Expression Of Subtypes Research Articles

PATH-13. APPLICATIONS OF SPATIAL TRANSCRIPTOMICS FOR THE DIAGNOSTICS OF GLIOMAS

Abstract Due to their infiltrative growth, diffuse gliomas can pose a major challenge for neuropathologists. Lack of specific immunohistochemistry markers for some gliomas like IDH-wildtype glioblastoma can make it hard to identify the tumour cell content or distinguish between tumour and reactive gliosis. Other hurdles are high fractions of DNA from healthy cells in molecular diagnostic analysis, or scarce tissue, which can result in a failed DNA extraction. To overcome these limitations, we evaluated the application of spatial transcriptomics on 14 glioblastoma bulk resections and 18 stereotactic biopsies of 7 glioblastoma, IDH wildtype, 4 oligodendroglioma IDH-mutant and 7 astrocytoma IDH-mutant samples with the aim to generate a diagnostic report containing all information needed by the neuropathologist and clinician. Using the SPATA2 R package, we were able to infer the copy number variation (CNV) profiles for each spot. When comparing the inferred CNVs to the CNVs calculated by the 850K methylome analysis, a correlation between CNVs identified from the 850K data and the inferred CNVs could be seen. For diagnostic relevant CNVs of chr. 7, 10, 1p and 19q, the inferred CNVs were concordant with the 850K data in 100% of the cases with confirmed tumour tissue. CNVs from spatial transcriptomics were able to reveal sub-clones, which could not be detected by the 850K array data. The CNVs could then be used in combination with a deconvolution approach to determine the tumour burden of the tissue. Using RNA expression, the immunohistochemistry staining of important diagnostic markers like NeuN, GFAP and Ki67 was predicted. In addition, the spatial gene expression could be used to identify the glioblastoma subtype based on the gene expression of different methylation subtypes. All the above-mentioned analysis also generated meaningful results for tissue fragments smaller than 1mm in diameter, which are too small for molecular diagnostic analysis.

EPCO-32. DISSECTING THE INTRA- AND INTER-TUMORAL HETEROGENEITY UNDERLYING GLIOBLASTOMA PATHOGENESIS UTILIZING MULTI-OMICS ANALYSIS

Abstract Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis. Considering its extensive molecular diversity underlies therapeutic resistance to multimodal treatment, it is imperative to reveal intra- and inter-tumoral heterogeneity, leading to further understanding of GBM pathogenesis. We analyzed 289 whole-genome sequencing data (WGS) including 159 unpublished deep WGS (≥ ×120 coverage) along with RNA-seq, DNA methylation array, whole-genome bisulfite sequencing, and assay for transposase-accessible chromatin with sequencing (ATAC-seq) to uncover the multi-omics molecular heterogeneity of GBM. Along with inter-tumoral heterogeneity of genetic driver alterations, deep WGS enables us to delineate a fine view of clonal architecture where mutational signatures differ across clonal and subclonal mutations, supporting that different mutational processes contribute to GBM pathogenesis depending on the developmental stage. As well, tumor cell differentiation status detected by transcriptional deconvolution analysis is associated with multi-layer profiles including genome, transcriptome, epigenome, and chromatin status. ATAC-seq demonstrated distinct features of genome-wide chromatin accessibility associated with gene expression subtypes of GBM. Motif enrichment analysis detects the differentially accessible regions where the proneural subtypes are enriched with the SOX10 motif, known as a transcription factor for mesenchymal transition whereas the mesenchymal and classical subtypes are enriched with the CREB/ATF motif, a regulator of TGF beta associated with a poor prognosis. Our findings support a model in which GBM evolves through generating genetic and epigenetic intra-tumoral heterogeneity suggesting that specific cell states may have distinct vulnerability to mutational processes and epigenetic modifications. Our analysis reveals the molecular mechanisms underlying the progression of GBM enhancing our understanding of the pathogenesis.

Targeting hyperactive platelet-derived growth factor receptor-β signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematologic malignancies. Current treatment consists of intensive chemotherapy leading to 80% overall survival but is associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient, and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient-derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL, and that screening T-ALL/T-LBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

Poor outcome of pediatric B-cell acute lymphoblastic leukemia associated with high level of CRLF2 gene expression in distinct molecular subtypes.

Overexpression of the cytokine receptor-like factor 2 (CRLF2) gene is the most common feature in the Philadelphia chromosome (Ph)-like subtype of B-cell acute lymphoblastic leukemia (B-ALL). However, the predictive value of CRLF2 overexpression for the prognosis of pediatric B-ALL patients remain controversial. The molecular mechanisms that upregulate CRLF2 expression level in patients has not been fully elucidated. In this study, the prognostic impact of CRLF2 expression level on molecular types of B-ALL in pediatric patients from Zhujiang Hospital (n = 111) was retrospectively analyzed. Youden index analysis was used to categorize CRLF2 expression into 3 groups, and these categories more precisely described the differences in the prognosis of patients with varying expression levels of CRLF2 in both the Zhujiang Hospital cohort and the TARGET cohort. We used the Zhujiang Hospital cohort as a discovery cohort to determine the cutoff value of CRLF2 expression. CRLF2-high patients accounted for approximately 6%. In addition, the percentage of bone marrow blast cells and initial white blood cell count in CRLF2-high patients were higher than those in CRLF2-low patients, and MRD turned negative slower. The results were validated in the TARGET cohort and indicated that CRLF2 overexpression could be subdivided by CRLF2 expression levels into 2 categories: CRLF2-high with a poor survival and CRLF2-medium with a good OS and EFS. Such heterogeneity was attributed to the different molecular mechanisms leading to CLRF2 upregulation, where the CRLF2 overexpression level was high in Ph-like B-ALL and medium in high hyperdiploid B-ALL. This study highlights the importance of the molecular mechanisms of the upregulation of CRLF2 expression in predicting the prognosis of pediatric B-ALL patients.

MicroRNA Landscape in Endometrial Carcinomas in an Asian population: Unraveling Subtype-Specific Signatures.

MicroRNAs (MiRNAs) are small, non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. We analyzed the differential expression of miRNAs in 119 endometrial carcinomas, measuring their expression in histological subtypes, molecular subtypes, and tumors with CTNNB1 mutations. Tumors were subdivided into histological and molecular subtypes as defined by The Cancer Genome Atlas. The expression levels of 352 miRNAs were quantified using the PanoramiR panel. Mir-449a, mir-449b-5p, and mir-449c-5p were the top three miRNAs showing increased expression in both endometrioid and de-differentiated carcinomas but were not significantly increased in serous and clear cell carcinomas. The miRNAs with the most increased expression in serous and clear cell carcinomas were miR-9-3p and miR-375, respectively. We also identified 62 differentially expressed miRNAs among different molecular subtypes. Using sequential forward selection, we built subtype classification models for some molecular subtypes of endometrial carcinoma, comprising 5 miRNAs for MMR-deficient tumors, 10 miRNAs for p53-mutated tumors, and 3 miRNAs for CTNNB1-mutated tumors, with areas under curves of 0.75, 0.85, and 0.78, respectively. Our findings confirm the differential expression of miRNAs between various endometrial carcinoma subtypes and may have implications for the development of diagnostic and prognostic tools.

Expression of Wilms tumor gene protein (WT1) in different histologic subtypes of ovarian carcinoma

Background: Ovarian carcinoma is the seventh most common cancer among women worldwide and the eighth leading cause of death. Ovarian cancer develops in the ovary's tissues. Methods: This cross-sectional descriptive study was carried out at the Department of Pathology, RMC and Department of Pathology, BSMMU, Dhaka, Bangladesh from July 2019 to June 2021 carried out with 31 histopathologically confirmed ovarian carcinomas. All cases were subjected to WT1 immunostaining followed by histopathological examination. The collected data were analyzed using Statistical Package for Social Sciences (SPSS) software, version-23.0. The ethical clearance of this study was obtained from the Institutional Review Board (IRB) of Rajshahi, Medical College, Rajshahi, Bangladesh. Results: A total of 31 histopathologically confirmed ovarian carcinoma patients were included in this cross-sectional descriptive study. All cases were subjected to WT1 immunostaining followed by histopathological examination. The age of the study sample ranged from 20 to 75 years. The mean age of the respondent was 45.10±13.78 years. According to the histologic subtypes, WT1 was mainly expressed in serous carcinoma. All the serous carcinoma (16/16) showed a positive reaction for WT1. Among them, 14 cases (87.5%) showed >50% (3+) WT1 score and two (2) cases (12.5%) showed 11% to 50 % (2+) scores. Regarding endometrioid carcinoma four (4) (66.6%) of six (6) were negative whereas one (1) (16.6%) showed 1%-10% (1+) and one (1) (16.6%) showed 11%-50% (2+) WT1 scores. All the mucinous (4/4) and all the clear cell carcinoma (3/3) were negative with less than 1 % of the tumor cells stained two (2) cases of undifferentiated carcinoma were also negative. There was a significant relation between WT1 expression and different histologic subtypes of epithelial ovarian carcinoma with a p value of 0.001 (p<0.05). Conclusions: WT1 can be applied by both the histopathologist and clinician as an effective marker regarding diagnosis and patient management.

UGDH promotes tumor-initiating cells and a fibroinflammatory tumor microenvironment in ovarian cancer

BackgroundEpithelial ovarian cancer (EOC) is a global health burden, with the poorest five-year survival rate of the gynecological malignancies due to diagnosis at advanced stage and high recurrence rate. Recurrence in EOC is driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that are supported by a complex extracellular matrix and immunosuppressive microenvironment. To target TICs to prevent recurrence, we identified genes critical for TIC viability from a whole genome siRNA screen. A top hit was the cancer-associated, proteoglycan subunit synthesis enzyme UDP-glucose dehydrogenase (UGDH).MethodsImmunohistochemistry was used to characterize UGDH expression in histological and molecular subtypes of EOC. EOC cell lines were subtyped according to the molecular subtypes and the functional effects of modulating UGDH expression in vitro and in vivo in C1/Mesenchymal and C4/Differentiated subtype cell lines was examined.ResultsHigh UGDH expression was observed in high-grade serous ovarian cancers and a distinctive survival prognostic for UGDH expression was revealed when serous cancers were stratified by molecular subtype. High UGDH was associated with a poor prognosis in the C1/Mesenchymal subtype and low UGDH was associated with poor prognosis in the C4/Differentiated subtype. Knockdown of UGDH in the C1/mesenchymal molecular subtype reduced spheroid formation and viability and reduced the CD133 + /ALDH high TIC population. Conversely, overexpression of UGDH in the C4/Differentiated subtype reduced the TIC population. In co-culture models, UGDH expression in spheroids affected the gene expression of mesothelial cells causing changes to matrix remodeling proteins, and fibroblast collagen production. Inflammatory cytokine expression of spheroids was altered by UGDH expression. The effect of UGDH knockdown or overexpression in the C1/ Mesenchymal and C4/Differentiated subtypes respectively was tested on mouse intrabursal xenografts and showed dynamic changes to the tumor stroma. Knockdown of UGDH improved survival and reduced tumor burden in C1/Mesenchymal compared to controls.ConclusionsThese data show that modulation of UGDH expression in ovarian cancer reveals distinct roles for UGDH in the C1/Mesenchymal and C4/Differentiated molecular subtypes of EOC, influencing the tumor microenvironmental composition. UGDH is a strong potential therapeutic target in TICs, for the treatment of EOC, particularly in patients with the mesenchymal molecular subtype.

Perturbation and stability of PAM50 subtyping in population-based primary invasive breast cancer

PAM50 gene expression subtypes represent a cornerstone in the molecular classification of breast cancer and are included in risk prediction models to guide therapy. We aimed to illustrate the impact of included genes and biological processes on subtyping while considering a tumor’s underlying clinical subgroup defined by ER, PR, and HER2 status. To do this we used a population-representative and clinically annotated early-stage breast tumor cohort of 6233 samples profiled by RNA sequencing and applied a perturbation strategy of excluding co-expressed genes (gene sets). We demonstrate how PAM50 nearest-centroid classification depends on biological processes present across, but also within, ER/PR/HER2 subgroups and PAM50 subtypes themselves. Our analysis highlights several key aspects of PAM50 classification. Firstly, we demonstrate the tight connection between a tumor’s nearest and second-nearest PAM50 centroid. Additionally, we show that the second-best subtype is associated with overall survival in ER-positive, HER2-negative, and node-negative disease. We also note that ERBB2 expression has little impact on PAM50 classification in HER2-positive disease regardless of ER status and that the Basal subtype is highly stable in contrast to the Normal subtype. Improved consciousness of the commonly used PAM50 subtyping scheme will aid in our understanding and interpretation of breast tumors that have seemingly conflicting PAM50 classification when compared to clinical biomarkers. Finally, our study adds further support in challenging the common misconception that PAM50 subtypes are distinct classes by illustrating that PAM50 subtypes in tumors represent a continuum with prognostic implications.

Integrated single-cell and bulk RNA sequencing analysis identified pyroptosis-related signature for diagnosis and prognosis in osteoarthritis

Osteoarthritis (OA), a degenerative disease of the joints, has one of the highest disability rates worldwide. This study investigates the role of pyroptosis-related genes in osteoarthritis and their expression in different chondrocyte subtypes at the individual cell level. Using OA-related datasets for single-cell RNA sequencing and RNA-seq, the study identified PRDEGs and DEGs and conducted Cox regression analysis to identify independent prognostic factors for OA. CASP6, NOD1, and PYCARD were found to be prognostic factors. Combined Weighted Gene Correlation Network Analysis with PPI network, a total of 15 hub genes related to pyroptosis were involved in the notch and oxidative phosphorylation pathways, which could serve as biomarkers for the diagnosis and prognosis of OA patients. The study also explored the heterogeneity of chondrocytes between OA and normal samples, identifying 19 single-cell subpopulation marker genes that were significantly different among 7 chondrocyte cell clusters. AGT, CTSD, CYBC, and THYS1 were expressed differentially among different cell subpopulations, which were associated with cartilage development and metabolism. These findings provide valuable insights into the molecular mechanisms underlying OA and could facilitate the development of new therapeutic strategies for this debilitating disease.

Inflammation induces α1-adrenoceptor expression in peripheral blood mononuclear cells of patients with complex regional pain syndrome

Persistent regional and systemic inflammation may promote pain and hyperalgesia in complex regional pain syndrome (CRPS). In this study, we investigated whether stimulation of α1-adrenoceptors (α1-AR) on peripheral blood mononuclear cells (PBMC) might contribute to this inflammatory state. PBMC were isolated from venous blood collected from 21 CRPS patients and 21 sex and age-matched controls. Lipopolysaccharide (LPS), a bacterial toxin, was administered to cultured PBMC for 24 h to trigger inflammation. Exposure to LPS resulted in heightened gene expression of α1-AR subtype B (α1B-AR) in PBMC of CRPS patients relative to controls. Interleukin (IL)-1β and IL-6 levels did not change when the α1-AR agonist phenylephrine was administered to naïve PBMC. However, α1-AR stimulation following LPS treatment increased IL-6 mRNA and protein levels in PBMC of patients and controls. To investigate the possible consequence of heightened IL-6 levels on immunoglobulin G antibody production, PBMC were stimulated with CD40 ligand and IL-21 to generate plasmablasts (B cells that secrete antibodies). This response was similar in patients and controls. Adding IL-6 to the cell culture medium increased plasmablast differentiation in controls and antibody production both in patients and controls. These findings suggest that the inflammatory cascade associated with elevated levels of IL-6 may generate α1B-AR expression in CRPS PBMC. A reciprocal interaction between heightened α1-AR expression in PBMC and IL-6 secretion may contribute to systemic inflammation and antibody production in CRPS.

Rac2 mediate foam cell formation and associated immune responses in THP-1 to promote the process of atherosclerotic plaques

Macrophages play an important role in the pathogenesis of atherosclerosis (AS) by mediating oxidative stress, inflammation and lipid metabolism, which can lead to the formation of vascular plaque. The Rac family isoforms of small molecules GTPase are active by binding to GTPase, but are inactivated by binding to GDP, and play a role in the switch of cell information conduction. This experiment adopts shRNA interference THP-1 cells respectively each subtype expression and inhibiting Rac1, Rac2, Rac3 activity, each subtype of Rac family on lipid metabolism, inflammatory reaction and oxidative stress. THP-1 cells were stimulated with Ox-LDL to establish AS cell models including lipid loading, adhesion, migration and chemotaxis. Oil Red O staining, cell immunofluorescence, scratching test, transwell, Western blot and other experiments were performed. To observe the different effects of three subtypes of Rac family on multiple links in the foaming process of THP-1 cells. ApoE-/- mice on a high-fat diet were used as animal models to examine the effects of Rac subtypes in vivo. The results showed that the activation of immune cells induced by ox-LDL was inhibited when Rac1, Rac2 and Rac3 in THP-1 were decreased, respectively. Thus, Rac1 and Rac3 act in combination with ox-LDL and are associated with cellular oxidative stress and inflammation. This study provides new means and ideas for finding potential intervention targets that have important regulatory effects on atherosclerosis, and provides a new direction for the development of clinical drugs.

Differential tyrosine and serine/threonine phosphorylation/dephosphorylation pathways regulate the expression of α7 versus α3β4 nicotinic receptor subtypes in mouse hippocampal neurons

We have recently reported that α7 and α3β4 nicotinic acetylcholine receptor (nAChR) subtypes are expressed in human chromaffin cells in the plasma membrane where they colocalize and physically interact. The present study was designed to evaluate whether those receptor subtypes also colocalize at the central nervous system to mutually interact, and whether their expression and colocalization are regulated by phosphorylation/dephosphorylation processes, as they are in human chromaffin cells. We have here found that in isolated and maintained in culture mouse hippocampal neurons, nAChR expression and colocalization of α7, but not α3β4, nAChR subtypes decreased by tyrosine (Tyr)- and serine/threonine (Ser/Thr)-phosphatase inhibition. However, Tyr-kinase inhibition or protein-phosphatase 2A (PP2A) activation increased α3β4 nAChR expression, diminishing receptor subtypes colocalization. Furthermore, colocalization is not recovered if the inhibitors of Tyr-phosphatase and kinases, or the inhibitor of Ser/Thr-phosphatases and the activator of PP2A are applied together. Therefore, regulation of α7 and α3β4 nAChR subtypes expression by Tyr- and Ser/Thr kinases and phosphatases exhibit differential mechanisms in mouse hippocampal neurons. Colocalization of nAChR subtypes, however, is altered by any maneuver that affects these kinases or phosphatases, which might have consequences in the functional activity of nAChR subtypes.

Identification the Low Oxidative Stress Subtype of Periodontitis

The aim of this research was to identify the low oxidative stress-related genes expression (L-OS) subtype in patients with periodontitis. Microarray data (MA) were retrieved from the Gene Expression Omnibus database. The different oxidative stress (OS) subtypes of periodontitis were identified by K-means clustering analysis and gene set variation analysis (GSVA). Differentially expressed genes (DEGs) (|Log fold change (FC)| >1, q < 0.05) amongst the OS subtypes and healthy controls (HCs) were identified by Limma R package. The genomic feature of L-OS subtype and corresponding medicines were evaluated and visualised with Drug-Gene Interaction Database and cytoscape-v3.7.2 software (Pearson correlation coefficient > 0.4). Finally, the LASSO-Logistic regression model was adopted to evaluate and predict patients' OS phenotype in routine clinical practice. The 241 periodontitis samples and 69 HCs were included. Thirty-three DEGs between the L-OS and high oxidative stress-related genes expression (H-OS) subtypes and 96 DEGs, including 8 transcription factors, between L-OS subtype and HCs were identified, respectively. Then, the network of TFs-Genes-Drugs was constructed to determine genomic feature of L-OS subtype. Finally, a 4-gene signature formula and the cutoff value were identified by ML with LASSO model to predict patients' classification. For the first time, we identified L-OS subtype of periodontitis and evaluated its genomic feature with MA.

FRI265 I123-MIBG Positive, CU64-Dotatate PET Negative Pheochromocytoma In A Patient With Bilateral Adrenal Incidentalomas

Abstract Disclosure: P. Pagadala: None. V. Patel: None. Bilateral incidental adrenal nodules represent 10-23% of all incidental adrenal nodules. Most are benign nonfunctioning adenomas however these can be catecholamine-secreting tumors that arise from chromaffin cells of the adrenal medulla called pheochromocytomas. Identifying pheochromocytoma using anatomic imaging alone in cases of bilateral adrenal nodules is challenging. Functional imaging with radioisotope tracers can be helpful such as MIBG (meta-iodobenzylguanidine) and DOTATATE scans. We describe a case of pheochromocytoma, which was negative on DOTATATE but positive on MIBG scan which localized the pheochromocytoma. A 65-year-old male with a history of hypertension, chronic kidney disease and obstructive sleep apnea was found to have bilateral adrenal nodules on routine imaging. MRI abdomen showed a right adrenal nodule 1.7cm x 1.8cm x 1.7cm and a left adrenal nodule 2.3 cm in maximum dimension. He had no apparent symptoms of catecholamine excess. Biochemical testing revealed elevated total plasma metanephrines on serial assessments along with elevated 24-hour urine dopamine levels. A CT scan with adrenal protocol further characterized the right adrenal nodule with precontrast density of 43 Hounsfield Units (HU) with a rapid washout of 75% within 5 minutes, consistent with benign adrenal adenoma. The left adrenal gland had 2 nodules. A smaller superior nodule 1.3 cm AP x 1.0 cm transverse x 0.9 cm craniocaudal in size, density of -3 HU on the precontrast images, with approximately 85% washout most consistent with a benign adrenal adenoma. A larger inferior nodule was 2.3 cm AP x 2.2 cm transverse x 2.3 cm craniocaudal in size and precontrast density of 32 HU, rising to 58 HU during the portal venous phase and remaining at about 58 HU on the delayed images, indicating no significant washout and is indeterminate. A Cu64 labeled DOTATATE scan showed the 1.6 cm right adrenal nodule demonstrated activity similar to that of contralateral background adrenal activity. There was no comment regarding the two left adrenal nodules. A I123 MIBG scan showed the larger of the two left adrenal nodules had a strong uptake of the MIBG highly likely to be a pheochromocytoma. The smaller left adrenal nodule and the right adrenal nodule had no significant MIBG uptake, making these adrenal adenomas. The patient underwent left robotic adrenalectomy and pathology confirmed a 2.3 cm pheochromocytoma in addition to adrenocortical nodular disease. Three months post-surgery, plasma and 24-hour urinary measurements for metanephrines and catecholamines had normalized. Further research is needed to assess the benefit of I123MIBG when DOTATATE is negative. A potential cause of false negatives on DOTATATE is variability in somatostatin receptor subtype expression. Better imaging modalities for pheochromocytomas and guidelines for the management of bilateral adrenal nodules would assist in treatment decisions. Presentation: Friday, June 16, 2023

Mistletoe Extracts from Different Host Trees Disparately Inhibit Bladder Cancer Cell Growth and Proliferation.

Extracts of European mistletoe (Viscum album) are popular as a complementary treatment for patients with many different cancer types. However, whether these extracts actually block bladder cancer progression remains unknown. The influence of different mistletoe extracts on bladder cancer cell growth and proliferation was investigated by exposing RT112, UMUC3, and TCCSup cells to mistletoe from hawthorn (Crataegi), lime trees (Tiliae), willow trees (Salicis), or poplar trees (Populi). The tumor cell growth and proliferation, apoptosis induction, and cell cycle progression were then evaluated. Alterations in integrin α and β subtype expression as well as CD44 standard (CD44s) and CD44 variant (CD44v) expressions were evaluated. Cell cycle-regulating proteins (CDK1 and 2, Cyclin A and B) were also investigated. Blocking and knock-down studies served to correlate protein alterations with cell growth. All extracts significantly down-regulated the growth and proliferation of all bladder cancer cell lines, most strongly in RT112 and UMUC3 cells. Alterations in CD44 expression were not homogeneous but rather depended on the extract and the cell line. Integrin α3 was, likewise, differently modified. Integrin α5 was diminished in RT112 and UMUC3 cells (significantly) and TCCSup (trend) by Populi and Salicis. Populi and Salicis arrested UMUC3 in G0/G1 to a similar extent, whereas apoptosis was induced most efficiently by Salicis. Examination of cell cycle-regulating proteins revealed down-regulation of CDK1 and 2 and Cyclin A by Salicis but down-regulation of CDK2 and Cyclin A by Populi. Blocking and knock-down studies pointed to the influence of integrin α5, CD44, and the Cyclin-CDK axis in regulating bladder cancer growth. Mistletoe extracts do block bladder cancer growth in vitro, with the molecular action differing according to the cell line and the host tree of the mistletoe. Integrating mistletoe into a guideline-based treatment regimen might optimize bladder cancer therapy.

Multi-channel Partial Graph Integration Learning of Partial Multi-omics Data for Cancer Subtyping

Background: The appearance of cancer subtypes with different clinical significance fully reflects the high heterogeneity of cancer. At present, the method of multi-omics integration has become more and more mature. However, in the practical application of the method, the omics of some samples are missing. Objective: The purpose of this study is to establish a depth model that can effectively integrate and express partial multi-omics data to accurately identify cancer subtypes. Methods: We proposed a novel partial multi-omics learning model for cancer subtypes, MPGIL (Multichannel Partial Graph Integration Learning). MPGIL has two main components. Firstly, it obtains more lateral adjacency information between samples within the omics through the multi-channel graph autoencoders based on high-order proximity. To reduce the negative impact of missing samples, the weighted fusion layer is introduced to replace the concatenate layer to learn the consensus representation across multi-omics. Secondly, a classifier is introduced to ensure that the consensus representation is representative of clustering. Finally, subtypes were identified by K-means. Results: This study compared MPGIL with other multi-omics integration methods on 16 datasets. The clinical and survival results show that MPGIL can effectively identify subtypes. Three ablation experiments are designed to highlight the importance of each component in MPGIL. A case study of AML was conducted. The differentially expressed gene profiles among its subtypes fully reveal the high heterogeneity of cancer. Conclusion: MPGIL can effectively learn the consistent expression of partial multi-omics datasets and discover subtypes, and shows more significant performance than the state-of-the-art methods.

Genomic Characterization of Cervical Tumors Driven by Rare High-Risk HPV Subtypes Utilizing a Deep Learning Framework

As many as 30-50% of patients with locally advanced cervical cancer (LACC) experience recurrence after chemoradiation therapy (CRT). More sophisticated prognostic schemes and targeted therapeutic options are urgently needed. Human papillomavirus (HPV) is responsible for the majority of invasive cervical carcinomas, most commonly HPV 16 and 18 subtypes. In large part due to their rarity, little is known about the biologic behavior of LACC driven by non-16/18 HPV genotypes. We hypothesized that rare HPV subtypes drive distinct gene expression patterns. To aid in the common dilemma of limited sample procurement, we developed a tool which leverages population data and generative adversarial neural network (GAN) modelling to exploit patterns of gene expression of small phenotypic groups for downstream analysis. Here, we employed a deep learning approach to identify gene expression patterns unique to tumors caused by rare HPV subtypes. Whole transcriptome analysis of tumor biopsies from an institutional LACC tumor bank were employed including 130 HPV16/18 positive, and at least 3 each of rare HPV subtypes 31, 33, 39, 45, 52, 58, and 59. A Wasserstein GAN model (WGAN) was trained for each rare HPV subtype, and used to generate augmented rare HPV population data for downstream differential expression testing between rare HPV subtypes and HPV16/18. K-means clustering analysis was performed using R stats package. Differential gene expression testing between rare HPV subtype populations was performed using the EdgeR R-package. Pathway Enrichment analysis was conducted using EnrichR software. By minimizing intergroup clustering distance, 3 clusters of HPV driven disease were demonstrated. The first cluster demonstrated biology similar to HPV16 and HPV18. Namely, pathway enrichment analysis performed on lists of differentially expressed genes between GAN augmented rare HPV subtypes (HPV45, HPV52, and HPV58) and classical HPV subtypes 16 and 18, yielded no significant statistical differences. The second cluster demonstrated enriched gene expression in proinflammatory IL-1 and IL-6 family cytokines and focal adhesion signaling (HPV31, HPV33, HPV39) (IL-1 regulation of extracellular matrix, p = 5.0e-6; Oncostatin M signaling, p = 2.0e-7; Integrin Signaling, 2.5e-7). The last cluster demonstrated an increased Th2/myeloid signature (HPV59) (T helper cell surface molecules 1.2e-7, myeloid cell signature 8.5e-9). Utilizing a deep learning architecture to model gene expression of rare HPV subtypes we demonstrate 3 groups of HPV driven cervical cancer biology. These groups include a classical subtype, a high IL-1/IL-6 high subtype, and a myeloid associated subtype. Knowledge of such tumor heterogeneity can aid in the future development of targeted treatment plans and patient prognosis.

Programmed cell death ligand 1 expression associated with subtypes of post-transplant lymphoproliferative disorder among pediatric kidney transplant recipients.

Programmed cell death ligand 1 (PD-L1) expression on tumor cells engages the PD-1 receptor on T cells, inhibiting anti-tumor responses. PD-L1 has been detected in cases of post-transplant lymphoproliferative disorder (PTLD) but reports are limited. Here we examine PD-L1 expression and evaluate for clinical correlations. Twenty-one cases of PTLD were identified among pediatric kidney transplant recipients at our institution from February 1996 to April 2017. Using paraffin-embedded tissue biopsies, we examined 21 primary tumors for expression using PD-L1 monoclonal antibody performed with PAX5 as a double stain. We scored expression of PD-L1 on lesional B-cells as a percentage of positive cells. Clinical course and outcome were obtained from retrospective chart review. Applying revised 2017 WHO PTLD classification showed five non-destructive, nine polymorphic, and seven monomorphic cases. Average PD-L1 expression based upon PTLD subtype was: non-destructive 11%, polymorphic 43%, and monomorphic 73% (p=.01). Two patients transferred shortly after diagnosis, five received chemotherapy, and three died from PTLD. Among the fatalities, all showed monomorphic PTLD and 90% of lesional B-cells expressed PD-L1. In this case series, significant differences in PD-L1 expression were seen among different subtypes, and monomorphic PTLD demonstrated the highest expression. Study of a larger cohort is needed, and if the correlation of PD-L1 expression and PTLD subtype is confirmed, this may highlight the potential utility of checkpoint inhibitor therapy in cases of severe or refractory disease among kidney transplant recipient in whom the risk of allograft loss is acceptable given the option of chronic dialysis.

Screening diagnostic markers of osteoporosis based on ferroptosis of osteoblast and osteoclast.

Osteoporosis is a negative balance of bone metabolism caused by the lower bone formation of osteoblasts than the bone absorption of osteoclasts. Ferroptosis plays an important role in osteoporosis, but its effects on osteoblasts and osteoclasts are still unclear. First, we compared the osteogenic differentiation potential of MSCs and osteoclast differentiation potential of monocytes between osteoporosis mice and control. Then, we obtained gene expression profiles of MSCs and monocytes, and screened differentially expressed genes for enrichment analysis. Next, we cluster the patients with osteoporosis according to genes related to osteogenesis inhibition and osteoclast promotion. Finally, according to the expression of different subtypes of ferroptosis genes, diagnostic markers were screened and verified. The osteogenic differentiation ability of MSCs in osteoporosis mice was decreased, while the osteoclast differentiation ability of monocytes was enhanced. The DEGs of MSCs are enriched in iron ion, oxygen binding and cytokine activity, while the DEGs of monocytes are enriched in iron ion transmembrane transport and ferroptosis. Compared with the osteogenic inhibition subtype, the osteoclast promoting subtype has a higher correlation with ferroptosis, and its functions are enriched in fatty acids, reactive oxygen species metabolism and oxidoreductase activity of metal ions. SLC40A1 may be the hub gene of ferroptosis in osteoporosis by promoting osteoclast differentiation. Ferroptosis may inhibit bone formation and promote bone absorption through oxidative stress, thus leading to osteoporosis. The study of ferroptosis on osteoblasts and osteoclasts provides a new idea for the diagnosis and treatment of osteoporosis.

Disulfidptosis-associated lncRNAs predict breast cancer subtypes

Disulfidptosis is a newly discovered mode of cell death. However, its relationship with breast cancer subtypes remains unclear. In this study, we aimed to construct a disulfidptosis-associated breast cancer subtype prediction model. We obtained 19 disulfidptosis-related genes from published articles and performed correlation analysis with lncRNAs differentially expressed in breast cancer. We then used the random forest algorithm to select important lncRNAs and establish a breast cancer subtype prediction model. We identified 132 lncRNAs significantly associated with disulfidptosis (FDR < 0.01, |R|> 0.15) and selected the first four important lncRNAs to build a prediction model (training set AUC = 0.992). The model accurately predicted breast cancer subtypes (test set AUC = 0.842). Among the key lncRNAs, LINC02188 had the highest expression in the Basal subtype, while LINC01488 and GATA3-AS1 had the lowest expression in Basal. In the Her2 subtype, LINC00511 had the highest expression level compared to other key lncRNAs. GATA3-AS1 had the highest expression in LumA and LumB subtypes, while LINC00511 had the lowest expression in these subtypes. In the Normal subtype, GATA3-AS1 had the highest expression level compared to other key lncRNAs. Our study also found that key lncRNAs were closely related to RNA methylation modification and angiogenesis (FDR < 0.05, |R|> 0.1), as well as immune infiltrating cells (P.adj < 0.01, |R|> 0.1). Our random forest model based on disulfidptosis-related lncRNAs can accurately predict breast cancer subtypes and provide a new direction for research on clinical therapeutic targets for breast cancer.