Heterogeneous Expression Profiles Research Articles

Heterogeneous Profile of ROR1 Protein Expression across Tumor Types.

The Wnt receptor ROR1 has generated increased interest as a cancer therapeutic target. Research on several therapeutic approaches involving this receptor is ongoing; however, ROR1 tissue expression remains understudied. We performed an immunohistochemistry analysis of ROR1 protein expression in a large cohort of multiple tumor and histologic types. We analyzed 12 anonymized multi-tumor tissue microarrays (TMAs), including mesothelioma, esophageal and upper gastrointestinal carcinomas, and uterine endometrioid carcinoma, among other tumor types. Additionally, we studied 5 different sarcoma types of TMAs and 6 patient-derived xenografts (PDX) TMAs developed from 19 different anatomic sites and tumor histologic types. A total of 1142 patient cases from different histologic types and 140 PDXs placed in TMAs were evaluated. Pathologists assessed the percentage of tumor cells in each case that were positive for ROR1 and the intensity of staining. For determining the prevalence of staining for each tumor type, a case was considered positive if >1% of its tumor cells showed ROR1 staining. Our immunohistochemistry assays revealed a heterogeneous ROR1 expression profile. A high prevalence of ROR1 expression was found in mesothelioma (84.6%), liposarcoma (36.1%), gastrointestinal stromal tumors (33.3%), and uterine endometrioid carcinoma (28.9%). Other histologic types such as breast, lung, renal cell, hepatocellular, urothelial carcinoma, and colon carcinomas; glioblastoma; cholangiocarcinoma; and leiomyosarcoma showed less ROR1 overall expression, ranging between 0.9 and 13%. No ROR1 expression was seen in mesenchymal chondrosarcoma, rhabdomyosarcoma, or gastric adenocarcinoma cases. Overall, ROR1 expression was relatively infrequent and low in most tumor types investigated; however, ROR1 expression was infrequent but high in selected tumor types, such as gastroesophageal GIST, suggesting that ROR1 prescreening may be preferable for those indications. Further, mesothelioma exhibited frequent and high levels of ROR1 expression, which represents a previously unrecognized therapeutic opportunity. These findings can contribute to the development of ROR1-targeted therapies.

Single-cell sequencing of tumour infiltrating T cells efficiently identifies tumour-specific T cell receptors based on the T cell activation score

Adoptively transferred T cell receptor-engineered T cells are a promising cancer treatment strategy, and the identification of tumour-specific TCRs is essential. Previous studies reported that tumour-reactive T cells and TCRs could be isolated based on the expression of activation markers. However, since T cells with different cell states could not respond uniformly to activation but show a heterogeneous expression profile of activation and effector molecules, isolation of tumour-reactive T cells based on single activation or effector molecules could result in the absence of tumour-reactive T cells; thus, combinations of multiple activation and effector molecules could improve the efficiency of isolating tumour-specific TCRs. We enrolled two patients with lung adenocarcinoma and obtained their tumour infiltrating lymphocytes (TILs) and autologous tumour cells (ATCs). TILs were cocultured with the corresponding ATCs for 12 h and subjected to single-cell RNA sequencing. First, we identified three TCRs with the highest expression levels of IFNG and TNFRSF9 mRNA for each patient, yet only the top one or two recognized the corresponding ATCs in each patient. Next, we defined the activation score based on normalized expression levels of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF1 mRNA for each T cell and then identified three TCRs with the highest activation score for each patient. We found that all three TCRs in each patient could specifically identify corresponding ATCs. In conclusion, we established an efficient approach to isolate tumour-reactive TCRs based on combinations of multiple activation and effector molecules through single-cell RNA sequencing.

Abstract PO3-26-07: Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer

Abstract The sodium iodide symporter (NIS, encoded by SLC5A5) is the sole transporter of iodide, and successfully exploited in thyroid cancer to uptake radioiodide (RAI). NIS is essentially undetected in normal mammary epithelial cells but elevated during lactation, and has a heterogeneous expression profile in breast cancer (BRCA), as evidenced by TCGA cohort and also in a ~120 sample TMA we have profiled. Furthermore, pre-clinical studies support an epigenetic mechanisms that involves Retinoic Acid Receptor alpha (RARa) to regulate SLC5A5 expression. Therefore, we have applied an orthogonal approach to both elucidate and target mechanisms that control NIS expression and function to deliver a therapeutic strategy that targets this transporter as a novel radiotherapy in BRCA. To understand the epigenetic control of expression we segregated samples in the TCGA-BRCA cohort into lower and upper quartile SLC5A5 expression and identified ~600 significant differentially expressed of genes (DEG). Interestingly, restricting analyses to either triple negative (TN) or tumors from African American (AA) patients doubled the number of DEGs. These DEGs were significantly associated with several transcription factors including LHX3, a pituitary development transcription factor regulated by thyroid signaling. The most significantly altered ~70 DEGs significantly clustered tumors by genomic ancestry and ERBB2/HER2 IHC status. The DEGs also supported a footprint of RARa signaling include PRAME, a known RARa co-repressor associated with histone deacetylase, and reflects the finding that NIS expression is highly responsive to SAHA treatment. To investigate if PRAME expression suppresses RARa regulation of SLC5A5 we have established a panel of BRCA cell lines (AU565, SKB3 SUM52 and MDA-MB-231) stably transfected with either CRISPR-VP64 (activator) and CRISPR-KRAB (repressor) to modulate PRAME and measure the epigenomic impact, the expression of SLC5A5 and the ability to transport RAI. In parallel we have applied a drug screen approach that targets non-canonical approaches to govern NIS expression, for example by targeting valosin-containing protein (VCP) that induces NIS proteolysis. In the first instance we have developed a therapeutic strategy to target NIS by inhibiting VCP. Specifically, we are screening a range of VCP inhibitors in combination with the HDAC inhibitor SAHA or the transcriptional potentiator, disulfiram metabolite Cu(DDC)2 in BRCA cell lines by testing the ability to augment RAI uptake. Remarkably, in MDA MB 231 cells, Cu(DDC)2 combined with the VCP inhibitor CB5339, resulted in a significant additive increase in RAI uptake and a prominent decrease in cell proliferation and survival. These studies combined with future murine and PDX testing of CRISPR-edited tumors and drug exposures will develop a comprehensive analyses of the mechanisms that control SLC5A5 expression across breast cancer cell types, how this can be most effectively targeted to restore NIS expression, and exploited to promote RAI as a novel targeted BRCA radiotherapy. Citation Format: Katie Brookes, Martin Read, Sajad Wani, Sissy Jhiang, Daniel Stover, Mathew Ringel, Christopher McCabe, Moray Campbell. Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-07.

Shedding light on PRAME expression in dysplastic nevi: a cohort study.

Dysplastic nevi represent one of the least agreed-upon entities in dermatopathology despite the existence of established criteria. This study explores preferentially expressed antigen in melanoma (PRAME) in dysplastic nevi, an uncharted area. We examined 22 common melanocytic nevi (CMN), 20 cutaneous melanomas (CM), 48 low-grade dysplastic nevi (LG-DN), and 40 high-grade dysplastic nevi (HG-DN). PRAME was immunohistochemically assessed using a five-tiered system (0 to 4 +). Among CMN, 59% scored 0, 32% scored 1 + , and 9% scored 2 + . CM had score 2 + and 4 + in 11% and 89% of cases, respectively. Among LG-DN, 38% presented score 0, 31% score 1 + , 17% score 2 + , 8% score 3 + , and 6% score 4 + . Thirty per cent of HG-DN demonstrated a score 0, 30% with score 1 + , 15% score 2 + , 10% score 3 + , and 15% score 4 + . Compared to CMN and CM, LG-DN and HG-DN showed heterogeneous expression profiles of PRAME. PRAME positivity effectively distinguished HG-DN from CM with 85% specificity and 80% sensitivity (p < 0.0001). Predictive values were 87% (negative) and 76% (positive). Furthermore, a trend of increased PRAME expression from LG-DN to HG-DN was observed. However, the applicability of PRAME in the differential diagnosis of dysplastic lesions remains unclear as can yield conflicting results with morphology, which remains the primary diagnostic tool for melanocytic lesions.

Abstract LB_B05: Mapping therapy resistant phenotypes in SCLC liquid biopsies with single-cell proteomics at a personalized level

Abstract Small-cell lung cancer (SCLC) accounts for ~15% cases of all lung cancer cases and is characterized by aggressive growth and dismal prognosis. Although patients initially respond favorably to platinum and immune checkpoint blockade therapy, most develop therapy resistance and relapse within 1 year. Leveraging liquid biopsies and circulating tumor cells (CTCs) for evaluating SCLC status is regarded as a promising approach especially in SCLC where biopsies are scarce. SCLC CTC numbers are higher compared to other cancers, presenting a unique opportunity to utilize them for assessing SCLC therapy response and resistance and identifying new targetable markers. In this study we leverage mass cytometry (i.e., CyTOF), a multiplex cytometric approach that enables interrogation of 30-50 protein markers per single cell, allowing us to identify and analyze a significant number of CTCs and their heterogeneous expression profiles. Our goal was to utilize CyTOF to help us map changes in CTC expression profiles in the treatment naïve and relapsed setting and thus be able to assess therapy response and resistance at a personalized level. To achieve this, we optimized a CyTOF antibody panel by using SCLC cell lines and blood specimens from healthy donors and SCLC patients. We show that by using this approach, we are able to identify and phenotype SCLC CTCs by using the combination of CD45 (negative expression) and CD56 positive expression with further validation achieved by TTF1 and SCLC subtype transcription factor expression (e.g. NEUROD1, POU2F3). This approach avoids enriching for specific CTC epithelial phenotypes that express surface markers that fluctuate during epithelial-mesenchymal transition (EMT) (e.g. EpCAM, a common marker for isolating CTCs). We also evaluated markers that describe immune suppression activity (PD-L1), EMT status (E-Cadherin, Vimentin, MUC1, Slug), as well as markers that can be therapeutically targeted/are indicative of resistance (e.g., p-YAP). For parallel immune profiling we assessed among others CD8/CD4 (T-cytotoxic/T-helpers respectively) and CD11b and HLA-DR (Myeloid-derived suppressor cells, MDSCs) expression. We found that p-YAP and MUC1 are significantly increased in CTCs detected in blood specimens from SCLC relapsed patients compared to treatment naïve patients. Both YAP and MUC1 have been shown to be play a role in therapy resistance and YAP inhibition is considered a promising therapeutic target. Our analysis also showed a significant increase of MDSC (CD33+CD11b+HLA-DR-) percentages and a decrease in the CD4/CD8 T cell ratio in the relapsed setting. Finally, when we used PHENOSTAMP, a previously developed reference EMT map for assessing EMT phenotypes in lung cancer clinical specimens at a personalized level, we observed an increase of EMT heterogeneity in CTCs in relapsed patients. Our data highlight the translational power of our approach in tracking therapy resistant cells during therapy directly in SCLC patient liquid biopsies towards assessing therapy resistance at a personalized level. Citation Format: Sayantan Bhattacharyya, Catherine A Stewart, Ashley M Victorian, Shafqat F Ehsan, Ester F Lujan, Alberto Duarte, John V Heymach, Jing Wang, Lauren A Byers, Loukia G Karacosta. Mapping therapy resistant phenotypes in SCLC liquid biopsies with single-cell proteomics at a personalized level [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B05.

A human mucosal melanoma organoid platform for modeling tumor heterogeneity and exploring immunotherapy combination options.

Mucosal melanoma (MM), an aggressive rare subtype of melanoma, is distinct from cutaneous melanoma and has poor prognoses. We addressed the lack of cell models for MM by establishing 30 organoids of human oral MM (OMM), which retained major histopathological and functional features of parental tumors. Organoid groups derived from chronologically or intratumorally distinct lesions within the same individual displayed heterogeneous genetics, expression profiles, and drug responses, indicating rapid tumor evolution and poor clinical response. Furthermore, transcriptome analysis revealed receptor tyrosine kinases (RTKs) signaling, particularly NGFR, a nerve growth factor receptor, was significantly up-regulated in OMMs and organoids from patients resistant to anti-programmed cell death protein 1 (anti-PD-1) therapy. Combining anti-PD-1 with anlotinib (a phase 2 multitarget RTK inhibitor for OMM) or NGFR knockdown enhanced the effective activity of immune cells in organoid-immune cell coculture systems. Together, our study suggested that OMM organoids serve as faithful models for exploring tumor evolution and immunotherapy combination strategies.

Structural and expression analysis of polyphenol oxidases potentially involved in globe artichoke (C. cardunculus var. scolymus L.) tissue browning

Globe artichoke capitula are susceptible to browning due to oxidation of phenols caused by the activity of polyphenol oxidases (PPOs), this reduces their suitability for fresh or processed uses. A genome-wide analysis of the globe artichoke PPO gene family was performed. Bioinformatics analyses identified eleven PPOs and their genomic and amino acidic features were annotated. Cis-acting element analysis identified a gene regulatory and functional profile associated to plant growth and development as well as stress response. For some PPOs, phylogenetic analyses revealed a structural and functional conservation with different Asteraceae PPOs, while the allelic variants of the eleven PPOs investigated across four globe artichoke varietal types identified several SNP/Indel variants, some of which having impact on gene translation. By RTqPCR were assessed the expression patterns of PPOs in plant tissues and in vitro calli characterized by different morphologies. Heterogeneous PPO expression profiles were observed and three of them (PPO6, 7 and 11) showed a significant increase of transcripts in capitula tissues after cutting. Analogously, the same three PPOs were significantly up-regulated in calli showing a brown phenotype due to oxidation of phenols. Our results lay the foundations for a future application of gene editing aimed at disabling the three PPOs putatively involved in capitula browning.

A novel glycolysis-related gene signature for predicting the prognosis of multiple myeloma.

Background: Metabolic reprogramming is an important hallmark of cancer. Glycolysis provides the conditions on which multiple myeloma (MM) thrives. Due to MM's great heterogeneity and incurability, risk assessment and treatment choices are still difficult. Method: We constructed a glycolysis-related prognostic model by Least absolute shrinkage and selection operator (LASSO) Cox regression analysis. It was validated in two independent external cohorts, cell lines, and our clinical specimens. The model was also explored for its biological properties, immune microenvironment, and therapeutic response including immunotherapy. Finally, multiple metrics were combined to construct a nomogram to assist in personalized prediction of survival outcomes. Results: A wide range of variants and heterogeneous expression profiles of glycolysis-related genes were observed in MM. The prognostic model behaved well in differentiating between populations with various prognoses and proved to be an independent prognostic factor. This prognostic signature closely coordinated with multiple malignant features such as high-risk clinical features, immune dysfunction, stem cell-like features, cancer-related pathways, which was associated with the survival outcomes of MM. In terms of treatment, the high-risk group showed resistance to conventional drugs such as bortezomib, doxorubicin and immunotherapy. The joint scores generated by the nomogram showed higher clinical benefit than other clinical indicators. The in vitro experiments with cell lines and clinical subjects further provided convincing evidence for our study. Conclusion: We developed and validated the utility of the MM glycolysis-related prognostic model, which provides a new direction for prognosis assessment, treatment options for MM patients.

Transcriptomic alterations underlying metaplasia into specific metaplastic components in metaplastic breast carcinoma

BackgroundMetaplastic breast carcinoma (MpBC) typically consists of carcinoma of no special type (NST) with various metaplastic components. Although previous transcriptomic and proteomic studies have reported subtype-related heterogeneity, the intracase transcriptomic alterations between metaplastic components and paired NST components, which are critical for understanding the pathogenesis underlying the metaplastic processes, remain unclear.MethodsFifty-nine NST components and paired metaplastic components (spindle carcinomatous [SPS], matrix-producing, rhabdoid [RHA], and squamous carcinomatous [SQC] components) were microdissected from specimens obtained from 27 patients with MpBC for gene expression profiling using the NanoString Breast Cancer 360 Panel on a NanoString nCounter FLEX platform. BC360-defined signatures were scored using nSolver software.ResultsHierarchical clustering and principal component analysis revealed a heterogeneous gene expression profile (GEP) corresponding to the NST components, but the GEP of metaplastic components exhibited subtype dependence. Compared with the paired NST components, the SPS components demonstrated the upregulation of genes related to stem cells and epithelial–mesenchymal transition and displayed enrichment in claudin-low and macrophage signatures. Despite certain overlaps in the enriched functions and signatures between the RHA and SPS components, the specific differentially expressed genes differed. We observed the RHA-specific upregulation of genes associated with vascular endothelial growth factor signaling. The chondroid matrix-producing components demonstrated the upregulation of hypoxia-related genes and the downregulation of the immune-related MHC2 signature and the TIGIT gene. In the SQC components, TGF-β and genes associated with cell adhesion were upregulated. The differentially expressed genes among metaplastic components in the 22 MpBC cases with one or predominantly one metaplastic component clustered paired NST samples into clusters with correlation with their associated metaplastic types. These genes could be used to separate the 31 metaplastic components according to respective metaplastic types with an accuracy of 74.2%, suggesting that intrinsic signatures of NST may determine paired metaplastic type. Finally, the EMT activity and stem cell traits in the NST components were correlated with specimens displaying lymph node metastasis.ConclusionsWe presented the distinct transcriptomic alterations underlying metaplasia into specific metaplastic components in MpBCs, which contributes to the understanding of the pathogenesis underlying morphologically distinct metaplasia in MpBCs.

Transcriptomic Heterogeneity of Skin Across Different Anatomic Sites

Multiomic studies, including RNA sequencing, single-cell RNA sequencing, and epigenomics, can provide insight into the connection between anatomically heterogeneous gene expression profile of the skin and dermatoses-predisposed sites, in which RNA sequencing is essential. Therefore, in this study, 159 skin samples collected mainly from discarded normal skin tissue during surgical treatment for benign skin tumors were used for RNA sequencing. On the basis of cluster analysis, the skin was divided into four regions, with each region showing specific physiological characteristics through differentially expressed gene analysis. The results showed that the head and neck region, perineum, and palmoplantar area were closely associated with lipid metabolism, hormone metabolism, blood circulation, and related neural regulation, respectively. Transcription factor enrichment indicated that different regions were associated with the development of adjacent tissues. Specifically, the head and neck region, trunk and extremities, perineum, and palmoplantar area were associated with the central nervous, axial, urogenital, and vascular systems, respectively. The results were imported into an open website (https://dermvis.github.io/) for retrieval. Our transcriptomic data elucidated that human skin exhibits transcriptomic heterogeneity reflecting physiological and developmental variation at different anatomic sites and provided guidance for further studies on skin development and dermatoses predisposed sites.

An improved graph Laplacian regularization method for identifying biomarkers of Alzheimer's disease

Alzheimer's disease (AD) is one of the most common dementia, and its pathogenesis has not been clarified. The failure of amyloid targeted therapy has led us to rethink the pathogenesis of AD. There is growing evidence that complex diseases usually involve the impairment of multiple biological functions, rather than focus on several single genes. Protein-protein interaction network (PPIN) has been recognized as an important tool for identifying and predicting disease biomarkers. It is a great challenge to design network-based classification method for identifying effective, stable and interpretable biomarkers to distinguish the disease phenotype based on gene expression profile data. In this study, we used graph Laplacian regularization method to introduce topology information of PPIN, which can reveal the damaged networks involved in disease from heterogeneous gene expression profile data and identify disease-related biomarkers. The results in three AD datasets showed that the biomarkers identified by our method can not only distinguish the sample categories more accurately, but also help researchers understand the biological meaning behind complex diseases.

Sarcomeric gene mutations in phenotypic positive hypertrophic cardiomyopathic patients in Indian population.

HCM is a monogenic cardiac disorder with a high risk of sudden cardiac death, heterogeneous phenotypic expression and genetic profile. HCM is expressed as autosomal dominant in fashion with the prevalence of 1:500 in the general population. The main objective of the current study was to unravel the mutation status in sarcomeric genes in urbanizing Pune population. HCM patients were recruited from Bharti hospital and Poona hospital and research centre, Pune after being screened by 2-D echocardiography. DNA was extracted from whole blood samples and PCR amplification was performed for selected exons from pre-selected genes, amplimers of >300 b.p were restriction digested and the SSCP technique was optimized for maximum result output. HCM patients shows the maximum prevalence of mitral regurgitation (23.3%) while the minimum prevalence was left auricular diameter (10%). Maximum variation spectrum was present in MYBPC3 genes as most of them were "benign" type as per Polyphen-2 tool status. Mutations in the MYH7 gene produce a prominent impact on splicing by the creation of a new SRP40 binding site (Exon Splicing Enhancer) as predicted by Human Splicing Finder 3.1. I736T mutation in the MYH7 gene results in replacement of β-strand by α-helix upstream from mutation site which may have a profound impact on protein tertiary structure as predicted by Polyphen-2 tool (probably damaging-1.00). Also, two 'novel' mutations and one 'novel' variation were reported in the present study. Thus, the MYBPC3 gene shows maximum mutation load among other sarcomeric genes. Double gene mutations do not represent much severe pathophysiology as compared to single gene mutated and genotypic negative HCM patients.

Expression and Clinical Significance of SARS-CoV-2 Human Targets in Neoplastic and Non-Neoplastic Lung Tissues

A higher incidence of COVID-19 infection was demonstrated in cancer patients, including lung cancer patients. This study was conducted to get insights into the enhanced frequency of COVID-19 infection in cancer. Using different bioinformatics tools, the expression and methylation patterns of ACE2 and TMPRSS2 were analyzed in healthy and malignant tissues, focusing on lung adenocarcinoma and data were correlated to clinical parameters and smoking history. ACE2 and TMPRSS2 were heterogeneously expressed across 36 healthy tissues with the highest expression levels in digestive, urinary and reproductive organs, while the overall analysis of 72 paired tissues demonstrated significantly lower expression levels of ACE2 in cancer tissues when compared to normal counterparts. In contrast, ACE2, but not TMPRSS2, was overexpressed in LUAD, which inversely correlated to the promoter methylation. This upregulation of ACE2 was age-dependent in LUAD, but not in normal lung tissues. TMPRSS2 expression in non-neoplastic lung tissues was heterogeneous and dependent on sex and smoking history, while it was downregulated in LUAD of smokers. Cancer progression was associated with a decreased TMPRSS2 but unaltered ACE2. In contrast, ACE2 and TMPRSS2 of lung metastases derived from different cancer subtypes was higher than organ metastases of other sites. TMPRSS2, but not ACE2, was associated with LUAD patients' survival. Comprehensive molecular analyses revealed a heterogeneous and distinct expression and/or methylation profile of ACE2 and TMPRSS2 in healthy lung vs. LUAD tissues across sex, age and smoking history and might have implications for COVID-19 disease.

Abstract 460: Cyclin Dependent Kinase Inhibitor A1 Identified as a Potential Risk Locus for Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by heterogeneous phenotypic expression, natural history, and genetic profile with numerous causative mutations. The relationship between genotype and phenotype in HCM is incompletely understood. Identifying genetic modifiers may expand understanding of the signaling pathways that are responsible for phenotypic expression. UK Biobank comprises clinical and genetic data for greater than 500,000 individuals. We compared control individuals to those with a diagnosis of HCM (identified via ICD-10 code I42.1) in a 20-to-1 fashion to identify genetic differences. Data were compared using Mixed Model Analysis for Pedigrees/Populations (MMAP) mixed-model regression. Variants were assessed using bioinformatics tools including linkage disequilibrium and Eigen scoring. Results were then compared to previously published genome-wide association datasets. Mixed-model regression identified 5 variants of cyclin-dependent kinase inhibitor 1A ( CDKN1A ) as statistically significant with one intronic variant (rs3176326) likely affecting a gene promoter region with an Eigen-PC score of 7.779. This variant was found to have a p-value of 5.08x10 -8 when contrasted to controls with a minor allele frequency of 0.199 in the affected individuals. The 4 other variants were in linkage disequilibrium (r 2 0.90-0.99) with rs3176326. The odds ratio of HCM with rs3176326 is 1.81.To our knowledge, CDKN1A has not previously been associated with HCM. By utilizing bioinformatic tools we were able to identify 5 variants that may be associated with the risk for HCM. One locus affects a promotor region and resulted in a notably high Eigan-PC score suggestive that it has an impact on HCM. When comparing these findings with previous genome-wide association studies utilizing the UK Biobank, we find that CDKN1A has a possible association with HCM, though those results were not statistically significant. CDKN1A has been previously associated with the p53 DNA repair and cell senescence pathway. Animal and human studies have implicated it in the development of fibrosis. A recent genome-wide association metanalysis identified it as a risk locus for heart failure. These findings support our conclusion that CDKN1A is a risk locus for HCM.

DSA Are Associated With More Graft Injury, More Fibrosis, and Upregulation of Rejection-associated Transcripts in Subclinical Rejection.

Subclinical T cell-mediated rejection (subTCMR) is commonly found after liver transplantation and has a good short-term prognosis, even when it is left untreated. Donor-specific antibodies (DSA) are putatively associated with a worse prognosis for recipient and graft after liver transplantation. To assess the immune regulation in subTCMR grafts, gene expression of 93 transcripts for graft injury, tolerance, and immune regulation was analyzed in 77 biopsies with "no histologic rejection" (NHR; n = 25), "clinical TCMR" (cTMCR; n = 16), and subTCMR (n = 36). In addition, all available subTCMR biopsies (n = 71) were tested for DSA with bead assays. SubTCMR showed heterogeneous and intermediate expression profiles of transcripts that were upregulated in cTCMR. Graft gene expression suggested a lower activation of effector lymphocytes and a higher activation of regulatory T cells in grafts with subTCMR compared to cTCMR. DSA positivity in subTCMR was associated with histological evidence of more severe graft inflammation and fibrosis. This more severe DSA+ associated graft injury in subTCMR was converged with an upregulation of cTCMR-associated transcripts. In nonsupervised analysis, DSA positive subTCMR mostly clustered together with cTCMR, while DSA negative subTCMR clustered together with NHR. T cell-mediated rejection seems to form a continuum of alloimmune activation. Although subTCMR exhibited less expression of TCMR-associated transcript, DSA positivity in subTCMR was associated with an upregulation of rejection-associated transcripts. The identification of DSA positive subclinical rejection might help to define patients with more inflammation in the graft and development of fibrosis.

MicroRNAs as Key Regulators of Ovarian Cancers.

The tumor microenvironment can be realistically viewed as an active battle ground between the host immune system and the growing tumor cells. This reactive space surrounding the tumor possesses several possibilities and facilitates the progression of a tumor from a neoplastic stage to that of metastasis. The contemporary approach of understanding the cancer biology from a “within the cell” perspective has been largely challenged with complex and intricate “outside the cell” events. Thus understanding the biology of the tumor microenvironment has been of scientific and clinical interest. Small non-coding microRNAs with a pleotropic and wide range of cellular gene targets can be reasonably hypothesized to regulate the events of carcinogenesis and progression. MicroRNAs have been investigated in different cancer models, and evidence of their involvement in the regulation of the tumor microenvironment has been of much interest. In particular, a major interest has been exploring the role of the tumor microenvironment in regulating the interaction of cancer cells with surrounding stromal components and the effect of such interactions on the cancer cells. Fine-tuned regulation by these microRNAs extends our contemporary understanding of these small biomolecules in epigenetic regulations. This review focuses on microRNAs that are dysregulated in ovarian carcinomas, their effect on the components of the tumor microenvironment, and the correlation of their heterogeneous expression profiles with disease severity and prognosis in patients. In addition, this paper also discusses the differential expression of exosomal microRNAs that are known to link the cancer cell with its microenvironment, facilitating the development of an improved prognostic/diagnostic marker and effective therapeutic regime.

The tumour phenotype of mycosis fungoides clusters into three heterogeneous surface expression profiles

The tumour phenotype of mycosis fungoides clusters into three heterogeneous surface expression profiles

Heterogeneous circRNA expression profiles and regulatory functions among HEK293T single cells

The single-cell analysis is becoming a powerful method for early detection of the abnormal variant in tissues, especially for profiling a small number of heterogeneous cells. With the advancement of sequencing technologies, many types of non-coding elements including miRNAs and lncRNAs which shed light on their heterogeneous patterns and functions among cells, have been profiled at the single-cell level. However, the complete picture of circRNA profile at single-cell level is still lacking. In this study, RNA-Seq data obtained from single HEK293T cells have been used to analyze expressions and functions of heterogeneous circRNA profiles. The enrichment patterns of circRNAs, interactions with miRNAs and pathways such as ErbB signaling pathway and protein processing in endoplasmic reticulum, have also been investigated. The results showed that circRNAs had a specific distribution pattern which was implicated with expression, miRNA and functional profiles at single-cell level. This assessment study of the expressions and functions of circRNAs at single-cell level shed light on heterogeneities among single cells.

Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions

The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites’ ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.

A phase II study of oral azacitidine (CC-486) in combination with pembrolizumab (PEMBRO) in patients with metastatic melanoma (MM).

TPS9594 Background: Immune checkpoint inhibitors have impressive response rates and improved survival for many pts with MM, offering durable responses in up to 35% of pts while others have short-lived or no response ( &gt; 40%). A potentially targetable mechanism for immune escape is for cancers to subvert the cellular epigenetic machinery, affecting multiple aspects of the immune response from suppression of tumor antigen expression [such as Cancer Testis Antigens (CTA)] to antigen processing and presentation, thus allowing tumor proliferation to continue undetected. In preclinical models, decitabine (DAC), a DNA hypomethylating agent (HMA), has been able to revert heterogeneous CTA expression profiles, increasing cancer cell immunogenicity. HMAs also increased TH1-chemokine expression, T cell tumor infiltration, T cell-mediated tumor killing, and have been shown to be synergistic with checkpoint inhibitors in preclinical models. This suggests that epigenetic therapy with checkpoint inhibition is a rational combination to target MM. Clinical advantages of the HMA CC-486 over DAC include oral bioavailability and potential versatility in dosing and schedule. We hypothesize that CC-486 + PEMBRO will be tolerated at biologically relevant doses; enhance response to PEMBRO in pts with mm who are PD-1 naïve; and reverse resistance to immunotherapy in pts refractory/resistant to PD-1 directed therapy. Methods: This study will evaluate the safety and efficacy of CC-486 + PEMBRO defined by Objective Response Rate (ORR) by RECIST 1.1 in pts with MM. Pts who are PD-1 naïve (Arm A, n = 36) and pts who have progressed on prior PD-1 directed therapy (Arm B, n = 35) will be enrolled. Unlimited prior systemic therapies will be allowed. Pts will receive 300mg PO of CC-486 on days 1-14 and 200mg IV of PEMBRO every 3 weeks. Continuous monitoring for toxicity and futility will be performed and assumes an ORR of &gt; 35% and &gt; 15% for Arms A and B, respectively (95% power). Tumor biopsies at baseline and post treatment are required. Effects of CC-486 + PEMBRO on CTAs, MDSCs and Tregs, and correlation between mutation burden and response will be studied. This study is open for enrollment. Clinical trial information: NCT02816021.