Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Gene-gene interactions in breast cancer susceptibility.

There have been few definitive examples of gene-gene interactions in humans. Through mutational analyses in 7325 individuals, we report four interactions (defined as departures from a multiplicative model) between mutations in the breast cancer susceptibility genes ATM and CHEK2 with BRCA1 and BRCA2 (case-only interaction between ATM and BRCA1/BRCA2 combined, P = 5.9 × 10(-4); ATM and BRCA1, P= 0.01; ATM and BRCA2, P= 0.02; CHEK2 and BRCA1/BRCA2 combined, P = 2.1 × 10(-4); CHEK2 and BRCA1, P= 0.01; CHEK2 and BRCA2, P= 0.01). The interactions are such that the resultant risk of breast cancer is lower than the multiplicative product of the constituent risks, and plausibly reflect the functional relationships of the encoded proteins in DNA repair. These findings have important implications for models of disease predisposition and clinical translation.

Tailoring therapy for locally advanced breast cancer using molecular profiles: are we there yet?

The term 'locally advanced breast cancer' covers a range of clinical scenarios, and has the implications that surgical clearance and local control will be difficult or impossible, and long-term survival rates will be poor. Treatment selection is particularly important in this group of patients to try to obtain maximum control of disease, and potentially improve surgical options and cure rates. Currently, assessment of estrogen receptor, progesterone receptor and human epidermal receptor 2 status in tumour samples remains the gold standard for prediction of response to endocrine therapy, chemotherapy or targeted agents such as trastuzumab. Progress has been made in identifying markers that can help select treatments likely to be associated with response and avoid those associated with resistance. These potential markers include Ki67 proliferation rate, cytochrome P450 (CYP) 2D6 expression, BRCA1/2 gene status and others.

Germline mutations in RAD51D confer susceptibility to ovarian cancer.

Recently, RAD51C mutations were identified in families with breast and ovarian cancer. This observation prompted us to investigate the role of RAD51D in cancer susceptibility. We identified eight inactivating RAD51D mutations in unrelated individuals from 911 breast-ovarian cancer families compared with one inactivating mutation identified in 1,060 controls (P = 0.01). The association found here was principally with ovarian cancer, with three mutations identified in the 59 pedigrees with three or more individuals with ovarian cancer (P = 0.0005). The relative risk of ovarian cancer for RAD51D mutation carriers was estimated to be 6.30 (95% CI 2.86-13.85, P = 4.8 × 10(-6)). By contrast, we estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59-2.96, P = 0.50). These data indicate that RAD51D mutation testing may have clinical utility in individuals with ovarian cancer and their families. Moreover, we show that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D mutation carriers.

Association of molecular markers with toxicity outcomes in a randomized trial of chemotherapy for advanced colorectal cancer: the FOCUS trial.

PURPOSE: Predicting efficacy and toxicity could potentially allow individualization of cancer therapy. We investigated putative pharmacogenetic markers of chemotherapy toxicity in a large randomized trial. PATIENTS, MATERIALS, AND METHODS: Patients were randomly assigned to different sequences of chemotherapy for advanced colorectal cancer. First-line therapy was fluorouracil (FU), irinotecan/FU (IrFU) or oxaliplatin/FU (OxFU). Patients allocated first-line FU had planned second-line irinotecan alone, IrFU, or OxFU. The primary toxicity outcome measure was toxicity-induced delay or dose reduction; the secondary outcome was Common Terminology Criteria of Adverse Events grade >or= 3 toxicity. DNA was analyzed in 1,188 patients; 1,036 were assessable for the primary outcome, including 688 treated with FU, 270 with IrFU (first or second line), 280 with OxFU (first or second line), 184 with irinotecan alone, and 454 with any irinotecan-containing regimen. Ten polymorphisms were assessed: thymidylate synthase-enhancer region (TYMS-ER), thymidylate synthase 1494 (TYMS-1494), dihydropyrimidine dehydrogenase (DPYD), methylenetetrahydrofolate reductase (MTHFR), mutL homolog 1 (MLH1), UDP glucuronyltransferase (UGT1A1), ATP-binding cassette group B gene 1 (ABCB1), x-ray cross-complementing group 1 (XRCC1), glutathione-S-transferase P1 (GSTP1), and excision repair cross-complementing gene 2 (ERCC2). Results Using the primary outcome measure, no polymorphism was significantly associated (P < .01) with the toxicity of any regimen or with the difference in toxicity of IrFU or OxFU versus FU alone. Trends (of doubtful significance) were seen for associations of XRCC1, ERCC2, and GSTP1 with toxicity during irinotecan regimens: XRCC1, primary end point, any irinotecan-containing regimen (P = .045); ERCC2, secondary end point, irinotecan alone (P = .003); GSTP1, secondary end point; IrFU (P = .039); and irinotecan alone (P = .05). There was no evidence of association of UGT1A1*28 with irinotecan toxicity. CONCLUSION: These results do not support the routine clinical use of the evaluated polymorphisms, including UGT1A1*28. Further investigation of XRCC1, ERCC2, and GSTP1 as potential predictors of irinotecan toxicity is warranted.

Predictive biomarkers of chemotherapy efficacy in colorectal cancer: results from the UK MRC FOCUS trial.

PURPOSE: Candidate predictive biomarkers for irinotecan and oxaliplatin were assessed in 1,628 patients in Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS), a large randomized trial of fluorouracil alone compared with fluorouracil and irinotecan and compared with fluorouracil and oxaliplatin in advanced colorectal cancer. METHODS: The candidate biomarkers were: tumor immunohistochemistry for MLH1/MSH2, p53, topoisomerase-1 (Topo1), excision repair cross-complementing gene 1 (ERCC1), O-6-methylguanine-DNA-methyltranserase (MGMT), and cyclooxygenase 2 (COX2); germline DNA polymorphisms in GSTP1, ABCB1, XRCC1, ERCC2, and UGT1A1. These were screened in more than 750 patients for interaction with benefit from irinotecan or oxaliplatin; two markers (Topo1 and MLH1/MSH2) met criteria to be taken forward for analysis in the full population. Primary end points were progression-free survival (PFS) and overall survival. RESULTS: One thousand three hundred thirteen patients (81%) were assessable for Topo1 immunohistochemistry (low, < 10%; moderate, 10% to 50%; or high, > 50% tumor nuclei). In patients with low Topo1, PFS was not improved by the addition of either irinotecan (hazard ratio [HR], 0.98; 95% CI, 0.78 to 1.22) or oxaliplatin (HR, 0.85; 95% CI, 0.68 to 1.07); conversely, patients with moderate/high Topo1 benefited from the addition of either drug (HR, 0.48 to 0.70 in all categories; interaction P = .005; overall, P = .001 for irinotecan; P = .05 for oxaliplatin). High Topo1 was associated with a major overall survival benefit with first-line combination chemotherapy (HR, 0.60; median benefit, 5.3 months); patients with moderate or low Topo1 did not benefit (HR, 0.92 and 1.09, respectively; interaction P = .005). MLH1/MSH2 did not show significant interaction with treatment, although the low rate of loss (4.4%) limits the power of the study for this biomarker. CONCLUSION: Topo1 immunohistochemistry identified subpopulations that did or did not benefit from irinotecan, and possibly also from oxaliplatin. If verified independently, this information will contribute to the individualization of treatment for colorectal cancer.

Prediction of the response of colorectal cancer to systemic therapy.

Adjuvant chemotherapy with fluorouracil and folinic acid improves overall survival for resected carcinoma of the colon of Dukes' stage C by 10-12%. In metastatic disease, response rates with fluorouracil-based regimens are about 25%. Combination with newer agents such as irinotecan and oxaliplatin can improve response rates to more than 50% in selected patients. New treatments with novel molecular targets will soon be entering clinical use. Despite these improvements, many patients undergo chemotherapy for resistant cancer, thus incurring side-effects without benefit. Expression of particular genes can be tested at the protein or RNA level and can be correlated with response or resistance to particular systemic therapies. Thus, predictive-factor testing of tumour biopsy samples may allow us to select chemotherapy or immunotherapy treatments with a high likelihood of benefit for the individual patient.