Coverage Policy Manual
Policy #: 2004029
Category: Laboratory
Initiated: August 2017
Last Review: June 2018
  Genetic Test: Assays of Genetic Expression in Tumor Tissue as a Technique to Determine Prognosis in Patients With Breast Cancer (Oncotype DX®, EndoPredict, the Breast Cancer Index and Prosigna, Mammaprint and BluePrint)

Description:
Newly Diagnosed Breast Cancer
Most women with newly diagnosed breast cancer in the United States present with early-stage or locally advanced (ie, nonmetastatic) disease. However, almost a third of women who are disease-free after initial local and regional treatment develop distant recurrences during follow-up (Colleoni, 2016). Current breast cancer treatment regimens involve systemic adjuvant chemotherapy, hormonal therapy, biologic therapy, or a combination, depending on patients’ baseline level of recurrence risk, hormonal markers, and risk tolerance.
 
Women whose tumors are positive for human epidermal growth factor receptor 2 (HER2) should receive adjuvant therapy with a HER2-directed therapy (trastuzumab with or without pertuzumab). Decision making about adjuvant biologic therapy for women with HER2-positive cancer is not discussed here. This review focuses on 3 decision points:
 
1. The decision to pursue adjuvant chemotherapy following locoregional therapy, with or without neoadjuvant chemotherapy, based on predicted risk of recurrence, for women who are hormone receptor-positive but HER2-negative. The use of adjuvant chemotherapy reduces the risk of breast cancer recurrence but carries risks of systemic toxicity. The risk:benefit ratio must be balanced for each patient, with a higher likelihood of net health benefits for patients with a greater baseline predicted the risk of recurrence. Some of the individual considerations are discussed below. HER2 expression independently confers an unfavorable prognosis, but assessing the independent effects of HER2 is complicated in the presence of targeted therapy; therefore, we focus specifically on patients without HER2 expression.
 
2. The decision to pursue adjuvant endocrine therapy from 5 to 10 years for women who are hormone receptor-positive but HER2-negative and who have survived without recurrence to 5 years. For patients with hormone receptor-positive tumors, the use of adjuvant endocrine therapy (tamoxifen and/or an aromatase inhibitor, with or without ovarian suppression) for 5 to 10 years after an initial diagnosis has support in clinical practice. The 2017 guidelines from the National Comprehensive Cancer Network (NCCN) recommend extended endocrine therapy (NCCN, 2017). The American Society for Clinical Oncology’s (ASCO) 2014 focused update to its guidelines on adjuvant endocrine therapy for women with hormone receptor‒positive breast cancer have recommended 10 years of tamoxifen for pre- or perimenopausal women, and a total of 7-8 to 10 years of endocrine therapy, following 1 of 4 regimens that include tamoxifen with or without an aromatase inhibitor for postmenopausal women (Burstein, 2010; Burstein, 2014)
 
3. The decision to pursue adjuvant radiotherapy in women with ductal carcinoma in situ (DCIS). Adjuvant radiotherapy reduces the risk of local recurrences but has not been shown to change the risk of distant recurrence or mortality. There may be a group of patients for whom the reduction in risk for local recurrence may not be large enough to justify the risks of radiotherapy.  
 
Selection Of Adjuvant Chemotherapy Based On Risk Of Recurrence
An important part of treatment planning for women with breast cancer involves determining which patients could benefit from adjuvant cytotoxic chemotherapy. For example, for women with early-stage invasive breast cancer (ie, cancer extending beyond the basement membrane of the mammary ducts into adjacent tissue), adjuvant cytotoxic chemotherapy consistently provides approximately a 30% relative risk reduction in 10-year breast cancer mortality regardless of patients’ baseline prognosis. However, the absolute benefit of chemotherapy depends on the underlying or baseline risk of recurrence. Women with the best prognosis have tumors that are small, early-stage, estrogen receptor‒positive, and lymph node negative. No single classifier is considered a criterion standard. As a result, a substantial number of patients are treated with chemotherapy who fail to benefit. Better predictors of recurrence risk could help women’s decision making, some of whom may prefer to avoid chemotherapy if assured their risk is low.
 
Selection Of Extended Endocrine Therapy
Randomized controlled trials have established that 5 years of tamoxifen improves mortality in women with hormone receptor-positive breast cancer. A 2011 individual patient data meta-analysis by the Early Breast Cancer Trialists' Collaborative Group, including 20 trials (total N=21,457 patients) found that 5 years of tamoxifen in estrogen receptor‒positive disease reduced the risk of recurrences by almost 50% over 10 years on the relative scale; breast cancer mortality was decreased by 29% through 15 years.6 For patients with early-stage, invasive breast cancer that is hormone receptor-positive, the use of endocrine therapy (tamoxifen and/or aromatase inhibitor, with or without ovarian suppression) for 5 to 10 years following initial diagnosis has support in national guidelines (NCCN, 2017; Burstein, 2010; Burstein, 2016). However, the regimens available and the evidence to support them vary.
 
Randomized controlled trials published recently have shown that extended endocrine therapy decreases the risk of recurrence. The ASCO and NCCN guidelines were informed primarily by results of the ATLAS trial, which compared 5 and 10 years of tamoxifen (Davies, 2014) and the subsequent aTTom trial (reported in abstract form) (Gray, 2013). In both trials, in women who were hormone receptor-positive and had completed 5 years of tamoxifen, 5 years of extended tamoxifen was associated with improvements in breast cancer-specific mortality; ATLAS showed improvements in overall survival .
 
Three previously reported randomized trials of extended tamoxifen treatment had mixed findings: Tormey et al (1996; total N=194 patients),(Tormey, 1996) the National Surgical Adjuvant Breast and Bowel Project (Fisher et al, 2001; total N=1172 patients),11 and the Scottish Cancer Trials Breast Group (Stewart et al, 2001; total N=342 patients) (Stewart, 2001).
 
Overall, the available trial evidence would suggest that 10 years of tamoxifen in pre- or postmenopausal women can be linked with improved survival while trials of extended aromatase inhibitors in different populations of hormone receptor-positive patients have had more mixed results. In addition to the trials published in full-length form, 3 trials presented in early 2017 evaluating extended endocrine therapy in postmenopausal women (NSABP-42 [NCT00382070]: 10 years vs 5 years of letrozole; DATA [NCT00301457]: 6 years vs 3 years of anastrozole; and IDEAL [NTR3077] 10 years vs 7.5 years of letrozole) did not meet their primary end points.
 
Clinical Uses Of Gene Expression Signatures For Breast Cancer
In other clinical scenarios involving breast cancer, accurate assessment of prognosis may affect the decision to offer certain treatments. Recently, several groups have identified panels of gene expression markers (“signatures”) that appear to predict the baseline risk of invasive breast cancer recurrence after surgery, radiotherapy, and endocrine therapy (for hormone receptor to positive tumors). Several gene expression tests commercially available in the United States are listed in Table 1. If these panels are more accurate risk predictors than current conventional classifiers, they could be used to aid decision making on adjuvant treatments without greatly affecting disease-free survival and overall survival (OS). This review focuses on gene expression profiling panels that have prognostic or predictive ability in individuals with early-stage, invasive breast cancer with known estrogen receptor and progesterone receptor and human epidermal growth factor receptor (HER2) status. The proposed clinical utility of these tests varies by the clinical context; these specific indications are discussed in this review:  
 
1. Prognosis and/or prediction of treatment response in patients with node-negative, early-stage, hormone receptor-positive, HER2-negative invasive breast cancer who will receive adjuvant hormonal therapy for the purpose of determining whether patients can avoid adjuvant cytotoxic chemotherapy.
2. Prognosis and/or prediction of treatment response in patients with node-positive (1-3 nodes), hormone receptor-positive, early-stage, HER2-negative invasive breast cancer who will receive adjuvant hormonal therapy for the purpose of determining whether patients can avoid adjuvant cytotoxic chemotherapy.
3. Prognosis and/or prediction of treatment response in patients with DCIS for the purpose of determining whether patients can avoid radiotherapy.
4. Prognosis and/or prediction of treatment response in patients with node-negative, early-stage, hormone receptor-positive, HER2-negative invasive breast cancer, receiving adjuvant hormonal therapy, who have survived without progression to 5 years post-diagnosis, for the purpose of determining whether patients will continue adjuvant hormonal therapy.
 
For each of these indications, clinical trials have shown that there is some clinical benefit to receiving the additional therapy under consideration. However, each additional treatment has potential adverse effects. If a patient subgroup can be defined that has an extremely low risk of distant recurrence, or a subgroup can be defined that does not respond to the treatment, then the additional treatment can be forgone with little effect on cancer outcome due to the low risk of poor outcome or lack of response to treatment.
 
Table 1. Available Gene Expression Tests Reporting Recurrence Risk for Breast Cancer Considered Herein
    • Oncotype DX® (Genomic Health (Redwood City, CA)-21-gene RT-PCR
    • Breast Cancer Index Prognostic SM  Biotheranostics (San Diego, CA)-Combines MGI and the HOXB13:IL17BR Indexmeasured using RT-PCR
    • EndoPredict®  Sividon Diagnostics (acquired by Myriad [Salt Lake City, UT] in 2016)- 12-gene real-time RT-PCR
    • Prosigna® NanoString Technologies (Seattle, WA)-Gene expression protein signature Predictive signature based on nCounter® digital analysis system based on PAM50 breast cancer intrinsic subtype classifier
 
Additional commercially available tests may provide some prognostic or predictive information for breast cancer. Tests intended to assess estrogen receptor, progesterone receptor, and HER2 status, such as TargetPrint® (Agendia; via quantitative microarray), are outside the scope of this policy. In addition, tests that do not provide a specific recurrence risk are outside the scope of this review.
 
Other commercially available biomarkers are designed to provide information about tumors’ molecular subtypes (ie, luminal A, luminal B, HER2 type, and basal type). Prosigna was initially offered a molecular subtype test. The BluePrint® 80-gene molecular subtyping assay is offered in combination with MammaPrint to augment predictive data about response to chemotherapy.
 
Decision Framework For Evaluating Breast Cancer Biomarkers
 
Simon et al Framework
Many studies have investigated individual biomarkers or combinations of biomarkers associated with breast cancer outcomes. Determining which studies constitute sufficient evidence that the test or biomarker is likely to be clinically useful depends on attributes of the test such as its performance and the quality of the study generating the results. Simon et al (2009) have described a framework to evaluate prognostic biomarker evidence (Simon, 2009). Study designs such as prospective clinical trials or previously conducted clinical trials with archived tumor samples constitute stronger evidence than studies with less planned and systematic patient recruitment and data collection. Randomized trials allow determination of treatment-biomarker interactions that may be clinically important. In some clinical scenarios, demonstration of a treatment-biomarker interaction is not critical, because the decision to withhold chemotherapy in a low-risk group (to avoid chemotherapy-related morbidity) does not require the presence of a biomarker-treatment interaction. The study must generate an absolute estimate of outcomes in the patient group of interest that would result in a change in management (eg, withholding of chemotherapy), and the study must have sufficient precision (narrow confidence intervals). Results of the same test across studies should show the consistency of results and more than 1 study demonstrating the desired result should be available. Simon has proposed that at least 2 Simon category B studies showing results consistent with clinical utility are necessary to demonstrate adequate evidence of a biomarker (Simon, 2009).
 
Breast Cancer-Specific Outcomes
The main outcome of interest for this review is 10-year distant recurrence-free survival. Distant recurrence is a hallmark of advanced breast cancer and thus more informative of OS than disease-free survival. Disease-free survival also includes local recurrence, which has a much better treatment prognosis than distant disease. For the extended endocrine indications in this review, the main outcome of interest is 10- year distant recurrence-free survival conditional on recurrence-free survival for 5 years.
 
Decisions to undergo or forgo adjuvant therapy (chemotherapy or endocrine) depend on how a woman values the potential benefit of lower recurrence risk relative to the harms of treatment. The balance of benefits and harms determines the thresholds that inform decisions (Pauker, 1975; Pauker, 1980). Most women will accept substantial adverse events for even modest benefit. For example, Simes et al (2001) interviewed 104 Australian women with breast cancer treated with cytotoxic chemotherapy and elicited preferences to undergo chemotherapy according to probable gain in survival (Simes, 2001). With an expected survival of 5 years without chemotherapy, 73% said they would accept chemotherapy for an increased survival of 6 months or less; with an expected survival of 15 years, 39% would accept treatment for a gain of 6 months. Duric et al (2005) found 64% to 84% of 97 women expressing a willingness to undergo chemotherapy for a 1-year improvement in life expectancy or 3% increase in survival rates (Duric, 2005). About half felt a single day would justify adjuvant chemotherapy. A major difference between the 2 studies was that the chemotherapy regimen in Duric et al was less toxic. Thewes et al (2005) adopted the same approach for adjuvant endocrine therapy preferences in 102 premenopausal women with early-stage breast cancers (Thewes, 2005). Among women having a baseline life expectancy of 5 years, 61% said they would accept endocrine therapy for a 6-month increase in life expectancy and 79% for 1 year; rates were similar if the baseline life expectancy was 15 years. These proportions are close to those for adjuvant chemotherapy found by Duric. How these estimates correspond to the distant recurrence rates reported in prognostic studies is imprecise, but Henderson (2015) has suggested that below a recurrence threshold of 10% many patients will not elect adjuvant chemotherapy owing to the small absolute benefit (Henderson, 2015). He also noted that a majority of those patients are older with small node-negative tumors. That interpretation is consistent with a recent study of 81 women by Hamelinck et al (2016) who found that 78% of women ages 40 to 49 years, 88% ages 50 to 59, 59% ages 60 to 69, and 40% age 70 or older would accept adjuvant chemotherapy for a 0% to 10% absolute decrease in recurrence risk (Hamelinck, 2016). There was a wide range of minimally required absolute benefits, with the majority accepting chemotherapy for an absolute benefit of 1% to 5%. At a given age range, fewer women expressed a willingness to accept adjuvant endocrine therapy than chemotherapy for a given mortality benefit.
 
Regulatory Status
Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests must meet the general regulatory standards of the Clinical Laboratory Improvement Amendments. Oncotype DX® and other tests listed herein are available under the auspices of the Clinical Laboratory Improvement Amendments. Laboratories that offer laboratory-developed tests must be licensed by the Clinical Laboratory Improvement Amendments for high-complexity testing. To date, the U.S. Food and Drug Administration (FDA) has chosen not to require any regulatory review of this test.
 
In February 2007, MammaPrint® (Agendia) was cleared for marketing by FDA through the 510(k) process for the prediction of breast cancer metastasis. In January 2015, MammaPrint® was cleared for marketing by FDA through the 510(k) process for use in fresh-frozen, paraffin-embedded breast cancer tissue. In September 2013, Prosigna® was cleared for marketing by FDA through the 510(k) process. FDA determined that Prosigna® was substantially equivalent to MammaPrint®. FDA product code: NYI.
 
Coding
 
Oncotype DX
There is a specific CPT multianalyte assay with algorithmic analysis (MAAA) code for Oncotype DX:
 
81519 Oncology (breast), mRNA, gene expression profiling by real-time RT-PCR of 21 genes, utilizing formalin-fixed paraffin-embedded tissue, algorithm reported as recurrence score.
 
Prosigna
There is a CPT MAAA administrative code specific to the Prosigna test:
 
0008M Oncology (breast), mRNA analysis of 58 genes using hybrid capture, on formalin-fixed paraffin-embedded (FFPE) tissue, prognostic algorithm reported as a risk score.
 
CPT 81520 can also be used to bill for the Prosigna test:
 
81520 Oncology (breast), mRNA gene expression profiling by hybrid capture of 58 genes (50 content and 8 housekeeping), utilizing formalin-fixed paraffin-embedded tissue, algorithm reported as a recurrence risk score
 
EndoPredict
There is a HCPCS S code for this testing:
 
S3854 Gene expression profiling panel for use in the management of breast cancer treatment.
There is not a specific CPT code for the EndoPredict test. The unlisted CPT codes  81599 or 81479 might be used to bill for this test.
 
Mammaprint
There is a specific CPT code for the Mammaprint test.
 
81521 Oncology (breast), mRNA, microarray gene expression profiling of 70 content genes and 465 housekeeping genes, utilizing fresh frozen or formalin-fixed paraffin-embedded tissue, algorithm reported as index related to risk of distant metastasis
 
Breast Cancer Index
Effective January 01, 2019 there is a specific code for the Breast Cancer Index:
 
81518 Oncology (breast), mRNA, gene expression profiling by real-time RT-PCR of 11 genes (7 content and 4 housekeeping), utilizing formalin fixed paraffin-embedded tissue, algorithms reported as percentage risk for metastatic recurrence and likelihood of benefit from extended endocrine therapy
 
Prior to January 2019, this test might have been billed using the unlisted CPT codes 81479 or 81599.

Policy/
Coverage:
Effective December 2018
 
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
OncotypeDX®, EndoPredict, the Breast Cancer Index and Prosigna
The use of the 21-gene reverse transcriptase polymerase chain reaction (RT-PCR) assay (ie, OncotypeDX®, EndoPredict, the Breast Cancer Index and Prosigna) to determine recurrence risk for deciding whether to undergo adjuvant chemotherapy meets member benefit certificate primary coverage criteria and is covered once per lifetime in women with primary, invasive breast cancer meeting all of the following characteristics:
 
    • unilateral tumor;
    • hormone receptor‒positive (ie, estrogen receptor‒positive or progesterone receptor‒positive);
    • human epidermal growth factor receptor 2‒negative;
    • tumor size 0.6 to 1 cm with moderate or poor differentiation or unfavorable features OR tumor size larger than 1 cm;
    • node-negative (lymph nodes with micrometastases [<2 mm in size] are considered node-negative for this policy statement);
    • who will be treated with adjuvant endocrine therapy (eg, tamoxifen, aromatase inhibitors);
    • when the test result aids the patient in deciding on chemotherapy (ie, when chemotherapy is a therapeutic option); AND
    • when ordered within 6 months after diagnosis, because the value of the test for making decisions regarding delayed chemotherapy is unknown.
 
NOTE: The 21-gene RT-PCR assay Oncotype DX should only be ordered on a tissue specimen obtained during surgical removal of the tumor and after subsequent pathology examination of the tumor has been completed and determined to meet the above criteria (ie, the test should not be ordered on a preliminary core biopsy). The test should be ordered in the context of a physician-patient discussion regarding risk preferences when the test result will aid in making decisions regarding chemotherapy.
 
MammaPrint
The use of the 70-gene signature assay (ie, MammaPrint) to determine recurrence risk for deciding whether to undergo adjuvant chemotherapy meets member benefit certificate primary coverage criteria and is covered once per lifetime in women with primary, invasive breast cancer meeting all of the following characteristics:
 
    • unilateral tumor;
    • hormone receptor‒positive (ie, estrogen receptor‒positive or progesterone receptor‒positive);
    • human epidermal growth factor receptor 2‒negative;
    • tumor size 0.6 to 1 cm with moderate or poor differentiation or unfavorable features OR tumor size larger than 1 cm;
    • node-negative (lymph nodes with micrometastases [<2 mm in size] are considered node-negative for this policy statement);
    • who will be treated with adjuvant endocrine therapy (eg, tamoxifen, aromatase inhibitors);
    • when the test result aids the patient in deciding on chemotherapy (ie, when chemotherapy is a therapeutic option); AND
    • when ordered within 6 months after diagnosis, because the value of the test for making decisions regarding delayed chemotherapy is unknown.
 
For patients who otherwise meet the above characteristics but who have multiple ipsilateral primary tumors, a specimen from the tumor with the most aggressive histologic characteristics should be submitted for testing. It is not necessary to conduct testing on each tumor; treatment is based on the most aggressive lesion.
 
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Oncotype DX), EndoPredict, the Breast Cancer Index, and Prosigna,
All other indications for the 21-gene RT-PCR assay (ie, Oncotype DX), EndoPredict, the Breast Cancer Index, and Prosigna, including determination of recurrence risk in invasive breast cancer patients with positive lymph nodes, patients with bilateral disease, or to consider the length of treatment with tamoxifen, do not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, all other indications for the 21-gene RT-PCR assay (ie, Oncotype DX), EndoPredict, the Breast Cancer Index, and Prosigna, including determination of recurrence risk in invasive breast cancer patients with positive lymph nodes, patients with bilateral disease, or to consider the length of treatment with tamoxifen, are considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Oncotype DX® Breast DCIS Score
Use of a subset of genes from the 21-gene RT-PCR assay for predicting recurrence risk in patients with noninvasive ductal carcinoma in situ (ie, Oncotype DX® Breast DCIS Score) to inform treatment planning after excisional surgery does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, Use of a subset of genes from the 21-gene RT-PCR assay for predicting recurrence risk in patients with noninvasive ductal carcinoma in situ (ie, Oncotype DX® Breast DCIS Score) to inform treatment planning after excisional surgery is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
MammaPrint
Use of 70-gene signature (MammaPrint) for any indication not meeting the above criteria does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, use of 70-gene signature (MammaPrint)  for any indication not meeting the above criteria is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
BluePrint
The use of BluePrint, alone or in conjunction with MammaPrint  does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, the use of BluePrint alone or in conjunction with MammaPrint is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Use in Men
Use of gene expression assays in men with breast cancer does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, use of gene expression assays in men with breast cancer is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective Prior to December 2018
 
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
OncotypeDX®, EndoPredict, the Breast Cancer Index and Prosigna
The use of the 21-gene reverse transcriptase polymerase chain reaction (RT-PCR) assay (ie, OncotypeDX®, EndoPredict, the Breast Cancer Index and Prosigna) to determine recurrence risk for deciding whether to undergo adjuvant chemotherapy meets member benefit certificate primary coverage criteria and is covered once per lifetime in women with primary, invasive breast cancer meeting all of the following characteristics:
 
    • unilateral tumor;
    • hormone receptor‒positive (ie, estrogen receptor‒positive or progesterone receptor‒positive);
    • human epidermal growth factor receptor 2‒negative;
    • tumor size 0.6 to 1 cm with moderate or poor differentiation or unfavorable features OR tumor size larger than 1 cm;
    • node-negative (lymph nodes with micrometastases [<2 mm in size] are considered node-negative for this policy statement);
    • who will be treated with adjuvant endocrine therapy (eg, tamoxifen, aromatase inhibitors);
    • when the test result aids the patient in deciding on chemotherapy (ie, when chemotherapy is a therapeutic option); AND
    • when ordered within 6 months after diagnosis, because the value of the test for making decisions regarding delayed chemotherapy is unknown.
 
NOTE: The 21-gene RT-PCR assay Oncotype DX should only be ordered on a tissue specimen obtained during surgical removal of the tumor and after subsequent pathology examination of the tumor has been completed and determined to meet the above criteria (ie, the test should not be ordered on a preliminary core biopsy). The test should be ordered in the context of a physician-patient discussion regarding risk preferences when the test result will aid in making decisions regarding chemotherapy.
 
For patients who otherwise meet the above characteristics but who have multiple ipsilateral primary tumors, a specimen from the tumor with the most aggressive histologic characteristics should be submitted for testing. It is not necessary to conduct testing on each tumor; treatment is based on the most aggressive lesion.
 
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Oncotype DX), EndoPredict, the Breast Cancer Index, and Prosigna,
All other indications for the 21-gene RT-PCR assay (ie, Oncotype DX), EndoPredict, the Breast Cancer Index, and Prosigna, including determination of recurrence risk in invasive breast cancer patients with positive lymph nodes, patients with bilateral disease, or to consider the length of treatment with tamoxifen, do not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, all other indications for the 21-gene RT-PCR assay (ie, Oncotype DX), EndoPredict, the Breast Cancer Index, and Prosigna, including determination of recurrence risk in invasive breast cancer patients with positive lymph nodes, patients with bilateral disease, or to consider the length of treatment with tamoxifen, are considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Oncotype DX® Breast DCIS Score
Use of a subset of genes from the 21-gene RT-PCR assay for predicting recurrence risk in patients with noninvasive ductal carcinoma in situ (ie, Oncotype DX® Breast DCIS Score) to inform treatment planning after excisional surgery does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, Use of a subset of genes from the 21-gene RT-PCR assay for predicting recurrence risk in patients with noninvasive ductal carcinoma in situ (ie, Oncotype DX® Breast DCIS Score) to inform treatment planning after excisional surgery is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
MammaPrint
Use of 70-gene signature (MammaPrint) for any indication does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, use of 70-gene signature (MammaPrint) for any indication is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
BluePrint
The use of BluePrint, alone or in conjunction with MammaPrint  does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, the use of BluePrint alone or in conjunction with MammaPrint is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Use in Men
Use of gene expression assays in men with breast cancer does not meet member benefit certificate primary coverage criteria.
 
For members with contracts without primary coverage criteria, use of gene expression assays in men with breast cancer is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
EFFECTIVE PRIOR TO NOVEMBER 2017
 
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
Based on the population for which Oncotype DX was developed and validated, the assay meets primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for deciding whether or not to undergo adjuvant chemotherapy in patients with newly diagnosed breast cancer meeting ALL of the following criteria:
    • stage I or II node negative tumors (this will exclude certain stage IIa and IIb node positive tumors). In cases where there are nodes with micrometastasis (i.e, pNI mi) the tumor will be considered node negative;
    • estrogen receptor-positive (ER-positive) or progesterone-receptor positive (PR-positive);  
    • who will be treated with hormone manipulation therapy;
    • human epidermal growth factor receptor 2 (HER2) negative;
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Repeat testing with Oncotype DX or testing of multiple tumor sites, does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes. For patients with multiple primary tumors, a specimen from the tumor with the most aggressive histological characteristics should be submitted for testing. For contracts without primary coverage criteria, repeat testing or testing of multiple tumor sites is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
The use of Oncotype DX testing in any circumstance not listed above as meeting primary coverage criteria, does not meet member benefit certificate primary coverage criteria that there by scientific evidence of effectiveness in improving health outcomes. This testing is currently being studied in clinical trials in other populations and for other indications. For contracts without primary coverage criteria, the use of assays of genetic expression in tumor tissue in any circumstance not listed above is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective May 2014 – September 2015
 
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
Based on the population for which Oncotype DX was developed and validated, the assay meets primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for deciding whether or not  to undergo adjuvant chemotherapy in patients with newly diagnosed breast cancer meeting ALL of the following criteria:
 
    • stage I or II breast cancer;
    • node-negative;
    • estrogen receptor-positive (ER-positive) or progesterone-receptor positive (PR-positive);
    • who will be treated with hormone manipulation therapy;
    • unilateral, non-fixed tumor
    • human epidermal growth factor receptor 2 (HER2) negative;
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Repeat testing with Oncotype DX or testing of multiple tumor sites, does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes. For patients with multiple primary tumors, a specimen from the tumor with the most aggressive histological characteristics should be submitted for testing.  For contracts without primary coverage criteria, repeat testing or testing of multiple tumor sites is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
The use of Oncotype DX testing in any circumstance not listed above as meeting primary coverage criteria, does not meet member benefit certificate primary coverage criteria that there by scientific evidence of effectiveness in improving health outcomes. This testing is currently being studied in clinical trials in other populations and for other indications. For contracts without primary coverage criteria, the use of assays of genetic expression in tumor tissue in any circumstance not listed above is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective July 2011 – April 2014
 Based on the population for which Oncotype DX was developed and validated, the assay meets primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes for deciding whether or not  to undergo adjuvant chemotherapy in patients with newly diagnosed breast cancer meeting ALL of the following criteria:
    • stage I or II breast cancer;
    • node-negative;
    • estrogen receptor-positive;
    • who will be treated with hormone manipulation therapy.
 
Repeat testing with Oncotype DX or testing of multiple tumor sites, does not meet primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes. For patients with multiple primary tumors, a specimen from the tumor with the most aggressive histological characteristics should be submitted for testing.  For contracts without primary coverage criteria, repeat testing or testing of multiple tumor sites is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Oncotype DX testing does not meet primary coverage criteria of effectiveness if the test result is low and the patient opts to proceed to adjuvant chemotherapy. For contracts without primary coverage criteria, the use of Oncotype DX testing if the test result is low and the patient opts to proceed to adjuvant chemotherapy is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
The use of Oncotype DX testing in any circumstance not listed above as meeting primary coverage criteria, does not meet member benefit certificate primary coverage criteria that there by scientific evidence of effectiveness in improving health outcomes. This testing is currently being studied in clinical trials in other populations and for other indications. For contracts without primary coverage criteria, the use of assays of genetic expression in tumor tissue in any circumstance not listed above is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective Prior to July 2011
Based on the population for which Oncotype DX was developed and validated, the assay meets primary coverage criteria for effectiveness and is covered for deciding whether or not  to undergo adjuvant chemotherapy in newly diagnosed patients whose breast cancer is stage I or II, node-negative, estrogen receptor-positive, and who will be treated with hormone manipulation therapy. Oncotype DX testing is not covered for repeat testing or for testing of multiple tumor sites.  Oncotype DX testing is not covered if the test result is low, but  the patient opts to proceed to adjuvant chemotherapy.
 
Clinical studies in other populations are currently under way.
 
The use of assays of genetic expression in tumor tissue in any circumstance not listed above is not covered based on benefit certificate primary coverage criteria that there by scientific evidence of effectiveness.
 
For contracts without primary coverage criteria, the use of assays of genetic expression in tumor tissue in any circumstance not listed above is considered investigational and is not covered.   Investigational services are an exclusion in the member certificate of coverage.

Rationale:
Due to the detail of the rationale, the complete document is not online. If you would like a hardcopy print, please email: codespecificinquiry@arkbluecross.com
 
 
Oncotype DX™ (21-Gene Assay)
The initial indication for the 21-gene expression profile (Oncotype DX®) was for patients newly diagnosed with stage 1 or 2, lymph node?negative, estrogen receptor (ER)?positive invasive breast cancer who would be treated with tamoxifen. Primary validation studies enrolled node-negative patients; this indication is reviewed first. More recently, Genomic Health has expanded indications for Oncotype DX® to include all stage 2 disease (tumor =2 cm with spread to axillary lymph nodes or 2-5 cm without lymph node involvement) and ductal carcinoma in situ (DCIS); these indications are reviewed separately.
 
Results from the Oncotype DX® 21-gene expression profile are combined into a recurrence score (RS). Based on a study of analytic validity, tissue sampling rather than technical performance of the assay is likely to be the greatest source of variability in results (Ahr, 2002).  The 21-gene expression profile was validated in studies using archived tumor samples from subsets of patients enrolled in already completed randomized controlled trials (RCTs) of early breast cancer treatment. Patients enrolled in the trial arms from which specimens were obtained had primary, unilateral breast cancer with no history of prior cancer and were treated with tamoxifen; tumors were ER-positive, most were human epidermal growth factor receptor 2 (HER2)?negative, and in the case of at least 1 trial (Paik, 2006), multifocal tumors were excluded.
 
Lymph Node?Negative Patients
Studies were published delineating the association between the 21-gene RS and recurrence risk (Paik, 2004a; Paik, 2004b; Bryant, 2005; Habel, 2006).  Results indicated strong, independent associations between the RS and distant disease recurrence or death from breast cancer (Paik, 2004a; Habel, 2006).  In secondary reclassification analyses of the Paik et al (2004b) data, Bryant et al (2005) (published in Tang et al. [2011]) classified individual patient risk levels by conventional risk classifiers, then reclassified patients by Oncotype DX®. Oncotype DX® added additional risk information to the conventional clinical classification of individual high-risk patients and identified a subset of patients who would otherwise be recommended for chemotherapy but who were actually at lower risk of recurrence (average 7%-9% risk at 10 years; upper 95% confidence interval [CI] limits, 11% to 15%). The analysis did not indicate significant erroneous reclassification given known outcomes. Thus, a woman who prefers to avoid the toxicity and inconvenience of chemotherapy and whose Oncotype DX® RS value shows that she is at very low risk of recurrence might reasonably decline chemotherapy. The lower the RS value, the greater the confidence the woman can have that chemotherapy will not provide net benefit; outcomes are improved by avoiding chemotherapy toxicity.
 
An additional study, in which samples from a RCT of ER-positive, node-negative breast cancer patients treated with tamoxifen versus tamoxifen plus chemotherapy were tested by Oncotype DX, provides supportive evidence. RS high-risk patients derived clear benefit from chemotherapy, whereas the average benefit for other patients was statistically not significant, although the confidence intervals were wide and included the possibility of a small benefit (Paik, 2006).
   
Papers related to the use of Oncotype DX® that have been published will be briefly mentioned.
Tzeng et al. (2010) examined how women receive and incorporate results of Oncotype DX® using mailed survey and chart review.  Approximately two-thirds of women believed they understood most or all of what they were told about their recurrence risk based on their test results; most who experienced test-related distress had intermediate or high estimated recurrence risks by RS result. The objective, recalled, and perceived recurrence risks by women in the study were surprisingly similar, and 95% agreed that the test gave them a better understanding of their treatment options and chances of success. However, approximately one-third of women believed they understood only a moderate amount or less during these discussions. The study was limited in generalizability in that participants were mostly white, well-educated women who had health insurance and came from urban areas.
 
Several studies have been published regarding the impact of RS results on chemotherapy recommendations by medical oncologists (Lo, 2010; Henry, 2009; Klang, 2010; Ademuyiwa, 2011; Prat, 2012; Kelly, 2010; Hassett, 2012; Joh, 2011; Carlson, 2013; Fried, 2014; Yamauchi, 2014; Bargallo et al, 2015; Frazier et al, 2015; Lee et al, 2015; McVeigh et al, 2014; Alvarado et al, 2015).   These studies generally reported that decisions changed for 25% to 40% of patients with physician knowledge of RS, most often from endocrine therapy plus chemotherapy to endocrine therapy alone. For example:
 
In a retrospective reclassification analysis, Joh et al (2011) found that addition of Oncotype DX® RS resulted in a 25% change in (after-the-fact) treatment recommendations, resulting in fewer patients projected to receive chemotherapy.
 
Hassett et al (2012) evaluated registry data from the National Comprehensive Cancer Network (NCCN) Breast Cancer Outcomes Database Project focusing on women diagnosed with hormone-receptor (HR)-positive stage 2 to 3 unilateral breast cancer during 2006 to 2008.  Compared with women who had Oncotype-determined intermediate-risk cancer, women who had Oncotype-determined high-risk cancers were more likely to receive chemotherapy (odds ratio [OR], 12.0; 95% CI, 6.7 to 21.3), and women with low-risk cancers were less likely to receive chemotherapy (OR=0.1; 95% CI, 0.1 to 0.2).
 
Carlson et al. (2013) conducted a systematic review of studies of Oncotype DX® used to inform actual adjuvant chemotherapy decisions in ER-positive, lymph node?negative patients with early stage breast cancer.  In 8 identified studies (total N=1437), Oncotype DX® RS changed the chemotherapy recommendation based on clinical-pathologic factors in 33% of patients. Compared with Oncotype DX® high risk patients, low risk patients were statistically more likely to follow Oncotype DX®-directed treatment (relative risk [RR], 1.07; 95% CI, 1.01 to 1.14).
 
Some view these studies as evidence of clinical utility because more patients avoid the toxicity of chemotherapy (Sparano, 2010); however, actual patient outcomes were not reported in these studies. Additionally, none of the studies formalized and described how information was delivered to patients, nor did they evaluate how patient preferences were incorporated into final treatment decisions.  Lo et al (2010) conducted a prospective multicenter study that examined both physician and patient treatment selection, as well as impact of RS results on patients’ anxiety, quality of life, and satisfaction with choice of treatment.  However, the study did not ensure that results were presented in a consistent format for all patients.
 
A prospective trial of Oncotype DX evaluating prognosis was published by Sparano et al (2015). Although the trial only evaluated outcomes at 5 years, it is among the few Simon category A studies available. In it, women with node-negative, estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-positive breast cancer were evaluated with Oncotype DX. Depending on the RS, women were assigned to endocrine therapy alone (low RS), randomized to adjuvant chemotherapy or no chemotherapy (middle category RS), or assigned to adjuvant chemotherapy (high RS). The published trial only reported the findings of the group at low risk of recurrence assigned to endocrine therapy. Of 10,253 subjects, 1629 patients had a RS of 0 to 10 and did not receive adjuvant chemotherapy. Note that the cutoff score of 10 is lower than that for other studies evaluating Oncotype DX and thus evaluates a group at lower predicted risk of distant recurrence than other Oncotype DX studies, which typically used a cutoff of 18. Consequently, only 15.9% of the study population was judged low risk, which is much lower than other studies. At 5 years, the distant recurrence rate was 0.7% (95% CI, 0.4% to 1.3%). Other outcomes at 5 years were rate of invasive disease-free survival (93.8%; 95% CI, 92.4% to 94.9%), rate of freedom from recurrence (98.7%; 95% CI, 97.9% to 99.2%), and overall survival (OS; 98%; 95% CI, 97.1% to 98.6%). Results from the randomized subjects in the trial are not available. The outcomes of these subjects, who were at higher predicted risk of recurrence, would demonstrate the risk of outcomes of subjects with higher scores and perhaps determine the magnitude of benefit of chemotherapy in these subjects.
 
Lymph Node-Positive Patients
Albain et al evaluated samples from the Southwest Oncology Group Trial 8814, in which randomized node-positive, ER-positive patients treated with tamoxifen for 5 years were compared to those treated with cyclophosphamide, doxorubicin, fluorouracil (CAF) chemotherapy followed by tamoxifen (CAF-T) for 5 years. Samples were available for determination of RS for 41% (n=148) and 39% (n=219) of the trial arms, respectively.
 
In this study, 10-year disease-free survival (DFS) and overall survival (OS) outcomes in the tamoxifen study arm differed by RS risk category (p=0.017 and 0.003, respectively), indicating that the RS is prognostic. When the 2 treatment arms were compared within RS risk categories, only patients in the high RS category significantly benefited from the addition of CAF to tamoxifen (for DFS, 42% [tamoxifen] vs. 55% [CAF-T], p=0.033; for OS, 51% [tamoxifen] vs. 68% [CAF-T], p=0.027), suggesting that RS is also predictive of response to chemotherapy.
 
A multivariable analysis of RS interaction with DFS, adjusted for number of positive nodes, was significant for the first 5 years of follow-up at p=0.029 and remained significant after adjusting for age, race, tumor size, progesterone receptor status, grade, p53, and HER2. However, the interaction was not significant (p=0.15) after adjusting for ER level (ER gene expression is a component of the 21-gene profile). Interaction results were similar for OS.
 
Dowsett et al (2010) included a separate evaluation of node-positive patients in their examination of the ATAC trial samples. Of 306 node-positive patients, 243 had 1-3 involved nodes, and 63 patients, 4 or more; these were not evaluated separately. Rates of distant recurrence at 9 years were 17% (95% CI: 12-24%), 28% (20-39%), and 49% (35-64%), respectively. It is not clear that the risk of distant recurrence in low-risk RS patients would be sufficiently low to forgo the choice of chemotherapy. The authors note that their study “did not directly evaluate the value of RS in predicting the benefit of chemotherapy.”
 
Goldstein et al (2008) evaluated samples from the Eastern Cooperative Oncology Group E2197 trial, which included patients with 0-3 positive lymph nodes and operable tumor greater than 1 cm in size. Patients were randomly assigned to doxorubicin plus cyclophosphamide or docetaxel plus 5 years of endocrine therapy; outcomes were not significantly different for the study arms. A case-control study of samples from this trial found that low-risk RS patients with 0-1 positive nodes had a recurrence risk of 3.3% (95% CI: 2.2-5%), and low-risk patients with 2-3 positive nodes had a recurrence risk of 7.9% (4.3-14.1%). RS was also a significant predictive of risk regardless of nodal status.
 
Brufsky (2014) reviewed the 3 studies above. The review was sponsored by Genomic Health, manufacturer of Oncotype DX®, and acknowledged the need for RxPONDER results (NCT01272037 clinical trial listed below) to confirm the findings of Albain et al (2010).
 
Chang et al (2008) reported that in women with locally advanced breast cancer treated with neoadjuvant docetaxel (n=97), a complete response was more likely in those with a high RS (p=0.008).  Gianni et al. (2005) studied 93 patients with locally advanced breast cancer who received neoadjuvant taxane chemotherapy, then post-surgery CMF treatment and tamoxifen (if ER-positive). The authors reported that pathological complete response was more likely in patients with high RS results than with low RS results (p<0.01).
 
One study surveyed oncologists ordering the 21-gene profile for lymph node-positive patients to determine the effect of assay results on treatment recommendations (Oratz, 2011).  Approximately half of oncologists who replied (16% response rate) changed their recommendations after receiving RS results, with 33% recommending endocrine therapy alone instead of endocrine plus chemotherapy. However, only medical oncologists who were already using the assay were surveyed, thus biasing the results. Finally, no outcomes were reported, providing no firm evidence of clinical utility.
 
Patients with DCIS
Ductal carcinoma in situ (DCIS) is breast cancer located in the lining of the milk ducts that has not yet invaded nearby tissues. It may progress to invasive cancer if untreated. The frequency of DCIS diagnosis in the U.S. has increased in tandem with the widespread use of screening mammography, accounting for about 20% of all newly diagnosed invasive plus noninvasive breast tumors. Recommended treatment is lumpectomy (mastectomy is also an option) with or without radiation treatment; post-surgical tamoxifen treatment is recommended for ER-positive DCIS, especially if excision alone is used. Because the overall rate of ipsilateral tumor recurrence (DCIS or invasive carcinoma) is about 25% at 10 years, it is believed many women are overtreated with radiation therapy. Thus, accurate prediction of recurrence risk may identify those women who may safely avoid radiation.
 
The Oncotype DX® DCIS test uses information from 12 of the 21 genes assayed in the standard Oncotype DX® test for early breast cancer to predict 10-year risk of local recurrence (DCIS or invasive carcinoma). The stated purpose is to help guide treatment decision-making in women with DCIS treated by local excision, with or without adjuvant tamoxifen therapy.
 
According to the Oncotype website, analyses from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 study (Paik, 2004a; Mamounas, 2010) and a case-control study by Habel et al. (2006) were used to select genes that predict the risk of recurrence independent of tamoxifen treatment and ER status. In a retrospective analysis of data and samples from patients in the prospective Eastern Cooperative Oncology Group E5194 study, the Oncotype DX® Score for DCIS was compared with 10-year recurrence risk in a subset of DCIS patients treated only with surgery and some with tamoxifen (n=327) (Solin, 2013).  Oncotype DX® DCIS Score was significantly associated with recurrence outcomes (HR=2.31; 95% CI, 1.15 to 4.49; p=0.02) whether or not patients were treated with tamoxifen. The standard Oncotype DX® Score for early breast cancer was not associated with DCIS recurrence outcomes. These studies addressed the development of the Oncotype DX® DCIS Score and clinical validity (association of the test result with recurrence outcomes). Whether women are better categorized as to their recurrence risk by Oncotype DX® DCIS Score compared with standard clinical indicators of risk has not been addressed.
 
In another retrospective analysis, Rakovitch et al (2015) evaluated 571 tumor specimens with negative margins from a convenience cohort of patients with DCIS treated by breast-conserving surgery (lumpectomy) alone. Patients were drawn from a registry of 5752 women in Ontario, Canada, who were diagnosed with DCIS between 1994 and 2003. Median follow-up of the 571 women was 9.6 years. There were 100 local recurrence events (18% prevalence); 43 were DCIS (8% prevalence), and 57 were invasive cancer (10% prevalence). Oncotype DX® DCIS score was significantly associated with local recurrence outcomes (HR=2.15; 95% CI, 1.43 to 3.22). Sixty-two percent of patients were classified as low-risk, 17% as intermediate risk, and 21% as high risk. Corresponding 10-year local recurrence estimates were 13% (95% CI, 10 to 17), 33% (95% CI, 24 to 45), and 28% (95% CI, 20 to 38), respectively. Corresponding 10-year estimates for DCIS recurrence (5% [95% CI, 3 to 9]; 14% [95% CI, 8 to 24]; 14% [95% CI: 9 to 22], respectively) and for invasive breast cancer recurrence (8% [95% CI, 6 to 12]; 21% [95% CI, 13 to 33]; 16% [95% CI, 9 to 25], respectively) were based on small numbers of events. It is unclear whether estimated recurrence risks for patients classified as low risk are low enough to forgo radiotherapy.
 
MammaPrint® (70-Gene Signature)
MammaPrint, also called the 70-gene signature, is a prognostic test for women with ER-positive or ER-negative, lymph node-negative invasive breast cancer. Twelve studies were reviewed and found insufficient evidence to determine whether MammaPrint is better than conventional risk assessment tools in predicting recurrence (van de Vijver, 2002; Drukker, 2014; van ‘t Veer, 2002; Espinosa, 2006; Glas, 2006; Haibe-Kains, 2008; Mook, 2010; Knauer/Cardoso, 2010; Knauer/Mook, 2010; Bueno-de-Mesquita, 2011)  Limited technical performance evaluation of the commercial version of the assay, using fresh frozen tumor samples, suggested good reproducibility. In 2014, Sapino et al published a validation study of MammaPrint® using FFPE tissue.(50) In a validation set of 221 tumor samples, concordance of FFPE and frozen tissue low- and high-risk classification was 91.5% (95% CI, 86.9 to 94.5). Concordance of repeat analyses of the same tumor was 96%, and interlaboratory reproducibility (ie, between labs in the Netherlands and in California) was 96%.
 
Studies of Primarily Node-Negative Disease
Because initial studies had been conducted on samples from younger patients (age younger than 61 years), Wittner et al. studied a cohort of 100 lymph node-negative patients with a median age of 62.5 years and a median follow-up of 11.3 years (Wittner, 2008). Twenty-seven low-risk patients by MammaPrint had distant metastasis-free survival at 10 years of 100%. However, the study was underpowered, and patients were heterogeneous in terms of ER-positivity (73%), endocrine therapy (25%), and chemotherapy (23%) making conclusions difficult. An additional small study of samples from women with lymph node-negative disease suggested that the 70-gene signature was an independent and significant predictor of distant metastases, but the small number of events limited conclusions (Bueno-de-Mesquita, 2007).
 
Original validation studies included patients with both node-negative and node-positive disease. Mook et al. (2010) retrospectively evaluated 148 consecutive, node-negative, post-menopausal patients, with primarily ER-positive tumors; only 18% received 2 years of adjuvant tamoxifen and none chemotherapy. For the 61% with good prognosis, 5-year distant metastasis-free survival (DMFS) probability was 93% (95% CI: 87-99%) whereas for those with poor prognosis DMFS was 72% (CI: 60-84%). The authors reported on concordance with Adjuvant! Online, but did not conduct a net reclassification analysis to determine additional impact of the MammaPrint signature on outcomes.
 
The Microarray Prognostics in Breast Cancer (RASTER) study was designed to assess feasibility of implementation and impact on treatment decisions of the MammaPrint 70-gene signature, as well as recurrence outcomes (Bueno-de-Mesquita, 2007). Five-year follow-up results of 427 node-negative, early-stage breast cancer patients who participated in the RASTER Study and had a 70-gene signature (MammaPrint), were published in 2013 (Drukker, 2013). Use of MammaPrint® to help direct postsurgery treatment decisions was compared with Adjuvant! Online.  Patients were aged 18 to 61 years and had a histologically-confirmed, unilateral, unifocal, primary operable, invasive adenocarcinoma of the breast. Median follow-up was 61.6 months. Eighty percent of patients were ER-positive. Discordant risk classifications occurred in 161 (38%) of 427 cases: 124 (29%) of 427 cases were discordant MammaPrint® low risk and Adjuvant! Online high risk, and 37 (9%) of 427 cases were discordant MammaPrint® high risk and Adjuvant! Online low risk. Use of MammaPrint® reduced the proportion of Adjuvant! Online high-risk patients by 20% (87/427). Five-year distant recurrence-free interval (DRFI) probabilities were excellent for patients who were clinically high risk but had a low-risk score with MammaPrint®, even in the absence of adjuvant systemic therapy.  Results suggested that MammaPrint® is a better prognostic classifier than standard clinical and pathologic classifiers. However, the study had several limitations. Patient numbers are low, and event numbers very low, making firm conclusions difficult. Actual treatment decisions were based on restrictive Dutch guidelines from 2004 and patients’ and doctors’ preferences. Additionally, Adjuvant! Online risk estimates were calibrated for 10-year outcomes, whereas RASTER outcomes were at 5 years. Because most clinical relapses in lymph node?negative, ER-positive breast cancers do not occur until 5 or even 10 years after diagnosis, with or without the use of adjuvant therapy, study data should be considered not yet mature.
 
Drukker et al (2014) reported additional comparisons between MammaPrint® and clinical risk classifiers in RASTER patients.  As measured by ROC analyses, MammaPrint® improved prognostic performance of all 6 clinical classifiers studied (Adjuvant! Online, Nottingham Prognostic Index [NPI], St. Gallen [2003], Dutch National guidelines [2004 and 2012], and PREDICT Plus, a clinicopathologic algorithm for estimating 5- and 10-year survival probabilities). However, area under the ROC curve for the best performing combination, MammaPrint® and PREDICT Plus, was only 0.662. Five-year distant recurrence-free survival estimates in 158 untreated patients were classified by MammaPrint® as low risk. Among MammaPrint® low-risk patients, 5-year survival estimates for patients classified as low or high risk by clinical risk classifiers were similar; only PREDICT Plus included the possibility of a less than 10% survival estimate for high-risk patients. Survival estimates for untreated patients classified as high risk by MammaPrint® were not reported, limiting full comparison of these risk stratifiers.
 
Studies of Mixed or Node-Positive Disease
In a study of node-positive disease, Mook et al. (2009) evaluated 241 patients with 1-3 positive nodes and primarily ER-positive, HER2-negative tumors treated variably. The 70-gene signature was a significant predictor of outcome. Reclassification analysis using Adjuvant! Online vs. MammaPrint showed significant additional discrimination of outcomes by the gene signature, but all were confounded by heterogeneous patient treatment. This study also updated the results of 106 patients with 1-3 positive nodes from the validation study, reporting 98% (95% CI: 94-100%) 10-year breast cancer-specific survival for good prognosis signatures vs. 64% (52-76%) for poor prognosis signatures; adjusted hazard ratio (HR): 3.63 (0.88–14.96), p=0.07. Based on these results, the ongoing MINDACT trial of MammaPrint was enlarged to include patients with 1-3 positive lymph nodes. Pilot phase results of the MINDACT trial were published in 2011 and showed successful implementation of the biomarker-stratified trial design and compliance with chemotherapy treatment according to the risk of recurrence according to MammaPrint (Rutgers, 2011).
 
The 2012 I-SPY trial evaluated 237 patients with locally advanced disease (node-positive) by correlating imaging and MammaPrint signatures with outcomes of pathologic complete response (pCR) and recurrence-free survival (RFS) (Esserman, 2012). Despite having locally advanced disease, patients with 70-gene low-risk profiles tended not to respond to chemotherapy and to have good short-term RFS.
 
Other studies comprise primarily small case series and pooled re-analyses of subgroups from previously published retrospective studies. A pooled analysis of 964 patients from previously reported studies with pT1 tumors (<2 cm) included 84% with ER-positive tumors, 68% with HER2-negative tumors (no HER2 information on 23%), 27% with node-positive disease, 68% given no adjuvant treatment, and the rest treated variably. In these patients, overall distant metastasis-free survival at 10 years was 87% (95% CI: 84-91%) for good prognosis patients and 72% (66-78%) for poor prognosis patients. The hazard ratio was 2.7 (95% CI: 1.88–3.88, p<0.001). Results are confounded by nodal status, HER2 status, and adjuvant therapy.
 
Kunz et al (2011) conducted a pooled re-analysis of a subgroup of patients aged 35-55 years from previously published studies. Patients were 75% ER-positive, 45% node-positive; 60% were untreated and the rest treated variably. The 70-gene signature categorized 39% of patients as good prognosis; for these patients, the 10-year distant metastasis-free survival was 88% (95% CI: 84–92%). Bighin et al. reported difficulties in that nearly 25% of samples from 21 prospectively studied patients were not assessable by the 70-gene signature and that results lead to a change in clinical decision in fewer than 20% of cases.
 
Retel et al (2010) reported a cost-effectiveness analysis that simulated the course of events in a hypothetical cohort of 1,000 patients aged 50 years with early, operable node-negative, ER-positive breast cancer, who are treated with 2.5 years of tamoxifen and 2.5 years of an aromatase inhibitor. The 70-gene signature was compared with Adjuvant! Online and St Gallen clinicopathologic classifiers.  While all three strategies were clinically equally effective, St Gallen was more costly and the 70-gene signature was most cost-effective when quality-adjusted life-years were taken into account.
 
Saghatchian et al (2013) evaluated MammaPrint® signatures of frozen tumor samples from patients who had 4 to 9 positive lymph nodes.  Approximately half of patients were ER-positive, half were HER2-positive, and half had received adjuvant radiotherapy or chemotherapy. Seventy (40%) of 173 samples were classified as low risk by MammaPrint®, and 103 (60%) were classified as high risk. With median follow-up of 8 years, 5-year breast cancer-specific survival in the low- and high-risk groups were 97% and 76%, respectively (log-rank test, p<0.01); 5-year distant metastasis-free survival was 87% and 63%, respectively (log-rank test, p=0.004). Survival estimates were reported without 95% CIs; it is therefore not possible to assess the degree of overlap between risk groups.
 
Ahn et al (2013) investigated the use of MammaPrint® to further risk-stratify 82 ER-negative patients (56% lymph node?negative) who had Oncotype DX® intermediate risk scores.  Although MammaPrint® risk classification was significantly associated with 10-year OS in multivariate analysis (log-rank test, p=0.013), this result was confounded by receipt of adjuvant chemotherapy, which also was significantly associated with OS (log-rank test, p=0.024).
 
To assess the impact of MammaPrint® on treatment decision making, Cusumano et al (2014) distributed clinical information about 194 patients in European countries to multidisciplinary teams in 2 other countries (e.g., data from the Netherlands was sent to Belgium and Italy) first without and then with MammaPrint® gene signatures.  Eighty-six percent of patients were ER-positive, 88% were HER2-negative, and 66% were lymph node?negative. With the addition of MammaPrint® signatures, treatment recommendations changed in 27% of patients, 22% from chemotherapy to no chemotherapy, and 35% from no chemotherapy to chemotherapy. In the subset of 453 ER-positive, HER2-negative patients, treatment advice changed in 32% of patients, with similar proportions changing from chemotherapy to no chemotherapy and vice versa. Other studies assessing the impacts of testing on treatment decision making also did not include survival or recurrence outcomes and are therefore considered uninformative for assessing clinical utility of MammaPrint® (Drukker et al, 2014; Exner et al, 2014).
 
Cardoso et al (2016) published results from the MINDACT trial which was a prospective trial evaluating MammaPrint, with some additional randomized components. Currently, only 5-year results are available. In this trial, women with early-stage breast cancer were evaluated with both MammaPrint and a clinical risk estimator. Women at low risk with both methods did not receive chemotherapy. Women with discordant risks were randomized to chemotherapy or to no chemotherapy. Women at high risk with both methods received chemotherapy. Although parts of the study are an RCT, the end point for this particular analysis was the distant recurrence rate among patients with high-risk clinical and low-risk genetic profile who did not receive chemotherapy. Investigators prespecified that the upper bound of the 95% confidence interval for distant recurrence was 8%, which they stated would be a sufficiently low risk that such patients could reasonably avoid chemotherapy. Declaring this to be the main end point implies a clinical strategy of using MammaPrint only in patients at high clinical risk, and deferring chemotherapy in those tested patients who have low genetic risk score. In this strategy, patients at low clinical risk are not tested with MammaPrint. Trial entry criteria included patients with either node-positive, estrogen receptor-positive, or HER2positive breast cancer. However, these patients constituted a minority of those in the study. The main results included these patients. The authors conducted supplemental analyses of various subgroups, including the subset who were node-negative, estrogen receptor-positive, or HER2-negative. To report results of patients most comparable to the other studies discussed herein, we abstracted the results of these supplemental analyses (see Table 6). The results are qualitatively similar to the published main results. In the main article, the principal objective of the study was met. The group at high clinical risk and low genomic risk who did not receive chemotherapy had a distant recurrence rate of 5.3% (95% CI, 3.8% to 7.5%). In the node-negative, estrogen receptor-positive, or HER2-negative subgroup analysis, this group has a distant recurrence rate of 4.5% (95% CI, 3.8% to 8.4%). In this subgroup, the confidence interval exceeded the prespecified bound of 8%. In the group with clinical low risk and high genomic risk, who were not considered in the main outcome, in both the main analysis and in the node-negative, estrogen receptor-positive, or HER2-negative subgroup, the results would indicate that the risk of distant recurrence is not low enough to avoid chemotherapy (main analysis distant recurrence, 5% [95% CI, 3% to 8.2%]; hazard ratio subgroup distant recurrence, 6.1% [95% CI, 3.9% to 9.4%]). In the testing strategy implied in this study, by not testing for genomic risk in the low clinical risk group, these patients would not be identified.  The groups randomized to chemotherapy showed no significant difference in 5-year distant recurrence, but the confidence intervals were wide and thus uninformative regarding whether chemotherapy is or is not beneficial in these patient groups. In the main study, the hazard ratio for chemotherapy in the high clinical risk/low genomic risk was 0.78 (95% CI, 0.5 to 1.21). The hazard ratio for chemotherapy in the low clinical risk/high genomic risk group was 1.17 (95% CI, 0.59 to 2.28).
 
Blueprint® and TargetPrint®
The BluePrint® molecular subtyping profile was developed using 200 breast cancer specimens that had concordant ER, PR, and HER2 protein levels by IHC and TargetPrint® mRNA readout (Krijgsman, 2012).  Using a 3-fold cross validation procedure, 80 genes thought to best discriminate the 3 molecular subtypes were identified. BluePrint® was confirmed on 4 independent validation cohorts (total N=784), which included patients from a consecutive series of patients seen at the Netherlands Cancer Institute and treated with adjuvant tamoxifen monotherapy (n=274), a group of patients from the RASTER trial (n=100), and 2 publicly available data sets (n=410). Additionally, in 133 patients treated with neoadjuvant chemotherapy, the molecular subtyping profile was tested as a predictor of chemotherapy response. The authors concluded that use of BluePrint® classification showed improved distribution of pCR among molecular subgroups compared with local pathology: 56% of patients had a pCR in the basal-type subgroup, 3% in the MammaPrint® low-risk, luminal-type subgroup, 11% in the MammaPrint® high-risk, luminal-type subgroup, and 50% in the HER2-type subgroup.  In a similar study, Whitworth et al (2014) reported reclassification of 94 (22%) of 426 patients with breast cancer who were classified by both IHC/fluorescence in situ hybridization (FISH) and BluePrint® and treated with neoadjuvant chemotherapy. Six percent of BluePrint® luminal-type patients achieved pCR compared with 10% of IHC/FISH hormone receptor?positive/HER2-negative patients; 53% of BluePrint® HER2-positive patients achieved pCR compared with 38% of IHC/FISH HER2-positive patients (the majority of HER2-positive patients by either method received trastuzumab); and 35% of BluePrint® basal-type patients achieved pCR compared with 37% of IHC/FISH “triple-negative” patients.
 
Nguyen et al (2012) undertook a comparison of molecular subtyping with Blueprint®, MammaPrint®, and TargetPrint® to locally assess clinical subtyping using IHC and fluorescence FISH.  The 3 gene expression assays were performed on fresh tumor tissue at Agendia Laboratories, blinded for pathologic and clinical data. IHC and FISH testing were performed according to local practice at 11 institutions in the U.S. and Europe. ER, PR, and HER2 assays were performed on 132 samples. Concordance between Blueprint® and IHC and FISH testing was 94% for both basal-type and luminal-type subgroups, and 95% for HER2-type. Concordance between Blueprint® and TargetPrint® was 98% for the basal-type, 96% for the luminal-type, and 97% for the HER2-type.
 
Viale et al (2014) reported concordance between TargetPrint® and IHC testing for ER, PR, and FISH for HER2 in the first 800 patients enrolled in the pilot phase of the MINDACT MammaPrint® trial.  For ER, positive and negative percent agreement between TargetPrint® and central testing were 98% and 96%, respectively; positive (PPV) and negative predictive value (NPV) were 99% and 87%, respectively. For PR, positive and negative percent agreement were 83% and 91%, respectively; PPV and NPV were 97% and 59%, respectively. For HER2, positive and negative percent agreement were 75% and 99%, respectively; PPV and NPV were 91% and 97%, respectively.
 
Grant et al (2015) compared HER2 testing results by IHC, FISH, and TargetPrint® in 127 tumor specimens from patients with early-stage breast cancer in South Africa. Tumor specimens were fresh frozen (32%) or FFPE (68%). Only specimens with IHC-positive results (n=23) underwent FISH testing, except for 1 IHC-negative specimen that had a positive TargetPrint® result, subsequently confirmed by reflex FISH. TargetPrint® improved HER2 testing compared with IHC/FISH in 4 (17%) of 24 cases that underwent both IHC and FISH testing. TargetPrint® performance in this study cannot be fully characterized in the absence of FISH testing of IHC-negative samples.
 
Breast Cancer Index SM (BCI)
Breast Cancer Index (BCI) is a simultaneous assessment of the HOXB13:IL17BR (H/I) ratio and the Molecular Grade Index (MGI). The H/I ratio indicates estrogen-mediated signaling; MGI assesses tumor grade by measuring the expression of 5 cell-cycle genes and provides prognostic information in ER-positive patients regardless of nodal status.  Review of five published studies (Goetz, 2006; Ma, 2006; Reid, 2005; Jansen, 2007; Jerevall, 2008) for the original component assays showed insufficient evidence to determine whether the H/I ratio is better than conventional risk assessment tools in predicting recurrence. Ten-year recurrence estimates of patients classified as low risk were 17% to 25%, likely too high for most patients and physicians to consider forgoing chemotherapy. Studies of the combination BCI are reviewed below.
 
Ma et al (2008) evaluated MGI along with H/I in 93 patients with lymph node–negative tumors who received adjuvant hormone therapy and found that each index modified the other’s predictive performance. High MGI was associated with significantly worse outcome only in patients with high H/I and vice versa. When the H/I Ratio and MGI were categorically combined into a single predictor, the estimates of 10-year distant metastasis-free survival were 98% (95% CI: 96-100%), 87% (77-99%), and 60% (47-78%) for the low, intermediate, and high-risk groups, respectively.
 
Jerevall et al (2011) combined the H/I Ratio and MGI into a continuous risk model using 314 ER-positive, node-negative postmenopausal patients from the tamoxifen-only arm of an RCT. The continuous model was also categorized, resulting in proportions of low-, intermediate-, and high-risk patients similar to those reported in the Ma et al. study. This continuous predictor was tested in patients from the no adjuvant treatment arm (n=274) of the same clinical trial, with estimates of rates of distant metastasis at 10 years in the low-, intermediate-, and high-risk groups of 8.3% (95% CI: 4.7–14.4), 22.9% (14.5–35.2), and 28.5% (17.9–43.6), respectively. The estimates of breast cancer-specific death were 5.1% (95% CI: 1.3–8.7), 19.8% (10.0–28.6), and 28.8% (15.3–40.2). An independent population of otherwise similar but tamoxifen-treated patients was not tested.
 
Jankowitz et al (2011) evaluated tumor samples from 265 ER-positive, lymph node (LN)-negative, tamoxifen-treated patients from a single academic institution’s cancer research registry. BCI categorized 55%, 21%, and 24% of patients as low, intermediate and high risk, respectively, for distant recurrence. The 10-year rates of distant recurrence were 6.6% (95% CI: 2.3-10.9%), 12.1% (95% CI: 2.7-21.5%), and 31.9% (95% CI: 19.9-43.9) and of breast cancer-specific mortality were 3.8%, 3.6% and 22.1% in low-, intermediate-, and high-risk groups, respectively. In a multivariate analysis, BCI was a significant predictor of distant recurrence and breast cancer-specific mortality. In a time-dependent (10-year) ROC curve analysis of recurrence risk, the addition of BCI to Adjuvant! Online risk prediction increased maximum predictive accuracy in all patients from 66% to 76% and in tamoxifen-only treated patients from 65% to 81%.
 
Sgroi et al (2013) examined 665 lymph node?negative, ER-positive, postmenopausal women receiving endocrine therapy but no chemotherapy in the ATAC trial.  For patients in the low- and intermediate-risk groups, 10-year distant recurrence risks were 5% and approximately 19%, respectively, regardless of endocrine treatment (tamoxifen, anastrozole, or both). In the high-risk group, recurrence risk was lowest (22%) for patients taking anastrozole only –22%, comparable to the intermediate-risk group, and highest for patients taking tamoxifen only (37%), although these groups were small (54 and 55 patients, respectively).
 
Zhang et al reported 2 sets of findings, one set deriving from a clinical trial and another from patient registries. Patients from the registry were only included if tissue samples were available. In all sets of findings, a BCI low-risk category classified more than half of the patients as low risk, and such patients had low risk of disease recurrence at 10 years. Sgroi et al found that the BCI-C and BCI-L showed a low risk of disease recurrence in the low-risk groups, with the confidence intervals not exceeding 10%. In the study by Zhang et al, patients in BCI low-risk categories also showed a low risk of distant disease recurrence, with confidence intervals not exceeding 10%.
 
Mammostrat™ Breast Cancer Test
Mammostrat is an immunohistochemistry (IHC) test intended to evaluate risk of breast cancer recurrence in postmenopausal, node-negative, ER-positive invasive breast cancer patients who will receive endocrine therapy and are considering adjuvant chemotherapy. The test employs 5 monoclonal antibodies to detect proteins biologically independent of each other and not involved in cell proliferation, hormone receptor status, or growth/differentiation, thus potentially allowing integration with clinically routine biomarkers. A proprietary diagnostic algorithm is used to calculate a risk score and to classify patients into high-, moderate-, or low-risk categories.
 
One published study described the development of the assay but provides no information on technical performance (analytic validity) (Welsh, 2011).  In a validation study in an independent cohort, a multivariable model predicted 50%, 70%, and 87% 5-year DFS for patients classified as high, moderate, and low prognostic risk, respectively, by Mammostrat® (p=0.0001).  An additional study of the same trial samples used for Oncotype DX validation (NSABP B-14 and B-20 trials) found that among patients with early, node-negative breast cancer treated only with tamoxifen, those stratified by Mammostrat into low-, moderate-, and high-risk groups had recurrence-free survival estimates of 85%, 85%, and 73%, respectively (Allred, 2009). Both low- and high-risk groups benefited significantly from chemotherapy treatment, but high-risk patients benefited to a greater degree. The moderate-risk group was not well-separated from the low-risk group and thus, moderate-risk results do not appear to provide clinically useful information. A test for an interaction between chemotherapy and the risk group stratification was not significant (p=0.13).
 
Bartlett et al (2010) used Mammostrat on 1,540 of 1,812 patient samples from a consecutive cohort for which minimum 9-year outcomes were available. The tested samples were from tamoxifen-treated patients; 568 of these were from node-negative patients treated only with tamoxifen and whose tumors were ER-positive. In the latter group, the distant recurrence rates at 10 years for low-, moderate-, and high-risk patients were 7.6% (95% CI: 4.6-10.5%), 16.3% (10.0-22.6%), and 20.9% (12.3-29.5%) respectively. In multivariable analysis, Mammostrat was not a significant predictor of recurrence-free survival in node-negative, ER-positive patients treated only with tamoxifen. However, when all patients (24% node-positive, 20% tumors >2.0 cm, 18% ER-negative, and 46% treated with chemotherapy) with complete Mammostrat data (n=1,300) were included in a multivariable analysis, Mammostrat scores were independent predictors of recurrence-free survival (p=0.0007). In exploratory analyses of various subpopulations (e.g. node-negative vs. node-positive, ER-negative), Mammostrat appeared to perform similarly in terms of identifying risk groups. However, numbers of subsets were small.
 
In 2014, Stephen et al assessed the ability of Mammostrat® and IHC4 to provide information on the risk of early (0-5 years) or late (5-10 years) distant recurrence. Tumor samples from 2 separate cohorts were analyzed: the Edinburgh Breast Conservation Series (n=1103) with median follow-up of 12.9 years, and the Tamoxifen Exemestane Adjuvant Multinational (TEAM) trial (n=3766) with median follow-up of 6.2 years. Patients had ER-positive disease and were treated with endocrine therapy without chemotherapy.
 
Within the first 5 years after diagnosis, HRs comparing Mammostrat® high- with Mammostrat® low-risk patients were statistically significant only in the TEAM cohort, which had greater risk for relapse (greater mean tumor size, larger proportion of higher grade tumors, and greater mean number of positive lymph nodes) compared with the Edinburgh cohort. Measures of calibration (slope) and discrimination (R2 statistic and index of discrimination) indicated that after 5 years (in the subset of patients who remained distant-recurrence free for at least 5 years, n=3920 [81%]), there was no evidence of an association between Mammostrat® scores and time to distant recurrence.
 
BreastOncPx™
The BreastOncPx test is an reverse transcriptase-polymerase chain reaction (RT-PCR) test performed on formalin-fixed, paraffin embedded tissue that measures the gene expression of 14 genes associated with key functions such as cell-cycle control, apoptosis, and DNA recombination and repair. The results are combined into a metastasis score, which is reported to be associated with the risk of distant metastases in patients who are node-negative and estrogen-receptor positive.
 
Tutt et al (2009) published information on the development and validation of the test; no information on analytic validity was provided. In order to develop a gene signature that was completely prognostic for distant recurrence and not confounded by treatment prediction, samples from untreated patients with early breast cancer were used. The training set (n=142) was derived from a cohort diagnosed with lymph node-negative, stage T1 and T2 breast cancer from 1975 to 1986; ER-positive samples from patients who had had no systemic treatment were selected for analysis. Fourteen genes were eventually selected as most prognostic of time-to-distant metastasis and were given equal weighting in a summary metastasis score (MS). Using a single cutoff, patients are separated into high- and low-risk groups.
 
The 14-gene signature was validated on ER-positive samples (n=279) from a separate cohort of patients diagnosed with lymph node-negative primary breast cancer between 1975 and 2001 (Tuft, 2008). The estimated rates of distant metastasis-free survival were 72% (95% CI: 64-78%) for high-risk patients and 96% (95% CI: 90-99%) for low-risk patients at 10 years’ follow up. Overall 10-year survival for high- and low-risk patients was 68% (95 CI: 61% to 75%) and 91% (95% CI: 84 to 95%), respectively. After adjusting for age, tumor size, and tumor grade in a Cox multivariate analysis, the HRs for distant metastasis-free survival for the high- versus low-risk group were 4.02 (95% CI: 1.91-8.44) and 1.97 (95% CI: 1.28 to 3.04) for distant metastasis-free survival and overall survival, respectively. However, this difference in risk between groups was not maintained when the analysis was restricted to patients with tumors larger than 2 cm (p value for interaction 0.012).
 
ROC analysis of the continuous MS for distant metastasis and for death at 10 years, compared to Adjuvant! resulted in slightly higher area under the curves (AUCs) for the MS in each case: 0.715 vs. 0.661 for distant metastases, and 0.693 vs. 0.655 for death. MS was not added to Adjuvant! and compared to Adjuvant! alone.
 
NexCourse Breast IHC4
NexCourse Breast IHC4 evaluates the protein expression of ER/PR, HER2, and Ki-67 to provide a combined recurrence risk score. The assay technology uses quantitative image analysis to measure immunofluorescent signals, with results that can be combined in an algorithm to generate the recurrence risk score. The use of quantitative immunofluorescence is said to increase sensitivity, be more reproducible, and allow specific measurement of tumor cells (Welsh, 2011; Allred, 2009).
 
Cuzick et al (2011) evaluated 1,125 ER-positive patients from the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial who did not receive adjuvant chemotherapy, already had the Oncotype DX Recurrence Score (RS) computed, and had adequate tissue for the IHC4 measurements.Of these, 793 were node-negative and 59 were HER2-positive (but were not treated with trastuzumab). A prognostic model that combined the 4 immunohistochemical markers was created (IHC4). In a model combining either IHC4 or Oncotype DX Rs with classical prognostic variables, the IHC4 score was found to be similar to the Oncotype DX RS, and little additional prognostic value was seen in the combined use of both scores. In a direct comparison the IHC4 score was modestly correlated with the Oncotype DX RS (r=0.72); the correlation was similar for node-negative patients (r=0.68). As an example, for a 1-2 cm, node-negative poorly differentiated tumor treated with anastrozole, 9-year distant recurrence at the 25th versus 75th percentiles for IHC4 and Oncotype DX were 7.6% versus 13.9% and 9.2% versus 13.4%, respectively. The IHC4 score was validated in a separate cohort of 786 ER-positive women, about half of whom received no endocrine treatment. The IHC4 score was significant for recurrence outcomes (HR: 4.1; 95% CI: 2.5-6.8).
 
Barton et al (2012) assessed the clinical utility of IHC4 plus clinicopathologic factors (IHC4 + C) by comparison with Adjuvant! Online and the Nottingham Prognostic Index (NPI). The study prospectively gathered clinicopathologic data for consecutively treated postmenopausal patients (n=101 evaluable) with hormone receptor-positive, HER2-negative, LN-negative or -positive with 1-2 nodes, resected early breast cancer. Of 59 patients classified as intermediate-risk group by the NPI, IHC4 reclassified 24 to low risk and 13 to high risk. IHC4 reclassified 13 of 32 Adjuvant! high-risk patients to intermediate risk, and 3 of 32 to low risk. In addition, 15 of 26 Adjuvant! intermediate-risk patients were reclassified to low risk. No Adjuvant! low-risk patients were reclassified high risk.
 
In the Stephen et al study (2014) described above (see Mammostrat®), HRs comparing the interquartile range of the continuous IHC4 score were statistically significant in both the Edinburgh and TEAM cohorts within the first 5 years after diagnosis. Measures of calibration and discrimination indicated that after 5 years, there was no evidence of an association between IHC4 scores and time to distant recurrence.
 
Prosigna™/PAM50 Breast Cancer Intrinsic Subtype Classifier
Initial development of the PAM50 breast cancer intrinsic classifier was reported in 2009 by Parker et al.  The authors developed a qRT-PCR test based on a panel of 50 genes to identify the breast cancer “intrinsic” subtypes luminal A, luminal B, HER2-enriched, and basal-like, and to generate risk-of-relapse scores in node-negative patients who had not had systemic treatment for their cancer. In an independent test set, the test using 3 categories of risk (low, intermediate, high) was significantly prognostic (log-rank test, p<0.001).
 
Nielsen et al (2010) compared the PAM50 classifier with standard clinicopathologic factors as represented by Adjuvant! Online and with models based on IHC for biomarkers of intrinsic subtypes.  The study used samples from patients diagnosed between 1986 and 1992 with ER-positive, node-negative or node-positive breast cancer at higher-risk (eg, with lymphovascular invasion), and treated with 5 years of tamoxifen but no adjuvant chemotherapy. In the node-negative population, Adjuvant! Online was inferior to all other biomarker models for predicting recurrence and disease-specific survival. A model including the PAM50 risk of recurrence score that also incorporated the influence of proliferation and tumor size identified patients with a greater than 95% chance of remaining alive and disease-free beyond 10 years. A slightly different gene expression model best fit the node-positive population but did not identify a population at sufficiently low-risk that adjuvant hormone therapy would likely be considered sufficient.
 
Because the cohort used to generate the models evaluated in this study was biased toward higher-risk early breast cancers, it is likely not generalizable. Nor did the authors clearly identify a final model for clinical use. Rather, the authors outlined potential additional studies.
 
Nielson et al (2014) assessed the analytical performance of Prosigna™ using the proprietary nCounter Analysis System (NanoString Technologies) at NanoString Technologies and 2 other laboratories.  Each tumor sample had been classified by a pathologist as invasive carcinoma (of any type), and all sample testing was blinded. Assay precision was assessed by testing 5 tumor RNA samples 36 times at the 3 labs. SD across labs was less than 1 ROR unit on the 0 to 100 ROR scale. Reproducibility was measured by testing 43 FFPE tumor samples in the 3 labs. Measured total standard deviation including all sources of variation (ie, tissue processing and RNA processing variability) was 2.9 ROR units, indicating that Prosigna™ measures a difference of 6.8 points between continuous 2 ROR scores with 95% confidence. Concordance across the 3 labs for risk categorization in node-negative patients ranged from 88% (95% CI, 73 to 96) to 93% (95% CI, 80 to 98), and in node-positive patients, from 90% (95% CI, 77 to 96) to 95% (95% CI, 84 to 99).
 
In a study that supported FDA clearance of Prosigna™, Gnant et al. (2014) evaluated tumor samples from 1047 lymph node?negative patients who participated in the Austrian Breast and Colorectal Cancer Study Group’s trial 8 (ABCSG-8); this represented 28% of the original trial sample.  ABCSG-8 randomized HR-positive, postmenopausal women with early stage breast cancer to 5 years of endocrine adjuvant therapy, either tamoxifen for 5 years or tamoxifen for 2 years followed by anastrozole for 3 years. Adjuvant or neoadjuvant chemotherapy was not allowed. In the Gnant et al (2014) study, both PAM50 subtype and Prosigna™ ROR class were associated with 10-year distant RFS, with CIs that overlapped slightly or not at all. Lower confidence limits for women in the luminal A and low-risk groups were around 94%, and upper confidence limits for luminal B and high-risk groups were approximately 90%. That is, the risk distinction seemed clinically useful.  Filipits et al. (2014) subsequently studied 919 patients who survived the first 5 years after treatment without recurrence.  Fifteen-year late-distant DRS (i.e., years 5-15) was 98%, 90%, and 86% in ROR low-, intermediate-, and high-risk groups, respectively.
 
Dowsett et al (2013) reported on groups from the ATAC trial stratified by subtype (luminal A or B) and by PAM50 ROR class, both with and without consideration of clinicopathologic factors.  Among 739 lymph node?negative patients, 10-year distant RFS was 94% in 529 luminal A patients and 75% in 176 luminal B patients and was comparable with low- and high-risk ROR groups with or without clinical factors: 95%, 85%, and 70% in low-, intermediate-, and high-risk groups, respectively. An ROC analysis in 649 lymph node?negative, HER2-negative patients showed that PAM50 plus clinical factors had greater discriminatory ability than either risk predictor alone. In this study, the commercial assay was performed on 46 of the PAM50 genes (ROR46). Because proliferation-associated genes are given special weighting to produce the Prosigna™ ROR score, it is unclear how closely ROR46 approximated the marketed test; the authors reported a correlation of 0.9989 between ROR50, which incorporated all PAM50 genes, and ROR46 risk classifications.
 
Two studies published in 2015 presented combined analyses of pretreatment FFPE tumor specimens from ABCSG-8 and ATAC trial monotherapy arms (TransATAC) (Sestak et al, 2015; Gnant et al, 2015).  Median follow-up was 10 years. Sestak et al (2015) examined the association between ROR score and late distant recurrence (5-10 years after diagnosis) in 2137 postmenopausal women (60% from ABCSG-8). Patients had hormone receptor?positive invasive breast cancer treated with only endocrine therapy (anastrozole or tamoxifen; no chemotherapy) for 5 years without recurrence. The majority of patients (74%) had node-negative disease (87% of patients with node-positive disease had 1 to 3 positive lymph nodes), and 92% were HER2-negative. ROR score was determined using a 46-gene subset of the PAM50 genes plus tumor size. Cut points differed from cut points used in the FDA-approved version of the test, designed to assess recurrence risk in the first 10 years after diagnosis (years 0-10). In this study, ROR score less than 26 identified patients with low risk of distant recurrence (<10% risk); ROR score 26 to 68 identified patients with intermediate risk (10%-20% risk); and ROR score greater than 68 identified patients with high risk (>20% risk) in both node-negative and node-positive patients. Fifty-five percent of women were categorized as low risk, 25% as intermediate risk, and 20% as high risk. Kaplan-Meier estimated risks for late distant recurrence in node-negative patients were 2.3% (95% CI, 1.3 to 3.5), 8.5% (95% CI, 5.9 to 12.1), and 9.3% (95% CI, 5.5 to 15.5), respectively. In node-positive patients, estimated risks were 3.3% (95% CI, 1.2 to 8.6), 7.8% (95% CI, 4.4 to 13.8), and 20.9% (95% CI, 16.1 to 26.9) in low-, intermediate-,and high-risk groups, respectively.
 
Gnant et al (2015) evaluated FFPE tissue specimens from 543 patients in the ABCSG-8 and ATAC trials who had 1 to 3 positive lymph nodes. The primary end point was distant recurrence-free survival, defined  as the interval from randomization until distant recurrence or death due to breast cancer.
 
Investigators developed a Clinical Treatment Score (CTS) that integrated nodal status, tumor size, histopathologic grade, patient age, and type of endocrine therapy received (anastrozole or tamoxifen) into a summary score.94 Risk classification by CTS was compared with and without ROR in subsets of patients with 1 positive lymph node (n=331) and with 2 to 3 positive lymph nodes (n=212). ROR cut points for defining risk groups differed from cut points used in the FDA-approved version of the test, which were defined by Gnant et al (2014) discussed above. Among patients with 1 positive node, 40% were categorized as low risk, 32% as intermediate risk, and 28% as high risk. Kaplan-Meier estimates for 10-year distant recurrence or death from breast cancer were 6.6% (95% CI, 3.3 to 12.8), 15.5% (95% CI, 9.5 to 25.0), and 25.5% (95% CI, 17.5 to 36.0), respectively. Because the upper bound of the 95% CI for patients categorized as low risk exceeded 10%, usefulness of these risk distinctions is uncertain. For patients with 2 to 3 positive nodes, low and intermediate risk groups were combined due to small numbers of patients and events in the low-risk group; 39% of patients were categorized as low/intermediate risk, and 61% were categorized as high risk. Ten-year distant RFS estimates were 12.5% (95% CI, 6.6 to 22.8) and 33.7% (95% CI, 25.5 to 43.8), respectively. When ROR, either as a continuous or a categorical variable, was added to CTS, prognostic information was improved (changes in likelihood ratios were statistically significant) compared with CTS alone for all nodal subgroups, including node-negative patients.
 
Liu et al (2015) assessed the prognostic and predictive value of PAM50–determined intrinsic subtypes and ROR scores in 1094 breast tumor samples from the National Cancer Institute of Canada’s MA.21 trial. MA.21 was an international, phase 3 trial that compared taxane and nontaxane chemotherapy in 2104 premenopausal or postmenopausal women 60 years of age or younger with node-positive or high- risk node-negative breast cancer. Patients were stratified by type of surgery (partial or total mastectomy), number of positive axillary lymph nodes, and ER status. Approximately 60% of patients were ER-positive, and approximately 60% received adjuvant endocrine therapy. PAM50 subtypes and ROR scores were determined using the nCounter Analysis system. Of all samples tested (52% of patients randomized), 3%, 18%, and 79% were classified as ROR low-, intermediate-, and high-risk, respectively. In multivariate analysis, ROR score on a continuous scale was statistically associated with RFS, but categorical ROR was associated with neither RFS nor survival by treatment group (ie, neither prognostic nor predictive).
Intrinsic subtypes were associated with RFS but were not predictive of treatment outcomes. The authors (Liu et al, 2015) stated:
 
“The characteristics of the study population of MA.21, which includes more high-risk breast cancer patients, are different from those used for the development and validation of the NanoString PAM50
ROR score classification. Thus, we suggest that researchers need to be cautious when applying the
ROR risk classification in different study populations. Compared with ROR score, intrinsic subtype is expected to be more reliable for predicting clinical outcome and response to therapies in different breast cancer populations as it is based on the fundamental biology of breast cancer, whereas the ROR algorithm was optimized against outcome in a specific population.”
 
Martin et al (2015) evaluated the impact of ROR on treatment decision making in patients with ER-positive, HER2-negative, node-negative breast cancer. Because survival or recurrence outcomes were not reported, the study is considered uninformative for assessing clinical utility of Prosigna®.
 
BreastPRS™
BreastPRS™ is a gene expression assay that analyzes 200 genes and was validated in a meta-analysis of publically available genomic datasets (Van Laar, 2011).  BreastPRS™ is a binary assay which stratifies patients into low- and high-risk groups (D’Alfonso, 2013).
 
D’Alfonso et al (2013) sought to translate a previously published validation study of BreastPRS, using fresh-frozen tissue, to FFPE tumor samples.  The authors compared BreastPRS™ to Oncotype DX® and correlated recurrence scores with clinicopathologic features. A linear relationship of BreastPRS™ prognostic scores between fresh-frozen and FFPE formats was observed. Using publically available whole genome profiles from a series of untreated ER-positive, node negative patients, investigators assessed the ability of BreastPRS™ to reclassify Oncotype DX® intermediate-risk patients into high- or low-risk categories with clinically significant differences in outcomes. BreastPRS™ prognosis scores were compared with Oncotype DX® recurrence scores in 246 patients with invasive breast carcinoma and known Oncotype DX® results. Using this series, a 120-gene Oncotype DX® approximation algorithm to predict Oncotype DX® risk groups was then applied to a series of untreated, ER-positive, node-negative patients from previously published studies with known clinical outcomes. Of 30 high-risk Oncotype DX® cases, 27 (90%) were classified as high-risk by BreastPRS™, and 95 low-risk Oncotype DX® cases (76%) were classified as low-risk by BreastPRS™. The correlation of recurrence score and risk group between Oncotype DX® and BreastPRS™ was statistically significant (p<0.001). Fifty-nine (23%) of 260 patients from 4 previously published studies were classified as intermediate-risk when the 120-gene Oncotype DX® approximation algorithm was applied. BreastPRS™ reclassified the 59 patients into binary risk groups (high vs low risk), with 23 (39%) patients classified as low risk and 36 (61%) as high risk (HR for a high-risk classification, 3.64; 95% CI, 1.40 to 9.50; p=0.029). Ten-year RFS was 90% in the low-risk group and 60% in the high-risk group. The authors concluded that BreastPRS™ prognosis score is comparable with Oncotype DX® recurrence score and can reclassify Oncotype DX® intermediate-risk patients into 2 groups with clinically significant differences in RFS.
 
EndoPredict™
Poremba et al (2014) assessed the impact of tissue handling on EndoPredict® (EP) test results. In analysis of 138 EndoPredict® assays, time to fixation up to 12 hours, fixation time up to 5 days, tumor cell content from 15% to 95%, and section storage time up to 12 months at 4°C or 20°C did not negatively impact results. This conclusion was based on correlations with test results from paired tissue samples with different storage conditions or different tumor cell content. It is unclear whether tests were conducted at more than 1 laboratory.
 
Bertucci et al (2014) evaluated 553 ER+/HER2-negative breast cancers treated with anthracycline-based neoadjuvant chemotherapy. Fifty-one percent of samples were classified as EndoPredict® low-risk with a  CR rate of 7%; 49% of samples were classified as EndoPredict® high-risk with a pCR rate of 17%. Estimated 5-year disease-free survival was 88% (95% CI, 81 to 95) in the EndoPredict® low-risk group and 73% (95% CI, 63 to 85) in the EndoPredict® high-risk group.
 

CPT/HCPCS:
0008MOncology (breast), mRNA analysis of 58 genes using hybrid capture, on formalin-fixed paraffin-embedded (FFPE) tissue, prognostic algorithm reported as a risk score
0045UOncology (breast ductal carcinoma in situ), mRNA, gene expression profiling by real-time RT-PCR of 12 genes (7 content and 5 housekeeping), utilizing formalin-fixed paraffin-embedded tissue, algorithm reported as recurrence score
81479Unlisted molecular pathology procedure
81518Oncology (breast), mRNA, gene expression profiling by real-time RT-PCR of 11 genes (7 content and 4 housekeeping), utilizing formalin-fixed paraffin-embedded tissue, algorithms reported as percentage risk for metastatic recurrence and likelihood of benefit from extended endocrine therapy
81519Oncology (breast), mRNA, gene expression profiling by real-time RT-PCR of 21 genes, utilizing formalin-fixed paraffin embedded tissue, algorithm reported as recurrence score
81520Oncology (breast), mRNA gene expression profiling by hybrid capture of 58 genes (50 content and 8 housekeeping), utilizing formalin-fixed paraffin-embedded tissue, algorithm reported as a recurrence risk score
81521Oncology (breast), mRNA, microarray gene expression profiling of 70 content genes and 465 housekeeping genes, utilizing fresh frozen or formalin-fixed paraffin-embedded tissue, algorithm reported as index related to risk of distant metastasis
81599Unlisted multianalyte assay with algorithmic analysis
S3854Gene expression profiling panel for use in the management of breast cancer treatment

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