Tissue Profile Research Articles

Comparative Analysis of mRNA and lncRNA Expression Profiles in Testicular Tissue of Sexually Immature and Sexually Mature Mongolian Horses.

Testicular development and spermatogenesis are tightly regulated by both coding and non-coding genes, with mRNA and lncRNA playing crucial roles in post-transcriptional gene expression regulation. However, there are significant differences in regulatory mechanisms before and after sexual maturity. Nevertheless, the mRNAs and lncRNAs in the testes of Mongolian horses have not been systematically identified. In this study, we first identified the testicular tissues of sexually immature and sexually mature Mongolian horses at the tissue and protein levels, and comprehensively analyzed the expression profiles of mRNA and lncRNA in the testes of 1-year-old (12 months, n = 3) and 10-year-old (n = 3) Mongolian horses using RNA sequencing technology. Through gene expression analysis, we identified 16,582 mRNAs and 2128 unknown lncRNAs that are commonly expressed in both sexually immature and sexually mature Mongolian horses. Meanwhile, 9217 mRNAs (p < 0.05) and 2191 unknown lncRNAs (p < 0.05) were identified as differentially expressed between the two stages, which were further validated by real-time fluorescent quantitative PCR and analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The analysis results showed that genes in the sexually immature stage were mainly enriched in terms related to cellular infrastructure, while genes in the sexually mature stage were enriched in terms associated with hormones, metabolism, and spermatogenesis. In summary, the findings of this study provide valuable resources for a deeper understanding of the molecular mechanisms underlying testicular development and spermatogenesis in Mongolian horses and offer new perspectives for future related research.

M6A Methylation Mediates the Function of the circRNA-08436/miR-195/ELOVL6 Axis in Regards to Lipid Metabolism in Dairy Goat Mammary Glands.

The nutritional value of goat milk is determined by the composition of its fatty acids, with particular importance placed on the role of unsaturated fatty acids in promoting human health. CircRNAs have been known to affect fatty acid metabolism through different pathways. In this study, high-throughput sequencing was employed to construct expression profiles of mammary tissue harvested during the dry period and peak lactation stages of dairy goats. Differentially expressed circRNAs and mRNAs were screened, revealing significantly higher expression levels of circRNA-08436 and ELOVL6 during the peak lactation period compared with the dry period. Thus, circRNA-08436 and ELOVL6 were chosen for subsequent studies. The findings demonstrated that circRNA-08436 not only promotes the synthesis of triglyceride (TAG) and cholesterol in goat mammary epithelial cells (GMECs), but also increases the concentrations of saturated fatty acids in the cells. Through the utilization of software prediction, the dual luciferase reporter system, and qRT-PCR, it was observed that circRNA-08436 binds to miR-195, with its overexpression reducing the expression levels of miR-195 and inhibiting TAG synthesis. In addition, circRNA-08436 upregulated the expression levels of the miR-195 target gene ELOVL6. The data also revealed that YTHDC1 facilitated the transport of circRNA-08436 from the nucleus to the cytoplasm, while YTHDC2 in the cytoplasm functioned as a "reader" to identify and degrade circRNA-08436. Taken together, these findings contribute to a better understanding of the molecular regulation of fatty acid metabolism in the mammary glands of dairy goats, thus offering a sound theoretical basis for the production of high-quality goat milk.

Evaluation of soft tissue profile changes following autogenous fat or onlay PEEK augmentation versus sliding genioplasty for correction of deficient chin: Randomized controlled clinical trial

AimThe study was conducted to evaluate soft tissue profile changes using autogenous fat augmentation or onlay PEEK versus sliding genioplasty for correction of deficient chin in patients with retruded chin. Material and methodsThirty-three patients with deficient chins were included in the study. The patients were distributed into 3 groups: the fat augmentation group as intervention I, the PEEK augmentation group as intervention II, and the osseous genioplasty group as control group. Preoperative and postoperative CBCT were performed for all patients. With the aid of MIMICS,3-MATIC, and PROPLAN software, diagnosis, virtual planning, and evaluation were performed. The Face-Q questionnaire was used to assess patient satisfaction. ResultsThere was a statistically significant difference regarding soft tissue relapse in the fat group after 6 months when compared to the control group (mean difference= 0.770), while there was no statistically significant difference regarding soft tissue relapse between PEEK and control group (mean difference= -0.060). Intragroup comparison has shown no statistically significant difference regarding soft tissue relapse within all groups between follow-up periods (P = 0.1389 for the fat group, P = 0.8739 for the peek group, and P = 0.8410 for the control group). All patients showed a statistically significant increase in scores of satisfaction with the chin between follow-up periods (P = 0.0165, P = 0.0150, and P = 0.0293) respectively. ConclusionsFat augmentation can be a good intervention choice in mild-moderate deficient cases. PEEK PSI has a stable surgical outcome.

Modeling of individual teleroentgenometric indicators using the "Cephalometrics for orthognathic surgery" method in Ukrainian young women with a wide face type and orthognathic bite

The study of individual normative cephalometric parameters in individuals of different sexes and ages is important for the "Cephalometrics for orthognathic surgery method", as it allows to accurately diagnose abnormalities and develop personalized treatment plans. This contributes to achieving better aesthetic results, reducing the risk of complications and increasing the effectiveness of surgical interventions. Taking into account age, sex, and face type helps predict long-term changes and adapt the treatment plan to obtain optimal results. In addition, it improves assessment accuracy and standardizes evidence-based approaches, making it easier to compare results between clinics. Thus, individual regulatory parameters are key to successful orthognathic surgery. The purpose of the study is to build and analyze regression models of teleroentgenometric indicators using the "Cephalometrics for orthognathic surgery" method in Ukrainian young women with a wide face type. 25 Ukrainian young women with an orthognathic bite and a wide face type underwent a cephalometric study using the "Cephalometrics for orthognathic surgery" (COGS-method) method. For the correct modeling of cephalometric parameters, their division into three groups was applied (Dmitriev M. O., 2016, 2017): the first group – basic metric characteristics of the skull; the second group – teleroentgenometric indicators by which it is possible to change the parameters of the upper and lower jaws with the help of orthognathic surgery; the third group – indicators that characterize the position of each tooth relative to each other, cranial structures and the profile of the soft tissues of the face. Construction of regression models was carried out in the license package "Statistica 6.0". Only reliable models with a coefficient of determination R2 of at least 0.60 were subject to further analysis. It was found that in young women with a wide face, using the COGS method, 6 models of teleroentgenometric indicators were built out of 33 possible, which were included in the second and third groups depending on the indicators of the first group (R²= from 0.601 to 0.705, p&lt;0.01-0.001); out of 19 possible, 16 indicator models were built, which were included in the third group depending on the indicators of the first and second groups (R²= from 0.614 to 0.983, p&lt;0.01-0.001). The analysis of the models showed that most often the regression equations of the indicators included in the second and third groups, depending on the indicators of the first group, include the distance P-PTV and N-СС according to Ricketts, N-Se according to Schwarz, N-S and S-Ar according to Roth-Jarabak, Ar-Pt and Pt-N according to the COGS method (7.69 % each), as well as the value of the H angles according to Schwarz and N-S-Ba according to Bjork; and to the indicator models that were included in the third group depending on the indicators of the first and second groups – the value of the distances ANS-Me, N-B, N-A, N-Pog, B-Pog, N-CC according to Ricketts, PNS-N, Ar-Go and ANS-PNS, as well as the magnitude of the angles N-A-Pog, N-S-Ba according to Bjork, MP-HP, as well as Por-NBa according to Ricketts.

Creatine and l-carnitine attenuate muscular laminopathy in the LMNA mutation transgenic zebrafish

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify l-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest l-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.

Molecular profiling in ABC: Is complementary testing with tissue and plasma necessary and clinically relevant?

e12566 Background: This retrospective analysis explores the concordance between molecular analyses of tissue and plasma amongst Advanced Breast Cancer (ABC) patients and investigates the utility of simultaneous profiling for enhanced clinical insights. Methods: A total of 187 (177 Cancertrack paired and 10 Guardant 360) patient samples with availability of both tissue and ctDNA specimens, were subjected to comparative molecular profiling. Median age was 52 years (Range 23 – 82 years), 174 (93%) patients had advanced breast cancer and 177 (95%) patients had received prior treatments. We assigned tier 1 status to predictive biomarkers with FDA approved or guideline recommended therapies in breast cancer. Tier 2 status was assigned to alterations for which therapies were approved in different tumor type. Tier 3 had either clinical evidence in small studies or had clinical trial inclusion criteria. Results: Paired tissue-ctDNA profiling showed 139/187 (74%) samples had at least one concordant molecular alteration. Simultaneous tissue and ctDNA profiling led to the opportunity to offer Tier 1 recommended therapies in 113/187 (60%) patients. Amongst these, 71/113 (63%) exhibited concordance in tissue and circulating free DNA (cfDNA). ctDNA missed Tier 1 alterations in 14 patients which were detected by tissue biopsy. On the contrary tissue biopsy failed to detect Tier 1 alterations in 18 patients which were identified by ctDNA only. When Tier 2 and 3 alterations were considered, ctDNA identified additional alterations compared to tissue in 29 patients (37 variants), whereas tissue identified additional alterations in 20 patients (24 variants). Complementary tissue and ctDNA profiling showed alterations with potential to impact clinical management (Tier1/2/3) in 82% patients. When including only Tier 1 and 2 alterations, simultaneous profiling approach gives an opportunity to offer personalized therapy in 68% patients. The Tier1 ctDNA only alterations were most frequent in ESR1 gene. In fact, a total of a total of 29 ESR1 mutations were detected in these patients, of which 10 mutations were detected in both tissue and liquid. In 3 patients’ tissues were positive for ESR1 mutations but the corresponding ctDNA was negative. Similarly, ctDNA detected 16 ESR1 variants which were negative in the tissue sample. The most common ESR1 variants were D538G and Y537S. Conclusions: The simultaneous profiling of tissue and ctDNA enhanced overall clinical utility of molecular analysis in advanced breast cancer by identifying predictive biomarkers. The integration of data from both sources allowed for comprehensive understanding of the molecular heterogeneity in breast cancer. It underscores the potential of simultaneous profiling to guide treatment decisions and improve patient outcomes. By detecting acquired resistance ESR1 mutations more effectively, ctDNA serves as a valuable tool for monitoring as well.

Small non-coding RNA signatures in atrial appendages of patients with atrial fibrillation.

The development of high-throughput technologies has enhanced our understanding of small non-coding RNAs (sncRNAs) and their crucial roles in various diseases, including atrial fibrillation (AF). This study aimed to systematically delineate sncRNA profiles in AF patients. PANDORA-sequencing was used to examine the sncRNA profiles of atrial appendage tissues from AF and non-AF patients. Differentially expressed sncRNAs were identified using the R package DEGseq 2 with a fold change >2 and p < 0.05. The target genes of the differentially expressed sncRNAs were predicted using MiRanda and RNAhybrid. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. In AF patients, the most abundant sncRNAs were ribosomal RNA-derived small RNAs (rsRNAs), followed by transfer RNA-derived small RNAs (tsRNAs), and microRNAs (miRNAs). Compared with non-AF patients, 656 rsRNAs, 45 miRNAs, 191 tsRNAs and 51 small nucleolar RNAs (snoRNAs) were differentially expressed in AF patients, whereas no significantly differentially expressed piwi-interacting RNAs were identified. Two out of three tsRNAs were confirmed to be upregulated in AF patients by quantitative reverse transcriptase polymerase chain reaction, and higher plasma levels of tsRNA 5006c-LysCTT were associated with a 2.55-fold increased risk of all-cause death in AF patients (hazard ratio: 2.55; 95% confidence interval, 1.56-4.17; p < 0.001). Combined with our previous transcriptome sequencing results, 32 miRNA, 31 snoRNA, 110 nucleus-encoded tsRNA, and 33 mitochondria-encoded tsRNA target genes were dysregulated in AF patients. GO and KEGG analyses revealed enrichment of differentially expressed sncRNA target genes in AF-related pathways, including the 'calcium signaling pathway' and 'adrenergic signaling in cardiomyocytes.' The dysregulated sncRNA profiles in AF patients suggest their potential regulatory roles in AF pathogenesis. Further research is needed to investigate the specific mechanisms of sncRNAs in the development of AF and to explore potential biomarkers for AF treatment and prognosis.

Effect of plasma-based molecular testing in patients with newly diagnosed advanced NSCLC on initiation of targeted agents in patients with targetable genomic alterations: A real world experience from the Middle East.

e20514 Background: The treatment of metastatic non-small cell lung cancer ( m-NSCLC) is personalized and driven by mutational profiling of the tumour tissue at diagnosis. This study evaluates the additional clinical utility of plasma based molecular testing towards shortening time to initiation of treatment and detection of targetable mutations in a cohort of patients with newly diagnosed m-NSCLC. Methods: This is a retrospective analysis of patients with newly diagnosed advanced NSCLC referred to the Thoracic Oncology Program between September 2021 to December 2023, who underwent “paired” plasma based circulating tumour ( ct-DNA ) and tissue based next generation sequencing (NGS) testing (done less than 4 weeks apart). ct-DNA testing was performed using the Oncomine Pancancer NGS Assay (Thermo Scientific) that screens for single nucleotide variants (SNV) in hotspot regions of 52 and common fusions involving 12 genes respectively. Tissue NGS testing was performed using Oncomine Comprehensive Assay Plus (Thermo Scientific); a panel covering ~500 genes capable of identifying all single nucleotide variants, indels, fusions and copy number variants (CNV). Patient demographics and clinicopathological characteristics were collected by retrospective review of electronic medical records (EMR). Results: Among 36 patients, the median age was 66.0 years (range: 32-66), 25 (69.4%) were males, and 16 (44.4%) were never smokers. Thirty (83.3%) had adenocarcinoma and 33 (91.67%) had stage IV NSCLC. The mutation detection rate of ct-DNA was 66.7% with a concordance of 58.3% between ct-DNA and tissue NGS testing. The median turnaround time (TAT) from sample collection to reporting were12 days (IQR:8-15) and 14 days (IQR:9-24.50) for ct-DNA and tissue NGS testing respectively. Among 31 patients who started treatment, the median time to initiation of 1st line treatment was 26 days from date of first visit (IQR:18-42). Fifteen(41.7%), 6(16.7%) and 10(27.8%) started first line targeted therapy, chemotherapy and chemoimmunotherapy respectively. Twenty-three(63.89%) had targetable mutations (EGFR Exon 19 or 21, EGFR Exon 20 insertion, ALK, ROS1, MET Exon1 4 skipping, ROS1 or KRAS12C detected either in ct-DNA or tissue). Fifteen (65.21%) out of these 23 started targeted therapy as their 1st line treatment and of these 8 (53.3%) were initiated on targeted agents prior to availability of tissue NGS reports. Conclusions: Though our TAT for ct-DNA and tissue based NGS testing was similar, ct-DNA based NGS testing enables earlier initiation of targeted treatment for newly diagnosed advanced NSCLC patients with targetable mutations. In the real world, this increases access to targeted agents and may translate to a subsequent improvement in survival outcomes.

A novel liquid biopsy assay with an 88% detection rate of genomic alterations across CNS tumors.

2077 Background: Diagnosis and treatment decisions for CNS tumors rely upon imaging with subsequent histological and genomic analyses of tissue to identify appropriate courses of therapy. Genomic profiling of tumor tissue for informative alterations is used to guide treatment, however contemporaneous tissue can sometimes be unavailable, inconclusive or overlook possible actionable alterations given the high amount of spatial heterogeneity in certain tumor types such as glioblastomas GBMs. Liquid biopsies may pose a possible solution, as they are readily accessible and provide a holistic snapshot of the tumoral genomic landscape. However, detection of ctDNAs from CNS tumors is impeded by its inherently low shedding biology and poor permeability across the BBB resulting in low plasma variant allele fractions (VAFs), with alteration detection rates ranging 27-55%. Thus, current assays do not yet have the sensitivity to be clinically useful in aiding decision making for CNS tumors. Methods: 62 cases of primary CNS tumors were submitted commercially for assessment with Northstar Select, a tumor-naive, plasma-based ctDNA liquid biopsy assay. Cases were obtained from a single clinic dating from January-December 2023. Results: See table. Conclusions: There remains an unmet clinical need for a highly sensitive liquid biopsy assay for use in determining the genomic landscape of CNS tumors to aid in clinical decision making. Current assays do not have a low enough limit of detection (LoD) to accurately detect ctDNAs at low VAFs. Herein, we demonstrate that Northstar Select has a detection rate of 88.7% in all cases and 91.4% in GBMs, nearly doubling current rates. This is due to the assay’s uniquely low LoD of 0.13-0.16% VAF, which is below the 0.17% median VAF demonstrated across CNS tumors in this study, allowing for more low-abundance alterations to be detected. Given the increased total variant detection rate, nearly 50% of which were actionable when alterations were detected, these data support the clinical utility of Northstar Select to inform clinical decisions either as a complement to imaging and tissue analyses or independently when tissue biopsies are infeasible. These data further suggest that the clinical utility of Northstar Select may be expanded into metastatic brain disease and other low ctDNA shedding tumors. [Table: see text]

Reproduction-associated pathways in females of gibel carp (Carassius gibelio) shed light on the molecular mechanisms of the coexistence of asexual and sexual reproduction

Gibel carp (Carassius gibelio) is a cyprinid fish that originated in eastern Eurasia and is considered as invasive in European freshwater ecosystems. The populations of gibel carp in Europe are mostly composed of asexually reproducing triploid females (i.e., reproducing by gynogenesis) and sexually reproducing diploid females and males. Although some cases of coexisting sexual and asexual reproductive forms are known in vertebrates, the molecular mechanisms maintaining such coexistence are still in question. Both reproduction modes are supposed to exhibit evolutionary and ecological advantages and disadvantages. To better understand the coexistence of these two reproduction strategies, we performed transcriptome profile analysis of gonad tissues (ovaries) and studied the differentially expressed reproduction-associated genes in sexual and asexual females. We used high-throughput RNA sequencing to generate transcriptomic profiles of gonadal tissues of triploid asexual females and males, diploid sexual males and females of gibel carp, as well as diploid individuals from two closely-related species, C. auratus and Cyprinus carpio. Using SNP clustering, we showed the close similarity of C. gibelio and C. auratus with a basal position of C. carpio to both Carassius species. Using transcriptome profile analyses, we showed that many genes and pathways are involved in both gynogenetic and sexual reproduction in C. gibelio; however, we also found that 1500 genes, including 100 genes involved in cell cycle control, meiosis, oogenesis, embryogenesis, fertilization, steroid hormone signaling, and biosynthesis were differently expressed in the ovaries of asexual and sexual females. We suggest that the overall downregulation of reproduction-associated pathways in asexual females, and their maintenance in sexual ones, allows the populations of C. gibelio to combine the evolutionary and ecological advantages of the two reproductive strategies. However, we showed that many sexual-reproduction-related genes are maintained and expressed in asexual females, suggesting that gynogenetic gibel carp retains the genetic toolkits for meiosis and sexual reproduction. These findings shed new light on the evolution of this asexual and sexual complex.

Tumor mutation burden and FAT3 mutation influence long-term survival in surgically resected small cell lung cancer.

Small cell lung cancer (SCLC) is highly malignant and has a higher risk of recurrence even in patients who undergo early surgery. However, a subgroup of patients survived for many years. So far, the factors that determine the long-term survivorship remain largely unknown. To determine the genetic characteristics of long-term survival (LTS) after surgery in SCLC, we performed comprehensive comparative genomic profiling and tumor mutation burden (TMB) analysis of resected tumor tissues from patients with LTS and short-term survival (STS) after surgery. The present study screened 11 patients from 52 patients with SCLC who underwent surgery at Zhejiang Cancer Hospital from April 2008 to December 2017. A total of six LTS patients (≥4 years) with stage IIB or IIIA SCLC and five STS patients (<2 years) with stage IA or IB SCLC were included in the study. The STS patients were used as a control. All the patients underwent resection without neoadjuvant therapy. We assessed the genomic profiles of the resected tumor tissues and calculated the TMB using next-generation sequencing. We then analyzed and compared the molecular characteristics between the LTS and STS groups. Our data indicated that tumor tissues from patients with LTS harbor a high TMB. The median TMB for LTS patients was high (approximately 16.4 mutations/Mb), while that for STS patients was low (approximately 8.5 mutations/Mb). The median TMB of patients with LTS and STS showed a trend of significant difference (P=0.08). Gene alterations characterized the survival differences between the two groups. The FAT3 mutation was only found in the LTS group, and the P value determined by Fisher's exact test was 0.06. A high non-synonymous TMB and the FAT3 mutation could potentially influence LTS after SCLC resection. This study provides valuable information about the molecular differences between LTS and STS patients. Studies with larger sample sizes need to be conducted to confirm our findings in the future.

LC3AB protein-based gene (PbG) signatures as an indicator of relapse even after achieving pathological complete response.

e12552 Background: The application of tissue-based quantitative protein and morphological profiling of breast cancer tissues combined with RNA sequencing provides a flexible and generally applicable and scalable method to develop Protein-based Gene (PbG) signatures with significant prognostic value. Methods: A racially and subtype diverse breast cancer cohort (N=555) with available subset of RNA-seq data was used to develop gene expression-based signatures by stratifying patients according to protein expression (IHC) based on survival information and tumor lymphocyte infiltration (TILs) using survival-derived cutoff values. The predictive value of these signatures was then compared with other popular biomarkers scoring systems such as ROR.score, and OncotypeDx (Gene 21). Here we describe the predictive value of these signatures is evaluated by logistic regression modeling of publicly available gene expression data (N=996) from patients receiving neoadjuvant chemotherapy (anthracycline +/- taxane) with a final validation in an independent cohort (N=443) receiving similar therapy. Results: A model of PbG-derived signature from Kaiso, LC3A/B IHC and quantitative TIL-measurements together with ROR score, MAPK, PTEN, Immune and Gene21 signatures show significant prediction of distant metastasis-free survival in all patients: (DMFS, AUC= 0.737, 95% CI: 0.665- 0.819); and in non-pCR patients DMSF (AUC= 0.71, 95% CI: 0.633-0.773). When examining the role of all those markers among the patients who achieved the pCR and had relapse, the LC3A/B PbG-derived score was found to be highly predictive of those patients who would not achieve pCR and ultimately have recurrent disease. Conclusions: PbG-based signatures utilizing on LC3A/B protein values offer a novel and clinically relevant predictive biomarker of response to therapy and relapse in breast cancer patients.

Proteomic profiling of renal tissue of normo- and hypertensive rats with the renalase peptide RP220 as an affinity ligand.

Renalase (RNLS) is a recently discovered protein that plays an important role in the regulation of blood pressure by acting inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase that oxidizes isomeric forms of β-NAD(P)H. Extracellular renalase lacking its N-terminal peptide and cofactor FAD exerts various protective effects via non-catalytic mechanisms. Certain experimental evidence exists in the literature that the RP220 peptide (a 20-mer peptide corresponding to the amino acid sequence RNLS 220-239) reproduces a number of non-catalytic effects of this protein, acting on receptor proteins of the plasma membrane. The possibility of interaction of this peptide with intracellular proteins has not been studied. Taking into consideration the known role of RNLS as a possible antihypertensive factor, the aim of this study was to perform proteomic profiling of the kidneys of normotensive and hypertensive rats using RP220 as an affinity ligand. Proteomic (semi-quantitative) identification revealed changes in the relative content of about 200 individual proteins in the kidneys of hypertensive rats bound to the affinity sorbent as compared to the kidneys of normotensive animals. Increased binding of SHR renal proteins to RP220 over the normotensive control was found for proteins involved in the development of cardiovascular pathology. Decreased binding of the kidney proteins from hypertensive animals to RP220 was noted for components of the ubiquitin-proteasome system, ribosomes, and cytoskeleton.

Amniotic fluid-derived small extracellular vesicles for predicting postnatal severe outcome of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a life-threatening condition with high morbidity and mortality rates. The survival rate of neonates with severe CDH is reportedly only 10%-15%. However, prenatal prediction of severe cases is difficult, and the discovery of new predictive markers is an urgent issue. In this study, we focused on microRNAs (miRNAs) in amniotic fluid-derived small EVs (AF-sEVs). We identified four miRNAs (hsa-miR-127-3p, hsa-miR-363-3p, hsa-miR-493-5p, and hsa-miR-615-3p) with AUC>0.8 to classify good prognosis group and poor prognosis group in human study. The AUC for hsa-miR-127-3p and hsa-miR-615-3p, for predicting the poor prognosis, were 0.93 and 0.91, respectively. In addition, in the in vivo study, the miRNA profiles of the lung tissues of CDH rats were different from those of control rats. Additionally, two elevated miRNAs (rno-miR-215-5p and rno-miR-148a-3p) in the lung tissues of CDH rats were increased in the AF-sEVs of CDH rats. Our results suggest that severe CDH neonates can be predicted prenatally with high accuracy using miRNAs contained in AF-sEVs. Furthermore, miRNA profile changes in AF-sEVs reflected the lung status in CDH. Our findings may contribute to the development of advanced perinatal care for patients with CDH.

CUPCOMP: A multi-site UK trial in carcinoma of unknown primary: A comparison across tissue and liquid biomarkers.

3059 Background: Cancer of Unknown Primary (CUP) is a difficult to treat cancer entity for which the tumour origins remain elusive. There is emerging evidence to support a precision medicine approach to aid treatment decisions, however scarcity of tumour tissue for molecular profiling remains a challenge. CUPCOMP sought to compare the feasibility of molecular profiling from both tissue and blood in patients (pts) diagnosed with CUP. Methods: A non-randomised prospective feasibility clinical trial conducted across 7 UK sites between 06/2021-02/2023. Eligible pts had a histological confirmed diagnosis of favourable/unfavourable CUP based on clinical, radiological, and pathological review at a CUP Multi-Disciplinary Team meeting and ESMO Guidelines for CUP. Pts had mutation profiling performed from circulating tumour DNA (ctDNA) (FoundationOne Liquid CDx) and tumour tissue (FoundationOne CDX or whole genome sequencing (WGS)). All results were discussed at a dedicated Molecular Tumour Board (MTB) to evaluate the activation state of oncogenic pathways. Results: Baseline characteristics of the 117 pts recruited are shown (Table). 114/117 (97%) pts had successful blood (109/117; 93% pts) or tissue (69/117; 59% pts) molecular profiling; both methods were performed in 64/117 (55%) pts. WGS was only successful in 15/21 pts where repeat biopsy was obtained. Where targeted tissue and blood profiling was performed using the same panel (49/117; 42%): 421/550 (77%) Single Nucleotide Variants (SNVs) found in tissue were concordant in ctDNA. ctDNA tumour fraction was ≥10% in 41/109 (35%) pts. In ctDNA, 1556 SNVs, across 271 genes, and 42 rearrangements were identified. 73/114 (64%) of cases carried an actionable alteration in blood or tissue, as determined by MTB. The most frequently reported actionable gene alterations were: KRAS (n=11; 10%); ATM (n=9; 8%); ARID1A (n=9; 8%); PIK3CA (n=10; 9%); NF2 (n=7; 6%); BRAF (n=7; 6%); FGFR2-fusion (n=7; 6%). A tumour mutational burden (TMB) of ≥10 mutations/Mb was detected in 25/117 (22%) of pts. Potential germline variants were found in 8% of pts. 80/114 (70%) pts were alive 6 months after consent to trial. Conclusions: Successful tissue-based molecular profiling is challenging in pts with CUP; achieved in only 59% of pts in this cohort. ctDNA analysis was feasible (93% successful) and reliably concordant with tissue. Potentially actionable alterations were found in 64% of pts. ctDNA molecular profiling should be considered as a reliable alternative to tissue-based testing in pts diagnosed with CUP. Clinical trial information: NCT04750109 . [Table: see text]

Genomic alterations (GA) in ESR1, PIK3CA, AKT1, and PTEN in HR(+)HER2(-) patients (pts) with metastatic breast cancer (MBC): Co-occurrence and prevalence along treatment course.

1060 Background: The standard of care for HR(+) HER2(-) MBC is evolving with new biomarker-guided drugs, including novel selective estrogen receptor degraders (SERDs) for pts with ESR1 mutations (mut) and PIK3CA and AKT inhibitors for pts with alterations in the PI3K pathway (PIK3CA, AKT1, and PTEN). This study aimed to evaluate the co-occurrence and the prevalence of these GA in different metastatic lines of therapy in real-world practice for MBC. Methods: This study included pts with HR(+) HER2(-) MBC who underwent genomic testing using Foundation Medicine tissue or liquid comprehensive genomic profiling assays, with specimens collected within 60 days of therapy initiation. Clinical data were obtained by the nationwide (US-based) de-identified Flatiron Health and Foundation Medicine real-world clinicogenomic breast cancer database (FH-FMI CGDB), originated from ~280 US cancer clinics (~800 sites of care) between 01/2011-06/2023. The prevalence of ESR1mut, PIK3CAmut, AKT1, PTENmut, and PTEN copy loss were calculated in tissue (TBx) and liquid biopsies (LBx) collected at time of 1st, 2nd, and 3rd lines of therapy. The co-occurrence of GA in TBx was evaluated. Results: Among patients with a TBx collected around therapy initiation (n=2,536), 81.5% (2,066) was collected at 1st, 10.2% (258) at 2nd, and 8.3% (212) at 3rd line. Among patients with a LBx collected around therapy initiation (n=545), 47% (256) was collected at 1st, 33.2% (181) at 2nd, and 19.8% (108) at 3rd line. In TBx, the prevalence of GA detected in 1st, 2nd, and 3rd lines were 58.6% (1,210), 67.4% (174), and 71.2% (151), respectively. In LBx, the prevalence was 42.7% (121), 65.7% (119), and 60.1% (65), respectively. Among LBx with tumor fraction ³1%, the prevalence was 67.9% (74/109), 89.1% (81/91), and 84.3% (43/51). Among patients with a GA detected in TBx 1st (n=1,210), 2nd (n=174), and 3rd (n=151) lines, both ESR1mut and a PIK3 pathway GA was detected in 7.4% (90), 12.1% (21), and 23.8% (36), respectively. Conclusions: In our dataset, 42-59% of patients with HR(+) HER2(-) MBC have at least one GA detected by TBx or LBx in 1st line, with prevalence increasing in later lines of therapy to 60-71% due to acquisition of an ESR1mut. PTEN copy loss is detected at much higher rates in TBx than LBx, consistent with the known inherent limitations of ctDNA testing. Serial genomic testing with LBx with reflex TBx if alterations are not identified should strongly be considered with the recent approvals of several molecularly targeted agents for HR(+) HER2(-) MBC. [Table: see text]

Measuring Accuracy of Dolphin Imaging Software in predicting upper jaw soft tissue profile changes in Class II Adult Patients treated with Extraction versus Non-Extraction

Measuring Accuracy of Dolphin Imaging Software in predicting upper jaw soft tissue profile changes in Class II Adult Patients treated with Extraction versus Non-Extraction

Matched tissue and circulating tumor DNA (ctDNA) analysis in renal cell carcinoma (RCC): Results from a multimodal real-world database.

4533 Background: Next generation sequencing (NGS) of ctDNA can complement tissue NGS and is a non-invasive test that can be conducted serially, with the potential to enhance assessment of spatial and temporal molecular tumor heterogeneity. Here, we investigated mutations in RCC patients from ctDNA and matched tissue genomic profiling. Methods: From the Tempus multimodal database, we retrospectively analyzed de-identified NGS data from patients (pts) with RCC that had dual tissue (Tempus xT, 648 genes) and ctDNA testing (Tempus xF, 105 genes). Pts with matched samples (collected +/- 90 days of one another) were included. Clinical characteristics and select pathogenic somatic short variants (PSSV) and copy number variants [(amplifications and deletions, two copy number losses (CNL)] were evaluated. Concordance analyses were restricted to the 105 genes tested on the ctDNA panel and further restricted to short variants, with the exception of amplifications and CNL detected by both xF and xT. Results: Among all pts (n=393), the median age was 61 years and 71% were male. Median time from tissue to blood collection was 21 days (IQR, 7, 39). 67% (n=265) and 68% (n=266) had metastatic disease at the time of tissue and blood collection, respectively. The most common tissue sites were kidney (49%, n=189), bone (11%, n=43), lung (9%, n=34), lymph node (8%, n=29), liver (6%, n=23), and brain/CNS (4%, n=17). Genes harboring the most common PSSV in tissue included VHL (59% n=232), PBRM1 (31%, n=123), SETD2 (23%, n=91), and TP53 (14%, n=54). Genes with common PSSV in ctDNA included TP53 (23%, n=91), VHL (18%, n=69), BAP1 (6%, n=23), and PBRM1 (5%, n=21). The combination of tissue and ctDNA testing increased detection of mutations (Table). There was higher concordance between somatic alterations in select genes among patients with metastases. Conclusions: This analysis shows that ctDNA profiling is complementary to tissue based NGS in RCC and can increase the detection of mutations. Concordance between ctDNA and tissue profiling increased in individuals with metastatic disease. Future research is warranted to understand how longitudinal ctDNA analysis can define biomarkers of response and resistance at the mutation and ctDNA fraction levels. [Table: see text]

Comparison of positive clinical and computerized visual treatment objectives in prediction of soft tissue profile of skeletal class II patients.

Comparison of positive clinical and computerized visual treatment objectives in prediction of soft tissue profile of skeletal class II patients.

Exploring variations in glycolytic and gluconeogenic enzymes and isoforms across breast cancer cell lines and tissues

It is well established that tumour cells undergo metabolic changes to acquire biological advantage over normal cells with activation of the glycolytic pathway, a process termed “Warburg effect”. Enzyme isoforms are alternative enzymatic forms with the same function but with different biochemical or epigenetic features. Moreover, isoforms may have varying impacts on different metabolic pathways. We challenge ourselves to analyse the glycolytic and gluconeogenic enzymes and isoforms in breast cancer, a complex and heterogeneous pathology, associated with high incidence and mortality rates especially among women.We analysed epithelial and tumour cell lines by RT-PCR and compared values to a publicly available database for the expression profile of normal and tumour tissues (Gepia) of enzymes and enzymatic isoforms from glycolytic and gluconeogenic pathways. Additionally, GeneMANIA was used to evaluate interactions, pathways, and attributes of each glycolytic/gluconeogenic steps.The findings reveal that the enzymes and enzymatic isoforms expressed in cell culture were somewhat different from those in breast tissue. We propose that the tumor microenvironment plays a crucial role in the expression of glycolytic and gluconeogenic enzymes and isoforms in tumour cells. Nonetheless, they not only participate in glycolytic and gluconeogenic enzymatic activities but may also influence other pathways, such as the Pentose-Phosphate-Pathway, TCA cycle, as well as other carbohydrate, lipid, and amino acid metabolism.