CRCDx RAS Mutation Detection Assay Kit

P220005 · Entrogen, Inc. · OWD · Sep 29, 2023 · Pathology

Device Facts

• Record ID: P220005
• Device Name: CRCDx RAS Mutation Detection Assay Kit
• Applicant: Entrogen, Inc.
• Product Code: OWD · Pathology
• Decision Date: Sep 29, 2023
• Decision: APPR
• Device Class: Class 3

Intended Use

The CRCdx RAS Mutation Detection Kit is a qualitative real-time PCR in vitro diagnostic test intended for the detection of 35 variants of KRAS and NRAS exon 2, 3, 4 somatic mutations in genomic DNA extracted from formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue samples. The test is intended as a companion diagnostic (CDx) to aid in the identification of colorectal cancer (CRC) patients who may benefit from treatment with Vectibix (panitumumab) based on a no mutation detected test result in accordance with the approved therapeutic product labeling.

Device Story

Real-time PCR assay detecting 35 KRAS/NRAS mutations in FFPE colorectal cancer tissue; utilizes Promega Maxwell instrument for DNA extraction; QuantStudio Dx (QSDx) instrument for PCR amplification/data collection; EntroGen PCR Analysis Software (EPAS) for result interpretation. Input: genomic DNA from FFPE tissue (min 20% tumor content). Operation: allele-specific primers/hydrolysis probes amplify mutant sequences and B2M internal control; fluorescence measured by QSDx; EPAS calculates Ct values to determine mutation status. Used in clinical laboratories by trained personnel. Output: qualitative mutation status (detected/not detected). Benefits: identifies patients eligible for Vectibix therapy; aids clinical decision-making by identifying RAS-wildtype patients.

Clinical Evidence

Retrospective external concordance study of 398 archived CRC FFPE specimens. Compared CRCdx results to Praxis Extended RAS Panel (comparator). PPA 99.6% (95% CI 97.5-99.99%), NPA 92.0% (95% CI 86.9-95.5%). Subset of 151 samples analyzed against two additional validated NGS assays showed PPA 96.3% and NPA 92.8%. Non-inferiority testing confirmed CRCdx is non-inferior to the comparator.

Technological Characteristics

Real-time PCR assay; 8 primer/probe mixes; FAM-labeled probes for mutant targets, VIC-labeled probes for B2M internal control. Uses Promega Maxwell CSC DNA FFPE Kit for extraction. QuantStudio Dx instrument for thermal cycling/fluorescence detection. EPAS software for analysis. 96-well format. Manual processing steps for DNA extraction.

Indications for Use

Indicated for CRC patients to identify those who may benefit from Vectibix (panitumumab) therapy based on absence of KRAS/NRAS exon 2, 3, 4 somatic mutations in FFPE tissue. No known contraindications.

Regulatory Classification

Identification

The somatic gene mutation detection system is an in vitro diagnostic nucleic acid amplification and real-time detection system for the identification of genetic mutations in DNA extracted from human tumor tissues.

Reference Devices

• Praxis Extended Ras Panel (Illumina)
• FoundationOne CDx (Foundation Medicine, Inc.)
• Cobas KRAS Mutation Test (Roche Molecular Systems, Inc.)
• therascreen KRAS RGQ PCR Kit (QIAGEN)
• xT CDx (Tempus)
• ONCO/Reveal Dx Lung and Colon Cancer Assay (O/RDx-LCCA) (Pillar Biosciences, Inc.)

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: Somatic Gene Mutation Detection System

Device Trade Name: CRCdx® RAS Mutation Detection Kit

Device Procode: OWD

Applicant’s Name and Address: EntroGen, Inc.
20950 Warner Center Ln
Woodland Hills, CA 91367
USA

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P220005

Date of FDA Notice of Approval: September 29, 2023

II. INDICATIONS FOR USE

The CRCdx RAS Mutation Detection Kit is a qualitative real-time PCR in vitro diagnostic test intended for the detection of 35 variants of KRAS and NRAS exon 2, 3, 4 somatic mutations in genomic DNA extracted from formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue samples. The test is intended as a companion diagnostic (CDx) to aid in the identification of colorectal cancer (CRC) patients who may benefit from treatment with Vectibix (panitumumab) based on a no mutation detected test result in accordance with the approved therapeutic product labeling.

The CRCdx RAS assay is performed on the QuantStudio Dx real-time PCR instrument (QSDx) for testing analyses and data collection. The data are analyzed by EntroGen’s PCR Analysis Software (EPAS) for result interpretation.

III. CONTRAINDICATIONS

There are no known contraindications.

IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the CRCdx RAS labeling.

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# V. DEVICE DESCRIPTION

The CRCdx RAS Mutation Detection Kit (CRCdx RAS) is a real-time PCR (polymerase chain reaction) assay designed to qualitatively detect 35 mutations in exons 2, 3, and 4 of KRAS and NRAS genes in DNA extracted from metastatic colorectal (CRC) tumor specimens. It utilizes the Promega Maxwell instrument and reagents for extracting DNA from FFPE tissue, Life Technologies QuantStudio Dx (QSDx) real-time PCR instrument for the PCR reaction and analysis, and EntroGen PCR Analysis Software (EPAS v2.0) for result interpretation.

The reagents in the kit include eight (8) vials of primer/probe mix designed to detect distinct groups of mutations (see kit content table below), five (5) vials of reaction mix (enzyme), and one (1) vial of positive control mix. The nuclease free water for the No Template Control (NTC) or negative control is required but not provided with the kit. The kit allows for 40 specimens to be tested. The kit contents are described in Table 1.

Table 1. Kit Content

| Tube / Reaction Mix | Number of Tubes | Detection |
| --- | --- | --- |
| Mutation Detection Reaction Mix (2X) | 5 | Not applicable |
| CRCdx Reaction 1 Mix | 1 | KRAS G12A, G12D, G12R, G12V, and G13D mutations and B2M internal control gene. |
| CRCdx Reaction 2 Mix | 1 | KRAS G12C and G12S mutations and B2M internal control gene. |
| CRCdx Reaction 3 Mix | 1 | KRAS Q61H, Q61H, Q61L, Q61R, A59E, A59G, and A59T mutations and B2M internal control gene |
| CRCdx Reaction 4 Mix | 1 | KRAS K117N(AAC), K117N(AAT), K117R, and K117E, and B2M internal control gene |
| CRCdx Reaction 5 Mix | 1 | KRAS A146T, A146P, and A146V, and B2M internal control gene |
| CRCdx Reaction 6 Mix | 1 | NRAS G12D, G12C, G12S, G13R, G13V and K117R, and B2M internal control gene |
| CRCdx Reaction 7 Mix | 1 | NRAS Q61H (CAC), Q61H (CAT), Q61L, Q61K, and Q61R mutations, and B2M internal control gene |
| CRCdx Reaction 8 Mix | 1 | NRAS A146T, NRAS A59D and A59T mutations and B2M internal control gene |
| CRCdx PC Mix§ | 1 | Not applicable |

§ The positive control (PC) mix contains a mixture of synthetic DNA sequences that correspond to each mutation detected by this kit and an internal control.

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# Principles of the Procedure

# Specimen Preparation

Genomic DNA (gDNA) is isolated from a minimum of eight (8) 5-μm CRC FFPE tissue sections containing at least $20\%$ tumor using the Promega Maxwell CSC DNA FFPE Kit. The amount of isolated DNA is quantified using fluorometric quantification, for a minimum of 192 ng gDNA to achieve the 24ng needed for each of the 8 reactions.

# PCR Amplification

The CRCdx RAS assay uses a mixture of allele specific (AS) primers and hydrolysis probes to amplify and detect the presence of mutations. Each reaction contains multiple mixtures of AS primers that are specific for the mutations targeted by the corresponding reaction. Additionally, an internal control (IC) primer and probe set is included in each reaction that amplifies an 82 base pair sequence of the beta-2-microglobulin (B2M) gene as loading control. The amplification of the mutant sequences is detected with FAM-labeled probes, while the amplification of the internal control gene is detected with a VIC-labeled probe to allow differentiation between the amplified mutant target(s) and the control gene. The listing of 35 mutations detected by the CRCdx RAS assay is presented in Table 2 below. The assay does not distinguish between different mutations detected within each reaction.

Table 2: Mutations detected by CRCdx RAS Mutation Detection Kit

| Gene | Exon | AA Change | Nucleotide Change | Cosmic ID (COSV) | Reaction Mix |
| --- | --- | --- | --- | --- | --- |
| KRAS | 2 | G12A | c.35G>C | 55497479 | 1 |
| | | G12D | c.35G>A | 55497369 | |
| | | G12R | c.34G>C | 55497582 | |
| | | G12V | c.35G>T | 55497419 | |
| | | G13D | c.38G>A | 55497388 | |
| | | G12C | c.34G>T | 55497469 | 2 |
| | | G12S | c.34G>A | 55497461 | |
| | 3 | Q61H | c.183A>T | 55499223 | 3 |
| | | Q61H | c.183A>C | 55498802 | |
| | | Q61L | c.182A>T | 55504296 | |
| | | Q61R | c.182A>G | 55498739 | |
| | | A59E | c.176C>A | 55568979 | |
| | | A59G | c.176C>G | 55604554 | |
| | | A59T | c.175G>A | 55499283 | |
| | 4 | K117N(AAC) | c.351A>C | 55545304 | 4 |
| | | K117N(AAT) | c.351A>T | 55504752 | |
| | | K117R | c.350A>G | 55937655 | |
| | | K117E | c.349A>G | 55716212 | |
| | | A146T | c.436G>A | 55501778 | 5 |
| | | A146P | c.436G>C | 55541748 | |

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| Gene | Exon | AA Change | Nucleotide Change | Cosmic ID (COSV) | Reaction Mix |
| --- | --- | --- | --- | --- | --- |
| | | A146V | c.437C>T | 55498939 | |
| NRAS | 2 | G12D | c.35G>A | 54736383 | 6 |
| | | G12C | c.34G>T | 54736487 | |
| | | G12S | c.34G>A | 54736621 | |
| | | G13R | c.37G>C | 54736550 | |
| | | G13V | c.38G>T | 54736480 | |
| | 4 | K117R | c.350A>G | 104682079 | |
| | 3 | Q61H (CAC) | c.183A>C | 54736320 | 7 |
| | | Q61H (CAT) | c.183A>T | 54736991 | |
| | | Q61L | c.182A>T | 54736624 | |
| | | Q61K | c.181C>A | 54736310 | |
| | | Q61R | c.182A>G | 54736340 | |
| | 3 and 4 | A146T | c.436G>A | 65731978 | 8 |
| | | A59D | c.176C>A | 54738004 | |
| | | A59T | c.175G>A | 54743124 | |

Each reaction contains AS primers specific for the mutations being targeted in that reaction. The AS primers anneal to their target sequences only when the mutation being targeted is present in the DNA sample. A derivative of Taq DNA polymerase extends the forward and reverse primer sequences in presence of magnesium ion and excess dNTPs, doubling the number of copies of the amplicons with each PCR cycle. Dual-labeled probes designed for common sequences of the amplicons within each reaction are hydrolyzed to separate the fluorescent dye (FAM) from the quencher (Black Hole Quencher or BHQ). Separation of the quencher and the dye allows the emission of fluorescent light at specific wavelengths. The increase in fluorescence due to probe hydrolysis upon amplification is measured by the QuantStudio Dx real-time PCR instrument at the end of the annealing/extension step of each PCR cycle and saved in the run data file (*.eds).

# Mutation Detection

Amplification of the internal control gene sequence occurs in every well that contains a sample, while amplification of KRAS and NRAS sequences occurs only in reactions where the mutation targeted by the reaction is present. The qualitative detection of mutations in each reaction depends on the detection of amplified product by the AS primers when sufficient amount of template DNA is present as measured by the internal control amplification. The fluorescence data of each reporter are recorded and converted to cycle threshold (Ct) values by the QSDx software based on prespecified parameters (i.e., baseline and threshold). The VIC and FAM Ct values of each reaction are then evaluated by the EntroGen PCR Analysis Software (EPAS), an accessory software to determine whether the Ct value meets the criteria for valid input and mutation status. Samples with VIC Ct value within a valid range and a FAM Ct value below the upper Ct cutoff are considered positive for one of the mutations in the group

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of mutations targeted by the reaction. Samples with VIC Ct value within a valid range and no FAM Ct value or FAM Ct value above the upper Ct cutoff are considered negative for mutations detected by the reaction. Samples with VIC Ct value outside of the valid range are considered invalid and the FAM Ct values are not evaluated.

## Instrumentation and Software

The CRCdx RAS assay uses Life Technologies, a real-time PCR instrument, which is a diagnostic device developed, manufactured, and distributed by Life Technologies. It comes with a 96-well block that enables testing of up to 10 samples with the CRCdx RAS assay. The instrument includes a touchscreen and a desktop computer that runs the QSDx software.

The QSDx real-time PCR instrument is controlled by the QSDx software which uses a test definition document (TDD), a locked file with predefined run, and analysis parameters to control the IVD test. The QSDx software maintains secure access to the instrument, enables assignment of specific software function privileges of users, records all actions completed by users, and signs data electronically.

The fluorescence data of each reporter are recorded and stored in real-time by the instrument using the QSDx software. Upon completion of the run, the analysis parameters defined in the TDD file are applied to the data to generate Ct values for each amplification curve detected by the instrument. The resulting Ct values are recorded in an analyzed data file (*.eds) and saved in a user-specified directory. The data file is transferred to a stand-alone workstation PC in a location appropriate for the individual lab. The data file is opened and analyzed by EPAS, EntroGen-developed software that provides interpretation and reporting of the results. EPAS first assesses run validity by evaluating the Ct values of positive and negative controls on the run. If the controls do not pass the validity evaluation, the run is flagged as 'Failed' and prompts the user to rerun the samples. If the controls meet the valid run criteria, EPAS evaluates the internal control (VIC) Ct values of each sample to ensure sample input validity followed by evaluation of target (FAM) Ct values of each reaction.

## Interpretation of Results

The EPAS software compares the FAM and VIC Ct values of positive and negative controls against Ct value ranges defined in a definition file to determine run validity. If EPAS determines that the run is valid, it assesses sample validity by evaluating the VIC Ct values of each sample. Specimens with a VIC Ct value within the assay's acceptable range are evaluated for presence of RAS mutations (Table 3). Each of the eight (8) reactions are evaluated independently for Ct values, then the reaction with the lowest Ct value that meets the criteria for a mutation positive status is reported as positive for the specified mutation group (Table 4). Specimens for which no FAM Ct value is detected in any of the 8 reactions, or the FAM Ct value does not meet the criteria for a mutation positive status are reported as "Negative". Specimens with an out-of-range VIC Ct value in any of the 8 reactions are flagged as 'Loading error' and users are instructed to repeat the testing with a modified specimen input.

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Table 3. CRCdx RAS Analytical Cutoffs for Mutation Amplicon Mutation Calls

| Reaction | Target (FAM) Ct | Internal Control (VIC) Ct |
| --- | --- | --- |
| KRAS 12/13 and KRAS 12C/S | < 38.0 | 24.0-26.5 |
| All other reactions | < 38.5 | 24.0-26.5 |

Table 4. Result Interpretation of the CRCdx RAS Mutation Detection Kit

| IC Status | EPAS Result | Interpretation |
| --- | --- | --- |
| Pass | Negative | No Mutation Detected |
| Pass | KRAS 12/13 | KRAS G12A, G12D, G12R, G12V, G13D |
| Pass | KRAS 12 | KRAS G12C, G12S |
| Pass | KRAS 59/61 | KRAS A59E, A59G, A59T, Q61H, Q61L, Q61R |
| Pass | KRAS 117 | KRAS K117N, K117R, K117E |
| Pass | KRAS 146 | KRAS A146T, A146P, A146V |
| Pass | NRAS 12/13/117 | NRAS G12D, G12C, G12S, G13R, G13V, K117R |
| Pass | NRAS 61 | NRAS Q61H, Q61L, Q61K, Q61R |
| Pass | NRAS 59/146 | NRAS A59D, A59T, A146T |
| Fail | Overloaded | VIC Ct values of all 8 reactions are below lower cutoff. |
| Fail | Underloaded | VIC Ct values of all 8 reactions are above upper cutoff. |
| Fail | Loading error | Results are invalid due to one or more reactions being over/under-loaded. Repeat the testing of specimen following instructions in the Package Insert. |

## Test Controls

### Positive Control

The CRCdx RAS assay includes one Positive Control (PC) mix to serve as an external run control. Runs with out-of-range FAM and VIC Ct values for the PC are deemed invalid ("Fail") and will not be deemed reportable. See Table 5 for the Ct ranges for valid results.

### No Template Control (No DNA Control)

The CRCdx RAS assay requires the inclusion of molecular grade water as No Template Control (NTC) on each run to identify potential contamination. The molecular grade water used for NTC is not provided with the kit. Each valid run must produce results with no Ct values detected within a range specified in the EPAS assay definition file. Runs that do not meet these criteria are considered invalid ("Fail") and will not be deemed reportable. Table 5 shows the Ct ranges for valid results.

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Table 5. Acceptance Pass/Fail Criteria for CRCdx Ras Runs

| Sample | Target (FAM) Ct | Internal Control (VIC) Ct |
| --- | --- | --- |
| Positive Control (PC) KRAS 12/13 and KRAS 12C/S | <38.0 | <38.5 |
| Positive Control (PC) All other reactions | <38.5 | <38.5 |
| Negative Control (NTC) All reactions | >38.5 or ND (Not Detected) | >36.0 or ND (Not Detected) |

ND - Not Detected. Ct value that is above the specified Ct cutoff or not present due to lack of amplification.

# VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are FDA-approved CDx alternatives for the detection of NRAS and KRAS biomarkers to direct the use of specific therapies using FFPE CRC specimens. The approved CDx tests are listed in Table 6 below.

Table 6. Alternative FDA-approved CDx assays for CDx biomarkers identified by CRCdx RAS

| Biomarker | Device | Company | Technology | Therapy |
| --- | --- | --- | --- | --- |
| NRAS Variants | Praxis Extended Ras Panel | Illumina | NGS | Vectibix (panitumumab) |
| | FoundationOne CDx | Foundation Medicine, Inc. | NGS | Vectibix (panitumumab) |
| KRAS Variants | Cobas KRAS Mutation Test | Roche Molecular Systems, Inc. | PCR | Erbitux (cetuximab) Vectibix (panitumumab) |
| | therascreen KRAS RGQ PCR Kit | QIAGEN | PCR | Erbitux (cetuximab) Vectibix (panitumumab) |
| | xT CDx | Tempus | NGS | Erbitux (cetuximab) Vectibix (panitumumab) |
| | FoundationOne CDx | Foundation Medicine, Inc. | NGS | Erbitux (cetuximab) Vectibix (panitumumab) |
| | Praxis Extended Ras Panel | Illumina | NGS | Erbitux (cetuximab) Vectibix (panitumumab) |

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| Biomarker | Device | Company | Technology | Therapy |
| --- | --- | --- | --- | --- |
| | ONCO/Reveal Dx Lung and Colon Cancer Assay (O/RDx-LCCA) | Pillar Biosciences, Inc. | NGS | Erbitux (cetuximab)
Vectibix (panitumumab) |

For additional details see the FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools.

## VII. MARKETING HISTORY

The CRCdx RAS Mutation Detection Kit has not been marketed in the United States or any foreign country.

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect CRCdx RAS test results, and consequently improper patient management decisions in treatment of colorectal cancer (CRC). A false positive test result may lead to withholding of Vectibix (panitumumab) treatment from a patient who might have benefited. A false negative test result may lead to a patient being treated with Vectibix which may result in occurrence of adverse side effects associated with the treatment without clinical benefit. There is also a risk of delayed results, which may lead to delay of treatment with indicated therapy.

No adverse side effects occurred in the studies conducted to support this PMA as all specimens used were acquired retrospective and archival CRC FFPE samples.

For the specific adverse events related to the approved therapeutics, please see approved FDA therapeutic product labeling.

## IX. SUMMARY OF NONCLINICAL STUDIES

### A. Laboratory Studies

To provide samples for evaluation of the 35 mutations identified by the CRCdx RAS, genomic DNA (gDNA) was extracted from clinical (FFPE) CRC tumor tissue specimens and cell lines (when necessary), using Maxwell CSC DNA FFPE Kit. Cell line gDNA or synthetic gene fragments were blended with wildtype gDNA from clinical specimens (CRC FFPE), for the very rare mutations, as applicable.

### 1. Accuracy – Comparison to Orthogonal Methods

The analytical accuracy of CRCdx RAS for the 35-variants detected by the test was evaluated using a multipronged approach, by comparing concordance results of

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CRCdx RAS with an FDA approved companion diagnostic (Praxis Extended RAS Panel), as well as two validated next generation sequencing (NGS) methods. A total of 662 archived CRC patient FFPE specimens were acquired from commercial biorepositories. Of the 662 specimens, 398 (60.1%) met the requirements to be included in the study and provided successful sequencing results by the comparator companion diagnostic (CCD) Panel. Of the 398 specimens, there were no invalid reactions reported by CRCdx RAS, and 151 specimens were also sequenced by 2 additional validated NGS-based assays to address the potential disparity in sensitivities between the different analytical platforms.

The resulting concordance between CRCdx and Praxis across all 398 samples are: Positive Percent Agreement (PPA) 99.6% with 95% Confidence Interval (CI) of 97.5% to 99.99%, and Negative Percent Agreement (NPA) 92.0%, with 95% CI 86.9% to 95.5% (Table 7).

Table 7. Positive and Negative Percent Agreement of Subject Level CRCdx Results - % Agreement with Praxis Reported Results

| | Praxis Extended RAS | | | PPA (95% CI) | NPA (95% CI) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Mutant | WT | | | Total |
| CRCdx | Mutant | 223 | 14 | 237 | 99.6% (97.5, 100.0) | 92.0% (86.8, 95.5) |
| | WT | 1 | 160 | 161 | | |
| | Total | 224 | 174 | 398 | | |

The mutation level accuracy of the CRCdx RAS compared to FDA approved CDx Praxis Extended Panel for each of the variants (as detected by orthogonal assay) was analyzed for all 398 specimens, and variant level comparison is shown in Table 8.

Table 8. Positive and Negative Percent Agreement KRAS and NRAS Accuracy Results by Variant as determined by Praxis Extended RAS Panel, calculated by CRCdx Reaction

| Gene | CRCdx Reaction | Amino Acid Change | Nucleotide Change | TP | FN | FP | TN | Total | PPA (95% CI*) | NPA (95% CI*) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| KRAS | 1 | G12A | c.35G>C | 10 | 0 | >1 | 387 | 398 | 100.0 (69.2-100.0) | 99.74 (98.5-100.0) |
| | | G12D | c.35G>A | 72 | 0 | >2 | 324 | 398 | 100.0 (95.0-100.0) | 99.39 (97.2-100.0) |
| | | G12R | c.34G>C | 2 | 0 | >0 | 396 | 398 | 100.0 (15.8-100.0) | 100.0 (99.1-100.0) |
| | | G12V | c.35G>T | 37 | 0 | >0 | 361 | 398 | 100.0 (90.5-100.0) | 100.0 (99.0-100.0) |
| | | G13D | c.38G>A | 34 | 0 | >1 | 363 | 398 | 100.0 (89.7-100.0) | 99.73 (98.1-100.0) |
| | | All Reaction 1 Mutations | | 155 | 0 | 11† | 232 | 398 | 100.0 (97.7-100.0) | 95.47 (91.4-97.12) |
| | 2 | G12C | c.34G>T | 11 | 0 | 0 | 387 | 398 | 100.0 (71.5-100.0) | 100.0 (99.1-100.0) |
| | | G12S | c.34G>A | 16 | 0 | 0 | 382 | 398 | 100.0 (79.4-100.0) | 100.0 (99.0-100.0) |
| | | All Reaction 2 Mutations | | 27 | 0 | 0 | 371 | 398 | 100.0 (87.2-100.0) | 100.0 (99.0-100.0) |
| | 3 | Q61H (CAT) | c.183A>T | 2 | 0 | 0 | 396 | 398 | 100.0 (15.8-100.0) | 100.0 (99.1-100.0) |
| | | Q61H (CAC) | c.183A>C | 3 | 0 | 0 | 395 | 398 | 100.0 (29.2-100.0) | 100.0 (99.1-100.0) |
| | | Q61L | c.182A>T | 1 | 0 | 0 | 397 | 398 | 100.0 (25.0-100.0) | 100.0 (99.1-100.0) |
| | | Q61R | c.182A>G | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| | | A59E | c.176C>A | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| | | A59G | c.176C>G | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |

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| Gene | CRCdx Reaction | Amino Acid Change | Nucleotide Change | TP | FN | FP | TN | Total | PPA (95% CI*) | NPA (95% CI*) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | A59T | c.175G>A | 0 | 0 | 1 | 397 | 398 | NE | 99.75 (98.5-100.0) |
| All Reaction 3 Mutations | 4 | 0 | 1 | 393 | 398 | 100.0 (39.8-100.0) | 99.75 (98.2-100.0) |
| 4 | K117N (AAC) | c.351A>C | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| K117N (AAT) | c.351A>T | 1 | 0 | 0 | 397 | 398 | 100.0 (25.0-100.0) | 100.0 (99.1-100.0) |
| K117R | c.350A>G | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| K117E | c.349A>G | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| All Reaction 4 Mutations | 1 | 0 | 0 | 397 | 398 | 100.0 (25.0-100.0) | 100.0 (99.1-100.0) |
| 5 | A146T | c.436G>A | 12 | 1 | 0 | 385 | 398 | 92.31 (53.9-100.0) | 100.0 (99.1-100.0) |
| A146P | c.436G>C | 2 | 0 | 0 | 396 | 398 | 100.0 (15.8-100.0) | 100.0 (99.1-100.0) |
| A146V | c.437C>T | 6 | 0 | 0 | 392 | 398 | 100.0 (54.1-100.0) | 100.0 (99.1-100.0) |
| All Reaction 5 Mutations | 20 | 1 | 0 | 377 | 398 | 95.24 (71.4,100.0) | 100.0 (99.0-100.0) |
| NRAS | 6 | G12D | c.35G>A | 4 | 0 | ≥0 | 394 | 398 | 100.0 (39.8-100.0) | 100.0 (99.1-100.0) |
| G12C | c.34G>T | 0 | 0 | ≥0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| G12S | c.34G>A | 0 | 0 | ≥0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| G13R | c.37G>C | 2 | 0 | ≥0 | 396 | 398 | 100.0 (15.8-100.0) | 100.0 (99.1-100.0) |
| G13V | c.38G>T | 0 | 0 | ≥0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| K117R | c.350A>G | 0 | 0 | ≥0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| All Reaction 6 Mutations | 6 | 0 | 2† | 390 | 398 | 100.0 (54.1-100.0) | 99.49 (97.7-100.0) |
| 7 | Q61H (CAC) | c.183A>C | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| Q61H (CAT) | c.183A>T | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| Q61L | c.182A>T | 3 | 0 | 0 | 395 | 398 | 100.0 (29.2-100.0) | 100.0 (99.1-100.0) |
| Q61K | c.181C>A | 3 | 0 | 0 | 395 | 398 | 100.0 (29.2-100.0) | 100.0 (99.1-100.0) |
| Q61R | c.182A>G | 2 | 0 | 0 | 396 | 398 | 100.0 (15.8-100.0) | 100.0 (99.1-100.0) |
| All Reaction 7 Mutations | 8 | 0 | 0 | 390 | 398 | 100.0 (63.1-100.0) | 100.0 (99.1-100.0) |
| 8 | A146T | c.436G>A | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| A59D | c.176C>A | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| A59T | c.175G>A | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| All Reaction 8 Mutations | 0 | 0 | 0 | 398 | 398 | NE | 100.0 (99.1-100.0) |
| Total | 223 | 1 | 14 | 13701 | 13930 | 99.55 (96.0-100.0) | 99.90 (99.8-100.0) |

NE = not estimable; NA=not applicable
†CRCdx does not differentiate between mutations in each reaction. False positives that were not resolved are accounted for in the 'All reaction' FP column
*For all agreements (PPA, NPA) not equal to 100%, bootstrap method was used to calculate the two-sided 95% confidence intervals (Cls); For all agreements equal to 100%, exact method was used to calculate the 95% CIs. Boot strap confidence intervals are obtained from bootstrap sampling of subjects (maintaining all mutation results nested within subject) with replacement 9,999 times

Of the 398 samples, accuracy was additionally assessed using a subset of 151 samples using two additional validated NGS-based assays. The 77 wildtype samples and 74 KRAS/NRAS mutant samples were screened by Praxis, CRCdx, and validated NGS for RAS mutations. To demonstrate that there was no bias between the evaluable and unevaluable sample populations with the external concordance study, the distribution of variants (determined by FDA approved comparator companion diagnostic, Praxis), stage, and the estimated tumor content were compared between the evaluable,

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unevaluable populations and the overall population. Table 9 shows the distribution of variants, Table 10 shows the distribution of stages, and Table 11 shows the distribution of estimated tumor content between the samples included or excluded from validated NGS analysis and the overall sample population.

Table 9. Distribution of Variants between Overall, Included, and Excluded from Validated NGS Sample Populations

| | Overall Samples n=398 | | Evaluable by NGS n=151 | | Unevaluable by NGS n=247 | |
| --- | --- | --- | --- | --- | --- | --- |
| | # of samples | % of total | # of samples | % of total | # of samples | % of total |
| KRAS G12A | 10 | 2.5% | 5 | 3.3% | 5 | 2.0% |
| KRAS G12D | 71 | 17.8% | 11 | 7.3% | 60 | 24.3% |
| KRAS G12R | 2 | 0.5% | 2 | 1.3% | 0 | 0.0% |
| KRAS G12V | 37 | 9.3% | 12 | 7.9% | 25 | 10.1% |
| KRAS G13D | 35 | 8.8% | 6 | 4.0% | 29 | 11.7% |
| KRAS G12C | 11 | 2.8% | 8 | 5.3% | 3 | 1.2% |
| KRAS G12S | 16 | 4.0% | 5 | 3.3% | 11 | 4.5% |
| KRAS Q61H (CAC) | 3 | 0.8% | 2 | 1.3% | 1 | 0.4% |
| KRAS Q61H (CAT) | 2 | 0.5% | 1 | 0.7% | 1 | 0.4% |
| KRAS Q61L | 1 | 0.3% | 1 | 0.7% | 0 | 0.0% |
| KRAS K117N (AAT) | 1 | 0.3% | 1 | 0.7% | 0 | 0.0% |
| KRAS A146P | 2 | 0.5% | 2 | 1.3% | 0 | 0.0% |
| KRAS A146T | 13 | 3.3% | 7 | 4.6% | 6 | 2.4% |
| KRAS A146V | 6 | 1.5% | 2 | 1.3% | 4 | 1.6% |
| NRAS G12D | 4 | 1.0% | 2 | 1.3% | 2 | 0.8% |
| NRAS G13R | 2 | 0.5% | 1 | 0.7% | 1 | 0.4% |
| NRAS Q61K | 3 | 0.8% | 3 | 2.0% | 0 | 0.0% |
| NRAS Q61L | 3 | 0.8% | 2 | 1.3% | 1 | 0.4% |
| NRAS Q61R | 2 | 0.5% | 1 | 0.7% | 1 | 0.4% |
| No mutation detected | 174 | 43.7% | 77 | 51.0% | 97 | 39.3% |

Table 10. Distribution of Stages between Overall, Included, and Excluded from Validated NGS Sample Populations

| | Overall Samples | | Samples included with NGS analysis | | Samples excluded with NGS analysis | |
| --- | --- | --- | --- | --- | --- | --- |
| Stag e | # of samples | % of total | # of samples | % of total | # of samples | % of total |
| I | 42 | 10.60% | 21 | 13.90% | 21 | 8.50% |
| II | 174 | 43.70% | 49 | 32.50% | 125 | 50.60% |
| III | 97 | 24.40% | 38 | 25.20% | 59 | 23.90% |
| IV | 15 | 3.80% | 6 | 4.00% | 9 | 3.60% |
| UNK | 70 | 17.60% | 37 | 24.50% | 33 | 13.40% |
| Total | 398 | | 151 | | 247 | |

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Table 11. Distribution of Estimated Tumor Content between Overall, Included, and Excluded from Validated NGS Sample Populations

| | Number of samples (n) | Average (%) | SD | Range (%) |
| --- | --- | --- | --- | --- |
| Overall | 398 | 65.6 | 18.5 | 18 - 100 |
| Included | 151 | 68.1 | 19.7 | 20 - 100 |
| Excluded | 247 | 64.0 | 17.6 | 18 - 100 |

A Wilcoxon Signed Rank Test was used to assess bias between the included and excluded sample populations compared to the overall sample population. The Wilcoxon Signed Rank Test is a non-parametric statistical test that informs if two populations have equal medians and distributions. The results, as shown in Table 12, demonstrate that there is no significant difference between the included and excluded cohorts and the overall population with respect to variant, estimated tumor content, and stage.

Table 12. Wilcoxon Signed Rank Test Results to Assess Population Bias during Validated NGS Sample Analysis

| Attribute | Included or Excluded from NGS Study | Sample Size | p-value | Pass or Fail |
| --- | --- | --- | --- | --- |
| Tumor proportion | Included | 151 | 0.91 | PASS |
| | Excluded | 247 | 0.91 | PASS |
| Stage Group | Included | 151 | 1.00 | PASS |
| | Excluded | 247 | 0.63 | PASS |
| Variant | Included | 151 | 0.16 | PASS |
| | Excluded | 247 | 0.16 | PASS |

Out of 151 samples tested using two validated NGS tests, the resulting concordance with aggregated results from two orthogonal NGS methods was: $\mathbf{PPA} = \mathbf{96.3\%}$ , with $95\%$ CI $89.7\%$ to $99.2\%$ , and $\mathbf{NPA} \times 92.8\%$ , with $95\%$ CI $83.9\%$ to $97.6\%$ (Table 13).

Table 13. CRCdx % Agreement with Validated NGS Aggregated Reported Results

| | Validated NGS | | | PPA (95% CI) | NPA (95% CI) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Mutant | WT | | | Total |
| CRCdx | Mutant | 79 | 5 | 84 | 96.3% (89.7, 99.2) | 92.8% (83.9, 97.6) |
| | WT | 3 | 64 | 67 | | |
| | Total | 82 | 69 | 151 | | |

Out of 118 samples tested using validated NGS test 1, the resulting concordance was: $\mathrm{PPA} = 100.0\%$ , with $95\%$ CI $94.0\%$ to $100.0\%$ , and NPA $93.1\%$ , with $95\%$ CI $83.3\%$ to $98.1\%$ .

Out of 37 samples tested using validated NGS test 2, the resulting concordance was: $\mathrm{PPA} = 96.0\%$ , with $95\%$ CI $79.7\%$ to $99.9\%$ , and NPA $91.7\%$ , with $95\%$ CI $61.5\%$ to $99.8\%$ .

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Of 398 samples tested in accuracy study, 383 (96.2%) results were concordant between CRCdx and Praxis, and 15 samples were discordant (14 false positives and 1 false negative). Using the data generated with the two validated NGS assays and comparative analysis with a composite method from all three orthogonal comparator methods used, EntroGen investigated 15 samples that had discordant results with Praxis, as shown in Table 14 below. Of the 15 discordant samples, three (3) did not have validated NGS assay results, and therefore no composite orthogonal result (one (1) sample with Praxis mutation positive was CRCDx positive but the mutation determined by Praxis is not one of the targeted mutations in CRCDx, therefore it was considered discordant; two (2) samples with Praxis mutation negative result were CRCDx positive), five (5) were classified as apparent negative status when assessed using composite orthogonal method, six (6) were classified as apparent positive status when assessed using composite orthogonal method, and one (1) sample with Praxis mutation positive but CRCDx mutation negative result was classified as apparent mutation positive. The agreement calculated using results for 151 samples tested with the composite orthogonal comparator is shown in the Table 15 below. When composite result of the orthogonal methods was used as the comparator, the resulting concordance was: $\mathrm{PPA} = 98.7\%$ , with $95\%$ CI $93.2\%$ to $100\%$ , and NPA $93.1\%$ , with $95\%$ CI $84.5\%$ to $97.7\%$ , as shown in the contingency table below.

Table 14. Results for Discordant Sample Results Between Praxis RAS and CRCdx Compared to Composite Orthogonal Result

| Praxis Call | CRCdx Call | Validated NGS Result | Status Classification |
| --- | --- | --- | --- |
| WT | KRAS 12/13 | Low %MAF KRAS G12D (< 5.0%) | Apparent Positive |
| WT | KRAS 12/13 | Low %MAF KRAS G12D (< 5.0%) | Apparent Positive |
| WT | KRAS 12/13 | Low %MAF KRAS G13D (< 5.0%) | Apparent Positive |
| WT | KRAS 12/13 | Low %MAF KRAS G12A (< 5.0%) | Apparent Positive |
| WT | KRAS 12/13 | High %MAF KRAS G13D (> 5.0%) | Apparent Positive |
| WT | KRAS 59/61 | High %MAF KRAS Q61L (> 5.0%) | Apparent Positive |
| KRAS A146T | WT | KRAS A146T 6.2% MAF | Apparent Positive |
| WT | KRAS 12/13 | No evidence of positive mutation | Apparent Negative |
| WT | KRAS 12/13 | No evidence of positive mutation | Apparent Negative |
| WT | KRAS 12/13 | No evidence of positive mutation | Apparent Negative |
| WT | KRAS 12/13 | No evidence of positive mutation | Apparent Negative |
| WT | NRAS 12/13/117 | No evidence of positive mutation | Apparent Negative |

Table 15. CRCdx % Agreement with Composite Aggregated Reported Results

| | Composite Orthogonal Method | | | PPA 95% CI | NPA 95% CI | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Mutant | WT | | | Total |
| CRCdx | Mutant | 78 | 5 | 83 | 98.7% | 93.1% |
| | WT | 1 | 67 | 68 | 93.2% to 100% | 84.5% to 97.7% |
| | Total | 79 | 72 | 151 | | |

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# 2. Analytical Sensitivity

a. Limit of Blank (LoB)

To determine the LoB and ensure that a KRAS or NRAS wildtype sample does not produce an analytical signal that might indicate a low concentrated mutant, five (5) CRC patient FFPE specimens that are wildtype at the KRAS and NRAS target codons were analyzed at the maximum allowable input as indicated by the internal control reference Ct of approximately 24. The specimens KRAS and NRAS genotypes were determined by Illumina's Praxis Extended RAS Panel. Three (3) reagent lots were used to evaluate each specimen (n=10, or 20 per specimen per lot) across eight (8) reactions. The results of the study (N=1600) showed an overall negative (wildtype at the KRAS and NRAS loci) call concordance rate of 99.6% (95% CI = 99.1 - 99.8). Seven (7) replicates from one (1) specimen were incorrectly called positive in Reaction 1 (KRAS G12/G13) with Ct values at 37.87, 37.46, 37.94, 37.69, 37.80, 37.89, 37.89.

b. Limit of detection (LoD)

The CRCdx RAS Mutation Detection Kit evaluates DNA input concentration using an internal control reaction Ct to indicate a sufficient load (internal control VIC Ct = 24 - 26.5). LoD was evaluated at the minimum amplifiable copy input (as indicated by a VIC Ct of 26.5). The LoD study was performed in 3 parts: (i) Primary dilution series, (ii) Secondary dilution series, and (iii) Clinical sample confirmation. The LoD was estimated as the lowest mutation allele frequency (MAF) where concordance was ≥95% for cell line or surrogate synthetic specimens and ≥95% concordance using clinical CRC FFPE DNA, when available, to confirm the estimated LoD. The primary dilution series included both cell lines and synthetic samples to represent 21 and 14 targets, respectively. All samples were diluted to the target MAF in a background of wildtype CRC FFPE DNA. Twenty replicates were performed at each level across two CRCdx device lots. The percentage of the replicates that were called "Positive" per acceptance criteria was reported for each target dilution as " correct call."

Clinical sample confirmation performed once the LoD was estimated by the dilution series for all thirty-five (35) targets, on a representative archived CRC clinical FFPE sample for each target per availability. From a pool of &gt;600 CRC FFPE blocks, specimens representing sixteen (16) targets met the requirements for the LoD study. The MAF values were determined for the extracted analyte using an NGS method for RAS mutations and blended with WT DNA from CRC FFPE tissue to the determined LoD and analyzed at 20 replicates across two CRCdx device lots. The dilution (% mutation) at which ≥95% of the calls were positive are shown in Table 16 below:

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Table 16. Limit of Detection for CRCdx RAS Mutation Detection Kit Targets

| Mutation Target | Reported LoD (% mutation) |
| --- | --- |
| KRAS G12A | 3.13 |
| KRAS G12D | 3.13 |
| KRAS G12R | 3.13 |
| KRAS G12V | 6.25 |
| KRAS G13D | 3.13 |
| KRAS G12C | 6.25 |
| KRAS G12S | 3.13 |
| KRAS Q61H CAT | 6.25 |
| KRAS Q61H CAC | 3.13 |
| KRAS Q61L | 3.13 |
| KRAS Q61R | 3.13 |
| KRAS A59E | 3.13 |
| KRAS A59G | 3.13 |
| KRAS A59T | 6.25 |
| KRAS K117N AAC | 6.25 |
| KRAS K117N AAT | 3.13 |
| KRAS K117R | 3.13 |
| KRAS K117E | 3.13 |
| KRAS A146T | 12.5 |
| KRAS A146P | 3.13 |
| KRAS A146V | 7.81 |
| NRAS G12D | 3.13 |
| NRAS G12C | 6.25 |
| NRAS G12S | 12.5 |
| NRAS G13R | 3.13 |
| NRAS G13V | 3.13 |
| NRAS K117R | 6.25 |
| NRAS Q61H CAC | 3.13 |
| NRAS Q61H CAT | 3.13 |
| NRAS Q61L | 3.13 |
| NRAS Q61K | 3.13 |
| NRAS Q61R | 3.13 |
| NRAS A146T | 3.13 |
| NRAS A59D | 3.13 |
| NRAS A59T | 7.81 |

# c. Sample Equivalency Study

In instances where clinical CRC FFPE tissue samples were not available, cell lines or synthetic DNA fragments harboring KRAS or NRAS mutations were blended with wildtype CRC FFPE DNA. Seven (7) CRC FFPE tissue specimens, seven (7) cell lines, four (4) FFPE cell lines, and eight (8) synthetic DNA fragment-based samples were included in the study. Each sample was serially diluted from $50\%$ , or less if the starting material was lower, to one level below LoD and analyzed. Five (5) operators tested two (2) replicates across two (2) CRCdx device lots for a total of 20 replicates at each dilution level for each sample. Functional equivalence was confirmed by demonstrating a non-statistically significant difference as compared to the clinical samples results by linear regression analysis within the acceptable predicted interval values and the respective $\%$ correct call ( $\geq 95\%$ ) for overall

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equivalent performance comparison. The linear regression analysis showed that all replicates for cell lines (n=698), FFPE cell line (n=452), and synthetic DNA fragment (n=743) specimens were within the predictive value or Ct acceptance criteria as compared to CRC FFPE DNA results (n=596). Furthermore, the study demonstrated 100.0% correct call or equivalence across all samples and sample types at dilutions at or above LoD. Contrived samples performed similarly to CRC FFPE DNA at one dilution level below LoD. These results suggest the three (3) surrogate specimen DNA formats (cell line, FFPE cell lines, and synthetic DNA fragment-based samples) perform similarly.

d. DNA Input Range

The DNA input concentration using an internal control reaction Ct to indicate a sufficient load (internal control VIC Ct = 24 - 26.5) was evaluated. The recommended starting input for the CRCdx RAS Mutation Detection Kit is 24 ng per amplification reaction to target the VIC Ct range. Nineteen (19) single mutant CRC FFPE specimens and sixteen (16) synthetic samples blended with wildtype CRC FFPE specimens (all 35 mutant targets represented) were analyzed at six (6) different inputs: 48, 24, 12, 6, 3, and 1.5 ng per amplification reaction. Each sample dilution was analyzed in duplicate (2) by two (2) reagent lots (n=24 per sample).

Internal control correlation coefficient across all dilutions for each sample range was 0.99-1.00 and reaction efficiency range was 87.2% - 104.3%. Input loads at 48 ng (n=140), 24 ng (n=140), 12 ng (n=140), and 6 ng (n=140) resulted in a 100.0% correct call rate. 139/140 (99.3%) were called correctly at 3 ng input and 136/140 (97.1%) were called correctly at 1.5 ng input. All 5 missed calls were outside of the acceptable VIC range (VIC Ct = 28.11 - 29.41).

e. Minimum Tumor Content

To evaluate the performance of CRCdx using specimens with various % tumor content, particularly at or near 20%, performance was evaluated based on data generated for the accuracy study. The 391 archived CRC patient FFPE specimens with tumor content ranging from 18-100% were evaluated by CRCdx and Illumina's Praxis. Nineteen (19) of the 35 targets are represented in the study. Samples were differentiated into at or near 20% (18-25%) and &gt;25% tumor content. CRCdx reported an overall concordance rate of 100.0% (8/8) for samples at or near 20% tumor content of the total samples of 391 with those &gt;25% tumor content yielded 97.9% concordance rate (375/383).

f. Number of Curls

Study was performed to evaluate the performance of the CRCdx assay using FFPE curl counts below the recommended eight (8) curls for DNA extraction. Genomic DNA isolated from two (2), four (4), and eight (8) curls from three (3) previously characterized CRC patient FFPE specimens and (3) FFPE cell lines were quantified and diluted to 4 ng/ul. Each extract was assessed using one (1) CRCdx reagent lot at 24 ng input in duplicate. The results of the study (N=288) support

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recommended 8 curls and show that overall percent mutation agreement for both specimen types within all curl counts was 100% (95% CI = 95.4% - 100.0%).

g. DNA Extraction Method Characterization

To ensure consistent DNA preparations, EntroGen has paired CRCdx with Maxwell CSC DNA FFPE kit for the required DNA isolation steps. A DNA extraction study was performed to establish Maxwell CSC FFPE DNA kit as a suitable pairing with CRCdx.

Data generated for this DNA Extraction method study was acquired from the Limit of Detection and Accuracy (in part) studies performed for CRCdx, in which thirty-five (35) CRC FFPE extraction were performed on nineteen (19) CRC FFPE blocks each representing a total of the nineteen (19) targeted mutations and analyzed with CRCdx assay. Twenty-two (22) Maxwell CSC FFPE DNA kit lots and nine (9) CRCdx device lots were used during the studies. All samples were previously characterized by Praxis Extended RAS NGS Panel. LoD samples were generated by diluting extracted CRC FFPE DNA to the previously determined LoD in a background of wildtype CRC FFPE DNA. Sixteen (16) samples each representing a different mutation targeted by CRCdx were analyzed by two (2) CRCdx device lots across twenty (20) replicates (n=317).

All DNA samples yielded &gt;95% Positive percent agreement (PPA = 99.8%), Negative Percent Agreement (NPA = 95.2%), and Overall Percent Agreement (OPA = 98.7%) for all replicates across both studies (N=710) for CRCdx performances (see Table 17). The results demonstrated that the Maxwell CSC DNA FFPE Kit was able to isolate gDNA of sufficient quality and quantity to perform the CRCdx RAS Mutation Detection analysis.

Table 17. Summary of Percent Agreement for LOD and Accuracy Studies

| | Praxis | | | |
| --- | --- | --- | --- | --- |
| | | Mutant | Wildtype | Total |
| CRCdx | Mutant | 543 | 8 | 551 |
| | Wildtype | 1 | 158 | 159 |
| | Total | 544 | 166 | 710 |
| | PPA (95% CI) | 543/544 = 99.8% (98.9% - 99.995%) | | |
| | NPA (95% CI) | 158/166 = 95.2% (90.7% - 97.9%) | | |
| | OPA (98% CI) | 701/710 = 98.7% (97.6 - 99.4%) | | |

3. Analytical Specificity

a. Cross-reactivity (Primer and Probe Specificity)

NCBI's Primer-BLAST (Basic Local Alignment Search Tool) was used to conduct a specificity test to ensure that the CRCdx primers and probes only amplify the targeted human genes KRAS or NRAS and no other human and non-human homologs or pseudogenes. The in silico modeling software identified no non-specific amplifications by the CRCdx oligonucleotides. In addition, no single nucleotide polymorphisms (SNPs) were identified (NCBI's dbSNP database) in the primer or probe binding regions that could lead to false calls.

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b. Cross Reactivity (Exclusivity)

Thirty-five (35) synthetic samples blended with wildtype CRC FFPE DNA specimens were analyzed by three (3) CRCdx reagent lots in duplicate. Each sample was loaded near the highest acceptable amplifiable DNA input (VIC Ct = 24.0) to best capture cross-reactive events. FAM Ct values were evaluated based on the assay's acceptance criteria.

Two instances of cross reactivity were identified: KRAS G12D showed signal in Reaction 1 (targeted) and Reaction 2 (non-specific) in 2/6 replicates and appeared to be lot specific. KRAS G12S resulted in signal in both Reaction 2 (targeted) and Reaction 1 (non-specific) 6/6 replicates. In all cross-reactive events, the targeted reaction was the earliest Ct. EntroGen's PCR Analysis Software (EPAS™) tool accounts for any cross-reactivity and reports only a single mutation. No additional cross reactivity was observed.

c. Interference – Effects of Necrotic Tissue and Exogenous Substances

Effects of Necrotic Tissue

Ten (10) wildtype and thirteen (13) KRAS or NRAS mutant CRC FFPE specimens that contained 30% - 70% necrotic tissue, as determined by a board-certified pathologist, were tested. The KRAS and NRAS genotype was determined by Illumina's Praxis Extended RAS Panel. Each specimen was analyzed by two (2) CRCdx device lots (N=46).

The PPA was 100% (26/26) and NPA was 100% (20/20) for all observations. The data supports that up to 70% necrotic tissue content does not interfere with the call results for the CRCdx RAS Mutation Detection Kit.

Exogenous Substances

Potentially interfering substances present in the manual processing steps of DNA extraction process were tested at 10-fold excess concentration (or the greatest excess concentration allowed by volume of the extraction tube) during the extraction process. Six (6) substances were tested and compared to the untreated control. Eight (8) wildtype and eight (8) KRAS or NRAS mutant CRC FFPE specimens were extracted by two (2) DNA extraction kit lots under each of seven (7) conditions and analyzed by two (2) CRCdx device lots (n=32 per condition). The specimens were genotyped by Praxis Extended RAS Panel. CRCdx correctly called 98.4% (188/191) of the treated specimens. Only three (3) samples (1.6% - (3/191)) – one extracted with excess paraffin, one extracted with excess blue dye, and one extracted with excess RNase A – were called incorrectly. One (1) sample extracted with excess lysis buffer did not yield sufficient DNA to be tested and was excluded from the study.

4. Precision / Reproducibility

Precision of the CRCdx RAS Mutation Detection Kit was assessed under the following conditions: Within-lot Variability, Within-Run Variability, Within-user/day

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Variability, Between-user Variability, Between lot Variability and Between-Site Variability.

The same batch of samples was used throughout the precision studies, consisting of sixteen (16) mutant CRC FFPE specimens, seven (7) mutant cell lines, and twelve (12) synthetic samples in a background of wildtype CRC FFPE DNA samples, and two (2) wildtype CRC FFPE specimens. Each mutant sample was diluted to 1X LoD, 1.5X LoD or 3X LoD and ran in duplicate (2) across three (3) CRCdx device lots by two (2) trained operators at each of the three (3) external sites, for all together six (6) users across all sites ( $n = 72$ replicates per sample; 36 samples). The results from each run were sent to EntroGen for Ct data analysis and mutation analysis using EPAS. A PC and NTC was included in each run to qualify each plate. Summary of positive control results in precision study is provided in Table 18.

Table 18. Summary of Results for Positive Controls in Reproducibility Study

| | | | Mean and SD | | CV | | Range | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| CRCdx Reaction | CRCdx Reacti on # | Sample Size (n)a | FAM | VIC | FAM | VIC | FAM | VIC |
| KRAS 12/13 | 1 | 285 | 27.96 ± 0.65 | 27.66 ± 0.6 | 2.34 % | 2.19 % | 26.16 - 35.52 | 26.00 - 35.13 |
| KRAS 12C/S | 2 | 285 | 26.56 ± 0.92 | 27.65 ± 0.71 | 3.50 % | 2.59 % | 24.82 - 34.18 | 25.63 - 36.30 |
| KRAS 59/61 | 3 | 285 | 27.67 ± 0.60 | 27.41 ± 0.62 | 2.17 % | 2.29 % | 25.87 - 33.77 | 25.75 - 35.06 |
| KRAS 117 | 4 | 285 | 26.77 ± 0.81 | 27.37 ± 0.61 | 3.06 % | 2.24 % | 24.66 - 34.69 | 25.71 - 34.66 |
| KRAS 146 | 5 | 285 | 27.95 ± 0.87 | 27.52 ± 0.69 | 3.11 % | 2.52 % | 25.85 - 35.81 | 25.67 - 36.65 |
| NRAS 12/13/117 | 6 | 285 | 27.93 ± 0.98 | 27.41 ± 0.63 | 3.54 % | 2.33 % | 25.52 - 37.60 | 25.34 - 34.91 |
| NRAS 61 | 7 | 285 | 27.57 ± 0.80 | 27.60 ± 0.71 | 2.92 % | 2.59 % | 24.94 - 35.58 | 25.35 - 35.74 |
| NRAS 59/61 | 8 | 285 | 27.00 ± 1.00 | 27.52 ± 0.75 | 3.72 % | 2.76 % | 24.73 - 36.15 | 25.15 - 35.50 |

The average cycle threshold (Ct) values of CRCdx RAS Mutation Detection Kit at 1.0X, 1.5X, and 3.0X LoD for each of the variants tested in the precision study are listed in Table 19.

Table 19. Average Ct Value of Samples Tested at ${1.0}\mathrm{X},{1.5}\mathrm{X}$ ,and ${3.0}\mathrm{X}$ LoD

| Gene & Mutation | 1.0X LoD | 1.5X LoD | 3.0X LoD |
| --- | --- | --- | --- |
| | AVG (Ct) | AVG (Ct) | AVG (Ct) |
| KRAS G12A | 33.77 | 31.80 | 30.76 |
| KRAS G12D | 36.00 | 34.57 | 33.45 |
| KRAS G12R | 36.76 | 34.59 | 33.47 |
| KRAS G12V | 36.91 | 34.69 | 33.36 |
| KRAS G13D | 34.12 | 32.95 | 31.84 |

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| KRAS
G12C | 36.99 | 34.65 | 33.61 |
| --- | --- | --- | --- |
| KRAS
G12S | 36.43 | 35.11 | 33.91 |
| KRAS
Q61H
CAT | 36.53 | 35.96 | 34.22 |
| KRAS
Q61H
CAC | 37.53 | 35.89 | 34.95 |
| KRAS
Q61L | 37.39 | 36.14 | 35.07 |
| KRAS
Q61R | 37.36 | 37.00 | 35.89 |
| KRAS
A59E | 34.38 | 33.04 | 31.84 |
| KRAS
A59G | 34.37 | 33.22 | 31.93 |
| KRAS
A59T | 37.88 | 37.01 | 35.88 |
| KRAS
K117N
AAC | 36.82 | 32.35 | 30.60 |
| KRAS
K117N
AAT | 35.09 | 35.34 | 33.94 |
| KRAS
K117R | 35.70 | 34.71 | 33.33 |
| KRAS
K117E | 35.19 | 34.45 | 33.10 |
| KRAS
A146T | 37.91 | 35.66 | 34.09 |
| KRAS
A146P | 35.18 | 35.59 | 34.30 |
| KRAS
A146V | 35.51 | 35.89 | 34.70 |
| NRAS
G12D | 34.05 | 31.98 | 30.80 |
| NRAS
G12C | 36.18 | 33.63 | 33.28 |
| NRAS
G12S | 37.26 | 32.90 | 30.81 |
| NRAS
G13R | 34.47 | 32.70 | 31.76 |
| NRAS
G13V | 36.94 | 34.76 | 33.30 |
| NRAS
K117R | 36.04 | 34.28 | 32.82 |
| NRAS
Q61H
CAC | 34.72 | 33.16 | 31.88 |
| NRAS
Q61H
CAT | 36.39 | 34.50 | 33.77 |

{20}

Overall, the study had a sample first pass rate of 90.0%, with 2.7% sample data having invalid PC results, 3.5% sample data invalid NTC results, and 3.8% user error results. Observed overall correct call rate (2499/2499 mutant and 72/72 wildtype samples) across all samples, dilutions, replicates, lots, users, sites, instruments, and days is listed in Table 20.

Table 20. Overall Agreement Correct Calls Per Site and Reaction

| Site | CRCdx Reaction | Correct Wildtype Call/Total Wildtype Samples | Correct Mutant Call/Total Mutant Samples | Percent Agreement |
| --- | --- | --- | --- | --- |
| 1 | 1 | 24/24 | 118/118 | 100.0 |
| | 2 | | 47/47 | 100.0 |
| | 3 | | 162/162 | 100.0 |
| | 4 | | 95/95 | 100.0 |
| | 5 | | 72/72 | 100.0 |
| | 6 | | 144/144 | 100.0 |
| | 7 | | 120/120 | 100.0 |
| | 8 | | 68/68 | 100.0 |
| 2 | 1 | 24/24 | 120/120 | 100.0 |
| | 2 | | 48/48 | 100.0 |
| | 3 | | 168/168 | 100.0 |
| | 4 | | 96/96 | 100.0 |
| | 5 | | 71/71 | 100.0 |
| | 6 | | 144/144 | 100.0 |
| | 7 | | 120/120 | 100.0 |
| | 8 | | 70/70 | 100.0 |
| 3 | 1 | 24/24 | 120/120 | 100.0 |
| | 2 | | 48/48 | 100.0 |
| | 3 | | 168/168 | 100.0 |
| | 4 | | 96/96 | 100.0 |
| | 5 | | 68/68 | 100.0 |
| | 6 | | 144/144 | 100.0 |
| | 7 | | 120/120 | 100.0 |
| | 8 | | 72/72 | 100.0 |
| Total | | 72/72 | 2499/2499 | 100.0 |

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The within-lot variability was $0.35\% - 3.37\%$ (N=2499 mutant samples). The within-run variability was $0.09\% - 1.53\%$ ((N=2499 mutant samples). The within-user/day variability was $0.11\% - 4.54\%$ (N=2499 mutant samples). The between-user variability was $0.19\% - 2.15\%$ (N=2499 mutant samples). The between lot variability was $0.30\% - 3.34\%$ (N=2499 mutant samples). The between-site variability was $0.35\%$ to $3.37\%$ (N=2499 mutant samples).

All precision variables in this study had $\% \mathrm{CV} &lt; 5.0\%$ , which shows consistency in calling and Ct results for Within-Site/ Lab, Within-Run, and Between-Site/ Lab with respect to user-to-user, run-to-run, lot-to-lot, day-to-day, instrument-to-instrument, and site-to-site variabilities for CRCdx RAS assay (N=2499 mutant samples and N=72 wildtype samples).

Reproducibility was determined by assessing the between-site and within-site variability (%CV). The Percent Correct Calls by variant and corresponding 95% two-sided confidence intervals was calculated using the Wilson score method. The Percent Correct Call across all mutant samples was 100% (lower bound 95% CI: 99.85%) when excluding samples with failed sample QC. Detailed results for each of the tested CDx variants across all replicates are presented in Table 21.

Table 21. Reproducibility Positive and Negative Percent Agreement by Variant

| Gene | Exon | Amino Acid Change | Nucleotide Change | N | Percent Correct Call (%) | 95% CI |
| --- | --- | --- | --- | --- | --- | --- |
| KRAS | 2 | G12A | c.35G>C | 72 | 72/72 (100.0) | (94.9,100.0) |
| G12D | c.35G>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| G12R | c.34G>C | 72 | 72/72 (100.0) | (94.9,100.0) |
| G12V | c.35G>T | 71 | 71/71 (100.0) | (94.9,100.0) |
| G13D | c.38G>A | 71 | 71/71 (100.0) | (94.9,100.0) |
| G12C | c.34G>T | 72 | 72/72 (100.0) | (94.9,100.0) |
| G12S | c.34G>A | 71 | 71/71 (100.0) | (94.9,100.0) |
| KRAS Exon 2 Total | 501 | 501/501 (100.0) | (99.2,100.0) |
| 3 | Q61H (CAT) | c.183A>T | 70 | 70/70 (100.0) | (94.8,100.0) |
| Q61H (CAC) | c.183A>C | 68 | 68/38 (100.0) | (94.7,100.0) |
| Q61L | c.182A>T | 72 | 72/72 (100.0) | (94.9,100.0) |
| Q61R | c.182A>G | 72 | 72/72 (100.0) | (94.9,100.0) |
| A59E | c.176C>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| A59G | c.176C>G | 72 | 72/72 (100.0) | (94.9,100.0) |
| A59T | c.175G>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| KRAS Exon 3 Total | 498 | 498/498 (100.0) | (99.2,100.0) |
| 4 | K117N (AAC) | c.351A>C | 71 | 71/71 (100.0) | (94.9,100.0) |
| K117N (AAT) | c.351A>T | 72 | 72/72 (100.0) | (94.9,100.0) |
| K117R | c.350A>G | 72 | 72/72 (100.0) | (94.9,100.0) |
| K117E | c.349A>G | 72 | 72/72 (100.0) | (94.9,100.0) |
| A146T | c.436G>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| A146P | c.436G>C | 72 | 72/72 (100.0) | (94.9,100.0) |
| A146V | c.437C>T | 67 | 67/67 (100.0) | (94.6,100.0) |
| KRAS Exon 4 Total | 498 | 498/498 (100.0) | (99.2,100.0) |
| All KRAS Exons | 1497 | 1497/1497 (100.0) | (99.7,100.0) |
| NRAS | 6 | G12D | c.35G>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| G12C | c.34G>T | 72 | 72/72 (100.0) | (94.9,100.0) |
| G12S | c.34G>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| G13R | c.37G>C | 72 | 72/72 (100.0) | (94.9,100.0) |
| G13V | c.38G>T | 72 | 72/72 (100.0) | (94.9,100.0) |
| NRAS Exon 2 Total | 360 | 360/360 (100.0) | (98.94,100.0) |
| 7 | Q61H (CAC) | c.183A>C | 72 | 72/72 (100.0) | (94.9,100.0) |

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| Gene | Exon | Amino Acid Change | Nucleotide Change | N | Percent Correct Call (%) | 95% CI |
| --- | --- | --- | --- | --- | --- | --- |
| | | Q61H (CAT) | c.183A>T | 72 | 72/72 (100.0) | (94.9,100.0) |
| | | Q61L | c.182A>T | 72 | 72/72 (100.0) | (94.9,100.0) |
| | | Q61K | c.181C>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| | | Q61R | c.182A>G | 72 | 72/72 (100.0) | (94.9,100.0) |
| | | A59D | c.176C>A | 70 | 70/70 (100.0) | (94.8,100.0) |
| | | A59T | c.175G>A | 72 | 72/72 (100.0) | (94.9,100.0) |
| | | NRAS Exon 3 Total | | 502 | 502/502 (100.0) | (99.2,100.0) |
| | 8 | A146T | c.436G>A | 68 | 68/68 (100.0) | (94.7,100.0) |
| | | K117R | c.350A>G | 72 | 72/72 (100.0) | (94.9,100.0) |
| | | NRAS Exon 4 Total | | 140 | 140/140 (100.0) | (97.3,100.0) |
| | All NRAS Exons Total | | | 1002 | 1002/1002 (100.0) | (99.0,100.0) |
| All KRAS and NRAS Exons Total | | | | 2499 | 2499/2499 (100.0) | (99.8,100.0) |

The Ct values data dispersion results show a very low $\% \mathrm{CV} &lt; 5.0\%$ across all sites $(\% \mathrm{CV} = 0.30\% - 3.34\%)$ for an overall average $\% \mathrm{CV}$ of $1.35\%$ (Table 22). The Ct range and variability for each target across all sites, users, lots, days and instruments for two concentrations tested in the precision study is shown in Table 23.

Table 22. Per-site and Overall Ct Variability

| Site | Obs (n) | CV | |
| --- | --- | --- | --- |
| | | AVG | RANGE |
| 1 | 827 | 1.04% | 0.30% - 3.21% |
| 2 | 836 | 1.18% | 0.36% - 3.04% |
| 3 | 836 | 1.29% | 0.37% - 3.34% |
| All | 2499 | 1.35% | 0.58% - 2.89% |

Table 23. Ct Range and Variability for Each Target Across All Variables

| Gene & Mutation | 1.5X LoD | | 3.0X LoD | |
| --- | --- | --- | --- | --- |
| | Range (Ct) | CV (%) | Range (Ct) | CV (%) |
| KRAS G12A | 31.11 - 32.28 | 0.88 | 30.2 - 31.31 | 0.87 |
| KRAS G12D | 33.88 - 35.19 | 0.01 | 32.69 - 34.03 | 0.89 |
| KRAS G12R | 33.73 - 35.25 | 0.01 | 32.71 - 34.73 | 1.22 |
| KRAS G12V | 33.96 - 35.48 | 0.01 | 32.65 - 34.15 | 1.11 |
| KRAS G13D | 32.38 - 33.64 | 0.96 | 31.32 - 32.57 | 0.96 |
| KRAS G12C | 33.92 - 36.25 | 1.33 | 32.83 - 34.96 | 1.27 |
| KRAS G12S | 33.77 - 36.15 | 1.28 | 32.91 - 34.88 | 1.22 |
| KRAS Q61H CAT | 35.2 - 36.16 | 0.66 | 33.68 - 34.77 | 0.67 |
| KRAS Q61H CAC | 35.41 - 36.37 | 0.58 | 34.48 - 35.48 | 0.79 |
| KRAS Q61L | 35.64 - 36.78 | 0.69 | 34.64 - 35.55 | 0.73 |
| KRAS Q61R | 36.48 - 37.51 | 0.77 | 35.35 - 36.44 | 0.69 |
| KRAS A59E | 32.13 - 33.68 | 1.36 | 30.93 - 32.46 | 1.26 |
| KRAS A59G | 32.28 - 33.9 | 1.14 | 31.04 - 32.51 | 1.34 |
| KRAS A59T | 36.32 - 37.51 | 0.88 | 35.24 - 36.47 | 0.90 |
| KRAS K117N AAC | 31.11 - 33.39 | 1.64 | 29.61 - 31.40 | 1.25 |
| KRAS K117N AAT | 34.45 - 36.59 | 1.40 | 33.26 - 34.9 | 1.12 |
| KRAS K117R | 33.89 - 35.95 | 1.30 | 32.37 - 34.06 | 1.13 |

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| KRAS K117E | 34.84 – 35.33 | 1.03 | 32.45 – 33.88 | 1.07 |
| --- | --- | --- | --- | --- |
| KRAS A146T | 33.98 – 38.05 | 2.29 | 32.58 – 35.25 | 2.16 |
| KRAS A146P | 34.83 – 36.74 | 1.29 | 33.72 – 34.98 | 0.99 |
| KRAS A146V | 34.72 – 37.29 | 1.75 | 33.48 – 35.58 | 1.75 |
| NRAS G12D | 31.48 – 32.48 | 0.75 | 30.41 – 31.14 | 0.59 |
| NRAS G12C | 32.72 – 35.7 | 2.41 | 32.02 – 35.04 | 2.89 |
| NRAS G12S | 33.48 – 36.33 | 1.75 | 32.24 – 34.09 | 1.36 |
| NRAS G13R | 31.96 – 34.01 | 1.31 | 31.12 – 32.29 | 0.99 |
| NRAS G13V | 31.62 – 34.86 | 2.60 | 29.87 – 32.06 | 2.17 |
| NRAS K117R | 33.13 – 34.98 | 1.14 | 31.95 – 33.4 | 1.20 |
| NRAS Q61H CAC | 32.24 – 34.43 | 1.89 | 31.0 – 33.17 | 2.29 |
| NRAS Q61H CAT | 34.06 – 36.1 | 1.47 | 32.76 – 34.78 | 1.39 |
| NRAS Q61L | 30.96 – 32.41 | 1.12 | 29.84 – 30.92 | 0.90 |
| NRAS Q61K | 34.51 – 36.66 | 1.58 | 33.47 – 38.08 | 2.15 |
| NRAS Q61R | 35.71 – 38.06 | 1.46 | 32.09 – 34.93 | 1.47 |
| NRAS A146T | 32.66 – 34.58 | 1.37 | 31.05 – 32.36 | 1.21 |
| NRAS A59D | 32.61 – 35.84 | 2.10 | 31.07 – 33.67 | 2.09 |
| NRAS A59T | 33.65 – 37.34 | 2.59 | 31.86 – 35.75 | 2.56 |

## 5. Guard Banding – Robustness

Thermal cycling temperatures, reaction composition, and proteinase K digestion methods were modified to evaluate the impact, if any, on mutation detection by CRCdx.

### a. Thermal Cycling Profile

The CRCdx thermal PCR cycling involves a denaturation step at 95°C and an annealing/extension step at 60°C. The robustness of the thermal cycling profile was evaluated by varying both the denaturation and annealing/extension temperatures by ±1°C. The study was conducted over a panel of seven (7) mutant CRC FFPE specimens at 3X LoD, one (1) mutant gene block in a background of wildtype CRC FFPE DNA at 3X LoD and one (1) wildtype CRC FFPE specimen. Each sample was run in triplicate across one CRCdx device lot (N=162).

Samples and replicates (162/162) were called correctly for all six (6) thermal cycling profiles. The results of the study showed that CRCdx RAS Mutation Detection Kit can tolerate thermal cycling temperature variations of ±1°C by means of 100% correct call.

### b. Reaction Composition

The robustness of the reaction composition (Mutation Detection Mix + Primer/Probe Mix + DNA sample) was evaluated by modifying the sample load volume ±1 μl changing the final concentrations of the master mix components ±3.0%. The study was conducted over a panel of seven (7) mutant CRC FFPE specimens at 3X LoD, one (1) mutant gene block in a background of wildtype

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CRC FFPE DNA at 3X LoD and one (1) wildtype CRC FFPE specimen. Each sample was run in triplicate across one CRCdx device lot (N=81).

All samples and replicates (81/81) were called correctly for all three (3) reaction composition profiles. The results of the study showed that CRCdx RAS Mutation Detection Kit is able to tolerate reaction composition variations of ±3.0% by means of 100% correct call.

## c. Proteinase K

CRCdx RAS Mutation Detection Kit is paired with Maxwell CSC FFPE DNA Extraction Kit for specimen preparation. During extraction, the specimen is lysed by incubation with Proteinase K and Lysis Buffer. The impact of modifying the amount of Proteinase K by ±20% on mutation call was evaluated. The study was conducted over a panel of seven (7) mutant CRC FFPE specimens at 3X LoD, one (1) mutant gene block in a background of wildtype CRC FFPE DNA at 3X LoD and one (1) wildtype CRC FFPE specimen. Each sample was run in triplicate across one CRCdx device lot (N=54).

All samples and replicates (54/54) were called correctly for both (2) Proteinase K concentrations. The results of the study showed that CRCdx® RAS Mutation Detection Kit is able to tolerate Proteinase K concentration variations of ±20% during extraction by means of 100% correct call.

## 6. Cross Contamination

A checkerboard patterned plate where wells containing high positive samples and NTC wells were alternated was used for this study. Eight replicates (8) of five (5) samples, two (2) CRC FFPE specimens, two (2) cell lines, and one (1) synthetic gene block in a background of wildtype CRC FFPA DNA, with high MAFs that ranged from 45.1% - 70.6% were analyzed by three (3) operators on non-consecutive days in two (2) checkerboard plate formats (N=480). A full plate of NTCs was also included.

All test wells (100%) displayed FAM and VIC signals that met the acceptance criteria demonstrating that there was no significant cross-contamination from neighboring wells containing high %MAF mutant specimens. The study results show that the manual processing steps are not vulnerable to cross contamination events resulting from well-to-well carryover by means of 100% correct call by three (3) different operators.

## 7. Stability Studies

### a. Extracted DNA from FFPE Specimens

The stability of DNA extracted from CRC FFPE tissue was determined by evaluating specimen extracts under three conditions: (i) Frozen (-15°C to -25°C) for up to 35 days, (ii) Refrigerated (2°C to 8°C) for up to 9 days, and (iii) Freeze thaw cycled up to 3 cycles (1 week/cycle). Acceptance criteria are defined as correct call &gt;95%; Ct value trends absent of significant measurand drift (p &gt;0.05 and/or ΔCt &lt;1.0 and %CV &lt;5.0).

i. Frozen DNA Extract Stability (-15°C to -20°C): Eight (8) KRAS or NRAS mutant containing CRC FFPE specimens, and one (1) wildtype CRC FFPE

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specimen was extracted by two (2) extraction kit lots and stored at -15°C to -25°C for the indicated duration. Seven of the eight (7/8 = 87.5%) CRCdx reactions are represented due to the rarity of some reaction targets (KRAS G13D, G12C, Q61H CAC, Q61H CAT, K117N AAT, A146T, and NRAS G12D, Q61K). Five (5) replicates per sample were analyzed by two (2) CRCdx device lots at six (6) timepoints: 0, 7, 14, 21, 28, and 35 days. The results of the study showed 100% correct call and no statistically significant mean difference of Ct measurand was observed, demonstrating that the extracted gDNA is stable for up to 35 days when stored at -15°C to -25°C.

ii. Refrigerated DNA Extract Stability (2°C to 8°C): Eight (8) KRAS or NRAS mutant containing CRC FFPE specimens, and one (1) wildtype CRC FFPE specimen was extracted by two (2) extraction kit lots and stored at 2°C to 8°C for the indicated duration. Five of the eight (5/8 = 62.5%) CRCdx reactions are represented due to the rarity of some reaction targets samples (KRAS G12V, G12A, G12S, G12C, Q61H CAT, Q61H CAC, A146T, and NRAS G12D). Three (3) replicates per sample were analyzed by three (3) CRCdx device lots at five (5) timepoints: 0, 3, 5, 7, and 9 days. The results of the study showed 100% correct call and no statistically significant mean difference of Ct measurand was observed. indicating that the isolated gDNA is stable up to 9 days when stored at 2°C to 8°C.

iii. Freeze-Thaw Cycling DNA Extract Stability: Eight (8) KRAS or NRAS mutant containing CRC FFPE specimens, and one (1) wildtype CRC FFPE specimen was extracted by two (2) extraction kit lots and placed at -15°C to -25°C prior to the starting the study. Each sample was thawed at room temperature (22°C to 28°C) at the indicated timepoint and placed back in the freezer (-15°C to -25°C) for the subsequent timepoint. Five of the eight (5/8 = 62.5%) CRCdx reactions are represented due to the rarity of some reaction targets samples (KRAS G12V, G12A, G12S, G12C, Q61H CAT, Q61H CAC, A146T, and NRAS G12D). Three (3) replicates per sample were analyzed by three (3) CRCdx device lots at four (4) timepoints: 0, 1, 2, and 3 weeks (1 cycle per week).

The results of the study showed 100% correct call and no statistically significant mean difference of Ct measurand was observed, indicating that the isolated DNA is stable up to three (3) freeze-thaw cycles.

b. Open Vial Stability

Study was performed to determine the stability of the of CRCdx RAS Mutation Detection Kit reagents after first use and stored in the refrigerator (2° to 8°C). Three (3) lots of reagents were used to test seven (7) KRAS or NRAS mutant CRC FFPE specimens, one (1) NRAS mutant cell line, and one (1) wildtype CRC FFPE specimen. CRC FFPE specimens were extracted and all mutant samples (KRAS G12A, G12C, Q61L, K117N AAT, A146V, and NRAS G12D, Q61L, A146T) were diluted to 3X and 9X LoD in wildtype CRC FFPE DNA background to be analyzed at a low DNA input to best capture false calls. Each sample dilution was

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run in triplicate per timepoint. All reagents for the study were thawed at room temperature (18°C – 26°C) and stored in the refrigerator (2° to 8°C) until the indicated timepoint, 0, 1, and 4 weeks. Reaction plates were assembled with master mixes and DNA and ran immediately. Acceptance criteria: correct call &gt;95%; Ct value trends absent of significant measurand drift (p &gt;0.05 and/or ΔCt &lt;1.0 and %CV &lt;5.0.

The results of the study showed 100% correct call and no statistically significant mean difference of Ct measurand was observed. CRCdx RAS Mutation Detection Kit reagents can be stored in the refrigerator (2° to 8°C), as necessary, for up to 3 weeks without compromising the performance characteristics of the assay.

c. Shelf Life Stability

Study was performed to determine the shelf-life stability of the of CRCdx® RAS Mutation Detection Kit reagents after manufacturing. Three (3) lots of reagents were used to test six (6) KRAS and NRAS mutant cell lines, two (2) synthetic surrogate NRAS mutant samples, and one (1) wildtype CRC FFPE specimen. Contrived samples were used for this Closed Vial stability study because of the lack of sufficient availability and reliability of CRC patient FFPE specimens, a consequence from the length of the study. All mutant samples (KRAS G12D, G12S, Q61H CAT, K117N AAT, A146P, and NRAS G13V, Q61R, A146T) were diluted to 3X and 9X LoD in wildtype CRC FFPE DNA background to be analyzed at a low DNA input to best capture false calls. Each sample dilution was run in triplicate per timepoint. All reagents for the study were stored at -15°C to -25°C and thawed at room temperature (18°C – 26°C) for the indicated timepoints, 0, 1, 2, 3, 4, 8, and 12 months. Reaction plates were assembled with master mixes and DNA and ran immediately. Acceptance criteria: correct call &gt;95%; Ct value trends absent of significant measurand drift (p &gt;0.05 and/or ΔCt &lt;1.0 and %CV &lt;5.0.

The results of the study showed 100% correct call and no statistically significant trend of the measurement drifts was observed. CRCdx RAS Mutation Detection Kit reagents can be stored frozen (-15° to -25°C) for up to 10 months.

d. Freeze Thaw Stability

Three (3) lots of reagents were used to test seven (7) KRAS or NRAS mutant CRC FFPE specimens, one (1) NRAS mutant cell line, and one (1) wildtype CRC FFPE specimen. CRC FFPE specimens were extracted and all mutant samples (KRAS G12A, G12S, Q61L, K117N AAT, A146P, and NRAS G12D, Q61L, A146T) were diluted to 3X and 9X LoD in wildtype CRC FFPE DNA background to be analyzed at a low DNA input to best capture false calls. Each sample dilution was run in triplicate per timepoint. CRCdx reagents were stored at -15°C to -25°C between uses. Reagents were removed from the freezer and thawed at room temperature (18°C – 26°C) at each timepoint, 0, 1, 2, 3, 4, and 5 weeks, and returned to the freezer after use for the subsequent timepoints. Reaction plates were assembled with master mixes and DNA and ran immediately. Acceptance criteria: Correct call &gt;95%; Ct value trends absent of significant measurand drift (p &gt;0.05 and/or ΔCt &lt;1.0 and %CV &lt;5.0.

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The results of the study showed 100% correct call and no statistically significant trend of the measurement drifts was observed. CRCdx RAS Mutation Detection Kit reagents can be freeze thawed as necessary for up to five (5) cycles without compromising the performance characteristics of the assay.

e. Master Mix Stability

Three (3) lots of reagents were used to test seven (7) KRAS or NRAS mutant CRC FFPE specimens, one (1) NRAS mutant cell line, and one (1) wildtype CRC FFPE specimen. CRC FFPE specimens were extracted and all mutant samples (KRAS G12A, G12S, Q61L, K117N AAT, A146P, and NRAS G12D, Q61L, A146T) were diluted to 3X and 9X LoD in wildtype CRC FFPE DNA background to be analyzed at a low DNA input to best capture false calls. Each sample dilution was run in triplicate per timepoint. Master mixes were prepared by mixing the appropriate amount of Mutation Detection Reaction Mix, Primer/Probe Mix, and Water. Master mixes were tested at four (4) incubation timepoints, 0, 30, 60, and 120 minutes, and at two (2) dark storage conditions (room temperature: 18°C – 26°C; or refrigerated: 2°C – 8°C). Following incubation, master mixes were aliquoted into 96-well reaction plates and DNA samples added just prior to the start of amplification. Acceptance criteria: Correct call &gt;95%; Ct value trends absent of significant measurand drift (p &gt;0.05 and/or ΔCt &lt;1.0 and %CV &lt;5.0). The results of the study showed 100% correct call and no statistically significant trend of the measurement drifts was observed. CRCdx master mixes can be stored in the dark at either room temperature (18°C – 26°C) or refrigerated (2°C – 8°C) for up to 100 minutes without compromising the performance characteristics of the CRCdx assay.

f. Plate Stability

Purpose of the study was to evaluate the stability of CRCdx RAS Mutation Detection Kit reagents plus DNA in an assembled 96-well reaction plate (Mutation Detection Reaction Mix + Primer/Probe Mix + Water + DNA). Three (3) lots of reagents were used to test seven (7) KRAS or NRAS mutant CRC FFPE specimens, one (1) NRAS mutant cell line, and one (1) wildtype CRC FFPE specimen. CRC FFPE specimens were extracted and all mutant samples (KRAS G12A, G12S, Q61L, K117N AAT, A146P, and NRAS G12D, Q61L, A146T) were diluted to 3X and 9X LoD in wildtype CRC FFPE DNA background to be analyzed at a low DNA input to best capture false calls. Each sample dilution was run in triplicate per timepoint. Master mixes were prepared by mixing the appropriate amount of Mutation Detection Reaction Mix, Primer/Probe Mix, and Water and aliquoted into 96-well reaction plates. DNA samples were added to the appropriate master mixes and the assembled plates were sealed and stored in the dark at either room temperature (18°C – 26°C) or refrigerated (2°C – 8°C) for 0, 2, or 6 hours. Acceptance criteria: Correct call &gt;95%; Ct value trends absent of significant measurand drift (p &gt;0.05 and/or ΔCt &lt;1.0 and %CV &lt;5.0). The results of the study showed 100% correct call and no statistically significant trend of the measurement drifts was observed. CRCdx assembled reaction plates

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can be stored in the dark at either room temperature (18°C – 26°C) or refrigerated (2°C – 8°C) for up to 5 hours without compromising the performance characteristics of the CRCdx assay.

g. Transportation Simulation

Three (3) lots of reagents were used to test seven (7) KRAS or NRAS mutant CRC FFPE specimens, one (1) NRAS mutant cell line, and one (1) wildtype CRC FFPE specimen. CRC FFPE specimens were extracted and all mutant samples (KRAS G12A, G12S, Q61L, K117N AAT, A146P, and NRAS G12D, Q61L, A146T) were diluted to 3X and 9X LoD in wildtype CRC FFPE DNA background to be analyzed at a low DNA input to best capture false calls. Each sample dilution was run in triplicate per timepoint. The reagents were subjected to one of the following four (4) treatments: No treatment control (-15° to -25°C); Refrigerated Treatment (2° to 8°C) for 24 hours; Room Temperature Treatment (18° to 26°C) for 24 hours; and Extreme Heat Treatment (32° to 42°C) for 24 hours. Reagents were placed in the freezer for a minimum of 24 hours (-15° to -25°C) following treatment. Reagents were removed from the freezer and thawed at room temperature (18°C – 26°C) and reaction plates were assembled with master mixes and DNA and run immediately. Acceptance criteria: Correct call &gt;95%; Ct value trends absent of significant measurand drift (p &gt;0.05 and/or ΔCt &lt;1.0 and %CV &lt;5.0). The results of this study showed 100% correct call and no statistically significant mean difference of Ct measurand was observed.

B. Animal Studies

N/A

C. Additional Studies

N/A

X. SUMMARY OF PRIMARY CLINICAL STUDY(IES)

The safety and effectiveness of the CRCdx RAS Mutation Detection Kit was evaluated in an external concordance study designed to demonstrate non-inferiority to the FDA approved companion diagnostic Praxis Extended RAS Panel for the purpose of validating the use of the follow-on companion diagnostic test for panitumumab (Vectibix). The objective of the study was to demonstrate the non-inferiority-equivalence of CRCdx to Praxis Extended RAS to infer treatment outcome efficacy of Vectibix as CRC patients without KRAS or NRAS mutations may benefit from treatment. Archived clinical CRC patient FFPE tissue acquired from biorepositories were tested with CRCdx RAS and FDA approved Praxis Extended RAS Panel. A summary of the clinical study is presented below.

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## A. Study Design

A multipronged approach on estimation of diagnostic accuracy on non-inferiority to Praxis Extended RAS NGS Panel was utilized in this study (analytical accuracy results presented in the non-clinical study section above).

### 1. Clinical Inclusion and Exclusion Criteria

The CRCdx study samples population in this study was based on the inclusion criteria for the Vectibix trial and passed the QC criteria for Praxis to include the Praxis' companion diagnostic intended use population (CCD-IU) with patient sample demography as close as possible. The study sample pool represents the CRCdx intended use population as candidate follow-on companion diagnostic (FCD-IU) covering the degrees of tumor, nodes and metastasis stage per TNM classification by National Cancer Institute (NCI). With limitation and constraints that includes no accessibility to Amgen's clinical trial samples to obtain samples post trial, in this CRCdx accuracy and correlation study, all CRC study samples were obtained from available biorepositories with the inclusion criteria set forth to encompass the Praxis' CCD-IU population as close as possible (refer to Samples Patient Demography below).

#### Inclusion Criteria:

1. Clinically and morphologically confirmed Primary Colorectal Carcinom (CRC)
2. Adenocarcinoma of colon or rectum with Location: ~60-65% left colon/rectum and ~30-35% right colon
3. Gender: ~60% male
4. Race: ~90% Caucasian
5. Median age (years): 60-65 years
6. Number of cases: N=500
7. Various tumor subtypes, stage and patient treatment status
8. Male or female over 18 years of age
9. Signed informed consent
10. FFPE tumor samples, prepared using 10% neutral formalin and 24h fixation
11. Median area of sample tissue (mm sq.): 300 (min &gt; 40)
12. Median tumor content in sample: 45-55% (min &gt; 10%)

#### Exclusion Criteria:

1. Samples that failed to meet the Inclusion Criteria
2. Samples that failed DNA prequalification for the comparator assay (Praxis Extended RAS Panel)

### 2. Follow-up Schedule

None required for this study. The CDx Clinical Validation study involved retrospective testing of samples; as such, no additional patient follow-up was conducted.

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3. Clinical Endpoints
None required for this study. The clinical validity was established by evaluating 398 CRC FFPE specimens, using a non-inferiority statistical testing approach.

B. Accountability of PMA Cohort

A total of 662 CRC FFPE specimens were screened for Illumina’s Praxis, 399 or 60.27% CRC FFPE samples qualified for Illumina’s Praxis. A total of 264 CRC FFPE samples were considered invalid because they failed to meet Illumina’s Praxis pre-qualification requirements. One CRC FFPE sample failed to produce a valid result on Illumina’s Praxis NGS assay after qualification. A total of 398 CRC samples were tested with both Praxis and CRCdx.

C. Study Population Demographics and Baseline Parameters

Demographics of the patients included in the study and their tumor characteristics of the CRCdx as follow-on companion diagnostic (FCD) are presented below in tables 24 - 27. The mean age of patient…