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Understanding the Role of EGFR in the Treatment of Head and Neck Squamous Cell Carcinoma
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck squamous cell carcinoma (HNSCC) originates from the epithelial lining of the upper aerodigestive tract. It accounts for over 90 % of the malignancies found in the head and neck region. 600,000 new cases of HNSCC occur each year worldwide. Apart from causing painful lesions, HNSCC directly impacts the patient’s fundamental functions such as breathing and eating and also can disrupt the patient’s senses such as smell, taste, speech and even vision. Most cases of HNSCC require a combination of different treatments such as surgery, chemotherapy (primarily cisplatin based), and radiotherapy. Treatment decisions are largely based on the size of the tumor, the involvement of local lymph nodes, and distant spread. Treatment resistance and local recurrence are significant problems and to date no form of clinical treatment sensitivity  prediction is available.

A majority of HNSCC tumors overexpresses the epidermal growth factor receptor (EGFR). This receptor is involved in proliferation and DNA repair and is the target of a monoclonal antibody named cetuximab that selectively binds and inhibits EGFR. It is the only targeted therapy available to HNSCC patients and reserved for late stage patients in  Sweden.

Numerous investigators have searched for predictive markers and we hypothesized that since HNSCC is a very heterogeneous disease a single factor would not be able to predict the treatment outcome. In paper I we explore a panel of predictive factors using a point system, called the number of negative points (NNP), in which we could combine both proteins and genetic variations in an attempt to find a set of markers that could predict the intrinsic cisplatin sensitivity (ICS). The expression level of EGFR, Hsp70, Bax, Bcl-XL, survivin, and COX-2 was determined in 39 HNSCC cell lines. Moreover, the p53, MDM2, FGFR4, XPC, XPD, XRCC1, and XRCC3 genes were analyzed for the presence of specific single nucleotide polymorphisms (SNPs). Pearson’s correlation tests showed that EGFR was the only protein that alone correlated to ICS (r=0.388, P=0.015). The strongest correlation to ICS was found when combining SNPs in XRCC3 and XPD with the expression of EGFR, Hsp70, Bax, and Bcl-2 using the NNP system (r=0.614 P≤0.001).

In paper II we assess the intrinsic radiosensitivity (IR), the ICS, and the intrinsic cetuximab sensitivity (ICmabS) as well as their combinations in 25 HNSCC cell lines established from HNSCC biopsies taken at the Department of Otorhinolaryngology and Head and Neck Surgery at Linköping University Hospital. Furthermore we investigate the EGFR status (consisting of EGFR gene copy number, EGFR mRNA, EGFR protein, pEGFR), pAkt and mRNA levels of the seven known EGFR ligands. No correlation was found between the different treatment sensitivities. Cetuximab treatment response was significantly correlated to epiregulin (EREG) mRNA expression (r=-0.408, P=0.043). Cetuximab resistant cell lines tended to have low levels of pEGFR (P=0.13) while resistant cell lines had a significantly lower expression of EGFR protein (P=0.04) and tended to have decreased levels of pAkt (P=0.13) and amphiregulin (AREG) mRNA (p=0.18).

In paper III the functional importance of EGFR ligands in relation to proliferation and cetuximab sensitivity was investigated. Here we tried to diminish the tumor heterogeneity by selecting three cell lines that are derived from the same anatomical location but display different ICmabS. Signaling through the EGFR was stimulated with recombinant EGF, AREG or EREG or reduced by siRNA-mediated silencing of the aforementioned EGFR ligands. EGF downregulation suppressed the proliferation of all investigated tumor cell lines whereas the response to an increased level of EGF differed between EGFR overexpressing and EGFR non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of EGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AREG and EREG were inconclusive.

In paper IV we wanted to validate in vitro drug sensitivity testing of HNSCC cell lines in an in vivo xenograft model, and to identify treatment-induced changes in the EGFR signaling pathway that could be used as markers for cetuximab treatment response. In vitro ICmabS for the HNSCC cell lines UT-SCC-14 and UT-SCC-45 was established using a crystal violet assay. In order to determine the corresponding in vivo sensitivity, UT-SCC-14 and UT-SCC-45 xenografts were generated in female BALB/c (nu/nu) nude mice. Mice were given three injections of intraperitoneal cetuximab or PBS and the tumor volume was recorded continuously. The expression of EGFR, pEGFR, pSrc, and Ki67 in the tumor tissue was investigated by immunohistochemistry. The in vitro sensitivity was reproduced in the in vivo model. Furthermore a clear reduction of EGFR, pEGFR, and pSrc after cetuximab treatment was noted in UT-SCC-14, the cetuximab sensitive cell line while the cetuximab resistant UT-SCC-45 showed a slight increase in EGFR, pEGFR and pSrc.

In conclusion, the EGFR ligand EGF is a potential predictive marker of poor cetuximab response and a possible treatment target. Moreover, treatment-induced downregulation of EGFR and pEGFR is associated with a good cetuximab response.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. , 74 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1432
National Category
Other Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-113744DOI: 10.3384/diss.diva-113744ISBN: 978-91-7519-176-8 (print)OAI: oai:DiVA.org:liu-113744DiVA: diva2:784460
Public defence
2015-03-06, Eken, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2015-01-29 Created: 2015-01-29 Last updated: 2015-01-29Bibliographically approved
List of papers
1. Proteins and single nucleotide polymorphisms involved in apoptosis, growth control, and DNA repair predict cisplatin sensitivity in head and neck cancer cell lines
Open this publication in new window or tab >>Proteins and single nucleotide polymorphisms involved in apoptosis, growth control, and DNA repair predict cisplatin sensitivity in head and neck cancer cell lines
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2009 (English)In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 24, no 4, 549-556 p.Article in journal (Refereed) Published
Abstract [en]

The present study was undertaken to evaluate the possibility of using a panel of proteins and single nucleotide polymorphisms (SNPs) involved in apoptosis, growth control, and DNA repair as predictive markers for cisplatin sensitivity. For this purpose the intrinsic cisplatin sensitivity (ICS) was determined in 39 cell lines derived from squamous cell carcinomas of the head and neck using a colony-forming assay. In these cell lines and in normal oral keratinocytes (NOK), the expression of epidermal growth factor receptor (EGFR), Hsp70, Bax, Bcl-2, Bcl-XL, survivin, and COX-2 was determined. Moreover, the p53, MDM2, FGFR4, XPC, XPD, XRCC1, and XRCC3 genes were analyzed for the presence of specific single nucleotide polymorphisms (SNPs). Pearsons correlation test showed that EGFR was the only protein that was significantly correlated to the ICS (r=0.388, p=0.015). The combination of EGFR, Hsp70, Bax, and Bcl-2 gave the strongest correlation (r=0.566, p andlt;= 0.001), whereas Bax alone had the second highest influence on the ICS. Furthermore, all four SNPs within genes involved in DNA repair, i.e. XPC, XPD, XRCC1, and XRCC3, tended to influence the ICS. In order to find the combination of factors, on both protein and gene levels, with the highest correlation to ICS, a multivariate statistical calculation was performed. Our results indicate that SNPs in DNA repair genes (XRCC3(241) and XPD751) influence the ICS and together with the expression of EGFR, Hsp70, Bax, and Bcl-2, they could predict the cisplatin sensitivity of head and neck cancer cell lines (r=0.614, p andlt;= 0.001).

Keyword
epidermal growth factor receptor, Bax, Bcl-2, heat shock protein 70, DNA repair genes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-21513 (URN)10.3892/ijmm_00000264 (DOI)
Available from: 2009-10-02 Created: 2009-10-02 Last updated: 2017-12-13
2. EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines
Open this publication in new window or tab >>EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines
2013 (English)In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, Vol. 42, no 1, 26-36 p.Article in journal (Refereed) Published
Abstract [en]

Background: Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. Methods: Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. Results: Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). Conclusions: Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

Place, publisher, year, edition, pages
John Wiley and Sons, 2013
Keyword
chemoradiotherapy, EGFR, epiregulin, Erbitux (R), HB-EGF, SCCHN, TGF-a, treatment response
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-87963 (URN)10.1111/j.1600-0714.2012.01177.x (DOI)000312998500004 ()
Note

Funding Agencies|Swedish Laryng Foundation||Foundation of Olle Engkvist, Building Contractor||Linkoping University Hospital||

Available from: 2013-01-28 Created: 2013-01-28 Last updated: 2017-12-06
3. Epidermal growth factor is a biomarker for poor cetuximab response in tongue cancer cells
Open this publication in new window or tab >>Epidermal growth factor is a biomarker for poor cetuximab response in tongue cancer cells
2016 (English)In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, Vol. 45, no 1, 9-16 p.Article in journal (Refereed) Published
Abstract [en]

Background: Epidermal growth factor receptor (EGFR) is a target for treatment in tongue cancer. Here, EGFR ligands were evaluated for their potential uses as predictive biomarkers of cetuximab treatment response.

Methods: In three tongue cancer cell lines the influences of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumour cell proliferation and cetuximab response were evaluated by the addition of recombinant human (rh) proteins or the siRNA-mediated downregulation of endogenous ligand production.

Results: EGF or AR downregulation suppressed the proliferation of all investigated cell lines. Furthermore, all cell lines displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing resulted in an improved cetuximab response in one cell line.

Conclusions: Our data suggest that EGF and AR are critical components of the EGFR signalling network required for full proliferative potential. Moreover, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2016
Keyword
Head and neck cancer; Tongue cancer; Erbitux; EGFR ligands; treatment response
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-100675 (URN)10.1111/jop.12310 (DOI)000369990100003 ()
Note

Funding agencies: Foundation of Ake Wiberg; Swedish Cancer Society [2008/552, 2010/545]; County Council of Ostergotland; Linkoping University Hospital; Foundation of Magnus Bergvall; Cancer Foundation of Ostergotland

Vid tiden för disputation förelåg publikationen endast som manuskript

Available from: 2013-11-11 Created: 2013-11-11 Last updated: 2017-05-03
4. Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction of EGFR, pEGFR, and pSrc
Open this publication in new window or tab >>Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction of EGFR, pEGFR, and pSrc
Show others...
2017 (English)In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, no 9, 717-724 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The aim of this study was to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC)cell lines in an in vivo xenograft model, and to identify treatment-induced changes in the EGFR signaling pathway that could be used as markersfor cetuximab treatment response.

METHODS: The in vitro cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UTSCC-45, was assessed using a crystal violet assay. In order to determine the corresponding in vivo sensitivity, UT-SCC-14 and UT-SCC-45 xenografts were generated in female BALB/c (nu/nu) nude mice. Mice were given three injections of intraperitoneal cetuximab or PBS and the tumor volume was recorded continuously. The expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki67 was investigated by immunohistochemistry.

RESULTS: The treatment sensitive UT-SCC-14 cells were found to have an intrinsic cetuximab sensitivity (ICmabS) of 0.15 whereas the ICmabS of the insensitive cell line UT-SCC-45 was 0.78. The corresponding size ratio between untreated and cetuximab treated xenografts was 0.22 and 0.83 for UT-SCC-14 and UT-SCC-45, respectively. UT-SCC-14 cells had a higher baseline expression of pEGFR as compared to UT-SCC-45. Furthermore, in UT-SCC-14 xenografts there was a decrease in EGFR, pEGFR and pSrc upon cetuximab treatment. In contrast, a slight cetuximab-induced increase in EGFR, pEGFR and pSrc was observed in treatment-resistant UT-SCC-45 xenografts.

CONCLUSIONS: The in vitro treatment sensitivity was reproduced in the in vivo model and cetuximab sensitivity was found to associate with a treatment-induced reduction in pEGFR and pSrc.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
National Category
Medical and Health Sciences Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-113742 (URN)10.1111/jop.12545 (DOI)000412303500009 ()28036101 (PubMedID)
Note

Funding agencies: Foundation of Ake Wiberg; County Council of Ostergotland; Research Funds of Linkoping University Hospital; Cancer Foundation of Ostergotland; Foundation of Magn. Bergvall; The Swedish Cancer Society [2010/545]

The previous status of this publication was Manuscript

Available from: 2015-01-29 Created: 2015-01-29 Last updated: 2017-10-24Bibliographically approved

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