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Chemical-based medicine that targets specific oncogenes or proteins often leads to cancer recurrence due to tumor heterogeneity and development of chemoresistance. This challenge can be overcome by mechanochemical disruption of cancer cells via focused ultrasound (FUS) and sensitizing chemical agents such as ethanol. We demonstrate that this disruptive therapy decreases the viability, proliferation rate, tumorigenicity, endothelial adhesion, and migratory ability of prostate cancer cells in vitro. It sensitized the cells to TNFR1-‐ and Fas-‐mediated apoptosis and reduced the expression of metastatic markers CD44 and CD29. Using a prostate cancer xenograft model, we observed that the mechanochemical disruption led to complete tumor regression in vivo. This switch to a nonaggressive cell phenotype was caused by ROS and Hsp70 overproduction and subsequent impairment of NFκB signaling. FUS induces mechanical perturbations of diverse cancer cell populations, and its combination with agents that amplify and guide remedial cellular responses can stop lethal cancer progression.Implications:Mechanochemical disruption therapy in which FUS is combined with ethanol can be curative for locally aggressive and castration-resistant prostate cancer.

Supplementary Methods, Figures 1-18

Background Up to 30% of thyroid nodules undergoing fine needle aspiration (FNA) yield an indeterminate result. Recent research efforts have suggested that nuclear morphometry and morphology may enhance the diagnostic accuracy of FNA as an objective adjunct. We applied nuclear morphometric analysis on a diverse cohort of patients to evaluate the association between nuclear morphometry and malignancy. Methods Forty-five randomly selected patients, who underwent thyroid surgery after an indeterminate FNA result (Bethesda III & IV) between 2012-2015, were reviewed. One hundred representative nuclei per FNA of a thyroid nodule were analyzed using ImageJ. Seven validated morphometric parameters were collected: nuclear area, perimeter, circularity, aspect ratio, roundness, and maximum/minimum Feret's diameter. L/S ratio was subsequently calculated. All 8 nuclear parameters were reported as averages with standard errors of the mean (SEM). A Student's t-test was used to assess the association of nuclear parameters with final surgical pathology. Results The mean age of all patients was 56.31±15.39 years, with female patients comprising 68.9% of the cohort. Twenty-two patients had malignant thyroid nodules. The mean perimeter of nuclei for the cohort was 18.48±0.45 µm, the mean area was 22.19±0.93 µm, and the mean maximum Feret's diameter was 6.67±0.13 µm. No significant differences in the 8 nuclear parameters were observed between the malignant and non-malignant groups. Conclusions In the population examined, our results suggest that nuclear morphometry is not yet a tool of reliable diagnostic value in accessing malignant and non-malignant thyroid nodules. Further investigation is necessary to identify objective parameters that will enhance diagnostic accuracy of indeterminate FNA cytology to minimize the number of diagnostic thyroid surgery.

The presence of mutant BRAF (V600E) correlates with the risk of recurrence in papillary thyroid cancer (PTC) patients. However, not all PTC patients with BRAF (V600E) are associated with poor prognosis. Thus, understanding the mechanisms by which certain PTC patients with nuclear BRAF (V600E) become aggressive and develop resistance to a selective BRAF inhibitor, PLX-4032, is urgently needed. The effect of nuclear localization of BRAF(V600E) using in vitro studies, xenograft mouse-model and human tissues was evaluated. PTC cells harboring a nuclear localization signal (NLS) of BRAF(V600E) were established and examined in nude mice implanted with TPC1-NLS-BRAF(V600E) cells followed by PLX-4032 treatment. Immunohistochemical (IHC) analysis was performed on 100 PTC specimens previously confirmed that they have BRAF(V600E) mutations. Our results demonstrate that 21 of 100 (21%) PTC tissues stained with specific BRAF(V600E) antibody had nuclear staining with more aggressive features compared to their cytosolic counterparts. In vitro studies show that BRAF(V600E) is transported between the nucleus and the cytosol through CRM1 and importin (α/β) system. Sequestration of BRAF(V600E) in the cytosol sensitized resistant cells to PLX-4032, whereas nuclear BRAF(V600E) was associated with aggressive phenotypes and developed drug resistance. Proteomic analysis revealed Arp2/3 complex members, actin-related protein 2 (ACTR2 aliases ARP2) and actin-related protein 3 (ACTR3 aliases ARP3), as the most enriched nuclear BRAF(V600E) partners. ACTR3 was highly correlated to lymph node stage and extrathyroidal extension and was validated with different functional assays. Our findings provide new insights into the clinical utility of the nuclear BRAF(V600E) as a prognostic marker for PTC aggressiveness and determine the efficacy of selective BRAF(V600E) inhibitor treatment which opens new avenues for future treatment options.

Prostate cancer continues to be the most diagnosed non-skin malignancy in men. While up to one in eight men will be diagnosed in their lifetimes, most diagnoses are not fatal. Better lesion location accuracy combined with emerging localized treatment methods are increasingly being utilized as a treatment option to preserve healthy function in eligible patients. In locating lesions which are generally

Current breast tumor margin detection methods are destructive, time-consuming, and result in significant reoperative rates. Dual-modality photoacoustic tomography (PAT) and ultrasound has the potential to enhance breast margin characterization by providing clinically relevant compositional information with high sensitivity and tissue penetration. However, quantitative methods that rigorously compare volumetric PAT and ultrasound images with gold-standard histology are lacking, thus limiting clinical validation and translation. Here, we present a quantitative multimodality workflow that uses inverted Selective Plane Illumination Microscopy (iSPIM) to facilitate image co-registration between volumetric PAT-ultrasound datasets with histology in human invasive ductal carcinoma breast tissue samples. Our ultrasound-PAT system consisted of a tunable Nd:YAG laser coupled with a 40 MHz central frequency ultrasound transducer. A linear stepper motor was used to acquire volumetric PAT and ultrasound breast biopsy datasets using 1100 nm light to identify hemoglobin-rich regions and 1210 nm light to identify lipid-rich regions. Our iSPIM system used 488 nm and 647 nm laser excitation combined with Eosin and DRAQ5, a cell-permeant nucleic acid binding dye, to produce high-resolution volumetric datasets comparable to histology. Image thresholding was applied to PAT and iSPIM images to extract, quantify, and topologically visualize breast biopsy lipid, stroma, hemoglobin, and nuclei distribution. Our lipid-weighted PAT and iSPIM images suggest that low lipid regions strongly correlate with malignant breast tissue. Hemoglobin-weighted PAT images, however, correlated poorly with cancerous regions determined by histology and interpreted by a board-certified pathologist. Nuclei-weighted iSPIM images revealed similar cellular content in cancerous and non-cancerous tissues, suggesting malignant cell migration from the breast ducts to the surrounding tissues. We demonstrate the utility of our nondestructive, volumetric, region-based quantitative method for comprehensive validation of 3D tomographic imaging methods suitable for bedside tumor margin detection.

Chemical-based medicine that targets specific oncogenes or proteins often leads to cancer recurrence due to tumor heterogeneity and development of chemoresistance. This challenge can be overcome by mechanochemical disruption of cancer cells via focused ultrasound (FUS) and sensitizing chemical agents such as ethanol. We demonstrate that this disruptive therapy decreases the viability, proliferation rate, tumorigenicity, endothelial adhesion, and migratory ability of prostate cancer cells in vitro. It sensitized the cells to TNFR1-- and Fas--mediated apoptosis and reduced the expression of metastatic markers CD44 and CD29. Using a prostate cancer xenograft model, we observed that the mechanochemical disruption led to complete tumor regression in vivo. This switch to a nonaggressive cell phenotype was caused by ROS and Hsp70 overproduction and subsequent impairment of NFκB signaling. FUS induces mechanical perturbations of diverse cancer cell populations, and its combination with agents that amplify and guide remedial cellular responses can stop lethal cancer progression. Implications: Mechanochemical disruption therapy in which FUS is combined with ethanol can be curative for locally aggressive and castration-resistant prostate cancer.

Background Approximately half of cutaneous melanoma tissues harbor BRAF V600E mutations, resulting in a constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Nuclear-cytoplasmic transport machinery is dysregulated in neoplastic cells and alters the key regulatory proteins that can lead to tumor progression and drug resistance. The significance of nuclear localization of BRAF V600E has not been fully understood. We examined the clinical significance of intracellular localization of BRAF V600E in cutaneous melanoma. Study Design Immunohistochemical analysis of BRAF V600E was performed on formalin-fixed, paraffin-embedded specimens of cutaneous melanoma (n = 91). Staining intensity was graded in a blinded manner. Correlations to clinical factors were analyzed by Fisher's exact test and 2-tailed t -test. Localization of BRAF V600E was determined in melanoma cells, and we investigated their resistance to BRAF V600E -specific inhibitor according to nuclear localization in both in vitro and in vivo models. Results We included 91 patients, of whom 32% (29 of 91) had cytoplasmic BRAF V600E . Nuclear BRAF V600E was observed in 30% (27 of 91). Overall, BRAF V600E expression correlated with TNM stage (p = 0.011), mitotic activity (p = 0.010), and ulceration (p = 0.045). Nuclear BRAF V600E expression correlated with overall clinical stage (p BRAF V600E was identified in the nucleus, and its translocation was serum dependent. Our in vitro and in vivo data revealed sequestration of BRAF V600E in the cytosol-sensitized resistant cells to vemurafenib; nuclear retention of BRAF V600E was associated with aggressiveness and drug resistance. Conclusions Nuclear localization of BRAF V600E is associated with melanoma aggressiveness. Further multi-institutional studies are warranted to confirm the clinical relevance of nuclear localization of BRAF V600E .

Although overall survival rate for patients with thyroid cancer (TC) is high, there is an alarming 10-year recurrence rate of up to 30% conferring a ~50% survival among these high-risk patients. The BRAFV600E mutation is estimated to be present in over 50% of papillary thyroid cancer (PTC) cases besides being associated with carcinogenesis and poor prognosis. We assessed the status of NF-κB, Ki-67, cyclin D1 and BRAFV600E in TC tissues and TC cell lines using immunohistochemistry and Western blot analysis. Concurrently, we evaluated the outcomes of combined targeting of the proteasome pathway in addition to selective BRAF inhibitors in cases of PTC. In this study, BRAFV600E-bearing TC cells were treated with BRAFV600E inhibitor, Vemurafenib alone or in combination with the proteasome inhibitor, Bortezomib. The combination of both drugs showed synergistic effects as evidenced by cell growth inhibition (P P P P

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis worldwide. Currently, no curative treatment is available for early-to-intermediate stage HCC, characterized by large and/or multifocal tumors. If left untreated, HCC rapidly progresses to a lethal stage due to favorable conditions for metastatic spread. Mechanochemical disruption of cellular structures can potentially induce phenotypic alterations in surviving tumor cells that prevent HCC progression. In this paper, HCC response to mechanical vibration via high-intensity focused ultrasound and a chemical disruptive agent (ethanol) was examined in vitro and in vivo. Our analysis revealed that mechanochemical disruption caused a significant overproduction of reactive oxygen species (ROS) in multiple HCC cell lines (HepG2, PLC/PRF/5, and Hep3B). This led to a decrease in cell viability and long-term proliferation due to increased expression and activity of death receptors TNFR1 and Fas. The cells that survived mechanochemical disruption had a reduced expression of cancer stem cell markers (CD133, CD90, CD49f) and a diminished colony-forming ability. Mechanochemical disruption also impeded HCC migration and their adhesion to vascular endothelium, two critical processes in hematogenous metastasis. The HCC transformation to a non-tumorigenic phenotype post mechanochemical disruption was confirmed by a lack of tumor spheroid formation in vitro and complete tumor regression in vivo. These results show that mechanochemical disruption inhibits uncontrolled proliferation and reduces tumorigenicity and aggressiveness of HCC cells through ROS overproduction and associated activation of TNF- and Fas-mediated cell death signaling. Our study identifies a novel curative therapeutic approach that can prevent the development of aggressive HCC phenotypes.

Objective: This case report will describe the first adjunct use of directly measuring the concentration of human chorionic gonadotropin (HCG) in fine-needle aspiration (FNA) washout for diagnosing metastatic non-semi-nomatous germ cell tumor (NSGCT) of the testicle in a patient with cervical lymphadenopathy.Methods: We present the clinical, laboratory, imaging, and pathologic findings, along with a review of the literature.Results: A 23-year-old, otherwise healthy man who first presented with left testicular discomfort and swelling was diagnosed with NSGCT after undergoing a left orchiectomy. A few years later, the patient presented with a 2-cm left supraclavicular mass. Upon ultrasound of the thyroid and soft tissues of the neck, a 1-cm left thyroid nodule was revealed, as well as a 2.8-cm left supraclavicular lymph node, which was cystic in nature and worrisome for metastatic disease given the patient's history of metastatic NSGCT. The results of the FNA of the left thyroid nodule were benign, however the results from the supraclavicular mass were nondiagnostic. Due to the nondiagnostic FNA results, another aspiration with cytopathology and HCG evaluation washout was performed. The HCG aspirate came back with a value of 162 mIU/mL, despite the patient's negative serum HCG results.Conclusion: This case demonstrates a novel way to diagnose metastatic testicular germ cell tumors utilizing FNA-HCG washout. Future prospective trials are needed to further elucidate this important finding.Abbreviations: FNA fine-needle aspiration HCG human chorionic gonadotropin NSGCT non-seminomatous germ cell tumor TG thyroglobulin

We present a urologic case report associated with retinoblastoma (RB1) mutation. A 65-year-old man, who has a history of bilateral retinoblastoma treated with primary radiation therapy at approximately 1 year of age. He presented with a 3-month history of gross hematuria and, on initial workup, was found to have synchronous renal and urothelial malignancies. The patient underwent complete transurethral resection of high grade Ta urothelial cancer and robotic-assisted partial nephrectomy for a pT3a leiomyosarcoma. He remains responsive to Bacillus Calmette-Guerin, and shows no recurrence of his renal malignancy. Through targeted sequencing, Rb mutations can predispose patients to several urologic malignancies.

Objective To present a novel imaging technique used for rapid, nondestructive histological assessment of renal neoplasias using a dual-component fluorescence stain and structured illumination microscopy (SIM). Materials and Methods After Institutional Review Board approval, 65 total biopsies were obtained from 19 patients undergoing partial or radical nephrectomy. Biopsies were stained with a dual-component fluorescent, and optically sectioned SIM images were obtained from the surface of the intact biopsies. Specimens were subsequently fixed and analyzed using hematoxylin and eosin (H&E) histopathologic methods and compared with SIM images. A single, board-certified pathologist blinded to specimens reviewed all SIM images and H&E slides, and determined the presence or absence of neoplasias. Results of blinded diagnosis of SIM were validated against traditional pathology. Results Of the 19 patients, 15 underwent robotic partial nephrectomies and 4 underwent laparoscopic nephrectomies. Indications included clinical suspicion of renal cell carcinoma. In total, 65 biopsy specimens were available for review. Twenty-one specimens were determined to be neoplastic on H&E, whereas 41 represented benign renal tissue. The final sensitivity and specificity of our study were 79.2% and 95.1%, respectively. Conclusion SIM is a promising technology for rapid, near-patient, ex vivo renal biopsy assessment. By improving the ability to rapidly assess sufficiency of biopsy specimens and enabling immediate diagnostic capability, SIM aids in more effective biopsy performance, tissue triage, and patient counseling regarding management options. Additionally, because tissue is preserved, effective utilization of downstream diagnostic tests and molecular assessments are possible.

OBJECTIVE To investigate tyrosine kinase inhibitors (TKI) and gold nanorods (AuNRs) paired with photothermal ablation in a human metastatic clear cell renal cell carcinoma (RCC) mouse model. Nanoparticles have been successful as a platform for targeted drug delivery in the treatment of urological cancers. Likewise, the use of nanoparticles in photothermal tumour ablation, although early in its development, has provided promising results. Our previous in vitro studies of nanoparticles loaded with both TKI and AuNRs and activated with photothermal ablation have shown significant synergistic cell kill greater than each individual arm alone. This study is a translation of our initial findings to an in vivo model. MATERIALS AND METHODS Immunologically naive nude mice (athymic nude-Foxn1nu ) were injected subcutaneously bilaterally in both flanks (n = 36) with 2.5 × 106 cells of a human metastatic renal cell carcinoma cell line (RCC 786-O). Subcutaneous xenograft tumours developed into 1-cm palpable nodules. AuNRs encapsulated in human serum albumin protein (HSA) nanoparticles were synthesised with or without a TKI and injected directly into the tumour nodule. Irradiation was administered with an 808-nm light-emitting diode laser for 6 min. Mice were humanely killed 14 days after irradiation; tumours were excised, formalin fixed, paraffin embedded, and evaluated for size and the percentage of necrosis by a genitourinary pathologist. The untreated contralateral flank tumours were used as controls. RESULTS In mice that did not receive irradiation, TKI alone yielded 4.2% tumour necrosis on the injected side and administration of HSA-AuNR-TKI alone yielded 11.1% necrosis. In the laser-ablation models, laser ablation alone yielded 62% necrosis and when paired with HSA-AuNR there was 63.4% necrosis. The combination of laser irradiation and HSA-AuNR-TKI had cell kill rate of 100%. CONCLUSIONS In the absence of laser irradiation, TKI treatment alone or when delivered via nanoparticles produced moderate necrosis. Irradiation with and without gold particles alone also improves tumour necrosis. However, when irradiation is paired with gold particles and drug-loaded nanoparticles, the combined therapy showed the most significant and synergistic complete tumour necrosis of 100% (P < 0.05). This study illustrates the potential of combination nanotechnology as a new approach in the treatment of urological cancers.

Background: Fine needle aspiration (FNA) can lead to changes that extensively replace cytological confirmed thyroid lesions. These lesions, so called “vanishing tumors” can be diagnostically challenging to pathologists and therapeutically challenging for endocrinologists and surgeons. We performed a retrospective analysis to identify these tumors. Methods: Data of 656 patients referred for thyroid surgery was reviewed. Patients with suspicious lesions on neck ultrasound (US) underwent FNA. We compared FNA cytological and surgical pathological findings to identify vanishing tumors. FNA-induced changes such as cystic degeneration, hemorrhage, calcification, cholesterol crystals, fibrosis and granulation tissue were identified. Results: Seventeen patients (2.5%) were identified with vanishing tumors. FNA cytology was indeterminate in seven (41.1%) and benign in ten (58.8%) patients. Surgical pathology in all nodules showed regressive changes partially or entirely replacing the tumor. The mean size of vanishing tumors was 2.4±1.5 cm in greatest dimension. Seven nodules (41.1%) were entirely replaced while remaining ten nodules showed partial replacement of tumors. Three (17.6%) nodules had focal areas of optically clear nuclei suspicious of papillary thyroid carcinoma (PTC); one showed an additional focus of follicular neoplasm (FN) of uncertain malignant potential. Conclusions: FNA-induced changes can lead to obliteration of nodules rendering pathological diagnosis with no evidence of confirmed lesions. Pathologists and surgeons should be aware of this challenging scenario.

Metadherin (MTDH) is widely recognized as a promising molecular marker for tumor recurrence and poor survival in many cancers. By multiple pathways, MTDH promotes oncogenesis, metastasis, and chemoresistance. This study investigated the role of MTDH in papillary thyroid carcinoma (PTC) to determine its potential association with aggressive clinical and pathologic features, including its relation in tumors harboring a BRAF V600E mutation. Expression of MTDH was assessed by immunohistochemistry in 96 cases of PTC, including primary thyroid malignancies and lymph node metastases. The status of BRAF V600E mutation was determined by real-time polymerase chain reaction. Overexpression of MTDH was observed in 26 % (23/88) of primary PTC cases. High-intensity staining was observed in 75 % (6/8) of lymph nodes with metastatic PTC and moderate staining in 25 % (2/8) of cases. Normal adjacent thyroid tissue and benign thyroid controls were found to have significantly lower MTDH expression than cancer tissue (p

diSPIM is used to render 3D digital histological images on large specimens. By comparing dual-view deconvolved results with the corresponding single-view images, we demonstrate that dual-view imaging can provide higher image accuracy.

Background Fine-needle aspiration (FNA) cytology has been the standard of care in the workup of cervical lymph nodes (LNs) in patients with recurrent papillary thyroid cancer (PTC) and suspicious cervical LNs. Recently, FNA thyroglobulin (TG) washout measurement has been proposed as an adjunct in the management of these patients. We hypothesize that using FNA-TG washout for suspicious cervical LNs would increase the accuracy of diagnosing metastatic disease especially in cystic and highly vascular cervical LN in patients with recurrent PTC. Methods This is a retrospective study of a prospectively collected database for patients with thyroid cancer who underwent preoperative FNA followed by selective neck dissection by one surgeon at an academic institution. FNA-cytology and FNA-TG washout were performed simultaneously. A total of 138 patients were included in our study, of which 92 (66.7%) had undergone surgical intervention. Results of both methods were then correlated with the final surgical pathology. Results FNA-cytology alone showed a sensitivity of 80.0%, specificity of 100.0% with a negative predictive value (NPV) of 60.0%. By contrast, FNA-TG washout had a sensitivity of 95.8%, specificity of 90.5% with a NPV of 86.4%. Combination of the FNA-cytology with FNA-TG washout of cystic/highly vascular LN increased the accuracy of diagnosis with a sensitivity of 98.2%, specificity of 100.00% with a NPV of 95.0%. All 14 malignant cervical LNs with false-negative FNA-cytology showed elevated FNA-TG washout, 10 (71.4%) of which were cystic in nature and 4 were highly vascular on ultrasonography. Conclusion FNA-TG washout increases the diagnostic accuracy in detecting metastatic disease in patients with recurrent thyroid cancer. FNA-TG washout may be of special diagnostic importance in cystic or highly vascular LNs, which might have falsely negative cytology. Level of evidence 2B.

Molecular mechanisms underlying the health disparity of prostate cancer (PCa) have not been fully determined. In this study, we applied bioinformatic approach to identify and validate dysregulated genes associated with tumor aggressiveness in African American (AA) compared to Caucasian American (CA) men with PCa. We retrieved and analyzed microarray data from 619 PCa patients, 412 AA and 207 CA, and we validated these genes in tumor tissues and cell lines by Real-Time PCR, Western blot, immunocytochemistry (ICC) and immunohistochemistry (IHC) analyses. We identified 362 differentially expressed genes in AA men and involved in regulating signaling pathways associated with tumor aggressiveness. In PCa tissues and cells, NKX3.1, APPL2, TPD52, LTC4S, ALDH1A3 and AMD1 transcripts were significantly upregulated (p

Increased utilization of transplantation as treatment for patients with end-stage hepatic disease has resulted in a shortfall of available livers. Efforts to expand the available donor pool have resulted in the inclusion of donors who might not have been considered in the past. This has resulted in more requests for frozen section biopsy evaluation of the liver from "marginal" donors with significant co-morbidities. The information gained from the biopsy analysis determines whether the organ is suitable for transplantation. Critical to determining the adequacy of donor livers is analyzing the lipid content for macrosteatosis; high lipid livers are not suitable for transplant. Frozen section analysis (FSA) creates artifacts that limit tissue evaluation, exhausts tissue for downstream histological analysis, and requires a specialized team to evaluate these procedures in the hospital 24/7. We have developed a fluorescence microscopy system that utilizes structured illumination (SIM) to produce images of liver biopsies within seconds of removal from a deceased organ donor. Liver biopsies that require evaluation for donation suitability are stained with fast-acting fluorescent histology dyes and lipid specific stains in order to differentiate the lipids on SIM. The SIM images are compared to the standard-of-care FSA and the final pathology report. Here, we present the results of this blinded review performed by a liver pathology specialist. Imaging liver biopsies with SIM provides a more direct and accurate tool for determining macrosteatosis compared to standard FSA. SIM offers minimal tissue processing complexity and remote viewing capabilities, creating the potential to revolutionize tissue donation evaluation.

The current system for evaluating prostate cancer architecture is the Gleason grading system which divides the morphology of cancer into five distinct architectural patterns, labeled 1 to 5 in increasing levels of cancer aggressiveness, and generates a score by summing the labels of the two most dominant patterns. The Gleason score is currently the most powerful prognostic predictor of patient outcomes; however, it suffers from problems in reproducibility and consistency due to the high intra-observer and inter-observer variability amongst pathologists. In addition, the Gleason system lacks the granularity to address potentially prognostic architectural features beyond Gleason patterns. We evaluate prostate cancer for architectural subtypes using techniques from topological data analysis applied to prostate cancer glandular architecture. In this work we demonstrate the use of persistent homology to capture architectural features independently of Gleason patterns. Specifically, using persistent homology, we compute topological representations of purely graded prostate cancer histopathology images of Gleason patterns 3,4 and 5, and show that persistent homology is capable of clustering prostate cancer histology into architectural groups through a ranked persistence vector. Our results indicate the ability of persistent homology to cluster prostate cancer histopathology images into unique groups with dominant architectural patterns consistent with the continuum of Gleason patterns. In addition, of particular interest, is the sensitivity of persistent homology to identify specific sub-architectural groups within single Gleason patterns, suggesting that persistent homology could represent a robust quantification method for prostate cancer architecture with higher granularity than the existing semi-quantitative measures. The capability of these topological representations to segregate prostate cancer by architecture makes them an ideal candidate for use as inputs to future machine learning approaches with the intent of augmenting traditional approaches with topological features for improved diagnosis and prognosis.

Current methods for breast tumor margin detection are invasive, time consuming, and typically result in a reoperative rate of over 25%. This marks a clear clinical need to develop improved tools to intraoperatively differentiate negative versus positive tumor margins. Here, we utilize photoacoustic tomography (PAT), ultrasound (US), and inverted Selective Plane Illumination Microscopy (iSPIM) to assess breast tumor margins in eight human breast biopsies. Our PAT/US system consists of a tunable Nd:YAG laser (NT 300, EKSPLA) coupled with a 40MHz central frequency US probe (Vevo2100, FUJIFILM Visual Sonics). This system allows for the delivery of 10Hz, 5ns pulses with fluence of 40mJ/cm2 to the tissue with PAT and US axial resolutions of 125μm and 40μm, respectively. For this study, we used a linear stepper motor to acquire volumetric PAT/US images of the breast biopsies using 1100nm light to identify bloodrich “tumor” regions and 1210nm light to identify lipid-rich “healthy” regions. iSPIM (Applied Scientific Instrumentation) is an advanced microscopy technique with lateral resolution of 1.5μm and axial resolution of 7μm. We used 488nm laser excitation and acridine orange as a general comprehensive histology stain. Our results show that PAT/US can be used to identify lipid-rich regions, dense areas of arterioles and arteries, and other internal structures such as ducts. iSPIM images correlate well with histopathology slides and can verify nuclear features, cell type and density, stromal features, and microcalcifications. Together, this multimodality approach has the potential to improve tumor margin detection with a high degree of sensitivity and specificity.

The MAP kinase and NF-κB signaling pathways play an important role in thyroid cancer tumorigenesis. We aimed to examine the therapeutic potential of dually targeting the two pathways using AZD6244 and Bortezomib in combination. We evaluated their effects on cell proliferation, cell-cycle progression, apoptosis, cell migration assay, and the activation of the MAPK pathway in vitro and the in vivo using tumor size and immunohistochemical changes of Ki67 and ppRB. We found inhibition of cell growth rate by 10%, 20%, and 56% (p < 0.05), migration to 55%, 61%, and 29% (p < 0.05), and induction of apoptosis to 10%, 15%, and 38% (p < 0.05) with AZD6244, Bortezomib, or combination, respectively. Induction of cell cycle arrest occurred only with drug combination. Dual drug treatment in the xenograft model caused a 94% reduction in tumor size (p < 0.05) versus 15% with AZD6244 and 34% with Bortezomib (p < 0.05) and also reduced proliferative marker Ki67, and increased pRb dephosphorylation. Our results demonstrate a robust therapeutic potential of combining AZD6244 and Bortezomib as an effective strategy to overcome drug resistance encountered in monotherapy in the treatment of thyroid cancer, strongly supporting clinical trials to further test this strategy.

Rapid assessment of prostate core biopsy pathology at the point-of-procedure could provide benefit in a variety of clinical situations. Even with advanced transrectal ultrasound guidance and saturation biopsy protocols, prostate cancer can be missed in up to half of all initial biopsy procedures. In addition, collection of tumor specimens for downstream histologic, molecular, and genetic analysis is hindered by low tumor yield due to inability to identify prostate cancer grossly. However, current point-of-procedure pathology protocols, such as frozen section analysis (FSA), are destructive and too time- and labor-intensive to be practical or economical. Ex vivo microscopy of the excised specimens, stained with fast-acting fluorescent histology dyes, could be an attractive nondestructive alternative to FSA. In this work, we report the first demonstration of video-rate structured illumination microscopy (VR-SIM) for rapid high-resolution diagnostic imaging of prostate biopsies in realistic point-of-procedure timeframes. Large mosaic images of prostate biopsies stained with acridine orange are rendered in seconds and contain excellent contrast and detail, exhibiting close correlation with corresponding hematoxylin and eosin histology. A clinically relevant review of VR-SIM images of 34 unfixed and uncut prostate core biopsies by two independent pathologists resulted in an area under the receiver operative curve (AUC) of 0.82–0.88, with a sensitivity ranging from 63% to 88% and a specificity ranging from 78% to 89%. When biopsies contained more than 5% tumor content, the sensitivity improved to 75% to 92%. The image quality, speed, minimal complexity, and ease of use of VR-SIM could prove to be features in favor of adoption as an alternative to destructive pathology at the point-of-procedure. Cancer Res; 75(19); 4032–41. ©2015 AACR.

We demonstrate the first fluorescent virtual-H&E of the entire surface of an intact radical prostatectomy margin via DRAQ5 and Eosin imaging with VR-SIM. We also confirm that D&E does not affect clinical downstream fluorescent assays.

Structured light microscopy, including structured illumination microscopy (SIM) and selective plane illumination microscopy (SPIM), has a number of compelling applications in clinical pathology, including large-area rapid 2D imaging for core biopsy and tumor margin assessment, rapid nondestructive 3D cytology, and comprehensive 3D analysis of tumor architecture with isotropic resolution. This talk will briefly introduce our work in these areas, focusing on the use of SIM for tumor margin imaging.

Background. BRAFV600E mutation is associated with poor prognosis in patients with papillary thyroid carcinoma (PTC). PTC is often multifocal, and there are no guidelines on how many tumors to test forBRAFmutation in multifocal PTC.Methods. Fifty-seven separate formalin-fixed and paraffin-embedded PTCs from twenty-seven patients were manually macrodissected and tested forBRAFmutation using a commercial allele-specific real-time polymerase chain reaction-based assay (Entrogen, Woodland Hills, CA). Data related to histologic characteristics, patient demographics, and clinical outcomes were collected.Results. All mutations detected wereBRAFV600E. Seventeen patients (63%) had concordant mutation status in the largest and second-largest tumors (i.e., both were positive or both were negative). The remaining ten patients (37%) had discordant mutation status. Six of the patients with discordant tumors (22% overall) had aBRAF-negative largest tumor and aBRAF-positive second-largest tumor. No histologic feature was found to help predict which cases would be discordant.Conclusions. Patients with multifocal PTC whose largest tumor isBRAF-negative can have smaller tumors that areBRAF-positive. Therefore, molecular testing of more than just the dominant tumor should be considered. Future studies are warranted to establish whether finding aBRAFmutation in a smaller tumor has clinical significance.

Approximately half of cutaneous melanoma tissues harbor BRAFImmunohistochemical analysis of BRAFWe included 91 patients, of whom 32% (29 of 91) had cytoplasmic BRAFNuclear localization of BRAF

Objective To examine the potential benefits of sedation in adults undergoing fine needle aspiration (FNA) of thyroid nodules. Methods This retrospective study compared the outcomes of sedated and non-sedated FNA patients. Results A total of 860 patients underwent 1698 FNAs of thyroid nodules. The mean patient age was 52.4±14.4 years, and 80.2% of patients were women. The non-sedated group consisted of 782 patients with 1543 (93.5%) FNA procedures. The sedated group consisted of 66 patients who underwent 107 (6.5%) FNAs. There was no statistical difference between these groups with respect to age, gender, nodule size, nodule vascularity, non-diagnostic sample rate and post FNA hematoma (P > .05). Conclusions Performing FNA of thyroid nodules in adult patients under sedation is not associated with a higher diagnostic yield or lower bleeding rate when compared to local anesthesia. Sedation should be judiciously used on only very anxious patients due to the increased overall cost.

Although overall survival rate for patients with thyroid cancer (TC) is high, there is an alarming 10-year recurrence rate of up to 30% conferring a ~50% survival among these high-risk patients. The BRAF(V600E) mutation is estimated to be present in over 50% of papillary thyroid cancer (PTC) cases besides being associated with carcinogenesis and poor prognosis. We assessed the status of NF-κB, Ki-67, cyclin D1 and BRAF(V600E) in TC tissues and TC cell lines using immunohistochemistry and Western blot analysis. Concurrently, we evaluated the outcomes of combined targeting of the proteasome pathway in addition to selective BRAF inhibitors in cases of PTC. In this study, BRAF(V600E)-bearing TC cells were treated with BRAF(V600E) inhibitor, Vemurafenib alone or in combination with the proteasome inhibitor, Bortezomib. The combination of both drugs showed synergistic effects as evidenced by cell growth inhibition (P < 0.05), increased G2-phase cell cycle arrest and induced apoptosis (P < 0.05). In our TC xenograft model, the combination of Vemurafenib and Bortezomib significantly reduced tumor size (P < 0.05) and expression of the markers of cell growth and proliferation, Ki-67 and cyclin D1 (P < 0.001), when compared to monotherapy. Further analysis demonstrated that treatment with Bortezomib sensitized TC cells to Vemurafenib via mitochondrial dysregulation and apoptosis of TC cells, as evidenced by the increase in the expression of p53, Noxa protein, the loss of mitochondrial membrane potential, cytochrome c release and Poly (ADP-ribose) polymerase cleavage. Our results demonstrate a strong clinical potential for the combination of the Bortezomib and the BRAF inhibitor Vemurafenib as an efficient therapeutic approach for the treatment of TC.

Papillary thyroid carcinoma (PTC) has two major types, classic (PTCC) and follicular variant (FVPTC), which correlate with molecular findings and have varying clinical implications. We assessed the cytologic findings and subsequent surgical pathology findings with the molecular mutations in these two groups, including microcarcinomas. Fourty-four patients with PTC resections over a one-year period were retrospectively examined in conjunction with previous cytologic diagnoses. BRAF, NRAS and TERT promoter mutations for the resected specimens were analyzed. Correlation with previous cytology in regard to molecular mutations and tumor size (microcarcinoma) were made. Significantly more BRAF V600E mutations were seen with PTCC, whereas significantly more NRAS mutations were seen with FVPTC. TERT mutations were only seen with PTCC. Molecular studies for thyroid carcninomas are becoming increasingly more common and influence treatment and patient prognosis. BRAF and or TERT mutations are associated with a worse prognosis. NRAS mutations associated with FVPTC and may lead to milder cytologic changes compared to the BRAF- and TERT-driven PTCC.

Introduction: We aim to investigate the significance of enlarged cervical lymph nodes (ECLN) identified by initial surgeon-performed ultrasound (US) as a tool for determining the risk of malignancy in the patients presenting with suspicious thyroid nodules. Methods: Radiological and surgical reports were retrospectively reviewed for the patients with suspicious thyroid nodules who underwent thyroidectomy and preoperative comprehensive neck US. Ultrasonographic features of the identified cervical lymph nodes were correlated with the final pathology report. Patients with malignancy other than papillary thyroid cancer (PTC) were excluded. Results: The study consisted of 440 patients. On final pathology, PTC was found in 142 patients (32.3%), the remaining 298 (67.7%) exhibited benign findings. ECLN (>1 cm) were found in 66 (46.5%) patient with PTC compared to only 53 (17.8%) patients with benign nodules (P < 0.001). Of the 119 patients with ECLN, 54.6% had benign appearing ECLN with no suspicious features, 26.1% had one suspicious feature, and 19.3% had more than one suspicious features. Benign appearing ECLN had a positive predictive value (PPV) of 41.54%, negative predictive value (NPV) of 59.02%, sensitivity of 51.92%, and specificity of 48.65% in predicting malignancy as opposed to the absence of ECLN. While as opposed to benign looking ECLN, ECLN with only one suspicious feature had a PPV of 70.97%, NPV of 50.00%, sensitivity of 33.33%, and specificity of 83.02%, and ECLN with two or more suspicious feature had a PPV of 73.91%, NPV of 48.96%, sensitivity of 25.76%, and specificity of 88.68%. Conclusion: ECLN are associated with an increased likelihood of thyroid malignancy in the patients undergoing evaluation of a suspicious nodule. The risk of malignancy in thyroid nodules increases with the presence of suspicious ultrasonographic features on cervical lymph nodes.

Achieving cancer-free surgical margins in oncologic surgery is critical to reduce the need for additional adjuvant treatments and minimize tumor recurrence; however, there is a delicate balance between completeness of tumor removal and preservation of adjacent tissues critical for normal post-operative function. We sought to establish the feasibility of video-rate structured illumination microscopy (VR-SIM) of the intact removed tumor surface as a practical and non-destructive alternative to intra-operative frozen section pathology, using prostate cancer as an initial target. We present the first images of the intact human prostate surface obtained with pathologically-relevant contrast and subcellular detail, obtained in 24 radical prostatectomy specimens immediately after excision. We demonstrate that it is feasible to routinely image the full prostate circumference, generating gigapixel panorama images of the surface that are readily interpreted by pathologists. VR-SIM confirmed detection of positive surgical margins in 3 out of 4 prostates with pathology-confirmed adenocarcinoma at the circumferential surgical margin, and furthermore detected extensive residual cancer at the circumferential margin in a case post-operatively classified by histopathology as having negative surgical margins. Our results suggest that the increased surface coverage of VR-SIM could also provide added value for detection and characterization of positive surgical margins over traditional histopathology.

To identify variations in renal function and histology between Caucasian Americans (CA) and African Americans (AA) undergoing robotic nephron-sparing surgery (NSS).A retrospective chart review was performed on patients who underwent NSS. Multivariate analysis identified factors affecting postoperative estimated glomerular filtration rate (eGFR). Histology was re-reviewed by pathology to confirm papillary type.A total of 331 patients underwent NSS: CA (n = 212), AA (n = 105), Hispanic (n = 10), and other (n = 4). AA average age (60.1 years) was lower than CA (62.3 years) (P .001), with a higher proportion of AA women (46%) than CA (37%) (P = .021). AA had a higher incidence of diabetes (58.2%) and hypertension (93.9%). Preoperative average eGFR was similar: 70.35 mL/min for AA versus 69.06 mL/min for CA. Average postoperative eGFR was 50.59 mL/min for AA and 57.85 mL/min for CA. Postoperative creatinine increased more in AA (0.44 mg/dL) versus CA (0.33 mg/dL) (P .001) even when stratified by pathological stage. Clear cell renal cell carcinoma (RCC) was the most common histology with AA (45%) and CA (60%). A greater than 2-fold higher incidence of papillary RCC was observed in AA (31%) versus CA (13%). AA exhibited a greater proportion of high-grade or type 2 papillary RCC (40% and 30%) versus CA (25% and 13%).AA patients were treated at a younger age, with a larger proportion of women. Postoperatively, AA experienced a greater increase in serum creatinine. Final histology demonstrated greater papillary RCC incidence in AA and increased likelihood for type 2 papillary RCC, a more aggressive histology.

Real-time on-site histopathology review of biopsy tissues at the point-of-procedure has great potential for significant clinical value and improved patient care. For instance, on-site review can aid in rapid screening of diagnostic biopsies to reduce false-negative results, or in quantitative assessment of biospecimen quality to increase the efficacy of downstream laboratory and histopathology analysis. However, the only currently available rapid pathology method, frozen section analysis (FSA), is too time- and labor-intensive for use in screening large quantities of biopsy tissues and is too destructive for maximum tissue conservation in multiple small needle core biopsies. In this work we demonstrate the spectrally-compatible combination of the nuclear stain DRAQ5 and the anionic counterstain eosin as a dual-component fluorescent staining analog to hematoxylin and eosin intended for use on fresh, unsectioned tissues. Combined with optical sectioning fluorescence microscopy and pseudo-coloring algorithms, DRAQ5 and eosin ("D&E") enables very fast, non-destructive psuedohistological imaging of tissues at the point-of-acquisition with minimal tissue handling and processing. D&E was validated against H&E on a one-to-one basis on formalin-fixed paraffin-embedded and frozen section tissues of various human organs using standard epi-fluorescence microscopy, demonstrating high fidelity of the staining mechanism as an H&E analog. The method was then applied to fresh, whole 18G renal needle core biopsies and large needle core prostate biospecimen biopsies using fluorescence structured illumination optical sectioning microscopy. We demonstrate the ability to obtain high-resolution histology-like images of unsectioned, fresh tissues similar to subsequent H&E staining of the tissue. The application of D&E does not interfere with subsequent standard-of-care H&E staining and imaging, preserving the integrity of the tissue for thorough downstream analysis. These results indicate that this dual-stain pseudocoloring method could provide a real-time histology-like image at the time of acquisition and valuable objective tissue analysis for the clinician at the time of service.

Currently available pathology techniques for obtaining a rapid tissue diagnosis, or for determining the adequacy of specimens intended for downstream analysis, are too slow, labor-intensive, and destructive for point-of-care (POC) applications. We previously demonstrated video-rate structured illumination microscopy (VR-SIM) for accurate, high-throughput, non-destructive diagnostic imaging of fluorescently-stained prostate biopsies in seconds per biopsy, with an area under the ROC curve of 0.82-0.88 after pathologist review. In addition, we have demonstrated that it is feasible to use VR-SIM to routinely image very large gross pathology specimens, such as entire prostate resection surfaces, in relatively short timeframes at subcellular resolution. However, our prior work has focused on applications in prostate cancer; the utility in other organ sites has not been explored. Here we extended our technology to varying size kidney, liver, and lung biopsies. We conducted a validation study of VR-SIM against histopathology on a variety of human tissues, including both small biopsies and large slices of tissue. We conducted a blinded study in which the study pathologist accurately identified the organs based on VR-SIM images alone. The results were then used to create a clinical atlas between VR-SIM and H and E images for the different tissues of interest. This clinical atlas will be used to aid in pathologist interpretation in future POC clinical applications of VR-SIM in kidney, liver, and lung. Such applications could include on-site identification of the presence of kidney glomeruli for to ensure successful downstream IHC analysis, or determination of the adequacy of lung cancer biopsies for genomic analysis.

A novel, dual-stain fluorescent analogue to H&E, applied directly to both previously fixed and frozen tissue, is demonstrated. DRAQ5 and Eosin Y (D&E), when applied to human tissue, imaged using a fluorescent microscope, and pseudocolored to match H&E, visualizes morphological features that a pathologist found to directly correlate with gold standard bright field H&E.

The phenomenon of heterotopic bone formation (osseous metaplasia) is defined as an abnormal ossification of non-skeletal tissues and does represent a rare occurrence in the renal cell carcinoma setting. We describe a case of a 40-year old man with bilateral renal cell carcinomas of the histological clear cell subtype, with the right-sided renal cell carcinoma demonstrating heterotopic bone formation, as well as the presence of intratumoral adipose tissue. The etiology of bone formation in a renal cell carcinoma is unclear, but possible explanations include a response to tissue ischemia and the expression of Bone Morphogenetic Protein 2. The detection of these rare morphologic variations is of paramount importance, not to be mistaken as sarcomatoid transformation and renal sinus fat invasion, which would advance the pathologic tumor stage and aggressiveness of the disease.