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Number of negative points: a novel method for predicting radiosensitivity in head and neck tumor cell lines.
Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Department of Otorhinolaryngology, Head and Neck Surgery, Central Hospital and University of Turku and Medical Biochemistry, University of Turku, Finland.
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2008 (English)In: Oncology Reports, ISSN 1021-335X, Vol. 20, no 2, 453-461 p.Article in journal (Refereed) Published
Abstract [en]

The present study was aimed at establishing a method that combines multiple factors of protein and genetic changes that enables prediction of radiosensitivity in the head and neck squamous cell carcinoma (HNSCC) cell lines. In nine HNSCC cell lines, the quantity of protein expression and the type of genetic alterations were translated into a point system, called the Number of Negative Points. The expression of 14 proteins involved in growth control and/or apoptosis was quantified using a densitometric assessment of Western blots. The blots were adjusted to actin and standardised to normal oral keratinocytes classifying them into four groups depending on the amount of protein expressed (0-3 points). Mutations of the p53 gene were classified into three groups and each mutation was given one point. Since the cell lines each had a known intrinsic radiosensitivity, a multivariate statistical calculation could then be performed to select for the combination of factors having the strongest correlation to radiosensitivity. The strongest correlation of the investigated factors was the combination of epidermal growth factor receptor, survivin and splice site/missense p53 mutations (R=0.990 and P<0.0001). No single factor had a significant correlation to the intrinsic radiosensitivity. Since a significant correlation to the intrinsic radiosensitivity was achieved only when two or more factors were combined, we conclude that a method such as the Number of Negative Points is necessary for prediction of treatment response. We present a novel method to combine factors which enables the prediction of radiosensitivity of HNSCC cell lines.

Place, publisher, year, edition, pages
2008. Vol. 20, no 2, 453-461 p.
Keyword [en]
Predictive markers, p53, epidermal growth factor receptor, survivin, squamous cell carcinoma
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-19558DOI: 10.3892/or_00000028PubMedID: 18636211OAI: oai:DiVA.org:liu-19558DiVA: diva2:225375
Available from: 2009-06-26 Created: 2009-06-26 Last updated: 2012-03-29Bibliographically approved
In thesis
1. On Predictive Factors of Treatment Response in Head and Neck Squamous Cell Carcinoma
Open this publication in new window or tab >>On Predictive Factors of Treatment Response in Head and Neck Squamous Cell Carcinoma
2008 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and yearly include 500 000 new cases worldwide. The outcomes for these patients have not been significantly improved over the last decades and the five year survival is still around 50 %. Establishing predictive markers of treatment response will have great impact on the clinical management of this disease.

The aim of this thesis was to elucidate markers of intrinsic response to radiotherapy and cisplatin. Combining expression patterns of 14 proteins and identifying mutations in the p53 gene, we were able to incorporate both protein and genetic changes to create a predictive model termed Number of Negative Points (NNP). We used the NNP model to statistically calculate the combination of factors that had the best correlation to intrinsic radiosensitivity (IR). We established that a panel of three markers, epidermal growth factor receptor (EGFR), survivin and splice site/missence mutations of p53, had the best correlation to IR (R=0.990, p<0.0001).

We also conducted gene expression analysis to investigate what genes and gene ontologies that are different between cell lines with varying IR. Furthermore, we wanted to identify key regulator genes with central positions of molecular networks, which were generated from the transcripts included in the deregulated gene ontologies. A transcriptional profile of 28 key regulator genes was generated. Immunoblot analysis supported deregulation at the protein level of three markers implicated from the transcriptional profile. We propose that these proteins, notch1, thrombospondin 1, and pai‐1 are predictive markers of IR.

Finally, on a subset of cell lines with sensitivity or resistance to cisplatin, we performed gene expression analysis. Markers of intrinsic cisplatin sensitivity (ICS) such as gene ontologies and key regulators of molecular networks were proposed and five genes, APOE, CTNNB1, MMP7, MMP13, and THBS1 were selected for further analysis. Quantitative polymerase chain reaction (qPCR) analysis of these genes in 25 cell lines established that MMP7 (p=0.0013) and MMP13 (p=0.058) are possible predictive markers of ICS.

The markers of IR and ICS presented here could, if confirmed in a clinical setting, guide clinicians in the choice of treatment and thus give a more individualized and effective therapy for patients with HNSCC.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 37 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 90
National Category
Otorhinolaryngology
Identifiers
urn:nbn:se:liu:diva-17781 (URN)978-91-7393-736-8 (ISBN)
Presentation
(English)
Supervisors
Available from: 2009-06-29 Created: 2009-04-20 Last updated: 2009-09-15Bibliographically approved
2. Predictive markers: for treatment sensitivity in head and neck squamous cell carcinoma
Open this publication in new window or tab >>Predictive markers: for treatment sensitivity in head and neck squamous cell carcinoma
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck cancer is the sixth most common cancer world wide. In Sweden approximately 850 new cases are diagnosed each year, and two thirds are men. The past decades of improved treatment strategies have unfortunately not significantly improved the five-year survival rates for this group of patients. Therefore, it is important to rapidly find combinations of new and strong predictive markers for treatment response. Different predictive markers have been investigated for decades, without succeeding in finding means to securely predict response to treatment. Models to combine markers are called for.

The aim of this thesis was to test multiple predictive markers on both gene and protein level to evaluate their predictive value for radiotherapy and cisplatin response. Furthermore, to combine, and correlate them to treatment response in order to extract the panel of markers that strongest correlated to the investigated treatment. Cell lines derived from 42 patients with head and neck squamous cell carcinoma (HNSCC) were used for protein quantification with Western blot and ELISA of the proteins survivin, Epidermal Growth Factor Receptor, Bcl-2, Bcl-XL, Bax, Bad, Bak, PUMA, Heat shock protein 70, MDM2, p53, SMAD4, Cyclooxygenase-2, and Cyclin D1. The expression of the selected proteins was related to the mean expression of normal oral keratinocytes (NOK) from healthy individuals. Furthermore, mutations in the p53 gene, along with single nucleotide polymorphisms in the genes of p53, MDM2, FGFR4, XRCC1, XRCC3, XPD, and XPC were analysed. To allow a large number of predictive markers on both protein and gene level to be combined and correlated to treatment response, the number of negative points (NNP) model was introduced. Both correlations of sensitivity to radiotherapy and to cisplatin treatment was analysed among the cell lines. In the first paper, including nine cell lines, the panel of EGFR, survivin, and splice site/missense p53 mutations correlated strongest to radioresponse. In paper II, 42 cell lines were used and the combination of survivin, Bcl-2, Bcl-XL, Bax, COX-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse. In paper IV, the panel correlating strongest with cisplatin sensitivity consisted of EGFR, Hsp70, Bax, and Bcl-2 in combination with SNPs in the DNA-repair genes XRCC3 and XPD.

The predisposition of the FGFR4 Gly388Arg polymorphism for the development of HNSCC was investigated in paper III. DNA was isolated from 110 tumour biopsies, and restriction fragment length polymorphism analysis showed that 58% of the individuals in the control group carried the FGFR4 Arg388 allele, whereas the frequency in the tumour group was 45%. The Gly388 allele gave a significantly higher risk of developing HNSCC, suggesting Gly388 to be the risk allele for cancer development. Furthermore, a novel mutation was found in the FGFR4 gene. The influence of this new mutation is however unknown.

In conclusion, predictive markers for treatment sensitivity need to be combined to receive an accurate prediction of treatment response.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2010. 84 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1205
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-61586 (URN)978-91-7393-319-3 (ISBN)
Public defence
2010-11-12, Elsa Brändströmsalen, Campus US, Linköpings universitet, Linköping, 09:00
Opponent
Supervisors
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2012-05-09Bibliographically approved
3. Predictive Markers of Treatment Resistance in Head and Neck Squamous Cell Carcinoma
Open this publication in new window or tab >>Predictive Markers of Treatment Resistance in Head and Neck Squamous Cell Carcinoma
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck cancer is a common malignancy with approximately 600 000 new cases yearly. Disappointingly, the overall survival has not increased over the last decades. The concept of personalized medicine, i.e. to treat every patient with an individually planned treatment regime has gathered increased interest, but requires the establishment of novel biomarkers that can predict treatment response.

The aim of this thesis is to propose novel predictive single markers or combinations of markers of response to radiation, cisplatin and cetuximab. The general methodology is to evaluate common differences of cell lines resistant to radiation, cisplatin or cetuximab compared to sensitive counterparts.

In paper I, we analysed the expression of 14 proteins involved in growth control and/or apoptosis by western blot and related them to intrinsic radiosensitivity (IR) in nine cell lines. No factor had a significant correlation to IR on its own. A combination of EGFR, survivin, Bak, Smad4, and Hsp70 had the best correlation to IR (R=0.886, p=0.001). Additionally, we analysed the presence of p53 mutations in the cell lines. All cell lines had at least one missense, splice site or loss of transcript mutation. To be able to combine protein expression and presence of p53 mutations we created a system designated the number of negative points (NNP). With this system we could extract that expression of EGFR, survivin, and p53 missense or splice site mutations had the best correlation to IR (R=0.990, p<0.001).

In paper II we conducted a gene expression microarray analysis of three cell lines, from which common deregulations in two cisplatin resistant cell lines was compared to a cisplatin sensitive cell line. From a bioinformatic approach of gene ontology and molecular network analysis, we defined a transcriptional profile of 20 genes. Finally, key findings were analysed in a larger panel of cell lines, where high MMP-7 expression correlated with higher cisplatin resistance.

Paper III compared 4 cell lines with high IR to a radiosensitive equivalent. Using a similar bioinformatic approach as paper II, we established a transcriptional profile of 14 genes. Analysis in a larger panel of cell lines revealed that FN1 expression predicts higher IR.

Paper IV establishes the cetuximab sensitivity of 35 cell lines of which 12 were resistant and five were sensitive to cetuximab. After whole genome gene copy number analysis of five cetuximab resistant and five cetuximab sensitive cell lines, and verification of key findings in a larger cell line panel, the results show that the amplification of the YAP1 gene is coupled to cetuximab resistance.

In summary, this thesis proposes a number of novel markers of resistance to radiation, cisplatin, and cetuximab which could influence treatment choice in the future, following verifications in primary tumor material.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 83 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1291
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-76152 (URN)978-91-7519-968-9 (ISBN)
Public defence
2012-04-27, Elsa Brändströmsalen, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2012-03-29 Created: 2012-03-29 Last updated: 2012-03-29Bibliographically approved

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Farnebo, LovisaJerhammar, FredrikVainikka, LindaNorberg-Spaak, LenaRoberg, Karin

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Oto-Rhiono-Laryngology and Head & Neck Surgery Faculty of Health SciencesDepartment of ENT - Head and Neck Surgery UHLCell Biology
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