TY  - THES
T1  - Functional validation of genetic changes discovered in high-throughput mutational and copy number screens with respect to carcinogenesis and treatment sensitivity for Head and Neck Cancer
A1  - Keck,Michaela-Kristina
Y1  - 2016/06/07
N2  - Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease regarding both anatomic location and molecular characteristics. It is comprised of two distinct disease entities - human papillomavirus (HPV)-positive and HPV-negative HNSCC - that differ regarding primary site of disease as well as aetiology/pathogenesis. Persistent infection with high-risk HPV is causally related to oropharyngeal HNSCC, whereas tobacco and alcohol consumption are linked to the development of HPV-negative head and neck cancers. HPV(+) and HPV(-) HNSCC differ regarding their clinical behavior as well as prognosis with HPV(+) tumors being associated with a more favorable prognosis than HPV(-) tumors. However, besides Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), which is used with modest success as no predictive biomarkers have been identified, no targeted treatments are available that consider the biologic heterogeneity and no classification is used in clinical practice that reflects the underlying biology of the disease. As a consequence, all patients are treated uniformly with a combination of surgery, radiation, and chemotherapy depending on stage and location of the tumor. In this study, a homogeneous and clinically annotated cohort of 134 treatment-naive HNSCC specimens, including 59 HPV(+) samples (44%), were processed, subjected to Agilent 4X44Kv2 gene expression arrays and Nanostring copy number assays, and analyzed together with all available HNSCC cohorts (n=938). As a result, a clinically meaningful classification was identified for HNSCC - which was hampered in previous studies due to a limited inclusion of HPV(+) samples, unknown HPV status, small sample size, or missing clinical annotation. Three HNSCC supergroups were identified - basal (BA), classical (CL), inflamed/mesenchymal (IMS) - and further subdivided by HPV status into five HNSCC subtypes - BA, CL-HPV, CL-nonHPV, IMS-HPV, IMS-nonHPV - that differ regarding survival and show different expression profiles and copy number (CN) aberrations. Importantly, two HPV-associated subtypes with different molecular profile and prognosis were identified - CL-HPV and IMS-HPV. The IMS subtypes show a strong immune phenotype with expression of immune response genes and may benefit from immune therapies such as programmed cell death protein 1 (PD-1) inhibitors. The BA subtype shows markedly increased expression of EGFR and hypoxia related genes, which might render these tumors susceptible to EGFR-targeting and hypoxia-targeting therapies. Besides differences regarding their biological and clinical behavior, HPV(+) and HPV(-) HNSCC show a distinct mutational landscape. The incidence of HPV(+) tumors is rising, however, studies are missing that investigate a sufficient number of HPV(+) tumors to derive meaningful information and assign targetable genomic changes to either disease entity. 120 (51 HPV(+), 69 HPV(-)) out of the cohort of 134 samples were further used to build DNA sequencing libraries and processed to next generation sequencing (NGS) on the Illumina platform (Illumina HiSeq 2000/2500 sequencers) to detect novel or targetable genomic changes. The overall mutational burden is similar in both disease entities, however, the mutational landscape differs. In addition to known aberrations, novel targetable aberrations with predicted driver potential are identified in various genes, among which are EGFR, CCND1, and FGFR1 in HPV(-) tumors, and FGFR2/FGFR3, KRAS, BRCA1/2 in HPV(+) tumors, as well as PIK3CA and PI3K pathway genes in both and rare aberrations in DDR2 and EPHA2. The fibroblast growth factor receptor 3 (FGFR3) S249C mutation is recurrently found in six HPV(+) tumors, which makes FGFR3 the second most mutated gene in HPV(+) tumors - after the well-known oncogene PIK3CA. Therefore, functional validation of FGFR3 S249C was performed in vitro in the two HNSCC cell lines SCC47 and 93VU147T. Two different FGFR3 isoforms were identified in all six FGFR3 mutated HNSCC tumor samples - FGFR3IIIb and the soluble FGFR3Delta8-10. Both isoforms were used to stably transfect the S249C mutation into SCC47 and 93VU147T and to study its effects on proliferation and migration as well as FGFR3 signaling. No effect on proliferation or migration rate is seen beyond an increase in proliferation rate in SCC47 cells transfected with wild type (WT) FGFR3IIIb. No constitutive activation of downstream pathways is seen in unstimulated SCC47 cells. Investigation of different downstream signaling pathways identifies the MAPK and the PI3K/Akt pathway as the two important signaling pathways downstream of FGFR3 in HNSCC cell lines. FGFR3 S249C cannot be confirmed as an oncogenic driver in SCC47 and 93VU147T. However, its possible role as an oncogenic co-driver besides other receptor tyrosine kinases and its function in drug resistance mechanisms - especially resistance to EGFR inhibitors - remain to be investigated.
KW  - Humanes Papillomavirus
KW  - Untergruppen
KW  - Sequenzierung
KW  - FGFR3
CY  - Hohenheim
PB  - Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim
AD  - Garbenstr. 15, 70593 Stuttgart
UR  - http://opus.uni-hohenheim.de/volltexte/2016/1194
ER  -