Peter Krippl

A common 936 C/T gene polymorphism of vascular endothelial growth factor is associated with decreased breast cancer risk

A common 936 C/T polymorphism in the gene for the vascular endothelial growth factor (VEGF) has been associated with VEGF plasma levels. In our case‐control study, we investigated the role of this polymorphism for breast cancer risk. VEGF genotype was determined in 500 women with breast cancer and 500 sex‐ and age‐matched healthy control subjects. Carriers of a 936T‐allele were more frequent among controls (29.4%) than among patients (17.6%; p = 0.000014). The odds ratio for carriers of a 936T‐allele for breast cancer was 0.51 (95% confidence interval 0.38–0.70). Additionally, VEGF plasma levels were determined in 21 nonsmoking post‐menopausal controls; carriers of a 936T allele had significantly lower levels (median 23 pg/ml; range 6–50 pg/ml) than noncarriers (37; 21–387; p = 0.034). We conclude that carriers of a VEGF 936T‐allele are at decreased risk for breast cancer, this, however, requiring further confirmation in a larger study.

Improvement of insulin sensitivity in insulin resistant subjects during prolonged treatment with the anti‐TNF‐α antibody infliximab

Sir, Insulin resistance (IR) is an important component in the pathophysiology of type-II diabetes, hypertension, hyperlipidaemia and finally cardiovascular disease with both genetic and environmental factors contributing to its development. Tumour necrosis factor alpha (TNFα ), a proinflammatory cytokine, may play a prominent role in obesity associated IR as well as β -cell dysfunction [1]. Elevated expression of TNFα in obese insulin resistant rodents and humans was observed along with the indication of elevated plasma TNFα in some studies. TNFα has tissue specific effects on glucose homeostasis and is known to impair insulin receptor signalling experimentally. Furthermore, cytokines that activate (NF)-kB (a nuclear transcription factor closely involved into regulation of cellular inflammatory response), such as TNFα , are thought to be a common denominator for β cell apoptosis in type 1 and type 2 diabetes [2]. In addition, it has been suggested that TNFα is a powerful regulator of adipose tissue [3]. Neutralizing TNFα in obese fa/fa-rats has shown increased insulin sensitivity [4]. So far, in humans two studies failed to demonstrate an effect of acute administration of either a chimeric anti-TNFα antibody [5] or a recombinant soluble human TNFα receptor [6] on insulin sensitivity in obese or type-II diabetic subjects. Prolonged administration of anti–TNFα antibody is used in the treatment of chronic inflammatory diseases such as rheumatic diseases [7] or Crohn’s disease [8]. Here we report observational findings of chronic treatment with infliximab (a chimeric anti-TNFα antibody) on insulin sensitivity as assessed by the homeostasis model assessment (HOMA: fasting plasma glucose (mmol L − 1 ) × fasting serum insulin (mU L − 1 ) divided by 225 [9]), in such patients. Our index case, a 31 years old male with a body-mass-index (BMI) of 31·2 kg m − 2 was diagnosed with type-II diabetes in November 1999 with an HbA 1c of 9·2%. The patient lost weight within 8 months to a BMI of 22·4 kg m − 2 and was treated with insulin (approx. 28 IU per day). As a result stable, good glycaemic control (fasting blood glucose: 6·05 mmol L − 1 ) in the absence of hypoglycaemia could be reached and maintained till 2001. In January 2001 treatment with infliximab (5 mg kg − 1 ) every 8 weeks, was started due to refractory psoriatic arthritis. At this time point he exhibited an unexpectedly severe insulin resistance with a HOMA of 36·98 despite maintenance of normal body weight (In healthy subjects the median HOMA is about 1·4 whereas only 10% of them exhibit a HOMA between 4·5 and 20). After 4–5 months, the patient noticed a decline in insulin requirements and the occurrence of hypoglycaemia. In October 2001, after a severe hypoglycaemic episode, the patient stopped insulin treatment. In July 2002, still on infliximab treatment glucose status was reevaluated. Fasting plasma glucose was 5·17 mmol L − 1 and 2-h after challenge with 75 g glucose 10·56 mmol L − 1 were observed indicating the presence of impaired glucose tolerance instead of insulin requiring diabetes. The respective serum insulin was 32·8 mU L − 1 and 96·4 mU L − 1 , HbA1c was 5·4%. Today the patient is still without insulin therapy. This observation prompted us to evaluate insulin sensitivity retrospectively, using the HOMA, in samples stored from patients treated with infliximab after obtaining informed consent (Fig. 1). Interestingly, with exception of our lean index patient, only the most obese patients (patients 3 and 5), available for analysis (Table 1), showed a pronounced insulin resistance at baseline and improved substantially. In contrast, the other patients (patients 2 and 4) with a lower BMI were insulin sensitive at baseline and did not change during infliximab Division of Rheumatology (B. Yazdani-Biuki, H. P. Brezinschek, J. Hermann, T. Mueller, W. Graninger), Diabetes and Metabolism Clinic (H. Stelzl, T. C. Wascher), and Division of Oncology ( P. Krippl), Department of Internal Medicine, Medical University Graz, Austria.

Immunohistochemical analysis of desmoid tumours.

Background/Aims: Although the standard treatment for desmoid tumours is complete surgical resection with wide margins, the optimal adjuvant treatment for recurrent or inoperable disease is unclear, often being based on sporadic immunohistochemical reports with a low number of cases. Therefore, a large immunohistochemical study was performed, to provide a theoretical basis for adjuvant treatment regimens. Methods: One hundred and sixteen tissue samples from 80 patients (49 female, 31 male; mean age, 34 years; range, 0–83) with desmoid tumours (46 extra-abdominal, 21 abdominal, 13 intra-abdominal) were tested for oestrogen receptors α and β, progesterone and androgen receptors, and somatostatin, in addition to HER2, cathepsin D, Ki-67, and c-KIT by immunohistochemistry. Results: All samples were negative for oestrogen receptor α, HER2, and the progesterone receptor. Positive staining for the androgen receptor was found in six extra-abdominal cases. Staining for oestrogen receptor β was positive in four extra-abdominal, two abdominal, and one intra-abdominal case. Staining for somatostatin was positive in six extra-abdominal, two abdominal, and one intra-abdominal case, and staining for cathepsin D was positive in all cases. Positive staining for Ki-67 was found in 14 extra-abdominal, three abdominal, and three intra-abdominal cases. C-KIT was detectable in one abdominal case only. Conclusions: The data from this immunohistochemical study show that the published effects of antioestrogens and imatinib mesylate in the treatment of aggressive fibromatoses may not be attributable to oestrogen receptor α or c-KIT expression.

INTERLEUKIN-10 PROMOTER POLYMORPHISM IS ASSOCIATED WITH DECREASED BREAST CANCER RISK

Interleukin-10 (IL-10) is an immunosuppressive cytokine which may facilitate development of cancer by supporting tumor escape from the immune response. A [TCATA] haplotype formed by polymorphisms at positions −3575, −2763, −1082, −819 and −592 in the promoter of the IL-10 gene is a strong determinant for IL-10 expression. The presence of this haplotype can be determined by analysis of the −592C > A polymorphism. Aim of the present study was to analyze the role of the IL-10 [TCATA] haplotype for breast cancer. We performed a case–control study including 500 female patients with histologically confirmed breast cancer and 500 female, age-matched, healthy control subjects from population-based screening studies. The −592C > A polymorphism was determined by a 5′-nuclease assay (TaqMan). Frequency of the homozygous −592 AA genotype, indicating homozygosity for the [TCATA] haplotype, was 4.2% among patients and 7.3% among controls (p=0.038; odds ratio 0.56; 95% confidence interval 0.32–0.97). IL-10 genotypes were not associated with tumor size, histological grading, estrogen or progesterone receptor status and age at diagnosis. Therefore we conclude that the IL-10 −592C > A promoter polymorphism may be associated with a reduced breast cancer risk.

The L10P polymorphism of the transforming growth factor-beta 1 gene is not associated with breast cancer risk

Transforming growth factor-beta 1 (TGF-beta1) is a potent inhibitor of proliferation of epithelial, endothelial and hematopoietic cells and acts as a tumor suppressor. The gene for TGF-beta1, TGFB1, carries a common T/C variation of nucleotide 29, resulting in a leucine (L) to proline (P) polymorphism at codon 10 (TGFB1 L10P). The less common 10P allele has repeatedly been linked to higher TGF-beta1 levels and in at least one study to a lower incidence of breast cancer. To further analyze the role of this polymorphism for breast cancer risk, 500 patients with histologically confirmed breast cancer and 500 sex-and age-matched healthy control subjects were genotyped for the TGFB1 L10P polymorphism by an allele-specific polymerase chain reaction assay. TGFB1 LL, LP and PP genotype frequencies were not significantly different for patients (39.6, 44.2, 16.2%) and controls (36.5, 45.9, 17.6%). We conclude that the TGFB1 L10P polymorphism is not associated with breast cancer risk.

The common 677C>T gene polymorphism of methylenetetrahydrofolate reductase gene is not associated with breast cancer risk.

Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and plays a role in DNA biosynthesis, methylation, and repair in actively dividing cells. A common 677C>T polymorphism in the gene for MTHFR, leading to a thermolabile enzyme with decreased activity, has been associated with reduced plasma folate levels and elevated homocysteine levels and could be a risk factor for breast cancer. In the present case-control study, MTHFR genotype was determined in 500 women with clinically verified breast cancer and 500 female age-matched healthy control subjects. The homozygous TT genotype was found in 13.0% patients and 13.1% controls (P = n.s.). The odds ratio of TT homozygotes for breast cancer was 0.99 (95% confidence interval 0.68–1.43). The MTHFR genotype was furthermore not associated with tumor size, histological grading, estrogen or progesterone receptor status and age at diagnosis. In a subgroup of 116 premenopausal patients, no increased frequency of the homozygous 677T genotype was found (13.8%). Therefore, we conclude that the MTHFR 677C>T polymorphism is not associated with individual susceptibility to breast cancer.

The 870G>A Polymorphism of the Cyclin D1 Gene is not Associated with Breast Cancer

A common 870G > A polymorphism in the gene for cyclin D1, CCND1, has been linked to alternative splicing and cancer susceptibility. To analyze its role for breast cancer, we determined the CCND1 genotype in 500 breast cancer patients and 500 controls. CCND1 genotype frequencies were similar among patients and controls. The CCND1 genotype was furthermore not associated with tumor characteristics. We conclude that the CCND1 870G > A polymorphism is not associated with breast cancer.

The 5A/6A Polymorphism of the Matrix Metalloproteinase 3 Gene Promoter and Breast Cancer

Purpose: The matrix metalloproteinase 3 (MMP3), also known as stromelysin-I, is a key-player for carcinogenesis and tumor growth. A 5A/6A promoter polymorphism is associated with differences in MMP3 activity and has been linked to cancer susceptibility in some studies. In the present study we evaluated the role of this polymorphism for breast cancer risk. Experimental Design: A case–control study was performed including 500 patients with histologically confirmed breast cancer and 500 female, age-matched, healthy control subjects from population-based screening studies. The MMP3 5A/6A polymorphism was determined by a 5′-nuclease (TaqMan) assay. Results: Prevalences of 5A/5A, 5A/6A, and 6A/6A genotypes were similar among patients (20.6, 51.8, and 27.6%, respectively) and controls (23.3, 47.3, and 29.4%, P = 0.34). The odds ratio of carriers of a MMP3 5A allele for breast cancer was 1.09 (95% confidence interval, 0.83–1.44). Patients with the 5A/5A genotype had a higher proportion of lymph-node metastases than those with a 5A/6A or 6A/6A genotype (P = 0.010). Conclusions: The MMP3 5A/6A promoter polymorphism does not appear to influence breast cancer susceptibility but may be linked to a higher risk for metastasizing among breast cancer patients.

Single nucleotide polymorphisms in the hypoxia-inducible factor-1 gene and colorectal cancer risk.

With an incidence of about 300 000 new cases colorectal cancer (CRC) is the second leading cause of cancer‐related death in Europe and the United States. Environmental and genetic factors influence CRC risk. Hypoxia‐inducible factor‐1 (HIF‐1), a heterodimeric protein composed of two subunits, HIF‐1 alpha and HIF‐1 beta, plays a critical role in oxygen homeostasis and is involved in angiogenesis and cell proliferation. The gene for the HIF‐1 alpha subunit (HIF1A) carries two common missense mutations—P582S (rs11549465) and A588T (rs11549467)—which both have been related to increased trans‐activation capacity of HIF1A. In our case–control study we investigated the association between these polymorphisms and CRC risk. We investigated 381 patients with histologically confirmed CRC and 2156 control subjects. HIF1A genotypes were determined by exonuclease (TaqMan) assays. For determination of microvessel density (MVD) tumor sections were stained using a mouse monoclonal antibody recognizing the pan‐endothelial marker CD31. In a multivariate logistic regression analysis including age and sex neither the HIF1A 582S allele (Odds ratio: 1.204; 95% confidence interval 0.911–1.592; P = 0.193) nor the 588T allele was significantly associated with CRC (Odds ratio: 0.851; 95% confidence interval 0.444–1.631; P = 0.626). However, in an exploratory analysis, the HIF1A 588T allele was associated with tumor localization (P = 0.016) and tumor size (P = 0.003). MVD was similar in tumors of patients carrying HIF1A 588T allele and patients without this rare allele. We conclude that functional polymorphisms in the HIF1A gene do not modify CRC risk but maybe associated with clinic‐pathological features of the disease.

Integrin alpha-2 and beta-3 gene polymorphisms and breast cancer risk

SummaryIntegrins are cell surface receptors, which mediate cell-to-cell and cell-to-extracellular matrix adhesion. Some of them, e.g. αVβ3, αIIbβ3 and α2β1, have been suggested as key players for cancer development and tumor metastasis. Two polymorphisms in the gene for the α2 component, ITGA2 807C>T and 1648G>A, have been associated with the cell-surface density of integrin α2β1. The 176T>C polymorphism in the ITGB3 gene, encoding the β3 subunit of integrins αIIbβ3 and αVβ3, modifies a variety of traits of β3 expressing cells. To analyze the role of ITGA2 and ITGB3 polymorphisms for breast cancer risk and prognosis, we performed a case-control study including 500 female breast cancer patients and 500 healthy female age-matched control subjects. All study participants were of Caucasian origin (Austria, Middle-Europe). The ITGA2 1648_AA genotype was significantly associated with breast cancer (odds ratio 3.12; 95% confidence interval 1.11–8.77). Carriers of the most common ITGA2 haplotype (807C_1648G, ‘wildtype’) were at decreased risk for breast cancer (odds ratio 0.72; 95% confidence interval 0.53–0.98). A histological grade of 3 or 4 was found more often in ITGA2 807TT subjects (p=0.039 compared to CC+CT genotypes) and carriers of an ITGA2 1648A allele (p=0.017 compared to GG genotype). Carriers of the ITGA2 807C_1648G haplotype were less likely to have a histological grade 3 or 4 compared to non-carriers (p=0.003). The ITGB3 176T>C polymorphisms was not associated with breast cancer susceptibility. In a Cox-regression analysis, carriers of the homozygous ITGB3 176-CC genotype had a higher risk for metastasis (relative risk 2.3; 95% CI 1.3–4.2; p=0.005). We conclude that functional polymorphisms in integrin genes ITGA2 and ITGB3 influence the development and progression of breast cancer, respectively. The precise mechanism remains to be determined, but likely involves dysregulated signaling pathways.