Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods 1 - 4 are limited by the loss of native modifications, short read length, high input requirements, low yield, or long protocols. Here, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adaptors for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants (SNVs), structural variations (SVs) and CpG methylation. We apply nCATS to four cell lines, a cell-line-derived xenograft, and normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675X using a minION flow cell and 34X using the smaller flongle flow cell. nCATS requires only ~3μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.

BACKGROUND: Human adenovirus-36 (Ad-36) increases adiposity and paradoxically lowers serum cholesterol and triglycerides in chickens, mice, and non-human primates. The role of Ad-36 in human obesity is unknown. OBJECTIVES: To determine the prevalence of Ad-36 antibodies in obese and nonobese humans. To evaluate the association of Ad-36 antibodies with body mass index (BMI) and serum lipids. DESIGN: Cohort study. Volunteers from obesity treatment programs, communities, and a research study. SUBJECTS: Obese and nonobese volunteers at the University of Wisconsin, Madison, WI, and the Bowen Center, Naples, Florida. Obese and thin volunteer research subjects and 89 twin pairs at Columbia University, New York. INTERVENTIONS: Study 1: 502 subjects; serum neutralization assay for antibodies to Ad-2, Ad-31, Ad-36, and Ad-37; serum cholesterol and triglycerides assays. Study 2: BMI and %body fat in 28 twin pairs discordant for Ad-36 antibodies. MAIN OUTCOME MEASURES: Presence of antibodies to adenoviruses, BMI, serum cholesterol and triglycerides levels. RESULTS: Significant (Po0.001) association of obesity and positive Ad-36 antibody status, independent of age, sex, and collection site. Ad-36 antibodies in 30% of obese, 11% of nonobese. Lower serum cholesterol and triglycerides (Po0.003) in Ad-36 antibody-positive vs -negative subjects. Twin pairs: antibody-positive twins had higher BMIs (24.575.2 vs 23.174.5 kg/m 2 , Po0.03) and %body fat (29.679.5% vs 27.579.9%, Po0.04). No association of Ad-2, Ad-31, or Ad-37 antibodies with BMI or serum lipids. CONCLUSIONS: Ad-36 is associated with increased body weight and lower serum lipids in humans. Prospective studies are indicated to determine if Ad-36 plays a role in the etiology of human obesity.

Background Adjuvant trastuzumab significantly improves outcomes for patients with HER2-positive early breast cancer. The standard treatment duration is 12 months but shorter treatment could provide similar efficacy while reducing toxicities and cost. We aimed to investigate whether 6-month adjuvant trastuzumab treatment is noninferior to the standard 12-month treatment regarding disease-free survival. Methods This study is an open-label, randomised phase 3 non-inferiority trial. Patients were recruited from 152 centres in the UK. We randomly assigned patients with HER2-positive early breast cancer, aged 18 years or older, and with a clear indication for chemotherapy, by a computerised minimisation process (1:1), to receive either 6-month or 12-month trastuzumab delivered every 3 weeks intravenously (loading dose of 8 mg/kg followed by maintenance doses of 6 mg/kg) or subcutaneously (600 mg), given in combination with chemotherapy (concurrently or sequentially). The primary endpoint was disease-free survival, analysed by intention to treat, with a non-inferiority margin of 3% for 4-year disease-free survival. Safety was analysed in all patients who received trastuzumab. This trial is registered with EudraCT (number 2006-007018-39), ISRCTN (number 52968807), and ClinicalTrials.gov (number NCT00712140).

Since there are no published data on breast cancer in British black women, we sought to determine whether, like African-American women, they present at a younger age with biologically distinct disease patterns. The method involved a retrospective review of breast cancer to compare age distributions and clinicopathological features between black women and white women in the UK, while controlling for socioeconomic status. All women presented with invasive breast cancer, between 1994 and 2005, to a single East London hospital. Black patients presented significantly younger (median age of 46 years), than white patients (median age of 67 years (P ¼ 0.001)). No significant differences between black and white population structures were identified. Black women had a higher frequency of grade 3 tumours, lymph node-positive disease, negative oestrogen receptor and progesterone receptor status and basal-like (triple negative status) tumours. There were no differences in stage at presentation; however, for tumours of p2 cm, black patients had poorer survival than white patients (HR ¼ 2.90, 95% CI 0.98 -8.60, P ¼ 0.05). Black women presented, on average, 21 years younger than white women. Tumours in younger women were considerably more aggressive in the black population, more likely to be basal-like, and among women with smaller tumours, black women were more than twice as likely to die of their disease. There were no disparities in socioeconomic status or treatment received. Our findings could have major implications for the biology of breast cancer and the detection and treatment of the disease in black women.

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BackgroundIntegrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer.MethodsProtein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student’s t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni’s Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided.ResultsHigh expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts.ConclusionsTargeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.

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Background:Molecular profiling has identified at least four subtypes of invasive breast carcinoma, which exhibit distinct clinical behaviour. There is good evidence now that DCIS represents the non-obligate precursor to invasive breast cancer and therefore it should be possible to identify similar molecular subtypes at this stage. In addition to a limited five-marker system to identify molecular subtypes in invasive breast cancer, it is evident that other biological molecules may identify distinct tumour subsets, though this has not been formally evaluated in DCIS.Methods:Tissue microarrays were constructed for 188 cases of DCIS. Immunohistochemistry was performed to examine the expression patterns of oestrogen receptor (ER), progesterone receptor (PR), Her2, EGFR, cytokeratin (CK) 5/6, CK14, CK17, CK18, β4-integrin, β6-integrin, p53, SMA, maspin, Bcl-2, topoisomerase IIα and P-cadherin. Hierarchical clustering analysis was undertaken to identify any natural groupings, and the findings were validated in an independent sample series.Results:Each of the intrinsic molecular subtypes described for invasive breast cancer can be identified in DCIS, though there are differences in the relative frequency of subgroups, in particular, the triple negative and basal-like phenotype is very uncommon in DCIS. Hierarchical cluster analysis identified three main subtypes of DCIS determined largely by ER, PR, Her2 and Bcl-2, and this classification is related to conventional prognostic indicators. These subtypes were confirmed in an analysis on independent series of DCIS cases.Conclusion:This study indicates that DCIS may be classified in a similar manner to invasive breast cancer, and determining the relative frequency of different subtypes in DCIS and invasive disease may shed light on factors determining disease progression. It also demonstrates a role for Bcl-2 in classifying DCIS, which has recently been identified in invasive breast cancer.