PROSIGNA BREAST CANCER PROGNOSTIC GENE SIGNATURE ASSAY

• K Number: K141771
• Device Name: PROSIGNA BREAST CANCER PROGNOSTIC GENE SIGNATURE ASSAY
• Manufacturer: NANOSTRING TECHNOLOGIES INC.
• Date Cleared: 2014-11-07 ( 129 days)
• Product Code: NYI CLA
• Regulation Number: 866.6040

Intended Use

The Prosigna® Breast Cancer Prognostic Gene Signature Assay is an in vitro diagnostic assay which is performed on the NanoString nCounter® Dx Analysis System using FFPE breast tumor tissue previously diagnosed as invasive breast carcinoma. This qualitative assay utilizes gene expression data, weighted together with clinical variables to generate a risk category and numerical score, to assess a patient's risk of distant recurrence of disease. 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 postmenopausal 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

Device Description

The required components for the Prosigna™ Breast Cancer Prognostic Gene Signature Assay include the RNA Isolation kit (manufactured by Roche), Prosigna reagents (Reference Sample, CodeSet, Prep Pack, Cartridge(s) and Prep Plate) and the instruments that comprise the nCounter Dx Analysis System; the Prep Station and Digital Analyzer. The assay requires microdissection of tumor from FFPE biopsies, isolation of RNA using a Roche RNA isolation kit, transfer of RNA to PCR tubes for hybridization before placing onto the prep station. Two sets of probes specific to each of 58 RNAs are added to the hybridization reaction. These consist of biotin-labeled magnetic probes to purify the RNAs and capture them on the assay cartridge and fluorescent "barcode" probes to detect and quantify individual RNAs. The patient sample and probes are pipetted automatically into the Prosigna test cartridge by the Prep Station. The prep station uses magnetic bead capture and washing to remove excess RNA and un-hybridized probes. The isolated and hybridized RNA species are then bound via biotin on the capture probe randomly to streptavidin on the cartridge. The fluorescent molecules are then aligned on the cartridge by addition of an electric current. The cartridge is then transferred to the Digital Analyzer where the cartridge is scanned and digital analysis software is used to count the number of each RNA species present. The amount of each RNA is then put into a proprietary algorithm to produce a Prosigna score. The test output is a patient specific report which includes a Prosigna score (0-100) and risk category (low/intermediate/high).

Predicate Devices

K130010

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description mentions a "proprietary algorithm" but provides no indication that it utilizes AI or ML techniques. The performance studies focus on comparing kit configurations, not validating an AI/ML model.

Therapeutic

No.
The device is an in vitro diagnostic assay used to assess a patient's risk of distant recurrence of breast cancer, not to provide therapy.

Diagnostic

Yes

The document explicitly states that the Prosigna® Breast Cancer Prognostic Gene Signature Assay is an "in vitro diagnostic assay" and details its use in assessing a patient's risk of distant recurrence of disease, which aligns with the definition of a diagnostic device.

SaMD (Software as a Medical Device)

No

The device description explicitly lists hardware components required for the assay, including the RNA Isolation kit, Prosigna reagents, and the nCounter Dx Analysis System (Prep Station and Digital Analyzer). While software is used for analysis, it is an integral part of a larger hardware-based system.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states: "The Prosigna® Breast Cancer Prognostic Gene Signature Assay is an in vitro diagnostic assay..."

This confirms that the device is intended to be used outside of the body to examine specimens (in this case, FFPE breast tumor tissue) to provide information for the diagnosis or treatment of a disease.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Prosigna® Breast Cancer Prognostic Gene Signature Assay is an in vitro diagnostic assay which is performed on the NanoString nCounter® Dx Analysis System using FFPE breast tumor tissue previously diagnosed as invasive breast carcinoma. This qualitative assay utilizes gene expression data, weighted together with clinical variables to generate a risk category and numerical score, to assess a patient's risk of distant recurrence of disease.

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 postmenopausal 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

Product codes (comma separated list FDA assigned to the subject device)

NYI, Classifier, prognostic, recurrence risk assessment, RNA gene expression, breast cancer

Device Description

The required components for the Prosigna™ Breast Cancer Prognostic Gene Signature Assay include the RNA Isolation kit (manufactured by Roche), Prosigna reagents (Reference Sample, CodeSet, Prep Pack, Cartridge(s) and Prep Plate) and the instruments that comprise the nCounter Dx Analysis System; the Prep Station and Digital Analyzer.

The assay requires microdissection of tumor from FFPE biopsies, isolation of RNA using a Roche RNA isolation kit, transfer of RNA to PCR tubes for hybridization before placing onto the prep station. Two sets of probes specific to each of 58 RNAs are added to the hybridization reaction. These consist of biotin-labeled magnetic probes to purify the RNAs and capture them on the assay cartridge and fluorescent "barcode" probes to detect and quantify individual RNAs. The patient sample and probes are pipetted automatically into the Prosigna test cartridge by the Prep Station. The prep station uses magnetic bead capture and washing to remove excess RNA and un-hybridized probes. The isolated and hybridized RNA species are then bound via biotin on the capture probe randomly to streptavidin on the cartridge. The fluorescent molecules are then aligned on the cartridge by addition of an electric current. The cartridge is then transferred to the Digital Analyzer where the cartridge is scanned and digital analysis software is used to count the number of each RNA species present. The amount of each RNA is then put into a proprietary algorithm to produce a Prosigna score.

The test output is a patient specific report which includes a Prosigna score (0-100) and risk category (low/intermediate/high).

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

breast tumor tissue

Indicated Patient Age Range

post-menopausal women

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

• Analytical performance: See Predicate Device K130010 for Analytical Performance Data.
• Comparison studies:
  • Method comparison with predicate device: A comparison of Prosigna Scores was conducted using the two available configurations of the kit (4 sample and 10 sample kits). Forty samples of RNA previously extracted from FFPE breast tissue, covering a range of Prosigna scores were tested with both configurations and the data plotted using Deming regression. The data was linear over the range of the assay with no outliers between the two methods indicating that the 4-kit and 10-kit assays produce substantially equivalent results. No value deviated by more than 2 units from the average score when run using either configuration. Bland Altman Analysis showed that variations in data using the 4-kit versus the 10-kit configuration did not bias results and no changes in risk categorization occurred.
  • Matrix comparison: Not Applicable. FFPE tissue is the only matrix indicated for this device.
• Clinical studies: See Predicate Device K130010 for Clinical Performance Data

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Not Found

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K130010

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.6040 Gene expression profiling test system for breast cancer prognosis.

(a)
Identification. A gene expression profiling test system for breast cancer prognosis is a device that measures the ribonucleic acid (RNA) expression level of multiple genes and combines this information to yield a signature (pattern or classifier or index) to aid in prognosis of previously diagnosed breast cancer.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Gene Expression Profiling Test System for Breast Cancer Prognosis.” See § 866.1(e) for the availability of this guidance document.

Summary

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized eagle with three lines forming its body and wings. The eagle is positioned to the right of a circular text that reads "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA". The logo is black and white.

Public Health Service

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

May 28, 2015

Nanostring Technologies, Inc. Sylva Krizan, Ph.D. Regulatory Affairs Manager 530 Fairview Avenue North, Suite 2000 Seattle, WA 98109

Re: K141771

Trade/Device Name: Prosignatin Breast Cancer Prognostic Gene Signature Assay Regulation Number: 21 CFR §866.6040 Regulation Name: Gene expression profiling test system for breast cancer prognosis Regulatory Class: Class II Product Code: NYI Dated: June 30, 2014 Received: July 1, 2014

Dear Dr. Krizan:

This letter corrects our substantially equivalent letter of November 7, 2014.

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA), You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration. Isting of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801); medical device reporting of medical

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Page 2 - Sylva Krizan, Ph.D.

device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours.

Reena Philip -S

Reena Philip, Ph.D. Director Division of Molecular Genetics and Pathology Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure: Corrected Indications for Use Statement

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510(k) Number (if known): K141771

Device Name: Prosigna™ Breast Cancer Prognostic Gene Signature Assay

Indications for Use:

The Prosigna® Breast Cancer Prognostic Gene Signature Assay is an in vitro diagnostic assay which is performed on the NanoString nCounter® Dx Analysis System using FFPE breast tumor tissue previously diagnosed as invasive breast carcinoma. This qualitative assay utilizes gene expression data, weighted together with clinical variables to generate a risk category and numerical score, to assess a patient's risk of distant recurrence of disease.

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 postmenopausal 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

Prescription Use ___x (Part 21 CFR 801 Subpart

Over-The-Counter Use (21 CFR 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostics and Radiological Health (OIR)

Division Sign-Off Office of In Vitro Diagnostics and Radiological Health

510(k) K141771

Page 1 of 1_

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE

A. 510(k) Number:

K141771

B. Purpose for Submission:

Modification of device configuration and software

C. Measurand:

58 gene RNA expression profile

D. Type of Test:

Gene expression profile system based upon non-amplified RNA hybridization, visualization, and image analysis

E. Applicant:

NanoString Technologies

F. Proprietary and Established Names:

Prosigna™ Breast Cancer Prognostic Gene Signature Assay

G. Regulatory Information:

• 1. Regulation section:
     21 CFR §866.6040 Gene expression profiling test system for breast cancer prognosis
• 2. Classification:
     Class II
• 3. Product code:
     NYI, Classifier, prognostic, recurrence risk assessment, RNA gene expression, breast cancer
• 4. Panel:
     Immunology (82)

H. Intended Use:

• 1. Intended use(s):
     The Prosigna™ Breast Cancer Prognostic Gene Signature Assay is an in vitro diagnostic assay which is performed on the NanoString nCounter Dx Analysis System using FFPE

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breast tumor tissue previously diagnosed as invasive breast carcinoma. This qualitative assay utilizes gene expression data, weighted together with clinical variables to generate a risk category and numerical score, to assess a patient's risk of distant recurrence of disease.

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 post-menopausal 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.
• 2. Indication(s) for use:

Same as intended use

• 3. Special conditions for use statement(s): For Prescription Use Only
• 4. Special instrument requirements: nCounter Dx Analysis System

I. Device Description:

The required components for the Prosigna™ Breast Cancer Prognostic Gene Signature Assay include the RNA Isolation kit (manufactured by Roche), Prosigna reagents (Reference Sample, CodeSet, Prep Pack, Cartridge(s) and Prep Plate) and the instruments that comprise the nCounter Dx Analysis System; the Prep Station and Digital Analyzer.

The assay requires microdissection of tumor from FFPE biopsies, isolation of RNA using a Roche RNA isolation kit, transfer of RNA to PCR tubes for hybridization before placing onto the prep station. Two sets of probes specific to each of 58 RNAs are added to the hybridization reaction. These consist of biotin-labeled magnetic probes to purify the RNAs and capture them on the assay cartridge and fluorescent "barcode" probes to detect and quantify individual RNAs. The patient sample and probes are pipetted automatically into the Prosigna test cartridge by the Prep Station. The prep station uses magnetic bead capture and washing to remove excess RNA and un-hybridized probes. The isolated and hybridized RNA species are then bound via biotin on the capture probe randomly to streptavidin on the cartridge. The fluorescent molecules are then aligned on the cartridge by addition of an electric current. The cartridge is then transferred to the Digital Analyzer where the cartridge

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is scanned and digital analysis software is used to count the number of each RNA species present. The amount of each RNA is then put into a proprietary algorithm to produce a Prosigna score.

The test output is a patient specific report which includes a Prosigna score (0-100) and risk category (low/intermediate/high).

J. Substantial Equivalence Information:

• 1. Predicate device name(s):
     ProsignaTM Breast Cancer Prognostic Gene Signature Assay
• 2. Predicate 510(k) number(s): K130010
• 3. Comparison with predicate:

Table 1: Comparison with Predicate

Item	Device	Predicate
Intended Use	The Prosigna™ Breast Cancer
Prognostic Gene Signature
Assay is an in vitro diagnostic
assay which is performed on the
NanoString nCounter Dx
Analysis System using FFPE
breast tumor tissue previously
diagnosed as invasive breast
carcinoma. This qualitative
assay utilizes gene expression
data, weighted together with
clinical variables to generate a
risk category and numerical
score, to assess a patient's risk
of distant recurrence of disease.

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: | Same |

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| | A prognostic indicator for

1. distant recurrence-free
   survival at 10 years in
   post-menopausal 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. | |
   |------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|
   | Prescription Use | Yes | Yes |
   | Device Description | Prosigna™ Breast Cancer
   Prognostic Gene Signature
   Assay and nCounterDx
   Analysis Platform; all elements
   cleared by FDA as a distributed
   test and platform | Same |
   | Test Sample | FFPE breast tumor tissue | Same |
   | Extraction/amplification
   reagents/amplification
   procedures | No amplification required;
   procedure for processing FFPE
   tumor samples provided;
   includes RNA isolation,
   multiplex
   hybridization in solution,
   automated purification on a | Same |

·

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| liquid handling robot and
analysis on an automated

epifluorescence microscope
Differences
Item	Device	Predicate
Kit Stability/Shelf Life	8 months, based upon real time
stability data available from
original testing protocol	7 months, based on testing
completed at time of
clearance
Device Configuration	Reagents configured and
software programmed to
prepare 2 reference samples and
10 test sample -10 test
configuration
And
Reagents configured and
software programmed to
prepare 2 reference samples and
4 test sample -4 test
configuration	Reagents configured and
software programmed to
prepare 2 reference
samples and 10 test sample
-10 test configuration
Instrument Software	Version 1.3	Version 1.0
Instrument Functionality	FLEX configuration allows for
IVD or research use of the
device with different modes
separated through user
permissions and required log-
out and log-in when changing
modes	IVD use only

K. Standard/Guidance Document Referenced (if applicable):

Class II Special Controls Guidance Document: "Gene Expression Profiling Test System for Breast Cancer Prognosis, issued on May 9, 2007"

L. Test Principle:

Used together, the Prosigna™ Breast Cancer Prognostic Gene Signature Assay and nCounter Dx Analysis System are a nucleic acid hybridization and image analysis system based upon coded probes designed to detect the messenger RNA transcribed from 58 genes. The test input is purified RNA from FFPE breast tumor specimens which are acquired from surgical resection. The Prosigna assay uses gene-specific probe pairs that hybridize directly to the mRNA transcripts in solution. The nCounter Dx Analysis System delivers direct, multiplexed measurements of gene expression through digital readouts of the relative abundance of the mRNA transcripts.

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Specifications are included as part of the Prosigna Assay to control for sample quality, RNA quality, and process quality. The Prosigna assay utilizes prototypical expression profiles (centroids) for breast cancer. Patients RNA signatures are categorized into one of four centroids (not reported) based upon how close their gene expression pattern is to each of the centroids. The software algorithm produces a Prosigna score based on the similarity of the expression profile to each centroid, as well as the pathological tumor size and a proliferation score computed from a subset of genes. Three risk categories (low, intermediate and high) were defined based on a study with over 1007 patient samples associating Prosigna score with long-term outcome, defined by distance recurrence free survival at 10 years (DRFS) (Table 2).

Nodal Status	Prosigna Score Range	Risk Classification
Node-Negative	0-40	Low
	41-60	Intermediate
	61-100	High
Node-Positive
(1-3 nodes)	0-40	Low
	41-100	High

Table 2: Risk Classification Scoring Algorithm Using Prosigna Score

Performance Characteristics (if/when applicable): M.

• 1. Analytical performance:
     See Predicate Device K130010 for Analytical Performance Data.

2. Comparison studies:

• a. Method comparison with predicate device:
  A comparison of Prosigna Scores was conducted using the two available configurations of the kit (4 sample and 10 sample kits). Forty samples of RNA previously extracted from FFPE breast tissue, covering a range of Prosigna scores were tested with both configurations and the data plotted using Deming regression (Figure 1 below).

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Image /page/9/Figure/0 description: This image is a scatter plot comparing two samples, labeled '10 sample' on the y-axis and '4 sample' on the x-axis. The plot shows a strong positive correlation between the two samples, with data points clustered tightly around a straight line. A Deming regression equation is provided, stating '10 sample = 1.094 + 0.982 * 4 sample', indicating the linear relationship between the two samples.

The data was linear over the range of the assay with no outliers between the two methods indicating that the 4-kit and 10-kit assays produce substantially equivalent results.

No value deviated by more than 2 units from the average score when run using either configuration.

Image /page/9/Figure/3 description: The image is a Bland Altman plot showing the average ROR vs. ROR difference. The x-axis represents the average ROR, ranging from 0 to 100. The y-axis represents the ROR difference, ranging from -4 to 4. The plot includes horizontal lines indicating the upper acceptance limit, upper limit of agreement, average bias, lower limit of agreement, and lower acceptance limit.

Bland-Allman Plot; Average ROR vs. ROR Difference

Figure 3: Bland-Altman comparison plo: of ROR scores obtained from each kit configuration for the analysis data set (40 samples).

Bland Altman Analysis (above) showed that variations in data using the 4-kit versus the 10-kit configuration did not bias results and no changes in risk categorization occurred.

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• b. Matrix comparison:
  Not Applicable. FFPE tissue is the only matrix indicated for this device.

• 3. Clinical studies:
     See Predicate Device K130010 for Clinical Performance Data
• 4. Clinical cut-off:
     Same as assay cut-off
• 5. Expected values/Reference range:
     Risk assessment is reported as Low Risk, Intermediate Risk, or High Risk for node negative patients or as Low Risk or High Risk for Node positive patients (see Table 3).

Table 3: Risk Classification Scoring Algorithm Using Prosigna Score

Nodal Status	Prosigna Score Range	Risk Classification
Node-Negative	0-40	Low
	41-60	Intermediate
	61-100	High
Node-Positive (1-3 nodes)	0-40	Low
	41-100	High

N. Instrument Name:

The nCounter Dx Analysis System consists of a liquid handling robot Prep Station 5s and an epifluorescent scanner Digital Analyzer 5s.

O. System Descriptions:

• 1. Modes of Operation:
     Automated
• 2. Software:
     The Digital Analyzer measures and sorts multiple signals (reporter probes bound to mRNA transcript) from the clinical sample to establish an indicator (Prosigna score and risk category) to aid in determining patient prognosis. The Prep Station automates posthybridization sample processing while the Digital Analyzer includes signal reading, raw data storage, data acquisition software and software to process the detected targets (algorithm).

The Software is a Visual C++ web-based application developed by Nanostring.

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The current version of the Software is v1.3 and includes validation for the 4-test kit configuration in a dual IVD/RUO mode.

FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes __x ____________________________________________________________________________________________________________________________________________________________________

3. Specimen Identification:

Specimen identifying information is entered into a computer application manually.

• 4. Specimen Sampling and Handling:
     Samples are handled individually until RNA is extracted from FFPE tissue. RNA samples are then handled in batches of 6 or 12 on the instrument. These include 2 control samples and either 4 or 10 test samples, depending upon the configuration of reagent plates used.
• 5. Calibration:
     Installation, calibration and preventative maintenance of instrumentation are performed by the instrument manufacturer. No user calibration required.
• 6. Quality Control:
     Quality control includes testing of the mixed Reporter CodeSet and Capture ProbeSet for the following performance characteristics:

• Signal level of the geometric mean of housekeeping gene probes .

• . Signal levels of each of the 50 classifier genes

• Background level of the negative controls .

• Linearity of positive controls .

• . Probe cross-contamination

P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

None

Q. Proposed Labeling:

• 1. Labeling was modified to reflect
  • . the new software version,
  • to indicate the presence of the 4-test kit configuration .
  • . to include acceptable specifications for low volume spectrophotometers to be used with the assay
  • . to alert physicians to a trend in the data noted by post-hoc analysis whereby most distant recurrence appears to occur after 5 years.

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• 2. The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

R. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

S. Other Supportive Device and Instrument Information:

• 1. See predicate device K130010 for additional information concerning device performance.
• 2. Sponsor also included an update to the specifications for spectrophotometers that may be used to prepare RNA for the Prosigna device. The data revealed that at least two low volume spectrophotometers would provide acceptable quantitation of RNA for later use in the Prosigna assay.
• 3. Additional Clinical Claims
  • a. Sponsor originally sought additional claims, through changes to the patient Case Report Form in order to claim the Prosigna device could distinguish risk groups by risk of early recurrence versus late recurrence. That is to say that breast cancer that is expected to recur within a 10 year period, but that had a higher risk of arising within 5 years after surgery could be distinguished from those at higher risk of arising between 5 and 10 years after surgery. The Sponsor's calculations and pre-defined acceptance criteria only included those patients in the 5-10 year (late) recurrence group and did not take into account the early recurrence group.
  • b. Sponsor indicated that this information was provided to avoid confusion and were only included for "descriptive" purposes. FDA noted that the Sponsor provided precise calculations of data that had overlapping confidence intervals and was not statistically significant.
  • c. FDA noted to Sponsor that the information that they intended to convey was already present in the Kaplan-Meier survival curves presented on the case report forms and that a textual instructions to take care in the interpretation of survival was truthful and accurate without being false or misleading.
  • d. Sponsor removed charts of early v. late recurrence from the patient case report form.
  • e. Sponsor has altered the final version of labeling to remove a specific claim of early v. late recurrence.
  • Sponsor did include a text description of the 0-5 year early versus 5-10 year late ﺕ recurrence and presented the Kaplan-Meier curve for this population
• 4. Additional Stability Claims
  • a. Sponsor took a retrospective look at their stability data that was based upon reduction of signal of the control material geomean value once the stability study was completed.
  • b. Sponsor noted that if the lot release criteria for the control material were increased from a geomean value of 2289 to 3308, the stability data previously collected would support a stability claim of 11 months.
  • c. FDA noted that while this hypothesis was scientifically sound, it was not properly

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validated as the new lot release criteria was neither specified, nor in place when the stability testing began. As such, the stability remains established at 8 months, as was established via an add to file for the original 510(k): K130010..

• 5. Additional Labeling Changes
  • Sponsor validated a new software package that allowed the nCounter elements a. instrument run in two modes. The first is the cleared "IVD" mode. The other was variously described as "non-IVD" or "life Sciences" mode.
  • b. FDA indicated to Sponsor that the change in software was acceptable but that all labeling and software must indicate that the FLEX device configuration is acceptable for IVD use only when used in "IVD" mode.

T. Administrative Information:

• 1. Applicant Contact Information:
  • a. Name of applicant: Nanostring Technologies
  • b. Mailing address: 530 Fairview Avenue North, Suite 2000

Seattle, Washington 98109

• c. Phone #: [phone]
• d. Fax #: [phone]
• e. E-mail address (optional): skrizan(@nanostring.com
• Contact: Sylva Krizan, Ph.D. f.

2. Review Documentation:

July 2, 2014	Special 510(k) received. Lead Reviewer assigned-Kevin Lorick.
July 14, 2014	RTAA designation. 510(k) accepted for review.
July 14, 2104	Special 510(k) converted to a traditional 510(k) due to new claims
July 31, 2014	Request for Additional Information
September 8, 2014	Supplement S001, Response to Request for Additional Information received
September 26, 2014	FDA sent Email Request for clarification.
September 29, 2014	FDA sent 2nd Email Request for clarification.

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• October 1, 2014 Sponsor sent response to September 26, 2014 FDA email
• October 2, 2014 Email and phone calls to Sponsor to discuss possible disallowance of claims.
• October 2, 2014 Phone call from Sponsor. Sponsor agreed in principle to attempt to modify submission by COB on October 3, 2014.
• October 9, 2014 Teleconference with Sponsor to discuss timing and content of their response to FDA inquiries.
• October 24, 2014 Email from Sponsor with preliminary responses to FDA inquiries.
• October 30, 2014 Sponsor request to discuss proposed "Special Indications for Use"
• November 3, 2014 FDA call to Sponsor to seek modification of redlines version of PI.
• Sponsor email providing updates to labeling removing changes to November 4, 2014 Special Conditions for Use
• November 4, 2014 FDA Email to Sponsor indicating where the new version was deficient
• November 5, 2014 Email to Sponsor requesting updated data comparison figures and validation for support instrument specifications.
• November 5, 2014 Sponsor email request for additional feedback based upon the November 3. PI.
• Email to Sponsor with suggested PI changes. November 6, 2014
• November 7, 2014 Sponsor email with Final Labeling
• November 8, 2014 SE determination made
• April 17, 2015 Amendment received by DCC
• April 17, 2015 Lead Reviewer Kevin Lorick Assigned
• Internal Discussion about necessary documents and procedures May 1, 2015
• May 6, 2015 510(k) Amendment determined to be appropriate.
• Updated labeling, 510(k) Summary and Indications for Use May 22, 2015 uploaded to DocMan.

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3. Substantial Equivalence Discussion:

		Yes	No
1.	Same Indication Statement?	x		If YES = Go To 3
2.	Do Differences Alter The Effect Or Raise
New Issues of Safety Or Effectiveness?			If YES = Stop NSE
3.	Same Technological Characteristics?	x		If YES = Go To 5
4.	Could The New Characteristics Affect
Safety Or Effectiveness?			If YES = Go To 6
5.	Descriptive Characteristics Precise
Enough?	x		If NO = Go To 8
If YES = Stop SE
6.	New Types Of Safety Or Effectiveness
Questions?			If YES = Stop NSE
7.	Accepted Scientific Methods Exist?			If NO = Stop NSE
8.	Performance Data Available?			If NO = Request Data
9.	Data Demonstrate Equivalence?			Final Decision: SE

Note: See

http://eroom.fda.gov/eRoomReq/Files/CDRH3/CDRHPremarketNotification510kProgra m/0 4148/FLOWCHART%20DECISION%20TREE%20.DOC for Flowchart to assist in decision-making process. Please complete the following table and answer the corresponding questions. "Yes" responses to questions 2, 4, 6, and 9, and every "no" response requires an explanation.

• a. Explain how the new indication differs from the predicate device's indication: NA
• b. Explain why there is or is not a new effect or safety or effectiveness issue: NA
• c. Describe the new technological characteristics:

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NA

• d. Explain how new characteristics could or could not affect safety or effectiveness: NA
• e. Explain how descriptive characteristics are not precise enough: NA
• f. Explain new types of safety or effectiveness question(s) raised or why the question(s) are not new: N/A
• g. Explain why existing scientific methods cannot be used: NA
• h. Explain what performance data is needed: Demonstration that 4 and 10-test kit configurations produce identical results on the device with the v1.3 software are sufficient. No additional performance data is required.
• ...: Explain how the performance data demonstrates that the device is or is not substantially equivalent:

The changes to the devices are minor software and configuration changes. These effect only the sample preparation steps in terms of reagent positioning. No changes to reagents, hardware, analysis, scoring algorithm or significant changes to patient disease state result from the device modification. One additional piece of information is clarified in package insert but this was done for physician information purposes in a manner that may reduce confusion and aid patient safety.

• U. Reviewer Name and Signature:
  Kevin

Lorick -S

Digitally signed by Kevin Lorick -S
DN: c=US, o=U.S. Government,
ou=HHS, ou=FDA, ou=People,
cn=Kevin Lorick -S,
0.9.2342.19200300.100.1.1=200035
0803
Date: 2015.05.29 13:59:27-04'00'

Kevin Lorick, Ph.D. CDRH/OIR/PACB