A novel patient derived orthotopic xenograft model of gastro-esophageal junction cancer: Key platform for translational discoveries.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 64-64
Author(s):  
Omkara Lakshmi Veeranki
Keyword(s):  
Next Generation Sequencing ◽  
Tumor Microenvironment ◽  
Gastroesophageal Junction ◽  
Lymphatic Vessels ◽  
Xenograft Model ◽  
Inflammatory Cells ◽  
Next Generation ◽  
Orthotopic Xenograft ◽  
Generation Sequencing ◽  
Efficacy Studies

64 Background: Mouse models of gastroesophageal junction (GEJ) cancer strive to recapitulate the intratumoral heterogeneity and cellular crosstalk within patient tumors to improve clinical translation. Current GEJ models have limited applications in tumor microenvironment, immune oncology and metastatic studies. Methods: A novel patient derived tumor orthotopic xenograft (PDOX) was established from GEJ cancer via surgical implantation. Patient tumor was compared to subcutaneously implanted PDX and PDOX by H&E, IHC, and next generation sequencing (T200.1 panel). Drug efficacy studies of 5-flurouracil with and without radiotherapy are being performed. Results: Mechanical abrasion of mouse GEJ prior to implantation of patient derived tumor in situ promotes tumor engraftment (100%, n = 6). Complete PDOX engraftment was observed with rapid intra and extra luminal tumor growth as evidenced by MRI. Patient derived stroma co-engrafts with tumor cells in GEJ-PDOX. PDOXs contain fibroblasts, immune and inflammatory cells, vascular and lymphatic vessels. Stromal hallmarks of aggressive GEJs are recapitulated in GEJ-PDOX mouse model. PDOXs demonstrates tumor invasion into vasculature. GEJ-PDOXs is a clinically relevant model for metastases and immunological studies. Next generation sequencing with the T200.1 revealed that the loss of heterozygosity of NOTCH3, TGFβ1, EZH2, and MLL3 are maintained with similar allelic frequency between the patient tumor and the xenografts. Additional somatic SNVs such as ARID1A, NSD1 (CDS157-158), NSD1 (CDS158-159), KDM6A, XPO1, MAPK1 and EGFR were found to be acquired in xenograft tumor tissues that were not observed in patient tumor. Drug and radiation efficacy studies are ongoing, tumor response to radiation was observed. Conclusions: A GEJ-PDOX model exhibits remarkable fidelity to human disease and captures the precise tissue microenvironment present within the local GEJ architecture facilitating it as a novel tool in translating to clinics. This model can be applied to address importance of tumor microenvironment in metastatic and immunological studies, and to develop novel therapeutic approaches for the treatment of GEJ cancer.

scholarly journals Next Generation Sequencing-Based Transcriptome Predicts Bevacizumab Efficacy in Combination with Temozolomide in Glioblastoma

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3046 ◽  
Author(s):  
Alimu Adilijiang ◽  
Masaki Hirano ◽  
Yusuke Okuno ◽  
Kosuke Aoki ◽  
Fumiharu Ohka ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Next Generation Sequencing ◽  
Immune Cell ◽  
Xenograft Model ◽  
Idh1 Mutation ◽  
World Health ◽  
In Vivo Studies ◽  
Next Generation ◽  
U87 Cells ◽  
Generation Sequencing

Glioblastoma (GBM), the most common and malignant brain tumor, is classified according to its isocitrate dehydrogenase (IDH) mutation status in the 2016 World Health Organization (WHO) brain tumor classification scheme. The standard treatment for GBM is maximal resection, radiotherapy, and Temozolomide (TMZ). Recently, Bevacizumab (Bev) has been added to basic therapy for newly diagnosed GBM, and monotherapy for recurrent GBM. However, the effect of IDH1 mutation on the combination of Bev and TMZ is unknown. In this study, we performed transcriptomic analysis by RNA sequencing with next generation sequencing (NGS), a newly developed powerful method that enables the quantification of the expression level of genome-wide genes. Extracellular matrix and immune cell migration genes were mainly upregulated whereas cell cycle genes were downregulated in IDH1-mutant U87 cells but not in IDH1-wildtype U87 cells after adding Bev to TMZ. In vitro and in vivo studies were conducted for further investigations to verify these results, and the addition of Bev to TMZ showed a significant antitumor effect only in the IDH1-mutant GBM xenograft model. Further studies of gene expression profiling in IDH1 mutation gliomas using NGS will provide more genetic information and will lead to new treatments for this refractory disease.

Next Generation Sequencing-Based Molecular Dissection Of Lineage-Specific Mutational Hierarchies In Oligoclonal Primary and Xenografted Myelodysplasia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 519-519
Author(s):  
Johann-Christoph Jann ◽  
Daniel Nowak ◽  
Florian Nolte ◽  
Claudia Haferlach ◽  
Marita Staller ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Xenograft Model ◽  
Disease Diagnosis ◽  
Hierarchical Architecture ◽  
Small Scale ◽  
Next Generation ◽  
Hematopoietic Stem ◽  
Generation Sequencing

Abstract Background The underlying molecular defects in myelodysplastic syndromes (MDS), which are a heterogeneous group of malignant clonal hematologic disorders, are not well understood. Recently, next generation sequencing (NGS) based whole genome and exome sequencing highlighted the oligoclonal nature of persistent MDS clones that are present already at early disease stages. The reconstruction of mutational hierarchies in MDS clones and distinction of primary founder from subsequently acquired lesions has yet to be thoroughly interrogated and is likely to aid dissecting the molecular pathogenesis of MDS. Methods An amplicon-based NGS assay using the Roche 454 GS Junior system was established within the IRON-II framework study in order to screen for 17 commonly mutated genes in MDS. Genomic DNA from purified mononuclear bone marrow (BM) cells of 23 MDS IPSS low/int1 risk subjects was screened for somatic mutations. Called variants were compared to dbSNP and COSMIC database entries to rule out germline polymorphisms. In addition, copy number variation analysis was performed by Affymetrix SNP 6.0 array profiling. Custom pyrosequencing assays and interphase-FISH were applied for sensitive quantification of lesion burdens in FACS-sorted myeloid, erythroid, lymphoid and stem/progenitor cells. These were isolated from patients’ primary BM as well as their long-term engrafted human xenotransplants using our recently established MDS xenograft model. Results In this work, we identified 12 oligoclonal BM samples with ≥2 molecular lesions. Of note, varying frequencies of individual mutations between different sorted cell subsets from primary or human xenografted BM support the notion that distinct MDS (sub-)clones from these subjects contributed to hematopoiesis simultaneously and lead to differential engraftment between xenografts. Comparison of variable subset-specific mutation burdens allowed deciphering the individual hierarchical architecture of the mutational landscape from 9 individuals. ASXL1, SF3B1 and SRSF2 were detected as a primary lesion for 2 patients each. In contrast, large-scale genomic alterations such as del(5q), del(RUNX1) or trisomy 8 occurred as late-end lesion or even defined distinct clones which coexist with others harboring different mutations as detected for 2 subjects. Surprisingly, CD19+ and CD3+ lymphocytes from primary and/or xenografted BM displayed significant mutational burden of at least 1 mutation in 50% of the MDS cohort (5/10). Moreover, mutations were detected simultaneously in lymphocytes (hCD19+) as well as myeloid (hCD33+) and erythroid (hCD235a+) cells from three xenografted samples indicating a potent multilineage engraftment capability of MDS hematopoietic stem cells. Interestingly, one individual presented with high RUNX1 mutational frequency in the primary early progenitor fraction (CD34+CD38+), which was absent in the stem-cell enriched fraction (CD34+CD38-), whereas TET2, ZRSR2 and ASXL1 mutations were detected in both fractions and their xenografts. Intriguingly, only xenotransplantation of primary CD34+38- BM cells lead to long-term engraftment of RUNX1 wild type human BM cells in mice, while CD34+CD38+ BM cells gave rise to short term engraftment of RUNX1 mutated human BM cells indicating that mutated RUNX1might originate in a more committed progenitor fraction with limited self-renewal potential. Conclusion Molecular characterization of oligoclonal mutation patterns in primary and xenograft BM allowed the establishment of individual mutational hierarchies and indicates a relatively random order in the mutational evolution of MDS clones, although spliceosome mutations appear as rather early events. Furthermore, our analysis revealed engraftment of independent MDS clones in different mice xenografted with the same subject material, which opens the door to the in vivo study of isolated clones with respect to their pathomechanisms and response to treatment. Our data also suggests that the occurrence of large-scale genomic aberrations is frequently preceded by small-scale gene mutations, emphasizing their potential role in disease diagnosis and risk stratification. Finally, detection of MDS specific mutations in the lymphocytic compartment might be involved in facilitating impaired immune functionality and needs to be investigated prospectively. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Staller:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria.

scholarly journals Targeting ERBB2 (HER2) Amplification Identified by Next-Generation Sequencing in Patients With Advanced or Metastatic Solid Tumors Beyond Conventional Indications

2019 ◽  
pp. 1-12 ◽  
Author(s):  
Ecaterina E. Ileana Dumbrava ◽  
Kavitha Balaji ◽  
Kanwal Raghav ◽  
Kenneth Hess ◽  
Milind Javle ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Next Generation Sequencing ◽  
Clinical Benefit ◽  
Gastroesophageal Junction ◽  
Multivariable Analysis ◽  
Progression Free Survival ◽  
Next Generation ◽  
Her2 Amplification ◽  
Tumor Types ◽  
Generation Sequencing

PURPOSE Human epidermal growth factor receptor 2 (HER2) is an effective therapeutic target in breast and gastric and gastroesophageal junction cancers. However, less is known about the prevalence of ERBB2 ( HER2) amplification and the efficacy of HER2-targeted treatment in other tumors. PATIENTS AND METHODS We assessed HER2 amplification status among 5,002 patients with advanced disease (excluding breast cancer) who underwent next-generation sequencing. We evaluated the clinical benefit of HER2-targeted therapy by measuring the time-dependent overall survival (OS) from the genomic testing results, progression-free survival (PFS), and PFS during HER2-targeted therapy (PFS2) compared with PFS during prior therapy (PFS1). RESULTS Overall, 122 patients (2.4%) had HER2 amplification, including patients with endometrial (5.3%), bladder (5.2%), biliary or gallbladder (4.9%), salivary (4.7%), and colorectal cancer (3.6%). Forty patients (38%) with nongastric, nongastroesophageal junction, or nonesophageal cancers received at least one line of HER2-targeted therapy. Patients receiving HER2-targeted therapy had a median OS of 18.6 months, compared with 10.9 months for patients who did not receive HER2-targeted therapy ( P = .070). On multivariable analysis, HER2-targeted therapy was significantly associated with increased OS (hazard ratio, 0.5; 95% CI, 0.27 to 0.93; P = .029), regardless of sex, age, or number of prior lines of treatment. The PFS2-to-PFS1 ratio was 1.3 or greater in 21 (57%) of 37 patients who received HER2-targeted therapy not in the first line of systemic treatment, and the median PFS2 and PFS1 times were 24 and 13 weeks, respectively ( P < .001). CONCLUSION HER2 amplifications using next-generation sequencing can be identified in a variety of tumor types. HER2-targeted therapy may confer clinical benefit in tumor types other than those for which HER2 inhibitors are approved.

Labordiagnostik bei gastrointestinalen Tumoren

2020 ◽  
Vol 11 (05) ◽  
pp. 232-238
Author(s):  
Marcus Kleber
Keyword(s):  
Next Generation Sequencing ◽  
Kolorektales Karzinom ◽  
Next Generation ◽  
Generation Sequencing

ZUSAMMENFASSUNGDas kolorektale Karzinom (KRK) ist einer der häufigsten malignen Tumoren in Deutschland. Einer frühzeitigen Diagnostik kommt große Bedeutung zu. Goldstandard ist hier die Koloskopie. Die aktuelle S3-Leitlinie Kolorektales Karzinom empfiehlt zum KRK-Screening den fäkalen okkulten Bluttest. Für das Monitoring von Patienten vor und nach Tumorresektion werden die Messung des Carcinoembryonalen Antigens (CEA) und der Mikrosatellitenstabilität empfohlen. Für die Auswahl der korrekten Chemotherapie scheint derzeit eine Überprüfung des Mutationsstatus, mindestens des KRAS-Gens und des BRAF-Gens, sinnvoll zu sein. Eine Reihe an neuartigen Tumormarkern befindet sich momentan in der Entwicklung, hat jedoch noch nicht die Reife für eine mögliche Anwendung in der Routinediagnostik erreicht. Den schnellsten Weg in die breite Anwendung können Next-Generation-Sequencing-basierte genetische Tests finden.

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