Multi-State Gene Cluster Switches Determine the Adaptive Mitochondrial And Metabolic Landscape of Breast Cancer.

Adaptive metabolic switches are proposed to underlie conversions between cellular states during normal development as well as in cancer evolution. Metabolic adaptations represent important therapeutic targets in tumors, highlighting the need to characterize the full spectrum, characteristics, and regulation of the metabolic switches. To investigate the hypothesis that metabolic switches associated with specific metabolic states can be recognized by locating large alternating gene expression patterns, we developed a method to identify interspersed gene sets by massive correlated biclustering (MCbiclust) and to predict their metabolic wiring. Testing the method on breast cancer transcriptome datasets revealed a series of gene sets with switch-like behavior that could be used to predict mitochondrial content, metabolic activity, and central carbon flux in tumors. The predictions were experimentally validated by bioenergetic profiling and metabolic flux analysis of 13C-labelled substrates. The metabolic switch positions also distinguished between cellular states, correlating with tumor pathology, prognosis, and chemosensitivity. The method is applicable to any large and heterogeneous transcriptome dataset to discover metabolic and associated pathophysiological states.

Comparative survival analysis of multiparametric tests-when molecular tests disagree-A TEAM Pathology study.

Multiparametric assays for risk stratification are widely used in the management of both node negative and node positive hormone receptor positive invasive breast cancer. Recent data from multiple sources suggests that different tests may provide different risk estimates at the individual patient level. The TEAM pathology study consists of 3284 postmenopausal ER+ve breast cancers treated with endocrine therapy Using genes comprising the following multi-parametric tests OncotypeDx®, Prosigna™ and MammaPrint® signatures were trained to recapitulate true assay results. Patients were then classified into risk groups and survival assessed. Whilst likelihood χ2 ratios suggested limited value for combining tests, Kaplan-Meier and LogRank tests within risk groups suggested combinations of tests provided statistically significant stratification of potential clinical value. Paradoxically whilst Prosigna-trained results stratified Oncotype-trained subgroups across low and intermediate risk categories, only intermediate risk Prosigna-trained cases were further stratified by Oncotype-trained results. Both Oncotype-trained and Prosigna-trained results further stratified MammaPrint-trained low risk cases, and MammaPrint-trained results also stratified Oncotype-trained low and intermediate risk groups but not Prosigna-trained results. Comparisons between existing multiparametric tests are challenging, and evidence on discordance between tests in risk stratification presents further dilemmas. Detailed analysis of the TEAM pathology study suggests a complex inter-relationship between test results in the same patient cohorts which requires careful evaluation regarding test utility. Further prognostic improvement appears both desirable and achievable.

Computational approaches to support comparative analysis of multiparametric tests: Modelling versus Training.

Multiparametric assays for risk stratification are widely used in the management of breast cancer, with applications being developed for a number of other cancer settings. Recent data from multiple sources suggests that different tests may provide different risk estimates at the individual patient level. There is an increasing need for robust methods to support cost effective comparisons of test performance in multiple settings. The derivation of similar risk classifications using genes comprising the following multi-parametric tests Oncotype DX® (Genomic Health.), Prosigna™ (NanoString Technologies, Inc.), MammaPrint® (Agendia Inc.) was performed using different computational approaches. Results were compared to the actual test results. Two widely used approaches were applied, firstly computational "modelling" of test results using published algorithms and secondly a "training" approach which used reference results from the commercially supplied tests. We demonstrate the potential for errors to arise when using a "modelling" approach without reference to real world test results. Simultaneously we show that a "training" approach can provide a highly cost-effective solution to the development of real-world comparisons between different multigene signatures. Comparisons between existing multiparametric tests is challenging, and evidence on discordance between tests in risk stratification presents further dilemmas. We present an approach, modelled in breast cancer, which can provide health care providers and researchers with the potential to perform robust and meaningful comparisons between multigene tests in a cost-effective manner. We demonstrate that whilst viable estimates of gene signatures can be derived from modelling approaches, in our study using a training approach allowed a close approximation to true signature results.

Capivasertib Plus Paclitaxel Versus Placebo Plus Paclitaxel As First-Line Therapy for Metastatic Triple-Negative Breast Cancer: The PAKT Trial.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently activated in triple-negative breast cancer (TNBC). The AKT inhibitor capivasertib has shown preclinical activity in TNBC models, and drug sensitivity has been associated with activation of PI3K or AKT and/or deletions of PTEN. The PAKT trial was designed to evaluate the safety and efficacy of adding capivasertib to paclitaxel as first-line therapy for TNBC. PATIENTS AND METHODS: This double-blind, placebo-controlled, randomized phase II trial recruited women with untreated metastatic TNBC. A total of 140 patients were randomly assigned (1:1) to paclitaxel 90 mg/m2 (days 1, 8, 15) with either capivasertib (400 mg twice daily) or placebo (days 2-5, 9-12, 16-19) every 28 days until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS), PFS and OS in the subgroup with PIK3CA/AKT1/PTEN alterations, tumor response, and safety. RESULTS: Median PFS was 5.9 months with capivasertib plus paclitaxel and 4.2 months with placebo plus paclitaxel (hazard ratio [HR], 0.74; 95% CI, 0.50 to 1.08; 1-sided P = .06 [predefined significance level, 1-sided P = .10]). Median OS was 19.1 months with capivasertib plus paclitaxel and 12.6 months with placebo plus paclitaxel (HR, 0.61; 95% CI, 0.37 to 0.99; 2-sided P = .04). In patients with PIK3CA/AKT1/PTEN-altered tumors (n = 28), median PFS was 9.3 months with capivasertib plus paclitaxel and 3.7 months with placebo plus paclitaxel (HR, 0.30; 95% CI, 0.11 to 0.79; 2-sided P = .01). The most common grade ≥ 3 adverse events in those treated with capivasertib plus paclitaxel versus placebo plus paclitaxel, respectively, were diarrhea (13% v 1%), infection (4% v 1%), neutropenia (3% v 3%), rash (4% v 0%), and fatigue (4% v 0%). CONCLUSION: Addition of the AKT inhibitor capivasertib to first-line paclitaxel therapy for TNBC resulted in significantly longer PFS and OS. Benefits were more pronounced in patients with PIK3CA/AKT1/PTEN-altered tumors. Capivasertib warrants further investigation for treatment of TNBC.

Tumour profiling tests to guide adjuvant chemotherapy decisions in early breast cancer: a systematic review and economic analysis.

BACKGROUND: Breast cancer and its treatment can have an impact on health-related quality of life and survival. Tumour profiling tests aim to identify whether or not women need chemotherapy owing to their risk of relapse. OBJECTIVES: To conduct a systematic review of the effectiveness and cost-effectiveness of the tumour profiling tests oncotype DX® (Genomic Health, Inc., Redwood City, CA, USA), MammaPrint® (Agendia, Inc., Amsterdam, the Netherlands), Prosigna® (NanoString Technologies, Inc., Seattle, WA, USA), EndoPredict® (Myriad Genetics Ltd, London, UK) and immunohistochemistry 4 (IHC4). To develop a health economic model to assess the cost-effectiveness of these tests compared with clinical tools to guide the use of adjuvant chemotherapy in early-stage breast cancer from the perspective of the NHS and Personal Social Services. DESIGN: A systematic review and health economic analysis were conducted. REVIEW METHODS: The systematic review was partially an update of a 2013 review. Nine databases were searched in February 2017. The review included studies assessing clinical effectiveness in people with oestrogen receptor-positive, human epidermal growth factor receptor 2-negative, stage I or II cancer with zero to three positive lymph nodes. The economic analysis included a review of existing analyses and the development of a de novo model. RESULTS: A total of 153 studies were identified. Only one completed randomised controlled trial (RCT) using a tumour profiling test in clinical practice was identified: Microarray In Node-negative Disease may Avoid ChemoTherapy (MINDACT) for MammaPrint. Other studies suggest that all the tests can provide information on the risk of relapse; however, results were more varied in lymph node-positive (LN+) patients than in lymph node-negative (LN0) patients. There is limited and varying evidence that oncotype DX and MammaPrint can predict benefit from chemotherapy. The net change in the percentage of patients with a chemotherapy recommendation or decision pre/post test ranged from an increase of 1% to a decrease of 23% among UK studies and a decrease of 0% to 64% across European studies. The health economic analysis suggests that the incremental cost-effectiveness ratios for the tests versus current practice are broadly favourable for the following scenarios: (1) oncotype DX, for the LN0 subgroup with a Nottingham Prognostic Index (NPI) of > 3.4 and the one to three positive lymph nodes (LN1-3) subgroup (if a predictive benefit is assumed); (2) IHC4 plus clinical factors (IHC4+C), for all patient subgroups; (3) Prosigna, for the LN0 subgroup with a NPI of > 3.4 and the LN1-3 subgroup; (4) EndoPredict Clinical, for the LN1-3 subgroup only; and (5) MammaPrint, for no subgroups. LIMITATIONS: There was only one completed RCT using a tumour profiling test in clinical practice. Except for oncotype DX in the LN0 group with a NPI score of > 3.4 (clinical intermediate risk), evidence surrounding pre- and post-test chemotherapy probabilities is subject to considerable uncertainty. There is uncertainty regarding whether or not oncotype DX and MammaPrint are predictive of chemotherapy benefit. The MammaPrint analysis uses a different data source to the other four tests. The Translational substudy of the Arimidex, Tamoxifen, Alone or in Combination (TransATAC) study (used in the economic modelling) has a number of limitations. CONCLUSIONS: The review suggests that all the tests can provide prognostic information on the risk of relapse; results were more varied in LN+ patients than in LN0 patients. There is limited and varying evidence that oncotype DX and MammaPrint are predictive of chemotherapy benefit. Health economic analyses indicate that some tests may have a favourable cost-effectiveness profile for certain patient subgroups; all estimates are subject to uncertainty. More evidence is needed on the prediction of chemotherapy benefit, long-term impacts and changes in UK pre-/post-chemotherapy decisions. STUDY REGISTRATION: This study is registered as PROSPERO CRD42017059561. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Breast cancer is the most commonly diagnosed cancer in women in England and Wales. Breast cancer, and its treatment, can have an impact on a person's health-related quality of life and survival. Tumour profiling tests are used before chemotherapy. They test small samples of a patient's tumour (removed during surgery) to find out whether the genes in it mean that a person has a high or low risk of the disease returning (relapse). If the risk is low, the patient may be able to avoid having chemotherapy and, therefore, avoid its side effects. Some tests might also be able to identify which patients will respond to chemotherapy. This study looked at the evidence for five tumour profiling tests. A total of 153 studies were identified. This study considered the results and the quality of the studies to find out if the tests are helpful. Most studies had design flaws (e.g. some patients had already had chemotherapy) that meant that the studies were of low quality overall. The results suggest that all of the tests can give information on the risk of relapse; however, some tests may be less useful in patients whose disease has spread to the lymph nodes (lymph node-positive disease). There was limited and varying evidence about whether or not two of these tests can also predict which patients will respond to chemotherapy. This study also looked at whether or not these tests represent good value for money for the NHS through cost-effectiveness analyses. The analyses showed that some of the tests may represent a good use of NHS resources for some patient groups; however, there was still a lot of uncertainty about this.

The QuinteT Recruitment Intervention supported five randomized trials to recruit to target: a mixed-methods evaluation.

OBJECTIVE: To evaluate the impact of the QuinteT Recruitment Intervention (QRI) on recruitment in challenging randomized controlled trials (RCTs) that have applied the intervention. The QRI aims to understand recruitment difficulties and then implements "QRI actions" to address these as recruitment proceeds. STUDY DESIGN AND SETTING: A mixed-methods study, comprising (1) before-and-after comparisons of recruitment rates and the numbers of patients approached and (2) qualitative case studies, including documentary analysis and interviews with RCT investigators. RESULTS: Five UK-based publicly funded RCTs were included in the evaluation. All recruited to target. Randomized controlled trial 2 and RCT 5 both received up-front prerecruitment training before the intervention was applied. Randomized controlled trial 2 did not encounter recruitment issues and recruited above target from its outset. Recruitment difficulties, particularly communication issues, were identified and addressed through QRI actions in RCTs 1, 3, 4, and 5. Randomization rates significantly improved after QRI action in RCTs 1, 3, and 4. Quintet Recruitment Intervention actions addressed issues with approaching eligible patients in RCTs 3 and 5, which both saw significant increases in the number of patients approached. Trial investigators reported that the QRI had unearthed issues they had been unaware of and reportedly changed their practices after QRI action. CONCLUSION: There is promising evidence to suggest that the QRI can support recruitment to difficult RCTs. This needs to be substantiated with future controlled evaluations.

Randomized Phase II Study Evaluating Palbociclib in Addition to Letrozole as Neoadjuvant Therapy in Estrogen Receptor-Positive Early Breast Cancer: PALLET Trial.

PURPOSE: CDK4/6 inhibitors are used to treat estrogen receptor (ER)-positive metastatic breast cancer (BC) in combination with endocrine therapy. PALLET is a phase II randomized trial that evaluated the effects of combination palbociclib plus letrozole as neoadjuvant therapy. PATIENTS AND METHODS: Postmenopausal women with ER-positive primary BC and tumors greater than or equal to 2.0 cm were randomly assigned 3:2:2:2 to letrozole (2.5 mg/d) for 14 weeks (A); letrozole for 2 weeks, then palbociclib plus letrozole to 14 weeks (B); palbociclib for 2 weeks, then palbociclib plus letrozole to 14 weeks (C); or palbociclib plus letrozole for 14 weeks. Palbociclib 125 mg/d was administered orally on a 21-days-on, 7-days-off schedule. Core-cut biopsies were taken at baseline and 2 and 14 weeks. Coprimary end points for letrozole versus palbociclib plus letrozole groups (A v B + C + D) were change in Ki-67 (protein encoded by the  MKI67 gene; immunohistochemistry) between baseline and 14 weeks and clinical response (ordinal and ultrasound) after 14 weeks. Complete cell-cycle arrest was defined as Ki-67 less than or equal to 2.7%. Apoptosis was characterized by cleaved poly (ADP-ribose) polymerase. RESULTS: Three hundred seven patients were recruited. Clinical response was not significantly different between palbociclib plus letrozole and letrozole groups ( P = .20; complete response + partial response, 54.3% v 49.5%), and progressive disease was 3.2% versus 5.4%, respectively. Median log-fold change in Ki-67 was greater with palbociclib plus letrozole compared with letrozole (-4.1 v -2.2; P < .001) in the 190 evaluable patients (61.9%), corresponding to a geometric mean change of -97.4% versus -88.5%. More patients on palbociclib plus letrozole achieved complete cell-cycle arrest (90% v 59%; P < .001). Median log-fold change (suppression) of cleaved poly (ADP-ribose) polymerase was greater with palbociclib plus letrozole versus letrozole (-0.80 v -0.42; P < .001). More patients had grade 3 or greater toxicity on palbociclib plus letrozole (49.8% v 17.0%; P < .001) mainly because of asymptomatic neutropenia. CONCLUSION: Adding palbociclib to letrozole significantly enhanced the suppression of malignant cell proliferation (Ki-67) in primary ER-positive BC, but did not increase the clinical response rate over 14 weeks, which was possibly related to a concurrent reduction in apoptosis.

Development of a framework to improve the process of recruitment to randomised controlled trials (RCTs): the SEAR (Screened, Eligible, Approached, Randomised) framework.

BACKGROUND: Research has shown that recruitment to trials is a process that stretches from identifying potentially eligible patients, through eligibility assessment, to obtaining informed consent. The length and complexity of this pathway means that many patients do not have the opportunity to consider participation. This article presents the development of a simple framework to document, understand and improve the process of trial recruitment. METHODS: Eight RCTs integrated a QuinteT Recruitment Intervention (QRI) into the main trial, feasibility or pilot study. Part of the QRI required mapping the patient recruitment pathway using trial-specific screening and recruitment logs. A content analysis compared the logs to identify aspects of the recruitment pathway and process that were useful in monitoring and improving recruitment. Findings were synthesised to develop an optimised simple framework that can be used in a wide range of RCTs. RESULTS: The eight trials recorded basic information about patients screened for trial participation and randomisation outcome. Three trials systematically recorded reasons why an individual was not enrolled in the trial, and further details why they were not eligible or approached, or declined randomisation. A framework to facilitate clearer recording of the recruitment process and reasons for non-participation was developed: SEAR - Screening, to identify potentially eligible trial participants; Eligibility, assessed against the trial protocol inclusion/exclusion criteria; Approach, the provision of oral and written information and invitation to participate in the trial, and Randomised or not, with the outcome of randomisation or treatment received. CONCLUSIONS: The SEAR framework encourages the collection of information to identify recruitment obstacles and facilitate improvements to the recruitment process. SEAR can be adapted to monitor recruitment to most RCTs, but is likely to add most value in trials where recruitment problems are anticipated or evident. Further work to test it more widely is recommended.

Value of Information Analysis of Multiparameter Tests for Chemotherapy in Early Breast Cancer: The OPTIMA Prelim Trial.

BACKGROUND: Precision medicine is heralded as offering more effective treatments to smaller targeted patient populations. In breast cancer, adjuvant chemotherapy is standard for patients considered as high-risk after surgery. Molecular tests may identify patients who can safely avoid chemotherapy. OBJECTIVES: To use economic analysis before a large-scale clinical trial of molecular testing to confirm the value of the trial and help prioritize between candidate tests as randomized comparators. METHODS: Women with surgically treated breast cancer (estrogen receptor-positive and lymph node-positive or tumor size ≥30 mm) were randomized to standard care (chemotherapy for all) or test-directed care using Oncotype DX™. Additional testing was undertaken using alternative tests: MammaPrintTM, PAM-50 (ProsignaTM), MammaTyperTM, IHC4, and IHC4-AQUA™ (NexCourse Breast™). A probabilistic decision model assessed the cost-effectiveness of all tests from a UK perspective. Value of information analysis determined the most efficient publicly funded ongoing trial design in the United Kingdom. RESULTS: There was an 86% probability of molecular testing being cost-effective, with most tests producing cost savings (range -£1892 to £195) and quality-adjusted life-year gains (range 0.17-0.20). There were only small differences in costs and quality-adjusted life-years between tests. Uncertainty was driven by long-term outcomes. Value of information demonstrated value of further research into all tests, with Prosigna currently being the highest priority for further research. CONCLUSIONS: Molecular tests are likely to be cost-effective, but an optimal test is yet to be identified. Health economics modeling to inform the design of a randomized controlled trial looking at diagnostic technology has been demonstrated to be feasible as a method for improving research efficiency.

Accelerated versus standard epirubicin followed by cyclophosphamide, methotrexate, and fluorouracil or capecitabine as adjuvant therapy for breast cancer in the randomised UK TACT2 trial (CRUK/05/19): a multicentre, phase 3, open-label, randomised, controlled trial.

BACKGROUND: Adjuvant chemotherapy for early breast cancer has improved outcomes but causes toxicity. The UK TACT2 trial used a 2×2 factorial design to test two hypotheses: whether use of accelerated epirubicin would improve time to tumour recurrence (TTR); and whether use of oral capecitabine instead of cyclophosphamide would be non-inferior in terms of patients' outcomes and would improve toxicity, quality of life, or both. METHODS: In this multicentre, phase 3, randomised, controlled trial, we enrolled patients aged 18 years or older from 129 UK centres who had histologically confirmed node-positive or high-risk node-negative operable breast cancer, had undergone complete excision, and were due to receive adjuvant chemotherapy. Patients were randomly assigned to receive four cycles of 100 mg/m2 epirubicin either every 3 weeks (standard epirubicin) or every 2 weeks with 6 mg pegfilgrastim on day 2 of each cycle (accelerated epirubicin), followed by four 4-week cycles of either classic cyclophosphamide, methotrexate, and fluorouracil (CMF; 600 mg/m2 cyclophosphamide intravenously on days 1 and 8 or 100 mg/m2 orally on days 1-14; 40 mg/m2 methotrexate intravenously on days 1 and 8; and 600 mg/m2 fluorouracil intravenously on days 1 and 8 of each cycle) or four 3-week cycles of 2500 mg/m2 capecitabine (1250 mg/m2 given twice daily on days 1-14 of each cycle). The randomisation schedule was computer generated in random permuted blocks, stratified by centre, number of nodes involved (none vs one to three vs four or more), age (≤50 years vs >50 years), and planned endocrine treatment (yes vs no). The primary endpoint was TTR, defined as time from randomisation to first invasive relapse or breast cancer death, with intention-to-treat analysis of standard versus accelerated epirubicin and per-protocol analysis of CMF versus capecitabine. This trial is registered with ISRCTN, number 68068041, and with ClinicalTrials.gov, number NCT00301925. FINDINGS: From Dec 16, 2005, to Dec 5, 2008, 4391 patients (4371 women and 20 men) were recruited. At a median follow-up of 85·6 months (IQR 80·6-95·9) no significant difference was seen in the proportions of patients free from TTR events between the accelerated and standard epirubicin groups (overall hazard ratio [HR] 0·94, 95% CI 0·81-1·09; stratified p=0·42). At 5 years, 85·9% (95% CI 84·3-87·3) of patients receiving standard epirubicin and 87·1% (85·6-88·4) of those receiving accelerated epirubicin were free from TTR events. 4358 patients were included in the per-protocol analysis, and no difference was seen in the proportions of patients free from TTR events between the CMF and capecitabine groups (HR 0·98, 95% CI 0·85-1.14; stratified p=0·00092 for non-inferiority). Compared with baseline, significantly more patients taking CMF than those taking capecitabine had clinically relevant worsening of quality of life at end of treatment (255 [58%] of 441 vs 235 [50%] of 475; p=0·011) and at 12 months (114 [34%] of 334 vs 89 [22%] of 401; p<0·001 at 12 months) and had worse quality of life over time (p<0·0001). Detailed toxicity and quality-of-life data were collected from 2115 (48%) of treated patients. The most common grade 3 or higher adverse events in cycles 1-4 were neutropenia (175 [16%]) and fatigue (56 [5%]) of the 1070 patients treated with standard epirubicin, and fatigue (63 [6%]) and infection (34 [3%]) of the 1045 patients treated with accelerated epirubicin. In cycles 5-8, the most common grade 3 or higher adverse events were neutropenia (321 [31%]) and fatigue (109 [11%]) in the patients treated with CMF, and hand-foot syndrome (129 [12%]) and diarrhoea (67 [6%]) in the 1044 patients treated with capcitabine. INTERPRETATION: We found no benefit from increasing the dose density of the anthracycline component of chemotherapy. However, capecitabine could be used in place of CMF without significant loss of efficacy and with improved quality of life. FUNDING: Cancer Research UK, Amgen, Pfizer, and Roche.

Comparing Breast Cancer Multiparameter Tests in the OPTIMA Prelim Trial: No Test Is More Equal Than the Others.

BACKGROUND: Previous reports identifying discordance between multiparameter tests at the individual patient level have been largely attributed to methodological shortcomings of multiple in silico studies. Comparisons between tests, when performed using actual diagnostic assays, have been predicted to demonstrate high degrees of concordance. OPTIMA prelim compared predicted risk stratification and subtype classification of different multiparameter tests performed directly on the same population. METHODS: Three hundred thirteen women with early breast cancer were randomized to standard (chemotherapy and endocrine therapy) or test-directed (chemotherapy if Oncotype DX recurrence score >25) treatment. Risk stratification was also determined with Prosigna (PAM50), MammaPrint, MammaTyper, NexCourse Breast (IHC4-AQUA), and conventional IHC4 (IHC4). Subtype classification was provided by Blueprint, MammaTyper, and Prosigna. RESULTS: Oncotype DX predicted a higher proportion of tumors as low risk (82.1%, 95% confidence interval [CI] = 77.8% to 86.4%) than were predicted low/intermediate risk using Prosigna (65.5%, 95% CI = 60.1% to 70.9%), IHC4 (72.0%, 95% CI = 66.5% to 77.5%), MammaPrint (61.4%, 95% CI = 55.9% to 66.9%), or NexCourse Breast (61.6%, 95% CI = 55.8% to 67.4%). Strikingly, the five tests showed only modest agreement when dichotomizing results between high vs low/intermediate risk. Only 119 (39.4%) tumors were classified uniformly as either low/intermediate risk or high risk, and 183 (60.6%) were assigned to different risk categories by different tests, although 94 (31.1%) showed agreement between four of five tests. All three subtype tests assigned 59.5% to 62.4% of tumors to luminal A subtype, but only 121 (40.1%) were classified as luminal A by all three tests and only 58 (19.2%) were uniformly assigned as nonluminal A. Discordant subtyping was observed in 123 (40.7%) tumors. CONCLUSIONS: Existing evidence on the comparative prognostic information provided by different tests suggests that current multiparameter tests provide broadly equivalent risk information for the population of women with estrogen receptor (ER)-positive breast cancers. However, for the individual patient, tests may provide differing risk categorization and subtype information.

OPTIMA prelim: a randomised feasibility study of personalised care in the treatment of women with early breast cancer.

BACKGROUND: There is uncertainty about the chemotherapy sensitivity of some oestrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers. Multiparameter assays that measure the expression of several tumour genes simultaneously have been developed to guide the use of adjuvant chemotherapy for this breast cancer subtype. The assays provide prognostic information and have been claimed to predict chemotherapy sensitivity. There is a dearth of prospective validation studies. The Optimal Personalised Treatment of early breast cancer usIng Multiparameter Analysis preliminary study (OPTIMA prelim) is the feasibility phase of a randomised controlled trial (RCT) designed to validate the use of multiparameter assay directed chemotherapy decisions in the NHS. OBJECTIVES: OPTIMA prelim was designed to establish the acceptability to patients and clinicians of randomisation to test-driven treatment assignment compared with usual care and to select an assay for study in the main RCT. DESIGN: Partially blinded RCT with adaptive design. SETTING: Thirty-five UK hospitals. PARTICIPANTS: Patients aged ≥ 40 years with surgically treated ER-positive HER2-negative primary breast cancer and with 1-9 involved axillary nodes, or, if node negative, a tumour at least 30 mm in diameter. INTERVENTIONS: Randomisation between two treatment options. Option 1 was standard care consisting of chemotherapy followed by endocrine therapy. In option 2, an Oncotype DX(®) test (Genomic Health Inc., Redwood City, CA, USA) performed on the resected tumour was used to assign patients either to standard care [if 'recurrence score' (RS) was > 25] or to endocrine therapy alone (if RS was ≤ 25). Patients allocated chemotherapy were blind to their randomisation. MAIN OUTCOME MEASURES: The pre-specified success criteria were recruitment of 300 patients in no longer than 2 years and, for the final 150 patients, (1) an acceptance rate of at least 40%; (2) recruitment taking no longer than 6 months; and (3) chemotherapy starting within 6 weeks of consent in at least 85% of patients. RESULTS: Between September 2012 and 3 June 2014, 350 patients consented to join OPTIMA prelim and 313 were randomised; the final 150 patients were recruited in 6 months, of whom 92% assigned chemotherapy started treatment within 6 weeks. The acceptance rate for the 750 patients invited to participate was 47%. Twelve out of the 325 patients with data (3.7%, 95% confidence interval 1.7% to 5.8%) were deemed ineligible on central review of receptor status. Interviews with researchers and recordings of potential participant consultations made as part of the integral qualitative recruitment study provided insights into recruitment barriers and led to interventions designed to improve recruitment. Patient information was changed as the result of feedback from three patient focus groups. Additional multiparameter analysis was performed on 302 tumour samples. Although Oncotype DX, MammaPrint(®)/BluePrint(®) (Agendia Inc., Irvine, CA, USA), Prosigna(®) (NanoString Technologies Inc., Seattle, WA, USA), IHC4, IHC4 automated quantitative immunofluorescence (AQUA(®)) [NexCourse BreastTM (Genoptix Inc. Carlsbad, CA, USA)] and MammaTyper(®) (BioNTech Diagnostics GmbH, Mainz, Germany) categorised comparable numbers of tumours into low- or high-risk groups and/or equivalent molecular subtypes, there was only moderate agreement between tests at an individual tumour level (kappa ranges 0.33-0.60 and 0.39-0.55 for tests providing risks and subtypes, respectively). Health economics modelling showed the value of information to the NHS from further research into multiparameter testing is high irrespective of the test evaluated. Prosigna is currently the highest priority for further study. CONCLUSIONS: OPTIMA prelim has achieved its aims of demonstrating that a large UK clinical trial of multiparameter assay-based selection of chemotherapy in hormone-sensitive early breast cancer is feasible. The economic analysis shows that a trial would be economically worthwhile for the NHS. Based on the outcome of the OPTIMA prelim, a large-scale RCT to evaluate the clinical effectiveness and cost-effectiveness of multiparameter assay-directed chemotherapy decisions in hormone-sensitive HER2-negative early breast would be appropriate to take place in the NHS. TRIAL REGISTRATION: Current Controlled Trials ISRCTN42400492. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 20, No. 10. See the NIHR Journals Library website for further project information. The Government of Ontario funded research at the Ontario Institute for Cancer Research. Robert C Stein received additional support from the NIHR University College London Hospitals Biomedical Research Centre.

Macroscopic handling and reporting of breast cancer specimens pre- and post-neoadjuvant chemotherapy treatment: review of pathological issues and suggested approaches.

Neoadjuvant chemotherapy (NACT) is used increasingly in the treatment of invasive breast cancer and presents challenges for the pathologist in the handling and interpretation of tissues. Potential issues include pathological identification and localization of the residual tumour site; how best to assess pathological response (given the diversity of scoring systems described); the timing and assessment of axillary node biopsy; and the value of retesting any residual tumour for dissonance between core biopsy and post-treatment residual cancer cells for biomarker expression such as oestrogen and progesterone receptors and human epidermal growth factor receptor 2 (HER2). The role of the pathologist is critical in modern NACT approaches to breast cancer and is likely to remain challenging as novel agents and newer biomarkers become available. In this manuscript we review these issues and describe some practical approaches to handling and reporting these samples in the routine histopathology laboratory.

Economic evaluation of genomic test-directed chemotherapy for early-stage lymph node-positive breast cancer.

BACKGROUND: Multi-parameter genomic tests identify patients with early-stage breast cancer who are likely to derive little benefit from adjuvant chemotherapy. These tests can potentially spare patients the morbidity from unnecessary chemotherapy and reduce costs. However, the costs of the test must be balanced against the health benefits and cost savings produced. This economic evaluation compared genomic test-directed chemotherapy using the Oncotype DX 21-gene assay with chemotherapy for all eligible patients with lymph node-positive, estrogen receptor-positive early-stage breast cancer. METHODS: We performed a cost-utility analysis using a state transition model to calculate expected costs and benefits over the lifetime of a cohort of women with estrogen receptor-positive lymph node-positive breast cancer from a UK perspective. Recurrence rates for Oncotype DX-selected risk groups were derived from parametric survival models fitted to data from the Southwest Oncology Group 8814 trial. The primary outcome was the incremental cost-effectiveness ratio, expressed as the cost (in 2011 GBP) per quality-adjusted life-year (QALY). Confidence in the incremental cost-effectiveness ratio was expressed as a probability of cost-effectiveness and was calculated using Monte Carlo simulation. Model parameters were varied deterministically and probabilistically in sensitivity analysis. Value of information analysis was used to rank priorities for further research. RESULTS: The incremental cost-effectiveness ratio for Oncotype DX-directed chemotherapy using a recurrence score cutoff of 18 was £5529 (US $8852) per QALY. The probability that test-directed chemotherapy is cost-effective was 0.61 at a willingness-to-pay threshold of £30 000 per QALY. Results were sensitive to the recurrence rate, long-term anthracycline-related cardiac toxicity, quality of life, test cost, and the time horizon. The highest priority for further research identified by value of information analysis is the recurrence rate in test-selected subgroups. CONCLUSIONS: There is substantial uncertainty regarding the cost-effectiveness of Oncotype DX-directed chemotherapy. It is particularly important that future research studies to inform cost-effectiveness-based decisions collect long-term outcome data.

Trastuzumab beyond progression: overall survival analysis of the GBG 26/BIG 3-05 phase III study in HER2-positive breast cancer.

BACKGROUND: Continuation of trastuzumab plus capecitabine (XH) showed a significantly improved overall response rate and time to progression compared with capecitabine (X) alone in women with HER2-positive breast cancer progressing during trastuzumab treatment. Here, we report the final analysis on overall survival. PATIENTS AND METHODS: Patients with HER2-positive, advanced breast cancer who progressed during treatment with trastuzumab with or without 1st-line metastatic chemotherapy were prospectively randomised to X (2500mg/m(2) on days 1-14, q3w) or XH (6 (8)mg/kg, q3w). Overall survival was a pre-specified secondary end-point. RESULTS: Median follow-up at June 2010 was 20.7months. Fifty nine of 74 and 60 of 77 patients died in the X and XH arm, respectively. Median overall survival was 20.6 and 24.9months with X and XH, respectively (HR=0.94 [0.65-1.35]; p=0.73). Performance status and metastatic site were independent prognosticators for overall survival. No difference between treatment arms was observed for patients who achieved clinical response or clinical benefit, respectively. Patients who continued/restarted anti-HER2 treatment (trastuzumab or lapatinib) after 2nd progression (N=52) had a post-progression survival of 18.8 compared with 13.3months for those who did not receive 3rd line treatment with anti-HER2 agents (N=88) (HR 0.63; p=0.02). CONCLUSIONS: Final overall survival analysis of the GBG-26 study did not demonstrate a significant survival benefit for treatment beyond progression with trastuzumab. However, in a post-hoc analysis, patients receiving anti-HER2 treatment as 3rd line therapy showed a better post-progression survival than those not receiving this targeted treatment.

Trastuzumab beyond progression in human epidermal growth factor receptor 2-positive advanced breast cancer: a german breast group 26/breast international group 03-05 study.

PURPOSE: Trastuzumab shows clinical activity in human epidermal growth factor receptor 2 (HER-2)-positive early and advanced breast cancer. In the German Breast Group 26/Breast International Group 03-05 trial, we investigated if trastuzumab treatment should be continued beyond progression. METHODS: Patients with HER-2-positive breast cancer that progresses during treatment with trastuzumab were randomly assigned to receive capecitabine (2,500 mg/m(2) body-surface area on days 1 through 14 [1,250 mg/m(2) semi-daily]) alone or with continuation of trastuzumab (6 mg/kg body weight) in 3-week cycles. The primary end point was time to progression. RESULTS: We randomly assigned 78 patients to capecitabine and 78 patients to capecitabine plus trastuzumab. Sixty-five events and 38 deaths in the capecitabine group and 62 events and 33 deaths in the capecitabine-plus-trastuzumab group occurred during 15.6 months of follow-up. Median times to progression were 5.6 months in the capecitabine group and 8.2 months in the capecitabine-plus-trastuzumab group with an unadjusted hazard ratio of 0.69 (95% CI, 0.48 to 0.97; two-sided log-rank P = .0338). Overall survival rates were 20.4 months (95% CI, 17.8 to 24.7) in the capecitabine group and 25.5 months (95% CI, 19.0 to 30.7) in the capecitabine-plus-trastuzumab group (P = .257). Overall response rates were 27.0% with capecitabine and 48.1% with capecitabine plus trastuzumab (odds ratio, 2.50; P = .0115). Continuation of trastuzumab beyond progression was not associated with increased toxicity. CONCLUSION: Continuation of trastuzumab plus capecitabine showed a significant improvement in overall response and time to progression compared with capecitabine alone in women with HER-2-positive breast cancer who experienced progression during trastuzumab treatment.

Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation.

Phosphatidylinositol-3-phosphate (PtdIns-3-P) is considered as a lipid constitutively present on endosomes; it does not seem to have a dynamic role in signalling. In contrast, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) plays a crucial role in different signalling pathways including translocation of the glucose transporter protein GLUT4 to the plasma membrane upon insulin receptor activation. GLUT4 translocation requires activation of two distinct pathways involving phosphatidylinositol 3-kinase (PI 3-K) and the small GTP-binding protein TC10, respectively. The contribution of each pathway remains to be elucidated. Here we show that insulin specifically induces the formation of PtdIns-3-P in insulin- responsive cells. The insulin-mediated formation of PtdIns-3-P occurs through the activation of TC10 at the lipid rafts subdomain of the plasma membrane. Exogenous PtdIns-3-P induces the plasma membrane translocation of both overexpressed and endogenous GLUT4. These data indicate that PtdIns-3-P is specifically produced downstream from insulin-mediated activation of TC10 to promote the plasma membrane translocation of GLUT4. These results give a new insight into the intracellular role of PtdIns-3-P and shed light on some aspects of insulin signalling so far not completely understood.

Cluster analysis of an extensive human breast cancer cell line protein expression map database.

In the current study, the protein expression maps (PEMs) of 26 breast cancer cell lines and three cell lines derived from normal breast or benign disease tissue were visualised by high resolution two-dimensional gel electrophoresis. Analysis of this data was performed with ChiClust and ChiMap, two analytical bioinformatics tools that are described here. These tools are designed to facilitate recognition of specific patterns shared by two or more (a series) PEMs. Both tools use PEMs that were matched by an image analysis program and locally written programs to create a match table that is saved in an object relational database. The ChiClust tool uses clustering and subclustering methods to extract statistically significant protein expression patterns from a large series of PEMs. The ChiMap tool calculates a differential value (either as percentage change or a fold change) and represents these graphically. All such differentials or just those identified using ChiClust can be submitted to ChiMap. These methods are not dependent on any particular commercial image analysis program, and the whole software package gives an integrated procedure for the comparison and analysis of a series of PEMs. The ChiClust tool was used here to order the breast cell lines into groups according to biological characteristics including morphology in vitro and tumour forming ability in vivo. ChiMap was then used to highlight eight major protein feature-changes detected between breast cancer cell lines that either do or do not proliferate in nude mice. Mass spectrometry was used to identify the proteins. The possible role of these proteins in cancer is discussed.

The application of 2D gel-based proteomics methods to the study of breast cancer.

The protein complement of breast cells consists of many thousands of proteins. Recent developments in 2D gel electrophoresis technology have made studies requiring the quantitative analysis of a differential proteome, such as comparison between normal and malignant cells or investigation of drug effects on cells, truly feasible. Computer software plays a central part in the comparisons between multiple gels required for such experiments. In addition, software tools allow patterns of coexpression of proteins to be studied, offering potential insights into protein regulation, interactions, and functions, especially when combined with complementary data on gene expression. In this paper, the technology and limitations of 2D gel-based proteomics are reviewed. Techniques for comparing sets of gels at a global level as well as identifying specific protein features that differentiate gels are discussed. Our own experience of studying the breast cell proteome is used to illustrate the difficulties and achievements of differential proteomics.