Refined cut-off for TP53 immunohistochemistry improves prediction of TP53 mutation status in ovarian mucinous tumors: implications for outcome analyses.

TP53 mutations are implicated in the progression of mucinous borderline tumors (MBOT) to mucinous ovarian carcinomas (MOC). Optimized immunohistochemistry (IHC) for TP53 has been established as a proxy for the TP53 mutation status in other ovarian tumor types. We aimed to confirm the ability of TP53 IHC to predict TP53 mutation status in ovarian mucinous tumors and to evaluate the association of TP53 mutation status with survival among patients with MBOT and MOC. Tumor tissue from an initial cohort of 113 women with MBOT/MOC was stained with optimized IHC for TP53 using tissue microarrays (75.2%) or full sections (24.8%) and interpreted using established criteria as normal or abnormal (overexpression, complete absence, or cytoplasmic). Cases were considered concordant if abnormal IHC staining predicted deleterious TP53 mutations. Discordant tissue microarray cases were re-evaluated on full sections and interpretational criteria were refined. The initial cohort was expanded to a total of 165 MBOT and 424 MOC for the examination of the association of survival with TP53 mutation status, assessed either by TP53 IHC and/or sequencing. Initially, 82/113 (72.6%) cases were concordant using the established criteria. Refined criteria for overexpression to account for intratumoral heterogeneity and terminal differentiation improved concordance to 93.8% (106/113). In the expanded cohort, 19.4% (32/165) of MBOT showed evidence for TP53 mutation and this was associated with a higher risk of recurrence, disease-specific death, and all-cause mortality (overall survival: HR = 4.6, 95% CI 1.5-14.3, p = 0.0087). Within MOC, 61.1% (259/424) harbored a TP53 mutation, but this was not associated with survival (overall survival, p = 0.77). TP53 IHC is an accurate proxy for TP53 mutation status with refined interpretation criteria accounting for intratumoral heterogeneity and terminal differentiation in ovarian mucinous tumors. TP53 mutation status is an important biomarker to identify MBOT with a higher risk of mortality.

Therapeutic options for mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. METHODS: We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117-166). RESULTS: Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). CONCLUSIONS: Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

Streamlined genetic education is effective in preparing women newly diagnosed with breast cancer for decision making about treatment-focused genetic testing: a randomized controlled noninferiority trial.

PURPOSE: Increasingly, women newly diagnosed with breast cancer are being offered treatment-focused genetic testing (TFGT). As the demand for TFGT increases, streamlined methods of genetic education are needed. METHODS: In this noninferiority trial, women aged <50 years with either a strong family history (FH+) or other features suggestive of a germ-line mutation (FH-) were randomized before definitive breast cancer surgery to receive TFGT education either as brief written materials (intervention group (IG)) or during a genetic counseling session at a familial cancer clinic (usual-care group (UCG)). Women completed self-report questionnaires at four time points over 12 months. RESULTS: A total of 135 women were included in the analysis, all of whom opted for TFGT. Decisional conflict about TFGT choice (primary outcome) was not inferior in the IG compared with the UCG (noninferiority margin of -10; mean difference = 2.45; 95% confidence interval -2.87-7.76; P = 0.36). Costs per woman counseled in the IG were significantly lower (AUD$89) compared with the UCG (AUD$173; t(115) = 6.02; P < 0.001). CONCLUSION: A streamlined model of educating women newly diagnosed with breast cancer about TFGT seems to be a cost-effective way of delivering education while ensuring that women feel informed and supported in their decision making, thus freeing resources for other women to access TFGT.Genet Med 19 4, 448-456.

Lynch syndrome and cervical cancer.

Carriers of germline mutations in DNA mismatch repair (MMR) genes are at increased risk of several cancers including colorectal and gynecologic cancers (Lynch syndrome). There is no substantial evidence that these mutations are associated with an increased risk of cervical cancer. A total of 369 families with at least one carrier of a mutation in a MMR gene (133 MLH1, 174 MSH2, 35 MSH6 and 27 PMS2) were ascertained via population cancer registries or via family cancer clinics in Australia, New Zealand, Canada, and USA. Personal and family histories of cancer were obtained from participant interviews. Modified segregation analysis was used to estimate the hazard ratio (incidence rates for carriers relative to those for the general population), and age-specific cumulative risks of cervical cancer for carriers. A total of 65 cases of cervical cancer were reported (including 10 verified by pathology reports). The estimated incidence was 5.6 fold (95% CI: 2.3-13.8; p = 0.001) higher for carriers than for the general population with a corresponding cumulative risk to 80 years of 4.5% (95% CI: 1.9-10.7%) compared with 0.8% for the general population. The mean age at diagnosis was 43.1 years (95% CI: 40.0-46.2), 3.9 years younger than the reported USA population mean of 47.0 years (p = 0.02). Women with MMR gene mutations were found to have an increased risk of cervical cancer. Due to limited pathology verification we cannot be certain that a proportion of these cases were not lower uterine segment endometrial cancers involving the endocervix, a recognized cancer of Lynch syndrome.

Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

Cancer risks for MLH1 and MSH2 mutation carriers.

We studied 17,576 members of 166 MLH1 and 224 MSH2 mutation-carrying families from the Colon Cancer Family Registry. Average cumulative risks of colorectal cancer (CRC), endometrial cancer (EC), and other cancers for carriers were estimated using modified segregation analysis conditioned on ascertainment criteria. Heterogeneity in risks was investigated using a polygenic risk modifier. Average CRC cumulative risks at the age of 70 years (95% confidence intervals) for MLH1 and MSH2 mutation carriers, respectively, were estimated to be 34% (25%-50%) and 47% (36%-60%) for male carriers and 36% (25%-51%) and 37% (27%-50%) for female carriers. Corresponding EC risks were 18% (9.1%-34%) and 30% (18%-45%). A high level of CRC risk heterogeneity was observed (P < 0.001), with cumulative risks at the age of 70 years estimated to follow U-shaped distributions. For example, 17% of male MSH2 mutation carriers have estimated lifetime risks of 0%-10% and 18% have risks of 90%-100%. Therefore, average risks are similar for the two genes but there is so much individual variation about the average that large proportions of carriers have either very low or very high lifetime cancer risks. Our estimates of CRC and EC cumulative risks for MLH1 and MSH2 mutation carriers are the most precise currently available.

Report of Endometrial Cancer in Australian BRCA1 and BRCA2 mutation-positive Families.

There is evidence that tamoxifen treatment of BRCA1 and BRCA2 carriers for prior breast cancer increases risk of endometrioid subtype endometrial cancer (EC), and suggestive evidence that BRCA1 and BRCA2 mutation carriers may be predisposed to EC in the absence of tamoxifen exposure. We assessed the association of EC with BRCA1 or BRCA2 mutation status in Australasian breast-ovarian families. Report of at least one case of EC was significantly greater in BRCA1-positive families (35/218 (16%); p = .03) and non-significantly greater in BRCA2-positive families (23/189 (12%); p = .6), compared to high-risk breast cancer families without a BRCA1/2 mutation (86/796 (11%)). EC was the first/concurrent cancer for 41% of EC cases with multiple cancer diagnoses from BRCA1/2 families, and early onset for most of these diagnoses. Mutation status was imputed for ungeno-typed individuals from 57 BRCA1/2 pedigrees reporting EC using BRCAPRO. Effects of genotype on EC diagnosis age, and interaction with tamoxifen therapy, were assessed using Cox proportional hazards regression analysis. EC risk was non-significantly marginally greater for BRCA1 carriers (hazard ratio = 1.25, 95%CI = 0.65-2.41), and BRCA2 carriers (HR = 1.12, 95%CI = 0.51-2.45), compared to non-carrier family members. Tamoxifen therapy was highly significantly associated with EC (HR = 6.68, 95%CI = 3.12-15.15; p = 1.7 x 10(-6)) in BRCA1/2 families, with no evidence for interaction between tamoxifen therapy and BRCA1/2 genotype. Our family-based study supports a 7-fold increase in EC risk with tamoxifen exposure for female family members from BRCA1/2 families. Early onset EC in carriers without tamoxifen use suggests that further study is required to assess association of modest EC risk with BRCA1/2 mutation status alone.

Loss of heterozygosity at the BRCA2 locus detected by multiplex ligation-dependent probe amplification is common in prostate cancers from men with a germline BRCA2 mutation.

PURPOSE: Prostate cancer risk is increased for men carrying a pathogenic germline mutation in BRCA2, and perhaps BRCA1. Our primary aim was to test for loss of heterozygosity (LOH) at the locus of the mutation in prostate cancers from men who a carry pathogenic germline mutation in BRCA1 or BRCA2, and to assess clinical and pathologic features of these tumors. EXPERIMENTAL DESIGN: From 1,243 kConFab families: (a) 215 families carried a pathogenic BRCA1 mutation, whereas 188 families carried a pathogenic BRCA2 mutation; (b) of the 158 men diagnosed with prostate cancer (from 137 families), 8 were confirmed to carry the family-specific BRCA1 mutation, whereas 20 were confirmed to carry the family-specific BRCA2 mutation; and (c) 10 cases were eliminated from analysis because no archival material was available. The final cohort comprised 4 and 14 men with a BRCA1 and BRCA2 mutation, respectively. We examined LOH at the BRCA1 and BRCA2 genes using multiplex ligation-dependent probe amplification of DNA from microdissected tumor. RESULTS: LOH at BRCA2 was observed in 10 of 14 tumors from BRCA2 mutation carriers (71%), whereas no LOH at BRCA1 was observed in four tumors from BRCA1 mutation carriers (P = 0.02). Under the assumption that LOH occurs only because the cancer was caused by the germline mutation, carriers of BRCA2 mutations are at 3.5-fold (95% confidence interval, 1.8-12) increased risk of prostate cancer. A high Gleason was the only distinct clinical feature. CONCLUSIONS: These observations are consistent with the idea that BRCA2, but not BRCA1, is a tumor suppressor of prostate cancer.

Psychological outcomes and surgical decisions after genetic testing in women newly diagnosed with breast cancer with and without a family history.

In patients with early breast cancer, personal and tumour characteristics other than family history are increasingly used to prompt genetic testing to guide women's cancer management (treatment-focused genetic testing, 'TFGT'). Women without a known strong family history of breast and/or ovarian may be more vulnerable to psychological sequelae arising from TFGT. We compared the impact of TFGT in women with (FH+) and without (FH-) a strong family history on psychological adjustment and surgical decisions. Women aged <50 years with high-risk features were offered TFGT before definitive breast cancer surgery and completed self-report questionnaires at four time points over 12 months. All 128 women opted for TFGT. TFGT identified 18 carriers of a disease-causing variant (50.0% FH+) and 110 non-carriers (59.1% FH+). There were no differences based on family history in bilateral mastectomy (BM) uptake, p = .190, or uptake of risk-reducing bilateral salpingo-oophorectomy (RRBSO), p = .093. FH- women had lower decreases in anxiety a year after diagnosis, p = .011, and regret regarding their decision whether to undergo BM, p = .022, or RRBSO, p = .016 than FH + women. FH- carriers reported significantly higher regret regarding their TFGT choice (p = .024) and test-related distress (p = .012) than FH + carriers, but this regret/distress could not be attributed to a concern regarding a possible worse prognosis. These findings indicate that FH- women may require additional counselling to facilitate informed decisions. Carriers without a family history may require additional follow-up counselling to facilitate psychological adjustment to their positive variant results, extra support in making surgical decisions, and counselling about how best to communicate results to family members.

Denosumab: Prevention and management of hypocalcemia, osteonecrosis of the jaw and atypical fractures.

Denosumab, a bone-modifying agent, reduces the risk of skeletal-related events in patients with bone metastases from solid tumors and is generally well tolerated. However, hypocalcemia, osteonecrosis of the jaw (ONJ) and atypical fracture are potential and important toxicities of denosumab therapy that require attention. In pivotal phase III trials in patients with bone metastases from solid tumors, the incidence of hypocalcemia was 9.6% in denosumab-treated patients, with most events being asymptomatic, grade 2 and resolving by week 4. Established hypocalcaemia requires additional short-term calcium and vitamin D supplementation and, if severe, administration of intravenous calcium. ONJ was reported in 1.8% of patients receiving denosumab over 3 years in these trials. Involvement of an experienced oro-maxillary surgeon is important if ONJ is suspected. Atypical fractures were rare in a large study of denosumab using the dose and scheduling approved for the treatment of osteoporosis. To prevent toxicities, patients should maintain calcium and vitamin D supplementation, good oral hygiene and regular dental reviews throughout treatment. This article presents case studies from our clinical practice and discusses the pathophysiology of these toxicities along with guidance on prevention, diagnosis and management.

Loss of heterozygosity analysis in ductal lavage samples from BRCA1 and BRCA2 carriers: a cautionary tale.

BACKGROUND: Loss of heterozygosity (LOH) in breast ductal lavage (DL) fluid has been reported to be a potential biomarker of malignant change. Interpretation of LOH is reliant on sufficient quality and quantity of DNA. We investigated LOH of the BRCA1/2 loci in DL samples from BRCA1/2 mutation carriers, while also assessing the effect of DNA quantity. METHODS: DNA yield was estimated using quantitative real-time PCR. Allelic status of DL DNA was determined using fluorescently tagged microsatellite markers with the subject's lymphocytic DNA serving as a control. Samples were scored as consistently heterozygous or as demonstrating LOH if the same result was observed in replicate experiments. Additionally, samples were scored as "discordant LOH" if they initially showed LOH, but in replicate experiments either showed heterozygosity or LOH of the opposite allele. RESULTS: In 11 BRCA1 carriers, 46 ducts were assessable, and 39 ducts from 14 BRCA2 carriers were assessable. LOH was observed in 17% and 18% of ducts from BRCA1 and BRCA2, respectively. Discordant results were seen in 23 BRCA1 (50%) and 15 BRCA2 (38%) samples. DNA yield was significantly greater in samples that were consistently heterozygous than those that were either discordant or showed LOH in replicate experiments for both BRCA1 (P = 0.003) and BRCA2 (P = 0.003). CONCLUSIONS: DNA quantity is highly variable between DL samples, with low yields likely to detrimentally affect the interpretation of LOH. In conclusion, LOH may not be an adequate method to detect the early stages of malignant change in samples obtained via DL.

Screening behavior in women at increased familial risk for breast cancer.

This multicenter study examined the adherence of high-risk women to screening recommendations for breast and ovarian cancer following consultation at a familial cancer clinic (FCC). Self-report questionnaires assessing recall of screening advice, tests undertaken, risk perception, anxiety (Impact of Events Scale) and demographics were mailed to 396 consecutive eligible women who had attended one of six FCCs a median of 3.6 years prior. Family history, genetic test results and screening recommendations were abstracted from medical records. 182/266 (68.4%) women responded with 130 lost to follow-up. The proportions of women undertaking at least the recommended frequency of screening tests were: breast self examination (BSE) 50.4%, clinical breast examination (CBE) 66.0%, mammography 82.2%, transvaginal ultrasound (TVUS) 70.0%, CA125 84.0%. Factors associated with adherence to screening were: higher anxiety for BSE and CBE, being BRCA1/2 positive for CBE, older age, method of arrangement and having at least one affected first degree relative for mammography. Factors significantly associated with over-adherence were higher scores for anxiety for BSE and CBE and younger age (< 40 years) for TVUS. Between 41.3% (BSE) and 57.6% (CBE) of women incorrectly recalled their screening recommendations. A substantial minority of high-risk women do not adhere to screening advice. Strategies to improve the accuracy of recall of recommendations and the uptake of recommended screening are required.

Nipple aspiration and ductal lavage in women with a germline BRCA1 or BRCA2 mutation.

INTRODUCTION: The aim of this study was to collect serial samples of nipple aspirate (NA) and ductal lavage (DL) fluid from women with germline BRCA1/2 mutations in order to create a biorepository for use in identifying biomarkers of breast cancer risk. METHODS: Between March 2003 and February 2005, 52 women with germline BRCA1 or BRCA2 mutations (median age 43 years, range 27 to 65 years) were scheduled for six-monthly NA, DL and venesection. DL was attempted for all NA fluid-yielding (FY) and any non-FY ducts that could be located at each visit. RESULTS: Twenty-seven (52%) women were postmenopausal, predominantly (19/27) from risk reducing bilateral salpingo-oophorectomy (BSO). FY ducts were identified in 60% of all women, 76% of premenopausal women versus 44% of postmenopausal (P = 0.026). Eighty-five percent of women had successful DL. Success was most likely in women with FY ducts (FY 94% versus non-FY 71% (P = 0.049). DL samples were more likely to be cellular if collected from FY ducts (FY 68% versus non-FY 43%; P = 0.037). Total cell counts were associated with FY status (FY median cell count 30,996, range 0 to >1,000,000 versus non-FY median cell count 0, range 0 to 173,577; P = 0.002). Four women (8%) had ducts with severe atypia with or without additional ducts with mild epithelial atypia; seven others had ducts with mild atypia alone (11/52 (21%) in total). Median total cell count was greater from ducts with atypia (105,870, range 1920 to >1,000,000) than those with no atypia (174, 0 to >1,000,000; P

Body mass index in early adulthood and endometrial cancer risk for mismatch repair gene mutation carriers.

OBJECTIVE: To investigate the association of body mass index (BMI) in early adulthood and endometrial cancer risk for carriers of a germline mutation in a DNA mismatch repair gene. METHODS: We estimated the association between BMI at age 18-20 years and endometrial cancer risk for mismatch repair gene mutation carriers and, as a comparison group, noncarriers using 601 female carriers of a germline mutation in a mismatch repair gene (245 MLH1, 299 MSH2, 38 MSH6, and 19 PMS2) and 533 female noncarriers from the Colon Cancer Family Registry using a weighted Cox proportional hazards regression. RESULTS: During 51,693 person-years of observation, we observed diagnoses of endometrial cancer for 126 carriers and eight noncarriers. For carriers, there was no evidence of an association between BMI at age 20 years and endometrial cancer (adjusted hazard ratio 0.73 per 5 kg/m²; 95% confidence interval [CI], 0.40-1.34; P=.31). For noncarriers, endometrial cancer risk increased by 74% for each 5-kg/m² increment in BMI (adjusted hazard ratio 1.74; 95% CI 1.27-2.37; P<.001). The hazard ratio for BMI and endometrial cancer for noncarriers was greater than for carriers (P=.04). CONCLUSION: The effect of body mass on endometrial cancer risk depends on the woman's mismatch repair gene mutation carrier status, suggesting obesity-independent endometrial carcinogenesis for carriers. LEVEL OF EVIDENCE: II.

Gene methylation in breast ductal fluid from BRCA1 and BRCA2 mutation carriers.

PURPOSE: Genomic alterations (including gene hypermethylation) are likely to precede the phenotypic changes associated with breast tumorigenesis. From a prospective collection of ductal lavage (DL) samples from women with a known mutation in BRCA1 or BRCA2, we have assessed promoter methylation with a comparison of results with several variables, including breast cancer (BC) outcome. EXPERIMENTAL DESIGN: Hypermethylation of p16, RASSF1A, twist, and RARbeta was assessed using a qualitative, real-time, nested PCR assay. Associations between methylation status and variables were tested using Fisher's exact test or logistic regression. Analyses were done at three levels: a single breast, a single duct (both over time), and each DL sample in isolation. RESULTS: A total of 168 samples from 93 ducts in 54 breasts have been analyzed in 34 women (16 BRCA1 and 18 BRCA2 mutation carriers). A median of 2 DL was done (range, 1-5), with 7 women developing BC on study, 1 bilateral. Methylation of p16 was associated with a known BRCA1 mutation (P = 0.001, P < 0.001, and P < 0.001 for breast, duct, and sample levels, respectively) and women with a history of contralateral BC (P = 0.001 and P < 0.001 for duct and sample levels, respectively). An association was seen for women who developed BC on study and RASSF1A methylation (P = 0.001 for sample level). CONCLUSIONS: Genetic methylation patterns could potentially be used to predict future BC risk. In addition, p16 methylation may be a predictor of BRCA1 mutation status. Further research is required to corroborate these findings.