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1.  Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test 
BMC Cancer  2012;12:456.
Background
EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory.
Methods
Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability.
Results
PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/μl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer’s laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units.
Conclusions
The EP test showed reproducible performance characteristics with good precision and negligible laboratory-to-laboratory variation. This study provides further evidence that the EP test is suitable for decentralized testing in specialized molecular pathological laboratories instead of a reference laboratory. This is a unique feature and a technical advance in comparison with existing RNA-based prognostic multigene expression tests.
doi:10.1186/1471-2407-12-456
PMCID: PMC3534340  PMID: 23039280
Breast cancer; Prognostic multigene expression test; Analytical validation; PCR; Pathology
2.  Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer 
Virchows Archiv  2014;465(4):409-417.
Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.
doi:10.1007/s00428-014-1652-0
PMCID: PMC4180906  PMID: 25218890
Breast cancer; Preanalytical; EndoPredict; Molecular pathology; Gene expression
3.  The EndoPredict Gene-Expression Assay in Clinical Practice - Performance and Impact on Clinical Decisions 
PLoS ONE  2013;8(6):e68252.
The validated EndoPredict assay is a novel tool to predict the risk of metastases of patients with estrogen receptor positive, HER2 negative breast cancer treated with endocrine therapy alone. It has been designed to integrate genomic and clinical information and includes clinico-pathological factors such as tumor size and nodal status. The test is feasible in a decentral setting in molecular pathology laboratories. In this project, we investigated the performance of this test in clinical practice, and performed a retrospective evaluation of its impact on treatment decisions in breast cancer. During one year, EndoPredict assays from 167 patients could be successfully performed. For retrospective evaluation of treatment decisions, a questionnaire was sent to the clinical partner. Regarding the molecular EP class, samples from 56 patients (33.5%) had a low-risk, whereas 111 patients (66.5%) showed a high-risk gene profile. After integration of the clinicopathological factors the combined clinical and molecular score (EPclin) resulted in a low-risk group of 77 patients (46.4%), while 89 (53.6%) had a high risk EPclin score. The EPclin-based estimated median 10-year-risk for metastases with endocrine therapy alone was 11% for the whole cohort. The median handling time averaged three days (range: 0 to 11 days), 59.3% of the tests could be performed in three or less than three days. Comparison of pre- and post-test therapy decisions showed a change of therapy in 37.7% of patients. 16 patients (12.3%) had a change to an additional chemotherapy while 25.4% of patients (n = 33) changed to an endocrine therapy alone. In 73 patients (56.2%) no change of therapy resulted. In 6.1% of patients (n = 8), the patients did not agree to the recommendation of the tumor board. Our results show that the EndoPredict assay could be routinely performed in decentral molecular pathology laboratories and the results markedly change treatment decisions.
doi:10.1371/journal.pone.0068252
PMCID: PMC3694878  PMID: 23826382
4.  Comparison of the RNA-based EndoPredict multigene test between core biopsies and corresponding surgical breast cancer sections 
Journal of Clinical Pathology  2012;65(7):660-662.
Aim
This study compared the perfomance of the RNA-based EndoPredict multigene test on core biopsies and surgical breast cancer specimens and analysed the influence of biopsy-induced tissue injuries on the test result.
Methods
80 formalin-fixed paraffin-embedded samples comprising paired biopsies and surgical specimens from 40 ER-positive, HER2-negative patients were evaluated. Total RNA was extracted and the EndoPredict score was determined.
Results
RNA yield was considerably lower in core biopsies, but sufficient to measure the assay in all samples. The EndoPredict score was highly correlated between paired samples (Pearson r=0.92), with an excellent concordance of classification into a low or high risk of metastasis (overall agreement 95%).
Conclusions
The measurements are comparable between core biopsies and surgical sections, which suggest that the EndoPredict assay can be performed on core biopsy tissue. Inflammatory changes induced by presurgical biopsies had no significant effect on the RNA-based risk assessment in surgical specimens.
doi:10.1136/jclinpath-2012-200716
PMCID: PMC3426896  PMID: 22447922
Breast; breast cancer; breast pathology; cancer; cancer genetics; cancer research; EGFR; endocrine pathology; gynaecological pathology; molecular oncology; molecular pathology; oncology; ovary; statistics; tumour markers
5.  Comparison of EndoPredict and Oncotype DX Test Results in Hormone Receptor Positive Invasive Breast Cancer 
PLoS ONE  2013;8(3):e58483.
Aim
Several multigene expression-based tests offering prognostic and predictive information in hormone-receptor positive early breast cancer were established during the last years. These tests provide prognostic information on distant recurrences and can serve as an aid in therapy decisions. We analyzed the recently validated reverse-transcription-quantitative-real-time PCR-based multigene-expression Endopredict (EP)-test on 34 hormone-receptor positive breast-cancer cases and compared the EP scores with the Oncotype DX Recurrence-scores (RS) obtained from the same cancer samples.
Methods
Formalin-fixed, paraffin-embedded invasive breast-cancer tissues from 34 patients were analyzed by the EP-test. Representative tumor blocks were analyzed with Oncotype DX prior to this study. Tumor tissue was removed from unstained slides, total-RNA was isolated and EP-analysis was performed blinded to Oncotype DX results.
Results
Extraction of sufficient amounts of RNA and generation of valid EP-scores were possible for all 34 samples. EP classified 11 patients as low-risk and 23 patients as high-risk. RS Score defined 15 patients as low-risk, 10 patients as intermediate-risk in and 9 patients as high-risk. Major-discrepancy occurred in 6 of 34 cases (18%): Low-risk RS was classified as high-risk by EP in 6 cases. Combining the RS intermediate-risk and high-risk groups to a common group, the concordance between both tests was 76%. Correlation between continuous EP and RS-scores was moderate (Pearson-coefficient: 0.65 (p<0.01).
Conclusion
We observed a significant but moderate concordance (76%) and moderate correlation (0.65) between RS and EP Score. Differences in results can be explained by different weighting of biological motives covered by the two tests. Further studies are needed to explore the clinical relevance of discrepant test results with respect of outcome.
doi:10.1371/journal.pone.0058483
PMCID: PMC3591350  PMID: 23505515
6.  Clinical validation of the EndoPredict test in node-positive, chemotherapy-treated ER+/HER2− breast cancer patients: results from the GEICAM 9906 trial 
Introduction
EndoPredict (EP) is an RNA-based multigene test that predicts the likelihood of distant recurrence in patients with estrogen receptor-positive (ER+), human epidermal growth factor receptor 2–negative (HER2−) breast cancer (BC) who are being treated with adjuvant endocrine therapy. Herein we report the prospective-retrospective clinical validation of EP in the node-positive, chemotherapy-treated, ER+/HER2− BC patients in the GEICAM 9906 trial.
Methods
The patients (N = 1,246) were treated either with six cycles of fluorouracil, epirubicin and cyclophosphamide (FEC) or with four cycles of FEC followed by eight weekly courses of paclitaxel (FEC-P), as well as with endocrine therapy if they had hormone receptor–positive disease. The patients were assigned to EP risk categories (low or high) according to prespecified cutoff levels. The primary endpoint in the clinical validation of EP was distant metastasis-free survival (MFS). Metastasis rates were estimated using the Kaplan-Meier method, and multivariate analysis was performed using Cox regression.
Results
The molecular EP score and the combined molecular and clinical EPclin score were successfully determined in 555 ER+/HER2− tumors from the 800 available samples in the GEICAM 9906 trial. On the basis of the EP, 25% of patients (n = 141) were classified as low risk. MFS was 93% in the low-risk group and 70% in the high-risk group (absolute risk reduction = 23%, hazard ratio (HR) = 4.8, 95% confidence interval (CI) = 2.5 to 9.5; P < 0.0001). Multivariate analysis showed that, in this ER+/HER2− cohort, EP results are an independent prognostic parameter after adjustment for age, grade, lymph node status, tumor size, treatment arm, ER and progesterone receptor (PR) status and proliferation index (Ki67). Using the predefined EPclin score, 13% of patients (n = 74) were assigned to the low-risk group, who had excellent outcomes and no distant recurrence events (absolute risk reduction vs high-risk group = 28%; P < 0.0001). Furthermore, EP was prognostic in premenopausal patients (HR = 6.7, 95% CI = 2.4 to 18.3; P = 0.0002) and postmenopausal patients (HR = 3.3, 95% CI = 1.3 to 8.5; P = 0.0109). There were no statistically significant differences in MFS between treatment arms (FEC vs FEC-P) in either the high- or low-risk groups. The interaction test results between the chemotherapy arm and the EP score were not significant.
Conclusions
EP is an independent prognostic parameter in node-positive, ER+/HER2− BC patients treated with adjuvant chemotherapy followed by hormone therapy. EP did not predict a greater efficacy of FEC-P compared to FEC alone.
doi:10.1186/bcr3642
PMCID: PMC4076639  PMID: 24725534
7.  Developing a new generation of breast cancer clinical gene expression tests 
When treatment decisions are based purely on clinicopathological factors, many women with estrogen receptor-positive/human epidermal growth factor receptor 2-negative cancers are overtreated. Gene expression profiles are valuable clinical tools that stratify the recurrence risk to identify patients most likely to benefit from adjuvant systemic therapies. Building upon greater understanding of tumor biology and more rigorous approaches to validation (including independent studies with a high level of evidence), several second-generation multigene tests have been developed. In the previous issue, Martin and colleagues report the third clinical validation study for EndoPredict, a distributed assay to assess risk of distant recurrences in estrogen receptor-positive/human epidermal growth factor receptor 2-negative women. The authors confirm the assay’s independent prognostic value in premenopausal and postmenopausal, node-positive women treated with contemporary chemotherapy followed by endocrine therapy. EndoPredict did not, however, predict benefit from adding paclitaxel. Predictive signatures for selecting among chemotherapy regimens remain an area needing further development.
doi:10.1186/bcr3688
PMCID: PMC4100317
8.  The EndoPredict score provides prognostic information on late distant metastases in ER+/HER2− breast cancer patients 
British Journal of Cancer  2013;109(12):2959-2964.
Background:
ER+/HER2− breast cancers have a proclivity for late recurrence. A personalised estimate of relapse risk after 5 years of endocrine treatment can improve patient selection for extended hormonal therapy.
Methods:
A total of 1702 postmenopausal ER+/HER2− breast cancer patients from two adjuvant phase III trials (ABCSG6, ABCSG8) treated with 5 years of endocrine therapy participated in this study. The multigene test EndoPredict (EP) and the EPclin score (which combines EP with tumour size and nodal status) were predefined in independent training cohorts. All patients were retrospectively assigned to risk categories based on gene expression and on clinical parameters. The primary end point was distant metastasis (DM). Kaplan–Meier method and Cox regression analysis were used in an early (0–5 years) and late time interval (>5 years post diagnosis).
Results:
EP is a significant, independent, prognostic parameter in the early and late time interval. The expression levels of proliferative and ER signalling genes contribute differentially to the underlying biology of early and late DM. The EPclin stratified 64% of patients at risk after 5 years into a low-risk subgroup with an absolute 1.8% of late DM at 10 years of follow-up.
Conclusion:
The EP test provides additional prognostic information for the identification of early and late DM beyond what can be achieved by combining the commonly used clinical parameters. The EPclin reliably identified a subgroup of patients who have an excellent long-term prognosis after 5 years of endocrine therapy. The side effects of extended therapy should be weighed against this projected outcome.
doi:10.1038/bjc.2013.671
PMCID: PMC3859949  PMID: 24157828
EndoPredict; endocrine therapy; late relapse
9.  Cost-Effectiveness Analysis of Prognostic Gene Expression Signature-Based Stratification of Early Breast Cancer Patients 
Pharmacoeconomics  2014;33:179-190.
Background
The individual risk of recurrence in hormone receptor-positive primary breast cancer patients determines whether adjuvant endocrine therapy should be combined with chemotherapy. Clinicopathological parameters and molecular tests such as EndoPredict® (EPclin) can support decision making in patients with estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative cancer.
Objective
Using a life-long Markov state transition model, we determined the health economic impact and incremental cost effectiveness of EPclin-based risk stratification in combination with clinical guidelines [German-S3, National Comprehensive Cancer Center Network (NCCN), and St. Gallen] to decide on chemotherapy use.
Methods
Information on overall and metastasis-free survival came from Austrian Breast & Colorectal Cancer Study Group clinical trials 6/8 (n = 1,619) and published literature. Effectiveness was assessed as quality-adjusted life-years (QALYs). Costs (2010) were assessed from a German third-party payer perspective.
Results
Lifetime costs per patient ranged from €28,268 (St.Gallen and EPclin) to €33,756 (NCCN). Due to an imperfect prognostic value and differences in chemotherapy use, strategies achieved between 13.165 QALYs (NCCN) and 13.173 QALYs (EPclin alone) per patient. Using German-S3 as reference, three strategies showed dominant results (St. Gallen and EPclin, German-S3 and EPclin, EPclin alone). Compared to German-S3, the addition of EPclin saved €3,388 and gained 0.002 QALYs per patient. Combining guidelines with EPclin remained preferable in sensitivity analysis.
Conclusion
Our study suggests that molecular markers can be sensibly combined with clinical guidelines to determine the risk profile of adjuvant breast cancer patients. Compared with the current German best practice (German-S3), combinations of EPclin with the St. Gallen, German-S3 or NCCN guideline and EPclin alone were dominant from the perspective of the German healthcare system.
Electronic supplementary material
The online version of this article (doi:10.1007/s40273-014-0227-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s40273-014-0227-x
PMCID: PMC4305105  PMID: 25404424
10.  Multigene prognostic tests in breast cancer: past, present, future 
There is growing consensus that multigene prognostic tests provide useful complementary information to tumor size and grade in estrogen receptor (ER)-positive breast cancers. The tests primarily rely on quantification of ER and proliferation-related genes and combine these into multivariate prediction models. Since ER-negative cancers tend to have higher proliferation rates, the prognostic value of current multigene tests in these cancers is limited. First-generation prognostic signatures (Oncotype DX, MammaPrint, Genomic Grade Index) are substantially more accurate to predict recurrence within the first 5 years than in later years. This has become a limitation with the availability of effective extended adjuvant endocrine therapies. Newer tests (Prosigna, EndoPredict, Breast Cancer Index) appear to possess better prognostic value for late recurrences while also remaining predictive of early relapse. Some clinical prediction problems are more difficult to solve than others: there are no clinically useful prognostic signatures for ER-negative cancers, and drug-specific treatment response predictors also remain elusive. Emerging areas of research involve the development of immune gene signatures that carry modest but significant prognostic value independent of proliferation and ER status and represent candidate predictive markers for immune-targeted therapies. Overall metrics of tumor heterogeneity and genome integrity (for example, homologue recombination deficiency score) are emerging as potential new predictive markers for platinum agents. The recent expansion of high-throughput technology platforms including low-cost sequencing of circulating and tumor-derived DNA and RNA and rapid reliable quantification of microRNA offers new opportunities to build extended prediction models across multiplatform data.
doi:10.1186/s13058-015-0514-2
PMCID: PMC4307898
11.  Systematic assessment of prognostic gene signatures for breast cancer shows distinct influence of time and ER status 
BMC Cancer  2014;14:211.
Background
The aim was to assess and compare prognostic power of nine breast cancer gene signatures (Intrinsic, PAM50, 70-gene, 76-gene, Genomic-Grade-Index, 21-gene-Recurrence-Score, EndoPredict, Wound-Response and Hypoxia) in relation to ER status and follow-up time.
Methods
A gene expression dataset from 947 breast tumors was used to evaluate the signatures for prediction of Distant Metastasis Free Survival (DMFS). A total of 912 patients had available DMFS status. The recently published METABRIC cohort was used as an additional validation set.
Results
Survival predictions were fairly concordant across most signatures. Prognostic power declined with follow-up time. During the first 5 years of followup, all signatures except for Hypoxia were predictive for DMFS in ER-positive disease, and 76-gene, Hypoxia and Wound-Response were prognostic in ER-negative disease. After 5 years, the signatures had little prognostic power. Gene signatures provide significant prognostic information beyond tumor size, node status and histological grade.
Conclusions
Generally, these signatures performed better for ER-positive disease, indicating that risk within each ER stratum is driven by distinct underlying biology. Most of the signatures were strong risk predictors for DMFS during the first 5 years of follow-up. Combining gene signatures with histological grade or tumor size, could improve the prognostic power, perhaps also of long-term survival.
doi:10.1186/1471-2407-14-211
PMCID: PMC4000128  PMID: 24645668
Breast cancer; Prognosis; Gene signature; Long-term survival prediction; Molecular subtype
12.  EndoPredict improves the prognostic classification derived from common clinical guidelines in ER-positive, HER2-negative early breast cancer 
Annals of Oncology  2012;24(3):640-647.
Background
In early estrogen receptor (ER)-positive/HER2-negative breast cancer, the decision to administer chemotherapy is largely based on prognostic criteria. The combined molecular/clinical EndoPredict test (EPclin) has been validated to accurately assess prognosis in this population. In this study, the clinical relevance of EPclin in relation to well-established clinical guidelines is assessed.
Patients and methods
We assigned risk groups to 1702 ER-positive/HER2-negative postmenopausal women from two large phase III trials treated only with endocrine therapy. Prognosis was assigned according to National Comprehensive Cancer Center Network-, German S3-, St Gallen guidelines and the EPclin. Prognostic groups were compared using the Kaplan–Meier survival analysis.
Results
After 10 years, absolute risk reductions (ARR) between the high- and low-risk groups ranged from 6.9% to 11.2% if assigned according to guidelines. It was at 18.7% for EPclin. EPclin reassigned 58%–61% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Women reclassified to low risk showed a 5% rate of distant metastasis at 10 years.
Conclusion
The EPclin score is able to predict favorable prognosis in a majority of patients that clinical guidelines would assign to intermediate or high risk. EPclin may reduce the indications for chemotherapy in ER-positive postmenopausal women with a limited number of clinical risk factors.
doi:10.1093/annonc/mds334
PMCID: PMC3574544  PMID: 23035151
adjuvant treatment; breast cancer; endocrine therapy; EndoPredict gene; expression
13.  Exquisite Sensitivity of TP53 Mutant and Basal Breast Cancers to a Dose-Dense Epirubicin−Cyclophosphamide Regimen 
PLoS Medicine  2007;4(3):e90.
Background
In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown.
Methods and Findings
In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m2 epirubicin and 1,200 mg/m2 cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status.
Conclusions
This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin–cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.
Hugues de The and colleagues report thatTP53 status is a predictive factor for responsiveness in breast cancers to a dose-dense epirubicin-cyclophosphamide chemotherapy regimen, and suggests that this regimen might be well suited for patientsTP53 mutant tumors.
Editors' Summary
Background.
One woman in eight will develop breast cancer during her life. As with other cancers, breast cancer arises when cells accumulate genetic changes (mutations) that allow them to grow uncontrollably and to move around the body. These altered cells are called malignant cells. The normal human breast contains several types of cell, any of which can become malignant. In addition, there is more than one route to malignancy—different sets of genes can be mutated. As a result, breast cancer is a heterogeneous disease that cannot be cured with a single type of treatment. Ideally, oncologists would like to know before they start treating a patient which therapeutic approach is going to be successful for that individual. Recently, researchers have begun to identify molecular changes that might eventually allow oncologists to make such rational treatment decisions. For example, laboratory studies in cell lines or animals indicate that the status of a gene called TP53 determines the chemotherapy agents (drugs that preferentially kill rapidly dividing cancer cells) to which cells respond. p53, the protein encoded by TP53, is a tumor suppressor. That is, in normal cells it prevents unregulated growth by controlling the expression of proteins involved in cell division and cell death. Consequently, p53 is often inactivated during cancer development.
Why Was This Study Done?
Although laboratory studies have linked TP53 status to chemotherapy responses, little is known about this relationship in human breast cancers. The clinical studies that have investigated whether TP53 status affects chemotherapy responses have generally found that patients whose tumors contain mutant TP53 have a poorer response to therapy and/or a shorter survival time than those whose tumors contain normal TP53. In this study, the researchers have asked whether TP53 status affects tumor responses to a dose-intense chemotherapy regimen (frequent, high doses of drugs) given to women with advanced noninflammatory breast cancer before surgery. This type of treatment is called neoadjuvant chemotherapy and is used to shrink tumors before surgery.
What Did the Researchers Do and Find?
The researchers collected breast tumor samples from 80 women before starting six fortnightly cycles of chemotherapy with epirubicin and cyclophosphamide. After this, each woman had her affected breast removed and examined to see whether the chemotherapy had killed the tumor cells. The researchers determined which original tumor samples contained mutated TP53 and used a technique called microarray expression profiling to document gene expression patterns in them. Overall, 28 tumors contained mutated TP53. Strikingly, all 15 tumors that responded completely to neoadjuvant chemotherapy (no tumor cells detectable in the breast tissue after chemotherapy) contained mutated TP53. Nine of these responsive tumors were basal-cell–like breast tumors, a particularly aggressive type of breast cancer; only one basal-cell–like, TP53-mutated tumor did not respond to chemotherapy. Patients whose tumors were unresponsive to the neoadjuvant chemotherapy but contained mutated TP53 tended to die sooner than those whose tumors contained normal TP53 or those with chemotherapy-responsive TP53-mutated tumors. Finally, expression profiling identified changes in the expression of many p53-regulated genes, but did not identify an expression profile in the TP53-mutated tumors unique to those that responded to chemotherapy.
What Do These Findings Mean?
These findings indicate that noninflammatory breast tumors containing mutant TP53—in particular, basal-cell–like tumors—are very sensitive to dose-dense epirubicin and cyclophosphamide chemotherapy. Intensive regimens of this type have rarely been used in previous studies, which might explain the apparent contradiction between these results and the generally poor response to chemotherapy of TP53-mutated breast tumors. More tumors now need to be examined to confirm the association between complete response, TP53 status and basal-cell–like tumors. In addition, although complete tumor responses generally predict good overall survival, longer survival studies than those reported here are needed to show that the tumor response to this particular neoadjuvant chemotherapy regimen translates into improved overall survival. If the present results can be confirmed and extended, dose-dense neoadjuvant chemotherapy with epirubicin and cyclophosphamide could considerably improve the outlook for patients with aggressive TP53-mutant, basal-cell–like breast tumors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040090.
The US National Cancer Institute provides patient and physician information on breast cancer and general information on understanding cancer
Cancer Research UK offers patient information on cancer and breast cancer
The MedlinePlus encyclopedia has pages on breast cancer
Emory University's CancerQuest discusses the biology of cancer, including the role of tumor suppressor proteins
Wikipedia has pages on p53 (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040090
PMCID: PMC1831731  PMID: 17388661
14.  Cleavage maps of the filamentous bacteriophages M13, fd, fl, and ZJ/2. 
Journal of Virology  1976;18(3):1024-1039.
The replicative form DNAs of bacteriophage M13, fd, f1, and ZJ/2 were found to be sensitive to cleavage by the restriction endonucleases endoR-HapII, endoR-HaeII, endoR-HaeIII, endoR-HindII, endoR-AluI, endoR-Hha, and endoR-Hinf. With respect to M13 DNA the number of cleavage sites varied from 21 for endoR-Hinf, 18 for endoR-AluI, 15 for endoR-Hha, 13 for endoR-HapII, 10 for endoR-HaeIII, 3 for endoR-HaeII, to only a single site for endoR-HindII. In contrast to M13, fd and f1, the ZJ/2 DNA molecule was not cleaved by the endoR-HindII endonuclease. No cleavage site on either phage DNA was detected for the endonucleases endoR-Hsu, endoR-EcoRI and endoR-Sma. When compared with M13 DNA, several differences were noted in the number and size of cleavage products obtained with DNA of phage fd, f1, and ZJ/2. From the results of these analyses, using the M13 enzyme cleavage maps as a reference, the endoR-HapII, endoR-HaeII, endoR-HaeIII, endoR-HindII and endoR-AluI maps of phage fd, f1, and ZJ/2 could be constructed. As is expected for very closely related phages, the enzyme cleavage patterns exhibit a high degree of homology. Phage f1 and ZJ/2 are most related since an identical pattern was obtained with seven different restriction endonucleases. Evidence is provided also that f1 is more similar to M13 than to fd. Furthermore, characteristic differences exist within the endoR-Hinf enzyme cleavage pattern of all the four phages tested. Digestion of phage DNA with this enzyme, therefore, provides a new and sensitive method of distinguishing these closely related filamentous coliphages .
Images
PMCID: PMC354802  PMID: 1271528
15.  Investigation of systemic folate status, impact of alcohol intake and levels of DNA damage in mononuclear cells of breast cancer patients 
British Journal of Cancer  2005;92(8):1524-1530.
Folate is required for DNA synthesis, repair and methylation. Low folate status has been implicated in carcinogenesis, possibly as a result of higher rate of genetic damage. The aim of this study is to compare folate status and levels of DNA damage between breast cancer and benign breast disease control patients. Fasting blood samples from 64 histologically confirmed untreated breast cancer patients (mean age 57 years) and 30 benign breast disease control patients (mean age 51 years) were obtained. Red cell folate (RCF) and plasma homocysteine were measured. Mononuclear cells (MNC) were isolated for genetic damage analysis using the basic alkaline comet assay. Results are expressed as tail moment. Data were log transformed as appropriate before analysis for normalisation purposes. The geometric mean (95% confidence interval) of RCF (ng ml−1) in breast cancer patients was 339.07 (333.3–404.6) vs 379.5 (335.8–505.2) in control patients (P=0.24). Corresponding plasma homocysteine concentrations (μmol l−1) were 11.9 (10.6–16.4) vs 10.1 (9.3–11.9) (P=0.073), respectively. The mean tail moment (s.d.) of DNA damage in MNC of breast cancer patients detected by the basic comet assay was 1.4 (0.66) vs –0.17 (0.79) in controls (P<0.0001, t-test), the modified comet assay ‘endonuclease III (Endo III)' was 1.7 (0.70) vs 0.86 (0.81) (P<0.0001, t-test), and the modified comet assay ‘formamidopyrimidine glycosylase (FPG)' was 1.6 (0.62) vs 0.99 (0.94) (P<0.0001, t-test). There was a significant negative correlation between RCF levels and DNA damage detected by modified comet assay ‘FPG' (Pearson Correlation Coefficient r2=−0.26, P=0.02) and DNA damage was found to be significantly higher in MNC of breast cancer patients compared to benign breast disease control patients. Breast cancer patients tended to have lower RCF levels and higher levels of plasma homocysteine, but these differences were not significant. The study provides preliminary evidence that reduced folate status may be implicated in the aetiology of breast cancer perhaps by increasing the in vivo level of genetic instability.
doi:10.1038/sj.bjc.6602530
PMCID: PMC2361990  PMID: 15812544
folate; breast cancer; DNA damage
16.  Test-retest reliability of neurophysiological tests of hand-arm vibration syndrome in vibration exposed workers and unexposed referents 
Background
Exposure to hand-held vibrating tools may cause the hand-arm vibration syndrome (HAVS). The aim was to study the test-retest reliability of hand and muscle strength tests, and tests for the determination of thermal and vibration perception thresholds, which are used when investigating signs of neuropathy in vibration exposed workers.
Methods
In this study, 47 vibration exposed workers who had been investigated at the department of Occupational and Environmental Medicine in Gothenburg were compared with a randomized sample of 18 unexposed subjects from the general population of the city of Gothenburg. All participants passed a structured interview, answered several questionnaires and had a physical examination including hand and finger muscle strength tests, determination of vibrotactile (VPT) and thermal perception thresholds (TPT). Two weeks later, 23 workers and referents, selected in a randomized manner, were called back for the same test-procedures for the evaluation of test-retest reliability.
Results
The test-retest reliability after a two week interval expressed as limits of agreement (LOA; Bland-Altman), intra-class correlation coefficients (ICC) and Pearson correlation coefficients was excellent for tests with the Baseline hand grip, Pinch-grip and 3-Chuck grip among the exposed workers and referents (N = 23: percentage of differences within LOA 91 – 100%; ICC-values ≥0.93; Pearson r ≥0.93). The test-retest reliability was also excellent (percentage of differences within LOA 96–100 %) for the determination of vibration perception thresholds in digits 2 and 5 bilaterally as well as for temperature perception thresholds in digits 2 and 5, bilaterally (percentage of differences within LOA 91 – 96%). For ICC and Pearson r the results for vibration perception thresholds were good for digit 2, left hand and for digit 5, bilaterally (ICC ≥ 0.84; r ≥0.85), and lower (ICC = 0.59; r = 0.59) for digit 2, right hand. For the latter two indices the test-retest reliability for the determination of temperature thresholds was lower and showed more varying results.
Conclusion
The strong test-retest reliability for hand and muscle strength tests as well as for the determination of VPTs makes these procedures useful for diagnostic purposes and follow-up studies in vibration exposed workers.
doi:10.1186/s12995-014-0038-1
PMCID: PMC4232643  PMID: 25400687
Work ability; Vibration exposure; Hand-arm vibration syndrome; Test-retest reliability; Neurophysiological findings
17.  P53 autoantibodies in 1006 patients followed up for breast cancer 
Breast Cancer Research : BCR  2000;2(6):438-443.
Serial plasma samples from 1006 patients with breast cancer revealed: (i) no correlation of p53 autoantibody status with disease status at the time of sample collection, or with menopausal status at time of primary diagnosis of breast cancer; (ii) 155 out of 1006 (15%) of patients were positive for p53 autoantibodies, and these patients tended to have a persistent autoantibody status throughout follow up, irrespective of disease behaviour; and (iii) where a negative autoantibody status was found at primary diagnosis of breast cancer, this negative status persisted throughout follow up, irrespective of later disease behaviour. We conclude that screening for p53 autoantibody status is not informative on residual tumour activity nor on therapeutic responsiveness.
Introduction:
Dysfunction of the tumour-suppressor protein, p53, may be due to either mutational or epigenetic factors, each of which may lead to accumulation of cytoplasmic p53. Abnormal accumulation of p53 in breast cancer tissue is predictive of poor prognosis [1,2]. Humoral studies [3,4] have shown that cancer patients may develop immunity to abnormally expressed p53, as revealed by p53 autoantibodies in the blood. Again, prognostic correlates have been noted, with presence of circulating p53 autoantibodies at diagnosis of breast cancer being associated with reduced overall survival [5,6] and with poor prognostic factors such as high histological grade and the absence of hormone receptors [5,7,8].
Little is known of the potential value of p53 autoantibody in follow up of cancer. In lung cancer there is evidence that autoantibodies to p53 may provide a useful tool to monitor response to therapy [9,10], whereas serial measurements of autoantibodies to p53 in 40 patients with advanced ovarian cancer were not found to be clinically useful [11]. In breast cancer some 30% of node-negative patients will relapse within 5 years, but there is no current means to predict those who are at risk.
We performed the present study to ask if the presence of autoantibodies to p53 has any association with breast cancer progression.
Materials and method:
A library of plasma samples were collected from all patients attending one general oncology clinic for postoperative follow up of breast cancer. The clinical status of each patient at the time of sampling was summarized. An average of eight plasma samples were cryopreserved for each patient over a period of 15 years.
The enzyme-linked immusorbent assay (ELISA) for p53 autoantibodies was developed in-house, based on the ELISA procedure of Lubin et al [3]. Our in-house method is detailed in the full text of this article. In one assay series we compared a commercial ELISA kit for p53 autoantibodies with our in-house ELISA. A total of 20 patients' samples were tested, representing a range of positive and negative readings. Two samples scored as strongly positive with the in-house assay, but only one of these two scored positive with the commercial assay. Having established sensitivity, specificity and reproducibility of the in-house assay, we judged that this was superior to the commercial assay both in terms of sensitivity and of cost (£1 per test compared with £23 per test). The in-house assay was thus used throughout the present study.
Results:
Serial plasma samples from 1006 patients with breast cancer revealed the following: (i) no correlation of p53 autoantibody status with disease status at the time of sample collection (Table 1), or with menopausal status at time of primary diagnosis of breast cancer (Table 2); (ii) 155 out of 1006 (15%) of patients were positive for p53 autoantibodies, and these patients tended to have a persistent autoantibody status throughout follow up, irrespective of disease behaviour; and (iii) where a negative autoantibody status was found at primary diagnosis of breast cancer, this negative status persisted throughout follow up, irrespective of later disease behaviour (Table 3).
Discussion:
As a working hypothesis, we proposed that levels of autoantibodies to p53 would reflect tumour behaviour. However, we found that the presence or absence of p53 autoantibodies was not predictive of presence or absence of recurrent disease. There was an equivalent incidence of active disease at the time of sampling in both the autoantibody-negative and autoantibody-positive groups, these being 25.2 and 28.7%, respectively. Thus, humoral immune activity against p53 appeared to be relatively restricted to a subgroup of patients in whom, once an autoantibody response had been generated, antibody was likely to persist regardless of tumour behaviour. Conversely, where no detectable p53 autoantibody was present at the time of primary diagnosis, these patients remained similarly negative for antibody, irrespective of subsequent disease activity (Table 3).
In contrast to shed markers that correlate with tumour mass, such as CA15.3 for cancer of the breast, any tumour-related immune response will be subject to complex regulation. Autoantibody responses to p53 will require appropriate primary immunization; initial low-dose antigen exposure may induce immune tolerance and lack of response. Higher antigen doses may activate either antibody-mediated immunity, or cellular immunity.
In breast cancer patients, our results suggest that, once an active humoral response against p53 is established, then this remains active. This persistent humoral reaction may be driven by persistent antigenic stimulation by p53 protein derived from overexpression of p53 at distant metastatic sites; alternatively, irradiated normal tissue may be a source of continued antigenic stimulation, because a long-term side effect of radiation therapy is an increased expression of p53 in normal breast tissue that persists for several years [12]. Since the great majority of our total patient cohort had received radiotherapy, humoral immunity to p53 associated with primary disease might persist, even in those patients who enter remission, due to tumour-independent antigenic stimulation.
Loss of p53 function is known to correlate with loss of efficacy of cancer therapy in vivo [13,14]. This raised the possibility that autoantibodies to p53 that develop during follow up might indicate those patients whose tumor has become resistant to therapy. However, the present results show that, if no immunity has been generated at the time of primary diagnosis, then later immunity is unlikely to occur. This corresponds to the finding that expression of p53 antigen in biopies of locally advanced breast cancer did not correlate with drug resistance [15,16]. Overall, the present observations show that screening for p53 autoantibody status is not informative on residual tumour activity, or on therapeutic responsiveness. We conclude that the potential value of p53 autoantibody screening in patients with breast cancer is limited to the prognostic information obtained at diagnosis.
PMCID: PMC13921  PMID: 11056691
breast; cancer; monitoring; p53 autoantibodies
18.  The Method of LDL Cholesterol Measurement influences Classification of LDL Cholesterol to Treatment Goals 
Background
Low density lipoprotein cholesterol (LDL-C) has been clearly associated with the risk of developing coronary heart disease (CHD). The best and most convenient method for determining LDL-C has come under increased scrutiny in recent years. We present comparisons of Friedewald’s calculated LDL-C (C-LDL-C) and direct LDL-C (D-LDL-C) using three different homogenous assays. This highlights differences between the two methods of LDL-C measurement, and how this affects the classification of samples into different LDL-C treatment goals as determined by NCEP ATP III guidelines thus potentially affecting treatment strategies.
Methods
Lipid profiles of a total of 2,208 clinic patients were retrieved from the Central Arkansas VA Healthcare System (CAVHS) clinical laboratory database. Samples studied were of one week period of time during the 3 periods studied, 2000 (period 1), 2002 (period 2) and 2005 (period 3). Different homogenous assays for D-LDL-C measurement were used for each of the 3 periods.
Results
There is a fundamental disagreement between D-LDL-C and C-LDL-C, even though Pearson’s correlation coefficients are 0.93, 0.97 and 0.98 for periods 1, 2 and 3 respectively. Using the model for period 1, when C-LDL-C is 70 mg/dl, the predicted D-LDL-C is 95 mg/dl (36% higher). The differences between C-LDL-C and predicted D-LDL-C progressively decrease at higher LDL-C cut points. In the assay used in period 3, there are 290 samples with D-LDL-C values between 100–130 mg/dl. Of these, only 182 samples show agreement with C-LDL-C values whereas 90 samples with a D-LDL-C in the 100–130 mg/dl range are in 70–100 mg/dl range using the C-LDL-C assay. While the kappa statistics suggests the LDL-C measures have relatively high levels of agreement, the significant generalized McNemar tests (p<0.01) provide additional evidence of disagreement between C-LDL-C and D-LDL-C during all the 3 periods.
Conclusion
Our results highlight D-LDL-C measurements using 3 different assays during 3 different time periods. In all assays there is substantial lack of agreement between D-LDL-C and C-LDL-C which in most cases resulted in higher D-LDL-C values than C-LDL-C. This leads to clinically significant misclassification of patient’s LDL-C to a different LDL-C treatment goal which would potentially result in more drug usage; thus exposing patients to more potential side effects and at a much greater cost with little evidence of benefit.
doi:10.231/JIM.0b013e3181fb7ca7
PMCID: PMC3992945  PMID: 20940623
direct LDL; calculated LDL; cholesterol
19.  Endo180 modulation by bisphosphonates and diagnostic accuracy in metastatic breast cancer 
British Journal of Cancer  2012;108(1):163-169.
Background:
Endo180 (CD280; MRC2; uPARAP)-dependent collagen remodelling is dysregulated in primary tumours and bone metastasis. Here, we confirm the release and diagnostic accuracy of soluble Endo180 for diagnosing metastasis in breast cancer (BCa).
Methods:
Endo180 was quantified in BCa cell conditioned medium and plasma from BCa patients stratified according to disease status and bisphosphonate treatment (n=88). All P-values are from two-sided tests.
Results:
Endo180 is released by ectodomain shedding from the surface of MCF-7 and MDA-MB-231 BCa cell lines. Plasma Endo180 was significantly higher in recurrent/metastatic (1.71±0.87; n=59) vs early/localised (0.92±0.37; n=29) BCa (P<0.0001). True/false-positive rates for metastasis classification were: 85%/50% for the reference standard, CA 15-3 antigen (28 U ml−1); ⩽97%/⩾36% for Endo180; and ⩽97%/⩾32% for CA 15-3 antigen+Endo180. Bisphosphonate treatment was associated with reduced Endo180 levels in BCa patients with bone metastasis (P=0.011; n=42). True/false-positive rates in bisphosphonate-naive patients (n=57) were: 68%/45% for CA 15-3 antigen; ⩽95%/⩾20% for Endo180; and ⩽92%/⩾21% for CA 15-3 antigen+Endo180.
Conclusion:
Endo180 is a potential marker modulated by bisphosphonates in metastatic BCa.
doi:10.1038/bjc.2012.540
PMCID: PMC3553530  PMID: 23257899
bisphosphonates; bone metastasis; breast cancer; collagen receptor; diagnostic marker; plasma marker
20.  Up-regulation of uPARAP/Endo180 during culture activation of rat hepatic stellate cells and its presence in hepatic stellate cell lines from different species 
BMC Cell Biology  2009;10:39.
Background
The urokinase plasminogen activator receptor associated protein (uPARAP)/Endo180 is a novel endocytic receptor that mediates collagen uptake and is implicated to play a role in physiological and pathological tissue-remodelling processes by mediating intracellular collagen degradation.
Result
This study investigates the expression of uPARAP/Endo180 protein and messenger RNA in primary rat hepatic stellate cell (HSC) cultures. The results show that uPARAP/Endo180 protein is not expressed in freshly isolated HSCs or during the first few days of culture while the cells still display quiescent features. In contrast, uPARAP/Endo180 protein is expressed early during HSC activation when cells are transdifferentiated into myofibroblast-like cells. Very low levels of uPARAP/Endo180 mRNA are detectable during the first days of culture but uPARAP/Endo180 mRNA is strongly up-regulated with increasing time in culture. Moreover, endocytic uptake of denatured collagen increases as transdifferentiation proceeds over time and correlates with increased expression of uPARAP/Endo180. Finally, analysis of uPARAP/Endo180 expression in four hepatic stellate cell lines from three different species showed that all these cell lines express uPARAP/Endo180 and are able to take up denatured collagen efficiently.
Conclusion
These results demonstrate that uPARAP/Endo180 expression by rat HSCs is strongly up-regulated during culture activation and identify this receptor as a feature common to culture-activated HSCs.
doi:10.1186/1471-2121-10-39
PMCID: PMC2689179  PMID: 19432973
21.  Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer 
Executive Summary
In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of published literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.
Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenomics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.
The following reports can be publicly accessed at the MAS website at: www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.html
Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer: An Evidence-Based and Economic Analysis
Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: An Evidence-Based and Ecopnomic Analysis
K-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based and Economic Analysis
Objective
To review and synthesize the available evidence regarding the laboratory performance, prognostic value, and predictive value of Oncotype-DX for the target population.
Clinical Need: Condition and Target Population
The target population of this review is women with newly diagnosed early stage (stage I–IIIa) invasive breast cancer that is estrogen-receptor (ER) positive and/or progesterone-receptor (PR) positive. Much of this review, however, is relevant for women with early stage (I and II) invasive breast cancer that is specifically ER positive, lymph node (LN) negative and human epidermal growth factor receptor 2 (HER-2/neu) negative. This refined population represents an estimated incident population of 3,315 new breast cancers in Ontario (according to 2007 data). Currently it is estimated that only 15% of these women will develop a distant metastasis at 10 years; however, a far great proportion currently receive adjuvant chemotherapy, suggesting that more women are being treated with chemotherapy than can benefit. There is therefore a need to develop better prognostic and predictive tools to improve the selection of women that may benefit from adjuvant chemotherapy.
Technology of Concern
The Oncotype-DX Breast Cancer Assay (Genomic Health, Redwood City, CA) quantifies gene expression for 21 genes in breast cancer tissue by performing reverse transcription polymerase chain reaction (RT-PCR) on formalin-fixed paraffin-embedded (FFPE) tumour blocks that are obtained during initial surgery (lumpectomy, mastectomy, or core biopsy) of women with early breast cancer that is newly diagnosed. The panel of 21 genes include genes associated with tumour proliferation and invasion, as well as other genes related to HER-2/neu expression, ER expression, and progesterone receptor (PR) expression.
Research Questions
What is the laboratory performance of Oncotype-DX?
How reliable is Oncotype-DX (i.e., how repeatable and reproducible is Oncotype-DX)?
How often does Oncotype-DX fail to give a useable result?
What is the prognostic value of Oncotype-DX?*
Is Oncotype-DX recurrence score associated with the risk of distant recurrence or death due to any cause in women with early breast cancer receiving tamoxifen?
What is the predictive value of Oncotype-DX?*
Does Oncoytpe-DX recurrence score predict significant benefit in terms of improvements in 10-year distant recurrence or death due to any cause for women receiving tamoxifen plus chemotherapy in comparison to women receiving tamoxifen alone?
How does Oncotype-DX compare to other known predictors of risk such as Adjuvant! Online?
How does Oncotype-DX impact patient quality of life and clinical/patient decision-making?
Research Methods
Literature Search
Search Strategy
A literature search was performed on March 19th, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 1st, 2006 to March 19th, 2010. A starting search date of January 1st, 2006 was because a comprehensive systematic review of Oncotype-DX was identified in preliminary literature searching. This systematic review, by Marchionni et al. (2008), included literature up to January 1st, 2007. All studies identified in the review by Marchionni et al. as well as those identified in updated literature searching were used to form the evidentiary base of this review. The quality of the overall body of evidence was identified as high, moderate, low or very low according to GRADE methodology.
Inclusion Criteria
Any observational trial, controlled clinical trial, randomized controlled trial (RCT), meta-analysis or systematic review that reported on the laboratory performance, prognostic value and/or predictive value of Oncotype-DX testing, or other outcome relevant to the Key Questions, specific to the target population was included.
Exclusion Criteria
Studies that did not report original data or original data analysis,
Studies published in a language other than English,
Studies reported only in abstract or as poster presentations (such publications were not sought nor included in this review since the MAS does not generally consider evidence that is not subject to peer review nor does the MAS consider evidence that lacks detailed description of methodology).
Outcomes of Interest
Outcomes of interest varied depending on the Key Question. For the Key Questions of prognostic and predictive value (Key Questions #2 and #3), the prospectively defined primary outcome was risk of 10-year distant recurrence. The prospectively defined secondary outcome was 10-year death due to any cause (i.e., overall survival). All additional outcomes such as risk of locoregional recurrence or disease-free survival (DFS) were not prospectively determined for this review but were reported as presented in included trials; these outcomes are referenced as tertiary outcomes in this review. Outcomes for other Key Questions (i.e., Key Questions #1, #4 and #5) were not prospectively defined due to the variability in endpoints relevant for these questions.
Summary of Findings
A total of 26 studies were included. Of these 26 studies, only five studies were relevant to the primary questions of this review (Key Questions #2 and #3). The following conclusions were drawn from the entire body of evidence:
There is a lack of external validation to support the reliability of Oncotype-DX; however, the current available evidence derived from internal industry validation studies suggests that Oncotype-DX is reliable (i.e., Oncotype-DX is repeatable and reproducible).
Current available evidence suggests a moderate failure rate of Oncotype-DX testing; however, the failure rate observed across clinical trials included in this review is likely inflated; the current Ontario experience suggests an acceptably lower rate of test failure.
In women with newly diagnosed early breast cancer (stage I–II) that is estrogen-receptor positive and/or progesterone-receptor positive and lymph-node negative:
There is low quality evidence that Oncotype-DX has prognostic value in women who are being treated with adjuvant tamoxifen or anastrozole (the latter for postmenopausal women only),
There is very low quality evidence that Oncotype-DX can predict which women will benefit from adjuvant CMF/MF chemotherapy in women being treated with adjuvant tamoxifen.
In postmenopausal women with newly diagnosed early breast cancer that is estrogen-receptor positive and/or progesterone-receptor positive and lymph-node positive:
There is low quality evidence that Oncotype-DX has limited prognostic value in women who are being treated with adjuvant tamoxifen or anastrozole,
There is very low quality evidence that Oncotype-DX has limited predictive value for predicting which women will benefit from adjuvant CAF chemotherapy in women who are being treated with adjuvant tamoxifen.
There are methodological and statistical limitations that affect both the generalizability of the current available evidence, as well as the magnitude and statistical strength of the observed effect sizes; in particular:
Of the major predictive trials, Oncotype-DX scores were only produced for a small subset of women (<40% of the original randomized population) potentially disabling the effects of treatment randomization and opening the possibility of selection bias;
Data is not specific to HER-2/neu-negative women;
There were limitations with multivariate statistical analyses.
Additional trials of observational design may provide further validation of the prognostic and predictive value of Oncotype-DX; however, it is unlikely that prospective or randomized data will become available in the near future due to ethical, time and resource considerations.
There is currently insufficient evidence investigating how Oncoytpe-DX compares to other known prognostic estimators of risk, such as Adjuvant! Online, and there is insufficient evidence investigating how Oncotype-DX would impact clinician/patient decision-making in a setting generalizable to Ontario.
PMCID: PMC3382301  PMID: 23074401
22.  The kSORT Assay to Detect Renal Transplant Patients at High Risk for Acute Rejection: Results of the Multicenter AART Study 
PLoS Medicine  2014;11(11):e1001759.
Minnie Sarwal and colleagues developed a gene expression assay using peripheral blood samples to detect patients with renal transplant at high risk for acute rejection.
Please see later in the article for the Editors' Summary
Background
Development of noninvasive molecular assays to improve disease diagnosis and patient monitoring is a critical need. In renal transplantation, acute rejection (AR) increases the risk for chronic graft injury and failure. Noninvasive diagnostic assays to improve current late and nonspecific diagnosis of rejection are needed. We sought to develop a test using a simple blood gene expression assay to detect patients at high risk for AR.
Methods and Findings
We developed a novel correlation-based algorithm by step-wise analysis of gene expression data in 558 blood samples from 436 renal transplant patients collected across eight transplant centers in the US, Mexico, and Spain between 5 February 2005 and 15 December 2012 in the Assessment of Acute Rejection in Renal Transplantation (AART) study. Gene expression was assessed by quantitative real-time PCR (QPCR) in one center. A 17-gene set—the Kidney Solid Organ Response Test (kSORT)—was selected in 143 samples for AR classification using discriminant analysis (area under the receiver operating characteristic curve [AUC] = 0.94; 95% CI 0.91–0.98), validated in 124 independent samples (AUC = 0.95; 95% CI 0.88–1.0) and evaluated for AR prediction in 191 serial samples, where it predicted AR up to 3 mo prior to detection by the current gold standard (biopsy). A novel reference-based algorithm (using 13 12-gene models) was developed in 100 independent samples to provide a numerical AR risk score, to classify patients as high risk versus low risk for AR. kSORT was able to detect AR in blood independent of age, time post-transplantation, and sample source without additional data normalization; AUC = 0.93 (95% CI 0.86–0.99). Further validation of kSORT is planned in prospective clinical observational and interventional trials.
Conclusions
The kSORT blood QPCR assay is a noninvasive tool to detect high risk of AR of renal transplants.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Throughout life, the kidneys filter waste products (from the normal breakdown of tissues and food) and excess water from the blood to make urine. If the kidneys stop working for any reason, the rate at which the blood is filtered decreases, and dangerous amounts of creatinine and other waste products build up in the blood. The kidneys can fail suddenly (acute kidney failure) because of injury or poisoning, but usually failing kidneys stop working gradually over many years (chronic kidney disease). Chronic kidney disease is very common, especially in people who have high blood pressure or diabetes and in elderly people. In the UK, for example, about 20% of people aged 65–74 years have some degree of chronic kidney disease. People whose kidneys fail completely (end-stage kidney disease) need regular dialysis (hemodialysis, in which blood is filtered by an external machine, or peritoneal dialysis, which uses blood vessels in the abdominal lining to do the work of the kidneys) or a renal transplant (the surgical transfer of a healthy kidney from another person into the patient's body) to keep them alive.
Why Was This Study Done?
Our immune system protects us from pathogens (disease-causing organisms) by recognizing specific molecules (antigens) on the invader's surface as foreign and initiating a sequence of events that kills the invader. Unfortunately, the immune system sometimes recognizes kidney transplants as foreign and triggers transplant rejection. The chances of rejection can be minimized by “matching” the antigens on the donated kidney to those on the tissues of the kidney recipient and by giving the recipient immunosuppressive drugs. However, acute rejection (rejection during the first year after transplantation) affects about 20% of kidney transplants. Acute rejection needs to be detected quickly and treated with a short course of more powerful immunosuppressants because it increases the risk of transplant failure. The current “gold standard” method for detecting acute rejection if the level of creatinine in the patient's blood begins to rise is to surgically remove a small piece (biopsy) of the transplanted kidney for analysis. However, other conditions can change creatinine levels, acute rejection can occur without creatinine levels changing (subclinical acute rejection), and biopsies are invasive. Here, the researchers develop a noninvasive test for acute kidney rejection called the Kidney Solid Organ Response Test (kSORT) based on gene expression levels in the blood.
What Did the Researchers Do and Find?
For the Assessment of Acute Rejection in Renal Transplantation (AART) study, the researchers used an assay called quantitative polymerase chain reaction (QPCR) to measure the expression of 43 genes whose expression levels change during acute kidney rejection in blood samples collected from patients who had had a kidney transplant. Using a training set of 143 samples and statistical analyses, the researchers identified a 17-gene set (kSORT) that discriminated between patients with and without acute rejection detected by kidney biopsy. The 17-gene set correctly identified 39 of the samples taken from 47 patients with acute rejection as being from patients with acute rejection, and 87 of 96 samples from patients without acute rejection as being from patients without acute rejection. The researchers validated the gene set using 124 independent samples. Then, using 191 serial samples, they showed that the gene set was able to predict acute rejection up to three months before detection by biopsy. Finally, the researchers used 100 blood samples to develop an algorithm (a step-wise calculation) to classify patients as being at high or low risk of acute rejection.
What Do These Findings Mean?
These findings describe the early development of a noninvasive tool (kSORT) that might, eventually, help clinicians identify patients at risk of acute rejection after kidney transplantation. kSORT needs to be tested in more patients before being used clinically, however, to validate its predictive ability, particularly given that the current gold standard test against which it was compared (biopsy) is far from perfect. An additional limitation of kSORT is that it did not discriminate between cell-mediated and antibody-mediated immune rejection. These two types of immune rejection are treated in different ways, so clinicians ideally need a test for acute rejection that indicates which form of immune rejection is involved. The authors are conducting a follow-up study to help determine whether kSORT can be used in clinical practice to identify acute rejection and to identify which patients are at greatest risk of transplant rejection and may require biopsy.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001759.
The US National Kidney and Urologic Diseases Information Clearinghouse provides links to information about all aspects of kidney disease; the US National Kidney Disease Education Program provides resources to help improve the understanding, detection, and management of kidney disease (in English and Spanish)
The UK National Health Service Choices website provides information for patients on chronic kidney disease and about kidney transplants, including some personal stories
The US National Kidney Foundation, a not-for-profit organization, provides information about chronic kidney disease and about kidney transplantation (in English and Spanish)
The not-for-profit UK National Kidney Federation provides support and information for patients with kidney disease and for their carers, including information and personal stories about kidney donation and transplantation
World Kidney Day, a joint initiative between the International Society of Nephrology and the International Federation of Kidney Foundations, aims to raise awareness about kidneys and kidney disease
MedlinePlus provides links to additional resources about kidney diseases, kidney failure, and kidney transplantation; the MedlinePlus encyclopedia has a page about transplant rejection
doi:10.1371/journal.pmed.1001759
PMCID: PMC4227654  PMID: 25386950
23.  The Western Ontario Shoulder Instability Index (WOSI): validity, reliability, and responsiveness retested with a Swedish translation 
Acta Orthopaedica  2009;80(2):233-238.
Background and purpose The WOSI score questionnaire is a tool designed for self-assessment of shoulder function for patients with instability problems. We made a translation into Swedish and retested the score by analyzing the psychometric properties validity, reliability, and responsiveness.
Patients and methods 3 patient materials were used for the assessment: (A) a follow-up on a group of 32 patients more than 8 years after having primary posttraumatic shoulder dislocation. Evaluation of Pearson’s correlation coefficient between WOSI and Rowe score and for test-retest reliability was made; (B) 22 patients, treated with a surgical stabilization of the shoulder at our department, were evaluated with Pearson’s correlation coefficient between WOSI and EQ-5D, and between WOSI and a VAS-scale of general shoulder function. Also, Cronbach’s alpha, effect size, and floor, and ceiling effects were analyzed; (C) 45 students with healthy shoulders (reference group) had their WOSI score determined.
Results The construct validity (Pearson’s correlation coefficient) was adequate (0.59) between the WOSI score and the Rowe score. The agreement with an ICC value (test-retest) for the WOSI score was excellent (0.94). Cronbach’s alpha (internal consistency) was satisfactory, with 0.89 preoperatively and 0.95 postoperatively. All 22 patients in group B reported improvement in the WOSI score (mean 29%). Responsiveness was excellent, with an effect size of 1.67 for the WOSI score. There were no floor or ceiling effects for the Swedish WOSI score. The mean WOSI score from group C with 45 normal healthy shoulders was 96%, with no floor but high ceiling effects.
Interpretation WOSI score does not require an examination of the patient and can be administered by mail. The high ICC and sensitivity makes it able to monitor an individual patient’s progress. At this retest, the WOSI score has good validity, a high degree of reliability, and a high degree of responsiveness, all at the same level as in the original publication. We recommend the WOSI when evaluating patients with instability problems.
doi:10.3109/17453670902930057
PMCID: PMC2823179  PMID: 19404809
24.  Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue 
BMC Cancer  2010;10:37.
Background
The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months.
Methods
RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score.
Results
Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses.
Conclusions
Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.
Trial Registration
Current Controlled Trials: NCT00004205
doi:10.1186/1471-2407-10-37
PMCID: PMC2829498  PMID: 20144231
25.  Endocardial Unipolar Voltage Mapping to Detect Epicardial VT Substrate in Patients with Nonischemic Left Ventricular Cardiomyopathy 
Background
Patients with non-ischemic left ventricular cardiomyopathy (LVCM) and ventricular tachycardia (VT) have complex three-dimensional substrate with variable involvement of the endocardium (ENDO) and epicardium (EPI). The purpose of this study was to determine if ENDO unipolar (UNI) mapping with a larger electrical field of view could identify EPI low bipolar (BIP) voltage regions in patients with LVCM undergoing VT ablation.
Methods and Results
The reference value for normal ENDO unipolar voltage was determined from 6 patients without structural heart disease. Consecutive patients undergoing VT ablation over an eight-year period with detailed (>100 point) LV ENDO and EPI mapping and normal LV ENDO BIP voltage were identified. From this cohort, we compared patients with structurally normal hearts and normal EPI BIP voltage (EPI-, Group 1) to patients with LVCM and low LV EPI BIP voltage regions present (EPI+, Group 2). Confluent regions of ENDO UNI and EPI BIP low voltage (> 2 cm2) were measured. The normal signal amplitude was >8.27mV for LV ENDO UNI electrograms. Detailed LV ENDO-EPI maps in 5 EPI- patients were compared to 11 EPI+ patients. Confluent ENDO UNI low voltage regions were seen in 9/11 (82%) of the EPI+ (Group 2) patients compared to 0/5 EPI- (Group 1) patients (P <0.001). In all 9 patients with ENDO UNI low voltage, the ENDO UNI low voltage regions were directly opposite to an area of EPI BIP low voltage (61% ENDO UNI-EPI BIP low voltage area overlap).
Conclusions
EPI arrhythmia substrate can be reliably identified in most patients with LVCM using ENDO UNI voltage mapping in the absence of ENDO BIP abnormalities.
doi:10.1161/CIRCEP.110.959957
PMCID: PMC3041847  PMID: 21131557
catheter ablation; ventricular tachycardia; electrophysiology mapping; cardiomyopathy

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