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(1) Background: Orbital rhabdomyosarcoma is a rare and aggressive soft tissue tumor that primarily occurs in the eye socket (orbit) of children. Treatment usually involves a combination of surgery, chemotherapy, and radiation therapy, aiming to remove the tumor and prevent metastasis. (2) Methods: An institutional retrospective study was conducted with data from 39 patients with primary orbital RMS treated between 1995 and 2016 at the Amsterdam University Medical Centers/Emma Children Hospital. (3) Results: The median age at presentation was 7 years (range, 9 months to 16 years). The median follow-up period was 9.4 years (range, 3 to 25 years). Ten underwent chemotherapy and excision without additional radiotherapy. A total of 29 patients received additional local treatment: Ablative surgery MOld technique with after loading brachytherapy and surgical REconstruction (AMORE) (N = 21), proton (N = 4) or external beam radiation treatment (EBRT; N = 4). We found 14 cases with recurrences, 9 of which underwent exenteration and two of which died. The 10-year overall survival rate was 95% and the EFS was 63%. (4) Conclusions: long-term follow-up with 10-year survival rate of orbital RMS in this series was 95% achieved by local tumor control and eye preservation in 77% of our study population.

Purpose: Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSC), though their origin and function remained unexplored. We demonstrate that cancer-secreted extracellular vesicles (EV) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function., Experimental Design: The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV-induced alterations of the MSC transcriptome were analyzed and compared with single-cell RNA sequencing data of biopsies from patients with osteosarcoma and multiple myeloma. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC-induced resistance in vivo., Results: We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGFβ signaling inhibition. Importantly, EV-induced iMSCs faithfully recapitulate the inflammatory single-cell RNA signature of stromal cells enriched in biopsies from patients with multiple myeloma and osteosarcoma. Mechanistically, cancer EVs act through two distinct mechanisms. EV-associated TGFβ induces IL6 production, whereas the EV-RNA cargo enhances TLR3-mediated chemokine production. We reveal that simultaneous blockade of downstream EV-activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC-induced resistance., Conclusions: Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as triggers of iMSC development, and highlight a promising combination strategy to improve therapy response in patients with bone cancer., (©2024 American Association for Cancer Research.)

Background: Distinguishing congenital pulmonary airway malformations (CPAMs) from pleuropulmonary blastoma (PPB) can be challenging. Previously diagnosed patients with CPAM may have been misdiagnosed and we may have missed DICER1-associated PPBs, a diagnosis with important clinical implications for patients and their families. To gain insight in potential misdiagnoses, we systematically assessed somatic DICER1 gene mutation status in an unselected, retrospective cohort of patients with a CPAM diagnosis., Methods: In the Amsterdam University Medical Center (the Netherlands), it has been standard policy to resect CPAM lesions. We included all consecutive cases of children (age 0-18 years) with a diagnosis of CPAM between 2007 and 2017 at this center. Clinical and radiographic features were reviewed, and DICER1 gene sequencing was performed on DNA retrieved from CPAM tissue samples., Results: Twenty-eight patients with a surgically removed CPAM were included. CPAM type 1 and type 2 were the most common subtypes (n = 12 and n = 13). For 21 patients a chest CT scan was available for reassessment by two pediatric radiologists. In 9 patients (9/21, 43%) the CPAM subtype scored by the radiologists did not correspond with the subtype given at pathology assessment. No pathogenic mutations and no copy number variations of the DICER1 gene were found in the DNA extracted from CPAM tissue (0/28)., Conclusions: Our findings suggest that the initial CPAM diagnoses were correct. These findings should be validated through larger studies to draw conclusions regarding whether systematic DICER1 genetic testing is required in children with a pathological confirmed diagnosis of CPAM or not., Level of Evidence: Level IV., Competing Interests: Conflicts of interest None., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Recently, follicle stimulating hormone (FSH) through interaction with its receptor (FSHR) has been proposed to play a role in postmenopausal osteoporosis and cardiovascular disease, rather than the loss of estrogen. To explore this hypothesis, unravelling which cells express extragonadal FSHR on protein level is key., Methods: We used two commercial anti-FSHR antibodies and validated them by performing immunohistochemistry on positive (ovary, testis) and negative controls (skin)., Results: The monoclonal anti-FSHR antibody could not identify the FSHR in ovary or testis. The polyclonal anti-FSHR antibody stained the granulosa cells (ovary) and Sertoli cells (testis), yet there was equally intense staining of other cells/extracellular matrix. Furthermore, the polyclonal anti-FSHR antibody also stained skin tissue extensively, suggesting that the antibody stains more than just FSHR., Discussion: The findings in this study may add accuracy to literature on extragonadal FSHR localization and warrants attention to the use of inadequate anti-FSHR antibodies to value the potential role of FSH/FSHR in postmenopausal disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tedjawirja, Hooijer, Savci-Heijink, Kovac, Balm and de Waard.)

Purpose: To assess the safety and technical feasibility of in-vivo needle-based forward-looking confocal laser endomicroscopy in prostate tissue. Methods: For this feasibility study, 2 patients with a suspicion of prostate cancer underwent transperineal needle-based confocal laser endomicroscopy during ultrasound-guided transperineal template mapping biopsies. After intravenous administration of fluorescein, needle-based confocal laser endomicroscopy imaging was performed with a forward-looking probe (outer diameter 0.9 mm) in 2 trajectories during a manual push-forward and pullback motion. A biopsy was taken in a coregistered parallel adjacent trajectory to the confocal laser endomicroscopy trajectory for histopathologic comparison. Peri- and postprocedural adverse events, confocal laser endomicroscopy device malfunction and procedural failures were recorded. Needle-based confocal laser endomicroscopy image quality assessment, image interpretation, and histology were performed by an experienced confocal laser endomicroscopy rater and uro-pathologist, blinded to any additional information. Results: In both patients, no peri- and post-procedural adverse events were reported following needle-based confocal laser endomicroscopy. No confocal laser endomicroscopy device malfunction nor procedural failures were reported. Within 1.5 min after intravenous administration of fluorescein, needle-based confocal laser endomicroscopy image quality was sufficient for interpretation for at least 14 min, yielding more than 5000 confocal laser endomicroscopy frames per patient. The pullback confocal laser endomicroscopy recordings and most of the push-forward recordings almost only visualized erythrocytes, being classified as non-representative. During the push-forward recordings, prostate tissue was occasionally visualized in single frames, insufficient for histopathologic comparison. Prostate carcinoma was identified by biopsy in one patient (Gleason score 4 + 3 = 7, >50%), while the biopsy from the other patient showed no malignancy. Conclusion: Needle-based confocal laser endomicroscopy imaging of in-vivo prostate tissue with a forward-looking confocal laser endomicroscopy probe is safe without device malfunctions or procedural failures. Needle-based confocal laser endomicroscopy is technically feasible, but the acquired confocal laser endomicroscopy datasets are non-representative. The confocal laser endomicroscopy images' non-representative nature is possibly caused by bleeding artifacts, movement artifacts and a lack of contact time with the tissue of interest. A different confocal laser endomicroscopy probe or procedure might yield representative images of prostatic tissue.

Background/aim/objective: Late side effects of radiotherapy (RT) in the treatment for head and neck (HN) malignancies involve an inadequate healing response of the distressed tissue due to RT-induced hypovascularity. The aim of this study was to develop a pilot model in which vascular alterations associated with the onset of late irradiation (IR) injury could be measured in rabbit oral mucosa and mandibular bone., Materials and Methods: Eight male New Zealand white rabbits were divided over four treatment groups. Group I-III received four fractions of RT (5.6 Gy, 6.5 Gy, and 8 Gy, respectively) and Group IV received 1 fraction of 30 Gy. Oral microcirculatory measurements were performed at baseline (before RT) and once a week during 11 consecutive weeks after RT assessing perfusion parameters, that is, total vessel density (TVD), perfused vessel density (PVD), proportion of perfused vessels (PPV), and microvascular flow index (MFI). Post-mortem histopathology specimens were analyzed., Results: Five weeks after RT, TVD, and PVD in all groups showed a decrease of >10% compared to baseline, a significant difference was observed for Groups I, II, and IV ( P <0.05). At T11, no lasting effect of decreased vessel density was observed. PPV and MFI remained unaltered at all-time points. Group IV showed a marked difference in scattered telangiectasia such as microangiopathies, histological necrosis, and loss of vasculature., Conclusion: No significant lasting effect in mucosal microcirculation density due to IR damage was detected. Observed changes in microcirculation vasculature and histology may align preliminary tissue transition towards clinical pathology in a very early state associated with late IR injury in the oral compartment., Relevance for Patients: Enhancing knowledge on the onset of late vascular IR injury in the HN region could help the development, monitoring, and timing of therapies that act on prevention, discontinuation, or repair of radiation pathology., (Copyright: © Whioce Publishing Pte. Ltd.)

Purpose: To determine the value of two-dimensional (2D) contrast-enhanced ultrasound (CEUS) imaging and the additional value of contrast ultrasound dispersion imaging (CUDI) for the localization of clinically significant prostate cancer (csPCa)., Methods: In this multicentre study, subjects scheduled for a radical prostatectomy underwent 2D CEUS imaging preoperatively. CUDI maps were generated from the CEUS recordings. Both CEUS recordings and CUDI maps were scored on the likelihood of presenting csPCa (any Gleason ≥ 4 + 3 and Gleason 3 + 4 larger than 0.5 mL) by five observers and compared to radical prostatectomy histopathology. An automated three-dimensional (3D) fusion protocol was used to match imaging with histopathology. Receiver operator curve (ROC) analysis was performed per observer and imaging modality., Results: 133 of 216 (62%) patients were included in the final analysis. Average area under the ROC for all five readers for CEUS, CUDI and the combination was 0.78, 0.79 and 0.78, respectively. This yields a sensitivity and specificity of 81 and 64% for CEUS, 83 and 56% for CUDI and 83 and 55% for the combination. Interobserver agreement for CEUS, CUDI and the combination showed kappa values of 0.20, 0.18 and 0.18 respectively., Conclusion: The sensitivity and specificity of 2D CEUS and CUDI for csPCa localization are moderate. Despite compressing CEUS in one image, CUDI showed a similar performance to 2D CEUS. With a sensitivity of 83% at cutoff point 3, it could become a useful imaging procedure, especially with 4D acquisition, improved quantification and combination with other US imaging techniques such as elastography.

Objectives: To compare and evaluate a multiparametric magnetic resonance imaging (mpMRI)-targeted biopsy (TBx) strategy, contrast-ultrasound-dispersion imaging (CUDI)-TBx strategy and systematic biopsy (SBx) strategy for the detection of clinically significant prostate cancer (csPCa) in biopsy-naïve men., Patients and Methods: A prospective, single-centre paired diagnostic study included 150 biopsy-naïve men, from November 2015 to November 2018. All men underwent pre-biopsy mpMRI and CUDI followed by a 12-core SBx taken by an operator blinded from the imaging results. Men with suspicious lesions on mpMRI and/or CUDI also underwent MRI-TRUS fusion-TBx and/or cognitive CUDI-TBx after SBx by a second operator. A non-inferiority analysis of the mpMRI- and CUDI-TBx strategies in comparison with SBx for International Society of Urological Pathology Grade Group [GG] ≥2 PCa in any core with a non-inferiority margin of 1 percentage point was performed. Additional analyses for GG ≥2 PCa with cribriform growth pattern and/or intraductal carcinoma (CR/IDC), and GG ≥3 PCa were performed. Differences in detection rates were tested using McNemar's test with adjusted Wald confidence intervals., Results: After enrolment of 150 men, an interim analysis was performed. Both the mpMRI- and CUDI-TBx strategies were inferior to SBx for GG ≥2 PCa detection and the study was stopped. SBx found significantly more GG ≥2 PCa: 39% (56/142), as compared with 29% (41/142) and 28% (40/142) for mpMRI-TBx and CUDI-TBx, respectively (P < 0.05). SBx found significantly more GG = 1 PCa: 14% (20/142) compared to 1% (two of 142) and 3% (four of 142) with mpMRI-TBx and CUDI-TBx, respectively (P < 0.05). Detection of GG ≥2 PCa with CR/IDC and GG ≥3 PCa did not differ significantly between the strategies. The mpMRI- and CUDI-TBx strategies were comparable in detection but the mpMRI-TBx strategy had less false-positive findings (18% vs 53%)., Conclusions: In our study in biopsy-naïve men, the mpMRI- and CUDI-TBx strategies had comparable PCa detection rates, but the mpMRI-TBX strategy had the least false-positive findings. Both strategies were inferior to SBx for the detection of GG ≥2 PCa, despite reduced detection of insignificant GG = 1 PCa. Both strategies did not significantly differ from SBx for the detection of GG ≥2 PCa with CR/IDC and GG ≥3 PCa., (© 2020 The Authors BJU International © 2020 BJU International Published by John Wiley & Sons Ltd.)

Objectives: To evaluate the diagnostic yield and concordance of upper tract urothelial carcinoma (UTUC) grading between ureterorenoscopic biopsies and surgical resections. Materials and Methods: The nationwide Dutch Pathology Registry (nationwide network and registry of histo- and cytopathology in the Netherlands [PALGA]) was searched for UTUC-positive renal units (RUs) with histopathology excerpts from ureterorenoscopic biopsies and surgical resections, matched for laterality and localization of the tumor, from 2011 until 2018. The positive predictive value (concordance) of the biopsy grade with regard to the final grade according to the World Health Organization (WHO) 2004 classification was calculated. Results: A total of 1002 UTUC-positive rental units were included, of which 776 UTUC-positive RUs were graded according to the WHO 2004 classification in the ureterorenoscopic biopsy, the localization-matched surgical resection, or in both. The diagnostic yield of biopsies for a classifying diagnosis was 89% with a sensitivity for UTUC of 84%. In case of UTUC, the diagnostic yield for biopsy-based grading and staging was 97% and 72%, respectively. The concordance of high-grade biopsies with regard to the final histopathology was 97% and 62% for low-grade biopsies. Upgrading to final high grade occurred in 33% of the low-grade biopsies. Downgrading to final low grade occurred in 2% of high-grade biopsies. Conclusions: This is the first study to portray the limitations of ureterorenoscopic biopsies for UTUC in a nationwide cohort. The diagnostic yield of ureterorenoscopic biopsies for a classifying diagnosis is suboptimal, but the diagnostic yield for grading according to the WHO 2004 classification is high. Yet, a worrisome amount of ureterorenoscopic biopsies are upgraded with regard to the surgical resection. Consequently, one-third of patients, who qualify for kidney-sparing treatment according to one of the criteria recommended for risk stratification, might be stratified incorrectly. These findings stress the importance of a timely and stringent ureterorenoscopic follow-up after kidney-sparing surgery and highlight the need for improvements in the diagnostic approach to optimize the risk stratification.

Accurate grading of non-muscle-invasive urothelial cell carcinoma is of major importance; however, high interobserver variability exists. A fully automated detection and grading network based on deep learning is proposed to enhance reproducibility. A total of 328 transurethral resection specimens from 232 patients were included, and a consensus reading by three specialized pathologists was used. The slides were digitized, and the urothelium was annotated by expert observers. The U-Net-based segmentation network was trained to automatically detect urothelium. This detection was used as input for the classification network. The classification network aimed to grade the tumors according to the World Health Organization grading system adopted in 2004. The automated grading was compared with the consensus and individual grading. The segmentation network resulted in an accurate detection of urothelium. The automated grading shows moderate agreement (κ = 0.48 ± 0.14 SEM) with the consensus reading. The agreement among pathologists ranges between fair (κ = 0.35 ± 0.13 SEM and κ = 0.38 ± 0.11 SEM) and moderate (κ = 0.52 ± 0.13 SEM). The automated classification correctly graded 76% of the low-grade cancers and 71% of the high-grade cancers according to the consensus reading. These results indicate that deep learning can be used for the fully automated detection and grading of urothelial cell carcinoma., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Cystoscopy enables the visualisation of suspicious bladder lesions but lacks the ability to provide real-time histopathologic information. Confocal laser endomicroscopy (CLE) is a probe-based optical technique that can provide real-time microscopic images. This high-resolution optical imaging technique may enable real-time tumour grading during cystoscopy., Objective: To validate and adapt CLE criteria for bladder cancer diagnosis and grading., Design, Setting, and Participants: Prospectively, 73 patients scheduled for transurethral resection of bladder tumour(s) were included. CLE imaging was performed intraoperatively prior to en bloc resection. Histopathology was the reference standard for comparison., Intervention: Cystoscopic CLE imaging., Outcome Measurements and Statistical Analysis: Three independent observers evaluated the CLE images to classify tumours as low- or high-grade urothelial carcinoma (UC), or benign lesions. Interobserver agreement was calculated with Fleiss kappa analysis and diagnostic accuracy with 2×2 tables., Results and Limitations: Histopathology of 66 lesions (53 patients) revealed 25 low-grade UCs, 27 high-grade UCs, and 14 benign lesions. For low-grade UC, most common features were papillary configuration (100%), distinct cell borders (81%), presence of fibrovascular stalks (79%), cohesiveness of cells (77%), organised cell pattern (76%), and monomorphic cells (67%). A concordance between CLE-based classification and histopathology was found in 19 cases (76%). For high-grade UC, pleomorphic cells (77%), indistinct cell borders (77%), papillary configuration (67%), and disorganised cell pattern (60%) were the most common features. A concordance with histopathology was found in 19 cases (70%). In benign lesions, the most prevalent features were disorganised cell pattern (57%) and pleomorphic cells (52%), and a concordance with histopathology was found in four cases (29%)., Conclusions: The CLE criteria enable identification of UC. CLE features correlate to histopathologic features that may enable real-time tumour grading. However, flat lesions remain difficult to classify., Patient Summary: Confocal laser endomicroscopy may enable real-time cancer differentiation during cystoscopy, which is important for prognosis and disease management., (Copyright © 2018. Published by Elsevier B.V.)

Background: Non-muscle-invasive bladder cancer (NMIBC) is the most common neoplasm of the urinary tract and requires life-long invasive surveillance to detect disease recurrence. Currently, there are no effective oral therapies that delay disease recurrence or progression. We recently demonstrated that in mice, metformin accumulates unchanged in the urine. Urothelial cells are exposed to metformin concentrations ~ 240-fold higher than in serum. This was effective in the treatment of mouse bladder cancer models., Methods: We describe the protocol of a multi-centre, open-label, phase II clinical trial of metformin in up to 49 evaluable patients with intermediate-risk NMIBC with the aim to determine the overall response to administration of oral metformin for 3 months on a marker tumour deliberately left following transurethral resection of multiple, papillary NMIBC tumours. All patients will receive metformin orally at doses up to 3000 mg per day. Metformin treatment will start within 2 weeks following transurethral resection of all tumours except one marker lesion. After 3 months of metformin treatment, the effect of metformin on the marker lesion is evaluated by cystoscopy and biopsy under anaesthesia. Residual tumour, if present at this evaluation, will be resected. In case of complete disappearance of the marker lesion, the former tumour area will be biopsied. The primary outcome is the complete response rate of the marker lesion, as determined by decentralised scoring of pre- and post-treatment cystoscopy images by expert independent urologists. Secondary outcomes are the partial response rate, overall safety of metformin and the duration of the time to recurrence., Discussion: Preclinical studies show the potential role of oral metformin treatment in the management of NMIBC. It could offer an alternative to current adjuvant intravesical treatment. If positive, the reported results of this study could warrant further phase III trials to compare the efficacy of metformin against current treatments of intravesical installations with chemotherapy or Bacillus Calmette-Guérin (BCG)., Trial Registration: This trial is registered in ClinicalTrials.gov under NCT03379909.

Purpose: Urothelial carcinoma of the bladder (UCB) is the most common urinary cancer. White-light cystoscopy (WLC) forms the corner stone for the diagnosis of UCB. However, histopathological assessment is required for adjuvant treatment selection. Probe-based confocal laser endomicroscopy (pCLE) enables visualization of the microarchitecture of bladder lesions during WLC, which allows for real-time tissue differentiation and grading of UCB. To improve the diagnostic process of UCB, computer-aided classification of pCLE videos of in vivo bladder lesions were evaluated in this study. Materials and Methods: We implemented preprocessing methods to optimize contrast and to reduce striping artifacts in each individual pCLE frame. Subsequently, a semiautomatic frame selection was performed. The selected frames were used to train a feature extractor based on pretrained ImageNet networks. A recurrent neural network, in specific long short-term memory (LSTM), was used to predict the grade of bladder lesions. Differentiation of lesions was performed at two levels, namely (i) healthy and benign vs malignant tissue and (ii) low-grade vs high-grade papillary UCB. A total of 53 patients with 72 lesions were included in this study, resulting in ∼140,000 pCLE frames. Results: The semiautomated frame selection reduced the number of frames to ∼66,500 informative frames. The accuracy for differentiation of (i) healthy and benign vs malignant urothelium was 79% and (ii) high-grade and low-grade papillary UCB was 82%. Conclusions: A feature extractor in combination with LSTM results in proper stratification of pCLE videos of in vivo bladder lesions.

Purpose: To compare the histologic features of the cartilage from the capitellum with 2 proposed alternative donor sites from the ipsilateral elbow in the treatment of capitellar osteochondritis dissecans (OCD): the nonarticulating part of the radial head and the nonarticulating lateral side of the olecranon tip., Methods: Ten human cadaveric elbow specimens with macroscopically normal articular surfaces were used to obtain 5-mm osteochondral grafts: 10 from the capitellum (60° anteriorly relative to the humeral shaft), 10 from the radial head (nonarticulating part at 80°), and 4 from the olecranon (lateral side of the olecranon tip). Grafts were fixated in formalin (4% formaldehyde), decalcified, and processed into standard 8-μm-thick hematoxylin and eosin-and Toluidine Blue-stained sections. These were assessed for cartilage thickness, shape of articular surface, and 13 histologic parameters of the International Cartilage Repair Society II. Olecranon scores were excluded from statistical analysis., Results: Mean cartilage thickness was 1.5 ± 0.22 mm at the capitellum; 1.3 ± 0.34 mm at the radial head; and 1.9 ± 1.0 mm at the olecranon. There was no difference in cartilage thickness between the capitellum and radial head (P = .062). All grafts demonstrated a convex articular surface. International Cartilage Repair Society II scores ranged from 82 to 100 for the capitellum, from 81 to 100 for the radial head, and from 67 to 87 for the olecranon tip. There was less chondrocyte clustering at the capitellum (84 ± 14) than in the radial head (94 ± 3.2; P = .019). Mid/deep zone assessment of the capitellum scored higher (97 ± 6.7) than the radial head (91 ± 4.6; P = .038)., Conclusions: This study demonstrates appropriate histologic similarities between the cartilage from the capitellum and 2 alternative donor sites of the ipsilateral elbow in the treatment of capitellar OCD: the nonarticulating part of the radial head and the nonarticulating lateral side of the olecranon tip., Clinical Relevance: From an histologic point of view, there seem to be no obstacles to use grafts from these alternative donor sites for reconstruction of the capitellum when performing osteochondral autologous transplantation., (Copyright © 2019 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Purpose: Confocal laser endomicroscopy (CLE) is a fluorescence-based fiber-optic imaging technique with the potential for intraoperative grading of upper tract urothelial carcinoma (UTUC). This study aims to (1) investigate the prevalence of the previously proposed CLE criteria for bladder cancer in papillary UTUC, (2) estimate the diagnostic value of CLE for UTUC grading and (3) propose a scoring system for a more quantifiable approach of CLE-based grading of UTUC., Materials and Methods: Ureteroscopic CLE was performed in patients with UTUC. Following CLE imaging, co-localized biopsies were taken for histopathologic comparison. Postoperatively, two blinded raters assessed the CLE images., Results: Fifty-three papillary UTUCs (34 low grade and 19 high grade) were imaged with CLE in 36 patients. All the previously described CLE criteria were identifiable in varying proportions. After excluding 10 non-diagnostic recordings (5 low grade and 5 high grade) due to insufficient image quality, the histopathologic grade was correctly identified with CLE in 26 low-grade UTUCs (90%) and in 12 high-grade UTUCs (86%). The most prevalent CLE criteria with the highest diagnostic potential were cellular organization, morphology and cohesiveness of cells. A scoring system was proposed with these criteria, which yielded similar diagnostic accuracies., Conclusions: Based on the previously proposed criteria, CLE enables accurate grading of papillary UTUC at a non-diagnostic rate of 19%. The most prevalent CLE criteria with the highest diagnostic potential for grading of papillary UTUC are cellular organization, morphology and cohesiveness of cells. The proposed scoring system may simplify the assessment of CLE images for UTUC grading but external validation is required.

Purpose: Histological grade is an important prognostic factor in patients with non-muscle-invasive bladder cancer (NMIBC). However, interobserver variability is high. Previous studies have suggested that quantification of histological features is useful to objectify grading. We evaluated whether quantification of the mean nuclear area of the 10 largest nuclei (MNA-10), degree of aneuploidy (DNA index or DI) and mitotic activity index (MAI) are of diagnostic value for NMIBC grade. Additionally, prognostic value of the 3 measures was assessed., Material and Methods: A consensus grade was determined by 3 uropathologists in 310 NMIBC tissues according to the World Health Organization (WHO) 1973 and the WHO2004. Logistic regression with forward selection was used to determine the optimal combination of measures (MNA-10, DI, and MAI) to diagnose grade 3 (G3) or high-grade (HG) NMIBC (WHO1973 and WHO2004, respectively)., Results: In 310 tumors of 215 patients at least 1 of the measures (MNA-10, DI, or MAI) had been determined. The combination of MNA-10 and MAI was selected as the most diagnostic combination and resulted in a sensitivity of 94% (95% confidence interval [CI]: 87-100) at a specificity of 72% (95% CI: 66-78) for G3 tumors. For the diagnosis of HG tumors sensitivity was 92% (95% CI: 86-97) at a specificity of 76% (95% CI: 70-93)., Conclusions: Determination of MNA-10 and MAI is promising for diagnosing G3 and HG bladder tumors. These findings warrant further studies on the diagnostic and prognostic value of proliferative and quantitative features in bladder cancer patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To demonstrate the safety and feasibility of clinical in vivo needle-based optical coherence tomography (OCT) imaging of the prostate., Materials and Methods: Two patients with prostate cancer underwent each two percutaneous in vivo needle-based OCT measurements before transperineal template mapping biopsy. The OCT probe was introduced via a needle and positioned under ultrasound guidance. To test the safety, adverse events were recorded during and after the procedure. To test the feasibility, OCT and US images were studied during and after the procedure. Corresponding regions for OCT and biopsy were determined. A uropathologist evaluated and annotated the histopathology. Three experts assessed all the corresponding OCT images. The OCT and biopsy conclusions for the corresponding regions were compared., Results: No adverse events during and following the, in total four, in vivo needle-based OCT measurements were reported. The OCT measurements showed images of prostatic tissue with a penetration depth of ~1.5 mm. The histological-proven tissue types, which were also found in the overlapping OCT images, were benign glands, stroma, glandular atrophy, and adenocarcinoma (Gleason pattern 3)., Conclusions: Clinical in vivo needle-based OCT of the prostate is feasible with no adverse events during measurements. OCT images displayed detailed prostatic tissue with a imaging depth up to ~1.5 mm. We could co-register four histological-proven tissue types with OCT images. The feasibility of in vivo OCT in the prostate opens the pathway to the next phase of needle-based OCT studies in the prostate. Lasers Surg. Med. 51:390-398, 2019. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc., (© 2019 The Authors Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.)

Histopathologic grading of prostate cancer using Gleason patterns (GPs) is subject to a large inter-observer variability, which may result in suboptimal treatment of patients. With the introduction of digitization and whole-slide images of prostate biopsies, computer-aided grading becomes feasible. Computer-aided grading has the potential to improve histopathological grading and treatment selection for prostate cancer. Automated detection of GPs and determination of the grade groups (GG) using a convolutional neural network. In total, 96 prostate biopsies from 38 patients are annotated on pixel-level. Automated detection of GP 3 and GP ≥ 4 in digitized prostate biopsies is performed by re-training the Inception-v3 convolutional neural network (CNN). The outcome of the CNN is subsequently converted into probability maps of GP ≥ 3 and GP ≥ 4, and the GG of the whole biopsy is obtained according to these probability maps. Differentiation between non-atypical and malignant (GP ≥ 3) areas resulted in an accuracy of 92% with a sensitivity and specificity of 90 and 93%, respectively. The differentiation between GP ≥ 4 and GP ≤ 3 was accurate for 90%, with a sensitivity and specificity of 77 and 94%, respectively. Concordance of our automated GG determination method with a genitourinary pathologist was obtained in 65% (κ = 0.70), indicating substantial agreement. A CNN allows for accurate differentiation between non-atypical and malignant areas as defined by GPs, leading to a substantial agreement with the pathologist in defining the GG.

Pycnodysostosis is an autosomal recessive disease caused by a gene mutation leading cathepsin K deficiency. Pathological fractures of the long bones are common, but guidelines on fracture treatment in these patients are still lacking. We have treated 5 fractures in 2 pediatric pycnodysostosis patients. We hypothesize that pycnodysostosis patients have an incomplete remodeling process in fracture healing because of cathepsin K deficiency. Therefore, to minimize the role of endochondral bone formation (indirect) after a fracture, it seems prudent to strive for direct bone healing (intramembranous) instead of indirect bone healing. Open reduction with internal fixation should be the goal.

Background: Visceral organ metastasis is associated with poor survival outcomes in terms of metastasis free- and overall survival in breast carcinomas. Identification of a gene expression profile in tumours that selects a subpopulation of patients that is more likely to develop visceral organ metastases will help elucidate mechanisms for the development of distant metastases and could be of clinical value. With this study we aimed to determine genomic predictors that would help to distinguish breast cancer patients with more likelihood to develop visceral metastasis., Methods: Gene expression profiling data of 157 primary tumours from breast cancer patients who developed distant metastases were analyzed and differentially expressed genes between the group of tumours with visceral metastasis and the those without visceral metastases were identified. Published data were used to validate our findings. Multivariate logistic regression tests were applied to further investigate the association between the gene-expression-signature and clinical variables. Survival analyses were performed by the Kaplan-Meier method., Results: Fourteen differentially expressed genes (WDR6, CDYL, ATP6V0A4, CHAD, IDUA, MYL5, PREP, RTN4IP1, BTG2, TPRG1, ABHD14A, KIF18A, S100PBP and BEND3) were identified between the group of tumours with and without visceral metastatic disease. Five of these genes (CDYL, ATP6V0A4, PREP, RTN4IP1 and KIF18A) were up-regulated and the other genes were down-regulated. This gene expression signature was validated in the training and in the independent data set (p 2.13e- 08 and p 9.68e- 06, respectively). Multivariate analyses revealed that the 14-gene-expression-signature was associated with visceral metastatic disease (p 0.001, 95% CI 1.43-4.27), independent of other clinicopathologic features. This signature has been also found to be associated with survival status of the patients (p < .001)., Conclusion: We have identified an unique gene expression signature which is specific to visceral metastasis. This 14-gene-expression-signature may play a role in identifying the subgroup of patients with potential to develop visceral metastasis.

Transition between differentiation states in development occurs swift but the mechanisms leading to epigenetic and transcriptional reprogramming are poorly understood. The pediatric cancer neuroblastoma includes adrenergic (ADRN) and mesenchymal (MES) tumor cell types, which differ in phenotype, super-enhancers (SEs) and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we unravel how a NOTCH3 intracellular domain reprogrammed the ADRN transcriptional landscape towards a MES state. A transcriptional feed-forward circuitry of NOTCH-family transcription factors amplifies the NOTCH signaling levels, explaining the swift transition between two semi-stable cellular states. This transition induces genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. Once established, the NOTCH feed-forward loop maintains the induced MES state. In vivo reprogramming of ADRN cells shows that MES and ADRN cells are equally oncogenic. Our results elucidate a swift transdifferentiation between two semi-stable epigenetic cellular states.

Background: Epithelial-to-mesenchymal transition (EMT) has been implicated as an important step in the development of distant metastases. We therefore wished to study EMT status of primary breast carcinomas from patients who during follow-up developed distant metastases., Methods: mRNA expression profiles of primary breast carcinoma samples (n = 151) from patients who developed metastatic disease were analyzed and EMT status was designated using a previously described EMT-core signature. EMT status of the primary tumor was correlated to clinicopathological characteristics, molecular subtypes, metastasis pattern, chemotherapy response and survival outcomes. In addition, using immunohistochemistry, the expression levels of several proteins implicated in EMT were studied (CDH1, CDH2, NAT1, SNAI2, TWIST1, VIM, and ZEB1) compared with the designated EMT status and survival., Results: Utilizing the 130-gene-EMT-core signature, 66.2% of the primary tumors in the current study was assessed as EMT-activated. In contrast to our expectations, analyses revealed that 84.6% of Luminal A tumors, 65.1% of Luminal B tumors, and 55.6% of HER2-like had an activated EMT status, compared to only 25% of the basal-type tumors (p < 0.001). EMT status was not correlated to the pattern of metastatic disease, metastasis-specific survival, and overall survival. Similarly, there was not a significant association between EMT status of the primary tumor and chemotherapy response in the metastatic setting. Immunostaining for NAT1 and TWIST1 correlated with the EMT status (p 0.003 and p 0.047, respectively). Multivariate analyses showed that NAT1 and TWIST1 staining was significantly associated with EMT status regardless of the estrogen receptor status of the tumors (p values: 0.020 and 0.027, respectively)., Conclusions: The EMT status of breast cancers, as defined by the presence of a core EMT gene expression signature is associated with non-basal-type tumors, but not with the pattern of distant metastasis. Of several potential immunohistochemical EMT markers, only NAT1 and TWIST1 expression levels were associated with the gene expression-based EMT status.

Introduction: Analysis of urinary catecholamine metabolites is one of the primary modalities to diagnose patients with neuroblastoma. Although catecholamine excretion patterns have been recognised in the past, their biological rationale and clinical relevance remain largely unknown. Therefore, this study was designed to identify unique catecholamine excretion patterns and elucidate their underlying biology and clinical relevance., Patients and Methods: A panel of 25 neuroblastoma cell lines was screened for catecholamine excretion. Detection of the catecholamine enzymes was performed using Western blot. Based on catecholamine enzymes presence and excreted catecholamine metabolites, excretion profiles were defined. The prevalence of these profiles was investigated in vivo using diagnostic urines from 301 patients with neuroblastoma and immunohistochemistry on primary tumours. The clinical relevance of the profiles was determined by linking the profiles to clinical characteristics and outcome of patients with neuroblastoma., Results: Four excretion profiles (A-D) were identified in vitro, which correlated with the relative protein expression of the catecholamine enzymes. These profiles were also identified in urine samples from patients with neuroblastoma and correlated with the presence of the catecholamine enzymes in the tumour. Strikingly, in 66% of the patients, homovanillic acid and vanillylmandelic acid excretions were discordant with the catecholamine profiles. Clinical characteristics and outcome gradually improved from patients with profile A (predominantly high risk) towards profile D (predominantly observation), with 5-years overall survival of 35% and 93%, respectively., Conclusions: Catecholamine profiles in vitro and in vivo reflect, to a large extent, the presence of the individual catecholamine enzymes and represent distinct subgroups of patients with neuroblastoma., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Histopathological analysis is the cornerstone in bladder cancer (BCa) diagnosis. These analysis suffer from a moderate observer agreement in the staging of bladder cancer. Three-dimensional reconstructions have the potential to support the pathologists in visualizing spatial arrangements of structures, which may improve the interpretation of specimen. The aim of this study is to present three-dimensional (3D) reconstructions of histology images., Methods: En-bloc specimens of transurethral bladder tumour resections were formalin fixed and paraffin embedded. Specimens were cut into sections of 4 μm and stained with Hematoxylin and Eosin (H&E). With a Phillips IntelliSite UltraFast scanner, glass slides were digitized at 20x magnification. The digital images were aligned by performing rigid and affine image alignment. The tumour and the muscularis propria (MP) were manually delineated to create 3D segmentations. In conjunction with a 3D display, the results were visualized with the Vesalius3D interactive visualization application for a 3D workstation., Results: En-bloc resection was performed in 21 BCa patients. Per case, 26-30 sections were included for the reconstruction into a 3D volume. Five cases were excluded due to export problems, size of the dataset or condition of the tissue block. Qualitative evaluation suggested an accurate registration for 13 out of 16 cases. The segmentations allowed full 3D visualization and evaluation of the spatial relationship of the BCa tumour and the MP., Conclusion: Digital scanning of en-bloc resected specimens allows a full-fledged 3D reconstruction and analysis and has a potential role to support pathologists in the staging of BCa.

Mass spectrometry imaging (MSI) has emerged as a powerful tool in biomedical research to reveal the localization of a broad scale of compounds ranging from metabolites to proteins in diseased tissues, such as malignant tumors. MSI is most commonly used for the two-dimensional imaging of tissues from multiple patients or for the three-dimensional (3D) imaging of tissue from a single patient. These applications are potentially introducing a sampling bias on a sample or patient level, respectively. The aim of this study is therefore to investigate the consequences of sampling bias on sample representativeness and on the precision of biomarker discovery for histological grading of human bladder cancers by MSI. We therefore submitted formalin-fixed paraffin-embedded tissues from 14 bladder cancer patients with varying histological grades to 3D analysis by matrix-assisted laser desorption/ionization (MALDI) MSI. We found that, after removing 20% of the data based on novel outlier detection routines for 3D-MSI data based on the evaluation of digestion efficacy and z-directed regression, on average 33% of a sample has to be measured in order to obtain sufficient coverage of the existing biological variance within a tissue sample. SIGNIFICANCE: In this study, 3D MALDI-MSI is applied for the first time on a cohort of bladder cancer patients using formalin-fixed paraffin-embedded (FFPE) tissue of bladder cancer resections. This work portrays the reproducibility that can be achieved when employing an optimized sample preparation and subsequent data evaluation approach. Our data shows the influence of sampling bias on the variability of the results, especially for a small patient cohort. Furthermore, the presented data analysis workflow can be used by others as a 3D FFPE data-analysis pipeline working on multi-patient 3D-MSI studies., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Lack of accuracy in preoperative imaging leads to overtreatment of benign renal masses (RMs) or indolent renal cell carcinomas (RCCs). Optical coherence tomography (OCT) is real time and high resolution, enabling quantitative analysis through attenuation coefficient (μ OCT , mm -1 )., Objective: To determine the accuracy and diagnostic yield of OCT and renal mass biopsy (RMB) for the differentiation of benign RMs versus RCC and oncocytoma versus RCC., Design, Setting, and Participants: From October 2013 to June 2016, 95 patients with solid enhancing RMs on cross-sectional imaging were prospectively included. All patients underwent subsequent excision or ablation., Intervention: Percutaneous, image-guided, needle-based OCT followed by RMB in an outpatient setting under local anaesthesia., Outcome Measurements and Statistical Analysis: Accuracy and diagnostic yield, μ OCT correlated to resection pathology or second biopsy during ablation. Tables (2×2) for RMB, receiver operating characteristic curve for OCT. Mann-Whitney test to differentiate μ OCT of RMs., Results and Limitations: RMB diagnostic yield was 79% with sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 100%, 89%, 99%, and 100%, respectively. Diagnostic yield and added value of OCT to differentiate RCC from benign was 99% and 15%, respectively. Significant difference was observed in median μ OCT between benign RMs (3.2mm -1 , interquartile range [IQR]: 2.65-4.35) and RCCs (4.3mm -1 , IQR: 3.70-5.00), p=0.0171, and oncocytomas (3.38mm -1 , IQR: 2.68-3.95) and RCCs (4.3mm -1 , IQR: 3.70-5.00), p=0.0031. OCT showed sensitivity, specificity, positive predictive value. and NPV of 91%, 56%, 91%, and 56%, respectively, to differentiate benign RMs from RCCs and 92%, 67%, 95%, and 55%, respectively, to differentiate oncocytoma from RCC. Limitations include two reference standards and heterogeneity benign RMs., Conclusions: Compared with RMB, OCT has a higher diagnostic yield. OCT accurately distinguishes benign RMs from RCCs, and oncocytoma from RCCs, although specificity and NPV are lower., Patient Summary: Optical coherence tomography, a new optical scan, exhibits similar sensitivity and positive predictive value than renal mass biopsy, although lower specificity and negative predictive value. Optical coherence tomography has a higher diagnostic yield for diagnosing renal cell carcinoma., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Kidney-sparing surgery of upper tract urothelial carcinoma (UTUC) requires a stringent follow-up with frequent ureteroscopies. Triage testing could reduce the number of follow-up ureteroscopies and hence minimize the invasiveness of follow-up. The use of urine-based markers for triage seems appealing but should be feasible with selective urine from outpatient cystoscopy to maximize the reduction of invasiveness. In this study, the feasibility of UroVysion ® fluorescence in situ hybridization (FISH) for the detection of UTUC in 1 mL of selective urine is investigated. Ten consecutive patients with biopsy-proven UTUC and five patients with negative diagnostic ureteroscopy findings were included in this case-control study. During ureteroscopy, 1 mL of selective urine was collected passively with a ureteral splint for Urovysion® FISH. The FISH rater was blinded to any clinical information. The results of FISH were compared to the findings of concomitantly collected selective urine cytology and the patients' UTUC status. FISH was feasible in all samples with a sensitivity of 90% and a specificity of 80% for UTUC. In comparison, selective cytology resulted in a diagnostic yield of 87% with a sensitivity of 80% and a specificity of 67%. In conclusion, UTUC detection is feasible with FISH in 1 mL of passively collected selective urine. Thus from a technical point of view, FISH could be used as an outpatient triage test to decide if follow-up ureteroscopy is necessary after kidney-sparing surgery of UTUC. Evaluation of the diagnostic accuracy of FISH for the suggested pathway deserves further attention.

Background: Histologic grade is an important prognosticator in patients with non-muscle-invasive bladder cancer (NMIBC). Currently, 2 classifications for grade are widely used; the World Health Organization (WHO) 1973 and the WHO 2004. We compare inter-observer variability of both classifications and investigate which histologic criteria cause this variability. Furthermore, the prognostic value of both classifications was assessed., Patients and Methods: Three pathologists reviewed 328 bladder tissue samples of 232 patients with NMIBC in a blinded manner. WHO 1973 grade, WHO 2004 grade, histologic criteria of both classifications, and T-category were evaluated. Reproducibility was analyzed using the weighted Fleiss κ, association between criteria scores and grade with the χ 2  test, and time-to-recurrence and time-to-progression with the log-rank test and Cox regression., Results: Reproducibility of both classifications was poor. The WHO 2004 showed better reproducibility (κ = 0.35; 95% confidence interval (CI), 0.29-0.42) compared with the WHO 1973 as a 3-tiered (κ = 0.24; 95% CI, 0.19-0.28), but not as a 2-tiered (G1 + G2 vs. G3) classification (κ = 0.36; 95% CI, 0.29-0.42). Reproducibility of individual criteria was poor (κ range, -0.05 to 0.25). All criteria were associated with grade (P < .05). After a median follow-up of 60 months, 33 of 232 and 112 of 232 patients developed progression and recurrence, respectively. In 1 out of the 3 pathologists, progression was predicted by both the WHO 1973 grade and the WHO 2004 grade in multivariable analysis. Recurrence was not predicted by grade (multivariable)., Conclusions: Reproducibility of the WHO 2004 and WHO 1973 classification for grade are poor. Scoring of individual criteria is poorly reproducible, suggesting that descriptions of these criteria for grade are not specific. The prognostic value of both the WHO 1973 and the WHO 2004 differ per pathologist., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Early diagnosis of deep tissue injury remains problematic due to the complicated and multifactorial nature of damage induction and the many processes involved in damage development and recovery. In this paper, we present a comprehensive assessment of deep tissue injury development and remodeling in a rat model by multiparametric magnetic resonance imaging (MRI) and histopathology. The tibialis anterior muscle of rats was subjected to mechanical deformation for 2 h. Multiparametric in vivo MRI, consisting of T 2 , T 2 *, mean diffusivity (MD), and angiography measurements, was applied before, during, and directly after indentation as well as at several time points during a 14-day follow-up. MRI readouts were linked to histological analyses of the damaged tissue. The results showed dynamic change in various MRI parameters, reflecting the histopathological status of the tissue during damage induction and repair. Increased T 2 corresponded with edema, muscle cell damage, and inflammation. T 2 * was related to tissue perfusion, hemorrhage, and inflammation. MD increase and decrease was reported on the tissue's microstructural integrity and reflected muscle degeneration and edema as well as fibrosis. Angiography provided information on blockage of blood flow during deformation. Our results indicate that the effects of a single damage-causing event of only 2 h of deformation were present up to 14 days. The initial tissue response to deformation, as observed by MRI, starts at the edge of the indentation. The quantitative MRI readouts provided distinct and complementary information on the extent, temporal evolution, and microstructural basis of deep tissue injury-related muscle damage. NEW & NOTEWORTHY We have applied a multiparametric MRI approach linked to histopathology to characterize damage development and remodeling in a rat model of deep tissue injury. Our approach provided several relevant insights in deep tissue injury. Response to damage, as observed by MRI, started at some distance from the deformation. Damage after a single indentation period persisted up to 14 days. The MRI parameters provided distinct and complementary information on the microstructural basis of the damage.

Background: The value of multiparametric magnetic resonance imaging (mpMRI) and targeted biopsy (TBx) remains controversial for biopsy-naïve men when compared to transrectal ultrasound (TRUS)-guided systematic biopsy (SBx). Risk-based patient selection could help to selectively identify men with significant prostate cancer (PCa) and thus reduce unnecessary mpMRI and biopsies., Objectives: To compare PCa detection rates for mpMRI TBx with SBx and to determine the rate of potentially avoided mpMRI and biopsies through risk-based selection using the Rotterdam Prostate Cancer Risk Calculator (RPCRC)., Design, Setting, and Participants: Two-hundred consecutive biopsy-naïve men in two centres underwent mpMRI scanning, 12-core SBx, and subsequent MRI-TRUS TBx in the case of suspicious lesion(s) (Prostate Imaging-Reporting and Data System v.2 score ≥3)., Outcome Measurements and Statistical Analysis: We measured the detection rate for high-grade (Gleason score ≥ 3+4) PCa for TBx and SBx. We carried out a retrospective stratification according to RPCRC biopsy advice to determine the rate of mpMRI and biopsies that could potentially be avoided by RPCRC-based patient selection in relation to the rate of high-grade PCa missed., Results and Limitations: TBx yielded high-grade PCa in 51 men (26%) and low-grade PCa in 14 men (7%), while SBx yielded high-grade PCa in 63 men (32%) and low-grade PCa in 41 men (21%). Four out of 73 men (5%) with negative RPCRC advice and 63 out of 127 men (50%) with positive advice had high-grade PCa. Upfront RPCRC-based patient selection for mpMRI and TBx would have avoided 73 out of 200 (37%) mpMRI scans, missing two out of 51 (4%) high-grade PCas. Limitations include the RPCRC definition of high- and low-grade PCa and different mpMRI techniques., Conclusions: mpMRI with TBx detected PCa with high Gleason score and avoided biopsy in low-grade PCa, but failed to detect all high-grade PCa when compared to SBx among biopsy-naïve men. Risk-based patient selection using the RPCRC can avoid one-third of mpMRI scans and SBx in biopsy-naïve men., Patient Summary: Men with a suspicion of prostate cancer are increasingly undergoing a magnetic resonance imaging (MRI) scan. Although promising, MRI-targeted biopsy is not accurate enough to safely replace systematic prostate biopsy for now. Individualised assessment of prostate cancer risk using the Rotterdam Prostate Cancer Risk Calculator could avoid one-third of MRI scans and systematic prostate biopsies., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: Focal therapy for prostate cancer has been proposed as an alternative treatment to whole-gland therapies in selected men to diminish side effects in localized prostate cancer. As nowadays imaging cannot offer complete prostate cancer disease characterization, multicore systematic biopsies are recommended (transrectal or transperineal). Optical imaging techniques such as confocal laser endomicroscopy and optical coherence tomography allow in vivo, high-resolution imaging. Moreover, they can provide real-time visualization and analysis of tissue and have the potential to offer additive diagnostic information., Objective: This study has 2 separate primary objectives. The first is to assess the technical feasibility and safety of in vivo focal imaging with confocal laser endomicroscopy and optical coherence tomography. The second is to identify and define characteristics of prostate cancer and normal prostate tissue in confocal laser endomicroscopy and optical coherence tomography imaging by comparing these images with the corresponding histopathology., Methods: In this prospective, in vivo feasibility study, needle-based confocal laser endomicroscopy and optical coherence tomography imaging will be performed before transperineal template mapping biopsy or radical prostatectomy. First, confocal laser endomicroscopy and optical coherence tomography will be performed in 4 patients (2 for each imaging modality) undergoing transperineal template mapping biopsy to assess the feasibility and safety of confocal laser endomicroscopy and optical coherence tomography. If proven to be safe and feasible, confocal laser endomicroscopy and optical coherence tomography will be performed in 10 patients (5 for each imaging modality) undergoing radical prostatectomy. Confocal laser endomicroscopy and optical coherence tomography images will be analyzed by independent, blinded observers. Confocal laser endomicroscopy- and optical coherence tomography-based qualitative and quantitative characteristics and histopathology will be compared. The study complies with the IDEAL (Idea, Development, Exploration, Assessment, Long-term study) stage 2a recommendations., Results: At present, the study is enrolling patients and results and outcomes are expected in 2019., Conclusions: Confocal laser endomicroscopy and optical coherence tomography are promising optical imaging techniques that can visualize and analyze tissue structure, possible tumor grade, and architecture in real time. They can potentially provide real-time, high-resolution microscopic imaging and tissue characteristics of prostate cancer in conjunction with magnetic resonance imaging or transrectal ultrasound fusion-guided biopsy procedures. This study will provide insight into the feasibility and tissue-specific characteristics of confocal laser endomicroscopy and optical coherence tomography for real-time optical analysis of prostate cancer., Trial Registration: ClinicalTrials.gov NCT03253458; https://clinicaltrials.gov/ct2/show/NCT03253458 (Archived by WebCite at http://www.webcitation.org/6z9owM66B)., Registered Report Identifier: RR1-10.2196/9813., (©Abel Swaan, Christophe K Mannaerts, Matthijs JV Scheltema, Jakko A Nieuwenhuijzen, C Dilara Savci-Heijink, Jean JMCH de la Rosette, R Jeroen A van Moorselaar, Ton G van Leeuwen, Theo M de Reijke, Daniel Martijn de Bruin. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 21.05.2018.)

Due to the growing field of digital pathology, more and more digital histology slides are becoming available. This improves the accessibility, allows teleconsultations from specialized pathologists, improves education, and might give urologist the possibility to review the slides in patient management systems. Moreover, by stacking multiple two-dimensional (2D) digital slides, three-dimensional volumes can be created, allowing improved insight in the growth pattern of a tumor. With the addition of computer-aided diagnosis systems, pathologist can be guided to regions of interest, potentially reducing the workload and interobserver variation. Digital (3D) pathology has the potential to improve dialog between the pathologist and urologist, and, therefore, results in a better treatment selection for urologic patients.

Background: Visual confirmation of a suspicious lesion in the urinary tract is a major corner stone in diagnosing urothelial carcinoma. However, during cystoscopy (for bladder tumors) and ureterorenoscopy (for tumors of the upper urinary tract) no real-time histopathologic information can be obtained. Confocal laser endomicroscopy (CLE) is an optical imaging technique that allows for in vivo high-resolution imaging and may allow real-time tumor grading of urothelial lesions., Objective: The primary objective of both studies is to develop descriptive criteria for in vivo CLE images of urothelial carcinoma (low-grade, high-grade, carcinoma in situ) and normal urothelium by comparing CLE images with corresponding histopathology., Methods: In these two prospective clinical trials, CLE imaging will be performed of suspicious lesions and normal tissue in the urinary tract during surgery, prior to resection or biopsy. In the bladder study, CLE will be performed in 60 patients using the Cystoflex UHD-R probe. In the upper urinary tract study, CLE will be performed in 25 patients during ureterorenoscopy, who will undergo radical treatment (nephroureterectomy or segmental ureter resection) thereafter. All CLE images will be analyzed frame by frame by three independent, blinded observers. Histopathology and CLE-based diagnosis of the lesions will be evaluated. Both studies comply with the IDEAL stage 2b recommendations., Results: Presently, recruitment of patients is ongoing in both studies. Results and outcomes are expected in 2018., Conclusions: For development of CLE-based diagnosis of urothelial carcinoma in the bladder and the upper urinary tract, a structured conduct of research is required. This study will provide more insight in tissue-specific CLE criteria for real-time tumor grading of urothelial carcinoma., Trial Registration: Confocal Laser Endomicroscopy: ClinicalTrials.gov NCT03013894; https://clinicaltrials.gov /ct2/show/NCT03013894?term=NCT03013894&rank=1 (Archived by WebCite at http://www.webcitation.org/6wiPZ378I); and Dutch Central Committee on Research Involving Human Subjects NL55537.018.15; https://www.toetsingonline.nl /to/ccmo&#95;search.nsf/fABRpop?readform&unids=6B72AE6EB0FC3C2FC125821F001B45C6 (Archived by WebCite at http://www.webcitation.org/6wwJQvqWh). Confocal Laser Endomicroscopy in the upper urinary tract: ClinicalTrials.gov NCT03013920; https://clinicaltrials.gov/ct2/show/NCT03013920? term=NCT03013920&rank=1 (Archived by WebCite at http://www.webcitation.org/6wiPkjyt0); and Dutch Central Committee on Research Involving Human Subjects NL52989.018.16; https://www.toetsingonline.nl/to/ccmo&#95;search.nsf/fABRpop?readform&unids=D27C9C3E5755CFECC12581690016779F (Archived by WebCite at http://www.webcitation.org/6wvy8R44C)., (©Esmee IML Liem, Jan Erik Freund, Joyce Baard, D Martijn de Bruin, M Pilar Laguna Pes, C Dilara Savci-Heijink, Ton G van Leeuwen, Theo M de Reijke, Jean JMCH de la Rosette. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 07.02.2018.)

Background: To better predict the likelihood of response to chemotherapy, we have conducted a study comparing the gene expression patterns of primary tumours with their corresponding response to systemic chemotherapy in the metastatic setting., Methods: mRNA expression profiles of breast carcinomas of patients that later developed distant metastases were analyzed using supervised and non-supervised classification techniques to identify predictors of response to chemotherapy. The top differentially expressed genes between the responders and non-responders were identified and further explored. An independent dataset which was generated to predict response to neo-adjuvant CT was utilized for the purpose of validation. Response to chemotherapy was also correlated to the clinicopathologic characteristics, molecular subtypes, metastatic behavior and survival outcomes., Results: Anthracycline containing regimens were the most common first line treatment (58.4%), followed by non-anthracycline/non-taxane containing (25.8%) and taxane containing (15.7%) regimens. Response was achieved in 41.6% of the patients to the first line CT and in 21.8% to second line CT. Response was not found to be significantly correlated to tumour type, grade, lymph node status, ER and PR status. Patients with HER2+ tumours showed better response to anthracycline containing therapy (p: 0.002). Response to first and second line chemotherapy did not differ among gene expression based molecular subtypes (p: 0.236 and p: 0.20). Using supervised classification, a 14 gene response classifier was identified. This 14-gene predictor could successfully predict the likelihood of better response to first and second line CT (p: <.0001 and p: 0.761, respectively) in the training set. However, the predictive value of this gene set in data of response to neoadjuvant chemotherapy could not be validated., Conclusions: To our knowledge, this is the first study revealing the relation between gene expression profiles of the primary tumours and their chemotherapy responsiveness in the metastatic setting. In contrast to the findings for neoadjuvant chemotherapy treatment, there was no association of molecular subtype with response to chemotherapy in the metastatic setting. Using supervised classification, we identified a classifier of chemotherapy response; however, we could not validate this classifier using neoadjuvant response data., Trial Registration: Non applicable. Subjects were retrospectively registered.

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma ( n = 46), Ewing sarcoma ( n = 32), alveolar rhabdomyosarcoma ( n = 20), embryonal rhabdomyosarcoma ( n = 77), synovial sarcoma ( n = 22) and desmoplastic small round cell tumors (DSRCT) ( n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described., Competing Interests: CONFLICTS OF INTEREST No conflicts of interest were disclosed

Purpose: Irreversible electroporation is a tissue ablation modality that uses high voltage electric energy to induce an increase in cell membrane permeability. This causes destabilization of the existing cellular transmembrane potential leading to cell death, due to the inability to maintain cellular homeostasis. This phase I-II study was designed to evaluate the histopathological outcomes of irreversible electroporation to prostate and surrounding tissue in radical prostatectomy specimens., Materials and Methods: Sixteen patients with prostate cancer underwent an irreversible electroporation ablation without curative intent, followed by radical prostatectomy scheduled 4 weeks later. For histopathological examination of the prostate, whole mounted tissue slices were examined by dedicated genitourinary pathologists. The borders of the ablation zone and residual tumor were outlined on the slides., Results: The irreversible electroporation ablation zones were characterized as areas of fibrosis, necrosis and loss of epithelial tissue in terms of denudation in the glandular structures. The ablation zone was well demarcated, showing trenchant delineations between viable and nonviable tissue. The ablated tissue showed mild to moderate inflammation, with atrophic cells in 1 case. The area was surrounded by hemorrhage at the location of the electrodes. No skip lesions or viable tissue was seen in the ablation zone. Fibrinoid necrosis of the neurovascular bundle was observed in 13 patients and denudation of the urothelium of the prostatic urethra was seen in 9., Conclusions: Histopathological assessment of the prostate 4 weeks after irreversible electroporation ablation showed sharply demarcated fibrotic and necrotic tissue in the ablation zone. No viable tissue was observed in the irreversible electroporation ablation zone., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objectives: Irreversible electroporation (IRE) is an ablative therapy with a low side-effect profile in prostate cancer. The objective was: 1) To compare the volumetric IRE ablation zone on grey-scale transrectal ultrasound (TRUS), contrast-enhanced ultrasound (CEUS) and multiparametric MRI (mpMRI) with histopathology findings; 2) To determine a reliable imaging modality to visualize the IRE ablation effects accurately., Methods: A prospective phase I-II study was performed in 16 patients scheduled for radical prostatectomy (RP). IRE of the prostate was performed 4 weeks before RP. Prior to, and 4 weeks after the IRE treatment, imaging was performed by TRUS, CEUS, and mpMRI. 3D-analysis of the ablation volumes on imaging and on H&E-stained whole-mount sections was performed. The volumes were compared and the correlation was calculated., Results: Evaluation of the imaging demonstrated that with T2-weighted MRI, dynamic contrast enhanced (DCE) MRI, and CEUS, effects of IRE are visible. T2MRI and CEUS closely match the volumes on histopathology (Pearson correlation r = 0.88 resp. 0.80). However, IRE is not visible with TRUS., Conclusions: mpMRI and CEUS are appropriate for assessing IRE effects and are the most feasible imaging modalities to visualize IRE ablation zone. The imaging is concordant with results of histopathological examination., Key Points: • mpMRI and contrast-enhanced ultrasound are appropriate imaging modalities for assessing IRE effects • mpMRI and CEUS are the most feasible imaging modalities to visualize IRE ablation zone • The imaging is concordant with results of histopathological examination after IRE • Grey-scale US is insufficient for assessing IRE ablations.

Purpose: Irreversible electroporation (IRE) is a novel minimally invasive therapy for prostate cancer using short electric pulses to ablate prostate tissue. The purpose of this study is to determine the IRE effects in prostate tissue and correlate electrode configuration with the histology of radical prostatectomy (RP) specimens. We hypothesize that the area within the electrode configuration is completely ablated and that the area within the electrode configuration is predictive for the ablated area after treatment., Methods: A prospective phase I/II study was conducted in 16 consecutive patients with histopathologically confirmed prostate cancer scheduled for RP. Focal or extended IRE treatment of the prostate was performed 4 weeks prior to RP. The locations of the electrodes were used to calculate the planned ablation zone. Following RP, the specimens were processed into whole-mount sections, histopathology (PA) was assessed and ablation zones were delineated. The area of the tissue alteration was determined by measuring the surface. The planned and the histological ablation zones were compared, analysed per individual patient and per protocol (focal vs. extended)., Results: All cells within the electrode configuration were completely ablated and consisted only of necrotic and fibrotic tissue without leaving any viable cells. The histological ablation zone was always larger than the electrodes configuration (2.9 times larger for the 3 electrodes configuration and 2.5 times larger for the ≥4 electrode configuration). These ablation effects extended beyond the prostatic capsule in the neurovascular bundle in 13 out of 15 cases., Conclusions: IRE in prostate cancer results in completely ablated, sharply demarcated lesions with a histological ablation zone beyond the electrode configuration. No skip lesions were observed within the electrode configuration., Clinical Trials: ClinicalTrials.gov Identifier: NCT01790451 https://clinicaltrials.gov/ct2/show/NCT01790451.

Purpose: We determine the ability of percutaneous needle based optical coherence tomography to differentiate renal masses by using the attenuation coefficient (μOCT, mm(-1)) as a quantitative measure., Materials and Methods: Percutaneous needle based optical coherence tomography of the kidney was performed in patients presenting with a solid renal mass. A pathology specimen was acquired in the form of biopsies and/or a resection specimen. Optical coherence tomography results of 40 patients were correlated to pathology results of the resected specimens in order to derive μOCT values corresponding with oncocytoma and renal cell carcinoma, and with the 3 main subgroups of renal cell carcinoma. The sensitivity and specificity of optical coherence tomography in differentiating between oncocytoma and renal cell carcinoma were assessed through ROC analysis., Results: The median μOCT of oncocytoma (3.38 mm(-1)) was significantly lower (p=0.043) than the median μOCT of renal cell carcinoma (4.37 mm(-1)). ROC analysis showed a μOCT cutoff value of greater than 3.8 mm(-1) to yield a sensitivity, specificity, positive predictive value and negative predictive value of 86%, 75%, 97% and 37%, respectively, to differentiate between oncocytoma and renal cell carcinoma. The area under the ROC curve was 0.81. Median μOCT was significantly lower for oncocytoma vs clear cell renal cell carcinoma (3.38 vs 4.36 mm(-1), p=0.049) and for oncocytoma vs papillary renal cell carcinoma (3.38 vs 4.79 mm(-1), p=0.027)., Conclusions: We demonstrated that the μOCT is significantly higher in renal cell carcinoma vs oncocytoma, with ROC analysis showing promising results for their differentiation. This demonstrates the potential of percutaneous needle based optical coherence tomography to help in the differentiation of renal masses, thus warranting ongoing research., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Metastatic cancer remains the leading cause of death for patients with breast cancer. To understand the mechanisms underlying the development of distant metastases to specific sites is therefore important and of potential clinical value. From 157 primary breast tumours of the patients with known metastatic disease, gene expression profiling data were generated and correlated to metastatic behaviour including site-specific metastasis, metastasis pattern and survival outcomes. We analysed gene expression signatures specifically associated with the development of bone metastases. As a validation cohort, we used a published dataset of 376 breast carcinomas for which gene expression data and site-specific metastasis information were available. 80.5 % of luminal-type tumours developed bone metastasis as opposed to 41.7 % of basal and 55.6 % of HER2-like tumours. A novel 15-gene signature identified 82.4 % of the tumours with bone metastasis, 85.2 % of the tumours which had bone metastasis as first site of metastasis and 100 % of the ones with bone metastasis only (p 9.99e-09), in the training set. In the independent dataset, 81.2 % of the positive tested tumours had known metastatic disease to the bone (p 4.28e-10). This 15-gene signature showed much better correlation with the development of bone metastases than previously identified signatures and was predictive in both ER-positive as well as in ER-negative tumours. Multivariate analyses revealed that together with the molecular subtype, our 15-gene expression signature was significantly correlated to bone metastasis status (p <0.001, 95 % CI 3.86-48.02 in the training set; p 0.001, 95 % CI 1.54-5.00 in the independent set). The 15 genes, APOPEC3B, ATL2, BBS1, C6orf61, C6orf167, MMS22L, KCNS1, MFAP3L, NIP7, NUP155, PALM2, PH-4, PGD5, SFT2D2 and STEAP3, encoded mainly membrane-bound molecules with molecular function of protein binding. The expression levels of the up-regulated genes (NAT1, BBS1 and PH-4) were also found to be correlated to epithelial to mesenchymal transition status of the tumour. We have identified a novel 15-gene expression signature associated with the development of bone metastases in breast cancer patients. This bone metastasis signature is the first to be identified using a supervised classification approach in a large series of patients and will help forward research in this area towards clinical applications.

Among breast cancer patients who develop distant metastases, there is marked variability in the clinical course, including metastasis pattern. Here, we present a retrospective study of breast cancer patients who all developed distant metastases focusing on the association between breast cancer subtype and clinical course, including organ-specific metastasis. Tissue microarrays (TMAs) were assembled and stained for ER, PR, HER2, EGFR, CK5/6, CK14, E-Cadherin, TP53 and Ki67 for 263 breast cancer patients with metastatic disease. Tumours were classified into ER+/HER2-/Ki67high, ER+/HER2-/Ki67low, ER+/HER2+, ER-/HER2+ and ER-/HER2- groups. Relevant data related to metastasis pattern, metastasis timeline, systemic treatment and survival were retrieved. Associations between site-specific relapse and patient/tumour characteristics were assessed with multivariate models using logistic regression. Median time for development of distant metastasis was 30 months (range 0-15.3 years); 75.8 % of the distance metastases developed in the first 5 years after treatment of the primary tumour. Patients with ER-/HER2- tumours had a median overall survival of 27 months; those with HER2+ tumours of 52 months; those with ER+/HER2-/Ki67high of 76 months and those with ER+/HER2-/Ki67low of 79 months. Bone was the most common site for distant metastasis (70.6 %) followed by liver (54.5 %) and lung (31.4 %), respectively. Visceral metastasis was found in 76.8 % of the patients. Patients with ER-/HER2- tumours developed visceral metastases in 81 % and bone metastases in 55.2 %; those with HER2+ tumours developed visceral metastases in 77.4 % and bone metastases in 69.8 %; those with ER+/HER2-/Ki67high developed visceral metastases in 75.7 % and bone metastases in 87.8 % and those with ER+/HER2-/Ki67low developed visceral metastases in 76.9 % and bone metastases in 73.1 %. In metastatic breast cancer patients, tumour subtypes are associated with survival and pattern of distant metastases. These associations are of help in choices for surveillance and therapy in individual patients.

Background: Electroporation is a novel treatment technique utilizing electric pulses, traveling between two or more electrodes, to ablate targeted tissue. The first in human studies have proven the safety of IRE for the ablation of renal masses. However the efficacy of IRE through histopathological examination of an ablated renal tumour has not yet been studied. Before progressing to a long-term IRE follow-up study it is vital to have pathological confirmation of the efficacy of the technique. Furthermore, follow-up after IRE ablation requires a validated imaging modality. The primary objectives of this study are the safety and the efficacy of IRE ablation of renal masses. The secondary objectives are the efficacy of MRI and CEUS in the imaging of ablation result., Methods/design: 10 patients, age ≥ 18 years, presenting with a solid enhancing mass, who are candidates for radical nephrectomy will undergo IRE ablation 4 weeks prior to radical nephrectomy. MRI and CEUS imaging will be performed at baseline, one week and four weeks post IRE. After radical nephrectomy, pathological examination will be performed to evaluate IRE ablation success., Discussion: The only way to truly assess short-term (4 weeks) ablation success is by histopathology of a resection specimen. In our opinion this trial will provide essential knowledge on the safety and efficacy of IRE of renal masses, guiding future research of this promising ablative technique., Trial Registration: Clinicaltrials.gov registration number NCT02298608 . Dutch Central Committee on Research Involving Human Subjects registration number NL44785.018.13.

Introduction: Current surgical and ablative treatment options for prostate cancer have a relatively high incidence of side effects, which may diminish the quality of life. The side effects are a consequence of procedure-related damage of the blood vessels, bowel, urethra or neurovascular bundle. Ablation with irreversible electroporation (IRE) has shown to be effective in destroying tumour cells and harbours the advantage of sparing surrounding tissue and vital structures. The aim of the study is to evaluate the safety and efficacy and to acquire data on patient experience of minimally invasive, transperineally image-guided IRE for the focal ablation of prostate cancer., Methods and Analysis: In this multicentre pilot study, 16 patients with prostate cancer who are scheduled for a radical prostatectomy will undergo an IRE procedure, approximately 30 days prior to the radical prostatectomy. Data as adverse events, side effects, functional outcomes, pain and quality of life will be collected and patients will be controlled at 1 and 2 weeks post-IRE, 1 day preprostatectomy and postprostatectomy. Prior to the IRE procedure and the radical prostatectomy, all patients will undergo a multiparametric MRI and contrast-enhanced ultrasound of the prostate. The efficacy of ablation will be determined by whole mount histopathological examination, which will be correlated with the imaging of the ablation zone., Ethics and Dissemination: The protocol is approved by the ethics committee at the coordinating centre (Academic Medical Center (AMC) Amsterdam) and by the local Institutional Review Board at the participating centres. Data will be presented at international conferences and published in peer-reviewed journals., Conclusions: This pilot study will determine the safety and efficacy of IRE in the prostate. It will show the radiological and histopathological effects of IRE ablations and it will provide data to construct an accurate treatment planning tool for IRE in prostate tissue., Trial Registration Number: Clinicaltrials.gov database: NCT01790451., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Aberrant expression of human leukocyte antigens (HLA) class I has prognostic importance in various cancers. Here, we evaluated the prognostic value of classical (A/B/C) and nonclassical (G/E) HLA expression in 169 high grade epithelial ovarian cancer samples and linked that to clinicopathological characteristics and survival. Expression of HLA-A, -B/C, or -E was not correlated with survival. Survival was prolonged when tumours expressed HLA-G (P = 0.008) and HLA-G was an independent predictor for better survival (P = 0.011). In addition, HLA-G expression was associated with longer progression-free survival (P = 0.036) and response to chemotherapy (P = 0.014). Accordingly, high expression of HLA-G mRNA was associated with prolonged disease-free survival (P = 0.037) in 65 corresponding samples. Elevated serum-soluble HLA-G levels as measured by enzyme-linked immunosorbent assay in 50 matched patients were not correlated to HLA-G protein expression or gene expression nor with survival. During treatment, sHLA-G levels declined (P = 0.038). In conclusion, expression of HLA-G is an independent prognostic factor for improved survival in high grade epithelial ovarian cancer and a predictor for platinum sensitivity.

The genetic alterations in breast cancer have in recent years been studied through a variety of techniques: analysis of alterations in individual oncogenes and tumor suppressor genes; gene expression profiling of both messenger RNA and microRNA; global analysis of DNA copy number changes; and most recently, whole-genome sequence analysis. Analysis of the association between genetic alterations and gene expression profiles with prognosis and response to specific treatments will lead to improved possibilities for patient-tailored treatment. Russnes et al. now add an additional view on the complex genetic makeup of breast carcinomas by developing algorithms that can be used to subclassify tumors based on their patterns of genome-wide DNA copy number gains and losses, which vary from very simple (only a few gains and losses) to complex. The algorithms provide indices that can be used in conjunction with results from other genetic analyses to subclassify breast cancer, with the aim of defining subgroups of patients that differ with respect to prognosis and response to therapy.

Results from several microarray-based studies have led to the identification of up-regulated expression levels of the DSG3 gene in pulmonary squamous cell carcinomas (SQCCs). The purpose of this study was to determine the role of DSG3 expression in the diagnosis of SQCCs of the lung and to compare DSG3 with p63, CK5, and CK6, as markers of squamous cell differentiation. Expression of DSG3 mRNA was evaluated in bulk laser capture microdissection-derived microarray data and by quantitative reverse transcription PCR on both SQCCs and adenocarcinomas. Expression levels of p63, CK5, and CK6 were evaluated in microarray data from the same set. An immunohistochemical study using antibodies directed against DSG3, p63, and CK5/6 was also performed. DSG3 was over-expressed in SQCCs but had very limited expression in both adenocarcinomas and non-neoplastic lungs. The microarray data showed that DSG3 had a sensitivity and specificity of 88% and 98%, respectively, in detecting SQCC versus adenocarcinoma. In comparison, sensitivity and specificity was 92% and 82% for p63, and 85% and 96% for CK5, respectively. The correlation coefficient between the microarray and immunohistochemical data for these genes was greater than or equal to 0.9. Using immunohistochemistry, sensitivity and specificity of DSG3 for lung cancers were 98% and 99%, respectively. Therefore, DSG3 can be a useful ancillary marker to separate SQCC from other subtypes of lung cancer.

Purpose: This paper describes a process for the identification of genes that can report on the aggressiveness of prostate tumors and thereby add to the information provided by current pathologic analysis., Materials and Methods: Expression profiling data from over 100 laser capture microdissection derived samples from nonneoplastic epithelium; Gleason patterns 3, 4, and 5 and node metastasis prostate cancer were used to identify genes at abnormally high levels in only some tumors. These variably overexpressed genes were stratified by their association with aggressive phenotypes and were subsequently filtered to exclude genes with redundant expression patterns. Selected genes were validated in a case-control study in which cases (systemic progression within 5 years) and controls (no systemic progression at 7 years of follow-up) were matched for all clinical and pathologic criteria from time of prostatectomy (n = 175). Both cases and controls, therefore, could have nodal invasion or seminal vesicle involvement at the time of initial treatment., Results: A number of candidate variably overexpressed genes selected for their association with aggressive prostate cancer phenotype were evaluated in the case control study. The most prominent candidates were SSTR1 and genes related to proliferation, including TOP2A., Conclusions: The process described here identified genes that add information not available from current clinical measures and can improve the prognosis of prostate cancer.

Background: To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data., Results: RNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II reverse transcriptase was replaced with SuperScript III, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip(R) IVT labeling kit was used rather than the Enzo BioArray HighYield RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 mug of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA., Conclusion: Unsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR) and hepsin was confirmed using quantitative PCR.