Search

Purpose: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. Experimental Design: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. Results: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. Conclusions: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

With early detection, breast conservation surgery with adequate surgical margins is the standard of care. The aim of this study was to evaluate the use of pre-operative duct endoscopy (DE) to target surgical resection, improve adequate margins and reduce re-excision operations. Women with DCIS, stage I and II breast cancer suitable for breast conservation were randomized to DE-assisted wide local excision versus standard wide local excision (without DE). The primary endpoint was margin re-excision rates between the two groups. Secondary end points were: (i) volume differences of the surgical specimen; (ii) whether an extensive in situ component (EIC) influenced successful DE-guided resection. 78 women were randomized: 44 patients to no-DE and 34 patients to the DE group. The median age was 59 (49–65) and 56 (48–64) years in the two groups respectively with mean follow-up of 9.1 (4.2–11.1) years. There were 23 positive findings in 17 women in 30 successful DE procedures (17/30 = 56.7%). The surgical specimen volume, no-DE (17 [IQR 10–29] cm3) and DE 20 [IQR 12–28] cm3), did not differ, p = 0.377. The overall re-excision rate was 20/78 (26%), 9 (20%) and 11 (32% in the no-DE and DE groups, respectively, p = 0.233. This randomized clinical trial was limited by incomplete accrual. DE did not contribute to improved margin excision rates whether a target lesion was visualized or not. The presence of EIC did not improve efficacy of DE.

This study was supported by Cancer Research UK, AstraZeneca and Pfizer UK. Introduction: Phase I of the Cancer Research UK Stratified Medicine Programme (SMP1) was designed to roll out molecular pathology testing nationwide at the point of cancer diagnosis, as well as facilitate an infrastructure where surplus cancer tissue could be used for research. It offered a non-trial setting to examine common UK cancer genetics in a real-world context. Methods: A total of 26 sites in England, Wales and Scotland, recruited samples from 7814 patients for genetic examination between 2011 and 2013. Tumour types involved were breast, colorectal, lung, prostate, ovarian cancer and malignant melanoma. Centralised molecular testing of surplus material from resections or biopsies of primary/metastatic tissue was performed, with samples examined for 3-5 genetic alterations deemed to be of key interest in site-specific cancers by the National Cancer Research Institute Clinical Study groups. Results: 10 754 patients (98% of those approached) consented to participate, from which 7814 tumour samples were genetically analysed. In total, 53% had at least one genetic aberration detected. From 1885 patients with lung cancer, KRAS mutation was noted to be highly prevalent in adenocarcinoma (37%). In breast cancer (1873 patients), there was a striking contrast in TP53 mutation incidence between patients with ductal cancer (27.3%) and lobular cancer (3.4%). Vast inter-tumour heterogeneity of colorectal cancer (1550 patients) was observed, including myriad double and triple combinations of genetic aberrations. Significant losses of important clinical information included smoking status in lung cancer and loss of distinction between low-grade and high-grade serous ovarian cancers. Conclusion: Nationwide molecular pathology testing in a non-trial setting is feasible. The experience with SMP1 has been used to inform ongoing CRUK flagship programmes such as the CRUK National Lung MATRIX trial and TRACERx. Publisher PDF

Introduction NAC has been used for downsizing of the tumour in breast and axilla to allow more conservative surgery. In the NAC setting, intraoperative assessment of sentinel lymph node(s) (SLN) is still considered necessary1. Current awareness of the prognostic value for axillary nodal down-staging has renewed interest in analysis of SLN post-NAC. In this study we want to examine the clinical utility of OSNA (based on CK19 mRNA detection) as a method of intra-operative analysis of SLN to assist real-time decision-making for axillary surgery post-NAC in early breast cancer (EBC). Methods Retrospective analysis of prospective data on 399 consecutive patients with EBC who received NAC followed by breast surgery with SLN biopsy (408 axillae) and assessment by OSNA, from September 2011 to January 2018 at the Royal Marsden Hospital (UK). OSNA readouts from the Sysmex RD-100i were collected separate to and blinded from clinico-pathological data. A negative or benign pre-treatment axillary ultrasound scan or indeterminate ultrasound with negative or benign axillary cytology/histology prior to NAC was considered cN0. Univariate analysis (significance at p Results The median age at diagnosis was 49 years, median BMI 26, 41 EBC (10%) were screen-detected, 292 (72%) were grade 3 and the most frequent phenotype was receptor triple negative (n=132, 32%). Of 408 axillae, 248 (60%) were initially cN0, of which 113 (46%) had a pathological complete response (pCR) in the breast. SLN in 54 (22%) cN0 patients were positive on OSNA, of which only 6 (9%) had further involved axillary nodes all 6 of which were ER+ Her2-. The remaining 160 (40%) axillae were cN1 of which 87 (54%) had conversion to ypN0 including 55 (34%) with both ypT0ypN0. Axillary lymphadenectomy (AL) was performed in 79 (19%) patients overall, of which n=22 (28%) were cN0 and 57 (72%) were cN1. Of these, 30 (53%) of the cN1 and 6 of 22 (45%) of cN0 had at least 1 additional positive AL node. Overall 59 (14.4%) patients relapsed. A significantly worse rate of relapse was observed in cN1 compared to cN0 patients (37/159 (23.3%) versus 22/244 (9%), p The mean of both the single highest node tumour load (and total nodal tumour load), as measured by CK19mRNA copies/ul on OSNA, were significantly higher at 90,000 (98,300) for those who relapsed versus 23,100 (25,100) for those without relapse (p=0.027). Conclusions The OSNA assay is an accurate tool for axillary SLN analysis in patients after NAC and was helpful in intra-operative axillary management. OSNA reduces the need for a second surgery for AL in 20% of breast cancer patients with a positive-SLN after NAC and might offer additional prognostic value. Reference 1. NCCN. National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology Breast Cancer.2016.Version 2.2016. Citation Format: Muscara F, Christaki G, Richardson C, O'Connell R, Padmanabhan P, Warwick J, Lee Y, Smith I, Nerurkar A, Osin P, Krupa K, Rusby J, Roche N, Gui G, MacNeil F, Barry P. Clinical utility of one-step nucleic acid amplification (OSNA) in axillary surgery after neoadjuvant chemotherapy (NAC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-14.

Purpose: Estrogen withdrawal by treatment with aromatase inhibitors is the most effective form of endocrine therapy for postmenopausal estrogen receptor–positive (ER+) breast cancer. However, response to therapy varies markedly and understanding of the precise molecular effects of aromatase inhibitors and causes of resistance is limited. We aimed to identify in clinical breast cancer those genes and pathways most associated with resistance to aromatase inhibitors by examining the global transcriptional effects of AI treatment. Experimental Design: Baseline and 2-week posttreatment biopsies were obtained from 112 postmenopausal women with ER+ breast cancer receiving neoadjuvant anastrozole. Gene expression data were obtained from 81 baseline and 2-week paired samples. Pathway analysis identified (i) the most prevalent changes in expression and (ii) the pretreatment genes/pathways most related to poor antiproliferative response. Results: A total of 1,327 genes were differentially expressed after 2-week treatment (false discovery rate < 0.01). Proliferation-associated genes and classical estrogen-dependent genes were strongly downregulated whereas collagens and chemokines were upregulated. Pretreatment expression of an inflammatory signature correlated with antiproliferative response to anastrozole and this observation was validated in an independent study. Higher expression of immune-related genes such as SLAMF8 and TNF as well as lymphocytic infiltration were associated with poorer response (P < 0.001) and validated in an independent cohort. Conclusions: The molecular response to aromatase inhibitor treatment varies greatly between patients consistent with the variable clinical benefit from aromatase inhibitor treatment. Higher baseline expression of an inflammatory signature is associated with poor antiproliferative response and should be assessed further as a novel biomarker and potential target for aromatase inhibitor-treated patients. Clin Cancer Res; 19(10); 2775–86. ©2013 AACR.

Background In a previous proof-of-principle study we demonstrated that detection of circulating tumour DNA (ctDNA) in the adjuvant setting, after completion of surgery and chemotherapy for early stage breast cancer, was associated with a high risk of early relapse. Here we present longer follow-up of the same series, to define the predictive power of ctDNA analysis for disease free survival, and assess the potential to predict overall survival. Methods We recruited a cohort of 55 women presenting with early stage, primary breast cancer, who were all scheduled to receive neo-adjuvant chemotherapy. The primary tumour was sequenced to identify somatic mutations, identifying at least one mutation in 43 patients. Mutations were tracked with digital PCR to identify ctDNA, in plasma samples taken either at a single post-surgical time point (2-6 weeks post-surgery) or with serial plasma samples taken every 6 months in the adjuvant setting. Results At a median 31.7 months follow-up, 42% (18/43) patients had relapsed. Detection of ctDNA at the single post-surgical time point was associated with poor disease free survival, HR=13.6 95%CI (4.5, 41.2) p Conclusion Detection of ctDNA in the adjuvant setting has a high predictive power for future relapse and death from breast cancer. Therapeutic trials are required to determine whether mutation tracking identifies relapse sufficiently early to allow for further adjuvant therapy. Citation Format: Turner NC, Garcia-Murillas I, Chopra N, Beaney M, Kilburn L, Cutts R, Osin P, Nerurkar A, Schiavon G, Hrebien S, Bliss J, Dowsett M, Smith I. Circulating tumor DNA analysis to predict relapse and overall survival in early breast cancer – Longer follow-up of a proof-of-principle study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-01.

The concept of an intraductal approach to evaluate the breast microenvironment assumes direct access to the cancer-containing duct. Central duct access to the cancer-affected lobe is essential if cytology or cell markers are to be useful indicators of pre-malignant change. Access to the cancer-bearing lobe would be less important if field change effects of malignant change were predominantly supra-lobar. The aim of this study was to determine how often duct lavage fluid drains the breast cancer-affected segment. 58 patients undergoing mastectomy for breast cancer were recruited among which 47 had at least one fluid-yielding duct. Following duct lavage, fluid-yielding ducts were perfused ex vivo with Polyurethane Elastomer (PU4ii) resin. Specimens were sliced sagittally, and the extent of resin perfusion and anatomical relationship to the cancer-affected segment was recorded. Computed tomography (CT) scanning was performed on selected mastectomies before cut-up for a feasibility study of 3D duct reconstruction. The median number of fluid-yielding ducts cannulated per cancer-affected breast was 2 (range 1-4). 35/47 (74%) mastectomy specimens were successfully cannulated for resin perfusion. 29/35 (83%) showed tracing of the cancer-affected duct system, 6/35 resin perfusions traced duct systems unaffected by cancer and 12/35 perfusions extravasated. The proportion of sagittal breast slices perfused by resin was 13-68% (median 43%). Volume rendering CT showed it is feasible to produce a simulated image of the perfused ducts. Duct access to the cancer-containing segment is feasible in the majority of patients. Fluid-yielding ducts proportionately drain a significant volume of the breast. Large symptomatic cancers may cause obstruction with distal collapse. Further quantitative study of breast perfusion CT scans may be helpful for estimating the volume fraction of breast tissue perfused by fluid-yielding ducts. The intraductal approach is a valid concept for biomarker assessment of cancer-containing breast segments.

Prostate cancer (PrCa) is a significant public health problem. In the European Union, approximately 200 000 men are diagnosed annually with the disease. There are 35 515 cases (Cancer Research UK, 2009a) per year in the United Kingdom and 10 239 deaths (Cancer Research UK, 2009b). It is now the commonest male non-cutaneous cancer diagnosed in the United Kingdom; the lifetime risk of being diagnosed with PrCa is 1 in 10 (Cancer Research UK, 2009a). Although the increase in population screening is leading to an increase in diagnosis, many men will not develop aggressive disease. However, it is recognised that some PrCa cases have a particularly poor prognosis. Although there are some histological and stage predictors of prognosis (Kattan and Scardino, 2002), until recently, none of them have been related to inherited factors. Multiple aetiologies have been proposed to contribute to the development of PrCa. There is strong evidence that inherited genetic factors are important and exhibit significant familial aggregation in some men, particularly when affected at a young age (Woolf, 1960; Edwards and Eeles, 2004). There is a recognised association of breast cancer with PrCa in families (Thiessen, 1974; Anderson and Badzioch, 1992; Tulinius et al, 1992). Male relatives in breast cancer families in Iceland have a 2–3-fold risk of PrCa (Sigurdsson et al, 1997). The breast cancer predisposition genes BRCA1 and BRCA2 have been reported to increase the risk of PrCa by three-fold and seven-fold, respectively, in male mutation carriers ascertained through a family history of breast cancer (Ford et al, 1994; Struewing et al, 1997; Breast Cancer Linkage Consortium, 1999). Analyses of PrCa relative risks (RR) in male mutation carriers in breast cancer families from the Breast Cancer Linkage Consortium showed an RR of 4.65 (95% CI: 3.48–6.22) of PrCa in male BRCA2 mutation carriers (the RR is 7.33 below the age of 65 years) and of 1.07 (0.75–1.54) in BRCA1 carriers (with an RR of 1.82 (1.01–3.29) for men under 65 years of age) (Thompson and Easton, 2001, 2002). The estimated cumulative incidence of PrCa by the age of 70 years is 7.5–33%. Recent studies have suggested that the risk of PrCa in BRCA2 mutation carriers may be as high as an RR of 23-fold at age 60 years (Edwards et al, 2003). Studies from Iceland have reported that germline mutations in BRCA2 may be involved not only in susceptibility to PrCa but also in the aggressiveness of the disease (Sigurdsson et al, 1997; Tryggvadottir et al, 2007). However, these individuals all carried a common founder mutation (999del5 in BRCA2). Narod et al (2008) have reported that PrCa survival in BRCA2 mutation carriers is much shorter (median survival from diagnosis was 4 years) when compared with BRCA1 carriers' survival (median survival from diagnosis was 8 years). In PrCa, in which the BRCA2 germline mutation status was unknown, allele loss at the BRCA2 locus has been shown to be a prognostic factor for survival on univariate analysis (Edwards et al, 1998), and loss of the wild-type allele would imply a tumour suppressor mechanism in predisposition to this disease in BRCA2 mutation carriers, but it is not known whether this is a surrogate for high grade or is due to mutation per se (Knudson, 1971; Willems et al, 2008). A BRCA2 genomic screening study has previously been undertaken by ourselves, and six potentially pathogenic germline BRCA2 mutations were found in a set of 263 PrCa patients diagnosed at ⩽55 years (2.3%) (Edwards et al, 2003). In this study we report clinical follow-up data and the results of loss of heterozygosity (LOH) analyses on PrCa tumours from the mutation carriers in this report. We then studied a second validation data set of men with germline mutations in the BRCA2 gene from a cancer genetics clinic and assessed their survival to confirm our results in a different UK data set of male BRCA2 mutation carriers with PrCa.

567Background: Neoadjuvant chemotherapy (NAC) is increasingly used in phenotype appropriate early operable breast cancer. Pathologic complete response (pCR) rates of up to 60% have been reported, s...

Purpose To determine whether plasma estradiol (E2) levels are related to gene expression in estrogen receptor (ER)–positive breast cancers in postmenopausal women. Materials and Methods Genome-wide RNA profiles were obtained from pretreatment core-cut tumor biopsies from 104 postmenopausal patients with primary ER-positive breast cancer treated with neoadjuvant anastrozole. Pretreatment plasma E2 levels were determined by highly sensitive radioimmunoassay. Genes were identified for which expression was correlated with pretreatment plasma E2 levels. Validation was performed in an independent set of 73 ER-positive breast cancers. Results The expression of many known estrogen-responsive genes and gene sets was highly significantly associated with plasma E2 levels (eg, TFF1/pS2, GREB1, PDZK1 and PGR; P < .005). Plasma E2 explained 27% of the average expression of these four average estrogen-responsive genes (ie, AvERG; r = 0.51; P < .0001), and a standardized mean of plasma E2 levels and ER transcript levels explained 37% (r, 0.61). These observations were validated in an independent set of 73 ER-positive tumors. Exploratory analysis suggested that addition of the nuclear coregulators in a multivariable analysis with ER and E2 levels might additionally improve the relationship with the AvERG. Plasma E2 and the standardized mean of E2 and ER were both significantly correlated with 2-week Ki67, a surrogate marker of clinical outcome (r = −0.179; P = .05; and r = −0.389; P = .0005, respectively). Conclusion Plasma E2 levels are significantly associated with gene expression of ER-positive breast cancers and should be considered in future genomic studies of ER-positive breast cancer. The AvERG is a new experimental tool for the study of putative estrogenic stimuli of breast cancer.

Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AI). We developed ultra-high sensitivity multiplexed digital PCR assays for ESR1 mutations in circulating tumor DNA (ctDNA) and used these to investigate the clinical relevance and origin of ESR1 mutations in a cohort of 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies, and could be assessed in samples shipped at room temperature in preservative tubes without loss of accuracy. ESR1 mutations were found exclusively in patients with estrogen receptor positive breast cancer previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy (HR 3.1, 95%CI 1.9-23.1, log rank p=0.0041). ESR1 mutation prevalence differed markedly between patients that were first exposed to AI during the adjuvant and metastatic settings (5.8% (3/52) vs 36.4% (16/44) respectively, p=0.0002). In an independent cohort, ESR1 mutations were identified in 0% (0/32, 95%CI 0-10.9%) tumor biopsies taken after progression on adjuvant AI. In a patient with serial samples taken during metastatic treatment, ESR1 mutation was selected during metastatic AI therapy, to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI therapy, but are commonly selected by therapy for metastatic disease, providing evidence that the mechanisms of resistance to targeted therapy may be substantially different between the treatment of micro-metastatic and overt metastatic cancer.

The identification of early-stage breast cancer patients at high risk of relapse would allow tailoring of adjuvant therapy approaches. We assessed whether analysis of circulating tumor DNA (ctDNA) in plasma can be used to monitor for minimal residual disease (MRD) in breast cancer. In a prospective cohort of 55 early breast cancer patients receiving neoadjuvant chemotherapy, detection of ctDNA in plasma after completion of apparently curative treatment-either at a single postsurgical time point or with serial follow-up plasma samples-predicted metastatic relapse with high accuracy [hazard ratio, 25.1 (confidence interval, 4.08 to 130.5; log-rank P < 0.0001) or 12.0 (confidence interval, 3.36 to 43.07; log-rank P < 0.0001), respectively]. Mutation tracking in serial samples increased sensitivity for the prediction of relapse, with a median lead time of 7.9 months over clinical relapse. We further demonstrated that targeted capture sequencing analysis of ctDNA could define the genetic events of MRD, and that MRD sequencing predicted the genetic events of the subsequent metastatic relapse more accurately than sequencing of the primary cancer. Mutation tracking can therefore identify early breast cancer patients at high risk of relapse. Subsequent adjuvant therapeutic interventions could be tailored to the genetic events present in the MRD, a therapeutic approach that could in part combat the challenge posed by intratumor genetic heterogeneity.

Introduction Aromatase inhibitors (AIs) are a vital component of estrogen receptor positive (ER+) breast cancer treatment. De novo and acquired resistance, however, is common. The aims of this study were to relate patterns of copy number aberrations to molecular and proliferative response to AIs, to study differences in the patterns of copy number aberrations between breast cancer samples pre- and post-AI neoadjuvant therapy, and to identify putative biomarkers for resistance to neoadjuvant AI therapy using an integrative analysis approach. Methods Samples from 84 patients derived from two neoadjuvant AI therapy trials were subjected to copy number profiling by microarray-based comparative genomic hybridisation (aCGH, n = 84), gene expression profiling (n = 47), matched pre- and post-AI aCGH (n = 19 pairs) and Ki67-based AI-response analysis (n = 39). Results Integrative analysis of these datasets identified a set of nine genes that, when amplified, were associated with a poor response to AIs, and were significantly overexpressed when amplified, including CHKA, LRP5 and SAPS3. Functional validation in vitro, using cell lines with and without amplification of these genes (SUM44, MDA-MB134-VI, T47D and MCF7) and a model of acquired AI-resistance (MCF7-LTED) identified CHKA as a gene that when amplified modulates estrogen receptor (ER)-driven proliferation, ER/estrogen response element (ERE) transactivation, expression of ER-regulated genes and phosphorylation of V-AKT murine thymoma viral oncogene homolog 1 (AKT1). Conclusions These data provide a rationale for investigation of the role of CHKA in further models of de novo and acquired resistance to AIs, and provide proof of concept that integrative genomic analyses can identify biologically relevant modulators of AI response. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0532-0) contains supplementary material, which is available to authorized users.

Female BRCA gene mutation carriers are at increased risk for developing breast cancer. Ductal lavage is a novel method for sampling breast ductal fluid, providing epithelial cells for cytologic assessment and a source of free DNA for molecular analyses. Loss of heterozygosity (LOH) at the BRCA loci in ductal lavage fluid is a potential biomarker of breast cancer risk. The LOH rate was measured at the BRCA1/2 loci and compared with that at a control locus (APC) using free DNA from the ductal lavage fluid of BRCA carriers and predictive test negative controls. We evaluated the reproducibility of these analyses. Free DNA sufficient for PCR amplification was obtained from 33 ductal lavage samples of 17 healthy women of known BRCA status (14 BRCA carriers and 3 controls). LOH rates of 36.4% to 56.3% at the BRCA1 locus and 45% to 61.5% at the BRCA2 locus were found among BRCA carriers. The LOH rate at the APC locus was lower (18.5%). The interaliquot reproducibility for the D17S855 marker of the BRCA1 locus was 66.7%. Intraaliquot reproducibility was 90%. Although we successfully isolated sufficient free DNA from ductal lavage fluid for PCR amplification, the degree of reproducibility of these LOH studies raises questions about the robustness of this technique as a risk assessment tool in the evaluation of high-risk women. Further studies are required to evaluate the specificity and predictive value of LOH in ductal lavage fluid for breast cancer development. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1399–402)

DNA methylation of tumor-suppressor genes occurs early in the molecular transformation of precursor events to breast cancer and is therefore of interest to screening in high-risk women. The aim of this study was to use tumor-suppressor genes that have previously been shown to be cancer predictive in tissue to evaluate the potential of DNA methylation assays in cells from duct lavage (DL) fluid. The frequency of target gene DNA methylation in tissue and DL of cancer and healthy control patients was assessed, and an association of DNA methylation between different duct systems in the same breast was explored. The cancer and control groups were identified in the outpatient clinic when surgical treatment was finalized. Tumor, adjacent tissue and bilateral DL samples for comparative DNA methylation studies were obtained during surgery from women with cancer. In the healthy control group, samples of tissue and DL were collected. Reverse transcriptase methylation-specific PCR was conducted on modified DNA purified from 42 cancer biopsies, 41 benign excision cavity biopsies (internal control), 29 benign biopsies (external control), and 119 DL specimens. A validated panel of cancer predictive genes was analyzed in the study bank of tissue and DL samples from cancer and healthy patients. The sensitivity of DNA methylation in DL samples compared with matched cancer tissue was highest for SCGB3A1 (90 %), CDH13 (91 %), and RARB (83 %). The genetic algorithm selected RASSF1A, RARB, and IGFBP7 as the optimum predictor set for detecting DNA methylation in cancer tissue. The optimum area under the ROC curve for DNA methylation in cancer compared with internal control healthy tissue from excision margins was 0.84. The area under the ROC curve for DNA methylation in cancer DL compared with contralateral benign DL was 0.76. DL cytology was not a helpful predictor of breast cancer. This study shows that relative patterns of tumor-suppressor gene hypermethylation in breast cancer tissue are significantly reflected in the DL from the cancer affected breast. Using DL, nonconcordant patterns of DNA methylation between different duct systems confer independent oncologic potential for distinct breast lobes. The approach of DNA methylation in DL may be substantiated by a larger trial of breast cancer biomarkers.

Aim Nipple aspiration (NA) and duct lavage (DL) are modalities for obtaining breast duct fluid for biomarker analyses. The aim of this study was to assess the feasibility of obtaining serial NA and DL samples at consecutive patient visits for cytology assessment and the creation of a biobank. Methods Seventy eligible subjects were enroled at a single institution in the United Kingdom as part of an international multicentre study. Entry criteria were based on a 5-year Gail model risk of ⩾2% or Claus score lifetime risk of ⩾26%. Women underwent NA and DL in an outpatient clinic under local anaesthesia. Results The mean patient age was 48 (range 41–69) years. Sixty seven out of 70 women (96%) attended three consecutive 6 monthly visits and follow-up for 2 years. Three women withdrew due to intolerance of the DL procedure. 56/67 (83%) women produced NA fluid from at least one duct. 204/264 (77%) of ducts declared by NA were cannulated for DL. 170/204 (83%) produced DL samples with adequate cellularity. By the final visit 52/67 (78%) women produced DL, 28/52 (54%) of whom were premenopausal and 24/52 (46%) were postmenopausal. 50/52 women (96%) underwent repeated DL of 81 ducts on 3 consecutive visits. Conclusion NA and DL are well tolerated for repeated assessment to obtain material for cytology and to create a biobank for future biomarker studies in women at high breast cancer risk.

Introduction Very few studies have investigated whether the time elapsed between surgical resection and tissue fixation or the difference between core-cut and excision biopsies impact on immunohistochemically measured biomarkers, including phosphorylated proteins in primary breast cancer. The aim of this study was to characterise the differences in immunoreactivity of common biomarkers that may occur (1) as a result of tissue handling at surgery and (2) between core-cuts and resected tumours. Methods Core-cuts taken from surgical breast cancer specimens immediately after resection (sample A) and after routine X-ray of the excised tumour (sample B) were formalin-fixed and paraffin-embedded and compared with the routinely fixed resection specimen (sample C). The variation in immunohistochemical expression of Ki67, oestrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor 2 (HER2), p-Akt and p-Erk1/2 were investigated. Results Twenty-one tissue sets with adequate tumour were available. Median time between collection of core-cuts A and B was 30 minutes (range, 20 to 80 minutes). None of the markers showed significant differences between samples A and B. Similarly, Ki67, ER, PgR and HER2 did not differ significantly between core-cuts and main resection specimen, although there was a trend for lower resection values for ER (P = 0.06). However, p-Akt and p-Erk1/2 were markedly lower in resections than core-cuts (median, 27 versus 101 and 69 versus 193, respectively; both P < 0.0001 [two-sided]). This difference was significantly greater in mastectomy than in lumpectomy specimens for p-Erk1/2 (P = 0.01). Conclusions The delay in fixation in core-cuts taken after postoperative X-ray of resection specimens has no significant impact on expression of Ki67, ER, PgR, HER2, p-Akt or p-Erk1/2. However, extreme loss of phospho-staining can occur during routine fixation of resection specimens. These differences are likely attributable to suboptimal fixation and may have major repercussions for clinical research involving these markers.

s / International Journal of Surgery 8 (2010) 501–578 532 Introduction: Touch Imprint Cytology (TIC) allows intra-operative assessment of axillary lymph nodes, determining progression to axillary lymph node dissection at the same operation and requires specialist cytopathologists. We present our experience in a District General Hospital. Methods: A prospective study of consecutive series of patients undergoing level II or III axillary lymph node dissection, from October 2006 to September 2007, was undertaken. One surgeon and three cytopathologists were involved. TIC of a random level I lymph node was compared with imprint node histology. Cytopathologists were blinded to the use of coated and uncoated slides, used for each case. Results: 38/42 consecutive cases were analysed (4 exclusions as uninterpretable).Mean axillary yield 13.7. 12 TIC nodes were positive, all imprint nodes positive in these cases. 26 TIC nodes negative, all imprint nodes negative in these cases. Sensitivity 1⁄4 100%, specificity 1⁄4 100%, false negative rate 0, P

Background: Analysis of estrogen receptor (ER), progesterone receptor (PgR) and HER2 status in early breast cancer (EBC) is increasingly being conducted in core needle biopsies (CNBs) taken at diagnosis but the concordance with the excisional biopsy (EB) is poorly documented. Patients and methods: Patients with EBC presenting to The Royal Marsden Hospital from June 2005 to September 2007 who had CNB and subsequent EB were included. ER and PgR were determined by immunohistochemistry (IHC) and graded from 0 to 8 (Allred score). HER2 was determined by IHC and scored from 0 to 3+. FISH analysis was carried out in HER2 2+ cases and in discordant cases. Results: In all, 336 pairs of samples were compared. ER was positive in 253 CNBs (75%) for 255 EBs (76%) and was discordant in six patients (1.8%). PgR was positive in 221 CNBs (66%) and 227 (67.6%) EBs being discordant in 52 cases (15%). HER2 was positive in 41 (12.4%) of the 331 CNBs in which it was determined compared with 44 (13.3%) EBs and discordant in four cases (1.2%). Conclusions: CNB can be used with confidence for ER and HER2 determination. For PgR, due to a substantial discordance between CNB and EB, results from CNB should be used with caution.

INTRODUCTION Sentinel lymph node biopsy is emerging as the new standard for axillary staging in breast cancer. Intra-operative assessment of the sentinel lymph nodes allows immediate completion of axillary dissection during the same anaesthetic. This project was a quality assurance practice to establish feasibility, time-to-report, as well as accuracy of performing intra-operative assessment of sentinel lymph nodes using touch imprint cytology in our centre. PATIENTS AND METHODS This prospective audit included 146 sentinel lymph nodes from 74 consecutive patients with invasive breast cancer. All patients underwent axillary sentinel lymph node biopsy using combined blue dye and radiocolloid technique. Results of intra-operative touch imprint cytology using haematoxylin and eosin staining were compared with the definitive histopathology results. RESULTS Mean time to report touch imprint cytology was 25.7 ± 6.4 min (range, 15–40 min). Histopathology demonstrated metastasis in 25 sentinel nodes from 17 (23%) patients. Intra-operative touch imprint cytology detected 15 nodes in 11 patients, giving a sensitivity of 60% (nodes) and 66.7% (patients) and specificity of 99.2% (nodes) and 98.2% (patients) based on the number of nodes and patients involved, respectively. Touch imprint cytology failed to show metastatic involvement in 10 nodes from 6 patients; of these, five nodes had micrometastasis (< 2 mm) and the other five had macrometastasis. One touch imprint cytology positive node contained isolated tumour cells only. Using intra-operative touch imprint cytology made a change in treatment of 11(14.9%) patients, and spared second axillary procedure in 7 (9.4%) patients. CONCLUSIONS Intra-operative sentinel lymph node assessment using touch imprint cytology is feasible within a busy NHS practice. We now offer touch imprint cytology to patients following appropriate counselling.

Background: The identification of early stage breast cancers that are at high risk of relapse after apparently curative treatment of the primary tumor would allow tailored adjuvant therapy approaches to prevent relapse. We sought to define whether high-depth targeted sequencing of circulating cell-free plasma DNA could be used to interrogate the genetics of residual micro-metastatic disease (RMD) persisting after neoadjuvant treatment in patients with early stage breast cancer. Methods: In a cohort of 31 patients with early breast cancer receiving neoadjuvant chemotherapy, with no evidence of metastasis at presentation, we collected tumor tissue at baseline with the metastatic relapse if it occurred, and serial plasma samples at baseline, post-surgery, and every 6 months in follow-up. Serial plasma samples were subject to digital PCR mutation tracking to examine for circulating tumor DNA (ctDNA). Four patients had ctDNA detectable prior to clinical relapse. Primary tumor-derived DNA (from 4 cases), serial plasma DNA samples (from 4 cases), and metastases derived DNA (from 2 cases) were subjected to massively parallel sequencing (Illumina HiSeq2000) targeting all exons of 273 genes frequently mutated in breast cancers and/ or a custom AmpliSeq cancer panel (IonTorrent). Results: Targeted sequencing revealed the presence of 1 to 20 somatic mutations in the early breast cancers at baseline, including 3 PIK3CA H1047L/R and 2 TP53 pathogenic mutations, all of which were detected in the subsequent plasma DNA samples taken prior to relapse. However, in one case an ESR1 E380Q mutation found in the baseline primary breast cancer was undetectable by targeted sequencing in all 3 subsequent plasma samples and in the metastasis; this mutation could not be identified in the metastasis by digital PCR. In two cases plasma DNA sequencing revealed no additional mutations to those identified in the primary tumor, whereas in the other two cases, plasma DNA targeted capture sequencing revealed divergence in the genetics of RMD. In these cases, 1 and 5 somatic mutations were found in the plasma DNA but were not detected in the respective baseline tumors. In particular, an activating FGFR1 K656E mutation, which was not detected by targeted capture sequencing in the primary breast cancer at 355x sequencing depth, was found at a mutant allele fraction (MAF) of 2.6% in the plasma 12 months post-surgery, and was subsequently detected in the distant metastasis at a MAF of 43.4% by targeted sequencing and digital PCR. Conclusions: Our results provide evidence of clonal shifts in response to neoadjuvant systemic therapy of early breast cancers. In addition, we demonstrate that high-depth targeted capture massively parallel sequencing analysis of plasma ctDNA may help predict the genotype of recurrence prior to the onset of clinically overt metastasis. Citation Format: Charlotte KY Ng, Britta Weigelt, Isaac Garcia-Murillas, Gaia Schiavon, Sarah Hrebien, Rosalind J Cutts, Peter Osin, Ashutosh Nerurkar, Iwanka Kozarewa, Javier Armisen Garrido, Mitch Dowsett, Ian E Smith, Jorge S Reis-Filho, Nicholas C Turner. High-depth sequencing of circulating tumor DNA to interrogate the genetics of residual micro-metastatic disease prior to relapse in early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-01.

Introduction Female germline BRCA gene mutation carriers are at increased risk for developing breast cancer. The purpose of our study was to establish whether healthy BRCA mutation carriers demonstrate an increased frequency of aberrant gene promoter hypermethylation in ductal lavage (DL) fluid, compared with predictive genetic test negative controls, that might serve as a surrogate marker of BRCA1/2 mutation status and/or breast cancer risk. Methods The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-β, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid. Results Fifty-one DL samples from 24 healthy women of known BRCA mutation status (7 BRCA1 mutation carriers, 12 BRCA2 mutation carriers and 5 controls) were available for methylation analysis. Eight of 19 (42.1%) BRCA mutation carriers were found to have at least one hypermethylated gene in the four-gene panel. Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified. No hypermethylation was found in DL samples from 5 negative controls(p = 0.13). Conclusion We found substantial levels of aberrant methylation, with the use of a four-gene panel, in the fluid from the breasts of healthy BRCA mutation carriers compared with controls. Methylation analysis of free DNA in DL fluid may offer a useful surrogate marker for BRCA1/2 mutation status and/or breast cancer risk. Further studies are required for the evaluation of the specificity and predictive value of aberrant methylation in DL fluid for future breast cancer development in BRCA1/2 mutation carriers.

PURPOSE: The morphologic and molecular phenotype of breast cancers may help identify patients who are likely to carry germline mutations in BRCA1 and BRCA2. This study evaluates the immunohistochemical profiles of tumors arising in patients with mutations in these genes. MATERIALS AND METHODS: Samples of breast cancers obtained from the International Breast Cancer Linkage Consortium were characterized morphologically and immunohistochemically using antibodies to estrogen receptor, progesterone receptor, HER-2 (c-erbB-2 oncogene), and p53 protein. RESULTS: Breast cancers in patients with BRCA1 germline mutations are more often negative for estrogen receptor, progesterone receptor, and HER-2, and are more likely to be positive for p53 protein compared with controls. In contrast, BRCA2 tumors do not show a significant difference in the expression of any of these proteins compared with controls. CONCLUSION: BRCA1 has a distinctive morphology and immunohistochemical phenotype. The combined morphologic and immunohistochemical data can be used to predict the risk of a young patient harboring a germline mutation in BRCA1. The BRCA2 phenotype is currently not well defined.

The structure and expression of the human Rad53 homologue Chk2 was analysed in breast cancer. The previously described silent polymorphism at nucleotide 252 in codon 84 (GAA>GAG) was observed in 5/141 cases. Somatic Chk2 coding mutations were detected in 7/141 cases, these occurring in 4/18 BRCA1-associated breast cancers, 1/78 sporadic breast cancers and 2/25 typical medullary carcinomas. Each of the BRCA1-associated cancers with Chk2 mutations also contained mutations in p53, whereas the single sporadic cancer with Chk2 mutation was wild-type for p53. Expression of Chk2 was ubiquitously detected in normal ductal epithelium of the breast, but there was loss of expression in a significant proportion of breast carcinomas, and this occurred in cancers both with and without p53 mutation. A CpG island was identified 5′ of the Chk2 transcriptional start site, but there was no evidence of cytosine methylation in any of the cancers with down-regulated Chk2 expression. Analysis of the germ-fine of 45 individuals with hereditary or early onset breast cancer revealed wild-type Chk2 sequence in all cases. Thus, despite the rarity of somatic mutations in Chk2 in sporadic breast carcinomas, our results nevertheless reveal that concomitant loss of function in Chk2 (via down-regulation of expression) and p53 (via mutation) occurs in a proportion of sporadic cases. However, consistent with other studies, we show that germ-line mutations in Chk2 are unlikely to account for a significant proportion of non BRCA1-, non BRCA2-associated hereditary breast cancers.

Introduction: Intra-operative assessment of sentinel nodes (SLNs) allows immediate completion axillary dissection (cALND) in breast cancer patients. Molecular assessment such as one-step nucleic acid amplication (OSNA) promises greater sensitivity and provides a more accurate quantitative assessment than traditional methods. Our unit policy is to proceed to cALND in patients with macrometastases but not for micrometastases. However, evidence of upstaging has led us to seek to raise the threshold for proceeding to cALND. The CK19 mRNA copy number is an expression of the metastatic burden in the SLN and may be related to the presence of additional disease in the cALND. Since the original copy number threshold between micro (250–5000 copies/microliter) and macrometastasis (>5000 copies/microliter) was based on few patients and serial pathological sections, we investigated the mRNA copy number in patients with and without additional disease in the cALND. Methods: All patients in our unit undergo pre-operative axillary ultrasound with fine needle aspiration cytology of any suspicious nodes. Those with malignant cytology proceed directly to ALND. Radiologically and cytologically node negative patients undergo sentinel lymph node biopsy (SLNB) and OSNA. Electronic records of consecutive patients with invasive breast cancer undergoing SLNB with OSNA from August 2011 to March 2012 were retrospectively reviewed. Two parameters of mRNA copy number were examined: Copy number of the highest copy number SLN and the summed copy numbers of all positive SLNs. Their relationship to the presence of further disease in the axilla was examined using Student's t test. Results: Of 201 SLNBs, 45 (22%) had macrometastasis-positive OSNA and therefore underwent cALND (1 patient declined). Twenty patients (45%) had no further positive nodes (a negative cALND) with a total axillary metastatic burden of 1–2 in 11–27 nodes. Twenty four (55%) showed further disease (a positive cALND) with a burden of 2–20 in 9–30 nodes, including the SLNs. There was no significant difference in tumour size or grade between patients with additional positive nodes in the cALND compared with those with no further disease. There was no significant difference in the copy number of the highest copy number positive SLN (p = 0.44) or in the summed copy number of all positive SLNs (p = 0.36) between the cALND positive and negative groups. Conclusion: OSNA CK19 mRNA copy number does not correlate with the cALND metastatic burden. Therefore, raising the copy number threshold may be too simplistic as a method to better select patients with high probability of a positive cALND. A predictive model will be derived based on multivariate analysis of the larger patient population (>400 patients) that will have undergone SLNB with OSNA by the time of SABCS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-11.

Breast duct lavage for analysis by methylation-specific PCR is a novel but established method for detecting the presence of cancer and may contribute additional information about prognosis and response to treatment. The aim of this consecutive series of breast cancer patients was a case-control study to evaluate qualitative methylation of five published tumour suppressor genes1 in breast cancer tissue, adjacent normal breast parenchyma, duct lavage fluid and plasma.

The concept of the intraductal approach to evaluating the breast microenvironment assumes direct access to the breast cancer containing duct. Previous studies on duct anatomy infusing a dye or contrast found fluid-yielding ducts to be associated with the cancer containing duct in approximately 50% of fluid-yielding breasts with cancer. A concordant anatomical relationship between accessible ductal sytems and the cancer-affected lobe is essential if cytology or other cell markers are to be successfully identified as indicators of cancer or of early cancerous change. The concordant relationship is less important if field change effects are considered to predictors of malignant change. The aim of this study was to determine how often duct lavage effluent drains the breast cancer affected segment.

Studies of families with breast cancer have indicated that male carriers of BRCA2 mutations are at increased risk of prostate cancer, particularly at an early age. To evaluate the contribution of BRCA2 mutations to early-onset prostate cancer, we screened the complete coding sequence of BRCA2 for germline mutations, in 263 men with diagnoses of prostate cancer who were ≤55 years of age. Protein-truncating mutations were found in six men (2.3%; 95% confidence interval 0.8%–5.0%), and all of these mutations were clustered outside the ovarian-cancer cluster region. The relative risk of developing prostate cancer by age 56 years from a deleterious germline BRCA2 mutation was 23-fold. Four of the patients with mutations did not have a family history of breast or ovarian cancer. Twenty-two variants of uncertain significance were also identified. These results confirm that BRCA2 is a high-risk prostate-cancer–susceptibility gene and have potential implications for the management of early-onset prostate cancer, in both patients and their relatives.