Deleterious Alterations Research Articles

DNA Damage Response and Repair Gene Alterations Increase Tumor Mutational Burden and Promote Poor Prognosis of Advanced Lung Cancer.

DNA damage response and repair (DDR) gene alterations increase tumor-infiltrating lymphocytes, genomic instability, and tumor mutational burden (TMB). Whether DDR-related alterations relate to therapeutic response and prognosis in lung cancer lacking oncogenic drivers remains unknown. Pretherapeutic cancer samples of 122 patients [86 non-small cell lung cancer and 36 small cell lung cancer (SCLC)] harboring no EGFR/ALK alterations were collected. Through whole-exome sequencing, we outlined DDR mutational landscape and determined relationships between DDR gene alterations and TMB or intratumoral heterogeneity. Then, we evaluated the impacts of DDR gene alterations on therapeutic response and prognosis and established a DDR-based model for prognosis prediction. In addition, we investigated somatic interactions of DDR genes and immunomodulatory genes, immune expression patterns, immune microenvironment, and immune infiltration characteristics between DDR-deficient and DDR-proficient samples. Samples from cBioportal datasets were utilized for verification. We found that deleterious DDR gene alterations were closely associated with higher TMB than proficient-types (p < 0.001). DDR mechanisms attach great importance to the determination of patients’ prognosis after chemotherapy, and alterations of base excision repair pathway in adenocarcinoma, nucleotide excision repair in squamous carcinoma, and homologous recombination pathway in SCLC tend to associate with worse progression-free survival to first-line chemotherapy (all p < 0.05). A predictive nomogram model was constructed incorporating DDR-related alterations, clinical stage, and smoking status, with the area under curve values of 0.692–0.789 for 1- and 2-year receiver operating characteristic curves in training and testing cohorts. Furthermore, DDR-altered tumors contained enhanced frequencies of alterations in various genes of human leukocyte antigen (HLA) class I pathway including TAP1 and TAP2 than DDR-proficient samples. DDR-deficient types had lower expressions of STING1 (p = 0.01), CD28 (p = 0.020), HLA-DRB6 (p = 0.014) in adenocarcinoma, lower TNFRSF4 (p = 0.017), and TGFB1 expressions (p = 0.033) in squamous carcinoma, and higher CD40 (p = 0.012) and TNFRSF14 expressions (p = 0.022) in SCLC. DDR alteration enhanced activated mast cells in adenocarcinoma (p = 0.044) and M2 macrophage in squamous carcinoma (p = 0.004) than DDR-proficient types. Collectively, DDR gene alterations in lung cancer without oncogenic drivers are positively associated with high TMB. Specific DDR gene alterations tend to associate with worse progression-free survival to initial chemotherapy.

Analysis of toxicity effects of delta-9-tetrahydrocannabinol on isolated rat heart mitochondria

Mitochondria have the main roles in myocardial tissue homeostasis, through providing ATP for the vital enzymes in intermediate metabolism, contractile apparatus and maintaining ion homeostasis. Mitochondria-related cardiotoxicity results from the exposure with illicit drugs have previously reported. These illicit drugs interference with processes of normal mitochondrial homeostasis and lead to mitochondrial dysfunction and mitochondrial-related oxidative stress. Cannabis consumption has been shown to cause ventricular tachycardia, to increase the risk of myocardial infarction (MI) and potentially sudden death. Here, we investigated this hypothesis that delta-9-tetrahydrocannabinol (Delta-9-THC) as a main cannabinoid found in cannabis could directly cause mitochondrial dysfunction. Cardiac mitochondria were isolated with mechanical lysis and differential centrifugation form rat heart. The isolated cardiac mitochondria were treated with different concentrations of THC (1, 5, 10, 50, 100 and 500 µM) for 1 hour at 37 °C. Then, succinate dehydrogenase (SDH) activity, mitochondrial swelling, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) collapse and lipid peroxidation were measured in the treated and nontreated isolated cardiac mitochondria. Our observation showed that THC did not cause a deleterious alteration in mitochondrial functions, ROS production, MMP collapse, mitochondrial swelling, oxidative stress and lipid peroxidation in used concentrations (5–100 µM), even in several tests, toxicity showed a decreasing trend. Altogether, the results of the current study showed that THC is not directly toxic in isolated cardiac mitochondria, and even may be helpful in reducing mitochondrial toxicity.

MP24-01 RESPONSE TO IMMUNE CHECKPOINT BLOCKADE IN PATIENTS WITH MICROSATELLITE INSTABLE AND HIGH TUMOR MUTATIONAL BURDEN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) II (MP24)1 Sep 2021MP24-01 RESPONSE TO IMMUNE CHECKPOINT BLOCKADE IN PATIENTS WITH MICROSATELLITE INSTABLE AND HIGH TUMOR MUTATIONAL BURDEN PROSTATE CANCER Andrew T. Lenis, Vignesh Ravichandran, Hong Truong, Peter Reisz, Barbara Nweji, Karen Autio, Michael Morris, Susan Slovin, Hebert Alberto Vargas, Vincent Laudone, Behfar Ehdaie, Victor Reuter, Agnes Viale, Nikolaus Schultz, Anuradha Gopalan, Mark Donoghue, Konrad Stopsack, David Solit, and Wassim Abida Andrew T. LenisAndrew T. Lenis More articles by this author , Vignesh RavichandranVignesh Ravichandran More articles by this author , Hong TruongHong Truong More articles by this author , Peter ReiszPeter Reisz More articles by this author , Barbara NwejiBarbara Nweji More articles by this author , Karen AutioKaren Autio More articles by this author , Michael MorrisMichael Morris More articles by this author , Susan SlovinSusan Slovin More articles by this author , Hebert Alberto VargasHebert Alberto Vargas More articles by this author , Vincent LaudoneVincent Laudone More articles by this author , Behfar EhdaieBehfar Ehdaie More articles by this author , Victor ReuterVictor Reuter More articles by this author , Agnes VialeAgnes Viale More articles by this author , Nikolaus SchultzNikolaus Schultz More articles by this author , Anuradha GopalanAnuradha Gopalan More articles by this author , Mark DonoghueMark Donoghue More articles by this author , Konrad StopsackKonrad Stopsack More articles by this author , David SolitDavid Solit More articles by this author , and Wassim AbidaWassim Abida More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002015.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancers with high microsatellite instability/deficient mismatch repair (MSI-H/dMMR) and high tumor mutational burden (TMB-H) are rare but recognized as molecular subgroups with therapeutic implications. Patients harboring either of these genomic features are candidates for treatment with immunotherapy (IO) based on tumor agnostic approvals. Given the potential for different underlying biology, we aimed to compare clinical outcomes in patients with MSI-H/dMMR and TMB-H prostate cancer treated with IO. METHODS: We identified men with prostate cancer who underwent paired tumor–normal genomic profiling with MSK-IMPACT, a targeted exome sequencing panel. MSI-H/dMMR prostate cancer was defined as MSIsensor score ≥10 or MSIsensor score of ≥3 and <10 with evidence of a deleterious somatic or germline alteration in a mismatch repair gene (MSH6, MSH2, MLH1, or PMS2). TMB-H was defined as ≥10 in the absence of microsatellite instability. Response to IO was defined as a 50% decrease in PSA (PSA50). Median time to IO discontinuation was estimated using the Kaplan-Meier method. RESULTS: Of 2,351 men with prostate cancer who underwent MSK-IMPACT testing between 2/2014 and 2/2021, 62 (2.6%) were identified as having MSI-H/dMMR prostate cancer and 29 (1.2%) as having TMB-H. Of the 62 patients with MSI-H/dMMR prostate cancer, 29 (32%) were treated with IO and 26 had metastatic castration resistant disease and evaluable PSA levels. PSA50 was observed in 65% (17/26); no significant difference in response between MSI-H/dMMR and TMB-H patients was observed. The median time to IO discontinuation in patients with MSI-H/dMMR and TMB-H tumors was 20 (IQR: 10–107) and 12 (IQR: 9–15) weeks, respectively. At a median follow-up of 90 weeks, 35% (7/20) of MSI-H/dMMR and 33% (2/6) of TMB-H tumors were still on IO. MSIsensor scores and TMB were not significantly different between IO responders and non-responders. CONCLUSIONS: MSI-H/dMMR and TMB-H prostate cancers are rare but up to two thirds of such patients respond to IO with many durable responses. MSIsensor score and TMB were not predictive of response to IO in these molecularly defined populations. Other clinical and genomic factors are being investigated to identify biomarkers to guide treatment selection. Source of Funding: This work was funded in part by the Marie-Josée and Henry R. Kravis Center for Molecular Oncology © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e411-e411 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andrew T. Lenis More articles by this author Vignesh Ravichandran More articles by this author Hong Truong More articles by this author Peter Reisz More articles by this author Barbara Nweji More articles by this author Karen Autio More articles by this author Michael Morris More articles by this author Susan Slovin More articles by this author Hebert Alberto Vargas More articles by this author Vincent Laudone More articles by this author Behfar Ehdaie More articles by this author Victor Reuter More articles by this author Agnes Viale More articles by this author Nikolaus Schultz More articles by this author Anuradha Gopalan More articles by this author Mark Donoghue More articles by this author Konrad Stopsack More articles by this author David Solit More articles by this author Wassim Abida More articles by this author Expand All Advertisement Loading ...

Biogenic and characterizations of new silver nanoparticles stabilized with indole acetic acid derived from Azospirillum brasilense MMGH-SADAT1, their bioactivity, and histopathological assessment in rats

An Egyptian rhizobacterium Azospirillum sp. isolated from Sadat city was able to produce indole acetic acid (IAA) up to (30.59 µg/ml). The isolate was identified biochemically and by 16S rRNA sequencing which showed 99.9% similarity to Azospirillum brasilense. The new isolate has been registered in Genbank with accession number MH179119.1. Extracted IAA was used as reducing or stabilizing agent of sliver nanoparticles (AgNPs). Successful fabrication of biogenic IAA-AgNPs was confirmed by Fourier Transform Infrared Spectrophotometer (FTIR) analysis of IAA which showed absorbance peak at 3434.78 cm−1 due to the N-H stretch of primary amines. Highly resolution Transmission Electron Microscopy (HR-TEM) showed AgNPs coating or capping with IAA in spherical shaped with size ranged from 6.01 to 44.02 nm. Energy dispersive X-ray (EDX) analysis revealed that Ag+ ions were attached to the surface of IAA-AgNPs particles. HR-TEM examination showed cell wall damage of Citrobacter freundii cells after exposure to IAA-AgNPs leading to cell death. In vivo results showed that C. freundii infection of rats induced significant increase in liver and kidney functions and deleterious histopathological alteration in rat’s tissues. However, treatment by extracted IAA and IAA-AgNPs could normalize the biochemical and histopathological alterations occurred in infected rats. This is the first study to prove that IAA extracted from Azospirillum brasilense is a hopeful capping agent for NPs which has potential to protect against pathogenic infections, nontoxic and/or safe on rat’s metabolisms.

Dietary Restriction for Kidney Protection: Decline in Nephroprotective Mechanisms During Aging.

Dietary restriction (DR) is believed to be one of the most promising approaches to extend life span of different animal species and to delay deleterious age-related physiological alterations and diseases. Among others, DR was shown to ameliorate acute kidney injury (AKI) and chronic kidney disease (CKD). However, to date, a comprehensive analysis of the mechanisms of the protective effect of DR specifically in kidney pathologies has not been carried out. The protective properties of DR are mediated by a range of signaling pathways associated with adaptation to reduced nutrient intake. The adaptation is accompanied by a number of metabolic changes, such as autophagy activation, metabolic shifts toward lipid utilization and ketone bodies production, improvement of mitochondria functioning, and decreased oxidative stress. However, some studies indicated that with age, the gain of DR-mediated positive remodeling gradually decreases. This may be an obstacle if we seek to translate the DR approach into a clinic for the treatment of kidney diseases as most patients with AKI and CKD are elderly. It is well known that aging is accompanied by impairments in a huge variety of organs and systems, such as hormonal regulation, stress sensing, autophagy and proteasomal activity, gene expression, and epigenome profile, increased damage to macromolecules and organelles including mitochondria. All these age-associated changes might be the reasons for the reduced protective potential of the DR during aging. We summarized the available mechanisms of DR-mediated nephroprotection and described ways to improve the effectiveness of this approach for an aged kidney.

Abstract LB035: B7-H3 as a therapeutic target in prostate cancer

Abstract Background: Advanced prostate cancer (PC) is invariably fatal and largely insensitive to established immune checkpoint inhibition (ICI). The immune checkpoint, PD-L1 (B7-H1; CD274), is infrequently overexpressed in PC; whilst other B7 family immunomodulatory glycoprotein, B7-H3 (CD276), is overexpressed in many PCs. B7-H3 targeted immunoconjugates are in clinical development. We therefore studied the longitudinal expression of B7-H3 in PC and its associations with advanced PC immunogenomics. Design: We analysed matching, same-patient, formalin-fixed paraffin-embedded (FFPE) metastatic castration resistant PC (mCRPC) (n=98), and treatment-naïve, castration-sensitive PC (CSPC) biopsies (n=43) from patients treated at The Royal Marsden Hospital (UK). Biopsies were analysed by: targeted next-generation sequencing (NGS) for deleterious DNA repair gene alterations; immunohistochemistry (IHC) for ATM, PTEN, B7-H3 and mismatch repair (MMR) proteins using validated antibodies; and multi-color immunofluorescence (IF) assays for T cell surface markers to determine the density of tumour infiltrating T lymphocyte (TIL) subsets. Wilcoxon signed-rank test and Mann-Whitney U test compared B7-H3 expression across matched and unmatched subsets, respectively. Spearman correlation determined associations between continuous variables. Results: Most CRPC and CSPC biopsies had both membranous B7-H3 (mB7-H3) expression (134/141, 95.0%) and cytoplasmic B7-H3 expression (137/141, 97.2%). Analysis of the 43 matched samples sets showed that there was no significant change in mB7-H3 expression as tumours progressed from CSPC to CRPC (median Histoscore [HS] [range]: 130 [5-300] for CSPC vs. 130 [5-290] for CRPC; p=0.6). There was significant interpatient and intratumor heterogeneity in mB7-H3 expression, but a subset of tumour biopsies had very high mB7-H3 expression (HS ≥200: 28/98 [28.6%] CRPC; 14/43 [32.6%] CSPC). mB7-H3 expression strongly associated with the presence of deleterious alterations of genes involved in homologous recombination (HR) repair (p&amp;lt;0.0001), including BRCA2 mutations and homozygous deletions (p=0.0003) as well as ATM loss (p=0.001). mB7-H3 expression did not associate with defective MMR. Interestingly, mB7-H3 expression inversely associated with the density of intra-tumor CD3+ TILs (median: 104.6 TILs/mm2 in B7-H3 low tumors (HS &amp;lt; median) vs. 39.2 TILs/mm2 in B7-H3 high tumours (HS ≥ median), p=0.004) but did not associate with stromal TIL density (p=0.4). Conclusion: mB7-H3 is highly expressed in advanced PC, with higher expression associating with BRCA2 and ATM loss of function alterations and low intratumor TILs. B7-H3 may be an actionable target for treating this disease subset. Citation Format: Christina Guo, Ines Figueiredo, Bora Gurel, Matues Crespo, Jan Rekowski, Suzanne Carreira, Antje Neeb, Adam Sharp, Maria D. Fenor de la Maza, Pasquale Rescigno, Khobe Chandran, Ana Ferreira, Ruth Riisnaes, Susana Miranda, Rita Pereira, Veronica Gil, George Seed, Claudia Bertan, Chloe Baker, Wei Yuan, Johann S. de Bono. B7-H3 as a therapeutic target in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB035.

Ancestry analysis indicates two different sets of essential genes in eukaryotic model species.

Essential genes are so-called because they are crucial for organism perpetuation. Those genes are usually related to essential functions to cellular metabolism or multicellular homeostasis. Deleterious alterations on essential genes produce a spectrum of phenotypes in multicellular organisms. The effects range from the impairment of the fertilization process, disruption of fetal development, to loss of reproductive capacity. Essential genes are described as more evolutionarily conserved than non-essential genes. However, there is no consensus about the relationship between gene essentiality and gene age. Here, we identified essential genes in five model eukaryotic species (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) and estimate their evolutionary ancestry and their network properties. We observed that essential genes, on average, are older than other genes in all species investigated. The relationship of network properties and gene essentiality convey with previous findings, showing essential genes as important nodes in biological networks. As expected, we also observed that essential orthologs shared by the five species evaluated here are old. However, all the species evaluated here have a specific set of young essential genes not shared among them. Additionally, these two groups of essential genes are involved with distinct biological functions, suggesting two sets of essential genes: (i) a set of old essential genes common to all the evaluated species, regulating basic cellular functions, and (ii) a set of young essential genes exclusive to each species, which perform specific essential functions in each species.

Molecular characterization of ctDNA from Chinese patients with advanced gastric adenocarcinoma reveals actionable alterations for targeted and immune therapy.

Circulating tumor DNA (ctDNA) is considered an ideal sample type for genotyping patients with advanced unresectable cancer to inform treatment decision. It may better capture tumor heterogeneity, especially in gastric adenocarcinoma (GAC). However, there exists little evidence regarding genomic profiling of Chinese advanced GAC patients from ctDNA. Blood samples were obtained from 200 advanced GAC patients. Next-generation sequencing (NGS) was performed on ctDNA using a validated 150-gene panel. Blood tumor mutation burden (bTMB) was calculated according to the NGS results. Blood microsatellite instability (bMSI) status was determined by targeted sequencing of 100 microsatellite loci. One hundred sixty-nine (84.5%) patients carried at least one genomic alteration and 138 (69%) patients had at least one deleterious or likely deleterious alteration (del-alteration). The clonal fraction of del-alterations was higher than that of non-del-alterations (80.1% vs 54.5%, P < 0.0001). The most frequently altered genes were TP53 (38%), LRP1B (20%), MYC (13.5%), ERBB2 (12.5%), and KRAS (11.5%). The alterations were most enriched in the TP53/cell cycle (52%) and the RTK-Ras-MAPK pathway (51.5%). The median bTMB was two (range 0 to 42). Eight patients were identified to be high bMSI, with higher median bTMB than the blood microsatellite stable (bMSS) patients (15 vs 2, P = 0.0062). Patients harboring del-alterations of the DDR pathway had significantly higher percentages of high bTMB and bMSI-H patients than the wild-type subgroup (61.1% vs 6.5%, P < 0.0001; 33.3% vs 1.7%, P = 0.0002). A total of 45.5% cases harbored at least one potentially actionable alteration and one patient achieved complete response after receiving matched targeted therapy. Our study uncovered the molecular characterization of Chinese patients with advanced GAC from ctDNA, including genomic alteration, bTMB, and bMSI status. The findings suggested that targeted NGS-based ctDNA analysis may help inform the clinical decision in advanced GAC. KEY MESSAGES: We report the molecular profiling of the largest Chinese advance stage GACs cohort using a CLIA-certified ctDNA assay. Potentially actionable genomic alterations were identified in 45.5% of patients, suggesting clinical utility for ctDNA NGS in advance stage GACs. There was evidence of clinical benefit in one GAC patient with MET amplification treated with MET inhibitor.

Whole-exome sequencing in eccrine porocarcinoma indicates promising therapeutic strategies

Malignant sweat gland tumours are rare, with the most common form being Eccrine porocarcinoma (EP). To investigate the mutational landscape of EP, we performed whole-exome sequencing (WES) on 14 formalin-fixed paraffin-embedded samples of matched primary EP and healthy surrounding tissue. Mutational profiling revealed a high overall median mutation rate. This was attributed to signatures of mutational processes related to ultraviolet (UV) exposure, APOBEC enzyme dysregulation, and defective homologous double-strand break repair. All of these processes cause genomic instability and are implicated in carcinogenesis. Recurrent driving somatic alterations were detected in the EP candidate drivers TP53, FAT2, CACNA1S, and KMT2D. The analyses also identified copy number alterations and recurrent gains and losses in several chromosomal regions including that containing BRCA2, as well as deleterious alterations in multiple HRR components. In accordance with this reduced or even a complete loss of BRCA2 protein expression was detected in 50% of the investigated EP tumours. Our results implicate crucial oncogenic driver pathways and suggest that defective homologous double-strand break repair and the p53 pathway are involved in EP aetiology. Targeting of the p53 axis and PARP inhibition, and/or immunotherapy may represent promising treatment strategies.

P078 Proteomics and Lipidomics Analysis Revealed Alterations of Complement Activation and Lipid Metabolism in peripheral blood mononuclear cells of Inflammatory Bowel Disease Patients

Abstract Background The lack of interaction between immune system cells and microbiota is considered a key driver of the pathogenesis of inflammatory bowel disease (IBD). The association of age-associated B cells (ABC) from antigen-experienced B cells and autoimmunity is now well established. The increased demands in lipid metabolism during immune response and cell activation upon autoimmune signals leads to lipid tag modifications of proteins. In IBD patients, we observed an impairment of B cell development, identifying a transitional B cell subset expressing CD38Hi, CD24Hi and CD19+. We hypothesize that metabolic alterations of proteins and lipids during immune cell activation might be a relevant mechanism of autoimmune disease. Methods An IBD cohort of patients in clinical remission under anti-TNF treatment was included in order to test metabolic and proteomic changes in peripheral blood mononuclear cells (PBMCs). Proteomic analysis was performed by Mass Spectrometry using a label free quantitative method (SWATH-MS analysis) and lead to identify a number of proteins underregulated (&amp;lt; 0.6) and overexpressed (&amp;gt; 1.5) fold change respect to control. Lipidomics of serum samples analyzed by proton Nuclear Magnetic Resonance spectroscopy (NMR)2 and multivariant statistics. Results Serum samples were obtained in Crohn disease (CD) n = 18, ulcerative colitis (UC) n = 9, before biological infusion and healthy controls (CTRL) n = 10. Proteins related with lipid metabolism like APOC2 for CD and APOB, APOA1 for UC, were differentially modulated. Significant differences in non-polar metabolites and lipids that phenotypically define CD and UC groups respect to CTRL were observed (Fig. 1) Fig. 1 Orthogonal-Partial Least Square-Discriminant Analysis (OPLS-DA) of the global NMR data of the serum samples of groups CD and CTRL. Indeed, proteins associated with complement activation like Complement Factor 1 and Complement C1q subcomponent subunit B were differentially expressed in CD group. These results were confirmed by lipidomic analysis, in which palmitic (Fig. 2) as well as other candidate lipids were more abundant in IBD groups than CTRL. Fig 2. Differences of palmitic acid concentration in CD and UC patients vs CTRL (Bonferroni test showed statistical difference between CTRL vs. CD and CTRL vs. UC with p = 0.02 and p = 0.004 respectively). Conclusion Our study detected high levels of proteins regulating lipid metabolism and palmitic in IBD patients. This observation can be interpreted as part of deleterious alterations of proteins through palmitoylation during B-cell activation and might be a relevant mechanism of disease. Further exploration of palmitate catabolism might shed light to the mechanisms that result in immunometabolic disorders.

Efficacy and safety of rucaparib in previously treated, locally advanced or metastatic urothelial carcinoma from a phase 2, open-label trial (ATLAS)

BackgroundATLAS evaluated the efficacy and safety of the PARP inhibitor rucaparib in patients with previously treated locally advanced/unresectable or metastatic urothelial carcinoma (UC).MethodsPatients with UC were enrolled independent of tumor homologous recombination deficiency (HRD) status and received rucaparib 600 mg BID. The primary endpoint was investigator-assessed objective response rate (RECIST v1.1) in the intent-to-treat and HRD-positive (loss of genome-wide heterozygosity ≥10%) populations. Key secondary endpoints were progression-free survival (PFS) and safety. Disease control rate (DCR) was defined post-hoc as the proportion of patients with a confirmed complete or partial response (PR), or stable disease lasting ≥16 weeks.ResultsOf 97 enrolled patients, 20 (20.6%) were HRD-positive, 30 (30.9%) HRD-negative, and 47 (48.5%) HRD-indeterminate. Among 95 evaluable patients, there were no confirmed responses. However, reductions in the sum of target lesions were observed, including 6 (6.3%) patients with unconfirmed PR. DCR was 11.6%; median PFS was 1.8 months (95% CI, 1.6–1.9). No relationship was observed between HRD status and efficacy endpoints. Median treatment duration was 1.8 months (range, 0.1–10.1). Most frequent any-grade treatment-emergent adverse events were asthenia/fatigue (57.7%), nausea (42.3%), and anemia (36.1%). Of 64 patients with data from tumor tissue samples, 10 (15.6%) had a deleterious alteration in a DNA damage repair pathway gene, including four with a deleterious BRCA1 or BRCA2 alteration.ConclusionsRucaparib did not show significant activity in unselected patients with advanced UC regardless of HRD status. The safety profile was consistent with that observed in patients with ovarian or prostate cancer.Trial registrationThis trial was registered in ClinicalTrials.gov (NCT03397394). Date of registration: 12 January 2018. This trial was registered in EudraCT (2017–004166-10).

Genomic characteristics of homologous recombination deficiency in prostate cancer.

e17004 Background: PARP inhibitors (PARPi) have been approved in the treatment of metastatic castration-resistant prostate cancer with deleterious or suspected deleterious alterations in homologous recombination repair (HRR) genes. Patients carrying alterations of different HRR genes showed diverse response to PARPi, while the underlying mechanism is unrevealed. Homologous recombination deficiency (HRD) score serves as a promising way to interpret the genomic instability by targeted next-generation sequencing (NGS). In this study, we analyzed HRD score and its relationship to genomic characteristics in a prostate cancer (PC) cohort. Methods: Tumor tissue and matched white blood cells from 295 PC patients were analyzed by NGS-based assays, which target over 10,000 single-nucleotide polymorphisms distributed across human genome to characterize genomic instability, as well as the whole coding region of 733 or 233 genes. HRD score was derived by the sum of three indexes, including loss-of-heterozygosity, telomeric allelic imbalance, and large-scale state transitions. Deleterious or suspected deleterious mutations were included for analysis. Tumor mutation burden (TMB) was calculated by the number of somatic single nucleotide variants and indels in examined coding regions, with driver mutations excluded. An ovarian and fallopian tube cancer cohort, carrying deleterious or suspected deleterious mutations in BRCA1/2, was also included. Results: In PC cohort, 25 patients (8.47%) had BRCA1/2 mutations, with additional 39 patients (13.2%) carrying mutations in HRR genes other than BRCA1/2. HRD score was similar among tumors with germline BRCA1/2 mutation (n = 9), somatic BRCA1/2 mutation (n = 14) or both (n = 2) (median HRD score 38.0, 32.5 and 49.5; P = 0.281). In BRCA1/2 mutation carriers, HRD score in PC cohort was lower than ovarian cancer (n = 222) and fallopian tube cancer (n = 8) (median HRD score 36, 54 and 67; P &lt; 0.001). PC samples with CDK12 (n = 35), ATM (n = 13) and PALB2 (n = 2) mutations had lower HRD score compared with BRCA1/2 mutants (median HRD score 24, 14, 11 and 36; P &lt; 0.001). In PC patients without HRR mutation, HRD score was higher in TP53 mutants compared with TP53 wildtype (median HRD score 25 and 14; P &lt; 0.001), while tumors with ERG fusion had lower HRD score than ERG fusion absent tumors (median HRD score 8 and 18.5; P = 0.029). In four TP53 mutated PC patients treated with PARPi in our center, three patients achieved PSA response. Furthermore, no linear correlation was discovered between TMB and HRD score, while tumors with higher HRD score (HRD score ≥ 30) had a bit lower TMB (median TMB 3.07 vs 3.91, P = 0.002). Conclusions: HRD score was affected by alterations in different HRR genes, TP53 mutation and ERG fusion, which should be taken into consideration of the future clinical trials and research on intrinsic mechanisms. Patients carrying alterations other than HRR genes might benefit from PARPi. Further studies are needed.

ATR and BRCA2 Simultaneous Mutation in a ccRCC With Sarcomatoid Differentiation and Extensive Metastases: A Case Report

The genomic landscape and driver-gene mutations differ significantly among diverse histological subtypes of clear cell renal cell carcinoma (ccRCC) due to the intratumoral heterogeneity. Frequent mutations in canonical DNA damage response genes, such as BRCA1/2 or ATR serine/threonine kinase (ATR) haven't been reported even in large-scale genomic profiling of ccRCC researches. Herein, we reported a rare ccRCC harboring ATR and BRCA2 simultaneous mutation with complicated morphologies and extensive metastases. Our case indicates that the deleterious alteration of DNA damage response genes, increasing CD8+ TILs, high PD1/PD-L1 expression and high TMB might contribute to this patient's tumor metastasis and aggressive biological behavior.

178 Expression of active matrix metalloproteinase-1 in dermal fibroblasts: A novel mouse model of accelerated human dermal aging

Fragmentation, disorganization, and depletion of the collagen-rich dermal extracellular matrix (ECM) are characteristic features of aged human skin. These deleterious alterations are thought to critically mediate many of the prominent clinical attributes of aged skin including thinning, fragility, impaired wound healing, and propensity for carcinoma. Matrix metalloproteinase-1 (MMP1), which is a secreted enzyme that initiates cleavage of collagen fibrils, is significantly increased in dermal fibroblasts in aged human skin. To investigate the potential role of elevated MMP1 in skin aging, we have generated a Cre-inducible mouse model, using CRISPR/Cas9 technology, that expresses full-length, catalytically-active human MMP1 (MMP1*) in dermal fibroblasts, driven by a Pdgfra-Cre transgene. Immunohistology of Pdgfra;MMP1* bitransgenic mice revealed MMP1* expression by fibroblasts throughout the dermis in dorsal, ear, tail, and volar skin. MMP1 activity in dorsal skin, measured by ELISA, was significantly greater (39-fold, n=6, p<0.01) in Pdgfra;MMP1* mice than littermate controls. MMP1* protein was similarly increased (7.6ng/g, n=6) vs. not detectable in controls. Importantly, by the age of six months, dorsal skin of Pdgfra;MMP1* mice revealed physical, histological, and molecular alterations that closely resembled those seen in aged human skin. These features included increased fragility, thinning of the dermis (reduced 35%, n=5, p<0.01), fragmentation and disorganization of collagen fibrils, visualized multiphoton confocal second harmonic generation microscopy, reduced fibroblast spreading, and altered gene expression, including reductions of type I, III, IV, and V collagens and upregulation of IL1-beta and IL-6. The above data demonstrate that elevated dermal expression of MMP1 is a key driver of many aspects of skin aging. Therapeutic intervention to reduce/inhibit MMP1 activity during aging may help to prolong skin health throughout life.

Protective properties of Spondias mombin Linn leaves on redox status, cholinergic dysfunction and electrolyte disturbance in cyanide-intoxicated rats.

Cyanide is an environmental neurotoxin which has been reported to arrest the normal functioning of the brain. This study investigated the protective properties of methanol and flavonoid-rich extracts of the leaves of Spondias mombin on redox status, cholinergic dysfunction and electrolyte disturbance in cyanide-induced neurotoxicity in rats. Male Wistar rats were orally pre-treated with Spondias mombin methanol leaf extract (SMC) (50, 100 and 150 mg/kg), flavonoid-rich extract (SMF) (25, 50 and 75 mg/kg) or quercetin (20 mg/kg), followed by intraperitoneal administration of 2 mg/kg potassium cyanide. Cyanide intoxication caused brain damage in rats as echoed in the deleterious alterations to activities/levels of endogenous antioxidants and biomarkers/enzymes linked with electrolyte imbalance and neurotoxicity. Pre-treatment with SMC and SMF significantly attenuated these KCN-induced imbalances (p < 0.05). The results suggested that the protection conferred by SMC and SMF probably involves attenuation of oxidative stress and regulation of ionic homeostasis. SMF displayed a better apparent ameliorative activity than SMC and 75 mg/kg SMF offered the best protection suggesting that flavonoids probably contributed to the protective effect of Spondias mombin leaf.

Oral Nigella sativa oil administration alleviates arsenic-induced redox imbalance, DNA damage, and metabolic and histological alterations in rat liver.

Arsenic, an omnipresent environmental contaminant, is regarded as a potent hepatotoxin. Nigella sativa oil (NSO) consumption has been shown to improve hepatic functions in various in vivo models of acute hepatic injury. The present study evaluates the protective efficacy of NSO against sodium arsenate (As)-induced deleterious alterations in the liver. Male Wistar rats were divided into four groups, namely, control, As, NSO, and AsNSO. After pre-treating rats in AsNSO and NSO groups with NSO (2 mL/kg bwt, orally) for 14 days, NSO treatment was further extended for 30 days, with and without As treatment (5 mg/kg bwt, orally), respectively. As induced an upsurge in serum ALT and AST activities indicating liver injury, as also confirmed by the histopathological findings. As caused significant alterations in the activities of membrane marker enzymes and carbohydrate metabolic enzymes, and in the vital components of antioxidant defense system. Marked DNA damage and hepatic arsenic accumulation were also observed in As-treated rats. Oral NSO administration ameliorated these deleterious alterations and improved overall hepatic antioxidant and metabolic status in As-treated rats. Prevention of oxidative damage could be the underlying mechanism of NSO-mediated protective effects. The results suggest that NSO could be a useful dietary supplement in the management of arsenic hepatotoxicity.

Octanoic acid a major component of widely consumed medium-chain triglyceride ketogenic diet is detrimental to bone

Octanoic acid is a medium-chained saturated fatty acid found abundantly in the ketogenic dietary supplements containing medium chained triglycerides (MCT) along with decanoic acid. The MCT ketogenic diet is commonly consumed for weight loss but has also showcased neuroprotective potential against neurodegenerative disorders. However, recent clinical findings have reported a critical disadvantage with the long-term consumption of ketogenic diet i.e. bone loss. The following study was employed to investigate whether the two major components of MCT diet also possess bone loss potential as observed with classical ketogenic diet. Swiss albino mice aged between 10 and 12 weeks, were divided into 3 treatment groups that were administered with oral suspensions of octanoic acid, decanoic acid and a combination of both for 4 weeks. Bone specific markers, microarchitectural parameters, using micro computed tomography, and biomechanical strength were analyzed. Remarkably deleterious alterations in the trabecular bone microarchitecture, and on bone markers were observed in the octanoic acid treated groups. Our results suggest significant negative effects on bone health by octanoic acid. These findings require further investigation and validation in order to provide significant clinically relevant data to possibly modify dietary composition of the MCT ketogenic diet.

A synonymous variant in a non-canonical exon of CDC45 disrupts splicing in two affected sibs with Meier-Gorlin syndrome with craniosynostosis

Disruption of the initiation of DNA replication is significantly associated with Meier-Gorlin syndrome (MGORS), an autosomal recessive condition of reduced growth, microtia and patellar a/hypoplasia. Biallelic mutations in CDC45, a member of the pre-initiation complex in DNA replication, cause a spectrum of phenotypes ranging from MGORS with craniosynostosis, through to isolated short stature and craniosynostosis. Here we report two affected sibs with MGORS and craniosynostosis, with biallelic variants in CDC45 identified by 10X Chromium whole genome sequencing. One variant is a frameshift mutation, predicted to be pathogenic, and is inherited in trans with a synonymous variant in a non-canonical exon (exon 7) of CDC45. An in vitro splicing assay showed that while the canonical CDC45 exon 6-exon 8 transcript (with skipping of exon 7; numbering as per NM001178010.2) remained as the predominant transcript, the variant allele induced the use of novel splice acceptor sites in intron 6, all of which produced transcripts harbouring premature stop codons. This perturbation of canonical splicing provides evidence that this synonymous variant is indeed a deleterious alteration in this family. This report adds to the initial patient cohort in which several synonymous variants were also described, further highlighting the contribution of this variant type in CDC45. It also reiterates the true potential pathogenicity of synonymous variants, which is a mutation type that is commonly ignored in variant prioritization strategies.

Association of co-occurring gene alterations and clinical activity of rucaparib in patients with BRCA1 or BRCA2 mutated (BRCA+) metastatic castration-resistant prostate cancer (mCRPC).

80 Background: The poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib was granted accelerated approval by the US Food and Drug Administration for patients with BRCA+ mCRPC based on results from the phase 2 TRITON2 study (NCT02952534). The TP53 tumor suppressor gene is among the most frequently mutated genes in human cancers, including mCRPC, and alterations in TP53, PTEN, and RB1 are associated with poor prognosis in patients with prostate cancer and other tumor types. We present data on co-occurring alterations in patients with BRCA+ mCRPC treated with rucaparib in TRITON2. Methods: Patients had progressed on 1–2 lines of androgen receptor-directed therapy and 1 taxane-based chemotherapy and were treated with rucaparib 600 mg BID. Tissue and/or cell-free DNA extracted from plasma samples were profiled comprehensively for genomic alterations using Foundation Medicine, Inc., next-generation sequencing assays. Objective response rate (ORR) was assessed per modified Response Evaluation Criteria in Solid Tumors and Prostate Cancer Working Group 3 criteria by independent radiologic review of patients with measurable disease. Prostate-specific antigen (PSA) response rate (≥50% decrease from baseline) was assessed in all patients. Results: Tissue and/or plasma samples were available for 114/115 patients with BRCA+ mCRPC (visit cutoff date: Dec. 23, 2019). Among patients with BRCA+ mCRPC who had samples available for comprehensive genomic profiling, 36.8% (42/114) had a co-occurring alteration in TP53. Deleterious alterations in PTEN were observed in 34.2% (39/114) of patients, 44% (17/39) of which were homozygous deletions of PTEN. RB1 loss was observed in 12.3% (14/114) of patients and was seen more frequently in patients with measurable disease (18.0%, 11/61) than in patients with non-measurable disease (5.7%, 3/53). Although patients with and without TP53 mutations had generally similar baseline demographics and disease characteristics, visceral disease was more prevalent in patients with TP53 mutations (54.8%; 23/42) than in those without them (29.2%; 21/72). Similar ORR and PSA response rates were seen in patients with BRCA+ mCRPC with or without TP53 mutation, with a non-significant trend towards lower response rates in patients with co-occurring TP53 alterations. Conclusions: Results from TRITON2 showed antitumor activity for rucaparib in patients with BRCA+ mCRPC associated with or without co-occurring alterations in TP53. Demographics and additional efficacy analyses in genomic subgroups with co-occurring alterations in TP53, PTEN, and RB1 will be reported. Clinical trial information: NCT02952534. [Table: see text]

Germline alterations in cancer susceptibility genes in women with high-risk bladder cancer: Implications for germline testing and clinical management.

418 Background: Gender differences exist in bladder cancer incidence, stage at diagnosis, and outcomes. Women have lower incidence of bladder cancer but are diagnosed with more advanced disease at presentation. They also have less favorable outcomes even after adjusting for tumor stage and treatment modality. The biologic mechanisms underlying gender disparities in bladder cancer remain unknown. Methods: We leveraged a prospective matched tumor-normal genomic profiling initiative to determine the prevalence and spectrum of pathogenic/likely pathogenic (P/LP) germline variants in women with bladder cancer. Germline DNA was tested for mutations in ≥77 cancer susceptibility genes using next-generation sequencing in 686 patients with bladder cancer. Mutation frequency and clinical characteristics were assessed by gender. Results: A total of 184 (27%) women and 502 (73%) men with bladder cancer underwent germline testing; median age of diagnosis was 66 ± 11.3 and 65 ± 11.3 years, respectively. Twenty-two women (12%) had bladder cancer diagnosis at age ≤ 50 years. Both groups had similar rate of tobacco exposure (57% vs 63%, p = 0.1), family history of bladder cancer (10% vs 10%, p = 0.5), and disease stage at diagnosis (non-muscle invasive bladder cancer [NMIBC] 54% vs 54%, MIBC 38% vs 39%, and metastatic disease 8% vs 6%, p = 0.7). Women had more non-urothelial carcinoma histology than men (adenocarcinoma 5% vs. 1%; squamous cell carcinoma 1% vs 0.2%, p = 0.001). More P/LP germline variants were found in women than men (38 [21%] vs. 70 [14%], p = 0.04). Twenty-eight women (15%) had P/LP variants in DNA-damage repair (DDR) genes; 23 (13%) carried moderate/high penetrance germline mutations, the most common were BRCA1/ 2, CHEK2, NBN, ATM, and MITF. Current clinical guideline for referral for genetic testing failed to identify 12 (52%) women with moderate/high penetrance germline mutations. Nine women (5%) carried germline mutations associated with increased risk of ovarian/endometrial cancers ( BRCA1/ 2 [5], ATM [2], MLH1 [1], TP53 [1]). Conclusions: Deleterious germline alterations are commonly present in women with high-risk bladder cancer. The presence of germline variants in some genes, such as BRCA1/2, can guide cancer screening and risk-reducing surgeries for patients and their families. Women with high-risk bladder cancer should be evaluated for suitability of germline testing, especially those who desire preservation of uterus and ovaries at the time of radical cystectomy, to rule out the presence of P/LP variants that increase risk of future gynecologic malignancies.