Original Tumor Tissue Research Articles

Abstract 6016: Patient-derived tumor organoid cultures identify a chemo-resistant sub-population in esophageal squamous cell carcinoma

Abstract Background: Patient-derived xenograft has been a gold standard for cancer modeling. However, low establishment efficiency and high maintenance cost have hindered the development in the field. In recent years, patient-derived tumor organoid culture has been developed as an efficient and economic model to mimic and recapitulate the original tumor tissue, in contrast to conventional cancer cell lines and patient-derived xenografts (Drost & Clevers, 2018). Due to the limited availability of current esophageal squamous cell carcinoma (ESCC) models, we aimed to establish a panel of ESCC patient-derived organoid (PDO) long-term cultures for ESCC study. Results: In the current project, we have established and biobanked a panel of ESCC PDO cultures in vitro from Chinese ESCC treatment-naïve patients undergoing an endoscopic examination or upfront surgery, or patients undergoing neoadjuvant chemoradiotherapy (CRT) followed by surgery in Hong Kong. We also established and maintained PDO-derived xenograft (PDOX) in immunocompromised mice. Immunohistochemical examination confirmed the squamous origin of the established PDOs and PDOXs. Genomic profiling showed consistent mutational landscapes of the established PDO, as compared to ESCC patient profiles. Transcriptomic profiling suggests organoid culture resembles patient tissue samples better than the cell line model in terms of global gene expression patterns. Notably, PDOs from CRT-treated patients show enhanced re-growth after recovery from cisplatin treatment, indicating a candidate cancer stem cell (CSC) sub-population enriched by neoadjuvant CRT. This sub-population can be further enriched in vitro by serial cisplatin treatment and recovery cycles. Conclusions: ESCC PDOs can be efficiently established from highly limited patient tissue samples and maintained long-term in vitro and can be feasibly integrated with mouse model. It preserves the original cell hierarchy and indicates existence of candidate CSC sub-population, which may be targeted therapeutically in future. Reference: Drost J, Clevers H. Organoids in cancer research. Nat Rev Cancer. 2018 Jul; 18(7). Acknowledgment: We acknowledge the Research Grants Council (Theme-based Research Scheme T12-701/17-R to MLL) and the Food and Health Bureau (Health and Medical Research Fund 06171566 to VZY) of Hong Kong Special Administrative Region for funding supports. We acknowledge DSMZ (German Collection of Microorganisms and Cell Culture) for the KYSE cell lines. We acknowledge the University of Hong Kong Faculty of Medicine Core Facility for providing imaging facilities. Citation Format: Valen Z. Yu, Bryan C. Lung, Ian Y. Wong, Claudia L. Wong, Simon Y. Law, Maria L. Lung. Patient-derived tumor organoid cultures identify a chemo-resistant sub-population in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6016.

Abstract 3092: Urine-derived urinary carcinoma organoids: A novel tool for providing new insights into human and canine bladder cancer treatment

Abstract The outcome of patients with Muscle Invasive Bladder Cancer (MIBC) is highly variable with many patients experiencing progressive disease despite multimodal therapy. A key variable that contributes to poor patient outcomes lies in our inability to predict therapeutic response due to the molecular heterogeneity of MIBC tumors. There is, therefore, a significant unmet clinical need to accurately predict effective drug combinations and therapeutic doses in individual patients with MIBC. Patient-derived tumor organoids (PDOs) have the potential to serve as an ex vivo model of urothelial carcinoma (UC) capable of functionally predicting patient-specific treatment responses. However, current culture procedures typically rely on the expansion of UC organoids from resected tumors or biopsy specimens, which is inherently invasive and hence limits clinical application. We hypothesized that culture of MIBC PDOs can be achieved from urine samples, as is the case in dogs with MIBC, since histopathological features, tumor heterogeneity and subtypes, metastatic activity, and gene expression profiles of canine MIBC mimic those of human MIBC. Herein, we successfully cultured MIBC organoids from both human and canine patients using first-catch urine. Briefly, after centrifuging urine samples to pellet cells, the cells were washed with basal culture media containing FBS and Pen/Strep before being cultured in canine or human culture media containing essential growth factors based on their species of origin. Paraffin-embedded samples were used for molecular characterization by RNA in situ hybridization or immunohistochemistry. Organoids from canine MIBC patients were analyzed for expression of the urothelial differentiation marker (FOXA1) associated with high grade bladder cancer, CD44, a non-kinase transmembrane glycoprotein expressed in UC cancer stem cells, and CK7, an epithelial cell marker that is upregulated in neoplastic UC cells. FOXA1 expression was undetectable in canine MIBC, although CD44 expression was enhanced compared to healthy dog bladder organoids. All canine MIBC organoids had significantly enhanced CK7 expression. Similarly, in human-derived UC organoids, GATA binding protein 3 (GATA3), transcription factor paired box 8 (Pax8), and tumor protein p63 were utilized as markers, with a Pax8-/p63+/GATA3+ profile indicating UC origin of the organoids. Functional response to chemotherapy (cisplatin and gemcitabine) was further assessed using cell viability assay in canine and human UC organoids. Our data suggest that organoids cultured from the urine of patients with MIBC can recapitulate organ structures and maintain marker expression profiles of the original tumor tissues. 3D organoids may provide novel insights to predict therapeutic response to chemotherapy and serve as early diagnostic biomarkers of bladder cancer in both canine and human patients. Citation Format: Dipak Kumar Sahoo, Allison P. Mosichuk, Fabrice Lucien, Igor Frank, John C. Cheville, Karin Allenspach, Jonathan P. Mochel. Urine-derived urinary carcinoma organoids: A novel tool for providing new insights into human and canine bladder cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3092.

Patient-Derived Organoid Model in the Prediction of Chemotherapeutic Drug Response in Colorectal Cancer.

As an emerging technology in precision medicine, the patient-derived organoid (PDO) technology has been indicated to provide novel modalities to judge the sensitivity of individual tumors to cancer drugs. In this work, an in vitro model of colorectal cancer (CRC) was established using the PDO culture, and it is demonstrated that the PDO samples preserved, to a great extent, the histologic features and marker expression of the original tumor tissues. Subsequently, cancer drugs 5-FU, oxaliplatin, and irinotecan were selected and screened on five CRC PDO samples, while the patient-derived organoid xenograft (PDOX) model was applied for comparison. The receiver operating characteristic (ROC) curve was drawn according to the IC50 data from the PDO model and the relative tumor proliferation rate (T/C%) from PDOX. Interestingly, the area under the ROC curve was 0.84 (95% CI, 0.64-1.04, P value = 0.028), which suggested that the IC50 of cancer drugs from the PDO model was strongly correlated with PDOX responses. In addition, the optimal sensitivity cutoff value for drug screening in CRC PDOs was identified at 10.35 μM, which could act as a reference value for efficacy evaluation of 5-FU, oxaliplatin, and irinotecan in the colorectal cancer drug screening. Since there are no unified criteria to judge the sensitivity of drugs in vitro, our work provides a method for establishing in vitro evaluation criteria via PDO and PDOX model using the patient tissues received from local hospitals, exhibiting potential in clinical cancer therapy and precision medicine.

Establishment and Characterization of Advanced Penile Cancer Patient-derived Tumor Xenografts: Paving the Way for Personalized Treatments

BackgroundSystemic treatments for penile squamous cell carcinoma (pSCC) are toxic and inefficient. Patient-based preclinical models are essential to study novel treatments. ObjectiveTo establish a library of patient-derived tumor xenograft (PDX) models of human papillomavirus-positive (HPV+) and -negative (HPV−) pSCC and characterize these at the genomic and histological levels. Design, setting, and participantsEighteen tumor samples from 14 patients with recurrent or metastatic pSCC were implanted in nude mice. A biobank of PDX tumors was established after passaging of patient samples (F0) for three generations (F1, F2, F3) and was characterized using histopathology and targeted next-generation sequencing (tNGS). Single-nucleotide polymorphism fingerprinting was used to confirm PDX genealogy. Outcome measurements and statistical analysisThe engraftment rate, overall growth rate, and pSCC histomorphology were checked for each PDX generation. Staining for p40 (a pSCC marker) and p16 (a surrogate for HPV infection) was performed for F0 samples. The mutational profile according to a validated panel of 96 cancer genes was determined for F0 and F3 samples and compared to a larger tNGS database. Results and limitationsIncluding a previously established pilot model, 11 out of 18 tumor samples (61%) successfully engrafted in F1. The mean time from implantation in F1 to completion of F3 was 36 wk (standard deviation 18). Histological fidelity was demonstrated across generations. The patient mutational profiles were preserved in F3 and were representative of 277 pSCC samples in the Foundation Medicine database. The rapid progression of pSCC in patients from our selected high-risk cohort impeded the use of PDXs as avatars. ConclusionsWe successfully established the first library of 11 PDX models of HPV− and HPV+ pSCC. Our PDX models showed high engraftment rates and histological and genomic fidelity to the tumor tissue of origin. These models may help in paving the way towards the development of novel treatments. Patient summaryWe established 11 animal models based on tumor tissue from patients with penile cancer. These models could play a vital role in selection of novel treatments according to genetic mutations. In the future, therapies with confirmed preclinical effects may have a profound impact on the development of personalized treatments in penile cancer.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Establishment and Characterization of Brain Cancer Primary Cell Cultures From Patients to Enable Phenotypic Screening for New Drugs

Aim: Desmoplastic infantile ganglioglioma (DIG), is a rare tumor arising mainly during the first 2 years of life. Molecular characterization of these benign yet rapidly proliferating tumors has been limited to evaluating a few mutations in few genes. Our aim was to establish a live cell culture to enable the understanding of the cellular processes driving the non-malignant growth of these tumors. Methods: Tumor tissue from a rare non-infantile 8-year-old female DIG patient was dissociated and digested using collagenase to establish live cultures. Both 2D monolayer and 3D neurospheres were successfully cultured and characterized for proliferative potential, intrinsic plasticity, presence of cancer stem cells and the expression of stem cell markers. Cells cultured as 3D were embedded as tissue blocks. Immunohistochemistry was performed in both tissue and 3D sections for markers including synaptophysin, vimentin, neurofilament and MIB-1. Mutation analysis by NGS was performed using a-100 gene panel. Results: Using immunohistochemistry, the 3D cultures were shown to express markers as in the original DIG tumor tissue indicating that the spheroid cultures were able to maintain the heterogeneity found in the original tumor. Cells continued proliferating past passage 10 indicative of immortalization. Enrichment of cancer stem cells was observed in neurospheres by FACS using CD133 antibody and RT-PCR. Mutation analysis indicated the presence of germline mutations in three genes and somatic mutations in two other genes. Conclusion: A spontaneous cell line-like cell culture with high percentage of stem cells has been established from a DIG tumor for the first time.

Clinical validation of a 90-gene expression test for tumor tissue of origin diagnosis: a large-scale multicenter study of 1417 patients

BackgroundOnce malignancy tumors were diagnosed, the determination of tissue origin and tumor type is critical for clinical management. Although the significant advance in imaging techniques and histopathological approaches, the diagnosis remains challenging in patients with metastatic and poorly differentiated or undifferentiated tumors. Gene expression profiling has been demonstrated the ability to classify multiple tumor types. The present study aims to assess the performance of a 90-gene expression test for tumor classification (i.e. the determination of tumor tissue of origin) in real clinical settings.MethodsFormalin-fixed paraffin-embedded samples and associated clinicopathologic information were collected from three cancer centers between January 2016 and January 2021. A total of 1417 specimens that met quality control criteria (RNA quality, tumor cell content ≥ 60% and so on) were analyzed by the 90-gene expression test to identify the tumor tissue of origin. The performance was evaluated by comparing the test results with histopathological diagnosis.ResultsThe 1417 samples represent 21 main tumor types classified by common tissue origins and anatomic sites. Overall, the 90-gene expression test reached an accuracy of 94.4% (1338/1417, 95% CI: 0.93 to 0.96). Among different tumor types, sensitivities were ranged from 74.2% (head&neck tumor) to 100% (adrenal carcinoma, mesothelioma, and prostate cancer). Sensitivities for the most prevalent cancers of lung, breast, colorectum, and gastroesophagus are 95.0%, 98.4%, 93.9%, and 90.6%, respectively. Moreover, specificities for all 21 tumor types are greater than 99%.ConclusionsThese findings showed robust performance of the 90-gene expression test for identifying the tumor tissue of origin and support the use of molecular testing as an adjunct to tumor classification, especially to those poorly differentiated or undifferentiated tumors in clinical practice.

CD44 Expression Intensity Marks Colorectal Cancer Cell Subpopulations with Different Extracellular Vesicle Release Capacity.

Extracellular vesicles (EV) are released by virtually all cells and they transport biologically important molecules from the release site to target cells. Colorectal cancer (CRC) is a leading cause of cancer-related death cases, thus, it represents a major health issue. Although the EV cargo may reflect the molecular composition of the releasing cells and thus, EVs may hold a great promise for tumor diagnostics, the impact of intratumoral heterogeneity on the intensity of EV release is still largely unknown. By using CRC patient-derived organoids that maintain the cellular and molecular heterogeneity of the original epithelial tumor tissue, we proved that CD44high cells produce more organoids with a higher proliferation intensity, as compared to CD44low cells. Interestingly, we detected an increased EV release by CD44high CRC cells. In addition, we found that the miRNA cargos of CD44high and CD44low cell derived EVs largely overlapped and only four miRNAs were specific for one of the above subpopulations. We observed that EVs released by CD44high cells induced the proliferation and activation of colon fibroblasts more strongly than CD44low cells. However, this effect was due to the higher EV number rather than to the miRNA cargo of EVs. Collectively, we identified CRC subpopulations with different EV releasing capabilities and we proved that CRC cell-released EVs have a miRNA-independent effect on fibroblast proliferation and activation.

RNA-seq of nine canine prostate cancer cell lines reveals diverse therapeutic target signatures

BackgroundCanine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount.MethodsThis study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin.ResultsIndependent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing.ConclusionComparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.

Multiplexed nanomaterial-assisted laser desorption/ionization for pan-cancer diagnosis and classification

As cancer is increasingly considered a metabolic disorder, it is postulated that serum metabolite profiling can be a viable approach for detecting the presence of cancer. By multiplexing mass spectrometry fingerprints from two independent nanostructured matrixes through machine learning for highly sensitive detection and high throughput analysis, we report a laser desorption/ionization (LDI) mass spectrometry-based liquid biopsy for pan-cancer screening and classification. The Multiplexed Nanomaterial-Assisted LDI for Cancer Identification (MNALCI) is applied in 1,183 individuals that include 233 healthy controls and 950 patients with liver, lung, pancreatic, colorectal, gastric, thyroid cancers from two independent cohorts. MNALCI demonstrates 93% sensitivity at 91% specificity for distinguishing cancers from healthy controls in the internal validation cohort, and 84% sensitivity at 84% specificity in the external validation cohort, with up to eight metabolite biomarkers identified. In addition, across those six different cancers, the overall accuracy for identifying the tumor tissue of origin is 92% in the internal validation cohort and 85% in the external validation cohort. The excellent accuracy and minimum sample consumption make the high throughput assay a promising solution for non-invasive cancer diagnosis.

A Review of Progress in the Construction and Application of Human-Derived Xenograft Models for Bladder Cancer

The patient-derived xenografts (PDX) model is an animal model established by transplanting primary tumors or fresh tumor tissues of patient origin directly into immunodeficient mice, which preserves the heterogeneity and survival microenvironment of the primary tumor and is widely used in preclinical and precision medicine research of tumors. This article reviews the construction of the PDX model of human bladder cancer and the progress of the application of the PDX model in bladder cancer.

The HROC-Xenobank-A High Quality Assured PDX Biobank of >100 Individual Colorectal Cancer Models.

Simple SummaryConsidering recent research, it was established that the best experimental models to conserve biological features of human tumors and to predict individual clinical treatment success are patient-derived xenografts (PDX). Their recognized and growing importance for translational research, especially for late-stage preclinical testing of novel therapeutics, necessitates a high number of well-defined PDX models from individual patients’ tumors. The starting platform for the Hansestadt Rostock colorectal cancer (HROC)-Xenobank was the assortment of colorectal tumor and normal tissue samples from patients stored in our university biobank.Based on our research group’s large biobank of colorectal cancers (CRC), we here describe the ongoing activity of establishing a high quality assured PDX biobank for more than 100 individual CRC cases. This includes sufficient numbers of vitally frozen (n > 30 aliquots) and snap frozen (n > 5) backups, “ready to use”. Additionally, PDX tumor pieces were paraffin embedded. At the current time, we have completed 125 cases. This resource allows histopathological examinations, molecular characterizations, and gene expression analysis. Due to its size, different issues of interest can be addressed. Most importantly, the application of low-passage, cryopreserved, and well-characterized PDX for in vivo studies guarantees the reliability of results due to the largely preserved tumor microenvironment. All cases described were molecularly subtyped and genetic identity, in comparison to the original tumor tissue, was confirmed by fingerprint analysis. The latter excludes ambiguity errors between the PDX and the original patient tumor. A cancer hot spot mutation analysis was performed for n = 113 of the 125 cases entities. All relevant CRC molecular subtypes identified so far are represented in the Hansestadt Rostock CRC (HROC)-Xenobank. Notably, all models are available for cooperative research approaches.

Establishment and characterization of NCC-MPNST6-C1: a novel patient-derived cell line of malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are a rare subtype of mesenchymal tumors that arise from the sheath of peripheral nerves. MPNSTs exhibit strong metastatic potential, leading to poor clinical outcomes. The clinical utility of radiation therapy and chemotherapy is marginal with respect to overall survival and effective systematic therapies for MPNSTs are still needed to improve patient outcome. Although patient-derived cell lines are an essential tool for the development of novel therapies, only a limited number of cell lines have been reported and are available from cell banks. Thus, we established the novel MPNST cell line, NCC-MPNST6-C1, using surgically resected MPNST tissue. The NCC-MPNST6-C1 cells retained copy-number alterations similar to those of the original tumors and demonstrated constant proliferation, spheroid formation, and invasion capability in vitro, which reflected the malignant features of the original tumor tissue. While the NCC-MPNST6-C1 cells did not exhibit tumorigenesis in nude mice, their use in drug screening resulted in anti-cancer agents with low IC50 values. Hence, we conclude that the NCC-MPNST6-C1 cell line is a useful resource for the study of MPNSTs.

Expression of estrogen and progesterone receptors, HER2 protein and Ki-67 proliferation index in breast carcinoma in both tumor tissue and tissue microarray

ABSTRACT Breast cancer treatment is tailored to molecular subtypes, which are classified by cell type and by presence of estrogen and progesterone receptors, HER2 overexpression and Ki-67 proliferation index. In routine pathological practice, these markers are detected in tumor tissue using immunohistochemistry, which requires four immunohistochemical antibodies for each patient. We developed a new tissue microarray procedure using a punch device with a 6 mm core diameter. The presence of estrogen and progesterone receptors, HER2 expression and the Ki-67 proliferation index of tumor tissues of 50 breast carcinoma patients had been determined using the conventional approach. We created three tissue microarray blocks, each containing samples from 14 main tumor tissues. One tissue microarray block was created with samples taken from eight main tumor tissues. Sections were cut from the four blocks and subjected to immunohistochemical staining; the original samples and the microarrays then were compared. We found significant agreement between estrogen receptor, progesterone receptor and HER-2 expression as well as Ki-67 proliferation index status of the original tumor tissues and the tissue microarray. Our tissue microarray technique using a single 6 mm core is a reliable and cost-effective method for determining estrogen and progesterone receptors, HER-2 status and Ki-67 proliferation index levels in patients with early breast carcinoma.

Establishment and characterization of NCC-ssRMS2-C1: a novel patient-derived cell line of spindle cell/sclerosing rhabdomyosarcoma.

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma (RMS) that has fascicular spindle cell and/or sclerosing morphology. SsRMS has a diverse molecular background and is categorized into three groups: congenital/infantile ssRMS with a gene fusion involving the NCOA2 and VGLL2, ssRMS with the MYOD1 mutation, and ssRMS with no recurrent identifiable genetic alterations. Because ssRMS is a newly defined disease concept of RMS, the optimal treatment methods have not been determined. This results in unfavorable prognosis and consequently signals the urgent need for continuous research. Patient-derived cell lines are essential tools in basic and translational research. However, only two ssRMS cell lines with the MYOD1 mutation have been reported to date. Thus, we established a novel ssRMS cell line named NCC-ssRMS2-C1 using a surgically resected tumor tissue from an adult ssRMS patient. NCC-ssRMS2-C1 cells retained the copy number alterations corresponding to the original tumor and are categorized into the group with no recurrent identifiable genetic alterations. NCC-ssRMS2-C1 cells demonstrated constant proliferation, spheroid formation, and capability for invasion in vitro, reflecting the malignant features of the original tumor tissue. In a drug screening test, ssRMS demonstrated remarkable sensitivity to romidepsin, trabectedin, actinomycin D, and bortezomib. Hence, we conclude that the NCC-ssRMS2-C1 cell line is the first ssRMS cell line which belongs to the group with no recurrent identifiable genetic alterations, and it will be a useful resource in both basic and translational studies for ssRMS.

Common pathways and functional profiles reveal underlying patterns in Breast, Kidney and Lung cancers

BackgroundCancer is a major health problem which presents a high heterogeneity. In this work we explore omics data from Breast, Kidney and Lung cancers at different levels as signalling pathways, functions and miRNAs, as part of the CAMDA 2019 Hi-Res Cancer Data Integration Challenge. Our goal is to find common functional patterns which give rise to the generic microenvironment in these cancers and contribute to a better understanding of cancer pathogenesis and a possible clinical translation down further studies.ResultsAfter a tumor versus normal tissue comparison of the signaling pathways and cell functions, we found 828 subpathways, 912 Gene Ontology terms and 91 Uniprot keywords commonly significant to the three studied tumors. Such features interestingly show the power to classify tumor samples into subgroups with different survival times, and predict tumor state and tissue of origin through machine learning techniques. We also found cancer-specific alternative activation subpathways, such as the ones activating STAT5A in ErbB signaling pathway. miRNAs evaluation show the role of miRNAs, such as mir-184 and mir-206, as regulators of many cancer pathways and their value in prognoses.ConclusionsThe study of the common functional and pathway activities of different cancers is an interesting approach to understand molecular mechanisms of the tumoral process regardless of their tissue of origin. The existence of platforms as the CAMDA challenges provide the opportunity to share knowledge and improve future scientific research and clinical practice.

Evaluating DNA Methylation, Gene Expression, Somatic Mutation, and Their Combinations in Inferring Tumor Tissue-of-Origin

Carcinoma of unknown primary (CUP) is a type of metastatic cancer, the primary tumor site of which cannot be identified. CUP occupies approximately 5% of cancer incidences in the United States with usually unfavorable prognosis, making it a big threat to public health. Traditional methods to identify the tissue-of-origin (TOO) of CUP like immunohistochemistry can only deal with around 20% CUP patients. In recent years, more and more studies suggest that it is promising to solve the problem by integrating machine learning techniques with big biomedical data involving multiple types of biomarkers including epigenetic, genetic, and gene expression profiles, such as DNA methylation. Different biomarkers play different roles in cancer research; for example, genomic mutations in a patient’s tumor could lead to specific anticancer drugs for treatment; DNA methylation and copy number variation could reveal tumor tissue of origin and molecular classification. However, there is no systematic comparison on which biomarker is better at identifying the cancer type and site of origin. In addition, it might also be possible to further improve the inference accuracy by integrating multiple types of biomarkers. In this study, we used primary tumor data rather than metastatic tumor data. Although the use of primary tumors may lead to some biases in our classification model, their tumor-of-origins are known. In addition, previous studies have suggested that the CUP prediction model built from primary tumors could efficiently predict TOO of metastatic cancers (Lal et al., 2013; Brachtel et al., 2016). We systematically compared the performances of three types of biomarkers including DNA methylation, gene expression profile, and somatic mutation as well as their combinations in inferring the TOO of CUP patients. First, we downloaded the gene expression profile, somatic mutation and DNA methylation data of 7,224 tumor samples across 21 common cancer types from the cancer genome atlas (TCGA) and generated seven different feature matrices through various combinations. Second, we performed feature selection by the Pearson correlation method. The selected features for each matrix were used to build up an XGBoost multi-label classification model to infer cancer TOO, an algorithm proven to be effective in a few previous studies. The performance of each biomarker and combination was compared by the 10-fold cross-validation process. Our results showed that the TOO tracing accuracy using gene expression profile was the highest, followed by DNA methylation, while somatic mutation performed the worst. Meanwhile, we found that simply combining multiple biomarkers does not have much effect in improving prediction accuracy.

A comprehensive characterization of the cell-free transcriptome reveals tissue- and subtype-specific biomarkers for cancer detection

Cell-free RNA (cfRNA) is a promising analyte for cancer detection. However, a comprehensive assessment of cfRNA in individuals with and without cancer has not been conducted. We perform the first transcriptome-wide characterization of cfRNA in cancer (stage III breast [n = 46], lung [n = 30]) and non-cancer (n = 89) participants from the Circulating Cell-free Genome Atlas (NCT02889978). Of 57,820 annotated genes, 39,564 (68%) are not detected in cfRNA from non-cancer individuals. Within these low-noise regions, we identify tissue- and cancer-specific genes, defined as “dark channel biomarker” (DCB) genes, that are recurrently detected in individuals with cancer. DCB levels in plasma correlate with tumor shedding rate and RNA expression in matched tissue, suggesting that DCBs with high expression in tumor tissue could enhance cancer detection in patients with low levels of circulating tumor DNA. Overall, cfRNA provides a unique opportunity to detect cancer, predict the tumor tissue of origin, and determine the cancer subtype.

Creation and Maintenance of a Living Biobank - How We Do It.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

NTRK Fusions in Sarcomas: Diagnostic Challenges and Clinical Aspects.

Tropomyosin receptor kinase (TK) is encoded by the neurotrophic tyrosine receptor kinase genes (NTRK) 1, 2, and 3, whose activation plays an important role in cell cycle proliferation and survival. Fusions of one of these genes can lead to constitutive activation of TRK, which can potentially be oncogenic. NTRK fusions are commonly present in rare histologic tumor types. Among sarcomas, infantile fibrosarcoma shows NTRK fusion in more than 90% of the cases. Many other sarcoma types are also investigated for NTRK fusions. These fusions are druggable alteration of the agnostic type, meaning that all NTRK fused tumors can be treated with NTRK-inhibitors regardless of tumor type or tissue of origin. TRK-inhibitors have shown good response rates, with durable effects and limited side effects. Resistance to therapy will eventually occur in some cases, wherefore the next-generation TRK-inhibitors are introduced. The diagnosis of NTRK fused tumors, among them sarcomas, remains an issue, as many algorithms but no guidelines exist to date. Given the importance of this diagnosis, in this paper we aim to (1) analyze the histopathological features of sarcomas that correlate more often with NTRK fusions, (2) give an overview of the TRK-inhibitors and the problems that arise from resistance to the therapy, and (3) discuss the diagnostic algorithms of NTRK fused tumors with emphasis on sarcomas.