Biomarker For Tumor Progression Research Articles

The oncogenic kinase TOPK upregulates in psoriatic keratinocytes and contributes to psoriasis progression by regulating neutrophils infiltration

BackgroundT-LAK cell-oriented protein kinase (TOPK) strongly promotes the malignant proliferation of cancer cells and is recognized as a promising biomarker of tumor progression. Psoriasis is a common inflammatory skin disease featured by excessive proliferation of keratinocytes. Although we have previously reported that topically inhibiting TOPK suppressed psoriatic manifestations in psoriasis-like model mice, the exact role of TOPK in psoriatic inflammation and the underlying mechanism remains elusive.MethodsGEO datasets were analyzed to investigate the association of TOPK with psoriasis. Skin immunohistochemical (IHC) staining was performed to clarify the major cells expressing TOPK. TOPK conditional knockout (cko) mice were used to investigate the role of TOPK-specific deletion in IMQ-induced psoriasis-like dermatitis in mice. Flow cytometry was used to analyze the alteration of psoriasis-related immune cells in the lesional skin. Next, the M5-induced psoriasis cell model was used to identify the potential mechanism by RNA-seq, RT-RCR, and western blotting. Finally, the neutrophil-neutralizing antibody was used to confirm the relationship between TOPK and neutrophils in psoriasis-like dermatitis in mice.ResultsWe found that TOPK levels were strongly associated with the progression of psoriasis. TOPK was predominantly increased in the epidermal keratinocytes of psoriatic lesions, and conditional knockout of TOPK in keratinocytes suppressed neutrophils infiltration and attenuated psoriatic inflammation. Neutrophils deletion by neutralizing antibody greatly diminished the suppressive effect of TOPK cko in psoriasis-like dermatitis in mice. In addition, topical application of TOPK inhibitor OTS514 effectively attenuated already-established psoriasis-like dermatitis in mice. Mechanismly, RNA-seq revealed that TOPK regulated the expression of some genes in the IL-17 signaling pathway, such as neutrophils chemokines CXCL1, CXCL2, and CXCL8. TOPK modulated the expression of neutrophils chemokines via activating transcription factors STAT3 and NF-κB p65 in keratinocytes, thereby promoting neutrophils infiltration and psoriasis progression.ConclusionsThis study identified a crucial role of TOPK in psoriasis by regulating neutrophils infiltration, providing new insights into the pathogenesis of psoriasis.

HIPK2 in Colon Cancer: A Potential Biomarker for Tumor Progression and Response to Therapies.

Colon cancer, one of the most common and fatal cancers worldwide, is characterized by stepwise accumulation of specific genetic alterations in tumor suppressor genes or oncogenes, leading to tumor growth and metastasis. HIPK2 (homeodomain-interacting protein kinase 2) is a serine/threonine protein kinase and a "bona fide" oncosuppressor protein. Its activation inhibits tumor growth mainly by promoting apoptosis, while its inactivation increases tumorigenicity and resistance to therapies of many different cancer types, including colon cancer. HIPK2 interacts with many molecular pathways by means of its kinase activity or transcriptional co-repressor function modulating cell growth and apoptosis, invasion, angiogenesis, inflammation and hypoxia. HIPK2 has been shown to participate in several molecular pathways involved in colon cancer including p53, Wnt/β-catenin and the newly identified nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2). HIPK2 also plays a role in tumor-host interaction in the tumor microenvironment (TME) by inducing angiogenesis and cancer-associated fibroblast (CAF) differentiation. The aim of this review is to assess the role of HIPK2 in colon cancer and the underlying molecular pathways for a better understanding of its involvement in colon cancer carcinogenesis and response to therapies, which will likely pave the way for novel colon cancer therapies.

Unprocessed snRNAs Are a Prognostic Biomarker and Correlate with a Poorer Prognosis in Colorectal Cancer.

The human Integrator complex is a set of 15 subunits that mediates processing of small nuclear RNAs (snRNAs), and which later participates in splicing messenger RNAs (mRNAs). In addition, it controls the pause and release of RNA polymerase II (RNA pol II) at specific gene promoters in response to growth factors. Mutations in Integrator-complex subunit 6 (INTS6) are associated with different types of tumors. However, the INTS6 gene product does not have a significant prognostic value as a biomarker for tumor progression. Here we show that Integrator-complex deregulation is involved in 8.3% of the colorectal cancer cases diagnosed from the population screen carried out in La Rioja (Spain) from the years 2017 to 2019. Lack of Integrator-complex function, measured by an increased level of unprocessed snRNA, is a prognostic biomarker and correlates with a poorer prognosis in colorectal-cancer patients. The transcriptomic profile of all analyzed colorectal tumors shows a strong alteration of the metabolic state of tumor cells, which compromises standard energy production through mitochondrial respiration, known as the Warburg effect. Furthermore, there is a significant upregulation of genes involved in extracellular matrix organization and collagen rearrangement. This is consistent with tissue reorganization in a growing tumor forming a polyp. Crossing the molecular data generated in this study with the follow-up of patients from population screening indicates that population screening combined with early typing of tumors appears to be the most efficient way to increase patient survival.

Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.

Proteomic Analysis of Spatial Heterogeneity Identifies HMGB2 as Putative Biomarker of Tumor Progression in Adult-Type Diffuse Astrocytomas.

Although grading is defined by the highest histological grade observed in a glioma, most high-grade gliomas retain areas with histology reminiscent of their low-grade counterparts. We sought to achieve the following: (i) identify proteins and molecular pathways involved in glioma evolution; and (ii) validate the high mobility group protein B2 (HMGB2) as a key player in tumor progression and as a prognostic/predictive biomarker for diffuse astrocytomas. We performed liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple areas of adult-type astrocytomas and validated our finding in multiplatform-omics studies and high-throughput IHC analysis. LC-MS/MSdetected proteomic signatures characterizing glioma evolution towards higher grades associated with, but not completely dependent, on IDH status. Spatial heterogeneity of diffuse astrocytomas was associated with dysregulation of specific molecular pathways, and HMGB2 was identified as a putative driver of tumor progression, and an early marker of worse overall survival in grades 2 and 3 diffuse gliomas, at least in part regulated by DNA methylation. In grade 4 astrocytomas, HMGB2 expression was strongly associated with proliferative activity and microvascular proliferation. Grounded in proteomic findings, our results showed that HMGB2 expression assessed by IHC detected early signs of tumor progression in grades 2 and 3 astrocytomas, as well as identified GBMs that had a better response to the standard chemoradiation with temozolomide.

Abstract 5152: Co-expression and prognostic significance of HER family members and EGFRvIII in patients with hepatocellular carcinoma

Abstract Hepatocellular carcinoma is one of the most common type of liver cancer. Despite advances in its treatment including the approval of several targeted agents in the past few decades, many patients do not respond or have a response of short duration to such therapeutic interventions. As a result, liver cancer is the third leading cause of cancer-related deaths worldwide. Therefore, it is essential to identify additional therapeutic targets and to develop more effective therapeutic interventions. In the past few decades, increased expression, and activation of the human epidermal growth factor receptor (HER) family members have been detected in various cancer types and of these, EGFR and HER2 are important therapeutic targets for the treatment of patients with a wide range of cancers, but currently not in liver cancer. In some studies, cross-talk between different members of the HER family has been associated with tumor progression and resistance to therapy with EGFR inhibitors but there has been no comprehensive study of the expression of all members of the HER family in patients with hepatocellular carcinoma. In this study, we examined the relative expression of all members of the HER family, type-III deletion mutant of EGFR (i.e.EGFRvIII), which is a tumor specific antigen, and two putative liver cancer cell biomarkers (i.e. CD44, EpCAM) in 43 patients with hepatocellular carcinoma by immunohistochemistry. The prognostic significance of such antigens was also investigated. Of these, the expression of EGFR, HER2, HER3, HER4 and EGFRvIII was found in tumor specimens from 35%, 58%, 0%, 19%, and 26% of the cases, respectively. Interestingly, at a cut off value of >5% of tumor cells with staining, 23%, 14% and 9% of the patients had co-expression of EGFR with HER2, EGFRvIII, and HER2/EGFRvIII. In addition, 40% and 33% of the cases were CD44 and EpCAM positive. Of these, the expression of EGFRvIII positivity at >5% ( HR=3.35, p=0.002) and 20% (HR=7.22, p=0.003) of tumour cells and membranous CD44 ( HR=2.12, p=0.04) were associated with a poorer survival in patients with hepatocellular carcinoma. In contrast, the expression of cytoplasmic CD44 was associated with a better overall survival (HR=0.30, p=0.02). These were found to be independent prognostic markers in multivariate analysis. Interestingly, the co-expression of EGFR with EGFRvIII was associated with a poorer overall survival (59.5 vs 21.0 months, p=0.004). Taken together, our results suggest that the expression of EGFRvIII occurs in patients with hepatocellular carcinoma and highlight its importance as biomarker for tumor progression and as a potential target for therapeutic intervention with drugs targeting EGFRvIII and other members of HER family. Citation Format: Ozlem Sherif, Said Khelwatty, Izhar Bagwan, Alan Seddon, Angus Dalgleish, Satvinder Mudan, Helmout Modjtahedi. Co-expression and prognostic significance of HER family members and EGFRvIII in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5152.

Abstract 3755: Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b

Abstract Background: Fibroblast growth factor receptor 2b (FGFR2b) is part of the FGFR transmembrane tyrosine kinase receptor family. This protein has gained more attention in recent years as a tumor progression biomarker in numerous solid tumors. The detection of FGFR2b by immunohistochemistry (IHC) has been used recently in immunotherapy clinical trials with multiple tumor types. During IHC assay development on formalin-fixed paraffin embedded tissue, a thorough study of antigen retrieval through cell conditioning or enzyme incubation, primary antibody incubation, and detection parameters is critical to evaluate staining performance. Design: A study of an investigational anti-FGFR2b (FPR2-D) assay developed on a BenchMark ULTRA automated staining platform explored the effect of antigen retrieval and detection parameters on staining performance. VENTANA System Software was used to modify steps in the IHC staining protocol resulting in 42 permutations. Ten different solid tumors were stained with these modifications to the assay and subsequently evaluated for FGFR2b expression. Result:s The results demonstrate that the staining clarity, intensity, and dynamic range within tissue cases of FGFR2b staining can vary widely between different solid tumor types. While most permutations of the initial assay parameters had minimal impact on the staining performance and readability by a pathologist, the order of the enzyme treatment and the cell conditioning steps (in addition to increased incubation time of primary antibody) had a significant impact when comparing across different solid tumors. Detection of FGFR2b seemed to have a greater susceptibility by tumor type to the antigen retrieval process than has traditionally been observed in other biomarkers. Conclusion: The overall results of this investigation show the importance of an extensive parameter screening approach when evaluating new antibody targets and indication combinations in IHC to ensure optimal staining performance. This is critical to downstream testing and the potential use of new assays in the context of in vitro diagnostics. Citation Format: Autumn Sky Watson, Catherine Le, Anne M. Wertheimer, Spencer Escobedo, Lisa So, Bhavani Bagevalu Siddegowda, John D. Palting, Susan Marion, Steven P. Stratton, Birte Aggeler. Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3755.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1566.

Risk Factors in Serrated Pathway Lesions: N-Glycosylation Profile as a Potential Biomarker of Progression to Malignancy

Introduction: The serrated pathway contributes to interval colorectal cancers, highlighting the need for new biomarkers to assess lesion progression risk. The β1,6-GlcNAc branched N-glycans expression in CRC cells was associated with an invasive phenotype and with immune evasion. Therefore, this study aims to identify potential risk factors for progression of serrated lesions (SLs) to malignancy, analyzing the N-glycosylation profile of epithelial/infiltrating immune cells. Methods: A retrospective cohort study was performed with data from 53 colonoscopies (48 patients). Sixty-three serrated pathway lesions (SPLs) were characterized based on N-glycosylation profile (lectin histochemistry/flow cytometry) and MGAT5 expression. Statistical analysis was performed to search for associations between the glycoprofile and clinical variables from each patient. Results: Increased β1,6-GlcNAc branched N-glycans expression in epithelial cells is found associated with age (p = 0.007 in SPL), smoking (p = 0.038 in SL), increased BMI (p = 0.036 in sessile serrated lesions [SSL]), and polyp dimensions ≥10 mm (p = 0.001 in SL), while increased expression of these structures on immune cells is associated with synchronous CA number (CD4⁺T cells: p = 0.016; CD8⁺T cells: p = 0.044 in SL) and female gender (p = 0.026 in SL). Moreover, a lower high-mannose N-glycans expression in immune cells is associated with smoking (p = 0.010 in SPL) and synchronous CA presence (p = 0.010 in SPL). Higher expression of these glycans is associated with female (p = 0.016 in SL) and male (p = 0.044 in SL) gender, left colon location (p = 0.028), dysplasia (p = 0.028), and adenocarcinoma (p = 0.010). Conclusions: We identified an association between an abnormal glycoprofile and several clinical risk factors, proposing the N-glycosylation profile as a potential biomarker of tumor progression in the serrated pathway. The N-glycosylation anatomopathological profile analysis could be further used to decide shorter interval follow-up in patients with SPL.

DNA methylation-mediated epigenetic regulation of oncogenic RPS2 as a novel therapeutic target and biomarker in hepatocellular carcinoma

Ribosomal Protein S2 (RPS2) has emerged as a potential prognostic biomarker due to its involvement in key cellular processes and its altered expression pattern in certain types of cancer. However, its role in hepatocellular carcinoma (HCC) has yet to be investigated. Herein, we analyzed RPS2 mRNA expression and promoter methylation in HCC patient samples and HepG2 cells. Subsequently, loss-of-function experiments were conducted to determine the function of RPS2 in HCC cells in vitro. Our results revealed that RPS2 mRNA expression is significantly elevated, and its promoter is hypomethylated in HCC patient samples compared to controls. In addition, 5-Azacytidine treatment in HepG2 cells decreased RPS2 promoter methylation level and increased its mRNA expression. RPS2 knockdown in HepG2 cells suppressed cell proliferation and promoted apoptosis. Functional pathway analysis of genes positively and negatively associated with RPS2 expression in HCC showed enrichment in ribosomal biogenesis, translation machinery, cell cycle regulation, and DNA processing. Furthermore, utilizing drug-protein 3D docking, we found that doxorubicin, sorafenib, and 5-Fluorouracil, showed high affinity to the active sites of RPS2, and in vitro treatment with these drugs reduced RPS2 expression. For the first time, we report on DNA methylation-mediated epigenetic regulation of RPS2 and its oncogenic role in HCC. Our findings suggest that RPS2 plays a significant role in the development and progression of HCC, hence its potential prognostic and therapeutic utility. Moreover, as epigenetic changes happen early in cancer development, RPS2 may serve as a potential biomarker for tumor progression.

Semaphorin 6 Family—An Important Yet Overlooked Group of Signaling Proteins Involved in Cancerogenesis

According to recent evidence, some groups of semaphorins (SEMAs) have been associated with cancer progression. These proteins are able to modulate the cellular signaling of particular receptor tyrosine kinases (RTKs) via the stimulation of SEMA-specific coreceptors, namely plexins (plexin-A, -B, -C, -D) and neuropilins (Np1, Np2), which share common domains with RTKs, leading to the coactivation of the latter receptors. MET, ERBB2, VEGFR2, PFGFR, and EGFR, among others, represent acknowledged targets of semaphorins that are often associated with tumor progression or poor prognosis. In particular, higher expression of SEMA6 family proteins in cancer cells and stromal cells of the cancer niche is often associated with enhanced tumor angiogenesis, metastasis, and resistance to anticancer therapy. Notably, high SEMA6 expression in malignant tumor cells such as melanoma, pleural mesothelioma, gastric cancer, lung adenocarcinoma, and glioblastoma may serve as a prognostic biomarker of tumor progression. To date, very few studies have focused on the mechanisms of transmembrane SEMA6-driven tumor progression and its underlying interplay with RTKs within the tumor microenvironment. This review presents the growing evidence in the literature on the complex and shaping role of SEMA6 family proteins in cancer responsiveness to environmental stimuli.

Tumor-Agnostic Circulating Tumor DNA Testing for Monitoring Muscle-Invasive Bladder Cancer

Circulating tumor DNA (ctDNA) has recently emerged as a real-time prognostic and predictive biomarker for monitoring cancer patients. Here, we aimed to ascertain whether tumor-agnostic ctDNA testing would be a feasible strategy to monitor disease progression and therapeutic response in muscle-invasive bladder cancer (MIBC) patients after radical cystectomy (RC). Forty-two MIBC patients who underwent RC were prospectively included. Blood samples from these patients were collected at different follow-up time points. Two specific mutations (TERT c.1-124C>T and ATM c.1236-2A>T) were analyzed in the patients’ plasma samples by droplet digital PCR to determine their ctDNA status. During a median follow-up of 21 months, 24% of patients progressed in a median of six months. ctDNA status was identified as a prognostic biomarker of tumor progression before RC and 4 and 12 months later (HR 6.774, HR 3.673, and HR 30.865, respectively; p < 0.05). Lastly, dynamic changes in ctDNA status between baseline and four months later were significantly associated with patient outcomes (p = 0.045). In conclusion, longitudinal ctDNA analysis using a tumor-agnostic approach is a potential tool for monitoring MIBC patients after RC. The implementation of this testing in a clinical setting could improve disease management and patients’ outcomes.

OR01-03 DNA Copy Number Profiles In Recurrent And Stable Non-functioning Pituitary Adenomas

Abstract Disclosure: G. Johannsson: None. M. Persson: None. S. Schlaffer: None. A. Fehr: None. C. Örndal: Employee; Self; Unilabs AB. W. Seager: None. M. Buchfelder: None. G. Stenman: None. D.S. Olsson: Consulting Fee; Self; Ipsen, Novo Nordisk, Sandoz. Grant Recipient; Self; Pfizer Global R&D. Employee; Self; AstraZeneca. Background: Residual tumors are common after primary surgery in patients with non-functioning pituitary adenoma (NFPA), 30-50% of these will experience tumor progression. Tumor control is of vital importance for patient outcome since tumor progression is strongly associated with excess morbidity and mortality. The aim of the study was to explore new potential biomarkers of tumor progression by studying the DNA copy number profiles of NFPAs with either a stable residual tumor behavior or marked tumor progression. Methods: In this case-controlled study, 72 patients operated for NFPA were selected. The progression group (n = 39) included patients [age at diagnosis (mean ± SD) 41.3±14.0 years] with marked tumor progression (30 patients required ≥3 surgical procedures). The stable group (n = 33; 61.1±10.2 years) had either a stable residual tumor without evidence of tumor progression during follow-up (n = 25) or no recurrence during follow-up (n = 8). The mean follow-up time in the stable group was 6.2 years (range 3.6-15 years). ArrayCGH analysis was performed on DNAs isolated from fresh frozen tumor tissue using the Human Genome CGH Microarray 244K and 180K oligonucleotide arrays. Copy number alterations (CNAs) were validated using FISH on formalin-fixed paraffin-embedded sections. Results: CNAs were identified in 29 tumors (40%). A total of 183 CNAs were detected in the 72 tumors, including 75 segmental alterations and 108 gains and losses of whole chromosomes and chromosome arms. The average number of CNAs per tumor was 2.5 (SD 5.9). CNAs were more common in the progression group compared to the stable group (Mean no. of CNAs 3.7 vs 1.2; P = 0.060). Twenty-four tumors (72%) in the stable group had normal profiles whereas only 19 (49%) in the progression group lacked CNAs. Eighteen CNAs were recurrent, defined as minimal common regions of deletion or gain in ≥4 tumors. The most frequently lost region was 11q21-q23.3 (n = 10). Deletions involving 10q23.21-q23.3 were seen in both the progression group (n = 5) and the stable group (n = 2) and included an approximately 2.8 Mb minimal common region harboring the tumor suppressor gene PTEN. The most frequent gains involved whole chromosomes 5 (n = 8), 7 (n = 10) and 12 (n = 7). Recurrent CNAs involving 10q (PTEN probe), 11q (MAML2 probe), and chromosomes 5 and 7 were confirmed using FISH. Conclusion: In this explorative study, we found a higher number of CNAs in NFPAs with marked tumor progression compared to stable adenomas without the need for reintervention during the long-term follow-up. Interestingly, segmental losses of chromosome 10q23.21-q23.3, including the tumor suppressor gene PTEN, were overrepresented in patients with marked tumor progression. Presentation: Thursday, June 15, 2023

CRISPR-Cas9 knockout screening identifies KIAA1429 as an essential gene in Ewing sarcoma

BackgroundEwing sarcoma (ES) is an aggressive childhood bone and soft tissue cancer. KIAA1429 is one type of N6-methyladenosine (m6A) writer that plays a tumor-progressive role in various cancers, but the role of KIAA1429 in ES remains to be elucidated. The aim of the study was to investigate the role of KIAA1429 in ES.MethodsWe performed a multi-omic screen including CRISPR-Cas9 functional genomic and transcriptomic approaches, and identified that KIAA1429 played a significant role in ES progression. Gene knockdown, quantitative real-time PCR (Q-RT-PCR), immunoblotting, CellTiter-Glo assays, clonogenic assays, a subcutaneous xenograft model and immunohistochemistry were used to assess the functional role of KIAA1429 in ES. We mainly conducted RNA sequencing (RNA-seq) in ES cells to analyze the downstream regulatory mechanism of KIAA1429. An integrative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq indicated the upstream regulatory mechanism of KIAA1429.ResultsIn vitro and in vivo CRISPR-Cas9 knockout screening identified KIAA1429 as an ES-dependent gene. Genetic suppression of KIAA1429 inhibited ES cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that KIAA1429 promotes ES tumorigenesis by regulating the ribosome-associated cell cycle and cancer-related inflammation. Interestingly, we found that STAT3 was a target of KIAA1429 and that a STAT3 inhibitor reduced KIAA1429 transcript levels, indicating positive feedback between KIAA1429 and STAT3. Finally, we found that NKX2-2 bound to the KIAA1429 promoter and transactivated KIAA1429.ConclusionOur study systematically analyzed ES-dependent epigenetic/transcriptional regulatory genes and identified KIAA1429 as a biomarker of tumor progression in ES, providing a potential therapeutic target for treating ES.

A convolutional neural network STIFMap reveals associations between stromal stiffness and EMT in breast cancer

Intratumor heterogeneity associates with poor patient outcome. Stromal stiffening also accompanies cancer. Whether cancers demonstrate stiffness heterogeneity, and if this is linked to tumor cell heterogeneity remains unclear. We developed a method to measure the stiffness heterogeneity in human breast tumors that quantifies the stromal stiffness each cell experiences and permits visual registration with biomarkers of tumor progression. We present Spatially Transformed Inferential Force Map (STIFMap) which exploits computer vision to precisely automate atomic force microscopy (AFM) indentation combined with a trained convolutional neural network to predict stromal elasticity with micron-resolution using collagen morphological features and ground truth AFM data. We registered high-elasticity regions within human breast tumors colocalizing with markers of mechanical activation and an epithelial-to-mesenchymal transition (EMT). The findings highlight the utility of STIFMap to assess mechanical heterogeneity of human tumors across length scales from single cells to whole tissues and implicates stromal stiffness in tumor cell heterogeneity.

Panel of miR-150 and linc00673, regulators of CCR6/CCL20 may serve as non-invasive diagnostic marker of non-small cell lung cancer

The C–C motif ligand 20 (CCL20) is a chemokine that specifically binds to the chemokine receptor 6 (CCR6) and the CCL20/CCR6 axis has been implicated in the non-small lung cancer (NSCLC) development and progression. Its expression is regulated by mutual interactions of non-coding RNAs (ncRNAs). This goals of presented study was to evaluate the expression level of CCR6/CCL20 mRNA in NSCLC tissue comparative to selected ncRNAs: miR-150, linc00673. The expression level of the studied ncRNAs was also assessed in serum extracellular vesicles (EVs). Thirty patients (n = 30) were enrolled as the study cohort. Total RNA was isolated from tumor tissue, adjacent macroscopically unchanged tissue and serum EVs. The expression level of studied genes and ncRNAs were estimated based on the qPCR method. Higher expression level of CCL20 mRNA but lower expression level of CCR6 mRNA were observed in tumor in comparison to control tissue. Relative to the smoking status, higher CCL20 (p < 0.05) and CCR6 mRNA (p > 0.05) expression levels were observed in current smokers than in never smokers. In serum EVs the expression level of miR-150 has a negative correlation with AJCC tumor staging, whereas the expression level of linc00673 positively correlated (p > 0.05). The lower expression level of miR-150 and higher expression level of linc00673 in serum EVs were observed in NSCLC patients with lymph nodes metastases (p > 0.05). Regarding the histopathological type, significantly lower expression level of miR-150 and higher expression level of linc00673 were observed in the serum EVs of patients with AC compared to patient with SCC. Our findings revealed that smoking significantly changed the expression level of CCL20 mRNA in NSCLC tissue. Changes in expression levels of miR-150 and linc00673 in the serum EVs of NSCLC patients in relation to presence of lymph node metastases and the stage of cancer development may serve as a non-invasive molecular biomarkers of tumor progression. Furthermore, expression levels of miR-150 and linc00673 may serve as non-intrusive diagnostic biomarkers differentiating adenocarcinoma from squamous cell carcinoma.

Clinical significance of long non-coding RNA ZEB2-AS1 and EMT-related markers in ductal and lobular breast cancer.

Breast cancer is considered the most prevalent type of cancer in women and accounts for a high rate of death. A body of research has demonstrated that lncRNAs have a regulatory function in human diseases, especially cancers. ZEB2-AS1 is known as an oncogenic lncRNA in various types of cancers, and its deregulation may contribute to cancer development and progression. Therefore, we aimed to reveal the association of ZEB2-AS1 expression with epithelial-mesenchymal transition (EMT) markers, as a hallmark of cancer progression, in a clinical setting. A recent study suggested that ZEB2-AS1 is significantly involved in EMT. Here we intended to explore the roles of lncRNA ZEB2-AS1 in breast cancer (BC) using bioinformatics tools and laboratory settings. We first evaluated the expression of ZEB2-AS1 mRNA in tumor and healthy control tissues by lnCAR database. Furthermore, ZEB2-AS1 expression level, ZEB2, E-cadherin, and vimentin was measured via qRT-PCR in 30 paired ductal and lobular carcinoma tissues from breast cancer patients and the normal adjacent ones. The correlation between the lncRNA ZEB2-AS1 expression and clinicopathological characteristics of the breast cancer patients was evaluated. ZEB2-AS1 showed an upregulation in breast cancer tissues (p = .04) compared to normal adjacent samples. In addition, its level was higher in breast cancer patients with advanced Stages (III & IV) (n = 18) compared to early Stages (I & II) (n = 12) (p = .04). Moreover, ZEB2 (p = .01) and vimentin (p = .02) expression were upregulated in the BC sample, but the expression level of E-cadherin (p = .02) was downregulated when compared with the adjacent normal tissues. By comparison of the expression of EMT-markers between different stages of breast cancer, overexpression of ZEB2 (p = .04) and vimentin (p = .04) and down expression of E-cadherin (p = .03) was observed in advance stages. Collectively, our findings suggest that ZEB2-AS1 expression is significantly upregulated in tumor tissues, especially in advanced stages and ZEB2-AS1 is associated with the aggressiveness of tumors by functioning as putative oncogenic lncRNA. In addition, a combination of ZEB2-AS1 and these EMT markers in breast cancer potentiates these genes as biomarkers for tumor progression.

Abstract 1265: Pan-cancer study of glycosylation-related gene expression: The emerging role of galectin-2 in tumor progression

Abstract Aberrant glycosylation has emerged as an important hallmark of cancer playing a critical role in the modulation of tumor cell aggressiveness, invasiveness, and modulation of the immune and vascular programs. However, the cellular and molecular mechanisms implicated in glycan-driven cancer progression remain poorly understood. In this study, we explored the expression profile of a glycosylation-related gene signature (Affymetrix, GlycoV4) and its association with cancer progression in tumors from different primary sites. First, we analyzed published transcriptomic data from patients’ tumor biopsies to identify pan-cancer molecular signatures associated with glycosylation-related genes including components of the glycosylation machinery and glycan-binding proteins or lectins, to determine unsupervised sample clusters with similar glyco-transcriptome profiles. Next, focusing on a melanoma transcriptomic database (TCGA-SKCM), a supervised machine learning algorithm based on Random Forest was applied to classify each biopsy according to its sample type (metastatic or primary tumor). Notably, we found that using 27 glycogenes out of the 570 included in the signature each biopsy could be classified with 87% accuracy as metastatic or primary tumor. These findings highlight the importance of components of the glycosylation machinery as novel biomarkers of tumor progression. To explore possible mechanisms underlying glycosylation changes, we focused on the study of lectins responsible for deciphering the biological information encoded by the glycome. Particularly, galectins, a family of endogenous lectins, are known to be involved in immunomodulation, inflammation, tumor escape, and metastasis. TCGA-SKCM data was used to study the implications of two galectins expression, galectin-2 (Gal-2) mostly unexplored, and galectin-1 (Gal-1), a known regulator of antitumor immune responses. In this dataset, we found that Gal-1 expression was associated with less survival, whereas higher expression of Gal-2 was linked to increased overall survival in melanoma patients. Furthermore, initial findings revealed that Gal-2 expression was significantly augmented in metastatic biopsies whereas Gal-1 was significantly increased in primary solid tumor biopsies. Finally, to study possible mechanisms underlying these correlations, we used an immunocompetent mouse melanoma model to challenge WT and Gal-2-deficient (Lgals2-/-) mice. Interestingly, tumor volume was significantly increased in Lgals2-/- mice (p&amp;lt;0.005) compared to their WT counterpart. To understand the molecular bases of Gal-2-driven tumor growth changes, we are studying the tumor microenvironment. Given the anti-inflammatory role of Gal-1 and the potential pro-inflammatory capacity of Gal-2, we propose that Gal-1 and Gal-2 may play opposing roles, regulating the outcome of tumor progression. Citation Format: Montana N. Manselle Cocco, Florencia Veigas, Yamil D. Mahmoud, Mora Massaro, Sabrina Gatto, Rosa M. Morales, Alejandro Cagnoni, Gabriel A. Rabinovich. Pan-cancer study of glycosylation-related gene expression: The emerging role of galectin-2 in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1265.

Abstract 5564: SerpinE2: A potential biomarker for urological cancers

Abstract SerpinE2, also known as protease nexin-1, is an extracellular serine protease inhibitor modulating the activity of serine proteases such as thrombin, plasmin, urokinase-type plasminogen activator and trypsin. Dysregulation between extracellular serine proteases and their cognate inhibitors has been implicated in malignant progression, making serpins promising biomarkers for tumor progression and prognosis. SerpinE2 is upregulated in many cancers and promotes tumor invasion and metastasis. In this study, we analyzed serpinE2 expression by immunohistochemistry on tissue microarrays comprising of samples from prostate, bladder and kidney cancers. The studied samples were clinically hard-to-risk stratify and thus relevant for biomarker studies. We aimed to study the association of serpinE2 expression with survival and clinicopathological features in all three cancers, and evaluate its potential as an immunohistochemical biomarker. In the case of bladder cancer, association of serpinE2 expression with response to neoadjuvant chemotherapy was also assessed. We found that although the strong expression of serpinE2 was related to more aggressive prostate cancers (p = 0.017) and high grade bladder cancers (p = 0.034), it was not associated with relapse, treatment response or survival of prostate and bladder cancer patients. In renal cancer, on the contrary, serpinE2 expression was significantly lower in patients with tumor relapse (p = 0.048) and high expression predicted favorable disease-specific survival (p = 0.013). Our findings help understanding the functional role of serpinE2 in cancer progression and guide the prognostic biomarker research of serpinE2, particularly for prediction of renal cancer-specific survival. Citation Format: Ruusu-Maaria Kovanen, Netta Koskinen, Timo-Pekka Lehto, Antti Rannikko, Tuomas Mirtti, Hannu Koistinen. SerpinE2: A potential biomarker for urological cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5564.

LBA03-11 CLINICAL UTILITY OF SERIAL CELL-FREE DNA ANALYSIS FOR MONITORING MUSCLE-INVASIVE BLADDER CANCER PATIENTS

You have accessJournal of UrologyCME1 Apr 2023LBA03-11 CLINICAL UTILITY OF SERIAL CELL-FREE DNA ANALYSIS FOR MONITORING MUSCLE-INVASIVE BLADDER CANCER PATIENTS Lourdes Mengual, Raquel Carrasco, Mercedes Ingelmo-Torres, Ascensión Gómez, Ramón Trullas, Fiorella L. Roldán, Tarek Ajami, Joan Sureda, Sandra Herranz, Antonio Alcaraz, and Laura Izquierdo Lourdes MengualLourdes Mengual More articles by this author , Raquel CarrascoRaquel Carrasco More articles by this author , Mercedes Ingelmo-TorresMercedes Ingelmo-Torres More articles by this author , Ascensión GómezAscensión Gómez More articles by this author , Ramón TrullasRamón Trullas More articles by this author , Fiorella L. RoldánFiorella L. Roldán More articles by this author , Tarek AjamiTarek Ajami More articles by this author , Joan SuredaJoan Sureda More articles by this author , Sandra HerranzSandra Herranz More articles by this author , Antonio AlcarazAntonio Alcaraz More articles by this author , and Laura IzquierdoLaura Izquierdo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003426.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cell-free DNA (cfDNA) has recently emerged as a real-time biomarker for diagnosis, monitoring and predicting therapy response of tumoral disease. Our aim was to evaluate cfDNA as a prognostic biomarker for monitoring muscle-invasive bladder cancer (MIBC) patients after radical cystectomy (RC) at different follow-up time points. METHODS: Blood samples from 37 MIBC patients who underwent RC and extended lymphadenectomy in our center were collected at cystectomy and one, four, 12 and 24 months later. cfDNA from plasma samples was quantified and fragmentation patterns were analyzed. Four mutations previously identified in BC tissue from these patients were studied in cfDNA samples by droplet digital PCR to identify presence of circulating tumor DNA (ctDNA) during patients’ follow-up. RESULTS: During a median follow-up of 36 months, 17 (46%) patients progressed and 14 (38%) died due to the BC. The median time to progression and cancer-specific survival (CSS) were 10 and 16 months, respectively. cfDNA level was significantly higher in progressive than non-progressive MIBC patients 12 months after RC (p=0.045). Of note, cfDNA level and ctDNA status four months after RC were identified as independent prognostic biomarkers of tumor progression (HR 5.290; p=0.033) and CSS (HR 4.199; p=0.038), respectively. By contrast, no correlation was found between cfDNA fragmentation and tumor progression nor CSS. ctDNA status detected tumor progression with a median anticipation period of 6 months compared with imaging techniques (p=0.024). Furthermore, ctDNA clearance four months after RC was significantly associated with patients’ clinical outcomes (p=0.033). CONCLUSIONS: cfDNA levels and ctDNA status four months after RC are prognostic biomarkers of tumor progression and CSS in MIBC patients. In addition, cfDNA monitoring is useful to predict patient outcomes after RC. The implementation of cfDNA analysis in the clinical setting could have an impact on disease management since patients could benefit from early treatment. Source of Funding: This work was supported by the Instituto de Salud Carlos III (ISCIII), project reference number PI17/01343 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1196 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lourdes Mengual More articles by this author Raquel Carrasco More articles by this author Mercedes Ingelmo-Torres More articles by this author Ascensión Gómez More articles by this author Ramón Trullas More articles by this author Fiorella L. Roldán More articles by this author Tarek Ajami More articles by this author Joan Sureda More articles by this author Sandra Herranz More articles by this author Antonio Alcaraz More articles by this author Laura Izquierdo More articles by this author Expand All Advertisement PDF downloadLoading ...