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Multiphoton microscopy in the evaluation of human bladder biopsies.

The American Cancer Society estimated that 70 530 new cases of bladder cancer were diagnosed in 2010 in the United States alone, accounting for 14 680 deaths. (1) Bladder cancer ranks as the fourth most common malignancy and ninth leading cause of death from cancer in men, who are nearly 3 times more likely to develop bladder cancer than are women. (1) The urothelium is the dominant type of epithelium lining the urinary bladder, ureters, and renal pelvis, and more than 90% of bladder cancers are urothelial carcinomas. (2,3) The World Health Organization/International Society of Urological Pathology (2004) classification of bladder lesions includes the following 3 broad diagnostic categories: (1) flat, intraurothelial lesions (ranging from benign to malignant); (2) papillary urothelial lesions (ranging from benign to malignant); and (3) invasive urothelial neoplasms (low grade and high grade). (2,4)

Approximately 70% of newly diagnosed cases of bladder cancer represent superficial disease, that is, nonmuscle-invasive (pathologic stage Ta, T1, or Tis). (3,5) The natural history of these superficial bladder cancers is difficult to predict because of tumor heterogeneity and multifocality, and patients with superficial bladder cancers are prone to disease recurrence and progression. The recurrence and progression rates of superficial bladder cancer vary according to several tumor characteristics, mainly tumor grade, tumor stage, and the presence or absence of carcinoma in situ (CIS). (2,5-7) Urothelial papillomas, papillary urothelial neoplasms of low malignant potential, and noninvasive low-grade papillary urothelial carcinomas have a low rate of disease progression, whereas all superficial high-grade urothelial carcinomas, whether flat or papillary, are at high risk for progression. (3,7,8)

Early detection, diagnosis, and treatment of these superficial bladder cancers would improve prognosis and possibly provide a cure for some patients. However, current diagnostic techniques, namely urine cytology and white-light cystoscopy, are unable to provide optimal sensitivity and specificity for early stage bladder cancers-- especially CIS--resulting in compromised patient care. (9) For instance, patients with suspicious or positive urine cytology but no obvious tumor during cystoscopic exam may be subjected to numerous (including many benign) biopsies to rule in or out carcinoma. Not infrequently, these patients must undergo repeat cystoscopy and biopsy when all initial biopsies are nondiagnostic or the findings are negative for carcinoma. Cystoscopy and biopsy procedures are not without potential complications, such as bleeding, infection, and occasionally, bladder perforation. In addition, repeat cystoscopy after nondiagnostic procedures increases both direct and indirect health care costs and negatively affects patient quality of life. (10,11)

High-magnification, high-resolution "optical biopsy techniques, such as multiphoton microscopy (MPM), are currently being explored as alternative and adjunctive approaches to diagnosis. Multiphoton microscopy relies on the simultaneous absorption of 2 (or 3) low-energy (near infrared) photons to cause nonlinear excitation equivalent to that created by a single photon of bluer light. Excitation only occurs where there is sufficient photon density (ie, at the point of laser focus), providing intrinsic optical sectioning with resolution equivalent to traditional confocal microscopy. Tissue penetration is greater than it is with standard confocal microscopy because absorption and scattering of the laser excitation is reduced at near-infrared wavelengths compared with visible or ultraviolet regions of the spectrum. (12,13)

Most importantly, by using 2-photon excitation in the 700 to 800 nm range, MPM enables both in vivo imaging and ex vivo imaging of fresh, unprocessed, and unstained tissue via intrinsic tissue emissions (ITEs). The ITE signal is composed of 2 components: (1) tissue auto fluorescence, in part from reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide in cells, elastin in the connective tissue, and lipofuscin in fat and other cells; and (2) second harmonic generation (SHG), a nonlinear scattering signal that arises from noncentrosymmetric structures, such as tissue collagen. (12-14) Multiphoton microscopy/ITE imaging is capable of generating distinctive optical signals that enable imaging of animal (13) and human (15-17) tissues at submicron resolution in 3 dimensions to a depth of up to 0.5 mm below the specimen surface, at acquisition rates of approximately 1 image/s, with greater than 105 pixels/image. These imaging parameters enable detailed visualization of cellular and subcellular structures, which is important because changes in cellular and subcellular morphology accompany the development of precancerous lesions and cancers. (18)

With in vivo MPM imaging techniques currently under investigation for assessment of pulmonary and other solid tumors, application of MPM to bladder or other hollow viscera is likely soon to follow. However, for MPM to become a clinically useful adjunct tool, the ability of MPM to identify relevant bladder lesions, compared with the current gold standard--histopathology of thin sections stained with hematoxylin-eosin (H&E)--must be assessed. Here, we report a prospective study of 77 human bladder biopsies that were first imaged (fresh, unfixed, and unstained) with MPM and were then processed for routine H&E histopathology. The MPM images were read by 2 trained surgical pathologists, and those diagnoses were compared with final histopathology results. Our results suggest that the MPM image sets alone, obtained from these unprocessed biopsies, can readily distinguish between normal bladder mucosa, benign inflammatory/ reactive lesions, CIS, papillary urothelial carcinomas, and to a lesser degree, invasive urothelial carcinomas. These observations indicate that MPM could be an important potential tool to provide immediate "intracystoscopic" diagnostic impressions that can guide urologists in biopsy and patient management.

MATERIALS AND METHODS

Study Cohort

The study included 77 adult men and women who reported to the clinical urology practice at Weill Cornell Medical College (New York, New York) with symptoms and cystoscopic findings consistent with bladder cancer and who were scheduled for a transurethral resection of a bladder tumor. All subjects consented to participate in an Institutional Review Board-approved study to image one of their biopsies with MPM before it was submitted to surgical pathology for diagnostic histopathologic examination. The nature of the detour of one of their biopsies to the MPM facility for 1 hour, before going to surgical pathology, was explained clearly to the subjects before consent was taken. Specimens for MPM were collected only from cases where sufficient number of biopsies had been obtained to ensure that diagnostic ability was not compromised because of participation in the research study.

Multiphoton Microscopy

Specimen Acquisition and Handling.--Seventy-seven fresh bladder biopsy specimens, obtained from transurethral resection of a bladder tumor or cold cup biopsies, were imaged with MPM. Immediately after excision, the specimens were collected in bottles containing normal saline, placed on ice, and brought directly to the MPM facility for imaging. Immediately after MPM imaging (up to a maximum of 1 hour), the specimens were placed in 10% buffered formalin and submitted for routine histopathology.

Specimen Imaging.--In the MPM imaging facility, each biopsy specimen was placed on a small tissue culture dish with a central well, with the urothelium oriented upwards. The specimen was then imaged using a custom-built MPM system consisting of an Olympus BX61 upright microscope (Olympus America, Center Valley, Pennsylvania) and a modified Bio-Rad 1024 scanhead (Bio-Rad, Hercules, California). Images were acquired at 2 different magnifications: (1) low magnification for overall architectural information (X4, 0.28 NA Olympus dry objective), allowing imaging of 3.1-m [m.sup.2] frames at 6-[micro]m/pixel resolution; and (2) high magnification for detailed cellular and local architectural information (X 20, 0.95 NA Olympus waterimmersion objective), allowing imaging of 614-[micro][m.sup.2] frames at 1.2-[micro]m/pixel resolution. If necessary, higher scanner (digital) zooms were used for further magnification. When imaging with the X 20 water immersion objective, a cover slip was placed on a normal buffered saline-moistened biopsy, and a drop of normal saline was placed on the cover slip to achieve water immersion. The specimens were excited using a tunable Ti-Sapphire laser (Mai Tai, Spectra-Physics Lasers, Newport Corporation, Irvine, California) tuned to 780 nm. The pulse width at the sample was estimated to be approximately 160 femtoseconds when delivered through the X 4 macro lens and approximately 200 femtoseconds through the X 20 objective, based on pulse autocorrelation measurements made on an identical MPM system. The laser power was controlled through a Pockels Cell (Conoptics, Danbury, Connecticut) and typically used a maximum power of 60 to 80 mW when acquiring data with the 20 X /0.95 water immersion objective. Images were collected in 2 channels: (1) the SHG signal 355 to 420; and (2) the broadband autofluorescence at 420 to 530 nm.

Typically, the collection of each image frame shown in the Figures took 1 to 3 seconds. Imaging through all the optical sections of a given area (20 to 40 images) took 1 to 1.5 minutes. The images can be evaluated by a pathologist during image acquisition. No postprocessing is necessary to identify essential architectural and cellular features.

MPM Image Evaluation.--All images were obtained as stacks of optical sections up to a tissue depth of 0.5 mm with the X4 objective and to approximately 0.25 mm with the X 20 objective. For better appreciation of the structures, the raw 8-bit grayscale images were color-coded red for SHG signals and green for autofluorescence signals. The 2 pathologists independently reviewed all MPM images, categorizing each lesion based on (1) architecture, and (2) cytologic grade. The lesions were scored numerically, as described in the section on data analysis below. The specific diagnostic criteria used in MPM, as compared with standard histopathology, are provided in the Table.

[FIGURE 1 OMITTED]

One of the pathologists began the project earlier and had a training period of 3 months, during which, she compared previously acquired MPM image sets with H&E slides from the same cases (not included in this study). The second pathologist, on the other hand, had a relatively brief "training phase," during which, he reviewed 10 comparative MPM and H&E image sets with the first pathologist, representing all classes of lesions included in this study.

Histopathology

After MPM imaging, the biopsies were processed per standard protocol in the surgical pathology laboratory (ie, formalin fixation, paraffin embedding, 5-mm-thick sectioning, and H&E staining, referred to briefly as formalin-fixed, paraffin-embedded [FFPE] specimens]. Cases were diagnosed by the attending pathology staff at Weill Cornell Medical College and reported using the World Health Organization/International Society of Urological Pathology (2004) grading system, with lesions classified as benign (including reactive conditions, such as cystitis cystica), CIS, noninvasive papillary urothelial carcinoma (low grade and high grade), and invasive carcinoma.

Data Analysis

All MPM diagnoses were scored by the 2 pathologists with the following architectural diagnoses: flat, 1; favor flat, 2; favor papillary, 3; or papillary, 4. Similarly, cytologic grade was reported as follows: benign/low grade, 1; favor benign/low grade, 2; favor high grade, 3; or high grade, 4. The MPM diagnoses were then compared with the gold standard histopathology diagnoses given by the attending pathologists, which were also coded numerically by the 2 pathologists using precisely the same scoring system described above for MPM diagnoses.

The following diagnostic criteria were evaluated: (1) ability to accurately diagnose neoplastic processes, and (2) ability to accurately diagnose cytologic grade of lesions. All cases that obtained scores of 1 or 2 for both architecture and grade were classified as benign, and those that obtained a score of 3 or 4 in either architecture or grade were classified as neoplastic. Regarding cytologic grade, all cases that obtained a grade of 1 or 2 were classified as benign/low grade, and those that obtained a score of 3 or 4 were classified as high grade.

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Statistical testing used a standard 2 X 2 contingency table comparing MPM with the gold standard H&E histopathology to determine the diagnostic test operating characteristics (accuracy, sensitivity, specificity, positive predictive value, and negative predictive value), as shown in Figure 1.

Visual comparisons were also made between images obtained from MPM image sets and the H&E slides of the corresponding bladder biopsy tissue, with the goal of identifying correlates and discrepancies in diagnosis, understanding the advantages and disadvantages of MPM, and determining whether MPM instrumentation improvements would improve diagnostic accuracy.

RESULTS

Based on MPM images obtained at low magnification, we were able to differentiate flat (nonpapillary) lesions from papillary lesions. Based on cytologic features (ie, nuclear to cytoplasmic [N:C] ratio, polarity, and nuclear pleomorphism) obtained from MPM images at high magnification, we were able to grade papillary tumors as either low or high grade, and flat lesions were categorized into benign/reactive or CIS.

Flat Intraurothelial Lesions and Invasive Urothelial Carcinoma

Normal Bladder Histology and Benign/Reactive Le sions.--At low magnification (Figure 2, A and B), the flat (nonpapillary) nature of the lesions was evident in benign urothelium. The unique structure of the urothelium, with its multiple (3-7) cell layers and surface umbrella cells (arrow), was recognized based on the cellular autofluorescence signal (color-coded green) at higher magnification. The underlying lamina propria consisted of collagen fibers, which generate SHG signal (color-coded red), interspersed with wavy, autofluorescent elastin fibers (color-coded green). These distinct signals allowed us to clearly identify features corresponding to normal bladder histology and to identify the junction of urothelium and lamina propria. Von Brunn nests in lamina propria, including cystitis cystica (Figure 2, E and F; arrow), were similarly straight forward to identify.

[FIGURE 3 OMITTED]

Flat Carcinoma In Situ.--In contrast to benign urothelium, in which cells have columnar to ovoid nuclei oriented perpendicular to the basement membrane, biopsies with CIS showed flat, urothelial lining and characteristic loss of polarity at low magnification (Figure 3, A and B). At higher magnification (Figure 3, C and D), CIS was diagnosed based on high-grade cytologic atypia, that is, large cells with marked pleomorphism and high N:C ratio (arrows).

Invasive Urothelial Carcinoma.--All cases of invasive urothelial carcinomas in this study (n = 7) were high-grade lesions. Invasion into lamina propria ranged from minimal to extensive, and 2 cases showed muscularis propria invasion. Most of these biopsy lesions, however, were superficial in nature, and muscularis propria was not present. One case could not be evaluated for invasion by MPM because of the lack of available high-magnification images. Lamina propria invasion was identified by MPM in only 1 of the remaining 6 invasive carcinoma cases (17%). That case (Figure 3, F) showed extensive invasion on light microscopic examination. At high magnification on MPM, we could appreciate clusters and nests of autofluorescent urothelial carcinoma cells (coded green; Figure 3, E and F, areas marked as a) infiltrating the SHG-signal-rich collagen bundles (coded red; Figure 3, E and F, areas marked as b) within the lamina propria. For the most part, however, invasion was difficult to assess by MPM. For instance, MPM did not provide sufficient information to identify microinvasion into lamina propria or invasion in cases with extensive cautery artifact. These limitations were due to the relatively small field and depth of view possible using the current MPM apparatus, as well as the low statistical probability of detecting micro invasion on any given section. Nevertheless, all 6 evaluable cases (100%) of invasive urothelial carcinoma were correctly identified on MPM as being high-grade neoplastic processes, whether invasion was detected or not.

Noninvasive Papillary Urothelial Carcinoma

At low magnification, the papillary nature of the urothelium was appreciated in all papillary urothelial neoplasms (Figures 4, A and B, and 5, A and B). The complex, arborizing papillae are composed of thin, fibrovascular cores containing collagen fibers (SHG signal, color-coded red) and lined by a thickened epithelial layer (autofluorescence, color-coded green). In some cases, the papillary cores become hyalinized, which can be seen as reddish, homogeneous areas on MPM (Figure 5, E and F).

The N:C ratio and degree of pleomorphism, seen at higher magnification, were evaluated in differentiating papillary lesions into low-grade and high-grade urothelial carcinomas.

Low-Grade Papillary Urothelial Carcinomas.--Lowgrade papillary lesions had thickened but orderly urothelium (color-coded green), sometimes appearing fused, and papillary fronds with thin, fibrovascular cores (colorcoded red; Figure 4, A, B, C, and D; arrows). At higher magnification, low-grade papillary urothelial carcinomas showed only mild variation in nuclear size and shape, with an overall low N:C ratio (Figure 4, E and F).

High-Grade Urothelial Carcinomas.--High-grade papillary urothelial carcinomas had low-power architecture similar to the low-grade papillary carcinomas, showing thickened papillary fronds that more frequently fused, forming sheets of tumor cells among the fronds (Figure 5, A through F). At higher magnification, these cells showed moderate to marked nuclear pleomorphism and high N:C ratios (Figure 5, E and F).

Assessment of MPM as a Diagnostic Tool

The distribution of the 65 cases evaluated were as follows: (1) benign urothelium (including reactive), n = 13 (20%); (2) CIS, n = 14 (22%); (3) papillary low-grade urothelial carcinoma, n = 17 (26%); (4) papillary highgrade urothelial carcinoma, n = 14 (22%); and (5) invasive urothelial carcinoma, n = 7 (11%).

The diagnostic accuracy of MPM was 88% (57 total accurate diagnoses, including 47 neoplastic cases [82%] and 10 benign cases [18%]). Of the 65 cases, 3 (5%) were called neoplastic on MPM that were diagnosed as benign on H&E, and 5 cases (8%) were called benign on MPM but showed neoplastic features on final FFPE pathologic examination. Overall, MPM showed a sensitivity of 90%, a specificity of 77%, a positive predictive value of 94%, and a negative predictive value of 67% as shown in Figure 1. The architecture of the lesion (flat or papillary) was correctly defined using MPM in 86% of cases. Determination of cytologic grade was accurate using MPM in 38 of 56 of cases (68%). Nine cases had to be excluded from the grading by MPM because the data sets had certain intrinsic limitations; specifically, of the 9 cases, 6 cases (67%) lacked high magnification images, 2 cases (22%) had specimens with largely denuded epithelium, and 1 case (11%) had a degree of cautery that allowed architectural analysis but not grading.

As discussed in the "Materials and Methods" (in the section titled "MPM Image Evaluation"), the training phase of the pathologists was quite brief. Even with this brief training, both pathologists agreed in their classification of lesions as benign or neoplastic in all 65 cases (100% concordance). They also agreed on the architecture of the lesion (flat versus papillary) in all cases (100%). In only 3 of 56 cases (5%) was there discordance in the grading of lesions (benign/low grade versus high grade), and all 3 of these cases (100%) were noninvasive papillary urothelial carcinomas.

COMMENT

Multiphoton microscopy image sets examined using ITE signals alone from fresh tissue can provide high magnification, high-resolution images of fresh, human bladder biopsies, allowing a high degree of diagnostic accuracy. In our study, we could identify benign bladder mucosa and submucosa by the flat architecture, bland urothelium (including superficial umbrella cells), and underlying lamina propria (including blood vessels and von Brunn nests). Furthermore, benign mucosa was distinguishable from flat CIS using cytologic detail apparent at high magnification. In fact, many of the same criteria used in light microscopy of H&E sections to diagnose CIS were present and evaluable in MPM (eg, N:C ratio, pleomorphism, polarity). Invasive carcinoma was also identifiable using MPM, although more rarely, where irregular nests of malignant urothelial cells could be seen infiltrating collagen fibers of the lamina propria.

Papillary lesions could be identified by the frondular structures that define these lesions, and further classification into low-grade and high-grade tumors was feasible using cytologic details at high magnification, similar to the differentiation possible between benign flat mucosa and flat CIS.

In addition to carcinomas, a variety of benign lesions were readily identifiable by MPM, which might appear cystoscopically suspicious for carcinoma, including cystitis cystica and florid proliferations of von Brunn nests.

Even with only a brief training phase, the 2 pathologists agreed on the classification of specimens as either benign or neoplastic in all cases (100%). Similarly, architectural classification of lesions was identical between the 2 pathologists (100%). A high level of concordance was seen in the interobserver grading of lesions, such that discrepancy in grading occurred in only 3 of the 56 cases (5%) of noninvasive papillary urothelial carcinoma. The high level of concordance with relatively minimal training is likely due, at least partially, to the strong similarities between MPM images and standard histopathologic examination of FFPE tissue.

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Current methods used in pathology to offer immediate impressions and evaluate specimen adequacy, such as frozen section analysis and Diff-Quick (Cardinal Health, Somerset, New Jersey) examination of fine needle aspirations, introduce irreversible freezing (19) and air-drying artifacts, respectively, often making a definitive final diagnosis on that specimen nearly impossible. For this reason, biopsy specimens are not routinely subjected to frozen section analysis because these small specimens may contain the suspicious lesion in its entirety. Multiphoton microscopy, on the other hand, introduces no artifacts during processing, and subsequent FFPE H&E sections of MPM-imaged tissue are indistinguishable from non-MPM-imaged tissue. Thus, MPM offers the ability to assess biopsy adequacy, ex vivo currently--and in vivo in the near future--without altering the tissue for permanent section diagnosis and ancillary analyses (eg, immunohistochemistry).

We expect the initial translation of MPM into the cystoscopy workflow to consist of fast, nondestructive, ex vivo evaluation of excised biopsies to achieve, among other things, the evaluation of biopsy adequacy in cases of suspected CIS, the confirmation of the presence of a papillary neoplasm, and potentially, immediate diagnostic impressions to alleviate patient anxiety in awaiting results and to expedite the scheduling of resection or other intravesical procedures in cases of carcinoma. The ability of MPM to provide real-time diagnoses of benign entities, such as cystitis cystica and proliferation of von Brunn nests, could direct urologists in further evaluation and sampling of the patient with urine cytology suspicious or diagnostic of carcinoma.

[FIGURE 5 OMITTED]

In this study, we used a laboratory-based MPM system built for research applications. Using this system, the imaging time for a 1-[mm.sup.3] biopsy specimen, covering the entire specimen surface and imaging up to approximately 0.5 mm below the surface at low magnification (X4 objective) and approximately 0.25 mm below the surface at higher magnification (X20 objective), took approximately 20 minutes. Although that time is already comparable with typical turnaround time for frozen sections, there are significant technical developments occurring in the field that are expected to bring that time down by an order of magnitude or more in the near future. (20-24) Also, custom designed MPM systems targeting a single type of clinical application will be more compact, cost-efficient, easy to operate and interpret, and usable as an in vivo tool, thus potentially preventing unnecessary benign biopsies. Such systems are already in existence and are being tested for use in the lung, among other organs. These improvements and customizations to MPM could include, for example, replacing the tunable laser with a portable, single wavelength fiber laser; incorporating automated sample handling and automated analysis; maximizing microscopic detail while minimizing imaging time (eg, by imaging the excised biopsy simultaneously from top and bottom); and providing a visual rendition of the images in a format most useful to the specific group of end users.

The use of MPM does have some limitations in its current state. For instance, MPM is able to image with meaningful contrast only up to 0.5 mm below the surface of human bladder tissue, which limits evaluation of lamina propria invasion. Muscularis propria evaluation is also limited for the same reason. Fortunately, many technologic developments are on the horizon, which are expected to improve the penetration depth of MPM imaging (eg, dispersion compensation for maintaining the shortest-possible laser pulse width at the specimen, adaptive optics to correct for aberrations, more efficient photodetectors, and new optics with better transmission profiles in the relevant wavelength regimes).

Another major limitation of MPM is that nuclei do not exhibit any ITE signals (autofluorescence or SHG) in the wavelength ranges used with the current imaging technique. Rather, they are seen as dark areas, devoid of any signal. These dark areas, surrounded by autofluorescent (color-coded green) cytoplasm, still allow one to define the nuclear contour and, thereby, the size of the nucleus and the N:C ratio. However, intranuclear details, such as the presence and size of nucleoli or the chromatin pattern, are indiscernible in MPM using ITE alone. This lack of intranuclear detail could pose significant problems, for instance, if the difference between dysplasia and CIS was necessary during cystoscopic examination. At least for ex vivo biopsy analysis, however, using exogenous nuclear stains, such as acridine orange, could largely circumvent this problem. In preliminary results using animal tissue (data not shown), we have obtained excellent staining of nuclei by incubation of several millimeter-thick tissues with acridine orange for 15 seconds at room temperature, followed by 2 to 3 buffered-saline washes. Acridine orange fluoresces at the same excitation wavelength as used in the current study (780 nm), and thus, can be easily combined with autofluorescence and SHG imaging. Such a staining protocol for biopsy analysis, if necessary, seems possible without a significant increase in time requirements.

Finally, current systems are prohibitively expensive for practical clinical application. Nevertheless, advances in the technology and design of these systems, as has occurred with almost all new and emerging technologies, are expected to decrease the costs drastically. We feel that validation of adjunctive diagnostic and screening tools, such as MPM, should be tested in parallel to potential development of clinical utility, so that access to these systems is not delayed and patients can benefit from potentially improved survival as well as quality of life.

Multiphoton imaging joins an array of other optical imaging techniques that are being assessed for real-time diagnosis and intrasurgical assistance. Specifically, 2 types of techniques are being assessed. The first category includes low-magnification, large field of view, primary detection or "red-flag" techniques, where the goal is to scan an entire organ, such as the bladder, for potentially suspicious lesions. (25,26) Techniques in this category include (1) photodynamic detection or fluorescence cystoscopy, where an exogenous contrast agent is instilled in the bladder to improve identification of neoplastic tissue over surrounding normal or benign mucosa; and (2) narrow band imaging, where the mucosa is illuminated with narrow bands of blue and green light by optical filters. These wavelengths are preferentially absorbed by hemoglobin, generating better visualization of neoplastic vasculature and other mucosal pattern changes. These techniques typically have high sensitivity but low specificity, primarily because similar signals are generated by benign inflammatory lesions in addition to neoplasia.

A second class of optical techniques are the so-called targeted imaging or optical biopsy techniques. (25,26) Here, the field of view is typically smaller, but the images are of higher magnification and resolution, approaching those used for traditional, postprocedural histopathology on excised tissues. These techniques are expected to work in conjunction with one or more red-flag techniques, which will direct the specific areas sampled. Techniques in this category include Raman spectroscopy, optical coherence tomography (OCT), and confocal microendoscopy. (25,26) Each of these techniques has unique advantages and limitations. Raman spectroscopy, as assessed clinically, is a nonimaging technique in which average Raman spectra are acquired from a relatively large area (approximately 1 [mm.sup.3]). However, this technique is able to assess specific chemical changes in the tissue and thereby diagnose neoplastic status. Raman spectroscopy has been evaluated in clinical trials for breast lumpectomy margin and atherosclerotic plaque assessments. (27,28) To our knowledge, it has not yet been studied in the context of bladder cancer assessments. Optical coherence tomography is a relatively mature technique (29) that has been assessed in vivo intransurethral procedures and has been found to improve the detection of early/flat urothelial lesions, especially when combined with fluorescence cystoscopy to identify areas to target. (30) Confocal microendoscopy has been used in the upper gastrointestinal tract to visualize Barrett esophagus, (31) and it has also been assessed in transurethral procedures, where it was able to distinguish between benign urothelium and low-grade and high-grade tumors. (32)

Multiphoton imaging has some advantages over both OCT and confocal microendoscopy. The latter technique requires instillation of a contrast agent (fluorescein) for imaging, whereas MPM can be contrast-free. Furthermore, because visible (blue) light is used for tissue illumination in confocal microendoscopy, depth of imaging is 2-fold to 3-fold shallower than it is with MPM, which uses more penetrating near-infrared illumination. Optical coherence tomography, on the other hand, can image 2-fold to 5-fold deeper into tissue than can MPM, but typically has a 5-fold to 20-fold lower lateral resolution. However, laboratory-based OCT systems are now available that approach the resolutions of MPM. Furthermore, traditional OCT is carried out in B-mode, similar to ultrasound imaging, generating XZ images that are somewhat less intuitive for intraprocedural interpretation. This problem can be easily overcome, however, by automated image analysis and by adequate training of the endoscopist.

Given the limitations and complementary nature of these techniques, the ultimate clinical tool will likely be multimodal in nature, combining one or more red-flag and/or optical biopsy systems. As a stand-alone tool, MPM currently offers the ability to make real-time diagnoses on excised tissue specimens without any processing of the tissue or introduction of permanent artifacts, such as those seen in tissue submitted for frozen section diagnosis. Although this study focused on the urinary bladder, our experience suggests that an immediate impression/diagnosis can be given on tissue from a variety of organs, including the kidney, lung, and colon, among others. One use of the technology as is now stands, therefore, would be as an adjunct or alternative to frozen section analysis for immediate diagnoses. Another potential application would be the assessment of endoscopic biopsies for specimen adequacy for future FFPE diagnoses. Future developments will likely lead to introduction of MPM as an endoscopic technique, allowing optical biopsy of tissue in vivo. Several such attempts at miniaturization of MPM into endoscopic formats are currently in progress. (33-36) The MPM images generated may be streamed over a secure server to a consulting pathologist for assessment. Such an endoscopic "biopsy" tool would prove invaluable, for instance, in lesions of the upper urinary tract, where the capability of biopsying a suspicious focus is often limited by the ability to introduce and navigate the necessary instruments through the ureter and into the renal pelvis. An optical biopsy tool would also likely minimize the removal of unnecessary biopsies (eg, for inflammatory lesions) and allow mapping of carcinomatous areas to ensure complete resection, thereby streamlining and improving the throughput of surgical pathology work flow and, ultimately, optimizing patient care.

In conclusion, we show that MPM image sets alone can readily distinguish between normal bladder mucosa, benign inflammatory/reactive lesions, CIS, papillary urothelial carcinomas, and to a lesser degree, invasive urothelial carcinomas. The ability of MPM to diagnose early stage bladder carcinoma and, more specifically, CIS, is important, especially because CIS is associated with poor prognosis and is frequently difficult to identify during routine cystoscopy. We expect that a future study with a larger sample size and use of the technical lessons learned from this work (such as imaging larger areas, always acquiring both low- and high-magnification images, avoiding regions of cautery, among other factors), will further improve our grading ability by MPM. We are currently planning a prospective study of the utility of MPM in individuals with abnormal results from urine cytology, where we will investigate the potential effect of immediate MPM diagnosis of a biopsy on the management and subsequent detection of bladder carcinoma. The initial evaluation will involve assessment of ex vivo biopsies in the cystoscopy suite to guide the urologist in assessing specimen adequacy in cases with a diagnosis of urothelial carcinoma on cytology. Meanwhile, new technologic developments, such as the miniaturization of MPM into endoscopic formats, as discussed above, are likely to bring optical biopsies into routine clinical use.

We acknowledge the following National Institutes of Health grants for financial support: National Cancer Institute R01 CA116583 (W.R.Z.), National Institute of Biomedical Imaging and Bioengineering (NIBIB) P41 RR04224 (W.R.Z.), and NIBIB 1 R01 EB006736 (W.W.W.). Significant aspects of imaging acquisition and processing were standardized with support from a K12 career development award to S.M. (1 KL2 RR024997-01) from the Clinical and Translational Science Center of the Weill Cornell Medical College.

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Manu Jain, MD *; Brian D. Robinson, MD *; Douglas S. Scherr, MD; Joshua Sterling, BS; Ming-Ming Lee, BA; James Wysock, MD; Mark A. Rubin, MD; Frederick R. Maxfield, PhD; Warren R. Zipfel, PhD; Watt W. Webb, ScD; Sushmita Mukherjee, PhD

Accepted for publication July 15, 2011.

* These authors contributed equally to this work.

From the Departments of Urology (Drs Jain, Scherr, and Wysock), Pathology and Laboratory Medicine (Drs Robinson and Rubin); and Biochemistry (Mr Sterling and Drs Maxfield and Mukherjee), Weill Cornell Medical College, New York, New York; Albert Einstein College of Medicine of Yeshiva University, Bronx, New York (Ms Lee); the Department of Biomedical Engineering (DrZipfel) and the School of Applied & Engineering Physics, Cornell University, Ithaca, New York (Dr Webb).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Sushmita Mukherjee, PhD, Department of Biochemistry, Weill Cornell Medical College, 1300 York Avenue, Room E 019A, New York, NY 10065 (e-mail: smukherj@med.cornell.edu).
Comparative Features Between Hematoxylin-Eosin (H&E) and Multiphoton
Microscopy (MPM) for Evaluation of Human Bladder Biopsies

 Diagnostic Features
Bladder Identifiable on MPM Limitations
Lesions Both H&E and MPM

Benign, flat 1. Multilayered Lack of
 intra urothelium with intranuclear
 -urothelial superficial details
 lesions umbrella cells
 2. Maintained
 cell polarity
 3. Underlying lamina
 propria with blood
 vessels, von
 Brunn nests

Carcinoma 1. Flat lesion Lack of
 in situ 2. Marked pleomorphism intranuclear detail
 3. High N:C may impair ability
 4. Loss of polarity to distinguish
 CIS from urothelial
 dysplasia or
 reactive urothelium

Low-grade 1. Papillary fronds Difficult to
 papillary with thin, differentiate
 urothelial fibrovascular cores between papilloma
 carcinoma 2. Overall orderly or malignant
 arrangement of potential and
 urothelial cells low-grade papillary
 with mild urothelial carcinoma
 pleomorphism
 3. Relatively low N:C

High-grade 1. Papillary fronds and Lack of intranuclear
 papillary occasional sheets of detail may impair
 urothelial tumor cells ability to
 carcinoma 2. Moderate to marked distinguish
 nuclear and cellular high-grade lesions
 pleomorphism from low-grade
 3. High N:C lesions
 4. Loss of polarity

Invasive Irregular nests of tumor 1. Invasion reliably
 urothelial cells or individual tumor identified only if
 carcinoma cells within the lamina extensive; micro
 propria (pT1) or the -invasion not
 muscularis propria (pT2) easily identified
 2. Difficult to
 assess presence
 of muscularis
 propria, including
 involvement by
 tumor, because of
 penetration-depth
 limitations in
 current MPM
 imaging
 instrumentation

Abbreviation: CIS, carcinoma in situ; N:C, nuclear to cytoplasmic
ratio.
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Author:Jain, Manu; Robinson, Brian D.; Scherr, Douglas S.; Sterling, Joshua; Lee, Ming-Ming; Wysock, James;
Publication:Archives of Pathology & Laboratory Medicine
Date:May 1, 2012
Words:6780
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