Paraffin-embedded Tissue Specimens Research Articles

A DNA copy number alteration classifier as a prognostic tool for prostate cancer patients.

Distinguishing between true indolent and potentially life-threatening prostate cancer is challenging in tumours displaying clinicopathologic features associated with low or intermediate risk of relapse. Several somatic DNA copy number alterations (CNAs) have been identified as potential prognostic biomarkers, but the standard cytogenetic method to assess them has a limited multiplexing capability. Multiplex ligation-dependent probe amplification (MLPA) targeting 14 genes was optimised to survey 448 tumours of patients with low or intermediaterisk (Grade Group 1-3, Gleason score ≤7) who underwent radical prostatectomy. A 6-gene CNA classifier was developed using random survival forest and Cox proportional hazard modelling to predict biochemical recurrence. The classifier score was significantly associated with biochemical recurrence after adjusting for standard clinicopathologic variables and the known prognostic index CAPRA-S score with a hazard ratio of 2.17 and 1.80, respectively (n = 406, P < 0.01). The prognostic value of this classifier was externally validated in published CNA data from three radical prostatectomy cohorts and one radiation therapy pre-treatment biopsy cohort. The 6-gene CNA classifier generated by a single MLPA assay compatible with the small quantities of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens has the potential to improve the clinical management of patients with low or intermediaterisk disease.

Targeted Transcriptional Analysis of IgA Vasculitis, IgA Nephropathy, and IgA-Dominant Infection-Related Glomerulonephritis Reveals Both Distinct and Overlapping Immune Signatures.

IgA vasculitis (IgAV), IgA nephropathy (IgAN), and IgA-dominant infection-related glomerulonephritis (IgA-IRGN) have shared histopathologic features but differences in clinical management and prognosis. The most serious IgAV organ involvement is in the kidneys (IgAV nephritis). In this study, we hypothesized that targeted immune transcript profiling could aid in 1) predicting development of IgAV nephritis in patients with cutaneous IgAV, and 2) differentiating IgAN, IgAV, and IgA-IRGN. RNA was extracted from 24 formalin-fixed paraffin-embedded tissue specimens (16 kidney, 8 skin) from 21 patients with IgAV nephritis (n=7), IgAN (n=5), and IgA-IRGN (n=4), and IgAV skin biopsies from patients with (n=3) and without (n=5) IgAV nephritis. Differential gene expression and gene set enrichment analysis were performed on a total of 594 transcripts (Nanostring immunology panel) profiled using the nCounter system. Skin biopsies in IgAV patients who develop kidney involvement exhibit reduced S100A8/S100A9, IL9, and KIR expression. The kidney tissue immune transcriptomes of IgAN, IgAV, and IgA-IRGN are largely overlapping. IgA-IRGN kidney biopsies are, however, uniquely enriched for transcripts involved in granulocyte chemotaxis. This study identifies immune transcript signatures that may predict IgAV nephritis in skin biopsies and distinguish IgA-IRGN from IgAN and IgAV in kidney biopsies.

Comparative evaluation of podoplanin in odontogenic cysts and tumours to determine their proliferative potential-An immunohistochemical study.

Odontogenic cysts and tumours are a wide array of complex pathological entities ranging from mild indolent to aggressive detrimental in nature, which occur as a result of anomalous alterations in normal odontogenesis. Hence, these odontogenic lesions need to be evaluated extensively by using potential immunohistochemical markers. To evaluate and compare the expression of podoplanin, a lymphoendothelial IHC marker in odontogenic cysts and odontogenic tumours to determine their proliferative potential. All the study samples were retrieved from the archives of the Department of Oral Pathology and Microbiology, PIDS&RC, Hyderabad. The study samples were selected as per the standard histopathological diagnostic criteria and subjected for IHC analysis using podoplanin. Seventy paraffin-embedded tissue specimens of OKC, OOC, dentigerous cyst (DC) and ameloblastoma (AM) include study sample, which were stained with podoplanin IHC marker and staining properties were evaluated. All the cases were categorized as high, moderate, weak or negatively reactive on the basis of the composite scoring. Statistical analysis was done using SPSS version 14, and then results were compared by ANOVA post hoc test and Kruskal Wallis Test. In the comparison of composite scores of OKCs and AM, there was no significant statistical difference. The present study contributes to the significant association of podoplanin expression with cellular proliferation, cystic expansion and local invasiveness of odontogenic cysts and tumours through cytoskeletal reorganization and cell migration.

PD04-01 EVALUATION OF COPY NUMBER AND ALLELIC IMBALANCE OF BRCA2 BY NEXT-GENERATION SEQUENCING TO PREDICT PROGNOSIS AND DRUG SENSITIVITY IN PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023PD04-01 EVALUATION OF COPY NUMBER AND ALLELIC IMBALANCE OF BRCA2 BY NEXT-GENERATION SEQUENCING TO PREDICT PROGNOSIS AND DRUG SENSITIVITY IN PROSTATE CANCER Makoto Sumitomo, Takuhisa Nukaya, Eiji Sugihara, Mayu Takeda, Sachio Nohara, Shigeki Tanishima, Masashi Takenaka, kenji Zennami, Kiyoshi Takahara, Ryoichi Shiroki, and Hideyuki Saya Makoto SumitomoMakoto Sumitomo More articles by this author , Takuhisa NukayaTakuhisa Nukaya More articles by this author , Eiji SugiharaEiji Sugihara More articles by this author , Mayu TakedaMayu Takeda More articles by this author , Sachio NoharaSachio Nohara More articles by this author , Shigeki TanishimaShigeki Tanishima More articles by this author , Masashi TakenakaMasashi Takenaka More articles by this author , kenji Zennami kenji Zennami More articles by this author , Kiyoshi TakaharaKiyoshi Takahara More articles by this author , Ryoichi ShirokiRyoichi Shiroki More articles by this author , and Hideyuki SayaHideyuki Saya More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although pathologic mutations in BRCA1 and BRCA2 are significantly associated with risk of prostate cancer (PC) initiation and metastatic castration-resistant PC (mCRPC), the details of the relationship between BRCA alterations and loss of BRCA function remain unclear. We aimed to establish a method for estimating BRCA function and to elucidate the details and clinical significance of BRCA alterations in PC. We further examined whether estimating BRCA function could be useful to predict PARP inhibitor sensitivity of poly ADP-ribose polymerase (PARP) inhibitors to mCRPC. METHODS: DNA was extracted from formalin-fixed paraffin-embedded tissue specimens from 220 clinically localized PC patients who underwent radical prostatectomy and from 18 mCRPC patients with BRCA2 alterations detected by comprehensive genomic profiling panel testing. Mutations and copy number (CN) alterations of 143 cancer genes was analyzed by the in-house PleSSision Rapid test using NGS, and CN changes were also assessed by droplet digital PCR (ddPCR). The allelic imbalance (AI) could be detected by measuring the proportion of one allele relative to the other in cells from individuals that are constitutionally heterozygous at a given locus. Objective response (OR) to a drug was defined as a 50% or greater decrease in serum PSA levels from baseline. RESULTS: NGS analysis revealed that in 220 localized PC patients, BRCA2 CN decreased in 29 (13.2%) and BRCA1 CN in 11 (5.0%). NGS-based CN values were shown to be highly correlated with ddPCR-based CN values. Twelve of the 29 patients with decreased BRCA2 CN were presumed to have somatic heterozygous or homozygous deletions. Pathogenic gene mutations other than deletion of BRCA2 were identified in 5 of 220 patients, while those of BRCA1 in one patient. Five (29.4%) of 17 patients with BRCA2 alterations developed biochemical recurrence within 6 months after surgery. Multivariate analyses revealed that BRCA2 CN and initial PSA levels were independent factors for biochemical recurrence. In the mCRPC study, 8 of 18 patients with BRCA2 alterations have shown to have BRCA2 deletions. Of the 8 patients, 4 patients with BRCA2 CN less than 0.8 had an OR to the PARP inhibitor olaparib, while the other 4 patients with BRCA2 CN greater than 0.8 did not have an OR to olaparib. As for the other 10 patients with frameshift or single nucleotide mutations in BRCA2, 3 of 4 (75.0%) patients without AI in BRCA2 have an OR to olaparib, while only 1 of 6 (16.7%) with AI had an OR to olaparib. CONCLUSIONS: Our findings show that estimating CN decrease in BRCA2 using NGS can predict the prognosis of localized PC. Furthermore, our results may provide new insight into the utility of assessing both CN and AI of BRCA2 to accurately predict response to PARP inhibitors in mCRPC patients with BRCA2 alterations. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e142 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Makoto Sumitomo More articles by this author Takuhisa Nukaya More articles by this author Eiji Sugihara More articles by this author Mayu Takeda More articles by this author Sachio Nohara More articles by this author Shigeki Tanishima More articles by this author Masashi Takenaka More articles by this author kenji Zennami More articles by this author Kiyoshi Takahara More articles by this author Ryoichi Shiroki More articles by this author Hideyuki Saya More articles by this author Expand All Advertisement PDF downloadLoading ...

Impact of CD34-dependent Micro Vessel Density on Periapical Odontogenic Cysts.

Odontogenic cysts belong to a type of lesions with endodontic origin that in some cases mimic even aggressive odontogenic tumors sharing with them similar radiographic features. Periapical cysts (PCs) belong to the inflammatory odontogenic cysts sub-category and rarely squamous cell carcinoma arises from their hyperplastic/ dysplastic epithelia. This study aimed to explore the impact of cluster differentiation 34 (CD34) protein expression combined with micro vessel density (MVD) on PCs. Forty-eight (n=48) archival, formalin-fixed, and paraffin-embedded PC tissue specimens were included in the study. Immunohistochemistry (IHC) was performed in the corresponding tissue sections using an anti- CD34 antibody. CD34 expression levels and also MVD in the examined cases were measured by implementing a digital image analysis protocol. CD34 over-expression (moderate to high staining intensity levels) were detected in 29/48 (60.4%) cases, whereas the rest of them (19/48-39.6%) were characterized by low levels of expression. Extended MVD was identified in 26/48 (50.1%) cases correlated with CD34 over-expression, epithelial hyperplasia (p-value=0.001), and marginally with inflammatory infiltration level in the examined lesions (p-value=0.056). CD34 over-expression combined with increased MVD is associated with a neoplastic-like (hyperplastic) phenotype in PCs as a result of increased neo-angiogenic activity. These histopathological characteristics rarely form an eligible substrate for squamous cell carcinoma onset in untended cases.

WD repeat protein 54-mediator of ErbB2-driven cell motility 1 axis promotes bladder cancer tumorigenesis and metastasis and impairs chemosensitivity

One of the most abundant protein-protein interaction domains in the human proteome is the WD40 repeat (WDR) domain. A Gene Expression Omnibus dataset revealed 37 differentially expressed WDR domain genes in bladder cancer (BC). WD repeat domain 54 (WDR54), an upregulated WDR domain gene, was selected for further investigation. Sixty pairs of frozen BC tumor and non-malignant bladder tissues and 83 paraffin-embedded BC tissue specimens were obtained. Loss-/gain-of-function experiments were carried out using BC and xenograft tumor models. WDR54 was overexpressed in BC cells, and its high expression was linked to tumor stage and lymph node metastases in patients. WDR54 contributed to the tumorigenesis and metastasis of BC and impaired its chemosensitivity. WDR54 prevented the degradation and ubiquitination of the mediator of ErbB2-driven cell motility 1 (MEMO1). WDR54 also promoted the interaction between MEMO1 and insulin receptor substrate 1 (IRS1) and activated the IRS1/AKT/β-catenin pathway in BC cells. Particularly, WDR54 depended on MEMO1 to exert its biological functions. Our study demonstrated the relevance of WDR54 in BC and provides insight into the molecular mechanism underlying BC.

Abstract C017: Detection of wild-type and truncated HMGA2 in prostate cancer tissues using RNA in situ hybridization

Abstract High mobility group A2 (HMGA2), a non-histone protein, is known to promote epithelial-mesenchymal transition (EMT), which plays a critical role in prostate cancer progression and metastasis. Both full-length HMGA2 (WT-HMGA2) and truncated (lacking the 3’UTR) HMGA2 (TR-HMGA2) isoforms are overexpressed in several cancers such as lung carcinomas, ovarian cancer, breast cancer, and gastric cancer. However, there is no study investigating the expression and differential roles of WT vs truncated HMGA2 isoforms in prostate cancer. Our previous results indicated that the overexpression of HMGA2 in LNCaP cells increased cell viability and migration for both wild-type and truncated HMGA2. Promotion of EMT was observed in wild-type, but not truncated HMGA2. Reactive oxygen species (ROS) levels were increased with truncated HMGA2 more than wild-type HMGA2. Therefore, wild-type and truncated HMGA2 may play different roles on cancer progression and metastasis. The goal of this study is to examine the expression of wild-type vs. truncated HMGA2 in prostate cancer patient tissue. We have developed a way to detect location and expression of wild-type and truncated HMGA2 using RNA in situ hybridization (RISH). The specific probes’ hybridization followed by a serial signal amplification process allows us to locate and quantify the HMGA2 isoforms in Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens from cancer patients. Several prostate tissue samples and a prostate cancer tissue microarray that includes 35 patients from different races, stages of cancer, and metastasis status was analyzed. Our results showed increased HMGA2 expression (both wild-type and truncated) in several prostate cancer samples with higher stage cancer. We also observed that most wild-type and truncated HMGA2 is located within the nucleus, while some wild-type isoforms were detected within the cytoplasm. We will further quantify the expression levels to examine whether they are associated with tumor stage and/or race. These studies are the first to examine HMGA2 isoform expression and localization in prostate patient tissue and may offer novel therapeutic intervention strategies based on HMGA2 isoform expression. Citation Format: Bor-Jang Hwang, Mojisoluwa Awolowo, Taaliah Campbell, Ohuod Hawsawi, Sharon Harrison, Denise Gibbs, Camille Ragin, Valerie Odero-Marah. Detection of wild-type and truncated HMGA2 in prostate cancer tissues using RNA in situ hybridization [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C017.

Over-expression of Long Non-coding RNA Urothelial Cancer-associated 1 as a Predictive Marker for Prostate Cancer.

To determine the expression of long non-coding RNA urothelial cancer-associated 1 (UCA1) by performing array-based quantitative polymerase chain reaction (PCR) and to identify the clinicopathological significance of UCA1 expression in prostate cancer using in situ hybridization (ISH) of surgically resected specimens. Array-based quantitative PCR was performed using 10 pairs of fresh malignant (prostate cancer) and normal tissue samples to determine UCA1 expression. Single-color RNA ISH of surgically resected prostate cancer specimens was performed using 70 formalin-fixed, paraffin-embedded tissue specimens to examine the clinicopathological significance of UCA1. Prostate cancer tissues exhibited higher levels of UCA1 expression than paired benign tissues. Furthermore, a correlation between high UCA1 expression and unfavourable clinicopathological characteristics, including advanced pathologic T stage, extraprostatic extension, presence of Gleason pattern 5, and involvement of the resection margins was observed. Notably, increased UCA1 expression significantly correlated with high- or very-high-risk patients, as defined by the 2023 National Comprehensive Cancer Network guidelines. UCA1 could be used as a novel diagnostic and prognostic biomarker for establishing an effective treatment protocol for prostate cancer.

The ALDH2, IGSF9, and PRDM16 Proteins as Predictive Biomarkers for Prognosis in Breast Cancer

IntroductionALDH2, IGSF9, and PRDM16 play crucial roles in regulating diverse cellular pathophysiologic functions. The current study was to evaluate the effect of the 3 proteins on clinicopathologic features and prognosis of patients with breast cancer. Materials and MethodsThe formalin-fixed and paraffin-embedded tissue specimens were collected from breast cancer patients by immunohistochemistry (IHC) were analyzed. ResultsOf the 216 patients enrolled, ALDH2 high expression was significantly correlated with the age (p = .040), larger tumor size (p = .001), LVI (p < .001), LNM (p < .001), advanced TNM staging (p < .001), PR (p = .027), HER2 status (p = .002), and molecular subtype (p = .003). IGSF9 low expression was significantly correlated with the LV1 (p = .024), LNM (p = .024), advanced TNM staging (p = .001). The low expression of PRDM16 was significantly correlated with age (p = .023), and LNM (p = .014). The A+I−P− expression (13.4%) were markedly correlated with lymphatic vessel invasion (LVI) (p < .001), lymph node metastasis (LNM) (p < .001), advanced TNM staging (p < .001). Furthermore, patients with A+I−P− expression had significantly advanced-stage breast cancer [stage III (72.4%) vs. (23.0%)]. Univariate and multivariate analysis identified variables (ie, larger tumor size, lymph node involvement, and A+I−P− expression) as independent prognostic factors for survival. ConclusionOur results reveal ALDH2 high expression, IGSF9 and PRDM16 low expression, A+I−P− expression was associated with advanced clinicopathological characteristics, and shorter OS and DFS in breast cancer patients. The 3 proteins may be potential prognosis markers and therapeutic targets for breast cancer patients.

Drug Resistance and Molecular Characteristics of Mycobacterium tuberculosis: A Single Center Experience.

In recent years, the incidence of tuberculosis (TB) and mortality caused by the disease have been decreasing. However, the number of drug-resistant tuberculosis patients is increasing rapidly year by year. Here, a total of 380 Mycobacterium tuberculosis (MTB)-positive formalin-fixed and paraffin-embedded tissue (FFPE) specimens diagnosed in the Department of Pathology of the Eighth Medical Center, Chinese PLA General Hospital were collected. Among 380 cases of MTB, 85 (22.37%) were susceptible to four anti-TB drugs and the remaining 295 (77.63%) were resistant to one or more drugs. The rate of MDR-TB was higher in previously treated cases (52.53%) than in new cases [(36.65%), p < 0.05]. Of previously treated cases, the rate of drug resistance was higher in females than in males (p < 0.05). Among specimens obtained from males, the rate of drug resistance was higher in new cases than in previously treated cases (p < 0.05). Of mutation in drug resistance-related genes, the majority (53/380, 13.95%) of rpoB gene carried the D516V mutation, and 13.42% (51/380) featured mutations in both the katG and inhA genes. Among the total specimens, 18.68% (71/380) carried the 88 M mutation in the rpsL gene, and the embB gene focused on the 306 M2 mutation with a mutation rate of 19.74%. Among the resistant INH, the mutation rate of −15 M was higher in resistance to more than one drug than in monodrug-resistant (p < 0.05). In conclusion, the drug resistance of MTB is still very severe and the timely detection of drug resistance is conducive to the precise treatment of TB.

Clinicopathological features, genetic alterations, and BRCA1 promoter methylation in Japanese male patients with breast cancer

PurposeMale breast cancer (MBC) is a rare cancer accounting for only 1% of all male cancers and is, therefore, poorly studied. We aimed to characterize the subtypes of MBC in Japanese patients based on genetic profiling, the presence of tumor-infiltrating cells, and the expression of immunohistochemical markers.MethodsThis retrospective study included 103 patients with MBC diagnosed between January 2009 and December 2019 at various hospitals in Japan. Clinicopathological patient characteristics were obtained from medical records, and formalin-fixed paraffin-embedded tissue specimens were analyzed for histological markers, mutations of 126 genes, BRCA1 methylation, and stromal tumor-infiltrating lymphocytes.ResultsThe median patient age was 71 (range 31–92) years. T1-stage tumors were the most frequent (47.6%), and most were node negative (77.7%). The majority of tumors were positive for estrogen receptor (98.1%), progesterone receptor (95.1%), and androgen receptor (96.1%), and BRCA2 was the most frequently mutated gene (12.6%). The most common treatment was surgery (99.0%), either total mastectomy (91.1%) or partial mastectomy (7.0%). Survival analysis showed a 5-year recurrence-free survival rate of 64.4% (95% confidence interval [CI] 46.7–88.8) and a 5-year overall survival rate of 54.3% (95% CI 24.1–100.0).ConclusionJapanese MBC is characterized by a high rate of hormonal receptor positivity and BRCA2 somatic mutation. Due to the observed clinicopathological differences in MBC between the Western countries and Japan, further prospective studies are needed to evaluate the most suitable treatment strategies.

Quality metrics for enhanced performance of an NGS panel using single-vial amplification technology

AimsTargeted next-generation sequencing (NGS) panels, which identify genomic alterations, are the stronghold of molecular oncology laboratories. In spite of technological advances, the quantity and quality of DNA from formalin-fixed paraffin-embedded...

Association of genomic instability of CDH1 gene with clinicopathological characteristics of gastric cancer

To assess the association of genomic instability of epithelial cadherin 1 (CDH1) gene and clinicopathological characteristics of gastric cancer. In total 120 paraffin-embedded gastric cancer tissue specimen were prepared, and genomic DNA was extracted. The genomic instability of the CDH1 gene was analyzed by immunohistochemistry and silver staining PCR-single-strand conformation polymorphism. The number of information individuals (heterozygotes) was 98 for the D16S752 locus. The detection rates for microsatellite instability (MSI) and loss of heterozygosity (LOH) at the D16S752 locus and the positive rate of CDH1 protein were 19.39%, 16.33% and 51.02%, respectively. The detection rate of MSI in TNM stages I or II was significantly higher than that in stages III or IV (P<0.05) while the detection rate of LOH was significantly lower than that in stages III or IV (P<0.05). The positive rate of CDH1 protein in TNM stages III or IV was significantly lower than that in stages I or II (P<0.05). The detection rate of MSI of cases with lymph node metastasis was significantly lower than that of without lymph node metastasis (P<0.05) while the detection rate of LOH was significantly higher than that without lymph node metastasis (P<0.05). The positive rate of CDH1 protein in patients with lymph node metastasis was significantly lower than that in patients without lymph node metastasis (P<0.05). The positive rate of CDH1 protein in MSI-positive group was significantly higher than that in MSI-negative group (P<0.05), and the positive rate of CDH1 protein in the LOH-positive group was significantly lower than that the LOH-negative group (P<0.05). The genomic instability of the CDH1 gene is associated with the progression of gastric cancer. MSI at the D16S752 locus may be used as a molecular marker for early gastric cancer, while LOH at this locus mostly occurs in advanced gastric cancer and can be regarded as an effective indicators for malignancy evaluation and prognosis.

Clinical Utility of Next-Generation Sequencing Panel Testing in the Evaluation of Arteriovenous Malformations

Abstract Arteriovenous malformations (AVMs) are vascular lesions in which an overgrowth of blood vessels of varying sizes develops with one or more direct connections between the arterial and venous circulation. AVMs are seen in sporadic and syndromic conditions that are caused by a variety of genomic alterations. Here, we performed a retrospective review of the cases sent for next-generation sequencing (NGS) analysis for diseases of somatic mosaicism (DoSM). Specimens from 54 patients with clinical indication of AVMs were submitted for the DoSM NGS panel between October 2013 and Dec 2021. DNA extraction and sequence analysis were performed on affected tissues, including fresh tissue, formalin-fixed paraffin-embedded tissue, fibroblast cultures, and buccal specimens. Single-nucleotide variants (SNVs) with variant allele fractions (VAFs) greater than 3% and small insertions and deletions (indels, &amp;lt;21 bp) were called using VarScan2, Genome Analysis Toolkit (GATK), and Pindel. Variants with VAFs between 1-3% were manually reviewed based on the sequencing data quality and the known clinical significance. 38/54 (69.1%) patients were females and 16/54 (30.9%) were males. Ages ranged from 1 month to 73 years (median age 17 years). Most of the patients were indicated with sporadic AVM (81%), although additional clinical indications were noticed, including unspecified congenital vascular malformation (9.1%), capillary malformation (5.5%), arteriovenous fistula, and AVM associated with capillary and lymphatic malformations (1.8% each). The biopsied lesion was most often located in the head (43.6%), followed by the limbs (30.9%), and unspecified areas of the body in the remaining 25.5%. Among the 54 cases, 37 (68.5%) cases had pathogenic and/or likely pathogenic (P/LP) variants identified, 2 cases (3.7%) had variants of unknown significance, and the remaining 15 cases (27.8%) had negative results. MAP2K1 variants were found in 12 samples, followed by KRAS (8), TEK (7), PTEN (5), BRAF (4), TSC2 (2), HRAS (2), RASA1 (2), and PDGFRB (1). Of note, four cases had two P/LP variants. Among the 37 positive cases, 32 cases had somatic alterations; the remaining 5 cases had at least one germline P/LP variant, including PTEN (4) and RASA1 (1). In summary, the diagnostic yield of cases with clinical indication of AVMs was 68.5% using our in-house DoSM NGS panel. This study demonstrates the clinical utility of the DoSM NGS panel testing in the evaluation of a cohort with clinical presentations of AVMs.

ODP559 The prognostic role of IL-17A-neutrophils crosstalk in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is characterized by extensive intratumoral heterogeneity. At this juncture, TNBCs are treated with conventional chemotherapy and have few targeted therapies. Due to the presence of increased tumor-infiltrating lymphocytes and immune-checkpoint molecules, TNBCs are considered highly immunogenic. This provides an opportunity to explore various immunotherapeutic options. The immune cells such as Th17 cells or neutrophils (tumor-associated neutrophils; TANs) can secrete a pro-inflammatory cytokine called IL-17A, which is known to be associated with poor outcomes in several cancers. TANs are related to tumor progression and are influenced by certain chemokines. Meanwhile, IL-17A can also regulate chemokine (C-X-C motif) ligand 1 (CXCL1), which is a strong chemoattractant for TANs and can lead to the growth of tumors in breast cancer. However, the significance of IL-17A and its interaction with TANs and CXCL1 has yet not been clearly understood in TNBC. For this purpose, formalin-fixed paraffin-embedded biopsy tissue specimens of 109 Japanese TNBC patients were included in this study. Immunohistochemistry was performed to assess the status of IL-17A, CXCL1, and CD66b (a neutrophil marker) and to understand their correlation among each other, with clinical parameters, and with the outcomes of patients. Also, in vitro studies were performed to evaluate the effect of recombinant IL-17A on TNBC cell lines proliferation, migration, and CXCL1 expression. Clinical results revealed that IL-17A was significantly correlated with CXCL1 and CD66b, which suggested potential crosstalk between them. On the other hand, CXCL1 significantly and CD66b tended to be correlated with tumor size. Also, CD66b was significantly correlated with the Ki-67 labeling index, showing that TANs may have a role in tumor cell proliferation. Kaplan-Meier survival curves showed that high IL-17A was significantly associated with poor disease-free and overall survival, indicating the poor outcome of the TNBC patients. To our great interest, in vitro results revealed that no difference was observed for proliferation or migration of TNBC cell lines (was only observed in the MDA-MB-231 cell line) after treatment with recombinant IL-17A. However, CXCL1 was highly up-regulated in a dose and time-dependent manner after exposure to IL-17A, indicating that IL-17A might be involved in the recruitment of neutrophils through up-regulation of CXCL1. Therefore, we concluded that IL-17A was a poor prognostic factor for TNBC patients, enhancing the chemokine-neutrophil recruitment to the tumor site, leading to tumor progression and aggression. In the future, we intend to explore different pathways for understanding the mechanism of CXCL1 up-regulation by IL-17A. Presentation: No date and time listed

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging: Diagnostic Pathways and Metabolites for Renal Tumor Entities

Background: Correct tumor subtyping of primary renal tumors is essential for treatment decision in daily routine. Most of the tumors can be classified based on morphology alone. Nevertheless, some diagnoses are difficult, and further investigations are needed for correct tumor subtyping. Besides histochemical investigations, high-mass-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can detect new diagnostic biomarkers and hence improve the diagnostic. Patients and Methods: Formalin-fixed paraffin embedded tissue specimens from clear cell renal cell carcinoma (ccRCC, n = 552), papillary renal cell carcinoma (pRCC, n = 122), chromophobe renal cell carcinoma (chRCC, n = 108), and renal oncocytoma (rO, n = 71) were analyzed by high-mass-resolution MALDI fourier-transform ion cyclotron resonance (FT-ICR) MSI. The SPACiAL pipeline was executed for automated co-registration of histological and molecular features. Pathway enrichment and pathway topology analysis were performed to determine significant differences between RCC subtypes. Results: We discriminated the four histological subtypes (ccRCC, pRCC, chRCC, and rO) and established the subtype-specific pathways and metabolic profiles. rO showed an enrichment of pentose phosphate, taurine and hypotaurine, glycerophospholipid, amino sugar and nucleotide sugar, fructose and mannose, glycine, serine, and threonine pathways. ChRCC is defined by enriched pathways including the amino sugar and nucleotide sugar, fructose and mannose, glycerophospholipid, taurine and hypotaurine, glycine, serine, and threonine pathways. Pyrimidine, amino sugar and nucleotide sugar, glycerophospholipids, and glutathione pathways are enriched in ccRCC. Furthermore, we detected enriched phosphatidylinositol and glycerophospholipid pathways in pRCC. Conclusion: In summary, we performed a classification system with a mean accuracy in tumor discrimination of 85.13%. Furthermore, we detected tumor-specific biomarkers for the four most common primary renal tumors by MALDI-MSI. This method is a useful tool in differential diagnosis and biomarker detection.

THE RELATIONSHIP BETWEEN THE USE OF SMOKELESS TOBACCO AND ORAL SQUAMOUS CELL CARCINOMA

Smokeless tobacco (ST) is a tobacco product that is used by means other than smoking. Evidence was available that ST products is a known risk factor for oral precancerous and cancers for long, with greater risk for oral cancer than others. The prospective, descriptive study has carried out to evaluate the relationship between the use of smokeless tobacco and oral cancer.Biopsy samples have collected from 60 patients, whose have attended to Al-Gamhouria Teaching Hospital and histo-pathological private labs in Aden using a questionnaire during the period of 12 months. The sections from paraffin-embedded tissue specimens were stained with hematoxylin and eosin (H &amp; E). A section containing the full thickness of the tumors have used for histopathological grading.The prevalence of oral squamous cell carcinoma (OSCC) lesions showed different grade of differentiation among 60 studied patients cases, the Well differentiation squamous cell carcinoma (WDSCC) was the highest percentage (70%). No significant difference noticed in well-differentiated SCCs (WDSCC) between men (36.7%) and women (33.3 %; P &lt; 0.05). The moderate differentiation SCC (MDSCC), and the poor differentiation SCC (PDSCC) represented (20% &amp; 10%) respectively. According to the age of patients, WDSCC, MDSCC and PDSCC highest percentage age group was (51-60) years. Male were predominant in both genders, male to female ratio 1.2: 1. Anatomical sites of all OSCC lesions had analyzed and revealed that tongue was the most prevalent site (33.3%), followed by the buccal mucosa, (23.3%) , floor of mouth (16.7%) , lip (13.3%), Gingiva (8.3%), and the least prevalent site was the hard palate represented (5%) of all cases., the chief etiological factor which we observed, was tobacco, in the form of smoking tobacco used by 75% of the patients followed by smokeless tobacco product users, in form of shamma chewers, (61.7%), and in form of Hot sachet (58.3%), and Zarda users represented 51.7% of cases.Our results indicate that the occurrence of OSCC among studied cases showed different grade of differentiation, age and gender have some relation with the presence of OSCC. and it is high association with risk habits of tobacco use in different forms particularly smokeless tobacco.

Alpha2,3 sialic acid processing enzymes expression in gastric cancer tissues reveals that ST3Gal3 but not Neu3 are associated with Lauren's classification, angiolymphatic invasion and histological grade.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Despite progress in the last decades, there are still no reliable biomarkers for the diagnosis of and prognosis for GC. Aberrant sialylation is a widespread critical event in the development of GC. Neuraminidases (Neu) and sialyltransferases (STs) regulate the ablation and addition of sialic acid during glycoconjugates biosynthesis, and they are a considerable source of biomarkers in various cancers. This study retrospectively characterized Neu3 and ST3Gal3 expression by immunohistochemistry in 71 paraffin-embedded GC tissue specimens and analyzed the relationship between their expression and the clinicopathological parameters. Neu3 expression was markedly increased in GC tissues compared with non-tumoral tissues (p<0.0001). Intratumoral ST3Gal3 staining was significantly associated with intestinal subtype (p=0.0042) and was negatively associated with angiolymphatic invasion (p=0.0002) and higher histological grade G3 (p=0.0066). Multivariate analysis revealed that ST3Gal3 positivity is able to predict Lauren's classification. No associations were found between Neu3 staining and clinical parameters. The in silico analysis of mRNA expression in GC validation cohorts corroborates the significant ST3Gal3 association with higher histological grade observed in our study. These findings suggest that ST3Gal3 expression may be an indicator for aggressiveness of primary GC.

Hippo-YAP signaling activation and cross-talk with PI3K in oral cancer: A retrospective cohort study.

This study aimed to investigate the clinical and prognostic relevance of the Hippo-YAP transactivators YAP1 and TAZ in oral squamous cell carcinoma, and their possible relationship with PI3K/mTOR pathway activation. Immunohistochemical analysis of YAP1, TAZ, PIK3CA (p110α), p-AKT (Ser473), and p-S6 (Ser235) was performed in paraffin-embedded tissue specimens from 165 OSCC patients. Correlations between protein expression and clinical data were further assessed. YAP1 expression was detected in both cytoplasm and nucleus of tumor cells, whereas TAZ expression was only found in the nucleus. Nuclear YAP1 was significantly associated with tumor size (p= 0.03), neck lymph node metastasis (p= 0.02), TNM stage (p= 0.02), and poor differentiation (p= 0.04). Nuclear TAZ was associated with tobacco (p= 0.03) and alcohol consumption (p= 0.04), and poor tumor differentiation (p= 0.04). There was a positive significant correlation between nuclear and cytoplasmic YAP1, nuclear TAZ, p110α expression, and mTORC1 activation p-S6 (S235). Combined expression of nuclear and cytoplasmic YAP1 was prognostic in both univariate and multivariate analyses. Active nuclear YAP1 was significantly and independently associated with poor disease-specific (p= 0.005, HR=2.520; 95%CI=1.319-4.816) and overall survival (p= 0.015, HR=2.126; 95%CI=1.155-3.916). Nuclear YAP1 is an independent predictor of poor survival in oral squamous cell carcinoma.

Lipopolysaccharide Inhibits Autophagy and Promotes Inflammatory Responses via p38 MAPK-Induced Proteasomal Degradation of Atg13 in Hepatic Stellate Cells.

Background Inflammation plays a critical role in the progression of acute-on-chronic liver failure (ACLF). Atg13 is a vital regulatory component of the ULK1 complex, which plays an essential role in the initiation of autophagy. Previously, hepatic stellate cells (HSCs) were considered to be noninflammatory cells that contribute only to hepatic fibrosis. Recently, it has been found that HSCs can secrete inflammatory cytokines and participate in hepatic inflammation. Autophagy and proteasome-mediated degradation constitute two major means of protein turnover in cells. Autophagy has been shown to regulate inflammation, but it is unclear whether ubiquitin (Ub)-proteasome system (UPS) is involved in inflammatory responses in HSCs during ACLF. Methods Clinical data were collected from ACLF patients, and surgically resected paraffin-embedded human ACLF liver tissue specimens were collected. The expression of Atg13 was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Secretion of IL-1β was assessed by ELISA. Atg13 was knocked down by siRNA in LX2 cells. Coimmunoprecipitation assay was used to detect protein binding and polyubiquitination of Atg13. In vitro tests with LX2 cells were performed to explore the effects and regulation of p38 MAPK, Atg13, UPS, autophagy, and inflammation. Results Serum lipopolysaccharide (LPS) was positively associated with disease severity in ACLF patients, and p38 MAPK was overexpressed in ACLF liver tissue. We evaluated the role of Atg13 in HSC inflammation and explored the possible underlying mechanisms. Inflammatory factors were upregulated via activation of p38 MAPK and inhibition of autophagy in LX-2 cells. Expression of Atg13 was decreased in LPS-incubated LX2 cells. Atg13 knockdown markedly inhibited autophagy and promoted LPS-induced inflammation in LX2 cells. Our in vitro experiments also showed that LPS induced depletion of Atg13 via UPS, and this process was dependent on p38 MAPK. Conclusions LPS induces proteasomal degradation of Atg13 via p38 MAPK, thereby participating in the aggravation of LPS-induced autophagy inhibition and inflammatory responses in LX2 cells. Atg13 serves as a mediator between autophagy and proteasome. Modulation of Atg13 or proteasome activity might be a novel strategy for treating HSC inflammation.