Andrea Tesoriero

Use of Molecular Tumor Characteristics to Prioritize Mismatch Repair Gene Testing in Early-Onset Colorectal Cancer

PURPOSE The relationships between mismatch repair (MMR) protein expression, microsatellite instability (MSI), family history, and germline MMR gene mutation status have not been studied on a population basis. METHODS We studied 131 unselected patients with colorectal cancer diagnosed younger than age 45 years. For the 105 available tumors, MLH1, MSH2, MSH6, and PMS2 protein expression using immunohistochemistry (IHC) and MSI were measured. Germline DNA was screened for hMLH1, hMSH2, hMSH6, and hPMS2 mutations for the following patients: all from families fulfilling the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer (HNPCC); all with tumors that were high MSI, low MSI, or that lacked expression of any MMR protein; and a random sample of 23 with MS-stable tumors expressing all MMR proteins. RESULTS Germline mutations were found in 18 patients (nine hMLH1, four hMSH2, four hMSH6, and one hPMS2); all tumors exhibited loss of MMR protein expression, all but one were high MSI or low MSI, and nine were from a family fulfilling Amsterdam Criteria. Sensitivities of IHC testing, MSI (high or low), and Amsterdam Criteria for MMR gene mutation were 100%, 94%, and 50%, respectively. Corresponding positive predictive values were 69%, 50%, and 75%. CONCLUSIONS Tumor IHC analysis of four MMR proteins and MSI testing provide a highly sensitive strategy for identifying MMR gene mutation-carrying, early-onset colorectal cancer patients, half of whom would have been missed using Amsterdam Criteria alone. Tumor-based approaches for triaging early-onset colorectal cancer patients for MMR gene mutation testing, irrespective of family history, appear to be an efficient screening strategy for HNPCC.

Genetic and Histopathologic Evaluation of BRCA1 and BRCA2 DNA Sequence Variants of Unknown Clinical Significance

Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on co-occurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1- and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that approximately 80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified variants.

Adaptive evolution of the tumour suppressor BRCA1 in humans and chimpanzees

Mutations in BRCA1 (ref. 1) confer an increased risk of female breast cancer. In a genome-wide scan of linkage disequilibrium (LD), a high level of LD was detected among microsatellite markers flanking BRCA1 (ref. 3), raising the prospect that positive natural selection may have acted on this gene. We have used the predictions of evolutionary genetic theory to investigate this further. Using phylogeny-based maximum likelihood analysis of the BRCA1 sequences from primates and other mammals, we found that the ratios of replacement to silent nucleotide substitutions on the human and chimpanzee lineages were not different from one another (P=0.8), were different from those of other primate lineages (P=0.004) and were greater than 1 (P=0.04). This is consistent with the historic occurrence of positive darwinian selection pressure on the BRCA1 protein in the human and chimpanzee lineages. Analysis of genetic variation in a sample of female Australians of Northern European origin showed evidence for Hardy-Weinberg (HW) disequilibrium at polymorphic sites in BRCA1, consistent with the possibility that natural selection is affecting genotype frequencies in modern Europeans. The clustering of between-species variation in the region of the gene encoding the RAD51-interaction domain of BRCA1 suggests the maintenance of genomic integrity as a possible target of selection.

BRCA1 mutations and other sequence variants in a population-based sample of Australian women with breast cancer

SummaryThe frequency, in women with breast cancer, of mutations and other variants in the susceptibility gene, BRCA1, was investigated using a population-based case–control-family study. Cases were women living in Melbourne or Sydney, Australia, with histologically confirmed, first primary, invasive breast cancer, diagnosed before the age of 40 years, recorded on the state Cancer Registries. Controls were women without breast cancer, frequency-matched for age, randomly selected from electoral rolls. Full manual sequencing of the coding region of BRCA1 was conducted in a randomly stratified sample of 91 cases; 47 with, and 44 without, a family history of breast cancer in a first- or second-degree relative. All detected variants were tested in a random sample of 67 controls. Three cases with a (protein-truncating) mutation were detected. Only one case had a family history; her mother had breast cancer, but did not carry the mutation. The proportion of Australian women with breast cancer before age 40 who carry a germline mutation in BRCA1 was estimated to be 3.8% (95% Cl 0.3–12.6%). Seven rare variants were also detected, but for none was there evidence of a strong effect on breast cancer susceptibility. Therefore, on a population basis, rare variants are likely to contribute little to breast cancer incidence.

BRCA1 and BRCA2 Mutation Carriers, Oral Contraceptive Use, and Breast Cancer Before Age 50

Background: Understanding the effect of oral contraceptives on risk of breast cancer in BRCA1 or BRCA2 mutation carriers is important because oral contraceptive use is a common, modifiable practice. Methods: We studied 497 BRCA1 and 307 BRCA2 mutation carriers, of whom 195 and 128, respectively, had been diagnosed with breast cancer. Case-control analyses were conducted using unconditional logistic regression with adjustments for family history and familial relationships and were restricted to subjects with a reference age under 50 years. Results: For BRCA1 mutation carriers, there was no significant association between risk of breast cancer and use of oral contraceptives for at least 1 year [odds ratio (OR), 0.77; 95% confidence interval (95% CI), 0.53-1.12] or duration of oral contraceptive use (Ptrend = 0.62). For BRCA2 mutation carriers, there was no association with use of oral contraceptives for at least 1 year (OR, 1.62; 95% CI, 0.90-2.92); however, there was an association of elevated risk with oral contraceptive use for at least 5 years (OR, 2.06; 95% CI, 1.08-3.94) and with duration of use (ORtrend per year of use, 1.08; P = 0.008). Similar results were obtained when we considered only use of oral contraceptives that first started in 1975 or later. Conclusions: We found no evidence overall that use of oral contraceptives for at least 1 year is associated with breast cancer risk for BRCA1 and BRCA2 mutation carriers before age 50. For BRCA2 mutation carriers, use of oral contraceptives may be associated with an increased risk of breast cancer among women who use them for at least 5 years. Further studies reporting results separately for BRCA1 and BRCA2 mutation carriers are needed to resolve this important issue. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1863–70)

De Novo BRCA1 Mutation in a Patient with Breast Cancer and an Inherited BRCA2 Mutation

We thank P. van der Spek, J. Hoeijmakers, S. Venitt, M. Stratton, S. Easteal, R. Sinden and T. Kunkel for discussions; L. Trute for technical assistance; M. Gardner for genetic counseling of patients; and E. Edkins for heteroduplex analysis. This work was funded by the National Health and Medical Research Council of Australia and by the Victorian Health Promotion Foundation.

Morphological predictors of BRCA1 germline mutations in young women with breast cancer

Background:Knowing a young woman with newly diagnosed breast cancer has a germline BRCA1 mutation informs her clinical management and that of her relatives. We sought an optimal strategy for identifying carriers using family history, breast cancer morphology and hormone receptor status data.Methods:We studied a population-based sample of 452 Australian women with invasive breast cancer diagnosed before age 40 years for whom we conducted extensive germline mutation testing (29 carried a BRCA1 mutation) and a systematic pathology review, and collected three-generational family history and tumour ER and PR status. Predictors of mutation status were identified using multiple logistic regression. Areas under receiver operator characteristic (ROC) curves were estimated using five-fold stratified cross-validation.Results:The probability of being a BRCA1 mutation carrier increased with number of selected histology features even after adjusting for family history and ER and PR status (P<0.0001). From the most parsimonious multivariate model, the odds ratio for being a carrier were: 9.7 (95% confidence interval: 2.6–47.0) for trabecular growth pattern (P=0.001); 7.8 (2.7–25.7) for mitotic index over 50 mitoses per 10 high-powered field (P=0.0003); and 2.7 (1.3–5.9) for each first-degree relative with breast cancer diagnosed before age 60 years (P=0.01).The area under the ROC curve was 0.87 (0.83–0.90).Conclusion:Pathology review, with attention to a few specific morphological features of invasive breast cancers, can identify almost all BRCA1 germline mutation carriers among women with early-onset breast cancer without taking into account family history.

The rs743572 common variant in the promoter of CYP17A1 is not associated with prostate cancer risk or circulating hormonal levels.

To use a large population‐based case‐control study to test the association between the common genetic variant rs743572 (−34 T to C), prostate cancer risk and circulating levels of several hormones.

The role of SMAD4 in early-onset colorectal cancer.

Objective  Chromosomal loss within the region of 18q and loss of SMAD4 expression have been reported to be frequent somatic events during colorectal cancer tumour progression; however, their associations with age at onset have not been widely studied.

Large genomic alterations in hMSH2 and hMLH1 in early-onset colorectal cancer: identification of a large complex de novo hMLH1 alteration.

To the Editor: The Victorian Colorectal Cancer Family Study is a population-based, case-family study of earlyonset colorectal cancer conducted in Australia during 1993–1997 (1). Eligible case patients comprised adult men and women living in the Melbourne metropolitan area who were under the age of 45 years when diagnosed with a histologically confirmed first primary adenocarcinoma of the colon or rectum (ICD-9 rubrics 153 and 154, respectively) who were identified through the population-wide Victorian Cancer Registry. Invasive tumour samples of primary colorectal adenocarcinoma were obtained from hospitals and private pathology laboratories for 118 case patients (90% of those recruited). The tumour protein expression of MLH1, MSH2, MSH6 and PMS2 was assessed as described and reported elsewhere (2). Microsatellite instability (MSI) was assessed utilizing a five-microsatellite marker panel (3, 4). All exonic and flanking intronic sequences of hMLH1, hMSH2, hMSH6 and hPMS2were screened for germline mutations using sequencing approaches, except for exon 4 of hMSH6 that was screened in eight overlapping fragments using denaturing high-performance liquid chromatography (DHPLC) (2). The multiplex ligation-dependent probe amplification (MLPA) assay (MRC-Holland, Amsterdam, The Netherlands) (5) was used to detect large genomic alterations in hMLH1 and hMSH2 and was performed on samples from 10 case patients that had tumours lacking expression of at least one mismatch repair (MMR) protein and for which no mutation had been identified byDNA-sequencing methods or DHPLC. Breakpoints were characterized by performing a long-range PCR encompassing the predicted genomic alteration, cloning and sequencing. PCR primer sequences and PCR product sizes are given in Table 1 panel a. The germline DNA from two individuals were found to have MLPA profiles consistent with having a large deletion in hMLH1. No alterations were identified in hMSH2. Case 1 was a male diagnosed with cancer of the transverse colon at the age of 24 years, with no reported family history of colorectal cancer in any firstor second-degree relative. The endoscopic biopsywasMSI high, displaying instability in four of the five markers tested (Bat25, Bat26, D5S345 and D17S250). The tumour expressed MSH2 and MSH6 protein, lacked the expression of PMS2, and some focal cells were negative for MLH1 as determined by immuno-histo-chemistry (IHC). MLPA analysis suggested that exon 15 of hMLH1 was deleted in the germline. Long-range PCR confirmed the deletion by identifying the breakpoint, a deletion of 1444 bp and a 264–269 bp insertion. Specifically, the genomic change was hMLH1, 50334del1444 and 50334ins264–269 (reference sequence accessionnumberAY217549).The insertion of 264–269 bp between the breakpoints identifiedhas99%homologyto theAlu-repeat subfamily Sb2 (accession number U14570). This complex mutation is illustrated in Fig. 1(a). A specific test was devised utilizing long-range PCR over the breakpoint region, and other participating family members were screened for hMLH1, 50334del1444ins264–269 (Table 1 panel a). Neither parent was found to carry this mutation. Two microsatellite markers, UT5013 and UT1699 (6), confirmed parentage, implying that thismutationwas de novo. Case 2 was a male diagnosed with cancer of the ascending colon at the age of 42 years, with a brother diagnosed with cancer of the caecum at age 36, a sister diagnosed with cancer of the colon at age 41, and father diagnosed with cancer of the stomach at age 67. This family history was sufficient to meet the Amsterdam II criteria for hereditary non-polyposis colorectal cancer