Association between single-nucleotide polymorphisms in growth factor genes and quality of life in men with prostate cancer and the general population.

PURPOSE: Improved survival for men with prostate cancer has led to increased attention to factors influencing quality of life (QOL). As protein levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) have been reported to be associated with QOL in people with cancer, we sought to identify whether single-nucleotide polymorphisms (SNPs) of these genes were associated with QOL in men with prostate cancer. METHODS: Multiple linear regression of two data sets (including approximately 750 men newly diagnosed with prostate cancer and 550 men from the general population) was used to investigate SNPs of VEGF and IGF-1 (10 SNPs in total) for associations with QOL (measured by the SF-36v2 health survey). RESULTS: Men with prostate cancer who carried the minor 'T' allele for IGF-1 SNP rs35767 had higher mean Role-Physical scale scores (≥0.3 SD) compared to non-carriers (p < 0.05). While this association was not identified in men from the general population, one IGF-1 SNP rs7965399 was associated with higher mean Bodily Pain scale scores in men from the general population that was not found in men with prostate cancer. Men from the general population who carried the rare 'C' allele had higher mean Bodily Pain scale scores (≥0.3 SD) than non-carriers (p < 0.05). CONCLUSIONS: Through identifying SNPs that are associated with QOL in men with prostate cancer and men from the general population, this study adds to the mapping of complex interrelationships that influence QOL and suggests a role for IGF-I in physical QOL outcomes. Future research may identify biomarkers associated with increased risk of poor QOL that could assist in the provision of pre-emptive support for those identified at risk.

A Kallikrein 15 (KLK15) single nucleotide polymorphism located close to a novel exon shows evidence of association with poor ovarian cancer survival.

BACKGROUND: KLK15 over-expression is reported to be a significant predictor of reduced progression-free survival and overall survival in ovarian cancer. Our aim was to analyse the KLK15 gene for putative functional single nucleotide polymorphisms (SNPs) and assess the association of these and KLK15 HapMap tag SNPs with ovarian cancer survival. RESULTS: In silico analysis was performed to identify KLK15 regulatory elements and to classify potentially functional SNPs in these regions. After SNP validation and identification by DNA sequencing of ovarian cancer cell lines and aggressive ovarian cancer patients, 9 SNPs were shortlisted and genotyped using the Sequenom iPLEX Mass Array platform in a cohort of Australian ovarian cancer patients (N = 319). In the Australian dataset we observed significantly worse survival for the KLK15 rs266851 SNP in a dominant model (Hazard Ratio (HR) 1.42, 95% CI 1.02-1.96). This association was observed in the same direction in two independent datasets, with a combined HR for the three studies of 1.16 (1.00-1.34). This SNP lies 15 bp downstream of a novel exon and is predicted to be involved in mRNA splicing. The mutant allele is also predicted to abrogate an HSF-2 binding site. CONCLUSIONS: We provide evidence of association for the SNP rs266851 with ovarian cancer survival. Our results provide the impetus for downstream functional assays and additional independent validation studies to assess the role of KLK15 regulatory SNPs and KLK15 isoforms with alternative intracellular functional roles in ovarian cancer survival.

Kallikrein-related peptidase 3 (KLK3/PSA) single nucleotide polymorphisms and ovarian cancer survival.

There is substantial evidence suggesting a role for hormone-regulated kallikrein-related peptidases (KLKs) in carcinogenesis and tumour metastasis. KLKs are considered to have potential as prognostic biomarkers for hormone dependent cancers, particularly ovarian cancer. The purpose of this study was to evaluate the association between Kallikrein-related peptidase 3 (KLK3) gene single nucleotide polymorphisms (SNPs) located in hormone response elements and ovarian cancer survival. DNA samples were analyzed from 304 Australian women diagnosed with epithelial ovarian cancer. The KLK3 rs266882 and rs11084033 SNPs were genotyped by the Sequenom iPLEX Mass Array platform. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression models. An association was observed with ovarian cancer survival for homozygote carriers of the rare allele of rs11084033 (adjusted HR 2.12, 95% CI 1.08-4.15). This finding is consistent with bioinformatic analysis predicting the rs11084033 rare allele to be responsible for the loss of a confirmed androgen response element, and with published expression data suggesting that aggressive ovarian cancers show decreased KLK3 tumor expression. The rs11084033 has potential prognostic significance in ovarian cancer. However, this finding requires replication, and further investigation regarding the functional significance of rs11084033 and correlated SNPs.

Vascular endothelial growth factor gene polymorphisms and ovarian cancer survival.

OBJECTIVES: We sought to evaluate the effect of polymorphisms in the VEGF (Vascular Endothelial Growth Factor) gene on overall survival in ovarian cancer patients. METHODS: A sample of 319 women diagnosed with primary invasive epithelial ovarian cancer in Australia between 1985 and 1997, recruited as incident cases, were genotyped for four VEGF single nucleotide polymorphisms (three tagSNPs and one functional SNP) using the Sequenom MassARRAY platform. A SNP found to be associated with ovarian cancer survival in this sample set was then evaluated in two independent datasets in an attempt to replicate the association. RESULTS: VEGF tagSNPs rs3025033 and rs2146323 were not associated with ovarian cancer survival in the Australian sample. Ovarian cancer patients homozygous for tagSNP rs833068 or the functional SNP rs2010963 displayed significantly shortened overall survival in the Australian sample (HR 2.09, 95% CI 1.16-3.78), an effect most apparent in the first 5years after diagnosis. This association was not replicated in two independent datasets. CONCLUSIONS: Findings from this study provide no evidence that rs3025033 and rs2146323 VEGF polymorphisms are associated with ovarian cancer survival. Although homozygous carriers of the tagSNP rs833068 experienced significantly worse survival in our Australian dataset, we were unable to replicate this in two independent datasets.

Kallikrein-related peptidase 10 (KLK10) expression and single nucleotide polymorphisms in ovarian cancer survival.

INTRODUCTION: Kallikrein-related peptidase 10 (KLK10) overexpression is a predictor of poor disease outcome in women with late-stage ovarian cancer. We aimed to identify whether KLK10 overexpression could be attributed to genetic variants, in particular, in hormone response elements or transcription factor binding sites. METHODS: Cox regression analysis was used to assess the association between 2 tag and 1 exonic KLK10 single nucleotide polymorphisms (SNPs) and the survival of 319 patients with ovarian cancer. Four different ovarian cancer cell lines were investigated for KLK10 expression after hormone stimulation, and sequence variation in the 3.6-Kb upstream of the KLK10 start site. In silico analyses of SNPs in cell lines and from published databases were undertaken to identify further research novel and potentially functional SNPs that are not covered by tag SNPs. RESULTS: The KLK10 SNPs investigated were not associated with ovarian cancer survival. However, steroid hormone treatment of ovarian cell lines showed KLK10 up-regulation in response to estrogen and estrogen plus progesterone treatments in the aggressive cell line PEO1 and affirmed a role for KLK10 in aggressive ovarian cancer. Potentially functional KLK10 SNPs were identified by cell line sequencing and bioinformatic analysis. CONCLUSION: Potentially functional candidate KLK10 SNPs require investigation in future association studies of ovarian cancer risk and survival, including rs3760738 identified in aggressive ovarian cancer cell lines and predicted to affect transcription factor binding sites.

Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors.

Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18-76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission.

Multiple novel prostate cancer predisposition loci confirmed by an international study: the PRACTICAL Consortium.

A recent genome-wide association study found that genetic variants on chromosomes 3, 6, 7, 10, 11, 19 and X were associated with prostate cancer risk. We evaluated the most significant single-nucleotide polymorphisms (SNP) in these loci using a worldwide consortium of 13 groups (PRACTICAL). Blood DNA from 7,370 prostate cancer cases and 5,742 male controls was analyzed by genotyping assays. Odds ratios (OR) associated with each genotype were estimated using unconditional logistic regression. Six of the seven SNPs showed clear evidence of association with prostate cancer (P = 0.0007-P = 10(-17)). For each of these six SNPs, the estimated per-allele OR was similar to those previously reported and ranged from 1.12 to 1.29. One SNP on 3p12 (rs2660753) showed a weaker association than previously reported [per-allele OR, 1.08 (95% confidence interval, 1.00-1.16; P = 0.06) versus 1.18 (95% confidence interval, 1.06-1.31)]. The combined risks associated with each pair of SNPs were consistent with a multiplicative risk model. Under this model, and in combination with previously reported SNPs on 8q and 17q, these loci explain 16% of the familial risk of the disease, and men in the top 10% of the risk distribution have a 2.1-fold increased risk relative to general population rates. This study provides strong confirmation of these susceptibility loci in multiple populations and shows that they make an important contribution to prostate cancer risk prediction.

First Australian experience of treating localised prostate cancer patients with CyberKnife stereotactic radiotherapy: early PSA response, acute toxicity and quality of life.

INTRODUCTION: This study is to evaluate biochemical response, acute toxicity and health-related quality-of-life (QOL) outcomes among prostate cancer patients following stereotactic body radiation therapy (SBRT) in the first Australian CyberKnife facility. METHODS: Forty-five consecutive patients with clinically localised prostate cancer were treated with SBRT using CyberKnife technology and enrolled in this study. Protocol treatment consisted of 36.25 Gy in five fractions. PSA and acute toxicity was assessed at each follow-up visit and QOL was assessed using the European Organisation for Research and Treatment of Cancer (EORTC) Global Health Status (GHS) C30 and PR25 questionnaires and the Karnofsky Performance Status (KPS). Distance of travel for treatment was recorded. RESULTS: The median prostate-specific antigen (PSA) level declined from the initial value of 6.9 ng/mL to 1.5 ng/mL at 6 months and 0.6 ng/mL at 18 months post-treatment. Results were similar in patients who did not receive hormone therapy. Acute grade 1 gastrointestinal (GI) and genitourinary (GU) toxicities were found in 11.1% and 24.4% of patients respectively. Acute grade 2 GI and GU toxicities were found in 2.2% and 11.1% of patients respectively. There were no grade 3 and grade 4 toxicities. Mean urinary symptom score was 14.8 at baseline, 17.2 at 6 weeks and 18.3 at 6 months (P > 0.05). Mean bowel symptom score was 2.7 at baseline, 4.2 at 6 weeks and 6.3 at 6 months (P > 0.05). The mean GHS score improved from 81.3 at baseline to 82.4 at 6 weeks, and was 75.6 at 6 months (P > 0.05, not significant). Compared to baseline KPS, there was a significant mean decrease from baseline of 96.7 to 93.3 at the 6-week follow-up (P = 0.0043), which then recovered to 94.3 at the 6-month follow-up (P = 0.1387). CONCLUSIONS: Early results show promising PSA response. Acute toxicity seemed comparable to results from conventionally fractionated radiotherapy and to international prostate SBRT studies. EORTC PR25 and C30 scores did not reveal any significant change from baseline, and although there was a decrease in KPS, the absolute decrease was small.

Participating in an International Stereotactic Radiotherapy Patient Registry: The Establishment of Data Collection Pathways.

Aim To describe data collection pathways and practical challenges experienced by an academic comprehensive cancer centre aiming to record clinical data for patients being treated with a novel radiotherapy treatment modality. Methods Various options to capture data from all patients treated with the CyberKnife Robotic Radiosurgery System at Sir Charles Gairdner Hospital (SCGH) in Western Australia were explored. An international multicenter web-based secure database established and maintained by the Radiosurgery Society the RSSearch® Patient Registry was selected. Data were collected and entered over four contiguous phases, with either opt-in or opt-out consent and the completion of Patient Reported Outcome questionnaires for specific sub-groups. Results Between April 2014 and June 2016, 461 patients at Sir Charles Gairdner Hospital were enrolled in the RSSearch® Patient Registry with the collection of over 17,500 data items. From 461 patients enrolled, 447 patients were treated with the CyberKnife Robotic Radiosurgery System. The majority of patients were treated for either a malignant primary (43.2%) or metastatic disease (39.4%). The establishment of matrix organisational processes for data collection led to the development of improved workflow patterns and data collection pathways. Conclusions This article describes the processes developed by a single centre to establish an efficient system for data collection and participation in an international registry. The opt-out approach was more efficient in terms of patient recruitment compared to the informed-consent method used in earlier phases. The experience of this single centre may help inform other institutions considering data collection options for assessments of new or novel treatments.

ADAM33 haplotypes are associated with asthma in a large Australian population.

The ADAM33 gene has recently been identified as being a potentially important asthma candidate gene, and polymorphisms in this gene have been shown to be associated with asthma and bronchial hyperresponsiveness in Caucasian individuals from several populations. We performed chip-based matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry using the MassARRAY system and multiplexed genotyping assays to investigate the association between 10 single nucleotide polymorphisms (SNPs) in the ADAM33 gene (F_+1, Q_-1, S_1, ST_+4, ST_+7, V_-2, V_-1, V_2, V_4, V_5) and asthma and asthma severity in a large Australian Caucasian population of nonasthmatic controls (n = 473), and patients with mild (n = 292), moderate (n = 238) and severe (n = 82) asthma. No significant association was found between any one of the 10 SNPs and asthma or asthma severity, however, there was a significant global haplotypic association with asthma (P = 0.0002) and disease severity (P = 0.0001), driven by the combination of two key SNPs, V_-1 and ST_+7. A meta-analysis of all the genetic studies conducted to date found significant between-study heterogeneity, likely to reflect population stratification. Our analysis of ADAM33 haplotypes further suggests a likely role for ADAM33 in the asthma phenotype, although it does not exclude an association with another locus in linkage disequilibrium with ADAM33.

Common variation in Kallikrein genes KLK5, KLK6, KLK12, and KLK13 and risk of prostate cancer and tumor aggressiveness.

The human tissue Kallikrein family consists of 15 genes with the majority shown to be differentially expressed in cancers and/or indicators of cancer prognosis. We sought to elucidate the role of common genetic variation in four of the Kallikrein genes, KLK5, KLK6, KLK12, and KLK13, in prostate cancer risk and tumor aggressiveness. Genotyping of all 22 tagging single nucleotide polymorphisms (tagSNPs) in the KLK5, KLK6, KLK12, and KLK13 genes was performed in approximately 1,000 prostate cancer cases and 1,300 male controls from Australia. Data from any positive results were also accessed for 1,844 cases and 1,886 controls from a previously published prostate cancer genome-wide association study set from the United Kingdom. For one SNP in KLK12, rs3865443, there was evidence for association with prostate cancer risk of similar direction and magnitude in the replication set to that seen in the Australian cohort. We conducted genotyping of a further 309 prostate cancer cases, and combined analyses revealed an increased risk of prostate cancer for carriers of the rare homozygous genotype for rs3865443 (OR 1.28, 95% CI 1.04-1.57; P = 0.018). No other tagSNPs in the KLK5, KLK6, and KLK13 genes were consistently associated with prostate cancer risk or tumor aggressiveness. Analysis of a combined sample of 3,153 cases and 3,199 controls revealed the KLK12 tagSNP rs3865443 to be marginally statistically significantly associated with risk of prostate cancer. Considering the total number of SNPs investigated in this study, this finding should be interpreted cautiously and requires additional validation from very large datasets such as those of the Prostate Cancer Association group to investigate cancer associated alterations (PRACTICAL) Consortium.

Possible genetic predisposition to lymphedema after breast cancer.

BACKGROUND: Known risk factors for secondary lymphedema only partially explain who develops lymphedema following cancer, suggesting that inherited genetic susceptibility may influence risk. Moreover, identification of molecular signatures could facilitate lymphedema risk prediction prior to surgery or lead to effective drug therapies for prevention or treatment. Recent advances in the molecular biology underlying development of the lymphatic system and related congenital disorders implicate a number of potential candidate genes to explore in relation to secondary lymphedema. METHODS AND RESULTS: We undertook a nested case-control study, with participants who had developed lymphedema after surgical intervention within the first 18 months of their breast cancer diagnosis serving as cases (n=22) and those without lymphedema serving as controls (n=98), identified from a prospective, population-based, cohort study in Queensland, Australia. TagSNPs that covered all known genetic variation in the genes SOX18, VEGFC, VEGFD, VEGFR2, VEGFR3, RORC, FOXC2, LYVE1, ADM, and PROX1 were selected for genotyping. Multiple SNPs within three receptor genes, VEGFR2, VEGFR3, and RORC, were associated with lymphedema defined by statistical significance (p<0.05) or extreme risk estimates (OR <0.5 or >2.0). CONCLUSIONS: These provocative, albeit preliminary, findings regarding possible genetic predisposition to secondary lymphedema following breast cancer treatment warrant further attention for potential replication using larger datasets.

Association between Prostinogen (KLK15) genetic variants and prostate cancer risk and aggressiveness in Australia and a meta-analysis of GWAS data.

BACKGROUND: Kallikrein 15 (KLK15)/Prostinogen is a plausible candidate for prostate cancer susceptibility. Elevated KLK15 expression has been reported in prostate cancer and it has been described as an unfavorable prognostic marker for the disease. OBJECTIVES: We performed a comprehensive analysis of association of variants in the KLK15 gene with prostate cancer risk and aggressiveness by genotyping tagSNPs, as well as putative functional SNPs identified by extensive bioinformatics analysis. METHODS AND DATA SOURCES: Twelve out of 22 SNPs, selected on the basis of linkage disequilibrium pattern, were analyzed in an Australian sample of 1,011 histologically verified prostate cancer cases and 1,405 ethnically matched controls. Replication was sought from two existing genome wide association studies (GWAS): the Cancer Genetic Markers of Susceptibility (CGEMS) project and a UK GWAS study. RESULTS: Two KLK15 SNPs, rs2659053 and rs3745522, showed evidence of association (p<0.05) but were not present on the GWAS platforms. KLK15 SNP rs2659056 was found to be associated with prostate cancer aggressiveness and showed evidence of association in a replication cohort of 5,051 patients from the UK, Australia, and the CGEMS dataset of US samples. A highly significant association with Gleason score was observed when the data was combined from these three studies with an Odds Ratio (OR) of 0.85 (95% CI = 0.77-0.93; p = 2.7×10(-4)). The rs2659056 SNP is predicted to alter binding of the RORalpha transcription factor, which has a role in the control of cell growth and differentiation and has been suggested to control the metastatic behavior of prostate cancer cells. CONCLUSIONS: Our findings suggest a role for KLK15 genetic variation in the etiology of prostate cancer among men of European ancestry, although further studies in very large sample sets are necessary to confirm effect sizes.

PSA/KLK3 AREI promoter polymorphism alters androgen receptor binding and is associated with prostate cancer susceptibility.

The proximal promoter of the kallikrein-related peptidase 3 gene (KLK3/PSA) contains a single-nucleotide polymorphism (G-158A) located within the second canonical half-site for the prostate-specific antigen (PSA) androgen response element 1 (AREI). Previous studies suggest that this polymorphism may be associated with higher PSA levels and increase prostate cancer risk. We have investigated the potential functional significance of this polymorphism and its association with prostate cancer susceptibility by genotyping the G-158A polymorphism in 209 men diagnosed with prostate cancer and 223 healthy control men in an Australian Caucasian population. Functional analyses of PSA AREI demonstrated that the A allele increased binding of AREI to the androgen receptor, as well as increasing transcriptional response to androgens. Association studies of the G-158A polymorphism demonstrated that men with an A/A genotype had a 3-fold increased risk for developing prostate cancer [95% confidence intervals (CIs) = 1.36-6.52] and men with an A/G genotype had a 2.4-fold increased risk (95% CIs = 1.23-4.81). Under a dominant model, the A allele conferred a 2.6-fold increased risk for prostate cancer (95% CIs = 1.37-4.96, P = 0.004). Taken together with the finding that the G-158A polymorphism is associated with an increased risk of prostate cancer in Australian men, our functional data suggest that the presence of the A allele in AREI may, in part, account for the altered PSA regulation seen in prostate cancer.

Mutation analysis of five candidate genes in familial breast cancer.

Most of the known breast cancer susceptibility genes (BRCA1, BRCA2, CHEK2 and ATM) are involved in the damage response pathway. Other members of this pathway are therefore good candidates for additional breast cancer susceptibility genes. ATR, along with ATM, plays a central role in DNA damage recognition and Chk1 relays checkpoint signals from both ATR and ATM. PPP2R1B and PPP2R5B code for subunits of protein phosphatase 2A (PP2A), which regulates autophosphorylation of ATM. In addition, EIF2S6/Int-6, which was originally identified as a common integration site for the mouse mammary tumour virus in virally induced mouse mammary tumours, is a candidate breast cancer susceptibility gene because of its putative role in maintaining chromosome stability. To investigate the role of ATR, CHK1, PPP2R1B, PPP2R5B and EIF2S6/Int-6, we carried out mutation analysis of these genes in the index cases from non-BRCA1/BRCA2 breast cancer families. We also screened sporadic breast tumours for somatic mutations in PPP2R1B and PPP2R5B. Although we identified many novel variants, we found no evidence that highly penetrant germline mutations in these five genes contribute to familial breast cancer susceptibility.

Characterization of two polymorphisms in the leukotriene C4 synthase gene in an Australian population of subjects with mild, moderate, and severe asthma.

BACKGROUND: The cysteinyl-leukotrienes (cys-LTs) are proinflammatory mediators that are important in the pathophysiology of asthma. LTC(4) synthase is a key enzyme in the cys-LT biosynthetic pathway, and studies in small populations have suggested that a promoter polymorphism (A(-444)C) in the gene might be associated with asthma severity and aspirin intolerance. OBJECTIVE: We sought to screen the LTC(4) synthase gene for polymorphisms and to determine whether there is an association between these polymorphisms and asthma severity or aspirin sensitivity in a large, well-phenotyped population and to determine whether this polymorphism is functionally relevant. METHODS: The coding regions of the LTC(4) synthase gene were screened for polymorphisms and the A(-444)C polymorphism was analyzed in a large Australian white adult population of mild (n=282), moderate (n=236), and severe asthmatic subjects (n=86) and nonasthmatic subjects (n=458), as well as in aspirin-intolerant asthmatic subjects (n=67). The functional activity of the promoter polymorphism was investigated by transient transfection of HL-60 cells with a promoter construct. RESULTS: A new polymorphism was identified in intron 1 of the gene (IVS1-10c>a) but was not associated with asthma. Association studies showed that the A(-444)C polymorphism was weakly associated with asthma per se, but there was no association between the C(-444) allele and chronic asthma severity or aspirin intolerance. A meta-analysis of all the genetic studies conducted to date found significant between-study heterogeneity in C(-444) allele frequencies within different clinical subgroups. In vitro functional studies showed no significant differences in transcription efficiency between constructs containing the A(-444) allele or the C(-444) allele. CONCLUSIONS: Our data confirm that, independent of transcriptional activity, the C(-444) allele in the LTC(4) synthase gene is weakly associated with the asthma phenotype, but it is not related to disease severity or aspirin intolerance.