David P. Atencio

Single nucleotide polymorphisms, apoptosis, and the development of severe late adverse effects after radiotherapy.

Purpose: Evidence has accumulated in recent years suggestive of a genetic basis for a susceptibility to the development of radiation injury after cancer radiotherapy. The purpose of this study was to assess whether patients with severe radiation-induced sequelae (RIS; i.e., National Cancer Institute/CTCv3.0 grade, ≥3) display both a low capacity of radiation-induced CD8 lymphocyte apoptosis (RILA) in vitro and possess certain single nucleotide polymorphisms (SNP) located in candidate genes associated with the response of cells to radiation. Experimental Design: DNA was isolated from blood samples obtained from patients (n = 399) included in the Swiss prospective study evaluating the predictive effect of in vitro RILA and RIS. SNPs in the ATM, SOD2, XRCC1, XRCC3, TGFB1, and RAD21 genes were screened in patients who experienced severe RIS (group A, n = 16) and control subjects who did not manifest any evidence of RIS (group B, n = 18). Results: Overall, 13 and 21 patients were found to possess a total of <4 and ≥4 SNPs in the candidate genes. The median (range) RILA in group A was 9.4% (5.3-16.5) and 94% (95% confidence interval, 70-100) of the patients (15 of 16) had ≥4 SNPs. In group B, median (range) RILA was 25.7% (20.2-43.2) and 33% (95% confidence interval, 13-59) of patients (6 of 18) had ≥4 SNPs (P < 0.001). Conclusions: The results of this study suggest that patients with severe RIS possess 4 or more SNPs in candidate genes and low radiation-induced CD8 lymphocyte apoptosis in vitro.

ATM mutations in female breast cancer patients predict for an increase in radiation-induced late effects

PURPOSE Mutation of the ATM gene may be associated with enhanced radiosensitivity and increased radiation-induced morbidity. Denaturing high performance liquid chromatography (DHPLC) is a powerful new technique proven to be sensitive and accurate in the detection of missense mutations, as well as small deletions and insertions. We screened female breast cancer patients for evidence of ATM gene alterations using DHPLC. This study attempted to determine whether breast cancer patients who develop severe radiotherapy (RT)-induced effects are more likely to possess ATM mutations than patients who display normal radiation responses. METHODS AND MATERIALS Forty-six patients with early-stage breast carcinoma underwent limited surgery and adjuvant RT. DNA was isolated from blood lymphocytes, and each coding exon of the ATM gene was amplified using polymerase chain reaction. Genetic variants were identified using DHPLC by comparing test patterns with a known wild-type pattern. All variants were subjected to DNA sequencing and compared with wild-type sequences for evidence of a mutation. A retrospective review was performed, and the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer acute and late morbidity scoring schemes for skin and subcutaneous normal tissues were applied to quantify the radiation-induced effects. RESULTS Nine ATM mutations were identified in 6 patients (8 novel and 1 rare). The median follow-up was 3.2 years (range 1.3-10.3). A significant correlation between ATM mutation status and the development of Grade 3-4 subcutaneous late effects was found. All 3 of the patients (100%) who manifested Grade 3-4 subcutaneous late sequelae possessed ATM mutations, whereas only 3 (7%) of the 43 patients who did not develop this form of severe toxicity harbored an ATM mutation (p = 0.001). One ATM mutation carrier developed Grade 4 soft tissue necrosis after RT and required hyperbaric oxygen. All 3 patients manifesting Grade 3-4 late subcutaneous responses in fact harbored 2 ATM mutations. In contrast, none of the 3 ATM carriers who had a single mutation developed a severe subcutaneous reaction. ATM mutation status did not predict for a significant increase in early effects. Of the 23 patients with Grade 2-3 moist desquamation, 4 (17%) had an ATM mutation compared with 2 (9%) of 23 patients without desquamation (p = 0.7). CONCLUSION Possession of an ATM mutation, particularly when 2 are present, may be predictive of an increase in subcutaneous late tissue effects after RT for breast cancer and may subsequently prove to be a relative contraindication to standard management. These patients may be better served with reduced doses of radiation. Equivalent local control remains to be tested, but this germline alteration may radiosensitize normal tissues, as well as the tumor itself. DHPLC is effective in the identification of these patients. A larger study is required to confirm these findings.

Association of Single Nucleotide Polymorphisms in SOD2, XRCC1 and XRCC3 with Susceptibility for the Development of Adverse Effects Resulting from Radiotherapy for Prostate Cancer

Abstract Burri, R. J., Stock, R. G., Cesaretti, J. A., Atencio, D. P., Peters, S., Peters, C. A., Fan, G., Stone, N. N., Ostrer, H. and Rosenstein, B. S. Association of Single Nucleotide Polymorphisms in SOD2, XRCC1 and XRCC3 with Susceptibility for the Development of Adverse Effects Resulting from Radiotherapy for Prostate Cancer. Radiat. Res. 170, 49–59 (2008). The objective of this study was to determine whether an association exists between certain single nucleotide polymorphisms (SNPs), which have previously been linked with adverse normal tissue effects resulting from radiotherapy, and the development of radiation injury resulting from radiotherapy for prostate cancer. A total of 135 consecutive patients with clinically localized prostate cancer and a minimum of 1 year of follow-up who had been treated with radiation therapy, either brachytherapy alone or in combination with external-beam radiotherapy, with or without hormone therapy, were genotyped for SNPs in SOD2, XRCC1 and XRCC3. Three common late tissue toxicities were investigated: late rectal bleeding, urinary morbidity, and erectile dysfunction. Patients with the XRCC1 rs25489 G/A (Arg280His) genotype were more likely to develop erectile dysfunction after irradiation than patients who had the G/G genotype (67% compared to 24%; P = 0.048). In addition, patients who had the SOD2 rs4880 T/C (Val16Ala) genotype exhibited a significant increase in grade 2 late rectal bleeding compared to patients who had either the C/C or T/T genotype for this SNP (8% compared to 0%; P = 0.02). Finally, patients with the combination of the SOD2 rs4880 C/T genotype and XRCC3 rs861539 T/C (Thr241Met) genotype experienced a significant increase in grade 2 late rectal bleeding compared to patients without this particular genotypic arrangement (14% compared to 1%; P = 0.002). These results suggest that SNPs in the SOD2, XRCC1 and XRCC3 genes are associated with the development of late radiation injury in patients treated with radiation therapy for prostate adenocarcinoma.

Variants in the ATM Gene Associated with a Reduced Risk of Contralateral Breast Cancer

Between 5% and 10% of women who survive a first primary breast cancer will subsequently develop a second primary cancer in the contralateral breast. The Womens Environment, Cancer, and Radiation Epidemiology Study was designed to identify genetic and environmental determinants of contralateral breast cancer (CBC). In this study, 708 women with asynchronous CBC served as cases and 1,397 women with unilateral breast cancer served as controls. ATM, a serine-threonine kinase, controls the cellular response to DNA double-strand breaks, and has been implicated in breast cancer risk. Complete mutation screening of the ATM gene in all 2,105 study participants identified 240 distinct sequence variants; only 15 were observed in >1% of subjects. Among the rare variants, deleterious alleles resulting in loss of ATM function were associated with a nonsignificant increase in risk of CBC. In contrast, carriers of common variants had a statistically significant reduction in risk of CBC. Four of these 15 variants were individually associated with a significantly decreased risk of second primary breast cancer [c.1899-55T>G, rate ratio (RR), 0.5; 95% confidence interval (CI), 0.3-0.8; c.3161C>G, RR, 0.5; 95% CI, 0.3-0.9; c.5558A>T, RR, 0.2; 95% CI, 0.1-0.6; c.6348-54T>C RR, 0.2; 95% CI, 0.1-0.8]. These data suggest that some alleles of ATM may exert an antineoplastic effect, perhaps by altering the activity of ATM as an initiator of DNA damage responses or a regulator of p53.

Combined subcarcinogenic benzo[a]pyrene and UVA synergistically caused high tumor incidence and mutations in H-ras gene, but not p53, in SKH-1 hairless mouse skin

Combined subcarcinogenic doses of benzo[a]pyrene (BaP) and UVA induced H‐ras, but not p53, gene mutations 8 weeks before tumor emergence in SKH‐1 mice. Neither UVA (40 kJ/m2) nor BaP (8 nmol) induced any tumors after mice were topically treated 3 times/week for 25 weeks. However, combined BaP‐UVA treatment synergistically increased tumor incidence and multiplicity. All tumors induced by BaP‐UVA were malignant. The epidermis was collected from mice treated for 2, 6 and 10 weeks. DNA from UVB‐ (0.3 kJ/m2) or BaP‐UVA‐(8 nmol and 40 kJ/m2‐induced tumors was isolated and screened for H‐ras and p53 mutations. Four types of point mutation, GGC→GAC, GCC, GTC and CGC, occurred in UVB‐induced tumors at H‐ras codon 13; and one type of point mutation, GGA→GAA, at codon 12. Treatment with either BaP alone or BaP‐UVA for 10 weeks caused GGA→GAA mutation at codon 12 or GGC→GAC mutation at codon 13 in nontumor skin, respectively, as well as in tumors induced by BaP‐UVA. All of the 10‐week samples treated with either BaP or BaP‐UVA showed detectable mutations at codons 12 and 13, but the genetic load was significantly higher in BaP‐UVA‐treated mice than in those exposed only to BaP. UVA alone induced mutations at codon 12 in only one‐third of samples. G→A mutations induced by BaP or BaP‐UVA at position 38 of codon 13 have not been reported previously. C→T transitions were detected in p53 hot spots of exon 8 in 2 of 19 BaP‐UVA‐induced tumors but were not found in nontumor skin.

p53 Gene Mutations in SKH-1 Mouse Tumors Differentially Induced by UVB and Combined Subcarcinogenic Benzo[a]pyrene and UVA†

We compared the frequency and spectra of p53 mutations in skin tumors from UVB‐irradiated and benzo(a)pyrene‐UVA‐treated SKH‐1 mice. Analysis of p53 mutations using a combination of polymerase chain reaction, denaturing high‐performance liquid chromatography, and sequencing shows that the frequency and spectrum of p53 mutations in BaP‐UVA‐induced tumors are quite different from those in UVB‐induced tumors. SKH‐1 mice were treated with BaP‐UVA or UVB for 30 weeks after which skin tumors were collected for analysis of p53 mutations. Among the 11 BaP‐UVA‐induced tumors with diameters of 5–10 mm, two displayed mutations in exon 8 yielding a mutation frequency of 18.2%. In contrast, the mutation frequency among BaP‐UVA‐induced tumors was 10.5%. In UVB‐induced tumors, the mutation frequency in exon 8 was highly correlated with tumor size. A total of 77.8% of tumors with diameters larger than 10 mm contained p53 mutations. The overall mutation frequency among UVB‐induced tumors was 17.9% in exon 8 and only 3.8% in exon 5. Hotspots for p53 mutation in UVB‐induced tumors were found at codons 128 and 149 (exon 5), and at codons 268, 270, 271 and 273–276 (exon 8). In addition to widely recognized C→T missense mutations, there were also tandem CC→AG changes coupled with either an insertion of T, a C→G substitution or G→C/T mutations. All of the mutations were found at tri‐ or tetra‐pyrimidine sites. Thirty‐nine per cent of all p53 mutations occurred at codons 274 and 275; 53% occurred at codons 268–271. Two multiple mutation clusters were located at codons 268–271 and 274–276. Both BaP‐UVA and UVB caused C→T transitions at codon 275 in exon 8. A C→T mutation at codon 294 was induced only by BaP‐UVA treatment. In contrast to UVB treatment, BaP‐UVA treatment did not induce any mutations in exon 5. We show that individually subcarcinogenic levels of BaP and UVA synergistically induce a novel p53‐mutation fingerprint. This fingerprint could serve as a prognostic indicator for the development of BaP‐UVA‐induced skin tumors.