Residual cancer burden after neoadjuvant chemotherapy and long-term survival outcomes in breast cancer: a multicentre pooled analysis of 5161 patients.

BACKGROUND: Previous studies have independently validated the prognostic relevance of residual cancer burden (RCB) after neoadjuvant chemotherapy. We used results from several independent cohorts in a pooled patient-level analysis to evaluate the relationship of RCB with long-term prognosis across different phenotypic subtypes of breast cancer, to assess generalisability in a broad range of practice settings. METHODS: In this pooled analysis, 12 institutes and trials in Europe and the USA were identified by personal communications with site investigators. We obtained participant-level RCB results, and data on clinical and pathological stage, tumour subtype and grade, and treatment and follow-up in November, 2019, from patients (aged ≥18 years) with primary stage I-III breast cancer treated with neoadjuvant chemotherapy followed by surgery. We assessed the association between the continuous RCB score and the primary study outcome, event-free survival, using mixed-effects Cox models with the incorporation of random RCB and cohort effects to account for between-study heterogeneity, and stratification to account for differences in baseline hazard across cancer subtypes defined by hormone receptor status and HER2 status. The association was further evaluated within each breast cancer subtype in multivariable analyses incorporating random RCB and cohort effects and adjustments for age and pretreatment clinical T category, nodal status, and tumour grade. Kaplan-Meier estimates of event-free survival at 3, 5, and 10 years were computed for each RCB class within each subtype. FINDINGS: We analysed participant-level data from 5161 patients treated with neoadjuvant chemotherapy between Sept 12, 1994, and Feb 11, 2019. Median age was 49 years (IQR 20-80). 1164 event-free survival events occurred during follow-up (median follow-up 56 months [IQR 0-186]). RCB score was prognostic within each breast cancer subtype, with higher RCB score significantly associated with worse event-free survival. The univariable hazard ratio (HR) associated with one unit increase in RCB ranged from 1·55 (95% CI 1·41-1·71) for hormone receptor-positive, HER2-negative patients to 2·16 (1·79-2·61) for the hormone receptor-negative, HER2-positive group (with or without HER2-targeted therapy; p<0·0001 for all subtypes). RCB score remained prognostic for event-free survival in multivariable models adjusted for age, grade, T category, and nodal status at baseline: the adjusted HR ranged from 1·52 (1·36-1·69) in the hormone receptor-positive, HER2-negative group to 2·09 (1·73-2·53) in the hormone receptor-negative, HER2-positive group (p<0·0001 for all subtypes). INTERPRETATION: RCB score and class were independently prognostic in all subtypes of breast cancer, and generalisable to multiple practice settings. Although variability in hormone receptor subtype definitions and treatment across patients are likely to affect prognostic performance, the association we observed between RCB and a patient's residual risk suggests that prospective evaluation of RCB could be considered to become part of standard pathology reporting after neoadjuvant therapy. FUNDING: National Cancer Institute at the US National Institutes of Health.

Pathological features of 11,337 patients with primary ductal carcinoma in situ (DCIS) and subsequent events: results from the UK Sloane Project.

BACKGROUND: The Sloane audit compares screen-detected ductal carcinoma in situ (DCIS) pathology with subsequent management and outcomes. METHODS: This was a national, prospective cohort study of DCIS diagnosed during 2003-2012. RESULTS: Among 11,337 patients, 7204 (64%) had high-grade DCIS. Over time, the proportion of high-grade disease increased (from 60 to 65%), low-grade DCIS decreased (from 10 to 6%) and mean size increased (from 21.4 to 24.1 mm). Mastectomy was more common for high-grade (36%) than for low-grade DCIS (15%). Few (6%) patients treated with breast-conserving surgery (BCS) had a surgical margin <1 mm. Of the 9191 women diagnosed in England (median follow-up 9.4 years), 7% developed DCIS or invasive malignancy in the ipsilateral and 5% in the contralateral breast. The commonest ipsilateral event was invasive carcinoma (n = 413), median time 62 months, followed by DCIS (n = 225), at median 37 months. Radiotherapy (RT) was most protective against recurrence for high-grade DCIS (3.2% for high-grade DCIS with RT compared to 6.9% without, compared with 2.3 and 3.0%, respectively, for low/intermediate-grade DCIS). Ipsilateral DCIS events lessened after 5 years, while the risk of ipsilateral invasive cancer remained consistent to beyond 10 years. CONCLUSION: DCIS pathology informs patient management and highlights the need for prolonged follow-up of screen-detected DCIS.

Accurate Estrogen Receptor Quantification in Patients with Negative and Low-Positive Estrogen-Receptor-Expressing Breast Tumors: Sub-Analyses of Data from Two Clinical Studies.

INTRODUCTION: Accurate assessment of estrogen receptor (ER) expression is crucial to ensure that patients with early breast cancer are accurately identified for appropriate treatment with endocrine therapy. Reverse transcriptase polymerase chain reaction (RT-PCR), compared with immunohistochemistry (IHC), may provide a more precise indication of ER status. Data were pooled and analyzed from two independent, but similarly designed, studies that examined ER status by IHC and the 21-gene Recurrence Score that employs RT-PCR-based methodology. METHODS: Tumor tissue from patients with early stage breast cancer where ER status could be determined by both IHC and RT-PCR was included. ER status by IHC staining was defined as ER-negative (< 1%), ER-low+ (1-10%), or ER+ (> 10%). ER status by RT-PCR was defined as ER-negative (≤ 6.5) or ER+ (> 6.5). Recurrence Score results from the 21-gene assay were reported on a continuous scale from 0 to 100. A sub-analysis examined the association between ER expression (Allred score 2-7) and response to a 14-day pre-surgery pulse with an aromatase inhibitor. A separate sub-analysis examined the association between ER expression and human epidermal growth factor receptor 2 (HER2) expression. RESULTS: Tumor specimens from 192 patients (aged 25-92 years) were included in the pooled analysis. Correlation between IHC- and RT-PCR-measured ER was strong for IHC-defined ER-negative and ER+ samples (r = 0.646 [95% CI 0.553-0.720]). There was 100% concordance for ER+ tumors; however, 56% of the ER-low+ tumors were negative by RT-PCR. Allred score correlated better with ER status measured by RT-PCR at pre-treatment (r = 0.83) than at post-treatment (r = 0.76). The majority (77%) of ER-negative and ER-low+ tumors were HER2-negative. CONCLUSIONS: RT-PCR provided a more accurate assessment of ER expression in patients with ER-low+ tumors, and data support dual testing for patients with ER-low+ status to ensure appropriate treatment planning as it pertains to endocrine therapy. FUNDING: Genomic Health, Inc.

Molecular changes during extended neoadjuvant letrozole treatment of breast cancer: distinguishing acquired resistance from dormant tumours.

BACKGROUND: The risk of recurrence for endocrine-treated breast cancer patients persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate; previous efforts have been limited to in-vitro or in-vivo approaches. In this study, sequential tumour samples from patients receiving extended neoadjuvant aromatase inhibitor therapy were characterised as a novel clinical model. METHODS: Consecutive tumour samples from 62 patients undergoing extended (4-45 months) neoadjuvant aromatase inhibitor therapy with letrozole were subjected to transcriptomic and proteomic analysis, representing before (≤ 0), early (13-120 days), and long-term (> 120 days) neoadjuvant aromatase inhibitor therapy with letrozole. Patients with at least a 40% initial reduction in tumour size by 4 months of treatment were included. Of these, 42 patients with no subsequent progression were classified as "dormant", and the remaining 20 patients as "acquired resistant". RESULTS: Changes in gene expression in dormant tumours begin early and become more pronounced at later time points. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to the resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. The DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared with dormant tissues after extended letrozole treatment. CONCLUSIONS: This is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumours continue to change during treatment whereas acquired resistant tumours more closely resemble their diagnostic samples. Global loss of DNA methylation was observed in resistant tumours under extended treatment. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients, supporting a potential role for therapy targeted at these epigenetic alterations in the management of resistance to oestrogen deprivation therapy.

Surgical Upstaging Rates for Vacuum Assisted Biopsy Proven DCIS: Implications for Active Surveillance Trials.

PURPOSE: This study was designed to determine invasive cancer upstaging rates at surgical excision following vacuum-assisted biopsy of ductal carcinoma in situ (DCIS) among women meeting eligibility for active surveillance trials. METHODS: Patients with vacuum-assisted, biopsy-proven DCIS at a single center from 2008 to 2015 were retrospectively reviewed. Imaging and pathology reports were interrogated for the imaging appearance, tumor grade, hormone receptor status, and presence of comedonecrosis. Subsequent surgical reports were reviewed for upstaging to invasive disease. Cases were classified by eligibility criteria for the COMET, LORIS, and LORD DCIS active surveillance trials. RESULTS: Of 307 DCIS diagnoses, 15 (5%) were low, 95 (31%) intermediate, and 197 (64%) high nuclear grade. The overall upstage rate to invasive disease was 17% (53/307). Eighty-one patients were eligible for the COMET Trial, 74 for the LORIS trial, and 10 for the LORD Trial, although LORIS trial eligibility also included real-time, multiple central pathology review, including elements not routinely reported. The upstaging rates to invasive disease were 6% (5/81), 7% (5/74), and 10% (1/10) for the COMET, LORIS, and LORD trials, respectively. Among upstaged cancers (n = 5), four tumors were Stage IA invasive ductal carcinoma and one was Stage IIA invasive lobular carcinoma; all were node-negative. CONCLUSIONS: DCIS upstaging rates in women eligible for active surveillance trials are low (6-10%), and in this series, all those with invasive disease were early-stage, node-negative. The careful patient selection for DCIS active surveillance trials has a low risk of missing occult invasive cancer and additional studies will determine clinical outcomes.

Current treatment trends and the need for better predictive tools in the management of ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) of the breast represents a group of heterogeneous non-invasive lesions the incidence of which has risen dramatically since the advent of mammography screening. In this review we summarise current treatment trends and up-to-date results from clinical trials studying surgery and adjuvant therapy alternatives, including the recent consensus on excision margin width and its role in decision-making for post-excision radiotherapy. The main challenge in the clinical management of DCIS continues to be the tailoring of treatment to individual risk, in order to avoid the over-treatment of low-risk lesions or under-treatment of DCIS with higher risk of recurring or progressing into invasion. While studies estimate that only about 40% of DCIS would become invasive if untreated, heterogeneity and complex natural history have prevented adequate identification of these higher-risk lesions. Here we discuss attempts to develop prognostic tools for the risk stratification of DCIS lesions and their limitations. Early results of a UK-wide audit of DCIS management (the Sloane Project) have also demonstrated a lack of consistency in treatment. In this review we offer up-to-date perspectives on current treatment and prediction of DCIS, highlighting the pressing clinical need for better prognostic indices. Tools integrating both clinical and histopathological factors together with molecular biomarkers may hold potential for adequate stratification of DCIS according to risk. This could help develop standardised practices for optimal management of patients with DCIS, improving clinical outcomes while providing only the amount of therapy required for each individual patient.

Tumour sampling method can significantly influence gene expression profiles derived from neoadjuvant window studies.

Patient-matched transcriptomic studies using tumour samples before and after treatment allow inter-patient heterogeneity to be controlled, but tend not to include an untreated comparison. Here, Illumina BeadArray technology was used to measure dynamic changes in gene expression from thirty-seven paired diagnostic core and surgically excised breast cancer biopsies obtained from women receiving no treatment prior to surgery, to determine the impact of sampling method and tumour heterogeneity. Despite a lack of treatment and perhaps surprisingly, consistent changes in gene expression were identified during the diagnosis-surgery interval (48 up, 2 down; Siggenes FDR 0.05) in a manner independent of both subtype and sampling-interval length. Instead, tumour sampling method was seen to directly impact gene expression, with similar effects additionally identified in six published breast cancer datasets. In contrast with previous findings, our data does not support the concept of a significant wounding or immune response following biopsy in the absence of treatment and instead implicates a hypoxic response following the surgical biopsy. Whilst sampling-related gene expression changes are evident in treated samples, they are secondary to those associated with response to treatment. Nonetheless, sampling method remains a potential confounding factor for neoadjuvant study design.

Estrogen-induced chromatin decondensation and nuclear re-organization linked to regional epigenetic regulation in breast cancer.

BACKGROUND: Epigenetic changes are being increasingly recognized as a prominent feature of cancer. This occurs not only at individual genes, but also over larger chromosomal domains. To investigate this, we set out to identify large chromosomal domains of epigenetic dysregulation in breast cancers. RESULTS: We identify large regions of coordinate down-regulation of gene expression, and other regions of coordinate activation, in breast cancers and show that these regions are linked to tumor subtype. In particular we show that a group of coordinately regulated regions are expressed in luminal, estrogen-receptor positive breast tumors and cell lines. For one of these regions of coordinate gene activation, we show that regional epigenetic regulation is accompanied by visible unfolding of large-scale chromatin structure and a repositioning of the region within the nucleus. In MCF7 cells, we show that this depends on the presence of estrogen. CONCLUSIONS: Our data suggest that the liganded estrogen receptor is linked to long-range changes in higher-order chromatin organization and epigenetic dysregulation in cancer. This may suggest that as well as drugs targeting histone modifications, it will be valuable to investigate the inhibition of protein complexes involved in chromatin folding in cancer cells.

Evaluation of carbonic anhydrase IX as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast cancer models.

Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

Accurate Prediction and Validation of Response to Endocrine Therapy in Breast Cancer.

PURPOSE: Aromatase inhibitors (AIs) have an established role in the treatment of breast cancer. Response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Accurate biomarkers are urgently needed to predict response in these settings and to determine which individuals will benefit from adjuvant AI therapy. PATIENTS AND METHODS: Pretreatment and on-treatment (after 2 weeks and 3 months) biopsies were obtained from 89 postmenopausal women who had estrogen receptor-alpha positive breast cancer and were receiving neoadjuvant letrozole for transcript profiling. Dynamic clinical response was assessed with use of three-dimensional ultrasound measurements. RESULTS: The molecular response to letrozole was characterized and a four-gene classifier of clinical response was established (accuracy of 96%) on the basis of the level of two genes before treatment (one gene [IL6ST] was associated with immune signaling, and the other [NGFRAP1] was associated with apoptosis) and the level of two proliferation genes (ASPM, MCM4) after 2 weeks of therapy. The four-gene signature was found to be 91% accurate in a blinded, completely independent validation data set of patients treated with anastrozole. Matched 2-week on-treatment biopsies were associated with improved predictive power as compared with pretreatment biopsies alone. This signature also significantly predicted recurrence-free survival (P = .029) and breast cancer -specific survival (P = .009). We demonstrate that the test can also be performed with use of quantitative polymerase chain reaction or immunohistochemistry. CONCLUSION: A four-gene predictive model of clinical response to AIs by 2 weeks has been generated and validated. Deregulated immune and apoptotic responses before treatment and cell proliferation that is not reduced 2 weeks after initiation of treatment are functional characteristics of breast tumors that do not respond to AIs.

Molecular changes in lobular breast cancers in response to endocrine therapy.

Invasive lobular carcinoma (ILC) accounts for approximately 10% to 15% of breast carcinomas, and although it responds poorly to neoadjuvant chemotherapy, it appears to respond well to endocrine therapy. Pre- and on-treatment (after 2 weeks and 3 months) biopsies and surgical samples were obtained from 14 postmenopausal women with estrogen receptor-positive (ER(+)) histologically confirmed ILC who responded to 3 months of neoadjuvant letrozole and were compared with a cohort of 14 responding invasive ductal carcinomas (IDC) matched on clinicopathologic features. RNA was extracted and processed for whole human genome expression microarray. Dynamic clinical response was assessed using periodic three-dimensional ultrasound measurements performed during treatment and defined as a reduction of >70% in tumor volume by 3 months. Pretreatment profiles of ILC and IDC tumors showed distinctive expression of genes associated with E-cadherin signaling, epithelial adhesion, and stromal rearrangement. The changes in gene expression in response to letrozole were highly similar between responding ILC and IDC tumors; genes involved in proliferation were downregulated and those involved with immune function and extracellular matrix remodeling were upregulated. However, molecular differences between the histologic subtypes were maintained upon treatment. This is the first study of molecular changes in ILC in response to endocrine therapy to date. The genes that change on letrozole are highly consistent between ILC and IDC. Differences in gene expression between ILC and IDC at diagnosis are maintained at each time point on treatment.

Increased STAT1 signaling in endocrine-resistant breast cancer.

Proteomic profiling of the estrogen/tamoxifen-sensitive MCF-7 cell line and its partially sensitive (MCF-7/LCC1) and fully resistant (MCF-7/LCC9) variants was performed to identify modifiers of endocrine sensitivity in breast cancer. Analysis of the expression of 120 paired phosphorylated and non-phosphorylated epitopes in key oncogenic and tumor suppressor pathways revealed that STAT1 and several phosphorylated epitopes (phospho-STAT1(Tyr701) and phospho-STAT3(Ser727)) were differentially expressed between endocrine resistant and parental controls, confirmed by qRT-PCR and western blotting. The STAT1 inhibitor EGCG was a more effective inhibitor of the endocrine resistant MCF-7/LCC1 and MCF-7/LCC9 lines than parental MCF-7 cells, while STAT3 inhibitors Stattic and WP1066 were equally effective in endocrine-resistant and parental lines. The effects of the STAT inhibitors were additive, rather than synergistic, when tested in combination with tamoxifen in vitro. Expression of STAT1 and STAT3 were measured by quantitative immunofluorescence in invasive breast cancers and matched lymph nodes. When lymph node expression was compared to its paired primary breast cancer expression, there was greater expression of cytoplasmic STAT1 (∼3.1 fold), phospho-STAT3(Ser727) (∼1.8 fold), and STAT5 (∼1.5 fold) and nuclear phospho-STAT3(Ser727) (∼1.5 fold) in the nodes. Expression levels of STAT1 and STAT3 transcript were analysed in 550 breast cancers from publicly available gene expression datasets (GSE2990, GSE12093, GSE6532). When treatment with tamoxifen was considered, STAT1 gene expression was nearly predictive of distant metastasis-free survival (DMFS, log-rank p = 0.067), while STAT3 gene expression was predictive of DMFS (log-rank p<0.0001). Analysis of STAT1 and STAT3 protein expression in a series of 546 breast cancers also indicated that high expression of STAT3 protein was associated with improved survival (DMFS, p = 0.006). These results suggest that STAT signaling is important in endocrine resistance, and that STAT inhibitors may represent potential therapies in breast cancer, even in the resistant setting.

Impact of screening and risk factors for local recurrence and survival after conservative surgery and radiotherapy for early breast cancer: results from a large series with long-term follow-up.

PURPOSE: To investigate conventional prognostic factors for ipsilateral breast tumor recurrence (IBTR), distant metastasis (DM), and survival after breast-conserving therapy (BCT) in screen-detected and symptomatic cases on surveillance up to 25 years. PATIENTS AND METHODS: A total of 1812 consecutive patients in three cohorts (1981-1989, 1990-1992, and 1993-1998) with T12N01M0 invasive breast cancer were treated with BCT (median follow-up, 14 years). Tumor type and grade were reviewed by a single pathologist. Hormone receptor status was measured by immunohistochemistry on tissue microarrays. A Cox proportional hazards model was used to assess independent prognostic variables for relapse and survival. RESULTS: A total of 205 IBTR occurred, with 5-, 10-, 15-, and 20-year actuarial relapse rates of 4.5% (95% confidence interval [CI] 3.35-5.5%), 8.4% (95% CI 7.1-9.8%), 14.1% (95% CI 12.0-16%), and 17.4% (95% CI 14.5-20.2%). Number of nodes, young age, pathologic tumor size, and multifocality were significant factors for IBTR. Three hundred seventy-eight patients developed DM. The actuarial metastatic rate was 12% at 5 years and 17.9% at 10 years. Young age, number of positive nodes, pathologic tumor size, and tumor grade were significant factors for DM relapse. When conventional prognostic indices were taken into account screen-detected cancers showed no improvement in overall relapse or survival rate compared with symptomatic cases but did show a reduced risk of DM after IBTR. After 10 years IBTR relapse continued at a constant rate of 0.87% per annum. CONCLUSIONS: The Edinburgh BCT series has shown that screen-detected invasive breast cancers do not have significantly different clinical outcomes compared with symptomatic cases when pathologic risk factors are taken into account. This suggests that these patients be managed in a similar way.

Determining tamoxifen sensitivity using primary breast cancer tissue in collagen-based three-dimensional culture.

We developed a three-dimensional assay prepared from primary breast cancer tissue and quantified tumor response to tamoxifen therapy. Freshly harvested breast cancer biopsies obtained at the time of curative surgical resection were fragmented and embedded into collagen I cushions. Changes in proliferation, apoptosis and tumor volume in response to tamoxifen treatment were quantified using image analysis software and optical projection tomography. Individual and collective invasion of epithelial cells into the surrounding collagen I was observed over the course of the experiment using phase contrast light microscopy and histopathological methods. Addition of tamoxifen to preparations derived from ER+ tumors demonstrated a range of response as measured by proliferative and apoptotic markers. In keeping with published data, tamoxifen reduced the percentage of apoptotic cells expressing cleaved caspase-3 (p = 0.02, Poisson regression analysis). Tamoxifen also reduced residual epithelial volume in ER+ tumors (p = 0.001, Mann-Whitney test), but not in ER low/- tumors (p = 0.78). Changes in tumor volume, as measured by optical projection tomography, allowed stratification into responsive and non-responsive tumors. The model mirrors observations of breast cancer response and histopathological changes to tamoxifen in neo-adjuvant trials. This assay provides a method of screening a battery of therapeutics against individual cancers, informing subsequent design of neo-adjuvant trials.

Two possible mechanisms of epithelial to mesenchymal transition in invasive ductal breast cancer.

Epithelial to mesenchymal transition (EMT) occurs in embryogenesis and normal development. It has been predominantly described in vitro and in animal studies, but EMT is also implicated in the progression of many cancers with proposed roles in invasion, metastasis and resistance to treatment. It is closely associated with loss of epithelial-specific protein expression and up-regulation of mesenchymal proteins, but several pathways are implicated in its execution. We explored what are the expression patterns of EMT proteins in human breast cancer. We interrogated two independent cohorts enriched for high-grade, invasive, ductal breast cancers. We used quantitative immunofluorescence to study the expression of key EMT proteins. Statistical associations to define protein profiles were based on Pearson's correlations. E-cadherin down-regulation in breast cancer was associated with ß-catenin down-regulation, but not with up-regulation of mesenchymal markers. While EMT-related transcription repressors were expressed in some breast cancers, their expression did not negatively correlate with E-cadherin. Instead, an additional EMT profile was identified, composing Snail and Slug. In conclusion, EMT occurs in human breast cancer in a manner distinct to that seen in vitro. Certain EMT events are uncoupled from E-cadherin down-regulation and may constitute a novel EMT profile, which warrants further exploration.

Gene expression profiling of response to mTOR inhibitor everolimus in pre-operatively treated post-menopausal women with oestrogen receptor-positive breast cancer.

There is growing evidence that uncontrolled activation of the PI3K/Akt/mTOR pathway contributes to the development and progression of breast cancer. Inhibition of this pathway has antitumour effects in preclinical studies and efficacy in combination with other agents in breast cancer patients. The aim of this study is to characterise the effects of pre-operative everolimus treatment in primary breast cancer patients and to identify potential molecular predictors of response. Twenty-seven patients with oestrogen receptor (ER)-positive breast cancer completed 11-14 days of neoadjuvant treatment with 5-mg everolimus. Core biopsies were taken before and after treatment and analysed using Illumina HumanRef-8 v2 Expression BeadChips. Changes in proliferation (Ki67) and phospho-AKT were measured on diagnostic core biopsies/resection samples embedded in paraffin by immunohistochemistry to determine response to treatment. Patients that responded to everolimus treatment with significant reductions in proliferation (fall in % Ki67 positive cells) also had significant decreases in the expression of genes involved in cell cycle (P = 8.70E-09) and p53 signalling (P = 0.01) pathways. Highly proliferating tumours that have a poor prognosis exhibited dramatic reductions in the expression of cell cycle genes following everolimus treatment. The genes that most clearly separated responding from non-responding pre-treatment tumours were those involved with protein modification and dephosphorylation, including DYNLRB2, ERBB4, PTPN13, ULK2 and DUSP16. The majority of ER-positive breast tumours treated with everolimus showed a significant reduction in genes involved with proliferation, these may serve as markers of response and predict which patients will derive most benefit from mTOR inhibition.

Correcting for intra-experiment variation in Illumina BeadChip data is necessary to generate robust gene-expression profiles.

BACKGROUND: Microarray technology is a popular means of producing whole genome transcriptional profiles, however high cost and scarcity of mRNA has led many studies to be conducted based on the analysis of single samples. We exploit the design of the Illumina platform, specifically multiple arrays on each chip, to evaluate intra-experiment technical variation using repeated hybridisations of universal human reference RNA (UHRR) and duplicate hybridisations of primary breast tumour samples from a clinical study. RESULTS: A clear batch-specific bias was detected in the measured expressions of both the UHRR and clinical samples. This bias was found to persist following standard microarray normalisation techniques. However, when mean-centering or empirical Bayes batch-correction methods (ComBat) were applied to the data, inter-batch variation in the UHRR and clinical samples were greatly reduced. Correlation between replicate UHRR samples improved by two orders of magnitude following batch-correction using ComBat (ranging from 0.9833-0.9991 to 0.9997-0.9999) and increased the consistency of the gene-lists from the duplicate clinical samples, from 11.6% in quantile normalised data to 66.4% in batch-corrected data. The use of UHRR as an inter-batch calibrator provided a small additional benefit when used in conjunction with ComBat, further increasing the agreement between the two gene-lists, up to 74.1%. CONCLUSION: In the interests of practicalities and cost, these results suggest that single samples can generate reliable data, but only after careful compensation for technical bias in the experiment. We recommend that investigators appreciate the propensity for such variation in the design stages of a microarray experiment and that the use of suitable correction methods become routine during the statistical analysis of the data.