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Invasive lobular carcinomas (ILC) are characterised by loss of the cell adhesion molecule E-cadherin. Approximately 15% of ILC are ER negative at the time of breast cancer diagnosis, or at relapse due to loss of ER expression. Less than 5% of classical ILC but up to 35% of pleomorphic ILC are HER2 positive (HER2+).Retrospective analysis of clinic-pathological data from patients with Triple negative (TN) or HER2+ ILC diagnosed 2004-2014 at the Royal Marsden Hospital. The primary endpoint was median overall survival (OS) in patients with metastatic disease. Secondary endpoints included response rate to neo-adjuvant chemotherapy (NAC), median disease-free interval (DFI) and OS for patients with early disease.Three of 16 patients with early TN ILC and 7/33 with early HER2+ ILC received NAC with pCR rates of 0/3 and 3/5 patients who underwent surgery, respectively. Median DFI was 28.5 months [95% Confidence interval (95%CI) 15-78.8] for TN ILC and not reached (NR) (111.2-NR) for HER2+ early ILC. Five-year OS was 52% (95%CI 23-74%) and 77% (95%CI 58-88%), respectively. Twenty-three patients with advanced TN ILC and 14 patients with advanced HER2+ ILC were identified. Median OS was 18.3 months (95%CI 13.0-32.8 months) and 30.4 months (95%CI 8.8-NR), respectively.In our institution we report a high relapse rate after treatment for early TN ILC, but median OS from metastatic disease is similar to that expected from TN IDC. Outcomes for patients with advanced HER2+ ILC were less favourable than those expected for IDC, possibly reflecting incomplete exposure to anti-HER2 therapies.ROLo (ClinicalTrials.gov Identifier: NCT03620643), ROSALINE (ClinicalTrials.gov Identifier: NCT04551495).

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.

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.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.We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations inOur research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of

Background Society of Surgical Oncology and American Society for Radiation Oncology guidelines define clear margins in breast-conserving therapy (BCT) as 'no ink on tumour', in contrast to the attainment of margins of at least 1 mm widely practised in the UK. The primary aim of this study was to explore clinical, surgical and tumour-related factors associated with local recurrence after BCT, with a secondary aim of assessing the impact of margin re-excision on the risk of local recurrence. Methods Patient demographics, surgical details, tumour characteristics and local recurrence were recorded for consecutive women with BCT undergoing surgery between January 1997 and January 2007. Margins were defined as clear (greater than 1 mm), close (less than 1 mm but no ink on tumour), reaches (ink on tumour) and clear after re-excision. Results A total of 1045 women of median age 54 (range 18-86) years were studied. Median follow-up was 89 (range 4-196) months. Local recurrence occurred in 52 patients (5·0 per cent). Ink on tumour was associated with local recurrence (hazard ratio (HR) 4·86, 95 per cent c.i. 1·49 to 15·79; P = 0·009). Risk of local recurrence was the same for close and clear margins (HR 1·03, 0·40 to 2·62; P = 0·954). In women with involved margins, re-excision was still associated with an increased local recurrence risk (HR 2·50, 1·32 to 4·72; P = 0·005). Oestrogen receptor negativity increased risk (HR 2·28, 1·28 to 4·06; P = 0·005). Conclusion Adequately excised margins, even when under 1 mm, provide equivalent outcomes to wider margins in BCT. Achieving complete excision at primary surgery achieves the lowest rates of local recurrence.

Background The majority of lesions resulting in pathological nipple discharge are benign. Conventional surgery is undirected and targeting the causative lesion by duct endoscopy may enable more accurate surgery with fewer complications. Methods Patients requiring microdochectomy and/or major duct excision were randomized to duct endoscopy or no duct endoscopy before surgery. Primary endpoints were successful visualization of the pathological lesion in patients randomized to duct endoscopy, and a comparison of the causative pathology between the two groups. The secondary endpoint was to compare the specimen size between groups. Results A total of 68 breasts were studied in 66 patients; there were 31 breasts in the duct endoscopy group and 37 in the no-endoscopy group. Median age was 49 (range 19–81) years. Follow-up was 5·4 (i.q.r. 3·3–8·9) years in the duct endoscopy group and 5·7 (3·1–9·0) years in no-endoscopy group. Duct endoscopy had a sensitivity of 80 (95 per cent c.i. 52 to 96) per cent, specificity of 71 (44 to 90) per cent, positive predictive value of 71 (44 to 90) per cent and negative predictive value of 80 (52 to 96) per cent in identifying any lesion. There was no difference in causative pathology between the groups. Median volume of the surgical resection specimen did not differ between groups. Conclusion Diagnostic duct endoscopy is useful for identifying causative lesions of nipple discharge. Duct endoscopy did not influence the pathological yield of benign or malignant diagnoses nor surgical resection volumes. Registered as INTEND II in CancerHelp UK clinical trials database (https://www.cancerresearchuk.org/about-cancer/find-a-clinical-trial/a-study-looking-at-changes-inside-the-breast-ducts-of-women-who-have-nipple-discharge).

Purpose To evaluate the efficacy of a BLES procedure as a primary excisional biopsy rather than a surgical wide local excision for treatment of a high risk or a malignant lesion. Methods 41 patients underwent a BLES procedure in order to attempt to remove a small breast lesion using a 15 mm or 20 mm wand from August 2007 to January 2009. The lesions were either proven on prior core biopsy to show high risk or malignant pathology or were considered to be indeterminate or suspicious on ultrasound or mammography. The pathology was reviewed to include the final status of lesion excision. If margin involvement was demonstrated then a formal surgical excision was subsequently recommended. Follow up mammography or ultrasound was performed annually in patients following the final pathological diagnosis. Results 9 patients had a primary diagnosis of atypia (columnar cell change with atypia or atypical ductal hyperplasia (ADH)), 23 patients had ductal carcinoma in situ (DCIS) and 9 had an invasive carcinoma (IC) at the original BLES pathology. Clear BLES margins of >1 mm were obtained in 3/9 atypia lesions, 15/23 DCIS and 0/9 IC. 12/13 low grade DCIS were completely excised. Subsequent surgical margin excisions were undertaken in 20 patients. After at least 5 years of follow up (mean 66 months), 1 lesion had recurred on imaging. Conclusion A BLES excision has potential as an alternative technique to traditional surgical wide local excision in the management of certain small breast lesions with high risk and low grade malignant potential.

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.

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.

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.

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)

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.

Purpose: Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number. Experimental Design: The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort. Results: In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86–0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2–positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2–negative, with a positive and negative predictive value of 70% and 92%, respectively. Conclusion: Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer. Clin Cancer Res; 19(12); 3276–84. ©2013 AACR.

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.

The examination of limited, potentially non-representative fragments of tumour tissue from a core biopsy can be misleading and misdirect subsequent treatment, especially in cases where a primary tumour has not been identified. This case report is of a 65-year-old woman presenting with a destructive sacral mass, diagnosed on radiological imaging and core biopsy as a hindgut neuroendocrine tumour, which on histopathological review of the subsequently resected tumour was found instead to represent a metastasis from an occult hormone-positive breast cancer with neuroendocrine features.

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.

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.

Inheritance of germ-line mutant alleles of BRCA1 and BRCA2 confers a markedly increased risk of breast cancer and we have previously reported a higher incidence of p53 mutations in these tumours than in grade matched sporadic tumours. We have now characterized these p53 mutants. The results of these studies identify a novel class of p53 mutants previously undescribed in human cancer yet with multiple occurrences in BRCA-associated tumours which retain a profile of p53-dependent activities in terms of transactivation, growth suppression and apoptosis induction which is close or equal to wild-type. However, these mutants fail to suppress transformation and exhibit gain of function transforming activity in rat embryo fibroblasts. These mutants therefore fall into a novel category of p53 mutants which dissociate transformation suppression from other wild-type functions. The rarity of these mutants in human cancer and their multiple occurrence in BRCA-associated breast tumours suggests that these novel p53 mutants are selected during malignant progression in the unique genetic background of BRCA1- and BRCA2-associated tumours.

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.