettes, similar to routine histologic stains. Additionally, IVM and EVM provide new views of tissue and disease. These include 3-dimensional microarchitecture, views across multiple tissue planes, features not overtly visible by standard microscopy, and new modes of tissue contrast. It is possible that these imaging technologies may lead to new diagnostic criteria or characterization above and beyond what has been described using 2-dimensional histology.

OPTICAL IVM

In Table 4, 3 clinical vignettes, which demonstrate the use and impact of clinical-grade IVM technologies, are presented.

Clinical Example 1: IVM-OCT of Esophagus: High-Grade Barrett Dysplasia With Invasive Adenocarcinoma

A 77-year-old man with a long history of gastroesophageal reflux, treated with pantoprazole, presented for endoscopic surveillance for Barrett esophagus. Although light endoscopy visualized circumferential changes consistent with Barrett esophagus, with a nonpolypoid lesion identified, endoscopic OCT with a balloon-centering catheter was performed during endoscopy with circumferential, volumetric scanning of the entire esophagus (Figure 1). In the upper esophagus, OCT visualized normal squamous mucosa of the esophagus with an orderly, densely layered arrangement of squamous epithelium, lamina propria, submucosa, inner muscularis propria, and outer muscularis propria. In the lower esophagus, OCT demonstrated numerous irregular glands with complex/cribriform contours and an intense signal at the mucosal surface, diagnostic of high-grade Barrett dysplasia. A non-polypoid lesion was identified that demonstrated destruction of the underlying architectural layers and a homogenized appearance of the esophageal wall, indicative of associated invasive adenocarcinoma. Biopsies were taken, which subsequently confirmed the OCT imaging interpretation.

This example demonstrates the use of IVM for targeted biopsy guidance. In this setting, the pathologist could provide a preliminary intraprocedural interpretation, similar in principle to rapid onsite evaluation or frozen section, to assist the gastroenterologist in biopsy site selection. This could be done either in the GI procedure suite or, potentially, via telepathology. Following the procedure, the imaging data could be sent to pathology along with the physical tissue biopsy for final pathologic interpretation.

Clinical Example 2: IVM-Reflectance Confocal Microscopy of Skin Lesion: Basal Cell Carcinoma

An 85-year-old woman presented to the dermatology clinic with a skin lesion on her posterior neck of unknown duration (Figure 2). Clinical skin exam revealed a 5-mm papule with irregular contour and pigmentation. Confocal microscopy images were obtained of the lesion in the clinic, which revealed disturbance of the normal honeycomb pattern of the epidermal surface in the region of the clinically observed papule. At an en face depth of 170 [micro]m, telangiectasia and a black tumor silhouette were observed. At a depth of 180 [micro]m, tumor islands were visualized with peripheral palisading and streaming. These confocal microscopy features were consistent with basal cell carcinoma. A biopsy was performed, which confirmed the confocal imaging interpretation of basal cell carcinoma.

This example demonstrates the use of IVM for primary diagnosis of skin lesions. Similar to clinical example 1, the pathologist could provide an intraprocedural interpretation, similar to frozen section, to assist the dermatologist in diagnosis of the lesion. Additionally, there is also the potential to use IVM for the identification of adequate margins during subsequent resection. This could be done either with an on-call pathologist in the clinic or via telepathology. Following the procedure, the imaging data could be sent to pathology along with the biopsy and/or tumor resection for final pathologic interpretation.

Clinical Example 3: IVM-OCT of Peripheral Lung: Usual Interstitial Pneumonitis

A 56-year-old man presented to a pulmonologist with a 4-month history of dyspnea. (20) Chest high-resolution computed tomography showed traction bronchiectasis and bibasilar, subpleural reticular opacities consistent with interstitial lung disease. However, the lack of honeycombing precluded a more specific diagnosis by high-resolution computed tomography. The patient underwent bronchoscopy and surgical lung biopsy for diagnosis, and endobronchial OCT was performed during bronchoscopy (Figure 3). OCT showed patchy loss of normal alveolated parenchyma in the peripheral lung. Destructive, signal-intense fibrosis was visualized with multifocal microscopic honeycombing, seen as irregular, cystic, signal-void spaces embedded within fibrosis. OCT visualized spatial heterogeneity as regions of preserved alveolar architecture intermixed with destructive fibrosis. The OCT imaging features were consistent with a diagnosis of usual interstitial pneumonitis. Subsequent surgical lung biopsy independently confirmed the diagnosis of usual interstitial pneumonitis, including the presence of peripheral destructive fibrosis, microscopic honeycombing, and spatial heterogeneity.

Many interstitial lung disease patients are unable to undergo surgical lung biopsy for diagnosis because of the risks of associated morbidity and mortality. This example demonstrates the use of IVM for primary diagnosis in an at-risk or compromised patient population where tissue biopsy may not be permissible or advisable. In this setting, IVM has the potential to replace traditional tissue biopsy. The IVM images may be interpreted by a pathologist intraprocedurally to ensure adequate diagnostic quality, much like the current frozen section of interstitial lung disease lung wedges is performed to ensure adequate diagnostic material is present. The IVM images could then be sent to pathology, in lieu of tissue, for final diagnostic interpretation.

OPTICAL EVM

In Table 5, 2 clinical vignettes, which demonstrate the use and impact of clinical-grade EVM technologies, are presented.

Clinical Example 4 (EVM, Confocal Microscopy): Adenocarcinoma of Lung

A 61-year-old woman with a 20-year smoking history presented to the emergency department with hemoptysis. She was found to have a mass involving the right upper and middle lobe by computed tomography scan without any evidence of involvement of mediastinal lymph nodes. She underwent surgical resection of the right upper and middle lobe of lung for removal of the mass. The surgical specimen showed a 5.0 X 4.8 X 3.4 cm tumor involving the right upper and middle lobe with direct invasion of the visceral pleura. A fragment of the lung tumor measuring 1.0 X 1.0 cm was imaged in the fresh state using a confocal fluorescence microscopy platform after staining the tissue with acridine orange for 6 seconds (Figure 4). Gray scale images, as well as images digitally psuedocolored to mimic histologic staining, were obtained for interpretation. Both types of images showed glandular features of invasive adenocarcinoma. The corresponding hematoxylin-eosinstained tissue section of the imaged tumor tissue confirmed features of moderately differentiated adenocarcinoma.

This EVM example is a prime illustration of using optical imaging to assist in selecting tissues for a variety of uses, including tissue block selection for frozen section, microscopic anatomic pathology assessment, and biobanking. The high resolution and pseudocolorization to more closely resemble traditional histologic staining provide a very close analog to traditional microscopy. The ability to visualize cellular detail in this manner would also have potential for rapid core biopsy adequacy assessment, without requiring consumption of tumor tissue.

Clinical Example 5 (EVM, OCT of Endomyometrium): Adenocarcinoma of Endometrium, Superficially Invasive

A 65-year-old woman presented with postmenopausal bleeding. The patient subsequently underwent total hysterectomy. Gross examination revealed a thickened, papillary endometrium without gross evidence of invasion into the myometrium. OCT imaging was performed ex vivo on the resection specimen to assess for depth of invasion (Figure 5). Based on imaging, the adenocarcinoma was superficially invasive into the myometrium, which was confirmed with traditional histologic sectioning.

This example illustrates the use of EVM to assist in gross assessment at the pathology bench, both for frozen section evaluation and for permanent section selection during grossing. This is applicable for tissue selection in other organ systems, such as prostate, gastroesophageal, breast, and lung resections for carcinoma.

WHY PATHOLOGISTS NEED TO EMBRACE THESE IMAGING TECHNOLOGIES

As demonstrated by the clinical vignettes, IVM and EVM have tremendous potential for clinical impact in a wide variety of applications. A summary of the Strengths, Weaknesses, Opportunities and Threats (SWOT analysis) for incorporating IVM and EVM into pathology practice is detailed in Table 6. Because of the similarities between IVM/EVM imaging features and traditional microscopy, and the overlapping skill sets required to interpret both, pathologists are ideally suited to assume the role of optical IVM/EVM expert. Compared with other clinical specialists who may consider introducing similar technologies into their practice, pathologists already have the skills and expertise to embrace such technologies. Pathologists are already experts at interpreting microscopic images. Pathologists are already trained to design and complete comprehensive validation procedures. Pathologists have been involved in numerous optical imaging validation studies, many of which included pathologist training on image interpretation and subsequent blinded validation assessment to determine interpretation sensitivity/specificity.* As clinical usage of these technologies develops further to assess a wider spectrum of disease, the pathologist's deep understanding of tissue microarchitecture and disease variability will be essential for accurate and clinically informative image interpretation and is a skill set no other clinical specialty currently has. Although not all of the imaging technologies provide direct 1:1 imaging to histology correlates, pathologists have the most comprehensive understanding of disease morphology from the gross to the ultramicroscopic level, which provides us with the best skill set to adapt our knowledge to optical image interpretation. Understanding biophysiologic correlates in tissue also allows pathologists to investigate additional diagnostic and prognostic markers that may be provided by other imaging modalities to complement those seen in thin, processed tissue sections under light microscopy. Embracing new optical imaging technologies could balance the reduction in surgical volumes associated with bundled payments, replace other labor-intensive procedures that have poor sensitivity and specificity (such as frozen sections), and reduce the number of repeat diagnostic biopsies due to inadequate tissue sampling. However, pathologists must be prepared to train themselves in the interpretation of new optical imaging modalities and consider the workflow and schedule changes that may accompany the introduction of these technologies into their daily practice. In vivo microscopy interpretation could be performed by pathologists intraprocedurally, similar to rapid assessment/frozen section, either with the pathologist in the procedure room or via telepathology. The IVM images could then be sent for final pathology interpretation, with or without accompanying tissue for traditional histology. Ex vivo microscopy could be performed and interpreted as part of the pathology workflow, either for rapid assessment of small tissue/biopsy or at the frozen section/grossing bench for large-area specimen imaging and targeted selection of tissue.

TRAINING OPPORTUNITIES FOR PATHOLOGISTS TO LEARN ABOUT IVM/EVM TECHNOLOGIES

In the same way that there is a learning curve for pathologists to interpret ultrasound images when performing fine-needle assessments on superficial lesions in the clinic setting or becoming comfortable with the software tools needed to diagnose and quantify digitized whole slide images, there is also a learning curve for interpreting IVM and EVM images. Numerous training and educational opportunities are available, including many resources provided by the College of American Pathologists and other professional associations, which are detailed in Table 7.

CONCLUSIONS

Pathologists today must actively embrace, learn about, and validate new technologies that can provide more cost-effective, standardized, and clinically relevant diagnoses for our patients. The College of American Pathologists, United States and Canadian Academy of Pathology, and Pathology Informatics associations have provided a wealth of resources and information to educate pathologists. Pathologists are already experts in interpreting microscopic images, particularly architectural and cytologic features as they relate to a specific diagnosis. They now need to master the interpretation of optical microscopy images. In addition to reflecting underlying patterns of disease understood from traditional microscopy, optical imaging comprises additional data that can potentially be leveraged to reveal previously unrealized features of disease and/or biochemical composition. Combining these features will allow pathologists to develop more comprehensive tissue and image-based signatures that may provide improved diagnostic accuracy and better predict patient prognosis and response to therapy.

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Wendy A. Wells, MD, MSc; Michael Thrall, MD; Anastasia Sorokina, MD; Jeffrey Fine, MD; Savitri Krishnamurthy, MD; Attiya Haroon, MD; Babar Rao, MD; Maria M. Shevchuk, MD; Herbert C. Wolfsen, MD; Guillermo J. Tearney, MD, PhD; Lida P Hariri, MD, PhD

Accepted for publication October 4, 2018.

Published online December 10, 2018.

From the Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire (Dr Wells); the Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, Texas (Dr Thrall); the Department of Pathology, University of Illinois at Chicago, Chicago (Dr Sorokina); the Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (Dr Fine); the Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston (Dr Krishnamurthy); the Department of Dermatology, Rutgers-Robert Wood Johnson Medical School, Somerset, New Jersey (Drs Haroon and Rao); the Department of Pathology, Weill Cornell Medical College, New York, New York (Dr Shevchuk); the Division of Gastroenterology & Hepatology, Mayo Clinic, Jacksonville, Florida (Dr Wolfsen); and the Wellman Center for Photomedicine (Dr Tearney) and the Department of Pathology (Drs Tearney and Hariri), Massachusetts General Hospital, Harvard Medical School, Boston.

Massachusetts General Hospital has a licensing arrangement with Terumo Corporation. Dr Tearney has the rights to receive royalties from this arrangement. Dr Tearney also receives royalties from iLumen, Nidek, and Abbott (through MIT). Dr Tearney has a financial/fiduciary interest in SpectraWave, a company developing an optical coherence tomography-near-infrared spectroscopy intracoronary imaging system and catheter. His financial/fiduciary interest was reviewed and is managed by the Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. Dr Tearney consults for NinePoint Medical and SpectraWave. Dr Tearney's laboratory receives materials from Terumo Corporation and sponsored research from Vivolight, iLumen, Boston Scientific, Ardea Biosciences, Vertex, and Canon Inc. Dr Rao is a consultant for CaliberlD, the maker of Vivascope. Dr Hariri is an author on a Massachusetts General Hospital-owned patent that has been licensed to LX Medical. Dr Hariri has the rights to receive royalties from the licensing arrangement. Dr Hariri is a consultant for LX Medical. The other authors have no relevant financial interest in the products or companies described in this article.

Corresponding author: Lida P. Hariri, MD, PhD, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, MA 02114 (email: Lhariri@partners.org).

Caption: Figure 1. In vivo microscopy optical coherence tomography (OCT) of esophagus for Barrett surveillance. A and B, OCT image of normal squamous mucosa of the esophagus, obtained using a balloon-centering probe in vivo. A, OCT circumferential scan of the entire esophagus. B, Magnified region of A showing an orderly, densely layered arrangement of squamous epithelium (EP), lamina propria (LP), submucosa (SM), inner muscularis propria (IMP), and outer muscularis propria (OMP). C and D, OCT circumferential scan of the entire esophagus with lesion (arrows). D, Magnified region showing numerous irregular glands with complex/ cribriform contours and an intense signal at the mucosal surface, diagnostic of high-grade Barrett dysplasia (HGBD). The destruction of the underlying architectural layers and a homogenized appearance of the esophageal wall are indicative of associated invasive adenocarcinoma (IA). This example and figures were contributed by authors H. C. W. and G.J. T.

Caption: Figure 2. In vivo microscopy reflectance confocal microscopy (CM) for assessment of skin papule. A, A pigmented papule was observed on the skin surface of the posterior neck. B through D, Reflectance CM mosaic of skin surface, en face view. B, Disturbance of the normal honeycomb pattern of the epidermal surface in the area of the clinical papule. C, Reflectance CM en face depth of 170 pm with telangiectasia (yellow arrows) and a black tumor silhouette (red box). D, Reflectance CM en face depth of 180 pm with tumor islands with peripheral palisading and streaming (white star). The tumor islands seen are consistent with basal cell carcinoma. A biopsy was performed, which confirmed the diagnosis of basal cell carcinoma. This example and figures were contributed by authors A.H. and B.R.

Caption: Figure 3. In vivo microscopy optical coherence tomography (OCT) for assessment of interstitial lung disease. A, High-resolution computed tomography showed traction bronchiectasis (arrowhead) and bibasilar, subpleural reticular opacities (arrows), but no honeycombing. B, Endobronchial OCT showed microscopic features of usual interstitial pneumonitis (UIP), including multifocal microscopic honeycombing (arrowsHC) embedded within peripheral, destructive fibrosis (f) beyond the bronchiolar epithelium (e). C, Subsequent surgical lung biopsy was independently diagnosed as UIP, and it confirmed the presence of peripheral fibrosis (f), microscopic honeycombing (arrows), and preserved alveoli (a), indicative of spatial heterogeneity (a). D, Example of peripheral lung with numerous, evenly spaced alveoli present (a), indicative of spatial heterogeneity (hematoxylin-eosin, original magnification X40). This example and figures were contributed by author L.PH. Figure modified and reprinted with the permission of the American Thoracic Society Copyright 2018 American Thoracic Society. (20) The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society.

Caption: Figure 4. Ex vivo confocal microscopy for assessment of lung carcinoma. A and B, Gray scale and digitally pseudocolored image of lung tumor tissue. The glandular differentiation of adenocarcinoma can be appreciated in the images. C, Corresponding hematoxylin-eosin-stained tissue section of the imaged tumor tissue confirms features of moderately differentiated adenocarcinoma (original magnification X200). This example and figures were contributed by author S.K.

Caption: Figure 5. Ex vivo microscopy optical coherence tomography (OCT) for the assessment of uterine endomyometrium. A, Ex vivo OCT image of fresh endomyometrium demonstrating endometrial adenocarcinoma with superficial invasion (white arrow). B, Corresponding routinely processed and hematoxylin-eosin-stained section of endomyometrium confirming the findings seen in OCT (black arrow) (original magnification X10). This example and the figures were contributed by author J.F.

* References 3, 4, 9, 24, 25, 29, 31, 34-36, 38, 42, 47, 54-59, 71, 72, 74-77, 80-83, 87, 91-93, 95, 97-103, 108-110, 116, 118, 119.
Table 1. Example Technologies Behind In Vivo
Microscopy and Ex Vivo Microscopy

1. OCT

Measures light reflectance, with contrast based on differences
   in endogenous tissue properties
Provides cross-sectional images with near-cellular resolution
   of ~10 [micro]m

Depth of penetration: 2-3 mm
Image acquisition rates: up to 200 frames per second,
   allowing for rapid, 3-dimensional imaging of large tissue
   volumes

OCT technology can be:

   Used for superficial applications, such as retinal imaging
   Catheter based for luminal organ imaging, with custom
     catheters for specific clinical applications
   Part of a swallowable capsule for upper GI tract screening
   Needle based or laparoscopic for solid organ imaging
Commercially available

Examples of use:

   GI tract (ie, Barrett esophagus, gastric lesions, colonic polyps,
     pancreatic cysts, inflammatory bowel disease)

   Coronary artery plaques

   Eye imaging (ie, retinal disease, choroidal disease)

   Pulmonary imaging (ie, lung nodules, interstitial lung disease,
     asthma)

2. CLE

Detects fluorescence to generate image contrast from:

   Endogenous tissue autofluorescence, from structures such as
     collagen

   Exogenous fluorescent agents, such as fluorescein, injected
     intravenously or applied topically
Provides images in the transverse, or "en face," plane with
   subcellular resolutions of 1-2 [micro]m
Depth of penetration: up to 300 [micro]m
FOV: up to 600 [micro]m
Image acquisition rates: up to 12 frames per second
Types of CLE probes:
   Probe-based devices with a fiber bundle that transmits the
     confocal image
   Endoscope-based devices with the confocal microscope
     scanner built into the endoscope
   Other endoscopic and needle-based confocal probes have
     also been developed
   Commercially available

Examples of use:

   Skin lesions (ie, pigmented lesions, Mohs surgery for margins)
   GI tract (ie, Barrett esophagus, gastric lesions, colonic polyps,
     inflammatory bowel disease)
   Pulmonary imaging (ie, lung nodules, parenchymal lung
     disease)
   Cystoscopy (ie, bladder lesions, upper urinary tract lesions)

Abbreviations: CLE, confocal laser endomicroscopy; FOV, field of view;
GI, gastrointestinal; OCT, optical coherence tomography.

Table 2. Current and Potential Applications of
In Vivo Microscopy

Organ Site         Disease or Application

Eye                Retinal disease
                   Choroidal disease

Gastrointestinal   Barrett esophagus and dysplasia
tract              Esophageal carcinoma, early stage
                   Eosinophilic esophagitis
                   Colorectal polyps
                   Biliary strictures
                   Pancreatic duct tumors
                   Pancreatic cysts
                   Inflammatory bowel disease
                   Celiac disease

Skin               Melanocytic/nonmelanocytic tumors
                   Melanocytic nevi
                   Inflammatory skin lesions
                   In vivo margin assessment

Heart              Plaque detection and morphology in
                   coronary arteries
                   Stent evaluation

Lung               Lung nodules/carcinoma
                   Interstitial lung disease
                   Asthma
                   Chronic obstructive pulmonary disease

Breast             Biopsy guidance
                   In vivo margin assessment
                   Sentinel lymph node assessment

Brain              Brain tumors

Genitourinary      Bladder and upper tract urothelial lesions
tract

Gynecologic        Cervical lesions
                   Endometrial lesions

Intraoperative     Biopsy guidance in multiple organ systems
or procedural      Tumor margin assessment

Table 3. Current and Potential Applications of Ex Vivo Microscopy

Clinical Setting      Pathology Application        Example of Use

Intraoperative or   Tumor margin assessment     Breast or lung
  Intraprocedural                                 carcinoma resection
                                                  margin assessment

                    Needle biopsy/aspirate      Rapid assessment
                                                  of adequacy
                    Transplant assessment       Assessment of donor
                                                  tissue suitability
                                                  for transplant

Gross examination   Block selection--identify    Esophageal, prostate,
                    tumor-containing regions      bladder, lung,
                                                  breast resections
                                                  for carcinoma

                    Block selection--depth of    Endometrial
                    invasion                      carcinoma resection
                                                Melanoma resection

Genomic-molecular   Tissue triaging--selection   Select tumor via
  studies                                         imaging and
                                                  conserve tissue

Biobanking          Tissue triaging--selection   Select tumor-rich
                                                 regions via imaging

Clinical Setting              Advantages

Intraoperative or   Alternative to
  Intraprocedural   freezing the tissue

                    No tissue consumption

                    No need to freeze
                    the tissue

Gross examination   More accurate selection
                    of tissue
                    sample

                    In situ assessment of
                    depth of invasion

Genomic-molecular   No need to freeze the tissue
  studies           or wait for permanent
                    sections

Biobanking          No need to freeze the
                    tissue or wait for
                    permanent sections

                    Microscopic tissue
                      documentation
                    Conservation of tissue

Table 4. Clinical Vignettes for In Vivo Microscopy (IVM)

Example                    1                         2

Clinical       Targeted biopsy              Primary diagnosis,
impact           guidance                     and potential for
                                              margin assessment

Organ system   Esophagus                    Skin
IVM modality     IVM-OCT                    IVM-RCM
Clinical         77-year-old man            85-year-old woman
history          with history of GE         with 5-mm skin
                 reflux treated with        lesion, posterior
                 pantoprazole               neck, unknown
                                            duration

Procedure      Endoscopic surveillance      Observed on
                 for Barrett                clinical exam
                 esophagus

Endoscopic/    Barrett, circumferential,    Papule, irregular
clinical/        with nonpolypoid           contour and
surgical         lesion at 35 cm,           pigmentation
findings         5 o'clock

IVM images     Figure 1                     Figure 2
Diagnosis        High-grade dysplasia       Basal cell
                 and invasive               carcinoma
                 adenocarcinoma

Example                  3

Clinical       Less invasive primary
impact           diagnosis in frail
                 patient population

Organ system   Lung
IVM modality   IVM-OCT
Clinical       56-year-old man
history          with interstitial
                 lung disease, and
                 nondiagnostic
                 chest HRCT

Procedure      Bronchoscopy and
                 VATS surgical
                 lung biopsy for
                 diagnosis

Endoscopic/    Abnormal lung
clinical/        parenchyma
surgical
findings

IVM images     Figure 3
Diagnosis      Usual interstitial
               pneumonitis

Abbreviations: GE, gastroesophageal; HRCT, high-resolution computed
tomography; OCT, optical coherence tomography; RCM, reflectance
confocal microscopy; VATS, video-assisted thoracoscopic surgery.

Table 5. Clinical Vignettes for Ex Vivo Microscopy (EVM)

Example        1                    2

Clinical       Tissue block         Depth of invasion:
application    selection:           endomyometrium
               lung cancer
EVM modality   EVM-CM               EVM-OCT

Clinical       61-year-old woman    65-year-old woman,
history        with lung mass       postmenopausal bleeding

Procedure      Bilobectomy          Total hysterectomy

Gross          5.0-cm lung mass     Thickened, papillary
findings                            endometrium, grossly
                                    noninvasive of myometrium
EVM images     Figure 4             Figure 5

Diagnosis      Adenocarcinoma       Endometrial adenocarcinoma,
               of lung              superficially invasive of
                                    myometrium

Abbreviations: CM, confocal microscopy; OCT, optical coherence
tomography.

Table 6. Strengths, Weaknesses, Opportunities, Threats Analysis for
In Vivo Microscopy (IVM) and Ex Vivo Microscopy (EVM)

Strengths                            Weaknesses

1. Pathologists are already tissue   1. Pathologists are currently not
imaging experts.                     trained in optical image
                                     interpretation.

2. Subspecialist pathologists are
already working with subspecialist   2. It can be difficult for busy
clinicians who can help validate     pathologists/trainees to embrace
new technology.                      and educate themselves about a new
                                     technology.

3. Pathologists must be involved     3. Workflow changes may require
in the design of a validation        coordinating schedules within the
process (how and what tissues) and   department, as well as the
new workflows.                       operating room, endoscopy
                                     suites, and outpatient clinics.
4. Pathologists can incorporate
new physiologic correlates           4. Pathologist reluctance to
(vascularity patterns, water         embrace digital images being sent
content, tumor oxygenation) for      to desktop workstations for rapid
additional diagnostic or             interpretation.
prognostic "value."

Opportunities                        Threats

1. Reduced surgical volumes--move     1. Resect and discard--faster
from fee/for/service to bundled      movement toward the "nonbiopsy
payments, balanced with IVM/EVM      diagnosis," supplanting the
technology.                          diagnostic biopsy gold standard.

2. Advantages to patients--less       2. Other clinicians
time in operating room, fewer        (gastroenterologists,
repeat biopsies, less inadequate     dermatologists, surgeons) will
tissue for diagnostic ancillary      "claim" expertise.
studies (molecular panels).

3. Replacement of other labor/       3. Attitude impediments from
intensive procedures with poor       clinicians and pathologists.
sensitivity/specificity (eg,
frozen sections).

4. Encourage new instrument          4. Perceived increased costs of
development by vendors (especially   new technologies (capital outlay,
in the field of EVM) and become      no immediate reimbursement).
involved in validation studies and
early adoption.

Table 7. Resources for Pathologists and Trainees to
Learn About In Vivo Microscopy (IVM)/Ex Vivo
Microscopy (EVM)

National societies with IVM focus areas
  CAP IVM/EVM Work Group
  International Society for Optics and Photonics
  The Optical Society of America
  DPA

CAP In Vivo Microscopy Resource Guide, including a complete
  bibliography and companion videos online
CAP IVM webinar series (upcoming and archived)

CAP IVM educational courses with practical demonstrations
CAP Short Presentation in Emerging Concepts on IVM and EVM
CAP IVM briefs on emerging technologies

Pathology conferences with IVM topics
  CAP annual meeting
  Molecular Med TriCon
  DPA

  Pathology Informatics Summit
  Pathology Visions
IVM fellowship programs

Abbreviations: CAP, College of American Pathologists; DPA, Digital
Pathology Association.
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Author:Wells, Wendy A.; Thrall, Michael; Sorokina, Anastasia; Fine, Jeffrey; Krishnamurthy, Savitri; Haroon
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Date:Mar 1, 2019
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