Breast-Conserving Surgery with or without Irradiation in Early Breast Cancer.

BACKGROUND: Limited level 1 evidence is available on the omission of radiotherapy after breast-conserving surgery in older women with hormone receptor-positive early breast cancer receiving adjuvant endocrine therapy. METHODS: We performed a phase 3 randomized trial of the omission of irradiation; the trial population included women 65 years of age or older who had hormone receptor-positive, node-negative, T1 or T2 primary breast cancer (with tumors ≤3 cm in the largest dimension) treated with breast-conserving surgery with clear excision margins and adjuvant endocrine therapy. Patients were randomly assigned to receive whole-breast irradiation (40 to 50 Gy) or no irradiation. The primary end point was local breast cancer recurrence. Regional recurrence, breast cancer-specific survival, distant recurrence as the first event, and overall survival were also assessed. RESULTS: A total of 1326 women were enrolled; 658 were randomly assigned to receive whole-breast irradiation and 668 to receive no irradiation. The median follow-up was 9.1 years. The cumulative incidence of local breast cancer recurrence within 10 years was 9.5% (95% confidence interval [CI], 6.8 to 12.3) in the no-radiotherapy group and 0.9% (95% CI, 0.1 to 1.7) in the radiotherapy group (hazard ratio, 10.4; 95% CI, 4.1 to 26.1; P<0.001). Although local recurrence was more common in the group that did not receive radiotherapy, the 10-year incidence of distant recurrence as the first event was not higher in the no-radiotherapy group than in the radiotherapy group, at 1.6% (95% CI, 0.4 to 2.8) and 3.0% (95% CI, 1.4 to 4.5), respectively. Overall survival at 10 years was almost identical in the two groups, at 80.8% (95% CI, 77.2 to 84.3) with no radiotherapy and 80.7% (95% CI, 76.9 to 84.3) with radiotherapy. The incidence of regional recurrence and breast cancer-specific survival also did not differ substantially between the two groups. CONCLUSIONS: Omission of radiotherapy was associated with an increased incidence of local recurrence but had no detrimental effect on distant recurrence as the first event or overall survival among women 65 years of age or older with low-risk, hormone receptor-positive early breast cancer. (Funded by the Chief Scientist Office of the Scottish Government and the Breast Cancer Institute, Western General Hospital, Edinburgh; ISRCTN number, ISRCTN95889329.).

Postoperative radiotherapy in women with early operable breast cancer (Scottish Breast Conservation Trial): 30-year update of a randomised, controlled, phase 3 trial.

BACKGROUND: Breast-conserving surgery, adjuvant systemic therapy, and radiotherapy are the standard of care for most women with early breast cancer. There are few reports of clinical outcomes beyond the first decade of follow-up of randomised trials comparing breast-conserving surgery with or without radiotherapy. We present a 30-year update of the Scottish Breast Conservation Trial. METHODS: In this randomised, controlled, phase 3 trial across 14 hospitals in Scotland, women aged younger than 70 years with early breast cancer (tumours ≤4 cm [T1 or T2 and N0 or N1]) were included. They underwent breast-conserving surgery (1 cm margin) with axillary node sampling or clearance. Oestrogen receptor (ER)-rich patients (≥20 fmol/mg protein) received 20 mg oral tamoxifen daily for 5 years. ER-poor patients (<20 fmol/mg protein) received chemotherapy (cyclophosphamide 600 mg/m2, methotrexate 50 mg/m2, and fluorouracil 600 mg/m2 every 21 days intravenously in eight courses). Stratification was by menstrual status (within or more than 12 months from last menstrual period) and ER status (oestrogen concentration ≥20 fmol/mg protein, <20 fmol/mg protein, or unknown) and patients were randomly assigned (1:1) to high-dose (50 Gy in 20-25 fractions) local or locoregional radiotherapy versus no radiotherapy. No blinding was possible due to the nature of the treatment. We report the primary endpoint of the original trial, ipsilateral breast tumour recurrence, and the co-primary endpoint, overall survival. Clinical outcomes were compared by the log-rank test. Hazard ratios (HRs) are reported, with no radiotherapy as the reference group. Failures of the proportional hazards assumption are reported if significant. All analyses are by intention to treat. FINDINGS: Between April 1, 1985, and Oct 2, 1991, 589 patients were enrolled and randomly assigned to the two treatment groups (293 to radiotherapy and 296 to no radiotherapy). After exclusion of four ineligible patients (two in each group), there were 291 patients in the radiotherapy group and 294 patients in the no radiotherapy group. Median follow-up was 17·5 years (IQR 8·4-27·9). Ipsilateral breast tumour recurrence was significantly lower in the radiotherapy group than in the no radiotherapy group (46 [16%] of 291 vs 107 [36%] of 294; HR 0·39 [95% CI 0·28-0·55], p<0·0001). Although there were differences in the hazard rate for ipsilateral breast tumour recurrence in the first decade after treatment (HR 0·24 [95% CI 0·15-0·38], p<0·0001), subsequent risks of ipsilateral breast tumour recurrence were similar in both groups (0·98 [0·54-1·79], p=0·95). There was no difference in overall survival between the two groups (median 18·7 years [95% CI 16·5-21·5] in the no radiotherapy group vs 19·2 years [16·9-21·3] in the radiotherapy group; HR 1·08 [95% CI 0·89-1 ·30], log-rank p=0·43). INTERPRETATION: Our findings suggest that patients whose biology predicts a late relapse a decade or more after breast-conserving surgery for early breast cancer might gain little from adjuvant radiotherapy. FUNDING: Breast Cancer Institute (part of Edinburgh and Lothian Health Foundation) and PFS Genomics (now part of Exact Sciences).

Radiation doses and fractionation schedules in non-low-risk ductal carcinoma in situ in the breast (BIG 3-07/TROG 07.01): a randomised, factorial, multicentre, open-label, phase 3 study.

BACKGROUND: Whole breast irradiation (WBI) after conservative surgery for ductal carcinoma in situ (DCIS) reduces local recurrence. We investigated whether a tumour bed boost after WBI improved outcomes, and examined radiation dose fractionation sensitivity for non-low-risk DCIS. METHODS: The study was an international, randomised, unmasked, phase 3 trial involving 136 participating centres of six clinical trials organisations in 11 countries (Australia, New Zealand, Singapore, Canada, the Netherlands, Belgium, France, Switzerland, Italy, Ireland, and the UK). Eligible patients were women aged 18 years or older with unilateral, histologically proven, non-low-risk DCIS treated by breast-conserving surgery with at least 1 mm of clear radial resection margins. They were assigned to one of four groups (1:1:1:1) of no tumour bed boost versus boost after conventional versus hypofractionated WBI, or randomly assigned to one of two groups (1:1) of no boost versus boost after each centre prespecified conventional or hypofractionated WBI. The conventional WBI used was 50 Gy in 25 fractions, and hypofractionated WBI was 42·5 Gy in 16 fractions. A boost dose of 16 Gy in eight fractions, if allocated, was delivered after WBI. Patients and clinicians were not masked to treatment allocation. The primary endpoint was time to local recurrence. This trial is registered with ClinicalTrials.gov (NCT00470236). FINDINGS: Between June 25, 2007, and June 30, 2014, 1608 patients were randomly assigned to have no boost (805 patients) or boost (803 patients). Conventional WBI was given to 831 patients, and hypofractionated WBI was given to 777 patients. Median follow-up was 6·6 years. The 5-year free-from-local-recurrence rates were 92·7% (95% CI 90·6-94·4%) in the no-boost group and 97·1% (95·6-98·1%) in the boost group (hazard ratio 0·47; 0·31-0·72; p<0·001). The boost group had higher rates of grade 2 or higher breast pain (10% [8-12%] vs 14% [12-17%], p=0·003) and induration (6% [5-8%] vs 14% [11-16%], p<0·001). INTERPRETATION: In patients with resected non-low-risk DCIS, a tumour bed boost after WBI reduced local recurrence with an increase in grade 2 or greater toxicity. The results provide the first randomised trial data to support the use of boost radiation after postoperative WBI in these patients to improve local control. The international scale of the study supports the generalisability of the results. FUNDING: National Health and Medical Research Council of Australia, Susan G Komen for the Cure, Breast Cancer Now, OncoSuisse, Dutch Cancer Society, Canadian Cancer Trials Group.

Radiation-induced prodrug activation: extending combined modality therapy for some solid tumours.

Combined chemoradiotherapy is the standard of care for locally advanced solid tumours. However, systemic toxicity may limit the delivery of planned chemotherapy. New approaches such as radiation-induced prodrug activation might diminish systemic toxicity, while retaining anticancer benefit. Organic azides have recently been shown to be reduced and activated under hypoxic conditions with clinically relevant doses of radiotherapy, uncaging pazopanib and doxorubicin in preclinical models with similar efficacy as the drug, but lower systemic toxicity. This approach may be relevant to the chemoradiation of glioblastoma and other solid tumours and offers potential for switching on drug delivery from implanted devices. The inclusion of reporters to confirm drug activation, avoidance of off-target effects and synchronisation of irradiation with optimal intratumoral drug concentration will be critical. Further preclinical validation studies of this approach should be encouraged.

Updated recommendations regarding the management of older patients with breast cancer: a joint paper from the European Society of Breast Cancer Specialists (EUSOMA) and the International Society of Geriatric Oncology (SIOG).

Breast cancer is increasingly prevalent in older adults and is a substantial part of routine oncology practice. However, management of breast cancer in this population is challenging because the disease is highly heterogeneous and there is insufficient evidence specific to older adults. Decision making should not be driven by age alone but should involve geriatric assessments plus careful consideration of life expectancy, competing risks of mortality, and patient preferences. A multidisciplinary taskforce, including members of the European Society of Breast Cancer Specialists and International Society of Geriatric Oncology, gathered to expand and update the previous 2012 evidence-based recommendations for the management of breast cancer in older individuals with the endorsement of the European Cancer Organisation. These guidelines were expanded to include chemotherapy toxicity prediction calculators, cultural and social considerations, surveillance imaging, genetic screening, gene expression profiles, neoadjuvant systemic treatment options, bone-modifying drugs, targeted therapies, and supportive care. Recommendations on geriatric assessment, ductal carcinoma in situ, screening, primary endocrine therapy, surgery, radiotherapy, adjuvant systemic therapy, and secondary breast cancer were updated.

Omission of sentinel node biopsy for breast cancer: Historical context and future perspectives on a modern controversy.

For older patients with clinically lymph node-negative breast cancer who have estrogen receptor-positive tumors and are treated with tamoxifen, randomized trials comparing axillary lymph node dissection (ALND) versus no ALND show that the omission of ALND improves patient quality of life and has no adverse effects on mortality. These results have served to justify sentinel node biopsy (SNB) omission in selected older patients with breast cancer. More recently, clinical trials were launched to assess SNB omission in younger patients, with recurrence and survival as the primary outcomes of interest. Three important considerations serve as the basis for these ongoing trials. First, it is assumed that SNB omission will improve patient quality of life, although, to date, there is no level I evidence to support this assumption. Second, axillary surgery has never been shown to reduce breast cancer mortality, but it does reduce the risk of axillary recurrences, although adjuvant systemic therapy and radiotherapy also reduce these recurrence risks. Finally, nodal status is losing importance as a guide for adjuvant systemic therapy decision making because these decisions are now increasingly predicated on tumor biomarkers and gene profiling, but it is gaining importance for adjuvant radiotherapy decision making. Because quality-of-life considerations are the primary motivation for abandoning SNB, there is a need for randomized trials comparing SNB versus no SNB/no axillary surgery, with quality of life as the primary end point (level I evidence). Moreover, suitable alternatives to guide adjuvant radiotherapy decision making will require validation before SNB omission can be justified for patients of all ages who have clinically node-negative breast cancer. LAY SUMMARY: In this review article, the authors provide a brief historical overview of the role of axillary surgery in breast cancer management and discuss additional studies and ramifications that should be considered before abandoning the sentinel node biopsy (SNB) procedure. Specifically, there is a need for level I evidence demonstrating that omission of the SNB procedure will improve patient quality of life and a need to validate suitable alternatives to SNB as a guide for adjuvant radiotherapy decision making.

Postmastectomy radiotherapy: a review.

PURPOSE OF REVIEW: We review the role of postmastectomy radiotherapy (PMRT) in the management of patients with early breast cancer. RECENT FINDINGS: PMRT in patients with 4 or more involved axillary lymph nodes is the current standard of care but the indications for PMRT in patients with 1-3 involved nodes remain controversial. The Early Breast Cancer Trialists' Collaborative Group meta-analysis of randomised trials of PMRT provides the most comprehensive level 1 evidence base. However, its applicability in contemporary practice in the context of recent multidisciplinary advances in surgery, radiation therapy and systemic therapy remains challenging. SUMMARY: The lack of consensus on the indications for PMRT in patients with 1-3 positive nodes underpins the variations in the national and international guidelines on PMRT. We emphasise the need for contemporary randomised trial data, and the potential to refine patient selection for PMRT using novel biomarkers of recurrence and radiosensitivity.

Preclinical Organotypic Models for the Assessment of Novel Cancer Therapeutics and Treatment.

The immense costs in both financial terms and preclinical research effort that occur in the development of anticancer drugs are unfortunately not matched by a substantial increase in improved clinical therapies due to the high rate of failure during clinical trials. This may be due to issues with toxicity or lack of clinical effectiveness when the drug is evaluated in patients. Currently, much cancer research is driven by the need to develop therapies that can exploit cancer cell adaptations to conditions in the tumor microenvironment such as acidosis and hypoxia, the requirement for more-specific, targeted treatments, or the exploitation of 'precision medicine' that can target known genomic changes in patient DNA. The high attrition rate for novel anticancer therapies suggests that the preclinical methods used in screening anticancer drugs need improvement. This chapter considers the advantages and disadvantages of 3D organotypic models in both cancer research and cancer drug screening, particularly in the areas of targeted drugs and the exploitation of genomic changes that can be used for therapeutic advantage in precision medicine.

In vivo validation of a miniaturized electrochemical oxygen sensor for measuring intestinal oxygen tension.

Recent advances in the fields of electronics and microfabrication techniques have led to the development of implantable medical devices for use within the field of precision medicine. Monitoring visceral surface tissue O2 tension (PTo2) by means of an implantable sensor is potentially useful in many clinical situations, including the perioperative management of patients undergoing intestinal resection and anastomosis. This concept could provide a means by which treatment could be tailored to individual patients. This study describes the in vivo validation of a novel, miniaturized electrochemical O2 sensor to provide real-time data on intestinal PTo2. A single O2 sensor was placed onto the serosal surface of the small intestine of anesthetized rats that were exposed to ischemic (superior mesenteric artery occlusion) and hypoxemic (alterations in inspired fractional O2 concentrations) insults. Control experiments demonstrated that the sensors can function and remain stable in an in vivo environment. Intestinal PTo2 decreased following superior mesenteric artery occlusion and with reductions in inspired O2 concentrations. These results were reversible after reinstating blood flow or by increasing inspired O2 concentrations. We have successfully developed an anesthetized rat intestinal ischemic and hypoxic model for validation of a miniaturized O2 sensor to provide real-time measurement of intestinal PTo2. Our results support further validation of the sensors in physiological conditions using a large animal model to provide evidence of their use in clinical applications where monitoring visceral surface tissue O2 tension is important.NEW & NOTEWORTHY This is the first report of real-time continuous measurements of intestinal oxygen tension made using a microfabricated O2 sensor. Using a developed rodent model, we have validated this sensor's ability to accurately measure dynamic and reversible changes in intestinal oxygenation that occur through ischemic and hypoxemic insults. Continuous monitoring of local intestinal oxygenation could have value in the postoperative monitoring of patients having undergone intestinal surgery.

Quality of life after postmastectomy radiotherapy in patients with intermediate-risk breast cancer (SUPREMO): 2-year follow-up results of a randomised controlled trial.

BACKGROUND: Postmastectomy radiotherapy in patients with four or more positive axillary nodes reduces breast cancer mortality, but its role in patients with one to three involved nodes is controversial. We assessed the effects of postmastectomy radiotherapy on quality of life (QOL) in women with intermediate-risk breast cancer. METHODS: SUPREMO is an open-label, international, parallel-group, randomised, controlled trial. Women aged 18 years or older with intermediate-risk breast cancer (defined as pT1-2N1; pT3N0; or pT2N0 if also grade III or with lymphovascular invasion) who had undergone mastectomy and, if node positive, axillary surgery, were randomly assigned (1:1) to receive chest wall radiotherapy (50 Gy in 25 fractions or a radiobiologically equivalent dose of 45 Gy in 20 fractions or 40 Gy in 15 fractions) or no radiotherapy. Randomisation was done with permuted blocks of varying block length, and stratified by centre, without masking of patients or investigators. The primary endpoint is 10-year overall survival. Here, we present 2-year results of QOL (a prespecified secondary endpoint). The QOL substudy, open to all UK patients, consists of questionnaires (European Organisation for Research and Treatment of Cancer QLQ-C30 and QLQ-BR23, Body Image Scale, Hospital Anxiety and Depression Scale [HADS], and EQ-5D-3L) completed before randomisation, and at 1, 2, 5, and 10 years. The prespecified primary outcomes within this QOL substudy were global QOL, fatigue, physical function, chest wall symptoms, shoulder and arm symptoms, body image, and anxiety and depression. Data were analysed by intention to treat, using repeated mixed-effects methods. This trial is registered with the ISRCTN registry, number ISRCTN61145589. FINDINGS: Between Aug 4, 2006, and April 29, 2013, 1688 patients were enrolled internationally and randomly assigned to receive chest wall radiotherapy (n=853) or not (n=835). 989 (79%) of 1258 patients from 111 UK centres consented to participate in the QOL substudy (487 in the radiotherapy group and 502 in the no radiotherapy group), of whom 947 (96%) returned the baseline questionnaires and were included in the analysis (radiotherapy, n=471; no radiotherapy, n=476). At up to 2 years, chest wall symptoms were worse in the radiotherapy group than in the no radiotherapy group (mean score 14·1 [SD 15·8] in the radiotherapy group vs 11·6 [14·6] in the no radiotherapy group; effect estimate 2·17, 95% CI 0·40-3·94; p=0·016); however, there was an improvement in both groups between years 1 and 2 (visit effect -1·34, 95% CI -2·36 to -0·31; p=0·010). No differences were seen between treatment groups in arm and shoulder symptoms, body image, fatigue, overall QOL, physical function, or anxiety or depression scores. INTERPRETATION: Postmastectomy radiotherapy led to more local (chest wall) symptoms up to 2 years postrandomisation compared with no radiotherapy, but the difference between groups was small. These data will inform shared decision making while we await survival (trial primary endpoint) results. FUNDING: Medical Research Council, European Organisation for Research and Treatment of Cancer, Cancer Australia, Dutch Cancer Society, Trustees of Hong Kong and Shanghai Banking Corporation.

Breast-conserving surgery with or without irradiation in women aged 65 years or older with early breast cancer (PRIME II): a randomised controlled trial.

BACKGROUND: For most older women with early breast cancer, standard treatment after breast-conserving surgery is adjuvant whole-breast radiotherapy and adjuvant endocrine treatment. We aimed to assess the effect omission of whole-breast radiotherapy would have on local control in older women at low risk of local recurrence at 5 years. METHODS: Between April 16, 2003, and Dec 22, 2009, 1326 women aged 65 years or older with early breast cancer judged low-risk (ie, hormone receptor-positive, axillary node-negative, T1-T2 up to 3 cm at the longest dimension, and clear margins; grade 3 tumour histology or lymphovascular invasion, but not both, were permitted), who had had breast-conserving surgery and were receiving adjuvant endocrine treatment, were recruited into a phase 3 randomised controlled trial at 76 centres in four countries. Eligible patients were randomly assigned to either whole-breast radiotherapy (40-50 Gy in 15-25 fractions) or no radiotherapy by computer-generated permuted block randomisation, stratified by centre, with a block size of four. The primary endpoint was ipsilateral breast tumour recurrence. Follow-up continues and will end at the 10-year anniversary of the last randomised patient. Analyses were done by intention to treat. The trial is registered on ISRCTN.com, number ISRCTN95889329. FINDINGS: 658 women who had undergone breast-conserving surgery and who were receiving adjuvant endocrine treatment were randomly assigned to receive whole-breast irradiation and 668 were allocated to no further treatment. After median follow-up of 5 years (IQR 3·84-6·05), ipsilateral breast tumour recurrence was 1·3% (95% CI 0·2-2·3; n=5) in women assigned to whole-breast radiotherapy and 4·1% (2·4-5·7; n=26) in those assigned no radiotherapy (p=0·0002). Compared with women allocated to whole-breast radiotherapy, the univariate hazard ratio for ipsilateral breast tumour recurrence in women assigned to no radiotherapy was 5·19 (95% CI 1·99-13·52; p=0·0007). No differences in regional recurrence, distant metastases, contralateral breast cancers, or new breast cancers were noted between groups. 5-year overall survival was 93·9% (95% CI 91·8-96·0) in both groups (p=0·34). 89 women died; eight of 49 patients allocated to no radiotherapy and four of 40 assigned to radiotherapy died from breast cancer. INTERPRETATION: Postoperative whole-breast radiotherapy after breast-conserving surgery and adjuvant endocrine treatment resulted in a significant but modest reduction in local recurrence for women aged 65 years or older with early breast cancer 5 years after randomisation. However, the 5-year rate of ipsilateral breast tumour recurrence is probably low enough for omission of radiotherapy to be considered for some patients. FUNDING: Chief Scientist Office (Scottish Government), Breast Cancer Institute (Western General Hospital, Edinburgh).

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Omission of radiation therapy after breast conserving surgery for older women at low-risk of local recurrence: One option among many.

This editorial discusses the evolving landscape of early-stage breast cancer treatment, emphasizing the need to tailor therapies based on disease biology and genomic approaches. The focus is on the reconsideration of postoperative radiation therapy (RT) for older patients with low-risk, hormone receptor-positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Recent trials show modest long-term local recurrence rates with the omission of RT after BCS in certain cases, challenging the traditional approach. The commentary calls for continued research on predictive tests for treatment response and advocates for a multidisciplinary approach to decision-making, considering factors like quality of life. The nuanced risk/benefit ratio of RT in older patients is explored, emphasizing the importance of comprehensive assessment for optimal therapy.

Proximity ligation assays for isoform-specific Akt activation in breast cancer identify activated Akt1 as a driver of progression.

The PI3K/Akt signal transduction pathway plays an important role in cancer progression and cell survival. Akt activation is associated with poor outcome in endocrine-treated breast cancer, whereas high levels of cytoplasmic Akt2 are associated with an improved overall survival. Proximity ligation assays (PLAs) were used to determine quantitative expression levels of isoform-specific activation (phosphorylation) of Akt1 and Akt2 in formalin-fixed, paraffin-embedded cell lines and breast cancer tumour tissues in situ. PLAs demonstrated a range of expression in breast cancer samples for total pAkt1 and pAkt2. High levels of pAkt1 were associated with reduced DRFS (HR: 1.45, 95% CI 1.14-1.83, p = 0.002) and OS (HR: 1.42, 95% CI 1.10-1.83, p = 0.007). When PLA results were combined, patients that had high levels of pAkt1 only had a significantly decreased DRFS (HR: 1.92, 95% CI 1.34-2.76, p = 0.005) and OS (HR: 1.94, 95% CI 1.32-2.86, p = 0.008) compared to other patients. Using PLAs to discriminate activation of Akt1 versus Akt2 suggests that Akt1 drives progression in early breast cancers. In cases where both Akt1/Akt2 are activated, Akt2 may act to reverse this effect. Using PLAs, we have measured activation of Akt1 and Akt2 proteins separately in situ in FFPE breast cancer samples.

GSK3ß and cyclin D1 expression predicts outcome in early breast cancer patients.

Glycogen synthase kinase 3ß (GSK3ß) is phosphorylated and inactivated by the phosphoinositide 3 kinase PI3K/Akt pathway. Activation of Akt phosphorylates GSK3ß preventing phosphorylation of cyclin D1 which leads to accumulation and nuclear localisation of cyclin D1, activation of CDK4/6 and cell cycle progression. The CCND1 gene found at chromosome 11q13 has been shown to be amplified in approximately 15 % of breast cancers. Cyclin D1, the product of the CCND1 gene, is one of the most commonly overexpressed proteins in breast cancer. Protein expression for GSK3ß, phosphorylated-GSK3ß (p-GSK3ß), cyclin D1 and gene expression of CCND1 were examined in tissue microarrays of 1,686 patients from the Edinburgh Breast Conservation Series. High GSK3ß expression was associated with reduced distant relapse-free survival (DRFS), while no association between p-GSK3ß and breast cancer-specific survival was seen. CCND1 amplification is also associated with poor DRFS. On the contrary, cyclin D1 overexpression is associated with an increase in DRFS. Multivariate analysis was performed. We suggest that analysis of both GSK3ß and cyclin D1 expressions can be considered as a marker of good prognosis in early breast cancer.

The p160 ER co-regulators predict outcome in ER negative breast cancer.

The SRC family of ER co-regulators are frequently overexpressed in breast cancer. Overexpression of AIB1 appears to be linked to hormone resistance in HER2 positive breast cancer. However, the role of these co-regulators in ER negative disease is poorly understood. SRC1, SRC2 and AIB1 expression was determined by immunohistochemical analysis of tissue microarrays constructed from tumours within the Edinburgh Breast Conservation Series (BCS). The BCS represents a fully documented consecutive cohort of 1,812 patients treated by breast conservation surgery in a single institution. Our results demonstrate tumours that overexpress both HER2 and AIB1 were associated with markedly reduced relapse free, distant relapse free and overall survival compared to HER2 and AIB1 only overexpressing tumours irrespective of ER status. In ER negative disease both SRC1 and AIB1 were linked to early relapse and death. The SRC family of ER co-regulators is involved in early relapse and resistance in both ER negative and ER positive breast cancer challenging the conventional concept that this effect is mediated solely via the ER.

Expression of activated type I receptor tyrosine kinases in early breast cancer.

Overexpression of EGFR, HER2 and HER3 are known to be associated with poor outcome in breast cancer. Few studies have examined the clinical impact of activation of these proteins. In the present study, we evaluated EGFR, HER2 and HER3 and the activated (phosphorylated) forms of these proteins in patients with early breast cancer. EGFR, HER2, HER3, pEGFR, pHER2 and pHER3 expression was determined by immunohistochemical analysis of tissue microarrays constructed from tumours within the Edinburgh Breast Conservation Series (BCS). The BCS represents a fully-documented consecutive cohort of 1,812 patients treated by breast conservation surgery in a single institution. Our results demonstrate overexpression of HER2 and pHER2 to be associated with a significant reduction in overall survival (OS) (HR: 1.66, 95 % CI 1.22-2.26, p = 0.001 and HR: 1.57, 95 % CI 1.22-2.03, p = 0.001, respectively) and distant relapse-free survival (DRFS) (HR: 1.63, 95 % CI 1.23-2.18, p = 0.001 and HR: 1.55, 95 % CI 1.23-1.97, p = 0.0002, respectively). Paradoxically, expression of pEGFR was associated with a significantly improved OS (HR: 0.67 95 % CI 0.50-0.91, p = 0.01) and DRFS (HR: 0.73, 95 % CI 0.56-0.96, p = 0.025). Expression of activated EGFR/HER2 provides additional information on ER positive breast cancer patients and suggests alternative treatment for those in this subgroup.

A Novel Approach for the Discovery of Biomarkers of Radiotherapy Response in Breast Cancer.

Radiotherapy (RT) is an important treatment modality for the local control of breast cancer (BC). Unfortunately, not all patients that receive RT will obtain a therapeutic benefit, as cancer cells that either possess intrinsic radioresistance or develop resistance during treatment can reduce its efficacy. For RT treatment regimens to become personalised, there is a need to identify biomarkers that can predict and/or monitor a tumour's response to radiation. Here we describe a novel method to identify such biomarkers. Liquid chromatography-mass spectrometry (LC-MS) was used on conditioned media (CM) samples from a radiosensitive oestrogen receptor positive (ER+) BC cell line (MCF-7) to identify cancer-secreted biomarkers which reflected a response to radiation. A total of 33 radiation-induced secreted proteins that had higher (up to 12-fold) secretion levels at 24 h post-2 Gy radiation were identified. Secretomic results were combined with whole-transcriptome gene expression experiments, using both radiosensitive and radioresistant cells, to identify a signature related to intrinsic radiosensitivity. Gene expression analysis assessing the levels of the 33 proteins showed that 5 (YBX3, EIF4EBP2, DKK1, GNPNAT1 and TK1) had higher expression levels in the radiosensitive cells compared to their radioresistant derivatives; 3 of these proteins (DKK1, GNPNAT1 and TK1) underwent in-lab and initial clinical validation. Western blot analysis using CM samples from cell lines confirmed a significant increase in the release of each candidate biomarker from radiosensitive cells 24 h after treatment with a 2 Gy dose of radiation; no significant increase in secretion was observed in the radioresistant cells after radiation. Immunohistochemistry showed that higher intracellular protein levels of the biomarkers were associated with greater radiosensitivity. Intracellular levels were further assessed in pre-treatment biopsy tissues from patients diagnosed with ER+ BC that were subsequently treated with breast-conserving surgery and RT. High DKK1 and GNPNAT1 intracellular levels were associated with significantly increased recurrence-free survival times, indicating that these two candidate biomarkers have the potential to predict sensitivity to RT. We suggest that the methods highlighted in this study could be utilised for the identification of biomarkers that may have a potential clinical role in personalising and optimising RT dosing regimens, whilst limiting the administration of RT to patients who are unlikely to benefit.

Mammostrat as a tool to stratify breast cancer patients at risk of recurrence during endocrine therapy.

INTRODUCTION: Patients with early-stage breast cancer, treated with endocrine therapy, have approximately 90% 5-year disease-free survival. However, for patients at higher risk of relapse despite endocrine therapy, additional adjuvant therapy, such as chemotherapy, may be indicated. The challenge is to prospectively identify such patients. The Mammostrat® test uses five immunohistochemical markers to stratify patients on tamoxifen therapy into risk groups to inform treatment decisions. We tested the efficacy of this panel in a mixed population of cases treated in a single center with breast-conserving surgery and long-term follow-up. METHODS: Tissue microarrays from a consecutive series (1981 to 1998) of 1,812 women managed by wide local excision and postoperative radiotherapy were collected following appropriate ethical review. Of 1,390 cases stained, 197 received no adjuvant hormonal or chemotherapy, 1,044 received tamoxifen only, and 149 received a combination of hormonal therapy and chemotherapy. Median age at diagnosis was 57, 71% were postmenopausal, 23.9% were node-positive and median tumor size was 1.5 cm. Samples were stained using triplicate 0.6 mm2 tissue microarray cores, and positivity for p53, HTF9C, CEACAM5, NDRG1 and SLC7A5 was assessed. Each case was assigned a Mammostrat risk score, and distant recurrence-free survival (DRFS), relapse-free survival (RFS) and overall survival (OS) were analyzed by marker positivity and risk score. RESULTS: Increased Mammostrat scores were significantly associated with reduced DRFS, RFS and OS in estrogen receptor (ER)-positive breast cancer (P < 0.00001). In multivariate analyses the risk score was independent of conventional risk factors for DRFS, RFS and OS (P < 0.05). In node-negative, tamoxifen-treated patients, 10-year recurrence rates were 7.6 ± 1.5% in the low-risk group versus 20.0 ± 4.4% in the high-risk group. Further, exploratory analyses revealed associations with outcome in both ER-negative and untreated patients. CONCLUSIONS: This is the fifth independent study providing evidence that Mammostrat can act as an independent prognostic tool for ER-positive, tamoxifen-treated breast cancer. In addition, this study revealed for the first time a possible association with outcome regardless of node status and ER-negative tumors. When viewed in the context of previous results, these data provide further support for this antibody panel as an aid to patient management in early-stage breast cancer.