Cancer-associated Markers Research Articles

Establishment and characterization of a novel multidrug-resistant pancreatic ductal adenocarcinoma cell line, PDAC-X1

Drug resistance remains a significant challenge in the treatment of pancreatic cancer. The development of drug-resistant cell lines is crucial to understanding the underlying mechanisms of resistance and developing novel drugs to improve clinical outcomes. Here, a novel pancreatic cancer cell line, PDAC-X1, derived from Chinese patients has been established. PDAC-X1 was characterized by the immune phenotype, biology, genetics, molecular characteristics, and tumorigenicity. In vitro analysis revealed that PDAC-X1 cells exhibited epithelial morphology and cell markers (CK7 and CK19), expressed cancer-associated markers (E-cadherin, Vimentin, Ki-67, CEA, CA19-9), and produced pancreatic cancer-like organs in suspension culture. In vivo analysis showed that PDAC-X1 cells maintained tumorigenicity with a 100% tumor formation rate. This cell line exhibited a complex karyotype, dominated by subtriploid karyotypes. In addition, PDAC-X1 cells exhibited intrinsic multidrug resistance to multiple drugs, including gemcitabine, paclitaxel, 5-fluorouracil, and oxaliplatin. In conclusion, the PDAC-X1 cell line has been established and characterized, representing a useful and valuable preclinical model to study the underlying mechanisms of drug resistance and develop novel drug therapeutics to improve patient outcomes.

Pentagalloyl Glucose (PGG) Exhibits Anti-Cancer Activity against Aggressive Prostate Cancer by Modulating the ROR1 Mediated AKT-GSK3β Pathway.

Androgen-receptor-negative, androgen-independent (ARneg-AI) prostate cancer aggressively proliferates and metastasizes, which makes treatment difficult. Hence, it is necessary to continue exploring cancer-associated markers, such as oncofetal Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1), which may serve as a form of targeted prostate cancer therapy. In this study, we identify that Penta-O-galloyl-β-D-glucose (PGG), a plant-derived gallotannin small molecule inhibitor, modulates ROR1-mediated oncogenic signaling and mitigates prostate cancer phenotypes. Results indicate that ROR1 protein levels were elevated in the highly aggressive ARneg-AI PC3 cancer cell line. PGG was selectively cytotoxic to PC3 cells and induced apoptosis of PC3 (IC50 of 31.64 µM) in comparison to normal prostate epithelial RWPE-1 cells (IC50 of 74.55 µM). PGG was found to suppress ROR1 and downstream oncogenic pathways in PC3 cells. These molecular phenomena were corroborated by reduced migration, invasion, and cell cycle progression of PC3 cells. PGG minimally and moderately affected RWPE-1 and ARneg-AI DU145, respectively, which may be due to these cells having lower levels of ROR1 expression in comparison to PC3 cells. Additionally, PGG acted synergistically with the standard chemotherapeutic agent docetaxel to lower the IC50 of both compounds about five-fold (combination index = 0.402) in PC3 cells. These results suggest that ROR1 is a key oncogenic driver and a promising target in aggressive prostate cancers that lack a targetable androgen receptor. Furthermore, PGG may be a selective and potent anti-cancer agent capable of treating ROR1-expressing prostate cancers.

Abstract 4238: ModelingRET-rearranged non-small cell lung cancer (NSCLC): Generation of lung cell progenitors from patient-derived induced pluripotent stem cells (iPSCs)

Abstract Rearranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has been described yet. Patient-derived iPSCs hold great promise for disease modeling. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the somatic mutation RETC634Y which is commonly associated with hereditary medullary thyroid carcinoma. Additionally, we have established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-days protocol and showed an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Ymutation potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor Pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets. Citation Format: Paul Marcoux, Jinwook Hwang, Christophe Desterke, Jusuf Imeri, Annelise Bennaceur Griscelli, Ali Turhan. ModelingRET-rearranged non-small cell lung cancer (NSCLC): Generation of lung cell progenitors from patient-derived induced pluripotent stem cells (iPSCs) [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 4238.

Modeling RET-Rearranged Non-Small Cell Lung Cancer (NSCLC): Generation of Lung Progenitor Cells (LPCs) from Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

REarranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has yet been described. Patient-derived iPSCs hold great promise for disease modeling and drug screening. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the mutation RETC634Y, commonly associated with medullary thyroid carcinoma. Additionally, we established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-day protocol and detected an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Y mutation, potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel patient-derived off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets.

Decoding Hidden Messengers: Proteomic Profiling of Exosomes in Mammary Cancer Research.

Cancer is a complex and heterogeneous disease, influenced by various factors that affect its progression and response to treatment. Although a histopathological diagnosis is crucial for identifying and classifying cancer, it may not accurately predict the disease's development and evolution in all cases. To address this limitation, liquid biopsy has emerged as a valuable tool, enabling a more precise and non-invasive analysis of cancer. Liquid biopsy can detect tumor DNA fragments, circulating tumor cells, and exosomes released by cancer cells into the bloodstream. Exosomes attracted significant attention in cancer research because of their specific protein composition, which can provide valuable insights into the disease. The protein profile of exosomes often differs from that of normal cells, reflecting the unique molecular characteristics of cancer. Analyzing these proteins can help identify cancer-associated markers that play important roles in tumor progression, invasion, and metastasis. Ongoing research and clinical validation are essential to advance and effectively utilize protein biomarkers in cancer. Nevertheless, their potential to improve diagnosis and treatment is highly promising. This review discusses several exosome proteins of interest in breast cancer, particularly focusing on studies conducted in mammary tissue and cell lines in humans and experimental animals. Unfortunately, studies conducted in canine species are scarce. This emphasis sheds light on the limited research available in this field. In addition, we present a curated selection of studies that explored exosomal proteins as potential biomarkers, aiming to achieve benefits in breast cancer diagnosis, prognosis, monitoring, and treatment.

Laminin 332 expression levels predict clinical outcomes and chemotherapy response in patients with pancreatic adenocarcinoma.

Poor outcomes and chemotherapy resistance for patients with pancreatic adenocarcinoma (PAAD) are a challenge worldwide, and new or improved prognostic biomarkers are urgently required. Individual laminin family members have been established as cancer-associated markers, predicting patient outcomes in many cancer types, including PAAD. Here, we used multiple modalities including RNAseq and gene chip, and genomic and proteomic data to examine the relationships of all laminin genes in PAAD with clinical outcomes. These analyses identified that LAMA3, LAMB3, and LAMC2 expression levels are increased at the mRNA and protein levels in PAAD tumours with evidence of co-regulation. Increased expression of all three genes was associated with decreased promoter methylation status, TP53 mutations, and altered receptor tyrosine kinase (RTK) pathways. Clinically, high LAMA3, LAMB3, and LAMC2 transcript abundance was each related to an advanced histological grade. Moreover, high expression of these genes individually predicted poor patient survival, while a signature of combined high expression of LAMA3, LAMB3, and LAMC2 was a stronger predictor of patient outcomes than each gene alone. Interestingly, cell lines with high expression of LM332 chains were not sensitive to the commonly used PAAD chemotherapy drugs paclitaxel and gemcitabine; however, increased sensitivity was evident for erlotinib, afatinib, gefitinib, and cetuximab epidermal growth factor (EGFR) RTK inhibitors. To explore possible mechanisms, we investigated co-expressed genes, identifying eight hub genes, namely, GJB3, ITGB6, SERPINB5, GPRC5A, PLEK2, TMPRSS4, P2RY2, and TRIM29, which are co-expressed with all three of LAMA3, LAMB3, and LAMC2. Of these, only SERPINB5 provided a stronger predictive value than the laminin-encoding genes. Together, these multiple integrated analyses suggest that the combined expression of LM332 is a useful prognostic biomarker for PAAD and could help patient stratification and therapeutic selection.

Plasma extracellular vesicle phenotyping for the differentiation of early-stage lung cancer and benign lung diseases.

The development of a minimally invasive technique for early-stage lung cancer detection is crucial to reducing mortality. Phenotyping of tumor-associated extracellular vesicles (EVs) has the potential for early-stage lung cancer detection, yet remains challenging due to the lack of sensitive, integrated techniques that can accurately detect rare tumor-associated EV populations in blood. Here, we integrated gold core-silver shell nanoparticles and nanoscopic mixing in a microfluidic assay for sensitive phenotypic analysis of EVs directly in plasma without EV pre-isolation. The assay enabled multiplex detection of lung cancer-associated markers PTX3 and THBS1 and canonical EV marker CD63 by surface-enhanced Raman spectroscopy, providing a squared correlation coefficient of 0.97 in the range of 103-107 EVs mL-1 and a limit of detection of 19 EVs mL-1. Significantly, our machine learning-based nanostrategy provided 92.3% sensitivity and 100% specificity in differentiating early-stage lung cancer from benign lung diseases, superior to the CT scan-based lung cancer diagnosis (92.3% sensitivity and 71.4% specificity). Overall, our integrated nanostrategy achieved an AUC value of 0.978 in differentiating between early-stage lung cancer patients (n = 28) and controls consisting of patients with benign lung diseases (n = 23) and healthy controls (n = 26), which showed remarkable diagnostic performance and great clinical potential for detecting the early occurrence of lung cancer.

CD44V3, an Alternatively Spliced Form of CD44, Promotes Pancreatic Cancer Progression

Pancreatic cancer is one of the most lethal malignant tumors. However, the molecular mechanisms responsible for its progression are little known. This study aimed to understand the regulatory role of CD44V3 in pancreatic cancer. A Kaplan–Meier analysis was performed to reveal the correlation between CD44/CD44V3 expression and the prognosis of pancreatic cancer patients. CD44V3 and U2AF1 were knocked down using shRNAs. The proliferation, migration, invasion, and stemness of two pancreatic cell lines, BxPC-3 and AsPC-1, were examined. The expression of CD44V3, cancer-associated markers, and the activation of AKT signaling were detected by qRT-PCR and Western blot. Both CD44 and CD44V3 expression levels were associated with a poor prognosis in pancreatic cancer patients. Interestingly, the expression of CD44V3, instead of CD44, was greatly increased in tumor tissues. CD44V3 knockdown inhibited the proliferation, migration, invasion, and stemness of cancer cells. CD44V3 splicing was regulated by U2AF1 and downregulation of U2AF1 enhanced CD44V3 expression, which promoted pancreatic cancer progression. CD44V3 is an important cancer-promoting factor, which may serve as a potential candidate for pancreatic cancer intervention.

β-Sheet Richness of the Circulating Tumor-Derived Extracellular Vesicles for Noninvasive Pancreatic Cancer Screening

Tumor-derived extracellular vesicles (EVs) are under intensive study for their potential as noninvasive diagnosis biomarkers. Most EV-based cancer diagnostic assays trace supernumerary of a single cancer-associated marker or marker signatures. These types of biomarker assays are either subtype-specific or vulnerable to be masked by high background signals. In this study, we introduce using the β-sheet richness (BR) of the tumor-derived EVs as an effective way to discriminate EVs originating from malignant and nonmalignant cells, where EV contents are evaluated as a collective attribute rather than single factors. Circular dichroism, Fourier transform infrared spectroscopy, fluorescence staining assays, and a de novo workflow combining proteomics, bioinformatics, and protein folding simulations were employed to validate the collective attribute at both cellular and EV levels. Based on the BR of the tumorous EVs, we integrated immunoprecipitation and fluorescence labeling targeting the circulating tumor-derived EVs in serum and developed the process into a clinical assay, named EvIPThT. The assay can distinguish patients with and without malignant disease in a pilot cohort, with weak correlations to prognosis biomarkers, suggesting the potential for a cancer screening panel with existing prognostic biomarkers to improve overall performance.

Abstract PO-069: Modeling the tumor microenvironment using tissue engineering technologies

Abstract Tissue engineering technologies provide controllable and reproducible approaches to reconstruct the extracellular and cellular elements of pancreatic cancer. In a reductionist approach they allow the modelling of the complex tumor microenvironment (TME) and the study of disease biology and anti-cancer treatments. Our objective is that an engineered TME model, that mimics the tissue and matrix composition, architecture and cell types, will behave like a real tumor and is suitable for preclinical drug testing. Using biomimetic materials, we recreated the desmoplastic tissue characteristics of the pancreatic TME. Our natural and synthetic biomaterials were tailored to achieve the mechanical properties that resembled the stiffness and viscoelasticity of patient-derived tissues, providing a supportive cell-matrix interface for 3D cell culture conditions. Pancreatic cancer cells grown embedded in the matrix scaffolds formed tumor spheroids. Cell-based assays and microscopic analysis indicated a high cell viability and proliferation of the tumor spheroids as well as the expression of cancer-associated markers. Incorporation of cancer-associated fibroblasts and myeloid cells led to a multicellular 3D systems and matrix stiffening due to the secretion of extracellular matrix proteins. Transcriptomic analysis of the 3D cell cultures identified differentially regulated pathways related to cell proliferation, mechano-transduction and the secretion of pro-inflammatory cytokines, indicative of a malignant behavior. Treatment with mechano-modulating inhibitors and anti-cancer compounds increased the efficacy of chemotherapeutics, thereby reducing matrix stiffness and the release of cytokines. Our engineered TME model provides an easily translatable technology to ease the burden of pancreatic cancer, allowing us to characterize combination treatments that slow down or reduce tumor growth. Citation Format: Rodrigo Curvello, Verena Kast, Daniela Loessner. Modeling the tumor microenvironment using tissue engineering technologies [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-069.

Dye-free spectrophotometric measurement of nucleic acid-to-protein ratio for cell-selective extracellular vesicle discrimination

Extracellular vesicles (EVs) can represent a novel source of disease biomarkers, and are under intensive study for their clinical potential. Most EV-based cancer diagnostic studies have focused on establishing EV assays that detect increased expression of a single cancer-associated marker or marker signatures based on multiplex detection of these biomarkers. EV biomarker readouts can be obscured by high background signal leading to false positives, and may markedly differ between analyses due to variation in sample purity during EV isolation. This can obstruct the comparisons among studies and lead to conflicting conclusions. This work reports that the nucleic acid to protein UV absorption ratio of an EV is a cell-specific EV characteristic. This EV collective attribute can be measured at low-cost to discriminate EVs derived from malignant and non-malignant cells rather than employing single markers that may be cancer- or subtype-specific. Our work also highlighted the application for accessing purity in EV preparations irrelevant to EV yield. It can be employed to distinguish from patients with and without malignant disease upon analysis of EVs isolated from their serum samples.

The prognostic value of combining CD133 and mismatch repair proteins in patients with colorectal cancer.

The prognostic value of cancer stem cells (CSCs) is a hot topic in colorectal carcinoma (CRC) research. CD133 has been identified as an important colorectal CSC marker, but its prognostic significance remains controversial. Recently, studies have reported a possible functional link between CSCs and DNA mismatch repair (MMR) system. However, the relationship between CRC stemness and MMR proteins remains little explored, and whether the predictive role of CD133 is affected by MMR proteins is still unknown. The aim of our study is to investigate the influence of MMR proteins on the predictive significance of CD133 in terms of CRC patient survival and to further analyze the correlation between MMR proteins and cancer stemness. In our study, we didn't observe the prognostic value of CD133 in CRC patients. However, we demonstrated that in patients with low expression of MSH6, MSH2, PMS2 and MLH1, especially MSH6, CD133 was an effective prognostic biomarker. Moreover, correlation analysis revealed a positive correlation between MSH6 and CD133 expression. In vitro studies supported our clinical data and showed that the expression of cancer-associated stemness markers CD133, BMI-1, OCT-4 and SOX-2 was significantly decreased in siRNA-MSH6/MLH1 CRC cells. Thus, our results demonstrated that MMR proteins might play an important role in modulating the stemness of CRC cells. MMR proteins might be a crucial determinant that can help to accurately identify tumour subclones that may benefit from using the CSC marker CD133 as a prognostic marker.

α‑enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

The expression levels of α-enolase, also known as enolase 1 (ENO1), in liver cancer tissues and the autoantibody levels of ENO1 in the sera of patients with liver cancer were detected to investigate the function of ENO1 in the invasion and metastasis of liver cancer, as well as its clinical diagnostic value. Small interfering RNA (siRNA) was used to disrupt ENO1 gene expression in HepG2 and Huh7 liver cancer cells. The proliferation ability of liver cancer cells was assessed using Cell Counting Kit-8 (CCK-8); the migration ability of liver cancer cells was assessed using scratch tests; and the migration and invasion abilities of liver cancer cells were assessed using Transwell assays. ENO1 expression in liver cancer tissues (43.8%) was significantly higher than that in benign liver lesions (15.2%) (P=0.005). The serum anti-ENO1 antibody levels in the liver cancer group were significantly higher than those in the control and benign liver lesion groups (P<0.001). After ENO1 gene interference, the proliferation, migration and invasion abilities of HepG2 and Huh7 liver cancer cells exhibited different degrees of suppression. The results revealed that ENO1 promotes liver cancer invasion and metastasis; ENO1 plays an important role in liver cancer and can be used as a potential liver cancer-associated marker.

A novel model of liver cancer stem cells developed from induced pluripotent stem cells

BackgroundLiver cancer is the second most common cause of cancer-related death. Every type of tumours including liver cancer contains cancer stem cells (CSCs). To date, the molecular mechanism regulating the development of liver CSCs remains unknown.MethodsIn this study, we tried to generate a new model of liver CSCs by converting mouse induced pluripotent stem cells (miPSCs) with hepatocellular carcinoma (HCC) cell line Huh7 cells conditioned medium (CM). miPSCs treated with CM were injected into the liver of BALB/c nude mice. The developed tumours were then excised and analysed.ResultsThe primary cultured cells from the malignant tumour possessed self-renewal capacity, differentiation potential and tumorigenicity in vivo, which were found rich in liver cancer-associated markers as well as CSC markers.ConclusionsWe established a model of liver CSCs converting from miPS and showed different stages of stemness during conversion process. Our CSC model will be important to assess the molecular mechanisms necessary to develop liver CSCs and could help in defeating liver cancer.

Relationship between epithelial cell adhesion molecule and wingless/integrated-β-catenin signaling in colon cancer and the effect of XAV939 on the expression of related markers

Objective To investigate whether activation of Wingless/integrated-β-catenin (Wnt-β-catenin) pathway regulates epithelial cell adhesion molecule (EpCAM) expression in colon cancer and analyze the effect of XAV939 on colon cancer-associated markers. Methods After Wnt-β-catenin signaling was activated, relative expression levels of related genes were determined by quantitative real-time polymerase chain reaction (RT-qPCR). After constructing a mouse colon cancer model, XAV939 and/or 5-Fu were intraperitoneally injected for 1 week. Detection of related molecules expression in colon tissue by Western blotting. Results Compared with the control group, LiCl could significantly stimulate the expression of Wnt-β-catenin signaling pathway genes, and the levels of TACSTD1, DKK1, BAMBI, CCND1 and MYC genes were significantly increased by 1.20-, 2.44-, 4.34-, 2.57-, 1.58 fold (t=12.179, 20.661, 54.378, 19.384, 20.341, P<0.05), respectively. ZnCl2 treatment significantly inhibited the expression of TACSTD1 and CCND1 in HCT116 cells (tTACSTD1=36.467, 9.682, tCCND1=16.130, 17.003, P<0.05), which were down-regulated by about 50.06% and 34.38%, respectively. In the APC gene-deficient cells, the inhibitory effect of ZnCl2 on TACSTD1 and CCND1 was reversed. XAV939 and XAV939+ 5-fluorouracil (5-Fu) treatment significantly up-regulated the expression of Axin in HCT116 cells, which was about 15.97 and 17.2 fold higher than that of the control group (t=19.502, 18.236, P<0.05); and expression of β-catenin, EpCAM, telomerase reverse transcriptase (TERT) and bicorticosteroids and calmodulin dependent protein kinase-like 1 (DCAMKL-1) were down-regulated by 0.75 and 0.72 fold, 0.68 and 0.25 fold, 0.74 and 0.73 fold, 0.27 and 0.34 fold (tβ-catenin=20.412, 22.862, tEpCAM=26.336, 27.111, 82.711, tTERT=19.636, 22.787, 23.678, tDCAMKL-1=81.637, 64.814, P<0.05), respectively. Conclusion XAV939 inhibits tumor cell proliferation by blocking Wnt-β-catenin signaling down-regulation of β-catenin, target gene EpCAM and other related factors of CSC to promote colon cancer prognosis. Key words: Wingless/Integrated-β-catenin pathway; Epithelial cell adhesion molecule; XAV939; Colon cancer

Aberrant expression of genes associated with stemness and cancer in endometria and endometrioma in a subset of women with endometriosis.

Is there molecular evidence for a link between endometriosis and endometriosis-associated ovarian cancers (EAOC)? We identified aberrant gene expression signatures associated with malignant transformation in a small subgroup of women with ovarian endometriosis. Epidemiological studies have shown an increased risk of EAOC in women with ovarian endometriosis. However, the cellular and molecular changes leading to EAOC are largely unexplored. CD73+CD90+CD105+ multipotent stem cells/progenitors (SC cohort) were isolated from endometrium (n = 18) and endometrioma (n = 11) of endometriosis patients as well as from the endometrium of healthy women (n = 14). Extensive phenotypic and functional analyses were performed in vitro on expanded multipotent stem cells/progenitors to confirm their altered characteristics. Aberrant gene signatures were also validated in paired-endometrium and -endometrioma tissue samples from another cohort (Tissue cohort, n = 19) of endometriosis patients. Paired-endometrial and -endometriotic biopsies were obtained from women with endometriosis (ASRM stage III-IV) undergoing laparoscopic surgery. Control endometria were obtained from healthy volunteers. Isolated CD73+CD90+CD105+ SC were evaluated for the presence of known endometrial surface markers, colony forming efficiency, multi-lineage differentiation, cell cycle distribution and 3D-spheroid formation capacity. Targeted RT-PCR arrays, along with hierarchical and multivariate clustering tools, were used to determine both intergroup and intragroup gene expression variability for stem cell and cancer-associated markers, in both SC+ and tissue cohorts. Isolated and expanded SC+ from both control and patient groups showed significantly higher surface expression of W5C5+, clonal expansion and 3D-spheroid formation capacity (P < 0.05) compared with SC-. The SC+ cells also undergo mesenchymal lineage differentiation, unlike SC-. Gene expression from paired-endometriosis samples showed significant downregulation of PTEN, ARID1A and TNFα (P < 0.05) in endometrioma compared with paired-endometrium SC+ samples. Hierarchical and multivariate clustering from both SC+ and tissue cohorts together identified 4 out of 30 endometrioma samples with aberrant expression of stem cell and cancer-associated genes, such as KIT, HIF2α and E-cadherin, altered expression ratio of ER-β/ER-α and downregulation of tumour suppressor genes (PTEN and ARID1A). Thus, we speculate that above changes may be potentially relevant to the development of EAOC. N/A. As the reported frequency of EAOC is very low, we did not have access to those samples in our study. Moreover, by adopting a targeted gene array approach, we might have missed several other potentially-relevant genes associated with EAOC pathogenesis. The above panel of markers should be further validated in archived tissue samples from women with endometriosis who later in life developed EAOC. Knowledge gained from this study, with further confirmation on EAOC cases, may help in developing screening methods to identify women with increased risk of EAOC. The study is funded by the Swedish Research Council (2012-2844), a joint grant from Stockholm County and Karolinska Institutet (ALF), RGD network at Karolinska Institutet, Karolinska Institutet for doctoral education (KID), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695), Horizon 2020 innovation program (WIDENLIFE, 692065), European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways funding (IAPP, SARM, EU324509) and MSCA-RISE-2015 project MOMENDO (691058). All authors have no competing interest.

Abstract 2959: Analysis of circulating tumor DNA reveals genomic alterations in metastatic prostate cancer patients treated with abiraterone acetate plus prednisone or enzalutamide

Abstract Background: Recent progress in the analysis of cell-free circulating tumor DNA (ctDNA) now allows for the monitoring of tumor genomes by noninvasive means. The introduction of abiraterone and enzalutamide for the treatment of metastatic castration-resistant prostate cancer (mCRPC) patients has led to a survival benefit. We analyzed genomic alterations as potential causes of disease progression using ctDNA in patients treated with abiraterone and enzalutamide. Furthermore, we investigated the correlation between cancer-associated clinical markers and the ctDNA detection in mCRPC patients. Patients and Methods: In order to identify variables associated with ctDNA detection, we analyzed two cohorts including 94 plasma samples from 25 treatment courses (23 patients) and 334 plasma samples from 125 patients, respectively. We performed low-pass whole-genome sequencing (plasma-Seq) for genome-wide profiling of somatic copy number alterations (SCNAs) and targeted resequencing of 31 prostate cancer-associated genes. Results: Applying plasma-Seq with targeted AR analyses we were able to identify significant genomic alterations in 16 of 25 (64%) cases in the first cohort. Our results indicated that AR amplification alone is not predictive for abiraterone and enzalutamide treatment outcome. In patients treated with abiraterone, low ctDNA levels at baseline were a significant determinant of progression-free survival. Next, we evaluated the determinants of ctDNA detection in an additional independent cohort of metastatic CRPC patients. Analysis of a total of 148 patients and 428 plasma samples from both cohorts revealed that increased levels of lactate dehydrogenase (LDH) highly correlated with the detection of ctDNA and could be used as the potential independent predictor of ctDNA release. Conclusion: Our results showed that AR amplification alone is an insufficient marker for the prediction of therapy response in patients treated with abiraterone and enzalutamide. Analyses of ctDNA in two independent cohorts have provided insights into unique patterns of response and the emergence of resistance to abiraterone and enzalutamide. Noninvasive analysis of ctDNA plays an important role in treatment monitoring and the prediction of therapy outcome in mCRPC patients. Citation Format: Jelena Belic, Ricarda Graf, Thomas Bauernhofer, Yauheniya Cherkas, Ulz Peter, Julie Waldispuehl-Geigl, Samantha Perakis, Michael Gormley, Jaymala Patel, Weimin Li, Jochen B. Geigl, Denis Smirnov, Ellen Heitzer, Mitchell Gross, Michael R. Speicher. Analysis of circulating tumor DNA reveals genomic alterations in metastatic prostate cancer patients treated with abiraterone acetate plus prednisone or enzalutamide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2959.

RKIP and peroxiredoxin 2 expression predicts the proliferative potential of gastric cancer stem cells.

Gastric cancer is associated with a high mortality rate, with the development of gastric cancer stem cells underlying this. Gastric cancer stem cells are responsible for tumor initiation, progression and recurrence. However, the link between gastric cancer and gastric cancer stem cells remains to be fully understood. Murine models mimic a human microenvironment more accurately than in vitro studies and are useful models for understanding the behavior of different markers. The present study compared the expression of cluster of differentiation 44 (CD44), a stem cell marker, with the expression of other cancer-associated markers, including Raf kinase inhibitor protein (RKIP) and peroxiredoxin 2, in different pathological conditions of gastric cancer development using histological, immunohistological and western blot analyses. Initially, the murine model of gastric cancer was established using N-methyl-N-nitrosourea, a chemical carcinogen. Following initiation of cancer, immunohistochemistry was used to compare the expression of CD44, RKIP and peroxiredoxin 2 at different stages of cancer development. The results suggested CD44 and peroxiredoxin 2 expression was upregulated as the tumor progressed. However, expression of RKIP, a metastasis suppressor, was elevated in the initial stage of gastric cancer and suppressed during the aggressive stages. In agreement with previous data suggesting higher expressions of RKIP in the initial stages of cancer and its downregulation in the advanced stage, the results of the present study revealed that RKIP exhibited a negative effect on initial tumor development, and that the downregulation of RKIP in the advanced stages of cancer facilitated CD44 and peroxiredoxin 2 overexpression.

Abstract 1719: Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels

Abstract Introduction: Liquid biopsy based on sub-micron or nanosized particles in human body fluid have been received vast attention due to their non-invasive characteristics and enabling multiple check-up. Circulating tumor cells (CTCs), as well as exosome are the most promising markers in liquid biopsy, however, dual isolation and profiling have been hampered due to their size difference and limited quantity for analysis. We proposed the novel and simple methods for both isolation and study their similarity between them. Using the label-free CTC filtration device and anti-CD63 antibody-conjugated degradable hydrogel, the CTCs and the CTCs-derived exosome are specifically isolated, and each samples were followed by molecular study after recovery. This versatile platform facilitates the comprehensive study of two biomarkers with reflecting their inherent characteristics, thus paving the way for revealing their roles in cancer progression and metastasis. Methods: In order to make stimuli-responsive degradable hydrogel, poly (vinyl alcohol) and alginate were mixed under constant stirring at 85 °C. The mixture was poured into the mold and dried for 24 hours. Then, the dried sheet was immersed into 100 mM calcium chloride solution to achieve gelation through ionic interaction. Subsequently, the anti-CD63 antibody was immobilized onto the prepared hydrogels via cross-linking. For the dual-profiling, the hydrogel and the filters containing the captured breast cancer cells by microfiltration were incubated with the exosome-depleted cell culture media for 6 hours. The captured cells were released from the device and the hydrogels were degraded by adding EDTA. The cell and exosome lysate were prepared using RIPA buffer at 4 °C. The supernatant was collected by centrifugation followed by western-blot assay. Four different markers, including exosome-specific marker (CD63), cancer-associated markers (EpCAM, vimentin), and a housekeeping marker (β-actin), were used. Results: All exosome and cell samples highly expressed the housekeeping marker. Especially, two exosome samples dissociated from the hydrogel showed CD63 predominantly, which support the secretion of exosome from the cancer cells. The samples from the released cancer cells from the device did not express CD63 remarkably. To verify the phenotypical similarity between cell and exosome, expressions of the epithelial marker (EpCAM) and mesenchymal marker (vimentin) were examined. The exosome and cell from MCF-7, epithelial cancer cell, showed higher expression of EpCAM then vimentin. On the contrary to this, the samples from MDA-MB-231, mesenchymal cell, showed higher vimentin expression then EpCAM. Discussion and conclusion: We showed that exosome follow the phenotypical characteristics of mother-cells. This dual profiling would be helpful for in-depth study of cancer with consideration of its heterogeneity and complexity. Citation Format: Yoon-Tae Kang, Young Jun Kim, Tae Hee Lee, Jae-Eul Shim, Young-Ho Cho. Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1719. doi:10.1158/1538-7445.AM2017-1719

Abstract 1764: Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories

Abstract Introduction: Reliable assessment of cancer-associated RNA markers in lung cancer produced by gene-fusions or exon skipping events by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We evaluated a comprehensive system for targeted RNA-Seq that includes reagents for nucleic acid quantification, library prep, run controls, and companion bioinformatics software. The reproducibility of this system was evaluated in a multi-phase study design at 5 independent laboratories. Methods: Total nucleic acid (TNA) was isolated from formalin-fixed, paraffin-embedded (FFPE) residual non-small cell lung cancer (NSCLC) tumor biopsies and cell-lines (RT-112, H596, HCC78). These TNA isolates were used to prepare a set of 30 test samples, including a dilution series to assess assay sensitivity. A non-template control and kit positive control were also included. The sample set was evaluated using the QuantideX® NGS RNA Lung Cancer Kit RUO (Asuragen) and sequenced on the MiSeq® system (Illumina) at Asuragen and 4 independent laboratories. Analyses were conducted using QuantideX® NGS Reporter RUO (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information into the fusion-caller algorithm and reporting tool. Results: Laboratories were trained on the assay workflow and companion bioinformatics software in less than two days followed by independent library preparation and sequencing workflows. A total of 266 sample libraries were evaluated from inputs down to &amp;lt;10 ng. A single library was excluded from sequencing due to a failed QC status. Specific targeted fusions (ALK, ROS1, FGFR3) and splice variants (MET exon 14 skipping) were detected in 132 libraries and were concordant at all sites. Designs to detect 3’/5’ expression imbalances reported the presence of gene fusions for ALK and ROS1 in a total of 96 libraries with one aberrant call and one missed call, which occurred in libraries flagged as “at risk” by the interpretive software. Conclusions: The accuracy of this novel targeted NGS assay for RNA fusions and splice variants in NSCLC was demonstrated in a multi-site laboratory evaluation using clinically-relevant specimens and low inputs of TNA. The ability of the panel to detect both common and rare gene fusion transcripts and exon skipping events within an integrated wet- and dry-bench workflow provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that can respond to current and emerging targeted therapies. This study highlighted the ease of implementation and consistent performance that can be achieved in different laboratories when the process from sample-to-report is highly integrated. Citation Format: Gary J. Latham, Richard Blidner, Brian C. Haynes, Shobha Gokul, Maria L. Aguirre, Stephen Hyter, Ziyan Y. Pessetto, Maria Curtis, Dan Su, Tom Halsey, Victor Weigman, Patrick Hurban, Andrew K. Godwin, Leon C. van Kempen. Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2017-1764