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Supplementary Figure 1 from RASSF1A Polymorphism A133S Is Associated with Early Onset Breast Cancer in BRCA1/2 Mutation Carriers

The tumor suppressor gene RASSF1A regulates cell cycle progression, apoptosis, and microtubule stability and is inactivated by promoter methylation in ∼50% of breast cancers. It has been shown previously that the polymorphism A133S in RASSF1A reduces its ability to regulate cell cycle progression and this polymorphism is associated with an increased risk of breast cancer. We analyzed the frequency of RASSF1A A133S in 190 Caucasian women without breast cancer and 653 patients with breast cancer including 138 BRCA1 and BRCA2 (BRCA1/2) mutation carriers, 395 non–BRCA1/2 mutations carriers, and 120 untested for BRCA1/2 mutations. Patients with breast cancer had a higher frequency of A133S than the controls [P = 0.017; odds ratios (OR), 1.71; 95% confidence intervals (95% CI), 1.10–2.66]. There is also a higher frequency of A133S in patients with higher familial breast cancer risk (P = 0.029; OR, 1.76; 95% CI, 1.06–2.92) and patients carrying BRCA1/2 mutations (P = 0.037, OR, 1.82; 95% CI, 1.04–3.18). Importantly, we found that the co-occurrence of a BRCA1 or BRCA2 mutation and A133S in RASSF1A was associated with earlier onset of breast cancer compared with those individuals with either a BRCA1/2 mutation or the A133S polymorphism alone (36.0 versus 42.0 years old, P = 0.002). Our data suggest that the presence of the RASSF1A A133S polymorphism is associated with breast cancer pathogenesis in general and modifies breast cancer age of onset in BRCA1/2 mutations carriers. Our results warrant a large-scale study to examine the effect of the A133S polymorphism in the development of breast and other types of cancers. [Cancer Res 2008;68(1):22–5]

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. The U.S. Surveillance Epidemiology and End Results (SEER) registry reports that Hispanic children have the highest incidence of ALL, however, it is unclear if this is due to genetic factors, unique environmental exposures, or both. Previous reports have shown an association between CYP1A1 variants and ALL. Methods: To explore the contribution of CYP1A1 polymorphisms to ALL susceptibility in different ethnic groups, we conducted a case–control analysis in Caucasian, Hispanic, and African-American children. Results: Increased risk of developing ALL was found in the whole sample group for homozygosity of variant alleles at CYP1A1*2C (OR 2.51, 95% CI 1.18–5.33, P = 0.016) and CYP1A1*2B (OR 3.24, 95% CI 1.43–7.34, P = 0.005). Stratified analyses showed increased risks in the Hispanic group (CYP1A1*2A, OR 2.70, 95% CI 1.27–5.74, P = 0.010; CYP1A1*2C, OR 2.47, 95% CI 1.13–5.38, P = 0.023; and CYP1A1*2B, OR 3.28, 95% CI 1.40–7.69, P = 0.006) but not for the other ethnic groups. Hispanic control subjects were significantly more likely to be carriers of variant alleles as compared to Caucasians (P < 0.0001) and African Americans (P = 0.005). Conclusions: Our study suggests that polymorphisms in CYP1A1 may contribute to the increased risk of ALL in Hispanic children due to both their impact on leukemia susceptibility and the increased prevalence of the at-risk alleles in the Hispanic population. Impact: Our study provides a novel and specific link between CYP1A1 polymorphisms and ethnic influence on ALL risk that may help explain varying susceptibilities across groups to environmental toxins. Cancer Epidemiol Biomarkers Prev; 20(7); 1537–42. ©2011 AACR.

Cyclin D1, encoded by the gene CCND1, is a major regulator of the cell cycle transition from G1 phase to S phase. A CCND1 polymorphism (G to A) at codon 242, the boundary of exon 4 and intron 4, affects splicing such that exon 5 is not expressed in the A allele. Since exon 5 is involved in rapid turnover, the variant cyclin D1 corresponding to the A allele may have a longer half-life. A previous study demonstrated that in families with hereditary nonpolyposis colorectal cancer, the age of onset of colorectal cancer varied according to variation at this polymorphic site. We examined this CCND1polymorphism in a series hepatoblastoma, a childhood liver cancer that shares other molecular features with colon cancer. We determined in an analysis of 84 children with hepatoblastoma that the G/A exon 4 polymorphism in CCND1 is correlated with the age of onset of hepatoblastomas. The A/A genotype is associated with an earlier age of onset compared to the G/A or G/G genotype. The median age of patients with the G/G genotype was 22 months, compared to 17 months in patients with the G/A genotype and 11 months for the A/A genotype. These findings suggest that the CCND1 A polymorphism may contribute to tumor development in children with hepatoblastoma.

Myeloperoxidase (MPO) is a major enzyme found in neutrophils. Oxidation by MPO produces free radicals that demonstrate genotoxic properties. A polymorphism (G to A) within the promotor region of the MPO gene reduces transcription and expression. This polymorphism is associated with a protective effect against some cancers in adults including lung cancer. The objective of our study was to investigate the effect of this MPO polymorphism on the risk of hepatoblastoma, the most common cancer of the liver in childhood. By using PCR-SSCP, we determined the genotype at this polymorphism in 48 cases of Caucasian children with hepatoblastoma and 180 normal controls. Genotypes were confirmed by a second method using Aci I restriction enzyme restriction. We found that A allele was associated with reduced risk of hepatoblastoma of 50% (OR, 0.51; 95%CI, 0.27-0.93) and G/A or A/A genotype reduced the risk by 56% (OR, 0.44; 95%CI, 0.21-0.90). Our data suggest that A allele is a protective factor with regard to the risk of hepatoblastoma, perhaps by altering genotoxic properties of xenobiotic substances which may act as carcinogens.

Hepatoblastoma is a pediatric malignancy characterized by the uncontrolled proliferation of immature hepatocytes (hepatoblasts). This disease is diagnosed primarily in children younger than 5 years and is disproportionately observed in former premature infants. Cytogenetically, hepatoblastoma is characterized by numerical aberrations, as well as unbalanced translocations involving the proximal region of chromosome 1q. The NOTCH2 gene has been mapped to this locus, and it is well established that the NOTCH gene family is an important regulator of several developmental pathways. Specifically, the NOTCH2 protein is known to delay hepatoblast maturation during early hepatic organogenesis, and the reduction of NOTCH2 expression correlates with the differentiation of hepatoblasts into hepatocytes and biliary cells in the developing liver. We hypothesized that NOTCH2 is involved in the pathogenesis of hepatoblastoma by maintaining a population of undifferentiated hepatoblasts. We studied the immunohistochemical expression of NOTCH2 and its isoforms NOTCH1, NOTCH3, and NOTCH4 and the NOTCH2 primary ligand JAGGED1 in hepatoblastomas. Compared with the normal liver, an increasedlevelofNOTCH2expressionwasseenin22of 24 (92%) hepatoblastomas. There was no significant staining for other NOTCH isoforms and JAGGED1 in hepatoblastomas. Therefore, we suggest that NOTCH2 expression and activation, independent of JAGGED1 expression, may contribute to the pathogenesis of hepatoblastoma. In the hepatoblastoma sinusoidal vasculature, we saw NOTCH3 and NOTCH1 expression. These observations have potential implications with regard to therapeutic targeting of the NOTCH signaling pathway in hepatoblastomas.

Background Hepatoblastoma is a rare malignancy of childhood. The scarcity of adequate cell models has limited our understanding of this tumor. Here we describe and characterize a new human liver tumor cell line, Hep293TT, derived from an aggressive childhood hepatoblastoma. Procedures Hep293TT cells were established using primary tumor tissues from a 5-year-old Caucasian female child. This cell line has been maintained for more than 34 months and over 20 subcultures, and was characterized by histopathology, ELISA, genotype, cytogenetics, CGH array, immunohistochemistry, and molecular sequence analyses. Results Cells were confirmed to originate from parental tumor cells, secrete α-fetoprotein, and express hepatic markers and β-catenin. Hep293TT cells were able to form colonies in soft agar. Tumorigenicity was demonstrated by induction of solid tumors after subrenal capsule injection in immunodeficient mice. Hep293TT cells demonstrated a highly aneuploid karyotype, and a whole genome CGH analysis revealed chromosomal imbalances in every chromosome. Allelotype analysis demonstrated loss of alleles at distal 11p15.5 as is typical of embryonal tumors. Both Hep293TT cells and the primary tumor contain a deletion of 351 nucleotides in β-catenin, as has been seen in other hepatoblastoma tumors. The cell line expressed β-catenin protein in both full-length and partially deleted forms, and expressed NOTCH2 protein characteristic of hepatoblasts. No mutation was detected in the APC, MYH, MLH1, or MSH2 genes. Conclusion This cell line, Hep293TT, is a valuable resource for the study of childhood liver cancer and may potentially provide a tool in the development of new agents. Pediatr Blood Cancer 2009;53:1040–1047. © 2009 Wiley-Liss, Inc.

The tumor suppressor gene RASSF1A regulates cell cycle progression, apoptosis, and microtubule stability and is inactivated by promoter methylation in ∼50% of breast cancers. It has been shown previously that the polymorphism A133S in RASSF1A reduces its ability to regulate cell cycle progression and this polymorphism is associated with an increased risk of breast cancer. We analyzed the frequency of RASSF1A A133S in 190 Caucasian women without breast cancer and 653 patients with breast cancer including 138 BRCA1 and BRCA2 (BRCA1/2) mutation carriers, 395 non–BRCA1/2 mutations carriers, and 120 untested for BRCA1/2 mutations. Patients with breast cancer had a higher frequency of A133S than the controls [P = 0.017; odds ratios (OR), 1.71; 95% confidence intervals (95% CI), 1.10–2.66]. There is also a higher frequency of A133S in patients with higher familial breast cancer risk (P = 0.029; OR, 1.76; 95% CI, 1.06–2.92) and patients carrying BRCA1/2 mutations (P = 0.037, OR, 1.82; 95% CI, 1.04–3.18). Importantly, we found that the co-occurrence of a BRCA1 or BRCA2 mutation and A133S in RASSF1A was associated with earlier onset of breast cancer compared with those individuals with either a BRCA1/2 mutation or the A133S polymorphism alone (36.0 versus 42.0 years old, P = 0.002). Our data suggest that the presence of the RASSF1A A133S polymorphism is associated with breast cancer pathogenesis in general and modifies breast cancer age of onset in BRCA1/2 mutations carriers. Our results warrant a large-scale study to examine the effect of the A133S polymorphism in the development of breast and other types of cancers. [Cancer Res 2008;68(1):22–5]

The dopamine receptor D2 (DRD2) gene has polymorphisms that have been linked to regulation of the dopamine system and to an increased prevalence of smoking. The present study examined the relationship of the DRD2 TaqI-A and -B polymorphisms with short-term clinical outcome (abstinence and withdrawal symptoms), collected from daily (14 pre-quit and 42 post-quit) diary data among smokers (n=116) treated with the nicotine patch plus either venlafaxine or placebo. The results showed that B1/B1 or B1/B2 smokers were slightly less likely to be abstinent on a given day than those homozygous for the TaqI-B2 allele. Significant DRD2 TaqI-B x time interactions were found for several of the withdrawal scales, indicating that those smokers with the B1/B1 or B1/B2 genotypes tended to report more symptoms over time compared to those with the B2/B2 genotype. No interactions or main effects were found for the DRD2 TaqI-A polymorphism. The findings demonstrate that smokers homozygous for the TaqI-B2 allele experience progressive improvement in self-reported withdrawal symptoms while smokers with the TaqI-B1 allele showing little change.