Breast Cancer Prognostic Gene Signature Assay (Prosigna®), IVD

The Prosigna® breast cancer prognostic assay is an FDA 510(k)-cleared assay that provides a risk category and numerical score to assess a patient's risk of distant recurrence of disease at 10 years in postmenopausal women with node-negative (Stage I or II) or node-positive (Stage II), hormone receptor-positive (HR+) breast cancer. The Prosigna® assay measures gene expression levels of RNA extracted from formalin-fixed paraffin-embedded (FFPE) breast tumor tissue previously diagnosed as invasive breast carcinoma.

The Prosigna® breast cancer prognostic assay is an in vitro diagnostic assay that is performed on the IVD enabled nCounter® system using formalin-fixed paraffin-embedded (FFPE) breast tumor tissue previously diagnosed as invasive breast carcinoma. This qualitative assay utilizes gene expression profiling on the PAM50 gene set to generate an intrinsic molecular subtype* (luminal A, luminal B, HER2-enriched or basal-like) and a proliferation score* that are weighted together with tumor size to provide a risk of recurrence score (ROR), and then lymph node status to generate an associated risk category. 

The risk category and ROR score are intended to be used to assess a patient's risk of distant recurrence of disease. The intrinsic molecular subtype and proliferation score are not intended to be used for treatment decisions.The Prosigna® Breast Cancer Prognostic Gene Signature Assay is indicated in female breast cancer patients who have undergone surgery in conjunction with locoregional treatment consistent with standard of care, either as:

1. A prognostic indicator for distant recurrence-free survival at 10 years in postmenopausal women with Hormone Receptor-Positive (HR+), lymph node negative, Stage I or II breast cancer to be treated with adjuvant endocrine therapy alone, when used in conjunction with other clinicopathological factors.

2. A prognostic indicator for distant recurrence-free survival at 10 years in post-menopausal women with Hormone Receptor-Positive (HR+), lymph node-positive (1-3 positive nodes), Stage II breast cancer to be treated with adjuvant endocrine therapy alone, when used in conjunction with other clinicopathological factors. The device is not intended for patients with 4 or more positive nodes.

* The intrinsic molecular subtype and proliferation score are only reported as constituent components of the ROR. Their correspondence with histopathological subtyping is not established. 

Prosigna® is not intended for diagnosis, to predict or detect response to therapy, or to help select the optimal therapy for patients. Performance characteristics of the Prosigna® assay have been established for postmenopausal women with hormone receptor-positive early stage breast cancer treated with five years of adjuvant endocrine therapy. Performance with other treatment regimens or in other patient populations has not been established. The interpretation of Prosigna® assay results (Prosigna® Score, risk category) should be evaluated within the context of other clinicopathological factors, the patient's medical history, and any other laboratory test results.

1. The Prosigna® Assay has been optimized to use purified RNA extracted from formalin-fixed paraffin-embedded human breast tissue. Other types of specimens or fixatives have not been tested and should not be used.

2. The performance of the Prosigna® assay was validated using the procedures provided in the package insert only. Modifications to these procedures may alter the performance of the test.

3. Performance characteristics of the Prosigna® Assay have been established for postmenopausal women with hormone receptor-positive early stage breast cancer treated with five years of adjuvant endocrine therapy. Performance with other treatment regimens or in other patient populations has not been established.

4. If RNA of insufficient quality or quantity is added to the assay, then the Prosigna® Assay may be unable to give a valid result and instead will report an assay failure.

5. The interpretation of Prosigna® Assay results (ROR Score risk category) should be evaluated within the context of other clinicopathological factors, the patient's medical history and any other laboratory test results.

6. The performance of the Prosigna® Assay has been established with RNA meeting the specifications defined in reference to the procedure above. Performance with isolated RNA that does not meet these specifications has not been established.

7. Known interfering substances to the Prosigna® Assay include genomic DNA and non-tumor tissue (e.g., normal tissue). Please refer to general assay considerations before starting the procedure. The area of viable invasive carcinoma must be clearly identified by a pathologist prior to running the procedure. Additionally, each RNA sample must be treated with DNase. 

The Prosigna® assay is performed on RNA isolated from FFPE breast tumor tissue. It simultaneously measures the expression levels of 50 genes used in the PAM50 classification algorithm, eight housekeeping genes used for signal normalization, six positive controls, and eight negative controls in a single hybridization reaction, using nucleic acid probes designed specifically for those genes. Prosigna® is a registered trademark of NanoString Technologies Inc.

Prosigna® simultaneously measures the expression levels of 50 genes used in the PAM50 classification algorithm, eight housekeeping genes used for signal normalization, six positive controls and eight negative controls in a single hybridization reaction, using nucleic acid probes designed specifically to those genes. In addition, a reference sample is included in the Prosigna® kit consisting of in-vitro transcribed RNA targets for each of the 58 genes. The reference sample is tested with each batch of patient RNA samples to qualify the run and normalize the signal from each gene. 

The Prosigna® assay is performed on RNA isolated from FFPE breast tumor tissue. A pathologist examines a hematoxylin and eosin (H&E) stained slide and identifies (and marks) the area of invasive breast carcinoma suitable for the test. The pathologist also measures the tumor surface area, which determines the number of unstained slides required for the test, and the tumor cellularity to ensure the presence of sufficient tumor tissue for the test. A trained technologist macro-dissects the area on the unstained slides corresponding to the marked tumor area on the H&E slide and isolates RNA from the tissue. The isolated RNA is then tested on the nCounter® Dx Analysis System to provide test results including the risk of recurrence (ROR) score and risk category.