Stina Garvin

Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells.

Angiogenesis is regulated by the balance between pro- and antiangiogenic factors. Vascular endothelial growth factor (VEGF), acting via the receptors VEGFR-1 and VEGFR-2, is a key mediator of tumour angiogenesis. The soluble form of the VEGF receptor-1 (sVEGFR-1) is an important negative regulator of VEGF-mediated angiogenesis. The majority of breast cancers are oestrogen dependent, but it is not fully understood how oestrogen and the antioestrogen, tamoxifen, affect the balance of angiogenic factors. Angiogenesis is a result of the interplay between cancer and endothelial cells, and sex steroids may exert effects on both cell types. In this study we show that oestradiol decreased secreted sVEGFR-1, increased secreted VEGF, and decreased the ratio of sVEGFR-1/VEGF in MCF-7 human breast cancer cells. The addition of tamoxifen opposed these effects. Moreover, human umbilical vein endothelial cells (HUVEC) incubated with supernatants from oestradiol-treated MCF-7 cells exhibited higher VEGFR-2 levels than controls. In vivo, MCF-7 tumours from oestradiol+tamoxifen-treated nude mice exhibited decreased tumour vasculature. Our results suggest that tamoxifen and oestradiol exert dual effects on the angiogenic environment in breast cancer by regulating cancer cell-secreted angiogenic ligands such as VEGF and sVEGFR-1 and by affecting VEGFR-2 expression of endothelial cells.

In vivo measurement of tumor estradiol and Vascular Endothelial Growth Factor in breast cancer patients

BackgroundAngiogenesis, crucial for tumor progression, is a process regulated in the tissue micro-environment. Vascular endothelial growth factor (VEGF) is a potent stimulatory factor of angiogenesis and a negative prognostic indicator of breast cancer. VEGF is biologically active in the extracellular space and hitherto, there has been a lack of techniques enabling sampling of angiogenic molecules such as VEGF in situ. The majority of breast cancers are estrogen-dependent, and estrogen has been shown to regulate VEGF in normal breast tissue and experimental breast cancer. We investigated if microdialysis may be applicable in human breast cancer for sampling of extracellular VEGF in situ and to explore if there is an association with local estradiol and VEGF levels in normal and cancerous breast tissue.MethodsMicrodialysis was used to sample VEGF and estradiol in tumors and adjacent normal breast tissue in postmenopausal breast cancer patients. VEGF and estradiol were also measured in plasma, and immunohistochemical staining for VEGF was performed on tumor sections.ResultsWe show that in vivo levels of extracellular VEGF were significantly higher in breast cancer tumors than in normal adjacent breast tissue. There was a significant positive correlation between estradiol and extracellular VEGF in normal breast tissue. However, no correlation was detected between estradiol and VEGF in tumors or between tumor VEGF and plasma VEGF.ConclusionWe conclude that VEGF and estradiol correlates significantly in normal breast tissue. Microdialysis may be used to provide novel insight in breast tumor biology and the regulation of molecules in the extracellular space of human breast tumors in vivo.

Fibronectin 1 is a potential biomarker for radioresistance in head and neck squamous cell carcinoma

Radiotherapy remains the backbone of head and neck cancer therapy but response is sometimes impeded by tumor radioresistance. Identifying predictive biomarkers of radiotherapy response is a crucial step towards personalized therapy. The aim of this study was to explore gene expression data in search of biomarkers predictive of the response to radiotherapy in head and neck squamous cell carcinoma (HNSCC). Microarray analysis was performed on five cell lines with various intrinsic radiosensitivity, selected from a panel of 29 HNSCC cell lines. The bioinformatics approach included Gene Ontology (GO) enrichment profiling and Ingenuity Pathway Analysis (IPA). The GO-analysis detected 16 deregulated categories from which development, receptor activity, and extracellular region represented the largest groups. Fourteen hub genes (CEBPA, CEBPB, CTNNB1, FN1, MYC, MYCN, PLAU, SDC4, SERPINE1, SP1, TAF4B, THBS1, TP53 and VLDLR) were identified from the IPA network analysis. The hub genes in the highest ranked network, (FN1, SERPINE1, THBS1 and VLDLR) were further subjected to qPCR analysis in the complete panel of 29 cell lines. Of these genes, high FN1 expression associated to high intrinsic radiosensitivity (p=0.047). In conclusion, gene ontologies and hub genes of importance for intrinsic radiosensitivity were defined. The overall results suggest that FN1 should be explored as a potential novel biomarker for radioresistance.

Strong expression of survivin is associated with positive response to radiotherapy and improved overall survival in head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy that is associated with severe mortality despite advances in therapy. Todays standard treatment most commonly includes radiotherapy, often combined with chemotherapy or surgery. There are so far no established biomarkers to predict response to radiation, and thus the aim of this study was to investigate a series of markers that could potentially identify HNSCC patients who would benefit from radiotherapy. The selected markers, both proteins (epidermal growth factor receptor, survivin and p53), and single nucleotide polymorphisms (SNPs) in the genes of XRCC3, XRCC1, XPC, XPD, MDM2, p53 and FGFR4 were correlated to the response to radiotherapy and overall survival. Investigations were performed on pretreatment tumor biopsies from patients classified as responders or nonresponders to radiotherapy. Protein expression was examined using immunohistochemistry and the genotyping of specific SNPs was analyzed using PCR‐RFLP or pyrosequencing. We found that survivin expression was significantly stronger in the responder group (p = 0.003) and that patients with a strong survivin expression had a significantly better overall survival (p < 0.001). Moreover, downregulation of survivin by siRNA in two HNSCC cell lines significantly decreased their sensitivity to radiation. Among the SNPs analyzed, patients with the XPD Lys751Gln SNP had a significantly shorter overall survival (p = 0.048), and patients with the FGFR4 Gly388Arg SNP had a significantly longer overall survival (p = 0.010). In conclusion, our results suggest that survivin plays an important role in the response to radiotherapy and may be a useful marker for predicting radiotherapy response in patients with HNSCC.

The number of identified lymph node metastases increases continuously with increased total lymph node recovery in pT3 colon cancer.

Background. The positive correlation between the number of recovered benign lymph nodes and patient prognosis is well established for stage II colon cancer patients. One theory explaining this correlation focuses on potential understaging of cancer specimen, implying that a specimen with few examined lymph nodes is likely to be assigned a lower N-stage than the correct one. Understaging may be the result of an insufficient examination of the specimen post-operatively, whereby few lymph nodes are recovered and potential lymph node metastases are overlooked. This study aims to investigate the association between the total lymph node harvest and the number of lymph node metastases in colon cancer specimen. Material and methods. We studied the original pathology reports of 649 patients diagnosed with T3 adenocarcinoma of the colon at the Department of Clinical Pathology and Genetics at Linköping University Hospital, Linköping, Sweden between the years 2000 and 2008. Patient demographics, specimen staging data, and lymph node recovery data were collected for each case. Results. We found a positive association between the total lymph node harvest and the number of lymph node metastases per specimen. For every additional recovered lymph node 0.17 (95% CI: 0.15–0.19) metastases were detected (p < 0.001). Discussion. Our results support the conclusion that there is no minimum number of recovered lymph nodes at which an accurate determination of nodal status can be assured. Rather than focusing on a recommended minimum number of nodes, efforts should be shifted towards developing methods assuring that colon cancer specimen are dissected in a standardized way that optimizes the lymph node harvest.

Germline SDHA Mutation Detected by Next-Generation Sequencing in a Young Index Patient With Large Paraganglioma

Department of Clinical and Experimental Medicine (J.W., S.G., P.S.), Linkoping University, SE-58183 Linkoping, Sweden; Departments of Pathology (S.G.), Radiology (R.B.), and Vascular Surgery (L.I.), County Council of Ostergotland, SE-58185 Linkoping, Sweden; Wisconsin National Primate Research Center (R.W.W.), University of Wisconsin-Madison, Madison, Wisconsin 53715; and Department of Clinical and Experimental Medicine (O.G.), Linkoping University, Department of Surgery, County Council of Ostergotland, SE-58183 Linkoping, Sweden

Nuclear expression of WRAP53β is associated with a positive response to radiotherapy and improved overall survival in patients with head and neck squamous cell carcinoma

OBJECTIVES Today there are no reliable predictive markers for radiotherapy response in head and neck squamous cell carcinoma (HNSCC), leading to both under- and over-treatment of patients, personal suffering, and negative socioeconomic effects. Inherited mutation in WRAP53β (WD40 encoding RNA Antisense to p53), a protein involved in intracellular trafficking, dramatically increases the risk of developing HNSCC. The purpose of this study was to investigate whether WRAP53β can predict response to radiotherapy in patients with HNSCC. MATERIALS AND METHODS Tumor biopsies from patients with HNSCC classified as responders or non-responders to radiotherapy were examined for the expression of the WRAP53β protein and single nucleotide polymorphisms in the corresponding gene employing immunohistochemistry and allelic discrimination, respectively. In addition, the effect of RNAi-mediated downregulation of WRAP53β on the intrinsic radiosensitivity of two HNSCC cell lines was assed using crystal violet and clonogenic assays. RESULTS Nuclear expression of WRAP53β was significantly associated with better response to radiotherapy and improved patient survival. Downregulation of WRAP53β with siRNA in vitro enhanced cellular resistance to radiation. CONCLUSIONS Our findings suggest that nuclear expression of WRAP53β promotes tumor cell death in response to radiotherapy and is a promising predictor of radiotherapy response in patients with HNSCC.

DNA repair genes XPC, XPD, XRCC1, and XRCC3 are associated with risk and survival of squamous cell carcinoma of the head and neck

Head and neck squamous cell carcinomas (HNSCC) are a heterogenous group of tumors with a high rate of early recurrences, second primary tumors, and mortality. Despite advances in diagnosis and treatment over the past decades, the overall 5-year survival rate remains around 50%. Since the head-and neck-region is continuously exposed to potentially DNA-damaging exogenous and endogenous factors, it is reasonable to expect that the DNA repair genes play a part in the development, progression, and outcome of HNSCC. The aim of this study was to investigate the SNPs XPC A499V, XPD K751Q, XRCC1 R399Q, and XRCC3 T241M as potential risk factors and indicators of survival among Caucasian patients. One-hundred-sixty-nine patients as well as 344 healthy controls were included and genotyped with PCR-RFLP. We showed that XPC A499V was associated with increased risk of HNSCC, especially laryngeal carcinoma. Among women, XPD K751Q was associated with increased risk of oral SCC. Furthermore, XPD homozygous mutant individuals had the shortest survival time, a survival time that increased however after full dose radiotherapy. Wild-type individuals of XRCC3 T241M demonstrated an earlier age of onset. HPV-positive never smokers had lower frequencies of p53 mutation. Among HNSCC patients, HPV-positivity was significantly associated with XRCC1 R399Q homozygous mutant genotype. Moreover, combinations of putative risk alleles seemed to act synergistically, increasing the risk of HNSCC. In conclusion, our results suggest that SNPs of the DNA repair genes XPC, XPD, XRCC1, and XRCC3 may affect risk and survival of HNSCC.

Fusion between M2-macrophages and cancer cells results in a subpopulation of radioresistant cells with enhanced DNA-repair capacity

Cell fusion is a natural biological process in normal development and tissue regeneration. Fusion between cancer cells and macrophages results in hybrids that acquire genetic and phenotypic characteristics from both maternal cells. There is a growing body of in vitro and in vivo data indicating that this process also occurs in solid tumors and may play a significant role in tumor progression. However, investigations of the response of macrophage:cancer cell hybrids to radiotherapy have been lacking. In this study, macrophage:MCF-7 hybrids were generated by spontaneous in vitro cell fusion. After irradiation, both hybrids and their maternal MCF-7 cells were treated with 0 Gy, 2.5 Gy and 5 Gy γ-radiation and examined by clonogenic survival and comet assays at three time points (0 h, 24 h, and 48 h). Compared to maternal MCF-7 cells, the hybrids showed increased survival fraction and plating efficiency (colony formation ability) after radiation. The hybrids developed less DNA-damage, expressed significantly lower residual DNA-damage, and after higher radiation dose showed less heterogeneity in DNA-damage compared to their maternal MCF-7 cells. To our knowledge this is the first study that demonstrates that macrophage:cancer cell fusion generates a subpopulation of radioresistant cells with enhanced DNA-repair capacity. These findings provide new insight into how the cell fusion process may contribute to clonal expansion and tumor heterogeneity. Furthermore, our results provide support for cell fusion as a mechanism behind the development of radioresistance and tumor recurrence.Cell fusion is a natural biological process in normal development and tissue regeneration. Fusion between cancer cells and macrophages results in hybrids that acquire genetic and phenotypic characteristics from both maternal cells. There is a growing body of in vitro and in vivo data indicating that this process also occurs in solid tumors and may play a significant role in tumor progression. However, investigations of the response of macrophage:cancer cell hybrids to radiotherapy have been lacking. In this study, macrophage:MCF-7 hybrids were generated by spontaneous in vitro cell fusion. After irradiation, both hybrids and their maternal MCF-7 cells were treated with 0 Gy, 2.5 Gy and 5 Gy γ-radiation and examined by clonogenic survival and comet assays at three time points (0 h, 24 h, and 48 h). Compared to maternal MCF-7 cells, the hybrids showed increased survival fraction and plating efficiency (colony formation ability) after radiation. The hybrids developed less DNA-damage, expressed significantly lower residual DNA-damage, and after higher radiation dose showed less heterogeneity in DNA-damage compared to their maternal MCF-7 cells. To our knowledge this is the first study that demonstrates that macrophage:cancer cell fusion generates a subpopulation of radioresistant cells with enhanced DNA-repair capacity. These findings provide new insight into how the cell fusion process may contribute to clonal expansion and tumor heterogeneity. Furthermore, our results provide support for cell fusion as a mechanism behind the development of radioresistance and tumor recurrence.

Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment‐induced reduction of EGFR, pEGFR, and pSrc

BACKGROUND The aims of this study were 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 epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. MATERIALS AND METHODS The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. RESULTS A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. CONCLUSION High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response.