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

The traditional surgical pathology assessment requires tissue to be removed from the patient, then processed, sectioned, stained, and interpreted by a pathologist using a light microscope. Today, an array of alternate optical imaging technologies allow tissue to be viewed at high resolution, in real time, without the need for processing, fixation, freezing, or staining. Optical imaging can be done in living patients without tissue removal, termed in vivo microscopy, or also in freshly excised tissue, termed ex vivo microscopy. Both in vivo and ex vivo microscopy have tremendous potential for clinical impact in a wide variety of applications. However, in order for these technologies to enter mainstream clinical care, an expert will be required to assess and interpret the imaging data. The optical images generated from these imaging techniques are often similar to the light microscopic images that pathologists already have expertise in interpreting. Other clinical specialists do not have this same expertise in microscopy, therefore, pathologists are a logical choice to step into the developing role of microscopic imaging expert. Here, we review the emerging technologies of in vivo and ex vivo microscopy in terms of the technical aspects and potential clinical applications. We also discuss why pathologists are essential to the successful clinical adoption of such technologies and the educational resources available to help them step into this emerging role.

In Vivo and Ex Vivo Microscopy: A Business Plan to Justify the Introduction of Similar Emerging Technologies Into Pathology Practice.

CONTEXT.­: Our patients are now demanding value for their medical diagnoses and treatment in terms of optimal costs, quality, and outcomes. The financial justification for the introduction of new emerging technologies that may better meet these needs will depend on many factors, even if there is an established reimbursement code. In vivo and ex vivo microscopic technologies (IVM and EVM, respectively) will be used as examples of potentially transforming technologies. OBJECTIVE.­: To describe the components of a business plan that ensures all of the ramifications of introducing a new technology into pathology practice have been considered. As well as the financial justification, such a plan should include strategic vision and congruence, the advantages and drawbacks of introducing such technology, and how plans for marketing, implementation, and verification can be operationalized. DATA SOURCES.­: Unlike many pathologists, administrative directors in clinical laboratories already know the components of a financially sound business plan. In addition to the financial justifications, other considerations of such a plan include expense reductions, multiyear buildups in revenue generation, the replacement of other technologies, improved productivity and workflows, additional space, new capital, retrained personnel, and the impact on other departments. CONCLUSIONS.­: Pathologists will learn a business plan format to improve their confidence in making the sound financial justifications needed to consider the introduction of an emerging technology into pathology practice, even when there is initially no obvious revenue stream because formal reimbursement codes have not been established.

Amelioration of Diabetic Nephropathy Using a Retinoic Acid Receptor ß2 Agonist.

Vitamin A (VA) and its derivatives, known as retinoids, play critical roles in renal development through retinoic acid receptor ß2 (RARß2). Disruptions in VA signaling pathways are associated with the onset of diabetic nephropathy (DN). Despite the known role of RARß2 in renal development, the effects of selective agonists for RARß2 in a high-fat diet (HFD) model of DN are unknown. Here we examined whether AC261066 (AC261), a highly selective agonist for RARß2, exhibited therapeutic effects in a HFD model of DN in C57BL/6 mice. Twelve weeks of AC261 administration to HFD-fed mice was well tolerated with no observable side effects. Compared with HFD-fed mice, HFD + AC261-treated mice had improved glycemic control and reductions in proteinuria and urine albumin-to-creatinine ratio. Several cellular hallmarks of DN were mitigated in HFD + AC261-treated mice, including reductions in tubule lipid droplets, podocyte (POD) effacement, endothelial cell collapse, mesangial expansion, and glomerular basement membrane thickening. Mesangial and tubule interstitial expression of the myofibroblast markers α-smooth muscle actin (α-SMA) and type IV collagen (Col-IV) was lower in HFD + AC261-treated mice compared with HFD alone. Ultrastructural and immunohistochemistry analyses showed that, compared with HFD-fed mice, HFD + AC261-treated mice showed preservation of POD foot process and slit-diaphragm morphology, an increase in the levels of slit-diagram protein podocin, and the transcription factor Wilms tumor-suppressor gene 1 in PODs. Given the need for novel DN therapies, our results warrant further studies of the therapeutic properties of AC261 in DN.

Role of NADH Dehydrogenase (Ubiquinone) 1 Alpha Subcomplex 4-Like 2 in Clear Cell Renal Cell Carcinoma.

PURPOSE: We delineated the functions of the hypoxia-inducible factor-1α (HIF1α) target NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) in clear cell renal cell carcinoma (ccRCC) and characterized NDUFA4L2 as a novel molecular target for ccRCC treatment. EXPERIMENTAL DESIGN: We evaluated normal kidney and ccRCC patient microarray and RNAseq data from Oncomine and The Cancer Genome Atlas for NDUFA4L2 mRNA levels and the clinical implications of high NDUFA4L2 expression. In addition, we examined normal kidney and ccRCC patient tissue samples, human ccRCC cell lines, and murine models of ccRCC for NDUFA4L2 mRNA and protein expression. Utilizing short hairpin RNA, we performed NDUFA4L2 knockdown experiments and analyzed the proliferation, clonogenicity, metabolite levels, cell structure, and autophagy in ccRCC cell lines in culture. RESULTS: We found that NDUFA4L2 mRNA and protein are highly expressed in ccRCC samples but undetectable in normal kidney tissue samples, and that NDUFA4L2 mRNA expression correlates with tumor stage and lower overall survival. In addition, we demonstrated that NDUFA4L2 is an HIF1α target in ccRCC and that NDUFA4L2 knockdown has a profound antiproliferative effect, alters metabolic pathways, and causes major stress in cultured RCC cells. CONCLUSIONS: Collectively, our data show that NDUFA4L2 is a novel molecular target for ccRCC treatment. Clin Cancer Res; 22(11); 2791-801. ©2016 AACR.

Multiphoton microscopy for rapid histopathological evaluation of kidney tumours.

OBJECTIVE: To explore the potential of multiphoton microscopy (MPM) for rapid evaluation and triaging of ex vivo kidney tissue. MATERIALS AND METHODS: Fresh neoplastic and non-neoplastic tissues from nephrectomy specimens (n = 40) were imaged with MPM and later submitted for routine histopathology. RESULTS: On MPM, normal kidney architecture was evident and clearly distinguishable from tumour. Forty malignant tumours (20 clear-cell renal cell carcinomas [RCCs], 10 papillary RCCs, five chromophobe RCCs and five papillary urothelial carcinomas [UCs], as diagnosed by haematoxylin and eosin staining) were imaged and subtyped as non-papillary and papillary, based on their architecture. Non-papillary tumours were further classified based on their unique cytoplasmic signatures. Clear-cell RCCs had a predominant population of cells with fat droplets in cytoplasm. Chromophobe RCCs had cells with non-fatty/homogeneous cytoplasm and distinct intra-cytoplasmic granules. Papillary RCCs had single-cell-lined papillae with often abundant histiocytes in their core, whereas PUC had multi-layered urothelium-lined papillae. The diagnostic accuracy of tumour subtyping by two independent uropathologists was 95%. CONCLUSIONS: We showed that MPM can reliably differentiate neoplastic from non-neoplastic kidney tissue and subtype kidney tumours in fresh, unprocessed tissue, MPM might therefore be useful as a rapid real-time diagnostic tool for the evaluation of kidney biopsies, and surgical margins in partial nephrectomies, to improve overall patient management.

Multiphoton microscopy: a potential intraoperative tool for the detection of carcinoma in situ in human bladder.

CONTEXT: Urothelial carcinoma in situ (CIS) is a precursor of invasive bladder cancer, which if left untreated, will likely progress to more aggressive disease. Approximately 50% of CIS lesions are missed on routine cystoscopy owing to their flat architecture. Furthermore, many benign but abnormal-appearing areas may be biopsied owing to lack of cellular resolution of cystoscopes. Multiphoton microscopy (MPM) is an optical imaging technique that generates subcellular-resolution three-dimensional images from unfixed tissue without using exogenous dyes. OBJECTIVE: To assess the diagnostic potential of MPM in identifying and differentiating benign from malignant flat bladder lesions, especially CIS. DESIGN: Seventy-eight specimens (benign = 46, CIS = 23, invasive = 9, as diagnosed on histopathology) were obtained from flat bladder mucosa via transurethral resection of bladder, cold cup biopsy, or cystectomy, imaged fresh with a commercial benchtop MPM, and submitted for routine histopathology. Multiphoton microscopy and hematoxylin-eosin diagnoses were compared. RESULTS: In 77 of 78 specimens (99%), accurate MPM diagnoses (benign/malignant) were given on the basis of their architectural and cytologic features (nuclear to cytoplasmic ratio, pleomorphism, polarity/organization of urothelial layers, etc). The sensitivity and specificity were 97% and 100%, respectively, with positive (malignant) and negative (benign) predictive values of 100% and 98%, respectively. The interobserver agreement, κ, was 0.93. CONCLUSIONS: Our study demonstrates the capability of MPM to identify and differentiate benign from malignant flat bladder lesions, especially CIS. With the advent of MPM endoscopes, we foresee their potential as a biopsy guidance tool for early detection and treatment of CIS, thus reducing the rate of biopsies with benign diagnoses and their associated complications.

Multiphoton gradient index endoscopy for evaluation of diseased human prostatic tissue ex vivo.

Multiphoton microscopy can instantly visualize cellular details in unstained tissues. Multiphoton probes with clinical potential have been developed. This study evaluates the suitability of multiphoton gradient index (GRIN) endoscopy as a diagnostic tool for prostatic tissue. A portable and compact multiphoton endoscope based on a 1-mm diameter, 8-cm length GRIN lens system probe was used. Fresh ex vivo samples were obtained from 14 radical prostatectomy patients and benign and malignant areas were imaged and correlated with subsequent H&E sections. Multiphoton GRIN endoscopy images of unfixed and unprocessed prostate tissue at a subcellular resolution are presented. We note several differences and identifying features of benign versus low-grade versus high-grade tumors and are able to identify periprostatic tissues such as adipocytes, periprostatic nerves, and blood vessels. Multiphoton GRIN endoscopy can be used to identify both benign and malignant lesions in ex vivo human prostate tissue and may be a valuable diagnostic tool for real-time visualization of suspicious areas of the prostate.

Prostate cancer with Paneth cell-like neuroendocrine differentiation has recognizable histomorphology and harbors AURKA gene amplification.

Aurora kinase A (AURKA) gene amplification has been documented in 67% of hormone-naive prostate cancer cases that progress to a highly aggressive variant of castrate-resistant disease, clinically referred to as "neuroendocrine" prostate cancer, "small cell" prostate carcinoma, or "anaplastic" prostate cancer. Therefore, AURKA amplification is a potential prognostic biomarker that may help to identify patients with prostate cancer who are at high risk for developing castrate-resistant disease with clinical features of small cell carcinoma. Furthermore, AURKA inhibitors are currently being tested in clinical trials. In a previous study, we found AURKA amplification in 6 cases of prostate cancer with Paneth cell-like cells. This morphologic pattern has been suggested to represent low-grade neuroendocrine differentiation (NED) with generally favorable prognosis. We sought to investigate the frequency of AURKA amplification and the histologic characteristics of prostate cancer with Paneth cell-like NED. Twenty-five cases from 172 prostatectomies were evaluated for the presence of 18 morphologic features and AURKA amplification. Most prostate cancers with Paneth cell-like NED had macronucleoli (92%), basophilic appearance (88%), perineural invasion (72%), and nuclear stratification (76%). The frequency of AURKA amplification was 45%, present throughout the examined tumor nodule including areas without Paneth cell-like cells. When histologically similar cases with and without AURKA amplification were compared, this gene alteration was associated with larger extent of Paneth cell-like NED identified at magnification ×20 (P = .015), higher percentage of Paneth cell-like NED throughout the tumor nodule (P = .033), ductal features (P = .02), and higher overall Gleason grade (P = .039). AURKA amplification was not associated with age, serum prostate specific antigen, or tumor stage. The high frequency of AURKA amplification (45%) in localized prostate cancer with Paneth cell-like NED and its potential prognostic significance warrant further investigation.

Multiphoton microscopy: a potential "optical biopsy" tool for real-time evaluation of lung tumors without the need for exogenous contrast agents.

CONTEXT: Multiphoton microscopy (MPM) is an emerging, nonlinear, optical-biopsy technique, which can generate subcellular-resolution images from unprocessed and unstained tissue in real time. OBJECTIVE: To assess the potential of MPM for lung tumor diagnosis. DESIGN: Fresh sections from tumor and adjacent nonneoplastic lung were imaged with MPM and then compared with corresponding hematoxylin-eosin slides. RESULTS: Alveoli, bronchi, blood vessels, pleura, smokers' macrophages, and lymphocytes were readily identified with MPM in nonneoplastic tissue. Atypical adenomatous hyperplasia (a preinvasive lesion) was identified in tissue adjacent to the tumor in one case. Of the 25 tumor specimens used for blinded pathologic diagnosis, 23 were diagnosable with MPM. Of these 23 cases, all but one adenocarcinoma (15 of 16; 94%) was correctly diagnosed on MPM, along with their histologic patterns. For squamous cell carcinoma, 4 of 7 specimens (57%) were correctly diagnosed. For the remaining 3 squamous cell carcinoma specimens, the solid pattern was correctly diagnosed in 2 additional cases (29%), but it was not possible to distinguish the squamous cell carcinoma from adenocarcinoma. The other squamous cell carcinoma specimen (1 of 7; 14%) was misdiagnosed as adenocarcinoma because of pseudogland formation. Invasive adenocarcinomas with acinar and solid pattern showed statistically significant increases in collagen. Interobserver agreement for collagen quantification (among 3 observers) was 80%. CONCLUSIONS: Our pilot study provides a proof of principle that MPM can differentiate neoplastic from nonneoplastic lung tissue and identify tumor subtypes. If confirmed in a future, larger study, we foresee real-time intraoperative applications of MPM, using miniaturized instruments for directing lung biopsies, assessing their adequacy for subsequent histopathologic analysis or banking, and evaluating surgical margins in limited lung resections.

Full-field optical coherence tomography for the analysis of fresh unstained human lobectomy specimens.

BACKGROUND: Full-field optical coherence tomography (FFOCT) is a real-time imaging technique that generates high-resolution three-dimensional tomographic images from unprocessed and unstained tissues. Lack of tissue processing and associated artifacts, along with the ability to generate large-field images quickly, warrants its exploration as an alternative diagnostic tool. MATERIALS AND METHODS: One section each from the tumor and from adjacent non-neoplastic tissue was collected from 13 human lobectomy specimens. They were imaged fresh with FFOCT and then submitted for routine histopathology. Two blinded pathologists independently rendered diagnoses based on FFOCT images. RESULTS: Normal lung architecture (alveoli, bronchi, pleura and blood vessels) was readily identified with FFOCT. Using FFOCT images alone, the study pathologists were able to correctly identify all tumor specimens and in many cases, the histological subtype of tumor (e.g., adenocarcinomas with various patterns). However, benign diagnosis was provided with high confidence in roughly half the tumor-free specimens (others were diagnosed as equivocal or false positive). Further analysis of these images revealed two major confounding features: (a) Extensive lung collapse and (b) presence of smoker's macrophages. On a closer inspection, however, the smoker's macrophages could often be identified as distinct from tumor cells based on their relative location in the alveoli, size and presence of anthracosis. We posit that greater pathologist experience, complemented with morphometric analysis and color-coding of image components, may help minimize the contribution of these confounders in the future. CONCLUSION: Our study provides evidence for the potential utility of FFOCT in identifying and differentiating lung tumors from non-neoplastic lung tissue. We foresee its potential as an adjunct to intra-surgical frozen section analysis for margin assessment, especially in limited lung resections.

Activation of HIF2α in kidney proximal tubule cells causes abnormal glycogen deposition but not tumorigenesis.

Renal cell carcinoma (RCC) is the most common primary cancer arising from the kidney in adults, with clear cell renal cell carcinoma (ccRCC) representing approximately 75% of all RCCs. Increased expression of the hypoxia-induced factors-1α (HIF1α) and HIF2α has been suggested as a pivotal step in ccRCC carcinogenesis, but this has not been thoroughly tested. Here, we report that expression of a constitutively activated form of HIF2α (P405A, P530A, and N851A, named as HIF2αM3) in the proximal tubules of mice is not sufficient to promote ccRCC by itself, nor does it enhance HIF1αM3 oncogenesis when coexpressed with constitutively active HIF1αM3. Neoplastic transformation in kidneys was not detected at up to 33 months of age, nor was increased expression of Ki67 (MKI67), γH2AX (H2AFX), or CD70 observed. Furthermore, the genome-wide transcriptome of the transgenic kidneys does not resemble human ccRCC. We conclude that a constitutively active HIF2α is not sufficient to cause neoplastic transformation of proximal tubules, arguing against the idea that HIF2α activation is critical for ccRCC tumorigenesis.

Functional outcomes following robotic prostatectomy using athermal, traction free risk-stratified grades of nerve sparing.

OBJECTIVE: To report our unique approach for individualizing robotic prostate cancer surgery by risk stratification and sub classification of the periprostatic space into 4 distinct compartments, and thus performing 4 precise different grades of nerve sparing based on neurosurgical principles and to present updated potency and continence outcomes data of patients undergoing robotic-assisted laparoscopic prostatectomy (RALP) using our risk-stratified approach based on layers of periprostatic fascial dissection. PATIENTS AND METHODS: (1) Between January 2005 and December 2010, 2,536 men underwent RALP by a single surgeon at our institution. (2) Included patients were those with ≥ 1-year follow-up and were preoperatively continent and potent, defined as having a SHIM questionnaire score of >21; thus, the final number of patient in the study cohort was 1,335. (3) Postoperative potency was defined as the ability to have successful intercourse (score of ≥ 4 on question 2 of the SHIM); continence was defined as the use of no pads per 24 h. RESULTS: (1) The potency and continence for NS grades 1, 2, 3, and 4 were found to be 90.6, 76.2, 60.5, and 57.1 % (P < 0.001) and 98, 93.2, 90.1, and 88.9 % (P < 0.001), respectively. (2) The overall PSM rates for patients with NS grades 1, 2, 3, and 4 were 10.5, 7, 5.8, and 4.8 %, respectively (P = 0.064). CONCLUSIONS: The study found a correlation between risk-stratified grades of NS technique and continence and potency. Patients with lesser grades of NS had higher rates of potency and continence.

Effect of a risk-stratified grade of nerve-sparing technique on early return of continence after robot-assisted laparoscopic radical prostatectomy.

BACKGROUND: The impact of nerve sparing (NS) on urinary continence recovery after robot-assisted laparoscopic radical prostatectomy (RALP) has yet to be defined. OBJECTIVE: To evaluate the effect of a risk-stratified grade of NS technique on early return of urinary continence. DESIGN, SETTING, AND PARTICIPANTS: Data were collected from 1546 patients who underwent RALP by a single surgeon at a tertiary care center from December 2008 to October 2011. Patients were categorized preoperatively by a risk-stratified approach into risk grades 1-4, with risk grade 1 patients more likely to receive NS grade 1 or complete hammock preservation. This categorization was also conducted for risk grades 2-4, with grade 4 patients receiving a non-NS procedure. INTERVENTION: Risk-stratified grading of NS RALP. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Univariate and multivariate analysis identified predictors of early return of urinary continence, defined as no pad use at ≤ 12 wk postoperatively. RESULTS AND LIMITATIONS: Early return of continence was achieved by 791 of 1417 men (55.8%); of those, 199 of 277 (71.8%) were in NS grade 1, 440 of 805 (54.7%) were in NS grade 2, 132 of 289 (45.7%) were in NS grade 3, and 20 of 46 (43.5%) were in NS grade 4 (p<0.001). On multivariate analysis, better NS grade was a significant independent predictor of early return of urinary continence when NS grade 1 was the reference variable compared with NS grade 2 (p<0.001; odds ratio [OR]: 0.46), NS grade 3 (p<0.001; OR: 0.35), and NS grade 4 (p=0.001; OR: 0.29). Lower preoperative International Prostate Symptom Score (p=0.001; OR: 0.97) and higher preoperative Sexual Health Inventory for Men score (p=0.002; OR: 1.03) were indicative of early return of urinary continence. Positive surgical margin rates were 7.2% (20 of 277) of grade 1 cases, 7.6% (61 of 805) of grade 2 cases, 7.6% (22 of 289) of grade 3 cases, and 17.4% (8 of 46) of grade 4 cases (p=0.111). Extraprostatic extension occurred in 6.1% (17 of 277) of NS grade 1 cases, 17.5% (141 of 805) of NS grade 2 cases, 42.5% (123 of 289) of NS grade 3 cases, and 63% (29 of 46) of NS grade 4 cases (p<0.001). Some limitations of the study are that the study was not randomized, grading of NS was subjective, and possible selection bias existed. CONCLUSIONS: Our study reports a correlation between risk-stratified grade of NS technique and early return of urinary continence as patients with a lower grade (higher degree) of NS achieved an early return of urinary continence without compromising oncologic safety.

Multiphoton tomographic imaging: a potential optical biopsy tool for detecting gastrointestinal inflammation and neoplasia.

Endoscopy is widely used to detect and remove premalignant lesions with the goal of preventing gastrointestinal (GI) cancers. Because current endoscopes do not provide cellular resolution, all suspicious lesions are biopsied and subjected to histologic evaluation. Technologies that facilitate directed biopsies should decrease both procedure-related morbidity and cost. Here we explore the use of multiphoton microscopy (MPM), an optical biopsy tool that relies on intrinsic tissue emissions, to evaluate pathology in both experimental and human GI specimens, using hematoxylin and eosin (H&E)-stained sections from these tissues for comparison. After evaluating the entire normal mouse GI tract, MPM was used to investigate disease progression in mouse models of colitis and colorectal carcinogenesis. MPM provided sufficient histologic detail to identify all relevant substructures in ex vivo normal GI tissue, visualize both acute and resolving stages of colitis, and show the progression of colorectal carcinogenesis. Next, ex vivo specimens from human subjects with celiac sprue, inflammatory bowel disease, and colorectal neoplasia were imaged by MPM. Finally, colonic mucosa in live anesthetized rats was imaged in vivo using a flexible endoscope prototype. In both animal models and human specimens, MPM images showed a striking similarity to the results of H&E staining, as shown by the 100% concordance achieved by the study pathologists' diagnoses. In summary, MPM is a promising technique that accurately visualizes histology in fresh, unstained tissues. Our findings support the continued development of MPM as a technology to enhance the early detection of GI pathologies including premalignant lesions.

Pilot study of the correlation of multiphoton tomography of ex vivo human testis with histology.

PURPOSE: Although microdissection testicular sperm extraction has become first line therapy for sperm retrieval in men with nonobstructive azoospermia, there are challenges to the procedure, including difficulty differentiating between seminiferous tubules with normal and abnormal spermatogenesis. Multiphoton microscopy illuminates tissue with a near infrared laser to elicit autofluorescence, which enables real-time imaging of unprocessed tissue without labels. We hypothesized that we could accurately characterize seminiferous tubular histology in humans using multiphoton microscopy. MATERIALS AND METHODS: Seven men with normal or abnormal spermatogenesis underwent testicular biopsies, which were imaged by multiphoton microscopy. We assessed these images in blinded fashion. The diagnosis rendered with multiphoton microscopy was then correlated with that of hematoxylin and eosin stained tissue. We evaluated the ability of multiphoton microscopy to differentiate normal from abnormal seminiferous tubules by examining autofluorescence characteristics and diameters, as imaged by multiphoton microscopy. Assessment was repeated with stained slides and results were compared. RESULTS: The overall concordance rate between multiphoton microscopy and stained slides was 86%. The seminiferous tubules of patients with nonobstructive azoospermia were smaller than those of controls when measured by multiphoton microscopy and staining (p <0.05). The proportion of normal tubules and the diameters obtained with multiphoton microscopy were not different from those obtained with hematoxylin and eosin (p >0.05). CONCLUSION: Multiphoton microscopy can be used to differentiate normal from abnormal spermatogenesis. Its characterization of seminiferous tubular architecture is similar to that provided by hematoxylin and eosin staining. Further investigation of the clinical applications of multiphoton microscopy may improve surgical sperm retrieval outcomes for patients with nonobstructive azoospermia.

Next-generation prostate cancer biobanking: toward a processing protocol amenable for the International Cancer Genome Consortium.

Next-generation DNA and RNA sequencing requires intact nucleic acids from high-quality human tissue samples to better elucidate the molecular basis of cancer. We have developed a prostate biobanking protocol to acquire suitable samples for sequencing without compromising the accuracy of clinical diagnosis. To assess the clinical implications of implementing this protocol, we evaluated 105 consecutive radical prostatectomy specimens from November 2008 to February 2009. Alternating levels of prostate samples were submitted to Surgical Pathology as formalin-fixed, paraffin-embedded blocks and to the institutional biobank as frozen blocks. Differences in reported pathologic characteristics between clinical and procured specimens were compared. Clinical staging and grading were not affected by the biobank protocol. Tumor foci on frozen hematoxylin and eosin slides were identified and high-density tumor foci were scored and processed for DNA and RNA extractions for sequencing. Both DNA and RNA were extracted from 22 cases of 44 with high-density tumor foci. Eighty-two percent (18/22) of the samples passed rigorous quality control steps for DNA and RNA sequencing. To date, DNA extracted from 7 cases has undergone whole-genome sequencing, and RNA from 18 cases has been RNA sequenced. This protocol provides prostate tissue for high-throughput biomedical research and confirms the feasibility of actively integrating prostate cancer into The Cancer Genome Atlas Program, a member of the International Cancer Genome Consortium.

Predictive models for worsening prognosis in potential candidates for active surveillance of presumed low-risk prostate cancer.

PURPOSE: Low-risk prostate cancer patients clinically eligible for active surveillance can also be managed surgically. We evaluated the pathologic outcomes for this cohort that was treated by radical prostatectomy and devised nomograms to predict patients at risk of upgrading and/or upstaging. MATERIALS AND METHODS: Seven hundred and fifty patients treated by radical prostatectomy from Jan 2005 to the present fulfilled conventional active surveillance criteria and formed the study cohort. Preoperative data on standard clinicopathologic parameters were available. The radical prostatectomy specimens were graded and staged, and any upgrading to Gleason sum >6 or upstaging to ≥pT3 ('worsening prognosis') were noted. Multivariable logistic regression models were used to develop predictive nomograms. RESULTS: Of the 750 patients, 303 (40.4%) patients were either upgraded or upstaged. Multivariable analysis found that preoperative PSA, number of positive cores, and prostate volume were significantly predictive of worsening prognosis and formed the nomogram criteria. CONCLUSIONS: Of patients deemed eligible for active surveillance based on conventional criteria, 40.4% have worse prognostic factors after radical prostatectomy. Current active surveillance criteria may be too relaxed, and the use of nomograms which we have devised, may aid in counseling primary prostate cancer patients considering active surveillance as their therapy of choice.

Pathological outcomes and strategies to achieve optimal cancer control during robotic radical prostatectomy in Asian-Indian men.

INTRODUCTION AND OBJECTIVES: There is a paucity of information in the literature about the characteristics of prostate cancer in the Asian-Indian population. We wanted to evaluate the oncological outcomes of Asian-Indians and Caucasians. We also derived a nomogram for prediction of extraprostatic extension (EPE) and presented biochemical recurrence (BCR) rates in the Asian-Indian population. MATERIALS AND METHODS: A total of 2367 D'Amico low-risk patients underwent robotic-assisted radical prostatectomy (RARP) for clinically localized prostate cancer between January 2005 and July 2010 by a single surgeon. Of these 56 (2.4%) patients were Asian-Indians and 2025 were Caucasians (85.6%). Univariate and multivariate models were created for predicting EPE. A multivariate logistic regression model was used to develop a predictive nomogram. BCR was defined as a prostate-specific antigen ≥0.2 at any postoperative time point. Kaplan-Meier survival analysis was used to investigate BCR rates. RESULTS: A significantly greater percentage of Asian-Indians compared to Caucasians had EPE (32.3 vs. 16.5; P = 0.01). In multivariate analysis adjusted for significant variables from univariate analyses, Asian-Indian race (P = 0.028), age (P = 0.050), maximum percentage cancer on biopsy (P < 0.001), and pathology prostate weight (P = 0.047) were independent predictors of EPE. Kaplan-Meier analysis demonstrated BCR free rates of 94.6% and 95.4%, for Asian-Indians and Caucasians, respectively, at a median follow-up of 16 months (range 2-70 months). There was no statistically significant difference in BCR rates across the two cohorts (log-rank P-value = 0.405). CONCLUSIONS: This study highlights that while Asian-Indians have more advanced cancer variables, their risk of BCR after surgery is similar to Caucasian patients. Further work is required to better understand the social, genetic and environmental factors that affect the biology of prostate cancer in men of Asian-Indian descent.