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Biomarkers in non-small cell lung carcinoma: methodological aspects and influence of gender, histology and smoking habits on estrogen receptor and epidermal growth factor family receptor signalling
Örebro University, School of Health and Medical Sciences.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Non-small cell lung carcinoma is a leading cause of cancer mortality worldwide. There are gender and smoking associated differences both in tumour types and clinical outcome. Squamous cell carcinomas (SCC) are more frequent among smoking men while females develop adenocarcinomas (ADCA). NSCLC among never smokers are mainly ADCA, and occurs mostly in females. The present thesis elucidates the role of estrogen receptor (ER) and epidermal growth factor receptor family (EGFR/HER2-4) in NSCLC in the perspective of gender and histology as well as the influence of smoking on those biomarkers.

A recently developed technique, tissue micro array (TMA), was employed.The question of how much of a tumour tissue that needed to be included in a TMA for biomarker analysis was analyzed by a statistical approach. Data indicates a sample size of three cylinders of tumour tissue with a diameter of 0.6 mm each as being appropriate and cost-effective. In order to optimally use the up to thousands of different tumour samples within a TMA, it would be optimal to serially cut and store slides before performing in situ detection of proteins and nucleic acids. Applying up to date methodology, and by evaluation with image analysis, data are presented that shows that such handling of TMA slides would be possible without any loss of biomarker information.

ERα is more frequently observed in ADCA and in females and a local estradiol synthesis is supported by the presence of aromatase. ERβ is identified as a positive prognostic marker in ADCA. Smoking is associated to increased levels of ERβ mRNA. EGFR over expression is associated with a ligand. Independent phosporylation of ERα. HER-4 intracellular domain may also act as a co-activator to ERα in ADCA, especially among neversmokers. The question of ER and EGFR family signalling crosstalk as a potential target for combined targeted therapy is raised.

Place, publisher, year, edition, pages
Örebro: Örebro universitet , 2011. , 86 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 61
Keyword [en]
Non-small cell lung carcinoma, estrogen receptor, epidermal growth factor receptor, HER-4, tissue microarray, immunohistochemistry, smoking habits, in situ hybridisation
National Category
Medical and Health Sciences Cancer and Oncology
Research subject
Oncology
Identifiers
URN: urn:nbn:se:oru:diva-19725ISBN: 978-91-7668-827-4 (print)OAI: oai:DiVA.org:oru-19725DiVA: diva2:446274
Public defence
2011-11-25, Hörsal P1, Örebro universitet, Örebro, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2011-10-06 Created: 2011-10-06 Last updated: 2012-01-09Bibliographically approved
List of papers
1. Tissue microarray validation: a methodologic study with special reference to lung cancer
Open this publication in new window or tab >>Tissue microarray validation: a methodologic study with special reference to lung cancer
2009 (English)In: Cancer Epidemiology, Biomarkers and Prevention, ISSN 1055-9965, E-ISSN 1538-7755, Vol. 18, no 7, 2014-2021 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Although tissue microarray (TMA) studies of histopathologic material have been frequently reported in studies of malignant diseases, the question of sample size (i.e., the diameter and the number of tissue cylinders investigated) has been rarely discussed. This study addresses the methodologic question of sample size in a variety of tumor types.

MATERIAL AND METHODS: Material from 29 cases of lung carcinoma (small cell, squamous cell, and adenocarcinomas) was examined immunohistochemically for Ki-67 and p53 expression in virtually constructed cylinders of different diameters. The influence of tissue sample size (i.e., different numbers of virtual cylinders) was also investigated. Results from Ki-67 evaluation were analyzed as a continuous variable, whereas p53 expression was scored. p53 evaluations based on scoring in cylinders versus scoring of whole sections were also compared. Furthermore, 10 cases of endometrial and breast carcinomas were evaluated for estrogen receptor, Ki-67, and HER2 by scoring up to five cylinders.

RESULTS AND CONCLUSIONS: Tissue cylinders of 0.6 and 1.0 mm diameters were compared and found equally informative about Ki-67 expression (intraclass correlation, 0.96). A statistical approach considering intraindividual and interindividual variation data is presented, indicating that in this specific setting three cylinders per case is an adequate sample size for TMA studies. Further sampling yields only a small gain in accuracy as determined by Ki-67 quantification and p53 scoring (kappa-coefficient, 0.9). For endometrial and breast tissues, TMA scoring of three cylinders yielded excellent agreement (kappa, >0.75) compared with whole-section scoring.

Place, publisher, year, edition, pages
American Association for Cancer Research, 2009
National Category
Medical and Health Sciences Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:oru:diva-12067 (URN)10.1158/1055-9965.EPI-08-0743 (DOI)000268059700010 ()19531681 (PubMedID)2-s2.0-67650462275 (Scopus ID)
Available from: 2010-10-05 Created: 2010-10-05 Last updated: 2017-02-23Bibliographically approved
2. Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides
Open this publication in new window or tab >>Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides
2011 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 59, no 12, 1113-1121 p.Article in journal (Refereed) Published
Abstract [en]

Storage of tissue slides has been claimed to induce dramatically reduced antigen detection particularly for immunohistochemistry (IHC). With tissue microarrays, the necessity to serially cut blocks in order to obtain as much material as possible is obvious. The presumed adverse effect of storage might hamper such an approach. The authors designed an experimental setting consisting of four different storage conditions with storage time of tissue slides of up to 1 year. Detection of proteins, DNA, and mRNA was performed using IHC and in situ hybridization techniques. Slight but significant changes in IHC occurred over time. The most important factor is the primary antibody used: four showed no significant changes, whereas limited decreases in 8 antibodies could be detected by image analysis. Whether the antigen was nuclear or cytoplasmic/membranous did not matter. No major differences between different storage conditions could be shown, but storage at 4C was overall the best procedure. Furthermore, gene copy number aberrations, chromosomal translocations, and the presence of mRNA could be detected on slides stored up to 1 year. In conclusion, in tissues optimally formalin fixed and using modern histological techniques, only minute changes in tissue antigenicity are induced by long-term storage.

Place, publisher, year, edition, pages
Sage Publications, 2011
Keyword
immunohistochemistry, heat-induced antigen retrieval, antigenicity, formalin-fixed paraffin-embedded, FISH, CISH, tissue microarray
National Category
Medical Biotechnology
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-20595 (URN)10.1369/0022155411423779 (DOI)000297649800006 ()22147608 (PubMedID)2-s2.0-84856012999 (Scopus ID)
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2017-02-08Bibliographically approved
3. Estrogen receptor β in NSCLC: prevalence, proliferative influence, prognostic impact and smoking
Open this publication in new window or tab >>Estrogen receptor β in NSCLC: prevalence, proliferative influence, prognostic impact and smoking
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In non-small cell lung carcinoma (NSCLC) there are gender differences. The female gender is associated with more adenocarcinomas (ADCA), among both smokers and non-smokers compared to men. Women with NSCLC have a better prognosis compared to men, regardless of other factors. A possible role for estrogen receptor (ER) signalling has been proposed and experimental as well as epidemiological data supports this view. In a material of NSCLC (n=262), ERβ and cyclins A and A2 were studied by immunohistochemistry on formalin-fixed paraffin embedded tissue. In 137 of those cases, frozen material was available, on which expression analysis of ESR2 (ERβ) and cyclin A1 were performed. Data was correlated to histology, gender, smoking habits, stage and clinical outcome.

ERβ was expressed in 86% of the cases. ERβ was most frequently expressed in Stage I ADCAs, especially in male subjects. A correlation between ERβ expression and cyclins was observed in ADCA, also with a male predominance. ERβ transcripts had a positive prognostic impact in ADCA. ERβ transcripts were also increased in NSCLC among smokers compared to non-smokers.

In conclusion, we report data supporting a role for ERβ in lung ADCAs where ERβ could be a biomarker for future targeted therapies.

Keyword
NSCLC, ERβ, immunohistochemistry, gene expression, gender, prognosis, smoking habits
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:oru:diva-20596 (URN)
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2016-12-07Bibliographically approved
4. Nuclear HER-4 (4ICD) and Estrogen Receptor α in non-small-cell lung carcinoma
Open this publication in new window or tab >>Nuclear HER-4 (4ICD) and Estrogen Receptor α in non-small-cell lung carcinoma
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In non-small cell lung carcinoma (NSCLC) squamous cell carcinomas (SCC) occurs more frequently among men and adenocarcinomas (ADCA) in females. The presence of estrogen receptors (ERs) in NSCLC have been proposed as a factor contributing to the gender associated differences. HER-4, a member of the epidermal growth factor receptor family, has recently been identified as a co-activator to ER by action of the shredded intracellular domain, 4ICD.

Our study was performed on tissue material from 262 NSCLC. In 137 cases, frozen material was available. HER-4/4ICD, ERα, PgR and aromatase was determined by immunohistochemistry. HER-4 gene copy number and HER-4 and ERα transcripts was also studied. Data was correlated to proliferation, histology, gender, smoking habits and clinical outcome.

We report the presence of n4ICD, significantly more frequently occurring in ADCA than in SCC. Furthermore, n4ICD and ERα expression was associated with the concomitant expression of aromatase. n4ICD was negatively correlated to proliferation in ADCA. Interestingly, a strong correlation between never smoking and n4ICD positivity was found among females and in the ADCA group.

In breast carcinomas, n4ICD is a biomarker for anti-ER treatment. A number of observations including our finding of n4ICD can constitute support for anti-ER treatment strategies in NSCLC.

Keyword
NSCLC, HER-4, ERα, gender, smoking habits
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:oru:diva-20597 (URN)
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2016-12-07Bibliographically approved
5. Estrogen receptor α phosphorylation and EGFR in non-small cell lung carcinoma
Open this publication in new window or tab >>Estrogen receptor α phosphorylation and EGFR in non-small cell lung carcinoma
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Non small cell lung carcinoma (NSCLC) occurs in two major histological types, squamous cell carcinoma (SCC) and adenocarcinoma (ADCA). There are well characterized differences in the carcinogenesis between these types. For example, epidermal growth factor receptor (EGFR) is frequently amplified and over expressed in SCC whilst mutations occur in ADCA. There are also gender associated differences in the relative incidence of SCC and ADCA. Estrogen receptor (ER) driven signalling have been proposed to play a role in this context. ER occurs in two different forms, α and β. The presence of ERα in NSCLC has been a matter of discussion. Immunohistochemical (IHC) analysis has reported divergent results, but the presence of ERα transcripts has been established to be widespread in NSCLC. ERα could be activated/phosphorylated in several ways, either ligand (estradiol) dependent or ligand independent. A potential crosstalk between EGFR and ERα has been proposed by a mechanism where EGFR signalling induces phosphorylation at specific sites within the ERα receptor.

We investigated in the present study, in a material of 262 NSCLC, by IHC the presence of a phosphorylated form of ERα, pERαS118, and correlated these findings to EGFR over expression as well as an ERα co-activator, CREB, which has been associated to ligand independent phosphorylation of ERα at that specific site.

We describe a widespread occurrence of pERαS118 in both SCC and ADCA. Furthermore, pERαS118is correlated to EGFR over expression in SCC. We propose a combined targeting of ERα and EGFR crosstalk as a possibility in the targeted therapy of SCC.

Keyword
NSCLC, ERα, ERβ, Immunohistochemistry, gene expression, gender, prognosis, smoking habits
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:oru:diva-20600 (URN)
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2016-12-07Bibliographically approved

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