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Purpose: It has been suggested that symptomatic UA requires surgical excision. However, the management of asymptomatic urachus is still controversial. We aimed to evaluate the clinical presentation, the efficacy of current modalities used, and postoperative pathology in patients with UA. Material and Methods: We have performed a retrospective review of all patients diagnosed with UA and treated surgically or conservatively over 18 years. Demographic data, clinical presentation, imaging modalities, pathology, treatment, and postoperative complications were analyzed. Results: Twenty-five symptomatic patients (18 males and seven females) with a median age of 13 years (one month to 37 years) were identified. 15 (60%) were diagnosed with a urachal cyst, 4(16%) with sinus, 3(12%) with urachal diverticulum, and the remaining 3(12%) with patent urachus. Of those, 20(80%) underwent surgical repair, and the remaining five (20%) patients were managed conservatively. 4 (20%) underwent laparotomy, 7 (35%) laparoscopic incision, and the remaining 9 (45%) laparoscopic robotic-assisted surgery. Nine patients required bladder cuff excision. The median operative time was 75 minutes (42-140 min). One patient developed Clavien Dindo grade IIIA complication resulting in infected hematoma, which resolved after drainage. Another patient with a complication of grade IIIB needed reoperation as a result of recurrent events of an abscess. 13 (65%) demonstrated epithelium lining of the urachus on postoperative pathology.Conclusions: Our data show that most of the patients with UA presented with epithelial lining, which might lead to the later malignant transformation. It might cause a shift from the conservative management of asymptomatic patients to surgical intervention. Robotic-assisted surgery appears beneficial in these patients, especially when the bladder cuff excision is required.

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity, two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

The use of hygroscopic sonographically detectable clips (HSDCs) has dramatically increased during the last years, especially in breast cancer patients who undergo neoadjuvant chemotherapy. The aims of this study are to define the appearance of HSDC sites in histopathological specimens, and to enable pathologists to recognize these sites and differentiate them from other lesions. We examined 124 breast cancer specimens in which the application of HSDCs was documented, 88 breast tissues and 36 lymph nodes, and analyzed the appearance of the clip site in these tissues. The clip site was clearly detected histologically in 79/88 (90%) of the breast specimens and in 29/36 (81%) of lymph node specimens. In most of the specimens, the HSDC site had a specific characteristic appearance of a pseudocyst, lined by layers of epithelioid histiocytes, sometimes with pseudopapillary formation, and with minimal or no fibrosis. This was the appearance in 69 of the breast specimens and in 23 of the lymph node specimens. In other specimens, scarring, scattered foamy macrophages and abundant siderophages were the predominant findings, as usually found in sites of other clips. As non-palpable breast lesions become more frequent, clips play a major role in the treatment of breast cancer, making them an important component of the communication among radiologists, surgeons, pathologists, and oncologists. HSDCs in tissues have a characteristic appearance with an epithelioid component. Pathologists should be able to recognize this finding, differentiate it from other breast lesions and include it in the pathology report.

Fyn kinase is implicated in prostate cancer. We illustrate the role of miR-125a-3p in cellular pathways accounted for motility and migration of prostate cancer cells, probably through its regulation on Fyn expression and Fyn-downstream proteins. Prostate cancer PC3 cells were transiently transfected with empty miR-Vec (control) or with miR-125a-3p. Overexpression of miR-125a-3p reduced migration of PC3 cells and increased apoptosis. Live cell confocal imaging indicated that overexpression of miR-125a-3p reduced the cells' track speed and length and impaired phenotype. Fyn, FAK and paxillin, displayed reduced activity following miR-125a-3p overexpression. Accordingly, actin rearrangement and cells' protrusion formation were impaired. An inverse correlation between miR-125a-3p and Gleason score was observed in human prostate cancer tissues. Our study demonstrated that miR-125a-3p may regulate migration of prostate cancer cells.

Renal cancers account for more than 3% of adult malignancies and cause more than 13,000 deaths per year in the US alone. The four most common types of kidney tumors include the malignant renal cell carcinomas; clear cell, papillary, chromophobe and the benign oncocytoma. These histological subtypes vary in their clinical course and prognosis, and different clinical strategies have been developed for their management. In some kidney tumor cases it can be very difficult for the pathologist to distinguish between tumor types on the basis of morphology and immunohistochemistry (IHC). In this publication we present the development and validation of a microRNA-based assay for classifying primary kidney tumors. The assay, which classifies the four main kidney tumor types, was developed based on the expression of a set of 24 microRNAs. A validation set of 201 independent samples was classified using the assay and analyzed blindly. The assay produced results for 92% of the samples with an accuracy of 95%.

Objectives High-grade nonmuscle-invasive urothelial tumors of the bladder that fail intravesical Bacillus Calmette-Guerin (BCG) immunotherapy are at the highest risk of progression. Initial evidence links heat shock protein expression levels and outcome of bladder cancer after BCG treatment. We aimed to determine the association between HSP60, 70, and 90 expression levels and long-term outcomes of T1 high-grade (T1HG) urothelial bladder tumors treated with BCG immunotherapy. Materials and Methods Data of 54 consecutive patients with primary T1HG bladder tumors who underwent transurethral resection between 2002 and 2008 and received at least an induction course of BCG were reviewed. Immunohistochemical staining for heat shock protein (HSP)60, 70, and 90 were performed on resected specimens. Study outcomes included disease recurrence and progression. The association between HSP expression levels and outcomes were evaluated with univariable and multivariable Cox proportional hazards models. Results During a median follow-up of 9.6 years, 25 patients had a disease recurrence and 14 patients a disease progression. Estimated 5-year recurrence and progression-free survival were 59% and 81%, respectively. On multivariable analyses, HSP60 staining >65% was associated with a higher risk for progression (hazard ratio [HR] = 3.96, 95% confidence interval [CI] 1.35–11.58, P = 0.012), and HSP70 staining >5% was associated with a decreased risk for progression (HR = 0.33, 95% CI 0.11–0.98, P = 0.045), and recurrence (HR = 0.29, 95% CI 0.13–0.65, P = 0.003). HSP90 expression was not associated with disease recurrence or progression. Five patients had both a HSP60 staining >65% and a HSP70 staining ≤5% all of whom recurred at a median time of 6 months (interquartile range 3, 16) and 80% of whom progressed at a median time of 26 months (interquartile range 5, 60). Conclusions HSP60 and 70 cellular expression levels are associated with long-term outcome following BCG treatment of T1HG urothelial bladder tumors. These findings, if further validated, may be used to better stratify the risk of disease recurrence and progression in this group of patients.

The detection of breast carcinoma cells in effusions is associated with rapidly fatal outcome, but these cells are poorly characterized at the molecular level. This study compared the gene array signatures of breast carcinoma cells in primary carcinomas and effusions. The genetic signature of 10 primary tumors and 10 effusions was analyzed using the Array-Ready Oligo set for the Human Genome platform. Results for selected genes were validated using PCR, Western blotting, and immunohistochemistry. Array analysis identified 255 significantly downregulated and 96 upregulated genes in the effusion samples. The majority of differentially expressed genes were part of pathways involved in focal adhesion, extracellular matrix-cell interaction, and the regulation of the actin cytoskeleton. Genes that were upregulated in effusions includedKRT8, BCAR1, CLDN4, VIL2, whileDCN, CLDN19, ITGA7, andITGA5were downregulated at this anatomic site. PCR, Western blotting, and immunohistochemistry confirmed the array findings forBCAR1, CLDN4, VIL2, andDCN. Our data show that breast carcinoma cells in primary carcinomas and effusions have different gene expression signatures, and differentially express a large number of molecules related to adhesion, motility, and metastasis. These differences may have a critical role in designing therapy and in prognostication for patients with metastatic disease localized to the serosal cavities.

Background: Renal cancers account for more than 3% of adult malignancies and result in more than 13,000 deaths per year in the US alone. The four most common types of kidney tumors include the malignant renal cell carcinomas: clear cell, papillary and chromophobe, as well as the benign oncocytoma. These histological subtypes vary in their clinical course and their prognosis, and different clinical strategies have been developed for their management. The differential diagnosis between the subtypes of kidney tumors based on morphology alone can be challenging, and is subjected to inter- and intra-observer variability. Even when utilizing immunohistochemistry (IHC) markers, the ability to differentiate between sub-types can be difficult, especially in the setting of uncommon morphology and biopsy sample with small amounts of tumor tissue. We present the development and validation of a microRNA-based test for classifying primary kidney tumors. Methods: 181 Formalin Fixed Paraffin Embedded (FFPE) samples from primary kidney tumors were collected and reviewed by pathologists from different institutes according to morphology and available IHC labeling data. High-quality total RNA, including the well-preserved microRNA fraction, was extracted from the FFPE samples using a proprietary protocol. Expression levels of hundreds of microRNAs were profiled using a custom microarray platform. Technical validation of the array results was performed using qRT-PCR. A diagnostic assay was developed using a K-nearest neighbor algorithm that searches for the 5 samples in the training database (181 samples used for assay development) that are most similar to the tested sample. The result for the tested sample is defined by a majority vote of the pre-determined subtypes of these 5 closest neighbors. A validation set of 201 independent samples was classified using the assay and analyzed blindly. Results: A set of 24 differentially expressed microRNAs were found to separate the four kidney tumor subtypes and were chosen as classifiers in the KNN algorithm. Clinical validation was performed using an independent, blinded sample set. The test was able to produce results for 92% of the validation set of 201 samples with accuracy of 95%. Conclusions: Expression levels of 24 microRNAs measured on a microarray platform were found to accurately differentiate the four main types of primary kidney tumors. These findings were the basis for the development and validation of a standardized diagnostic assay for the classification of renal cell tumors in FFPE samples from resections or biopsies. This assay can serve as a reliable diagnostic tool to aid physicians with the growing unmet need for kidney tumor classification. Citation Format: Robert Wassman, Brianna St. Cyr, Eddie Fridman, Iris Barshack, Yajue Huang, Sofia Zilber, Mats Sanden, Hila Benjamin, Noga Yerushalmi, Yael Spector. Development and validation of a microRNA-based diagnostic assay for the classification of renal cell tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 802. doi:10.1158/1538-7445.AM2013-802

Background: The SRC-family of kinases (SFKs) has been broadly implicated in the pathogenesis of prostate cancer. Fyn, a member of the SFKs, mediates mitogenic signals and regulates proliferation, adhesion and motility. Cumulative data indicate that Fyn serves as a proto-oncogene and is known to be over-expressed in several types of malignancies. MicroRNAs are noncoding RNAs that are aberrantly expressed in cancer and seem to influence tumor behavior and progression. In a previous study performed in human embryonic kidney (HEK 293T) cells, we have validated the binding specificity of miR-125a-3p and Fyn 3’UTR using Luciferase assay and established the regulation of Fyn expression and signaling by miR-125a-3p. In the current study we aimed to characterize the interplay between miRNA-125a-3p and Fyn in prostate cancer. Methods: Prostate cancer (PC3) cell line, were transiently transfected with empty miR-vec (control) or with miR-125a-3p and their cell cycle and apoptotic rate were measured by fluorescence activated cell sorting (FACS). The migration ability was measured by transwell assay and scratch assay. Proliferation was assessed by MTT assay. Quantitative PCR and western blot analysis were implicated to determine the effect of miR-125a-3p on Fyn expression as well as on the activation state of its downstream effectors/proteins FAK, Akt and paxillin. Extraction of miRNA from formalin fixed paraffin-embedded (FFPE) cancerous and matched adjacent prostate tissues was performed with miRNeasy FFPE Kit on a cohort of 20 human prostate cancer specimens. Results: Overexpression of miR-125a-3p in PC3 cells reduced significantly the expression of Fyn mRNA and protein and led to impaired cell viability to an arrest at the G2/M phase of the cell cycle. Moreover, overexpressing of miR-125a-3p reduced the activity of FAK, Akt and paxillin and the secretion of VEGF to the culture media. In miRNA125a-3p-overexpressing PC3 cells the migration was compromised by 50% and cell viability decreased by 50%. Moreover, we observed an inverse correlation between the expression of miR-125a-3p and the Gleason score of tissue samples obtained from patients diagnosed with prostate cancer, whereas the expression of miR-125a-3p was extremely reduced in Gleason score higher than 8. Conclusions: Our study indicates miR-125a-3p signature in cellular pathways accounted for cell viability and migration of prostate cancer cells. The interplay between miR-125a-3p and Fyn may represent a new plausible therapeutic target in prostate cancer. Citation Format: Lihi Ninio Many, Irit Ben-Aharon, Sofia Zilber, Mattan Levy, Salomon M. Stemmer, Ruth Shalgi. The role of miRNA-125a-Fyn interplay in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4168. doi:10.1158/1538-7445.AM2013-4168