Quantifying Y chromosome loss in primary and metastatic prostate cancer by chromosome painting.

Somatic Y chromosome loss in hematopoietic cells is associated with higher mortality in men. However, the status of the Y chromosome in cancer tissue is not fully known due to technical limitations, such as difficulties in labelling and sequencing DNA from the Y chromosome. We have developed a system to quantify Y chromosome gain or loss in patient-derived prostate cancer organoids. Using our system, we observed Y chromosome loss in 4 of the 13 (31%) patient-derived metastatic castration-resistant prostate cancer (mCRPC) organoids; interestingly, loss of Yq (long arm of the Y chromosome) was seen in 38% of patient-derived organoids. Additionally, potential associations were observed between mCRPC and Y chromosome nullisomy. The prevalence of Y chromosome loss was similar in primary and metastatic tissue, suggesting that Y chromosome loss is an early event in prostate cancer evolution and may not a result of drug resistance or organoid derivation. This study reports quantification of Y chromosome loss and gain in primary and metastatic prostate cancer tissue and lays the groundwork for further studies investigating the clinical relevance of Y chromosome loss or gain in mCRPC.

SLCO1B3 and SLCO2B1 genotypes, androgen deprivation therapy, and prostate cancer outcomes: a prospective cohort study and meta-analysis.

Solute carrier organic anion (SLCO) transporters (OATP transporters) are involved in cellular uptake of drugs and hormones. Germline variants in SLCO1B3 and SLCO2B1 have been implicated in prostate cancer progression and therapy response, including to androgen deprivation and statin medications, but results have appeared heterogeneous. We conducted a cohort study of five single-nucleotide polymorphisms (SNPs) in SLCO1B3 and SLCO2B1 with prior evidence among 3208 men with prostate cancer who participated in the Health Professionals Follow-up Study or the Physicians' Health Study, following participants prospectively after diagnosis over 32 years (median, 14 years) for development of metastases and cancer-specific death (lethal disease, 382 events). Results were suggestive of, but not conclusive for, associations between some SNPs and lethal disease and differences by androgen deprivation and statin use. All candidate SNPs were associated with SLCO mRNA expression in tumor-adjacent prostate tissue. We also conducted a systematic review and harmonized estimates for a dose-response meta-analysis of all available data, including 9 further studies, for a total of 5598 patients and 1473 clinical events. The A allele of the exonic SNP rs12422149 (14% prevalence), which leads to lower cellular testosterone precursor uptake via SLCO2B1, was associated with lower rates of prostate cancer progression (hazard ratio per A allele, 0.80; 95% confidence interval, 0.69-0.93), with little heterogeneity between studies (I2, 0.27). Collectively, the totality of evidence suggests a strong association between inherited genetic variation in SLCO2B1 and prostate cancer prognosis, with potential clinical use in risk stratification related to androgen deprivation therapy.

Polymer-mediated tuning of the monomer-aggregate equilibrium of a coumarin derivative for ratiometric sensing of protamine.

Amongst the various existing methods of analyte quantification, fluorescent-based methods, especially the ratiometric methods, continue to gain significant attention due to their high reproducibility, low environmental influence, and self-calibrating behavior. This paper presents the modulation in a monomer-aggregate equilibrium of coumarin-7 (C7) dye at pH ∼ 3, under the influence of a multi-anionic polymer, poly(styrene sulfonate) (PSS), leading to a significant modification in the ratiometric optical signal of the dye. At pH ∼ 3, cationic C7 formed aggregates in the presence of PSS via a strong electrostatic interaction, resulting in the development of a new emission peak at 650 nm at the expense of the monomer emission at 513 nm. Such contradicting changes in fluorescence intensities at two different wavelengths gave rise to a ratiometric signal, which was found to be highly sensitive towards external stimuli such as pH, and ionic strength. The stability of the C7-PSS complex was found to decrease as the pH of the solution was increased beyond 5, which indicated the decline in the electrostatic attraction between C7 and PSS due to the deprotonation of the C7 dye. Furthermore, an increase in the monomeric peak and a concomitant decrease of the aggregate peak with added salt in the solution (at pH ∼ 3) clearly justified the presence of an electrostatic attraction between C7 and PSS for the complex formation. This was further validated by the excited-state lifetime measurement of the C7-PSS complex, which showed a systematic increase in lifetime contribution from the monomeric species at the expense of aggregated species, as the concentration of NaCl increased in the solution. Thus, protamine (Pr), being a highly positively charged polypeptide, largely affected the monomer-aggregate equilibrium of the C7-PSS system, leading to a phenomenal change in the ratiometric signal, which was utilized to quantify with LOD as low as ∼2.8 nM in buffer for the bio-analyte Pr. Moreover, the ratiometric response of the C7-PSS assembly demonstrated excellent selectivity towards Pr, facilitating its practical relevance for the quantification of Pr in a 1% human serum matrix. Therefore, the studied C7-PSS can be utilized as a potential candidate for the quantification of the protamine even in complex biological media.

Grading of HCC Biopsy Images Using Nucleus and Texture Features.

Hepatocellular carcinoma (HCC) is one of the most critical health problems in the world. For proper treatment, it is important to identify the grade of cancer morbidity from HCC biopsy image. The diagnostic work is not only time-consuming but also subjective. The same biopsy image may be diagnosed as of different grades by different doctors, due to lack of experience or difference in opinion. In this work, we proposed an automatic grading system with classification accuracy matching to an experienced doctor, to help augment the diagnosis process. First, we proposed a segmentation method to isolate all nucleus-like objects present in a biopsy image. Non-target objects (here the target is a single HCC nucleus) present in the biopsy image are isolated too in the segmentation process. To eliminate such non-target objects, we proposed clustering of segmented images and a novel method to filter out target objects. Next, we proposed a two track neural network, where input consists of 2 different images. It combines a single segmented nucleus and a random cropped texture patch of the biopsy image to which the nucleus belongs. At this classifier output, we grade the single nucleus. Finally, a majority voting method is used to identify the grade of the whole biopsy image. We achieved an accuracy of 99.03% for nucleus image grading and 99.66% accuracy for grading biopsy images.

Increased MYBL2 expression in aggressive hormone-sensitive prostate cancer.

Loss of the histone demethylase KDM5D (lysine-specific demethylase 5D) leads to in vitro resistance of prostate cancer cells to androgen deprivation therapy (ADT) with and without docetaxel. We aimed to define downstream drivers of the KDM5D effect. Using chromatin immunoprecipitation sequencing (ChIP-seq) of the LNCaP cell line (androgen-sensitive human prostate adenocarcinoma) with and without silenced KDM5D, MYBL2-binding sites were analyzed. Associations between MYBL2 mRNA expression and clinical outcomes were assessed in cohorts of men with localized and metastatic hormone-sensitive prostate cancer. In vitro assays with silencing and overexpression of MYBL2 and KDM5D in androgen receptor (AR)-positive hormone-sensitive prostate cancer cell lines, LNCaP and LAPC4, were used to assess their influence on cellular proliferation, apoptosis, and cell cycle distribution, as well as sensitivity to androgen deprivation, docetaxel, and cabazitaxel. We found that silencing KDM5D increased histone H3 lysine K4 (H3K4) trimethylation and increased MYBL2 expression. KDM5D and MYBL2 were negatively correlated with some but not all clinical samples. Higher MYBL2 expression was associated with a higher rate of relapse in localized disease and poorer overall survival in men with metastatic disease in the CHAARTED trial. Lower MYBL2 levels enhanced LNCaP and LAPC4 sensitivity to androgen deprivation and taxanes. In vitro, modifications of KDM5D and MYBL2 altered cell cycle distribution and apoptosis in a cell line-specific manner. These results show that the transcription factor MYBL2 impacts in vitro hormone-sensitive prostate cancer sensitivity to androgen deprivation and taxanes, and lower levels are associated with better clinical outcomes in men with hormone-sensitive prostate cancer.

The Impact of PIK3R1 Mutations and Insulin-PI3K-Glycolytic Pathway Regulation in Prostate Cancer.

PURPOSE: Oncogenic alterations of the PI3K/AKT pathway occur in >40% of patients with metastatic castration-resistant prostate cancer, predominantly via PTEN loss. The significance of other PI3K pathway components in prostate cancer is largely unknown. EXPERIMENTAL DESIGN: Patients in this study underwent tumor sequencing using the MSK-IMPACT clinical assay to capture single-nucleotide variants, insertions, and deletions; copy-number alterations; and structural rearrangements, or were profiled through The Cancer Genome Atlas. The association between PIK3R1 alteration/expression and survival was evaluated using univariable and multivariable Cox proportional-hazards regression models. We used the siRNA-based knockdown of PIK3R1 for functional studies. FDG-PET/CT examinations were performed with a hybrid positron emission tomography (PET)/CT scanner for some prostate cancer patients in the MSK-IMPACT cohort. RESULTS: Analyzing 1,417 human prostate cancers, we found a significant enrichment of PIK3R1 alterations in metastatic cancers compared with primary cancers. PIK3R1 alterations or reduced mRNA expression tended to be associated with worse clinical outcomes in prostate cancer, particularly in primary disease, as well as in breast, gastric, and several other cancers. In prostate cancer cell lines, PIK3R1 knockdown resulted in increased cell proliferation and AKT activity, including insulin-stimulated AKT activity. In cell lines and organoids, PIK3R1 loss/mutation was associated with increased sensitivity to AKT inhibitors. PIK3R1-altered patient prostate tumors had increased uptake of the glucose analogue 18F-fluorodeoxyglucose in PET imaging, suggesting increased glycolysis. CONCLUSIONS: Our findings describe a novel genomic feature in metastatic prostate cancer and suggest that PIK3R1 alteration may be a key event for insulin-PI3K-glycolytic pathway regulation in prostate cancer.

Mesenchymal and stem-like prostate cancer linked to therapy-induced lineage plasticity and metastasis.

Bioinformatic analysis of 94 patient-derived xenografts (PDXs), cell lines, and organoids (PCOs) identifies three intrinsic transcriptional subtypes of metastatic castration-resistant prostate cancer: androgen receptor (AR) pathway + prostate cancer (PC) (ARPC), mesenchymal and stem-like PC (MSPC), and neuroendocrine PC (NEPC). A sizable proportion of castration-resistant and metastatic stage PC (M-CRPC) cases are admixtures of ARPC and MSPC. Analysis of clinical datasets and mechanistic studies indicates that MSPC arises from ARPC as a consequence of therapy-induced lineage plasticity. AR blockade with enzalutamide induces (1) transcriptional silencing of TP53 and hence dedifferentiation to a hybrid epithelial and mesenchymal and stem-like state and (2) inhibition of BMP signaling, which promotes resistance to AR inhibition. Enzalutamide-tolerant LNCaP cells re-enter the cell cycle in response to neuregulin and generate metastasis in mice. Combined inhibition of HER2/3 and AR or mTORC1 exhibits efficacy in models of ARPC and MSPC or MSPC, respectively. These results define MSPC, trace its origin to therapy-induced lineage plasticity, and reveal its sensitivity to HER2/3 inhibition.

Molecular Imaging of Neuroendocrine Prostate Cancer by Targeting Delta-Like Ligand 3.

Treatment-induced neuroendocrine prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer. Using the 89Zr-labeled delta-like ligand 3 (DLL3) targeting antibody SC16 (89Zr-desferrioxamine [DFO]-SC16), we have developed a PET agent to noninvasively identify the presence of DLL3-positive NEPC lesions. Methods: Quantitative polymerase chain reaction and immunohistochemistry were used to compare relative levels of androgen receptor (AR)-regulated markers and the NEPC marker DLL3 in a panel of prostate cancer cell lines. PET imaging with 89Zr-DFO-SC16, 68Ga-PSMA-11, and 68Ga-DOTATATE was performed on H660 NEPC-xenografted male nude mice. 89Zr-DFO-SC16 uptake was corroborated by biodistribution studies. Results: In vitro studies demonstrated that H660 NEPC cells are positive for DLL3 and negative for AR, prostate-specific antigen, and prostate-specific membrane antigen (PSMA) at both the transcriptional and the translational levels. PET imaging and biodistribution studies confirmed that 89Zr-DFO-SC16 uptake is restricted to H660 xenografts, with background uptake in non-NEPC lesions (both AR-dependent and AR-independent). Conversely, H660 xenografts cannot be detected with imaging agents targeting PSMA (68Ga-PSMA-11) or somatostatin receptor subtype 2 (68Ga-DOTATATE). Conclusion: These studies demonstrated that H660 NEPC cells selectively express DLL3 on their cell surface and can be noninvasively identified with 89Zr-DFO-SC16.

Waste derived approach towards wealthy fluorescent N-doped graphene quantum dots for cell imaging and H2O2 sensing applications.

Herein, we report the synthesis of a highly fluorescent nitrogen doped graphene quantum dots (N-GQDs) from waste precursors such as melamine sponge and arjuna bark via a microwave treatment and its functional and morphological characterization using various spectroscopy techniques such as optical, FTIR, XPS and TEM. The as-prepared aqueous N-GQD (dia. 2-3 nm) was used for the bio-imaging application using breast carcinoma cell line (MDA-MB-231) as a model, and the locations of all cells in the cytoplasm as well as nuclei were observed to stain brightly in blue fluorescent color successfully. In addition to that, the aqueous N-GQD showed fluorescence quenching behavior in the presence of hydrogen peroxide, which was exploited to sense H2O2, a probable toxin generated in the diseased cells. Importantly, the cell cytotoxicity was measured and found to be non-toxic (70% survival) to the MDA-MB-231 cells even at very high concentration (∼1.8 mg/ml) of the synthesized N-GQD. This study revealing excellent biocompatibility and imaging of the model cancer cells, and sensing of H2O2 by fluorescent quenching, indicates potential in-vivo cell culture applications of the prepared fluorescent N-GQD.

Prognostic and therapeutic significance of COP9 signalosome subunit CSN5 in prostate cancer.

Chromosome 8q gain is associated with poor clinical outcomes in prostate cancer, but the underlying biological mechanisms remain to be clarified. CSN5, a putative androgen receptor (AR) partner that is located on chromosome 8q, is the key subunit of the COP9 signalosome, which deactivates ubiquitin ligases. Deregulation of CSN5 could affect diverse cellular functions that contribute to tumor development, but there has been no comprehensive study of its function in prostate cancer. The clinical significance of CSN5 amplification/overexpression was evaluated in 16 prostate cancer clinical cohorts. Its oncogenic activity was assessed by genetic and pharmacologic perturbations of CSN5 activity in prostate cancer cell lines. The molecular mechanisms of CSN5 function were assessed, as was the efficacy of the CSN5 inhibitor CSN5i-3 in vitro and in vivo. Finally, the transcription cofactor activity of CSN5 in prostate cancer cells was determined. The prognostic significance of CSN5 amplification and overexpression in prostate cancer was independent of MYC amplification. Inhibition of CSN5 inhibited its oncogenic function by targeting AR signaling, DNA repair, multiple oncogenic pathways, and spliceosome regulation. Furthermore, inhibition of CSN5 repressed metabolic pathways, including oxidative phosphorylation and glycolysis in AR-negative prostate cancer cells. Targeting CSN5 with CSN5i-3 showed potent antitumor activity in vitro and in vivo. Importantly, CSN5i-3 synergizes with PARP inhibitors to inhibit prostate cancer cell growth. CSN5 functions as a transcription cofactor to cooperate with multiple transcription factors in prostate cancer. Inhibiting CSN5 strongly attenuates prostate cancer progression and could enhance PARP inhibition efficacy in the treatment of prostate cancer.

Carbon nano-dot for cancer studies as dual nano-sensor for imaging intracellular temperature or pH variation.

Cellular temperature and pH govern many cellular physiologies, especially of cancer cells. Besides, attaining higher cellular temperature plays key role in therapeutic efficacy of hyperthermia treatment of cancer. This requires bio-compatible, non-toxic and sensitive probe with dual sensing ability to detect temperature and pH variations. In this regard, fluorescence based nano-sensors for cancer studies play an important role. Therefore, a facile green synthesis of orange carbon nano-dots (CND) with high quantum yield of 90% was achieved and its application as dual nano-sensor for imaging intracellular temperature and pH was explored. CND was synthesized from readily available, bio-compatible citric acid and rhodamine 6G hydrazide using solvent-free and simple heating technique requiring purification by dialysis. Although the particle size of 19 nm (which is quite large for CND) was observed yet CND exhibits no surface defects leading to decrease in photoluminescence (PL). On the contrary, very high fluorescence was observed along with good photo-stability. Temperature and pH dependent fluorescence studies show linearity in fluorescence intensity which was replicated in breast cancer cells. In addition, molecular nature of PL of CND was established using pH dependent fluorescence study. Together, the current investigation showed synthesis of highly fluorescent orange CND, which acts as a sensitive bio-imaging probe: an optical nano-thermal or nano-pH sensor for cancer-related studies.

A simple strategy to reduce the salivary gland and kidney uptake of PSMA-targeting small molecule radiopharmaceuticals.

PURPOSE: Peptide-based prostate-specific membrane antigen (PSMA) targeted radionuclide therapy (TRT) agent [177Lu]-PSMA-617 has emerged as leading TRT candidate for treatment of castration-resistant prostate cancer (mCRPC). [177Lu]-PSMA-617 and other small molecule-based PSMA ligands have shown efficacy in reducing the tumor burden in mCRPC patients but irradiation to the salivary gland and kidneys is a concern and dose-limiting factor. Therefore, methods to reduce non-target organ toxicity are needed to safely treat patients and preserve their quality of life. Herein, we report that addition of cold PSMA ligand PSMA-11 can aid in reducing the uptake of [177Lu]-PSMA-617 in the salivary glands and kidneys. METHODS: Groups of athymic nude mice (n = 4) bearing PC3-PIP (PSMA+) tumor xenografts were administered with [177Lu]-PSMA-617 along with 0, 5, 100, 500, 1000, and 2000 pmoles of PSMA-11 and biodistribution studies were performed at 1 h. RESULTS: Biodistribution studies at 1 h post-administration revealed that [177Lu]-PSMA-617 uptake in PC3-PIP tumors was 21.71 ± 6.13, 18.7 ± 2.03, 26.44 ± 2.94, 16.21 ± 3.5, 13.52 ± 3.68, and 12.03 ± 1.96 %ID/g when 0, 5, 100, 500, 1000, and 2000 pmoles of PSMA-11 were added, respectively. Corresponding uptake values in kidney were 123.14 ± 52.52, 132.31 ± 47.4, 84.29 ± 78.25, 2.12 ± 1.88, 1.16 ± 0.36, and 0.64 ± 0.23 %ID/g, respectively. Corresponding salivary gland uptake values were 0.48 ± 0.11, 0.45 ± 0.15, 0.38 ± 0.3, 0.08 ± 0.03, 0.09 ± 0.07, and 0.05 ± 0.02 % ID/g, respectively. CONCLUSION: The uptake of [177Lu]-PSMA-617 in the salivary gland and kidney can be substantially reduced without significantly impacting tumor uptake by adding cold PSMA-11.

Gastric pull-up by the retrosternal route for esophageal replacement: Feasibility in a limited-resource scenario.

OBJECTIVES: The authors herein report the results of esophageal replacement by gastric pull-up technique through the retrosternal route as an option for esophageal replacement in a resource-constrained setup. METHOD: Prospectively collected data upon twenty-two consecutive patients (male:female = 17:5) with mean age 24.9 months (7 months-12 years) and mean weight 7.9 kg (4.2-32 kg) who underwent retrosternal gastric pull-up for esophageal atresia (n = 18; 16 atresia with distal fistula & 2 pure atresia) and corrosive injuries to the esophagus (n = 4) over the past 8 years are presented. The management protocol and surgical technique have been described. Observations parameters included indication for esophageal replacement, age at surgery, sex of the child and other demographic details, clinical and operative findings, post-operative outcomes and follow-up details. RESULTS: Retrosternal gastric pull-up could be performed in all cases with no mortality or graft loss. Of 22, 20 cases were extubated on-table and 2 cases were extubated within 48 hours of surgery. Mean operative duration was 265 min (range: 175 min to 310 min) and blood loss was 115.3 ml (range: 80-400 ml). Dense vascular adhesions in the region of the esophageal hiatus were encountered in patients with abdominal esophagostomy (n = 4) which were probably related to the local dissection at the time of previous surgery. Minor anastomotic leak was observed in 8 of 22 patients which settled spontaneously over 21 days mean period (range: 18 to 31 days). Antegrade dilatation was required in 3 of 8 cases with minor leak. None of them required revision of anastomosis. Mean follow-up duration is 63 months (range: 11 months - 94 months). Weight gain after surgery was close to or beyond the 25th centile. Symptoms of dumping syndrome or GER were not observed in our cohort. CONCLUSION: Our data have demonstrated the safety and feasibility of esophageal replacement by gastric transposition through the retrosternal route in a resource-limited setup. No significant difference has been observed from the results and complications reported in literature for the same procedure. TYPE OF STUDY: Prospective observational study / treatment study. LEVEL OF EVIDENCE: Level III.

Primary anastomosis in difficult cases of type "C" esophageal atresia: The atraumatic microvascular clamp technique of minimal tension with good outcome.

BACKGROUND: Surgical maneuvers for esophageal anastomosis in difficult cases of Gross type "C" esophageal atresia (EA) are challenging. The methods of early primary anastomosis are technically difficult and staged surgeries expose the child to repeated general anesthesia with problems of nursing care. We describe a simple method of partial disconnection of the lower esophagus from the fistula followed by approximation by an atraumatic microvascular clamp. The suitability of this method and its outcomes are discussed. METHODS: It was a prospective observational study that included 32 patients of type "C" EA between January 2014 and December 2016. Babies with birth weight more than 2 kg without cyanotic heart defects and requirement of intensive care were included. An early primary anastomosis using this technique was tried in all. A cervical esophagostomy with feeding gastrostomy was done where it was not possible. Analysis of the gap and post operative outcomes i.e. gastroesophageal reflux (GER), stricture, tracheomalacia, dysmotility, recurrence and survival were analyzed. RESULTS: The mean gap between esophageal ends was 4.3 cm. Primary anastomosis was possible in 26 (81.25%). Minor and major leak occurred in 3 (11.54%) and 1 (3.85%) patients respectively. Survival was 84.62% (22/26). All mortalities were early post operative. During mean follow up of 23.73 months (till December 2019), GER decreased from 63.64% (14/22) to 13.64% (3/22), partial stricture was seen in 18.18% (4/22), tracheomalacia in 36.36% (8/22) and dysmotility in 77.27% (17/22). There was no recurrence of fistula. Complications with this method did not show any significant difference as mentioned with other methods. CONCLUSION: This technique seems to be physiologically suitable as it enables anastomosis with minimal trauma to the esophageal ends. It is easy, reproducible and produces favorable outcomes comparable with other methods for difficult cases of type "C" esophageal atresia (EA).

Attenuation of SRC Kinase Activity Augments PARP Inhibitor-mediated Synthetic Lethality in BRCA2-altered Prostate Tumors.

PURPOSE: Alterations in DNA damage repair (DDR) pathway genes occur in 20%-25% of men with metastatic castration-resistant prostate cancer (mCRPC). Although PARP inhibitors (PARPis) have been shown to benefit men with mCRPC harboring DDR defects due to mutations in BRCA1/2 and ATM, additional treatments are necessary because the effects are not durable. EXPERIMENTAL DESIGN: We performed transcriptomic analysis of publicly available mCRPC cases, comparing BRCA2 null with BRCA2 wild-type. We generated BRCA2-null prostate cancer cells using CRISPR/Cas9 and treated these cells with PARPis and SRC inhibitors. We also assessed the antiproliferative effects of combination treatment in 3D prostate cancer organoids. RESULTS: We observed significant enrichment of the SRC signaling pathway in BRCA2-altered mCRPC. BRCA2-null prostate cancer cell lines had increased SRC phosphorylation and higher sensitivity to SRC inhibitors (e.g., dasatinib, bosutinib, and saracatinib) relative to wild-type cells. Combination treatment with PARPis and SRC inhibitors was antiproliferative and had a synergistic effect in BRCA2-null prostate cancer cells, mCRPC organoids, and Trp53/Rb1-null prostate cancer cells. Inhibition of SRC signaling by dasatinib augmented DNA damage in BRCA2-null prostate cancer cells. Moreover, SRC knockdown increased PARPi sensitivity in BRCA2-null prostate cancer cells. CONCLUSIONS: This work suggests that SRC activation may be a potential mechanism of PARPi resistance and that treatment with SRC inhibitors may overcome this resistance. Our preclinical study demonstrates that combining PARPis and SRC inhibitors may be a promising therapeutic strategy for patients with BRCA2-null mCRPC.

Ribonucleotide reductase small subunit M2 is a master driver of aggressive prostate cancer.

Although there are molecularly distinct subtypes of prostate cancer, no molecular classification system is used clinically. The ribonucleotide reductase small subunit M2 (RRM2) gene plays an oncogenic role in many cancers. Our previous study elucidated comprehensive molecular mechanisms of RRM2 in prostate cancer (PC). Given the potent functions of RRM2, we set out to determine whether the RRM2 signature can be used to identify aggressive subtypes of PC. We applied gene ontology and pathway analysis in RNA-seq datasets from PC cells overexpressing RRM2. We refined the RRM2 signature by integrating it with two molecular classification systems (PCS and PAM50 subtypes) that define aggressive PC subtypes (PCS1 and luminal B) and correlated signatures with clinical outcomes in six published cohorts comprising 4000 cases of PC. Increased expression of genes in the RRM2 signature was significantly correlated with recurrence, high Gleason score, and lethality of PC. Patients with high RRM2 levels showed higher PCS1 score, suggesting the aggressive PC feature. Consistently, RRM2-regulated genes were highly enriched in the PCS1 signature from multiple PC cohorts. A simplified RRM2 signature (12 genes) was identified by intersecting the RRM2 signature, PCS1 signature, and the PAM50 classifier. Intriguingly, inhibition of RRM2 specifically targets PCS1 and luminal B genes. Furthermore, 11 genes in the RRM2 signature were correlated with enzalutamide resistance by using a single-cell RNA-seq dataset from PC circulating tumor cells. Finally, high expression of RRM2 was associated with an immunosuppressive tumor-immune microenvironment in both primary prostate cancer and metastatic prostate cancer using CIBERSORT analysis and LM22, a validated leukocyte gene signature matrix. These data demonstrate that RRM2 is a driver of aggressive prostate cancer subtypes and contributes to immune escape, suggesting that RRM2 inhibition may be of clinical benefit for patients with PC.

Outcomes of preputioplasty in patients undergoing TIP urethroplasty (tubularization of incised urethral plate) for distal and mid penile hypospadias.

BACKGROUND: Preputioplasty as a part of hypospadias repair restores the normal appearance of phallus, which is especially important in distal and mid penile hypospadias. However possibility of its inherent complications such as iatrogenic phimosis or preputial breakdown are the cause of controversy and reluctance regarding this procedure.This study evaluates the results of preputial reconstruction with TIP urethroplasty in distal and mid penile hypospadias repair and analyses if preputioplasty may be offered to these patients. MATERIALS & METHODS: In this prospective observational study, 48 cases of distal and mid penile hypospadias underwent TIP urethroplasty and preputioplasty and results were assessed at 2 weeks, 3 months and 6 months. Major complications included preputial dehiscence, tight prepuce (iatrogenic phimosis) and minor complications included ventral tethering, persistent dorsal whorls and redundant prepuce. Data was analysed with Microsoft Excel spreadsheet where descriptive statistics were obtained. RESULTS: Preputioplasty was performed in 48 children with a mean age of 5.1 years. Preputioplasty dehiscence was seen in three (6%) patients, which gave an appearance of irregular prepuce on 6 m follow up. Two patients (4%) were confirmed to have preputial tightness at 3 months but this resolved conservatively in one patient and only one patient (2%) required circumcision for a tight prepuce. Minor complications included ventral tethering, persistence of dorsal whorls and redundant prepuce. Ventral tethering was present in 3 patients (6.25%). Redundant prepuce was observed in 2 patients (4.16%). Additionally, unsightly dorsal whorls were found to be persistent in 2 children (4.16%). None of these patients opted for circumcision. The rest of the children had a cosmetically and functionally normal prepuce. Two patients (4%) developed urethrocutaneous fistula at 3 months' follow-up. CONCLUSION: Preputial reconstruction is feasible with a good cosmetic outcome and minimal complications in patients of distal and mid penile hypospadias undergoing TIP urethroplasty. Mild preputial tightness evolves over time and resolves with conservative measures. In patients with very prominent dorsal whorls and underlying bulky tissues the preputioplasty does not appear to be of satisfactory cosmesis. To help the patient and parents take a well informed decision, it would be useful to explain all possible major and minor foreskin complications, and their rectification.

Novel approach towards the synthesis of highly efficient flame retardant electrode and oil/organic solvent absorber.

We have developed a facile one pot process to synthesize an ultra-light functionalized spongy graphene (FSG). This is the first approach to use carbon based flame retardant spongy material as an electrode to build completely flame retardant supercapacitor (FRS) also as an oil/organic solvent absorber. The fully FRS concept has created by the compilation of as-prepared FSG with flame retardant separator and electrolyte. As-prepared FSG contained high amount of phosphorus and nitrogen functional groups, which makes it potent flame retardant electrochemical material, to use it as an efficient FR electrode. Flame test of FSG revealed that it doesn't catch fire for ∼1500 s. Also, FSG was able to sustain flame retardancy at a temperature as high as 1500 °C for continuous exposure of ∼300 s. FSG used as an electrode for symmetric capacitor possessing maximum specific capacitance of 494.3 F g-1 at a current density 1 A g-1. Corresponding high energy density and power density values are 55.6 Wh kg-1 and 1799 W kg-1. It shows cycling stability of 86.1% after 5000 cycles at current density of 10 A g-1. The electrochemical property of FSG was also confirmed using three electrode system. Flame retardant FSG material was also used for the absorption and recovery of oil and organic solvents. FSG has high oil and organic solvent sorption capacity in the range of 40-70 g/g, also can be reused for minimum 10 cycles. Such approach has great significance for multifunctional graphene based nanocomposites will open the new window for large-scale applications.

Significance of BRCA2 and RB1 Co-loss in Aggressive Prostate Cancer Progression.

PURPOSE: Previous sequencing studies revealed that alterations of genes associated with DNA damage response (DDR) are enriched in men with metastatic castration-resistant prostate cancer (mCRPC). BRCA2, a DDR and cancer susceptibility gene, is frequently deleted (homozygous and heterozygous) in men with aggressive prostate cancer. Here we show that patients with prostate cancer who have lost a copy of BRCA2 frequently lose a copy of tumor suppressor gene RB1; importantly, for the first time, we demonstrate that co-loss of both genes in early prostate cancer is sufficient to induce a distinct biology that is likely associated with worse prognosis. EXPERIMENTAL DESIGN: We prospectively investigated underlying molecular mechanisms and genomic consequences of co-loss of BRCA2 and RB1 in prostate cancer. We used CRISPR-Cas9 and RNAi-based methods to eliminate these two genes in prostate cancer cell lines and subjected them to in vitro studies and transcriptomic analyses. We developed a 3-color FISH assay to detect genomic deletions of BRCA2 and RB1 in prostate cancer cells and patient-derived mCRPC organoids. RESULTS: In human prostate cancer cell lines (LNCaP and LAPC4), loss of BRCA2 leads to the castration-resistant phenotype. Co-loss of BRCA2-RB1 in human prostate cancer cells induces an epithelial-to-mesenchymal transition, which is associated with invasiveness and a more aggressive disease phenotype. Importantly, PARP inhibitors attenuate cell growth in human mCRPC-derived organoids and human CRPC cells harboring single-copy loss of both genes. CONCLUSIONS: Our findings suggest that early identification of this aggressive form of prostate cancer offers potential for improved outcomes with early introduction of PARP inhibitor-based therapy.See related commentary by Mandigo and Knudsen, p. 1784.