Abstract Objectives The PI3K/AKT pathway is frequently activated in endometrial carcinoma (EC) mainly due to mutations in the PIK3CA and PTEN genes. These events are common and believed to be the key ...to endometrial carcinogenesis. Recently, a somatic activating mutation in the AKT1 gene (E17K) was identified in several cancer types. In this study we explored the frequency of this AKT1 mutation in endometrial carcinoma. Methods Tumor DNA, extracted from 73 EC was analyzed for AKT1 E17K mutation (G49A) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, the tumors were screened for coexisting common mutations in PTEN, PIK3CA and KRAS. Results The AKT1 E17K mutation was detected in 4% of EC. One of the AKT1-mutated tumors showed coexisting PTEN loss-of-function mutation. Conclusion We identified the AKT1 E17K mutation in 4% of endometrial carcinomas. The presence of double AKT1/ PTEN mutants is in accord with the hypothesis that in EC more than one hit is required to completely activate the PI3K pathway. Furthermore, AKT1 mutations were limited to high grade, advanced stage tumors suggesting that this mutation confers a more aggressive tumor behavior.
Hyaline fibromatosis syndrome (HFS), resulting from
mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the ...gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious
mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9
knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or
organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.
Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate ...toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.
Abstract In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs) has gained evidence. However, the mechanistic ...pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID) as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection.
Paclitaxel, the most commonly used form of chemotherapy, is utilized in curative protocols in different types of cancer. The response to treatment differs among patients. Biological interpretation of ...a mechanism to explain this personalized response is still unavailable. Since paclitaxel is known to target BCL2 and TUBB1, we used pan-cancer genomic data from hundreds of patients to show that a single-nucleotide variant in the BCL2 sequence can predict a patient's response to paclitaxel. Here, we show a connection between this BCL2 genomic variant, its transcript structure, and protein abundance. We demonstrate these findings in silico, in vitro, in formalin-fixed paraffin-embedded (FFPE) tissue, and in patient lymphocytes. We show that tumors with the specific variant are more resistant to paclitaxel. We also show that tumor and normal cells with the variant express higher levels of BCL2 protein, a phenomenon that we validated in an independent cohort of patients. Our results indicate BCL2 sequence variations as determinants of chemotherapy resistance. The knowledge of individual BCL2 genomic sequences prior to the choice of chemotherapy may improve patient survival. The current work also demonstrates the benefit of community-wide, integrative omics data sources combined with in-lab experimentation and validation sets.
Abstract The prognostic value of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in melanoma was demonstrated more than a decade ago as superior to Breslow score. We have previously ...shown that intercellular homophilic CEACAM1 interactions protect melanoma cells from lymphocyte-mediated elimination. Here, we study the direct effects of CEACAM1 on melanoma cell biology. By employing tissue microarrays and low-passage primary cultures of metastatic melanoma, we show that CEACAM1 expression gradually increases from nevi to metastatic specimens, with a strong dominance of the CEACAM1-Long tail splice variant. Using experimental systems of CEACAM1 knockdown and overexpression of selective variants or truncation mutants, we prove that only the full-length long tail variant enhances melanoma cell proliferation in vitro and in vivo . This effect is not reversed with a CEACAM1-blocking antibody, suggesting that it is not mediated by intercellular homophilic interactions. Downstream, CEACAM1-Long increases the expression of Sox-2, which we show to be responsible for the CEACAM1-mediated enhanced proliferation. Furthermore, analysis of the CEACAM1 promoter reveals two single-nucleotide polymorphisms (SNPs) that significantly enhance the promoter's activity compared with the consensus nucleotides. Importantly, case-control genetic SNP analysis of 134 patients with melanoma and matched healthy donors show that patients with melanoma do not exhibit the Hardy-Weinberg balance and that homozygous SNP genotype enhances the hazard ratio to develop melanoma by 35%. These observations shed new mechanistic light on the role of CEACAM1 in melanoma, forming the basis for development of novel therapeutic and diagnostic technologies.
BackgroundImmunotherapy revolutionized the treatment of melanoma, however, a significant proportion of patients fail to respond. Thus, reliable biomarkers are needed to predict treatment response. ...Spatial biology, which investigates the spatial distribution and interactions of cells within tissues, has emerged as a powerful approach to unravel the complexity of the tumor-microenvironment (TME). In this study, we analyzed multiplex immunofluorescence (mIF) images from melanoma patients treated with immunotherapy, aiming to identify spatially resolved biomarkers associated with response.MethodsTwo consecutive slides from pretreatment biopsies of 47 melanoma patients who received PD-1 blockade, as a single-agent, or combined with ipilimumab, were collected and stained with two 6-plex mIF antibody panels using PhenoImager, followed by hematoxylin and eosin (H&E) stain on the same slide (figure 1). Utilizing our deep learning-based multiplex analysis pipeline1 we were able to identify 9 distinct cell types across the two panels (figure 2). These predictions were evaluated against expert annotations. Corresponding sections stained with H&E provided additional cellular information such as the identification of fibroblasts and granulocytes, and tumor or TME area region assignment. More than 1,300 spatial features were calculated using a combination of cell type, marker positivity and area assignments, as well as cell interactions, and were compared between responders and non-responders to immunotherapy using Welch’s t-test in lymph node (LN) and non-LN tumor biopsies.ResultsOur analysis pipeline demonstrated excellent performance both in accurately defining cell types and single-markers positivity (figures 1 and 2). As the TME is vastly different in LN and non-LN biopsies, we aimed to characterize different spatial features which are associated with response to treatment in each biopsy type. In LN biopsies, we observed CD8+ FOXP3+ T-cells upregulation in the TME of responders to PD-1 blockade, while fibroblast and granulocyte interactions in the TME correlated with poor response (figure 3). However, these associations were not observed in non-LN biopsies, where a high number of B-cell and T-cell interactions in the tumor invasive margin, and tumor cells and lymphocyte interactions in the tumor core were associated with treatment response. Additionally, a high number of granulocytes in the TME of non-LN biopsies was associated with treatment resistance (figure 4).ConclusionsOur study demonstrates that the tumor-microenvironment in LN and non-LN melanoma biopsies exhibits distinct spatial characteristics associated with immunotherapy response. These findings highlight the importance of spatial features, cell composition, and biopsy site when identifying spatial biomarkers for immunotherapy response prediction.ReferenceMarkovits E, Dankovich T, Gluskin R, et al. A novel deep learning pipeline for cell typing and phenotypic marker quantification in multiplex imaging. bioRxiv, 2022.Ethics ApprovalThis single-center, retrospective study of medical records was approved by the Institutional Review Board of the Sheba Medical Center (4387–17-SMC).Abstract 108 Figure 1Deep learning-based multiplex imaging analysis pipeline performance in single-markers positivity classification in panel 1 (A) and panel 2 (B).Abstract 108 Figure 2Deep learning-based multiplex imaging analysis pipeline performance in cell typing in panel 1 (A) and panel 2 (B).Abstract 108 Figure 3(A) The top differentially expressed features in between responders (n=7) and non-responders (n=6) to PD-I blockade in lymph node biopsies and their distribution in responders (n=19) and non-responders (n=15) in non-lymph node biopsies. (B) A representative image of a tumor-microenvironment enriched with CD8+ FOXP3+ T-cells (white arrows).Abstract 108 Figure 4(A) The top differentially expressed features in between responders (n=19) and non-responders (n=15) to PD-1 blockade in non-lymph node biopsies, and their distribution in responders (n=7) and non-responders (n=6) in lymph node biopsies. (B) A representative image of a tumor infiltrating T-cells.
Ulcerative colitis (UC), Crohn's disease (CD), and celiac disease are prevalent intestinal inflammatory disorders with nonsatisfactory therapeutic interventions. Analyzing patient data-driven cohorts ...can highlight disease pathways and new targets for interventions. Long noncoding RNAs (lncRNAs) are attractive candidates, since they are readily targetable by RNA therapeutics, show relative cell-specific expression, and play key cellular functions. Uniformly analyzing gut mucosal transcriptomics from 696 subjects, we have highlighted lncRNA expression along the gastrointestinal (GI) tract, demonstrating that, in control samples, lncRNAs have a more location-specific expression in comparison with protein-coding genes. We defined dysregulation of lncRNAs in treatment-naive UC, CD, and celiac diseases using independent test and validation cohorts. Using the Predicting Response to Standardized Pediatric Colitis Therapy (PROTECT) inception UC cohort, we defined and prioritized lncRNA linked with UC severity and prospective outcomes, and we highlighted lncRNAs linked with gut microbes previously implicated in mucosal homeostasis. HNF1A-AS1 lncRNA was reduced in all 3 conditions and was further reduced in more severe UC form. Similarly, the reduction of HNF1A-AS1 ortholog in mice gut epithelia showed higher sensitivity to dextran sodium sulfate-induced colitis, which was coupled with alteration in the gut microbial community. These analyses highlight prioritized dysregulated lncRNAs that can guide future preclinical studies for testing them as potential targets.
Omics analyses often result in dozens to hundreds of potential targets, requiring validation for their biological relevance. Current high-throughput functional investigation methods are frequently ...labor-intensive, expensive, and display low reproducibility. The Immune Co-Culture Cell Microarray (ICCM) is a formalin-fixed paraffin-embedded cell block microarray based on co-cultures of patient-derived tumor-infiltrating lymphocytes and their autologous melanoma cells. Each ICCM slide represents the same experiment and can be stained using standard immunohistochemistry and immunofluorescence techniques. Functional dynamics assessment of both proteins and microRNAs using ICCM stained slides demonstrated similar findings to flow cytometry assays and to previously published patient-derived biopsy reports.
Triple-negative breast cancer (TNBC) is primarily treated via chemotherapy; in parallel, efforts are made to introduce immunotherapies into TNBC treatment. CD4+ TNFR2+ lymphocytes were reported as ...Tregs that contribute to tumor progression. However, our published study indicated that TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs) were associated with improved survival in TNBC patient tumors. Based on our analyses of the contents of CD4+ and CD8+ TILs in TNBC patient tumors, in the current study, we determined the impact of chemotherapy on CD4+ and CD8+ TIL subsets in TNBC mouse tumors. We found that chemotherapy led to (1) a reduction in CD4+ TNFR2+ FOXP3+ TILs, indicating that chemotherapy decreased the content of CD4+ TNFR2+ Tregs, and (2) an elevation in CD8+ TNFR2+ and CD8+ TNFR2+ PD-1+ TILs; high levels of these two subsets were significantly associated with reduced tumor growth. In spleens of tumor-bearing mice, chemotherapy down-regulated CD4+ TNFR2+ FOXP3+ cells but the subset of CD8+ TNFR2+ PD-1+ was not present prior to chemotherapy and was not increased by the treatment. Thus, our data suggest that chemotherapy promotes the proportion of protective CD8+ TNFR2+ TILs and that, unlike other cancer types, therapeutic strategies directed against TNFR2 may be detrimental in TNBC.
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