SimpleScreen™ CRC

{0} # SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED) # I. GENERAL INFORMATION Device Generic Name: Blood-based Qualitative Colorectal Cancer Screening Test Device Trade Name: SimpleScreen™ CRC Device Procode: PHP Applicant's Name and Address: Freenome, LLC 3300 Marina Blvd Brisbane, CA 94005 Date(s) of Panel Recommendation: None Premarket Approval Application (PMA) Number: P250029 Date of FDA Notice of Approval: July 24, 2026 # II. INDICATIONS FOR USE Intended Use: The SimpleScreen™ CRC test is a qualitative, in vitro diagnostic test intended to detect molecular signals associated with colorectal cancer. SimpleScreen CRC utilizes circulating cell-free DNA from plasma of peripheral whole blood collected in the SimpleScreen CRC blood collection kit. SimpleScreen CRC is indicated for colorectal cancer screening in adults aged 45 years and older who are at average risk for the disease. Patients with a positive result should be followed by colonoscopy. SimpleScreen CRC is not a replacement for diagnostic colonoscopy or for surveillance colonoscopy in high-risk individuals. # III. CONTRAINDICATIONS SimpleScreen CRC is NOT indicated for use for patients that have the following: - Personal history of colorectal cancer (CRC), advanced precancerous lesions (APL), or other related cancers - Family history of CRC, defined as having one or more first-degree relatives (parent, sibling, or child) diagnosed with CRC at any age 1 of 50 {1} ○ A fecal occult blood test (FOBT) or a fecal immunochemical test (FIT) within the last 12 months ○ A FIT-DNA test within the last 36 months • Personal history of any of the following high-risk conditions for CRC: ○ Inflammatory bowel disease (IBD), including chronic ulcerative colitis (CUC) and Crohn's disease ○ Relevant hereditary cancer syndromes including but not limited to: hereditary non-polyposis colorectal cancer syndrome (HNPCC) or Lynch syndrome, familial adenomatous polyposis (FAP or Gardner's), Familial Hyperplastic Polyposis, Peutz-Jeghers syndrome, MUTYH-associated polyposis (MAP), Cowden's syndrome, Juvenile polyposis syndrome, serrated polyposis syndrome, Turcot's/Crail's syndrome, Cronkhite-Canada syndrome, or Neurofibromatosis. # IV. WARNINGS AND PRECAUTIONS Based on data from clinical studies, SimpleScreen CRC has limited detection of Stage I (57%) colorectal cancer (64% when U.S. 2020 Census age-sex adjusted) and does not detect 87% of advanced precancerous lesions (86% when U.S. 2020 Census age-sex adjusted). One out of 10 patients with a negative SimpleScreen CRC result may have a precancer that would have been detected by a screening colonoscopy. # LIMITATIONS • Providers should discuss the most appropriate screening test to use with patients depending on their medical history and individual circumstances. SimpleScreen CRC is not intended as a screening test for individuals who are at high risk for CRC. • SimpleScreen CRC has limited ability to prevent the development of CRC from advanced precancerous lesions (APLs) and has lower detection rates for Stage I CRC, given the current data available. ○ SimpleScreen CRC has lower performance of Stage I CRC [57.1% (16/28); 95% CI: (39.1% - 73.5%)]. ○ SimpleScreen CRC may fail to detect as many as 87.5% of patients with advanced precancerous lesions which can later become neoplastic because of its limited detection of advanced precancerous lesions [12.5% (321/2567); 95% CI: (11.3% - 13.8%)]. • The SimpleScreen CRC test may produce false positive or false negative results. ○ A false positive result occurs when the test produces a positive result even though a colonoscopy will not find colorectal cancer or advanced precancerous lesions. SimpleScreen CRC has a false positive rate of 10%, 2 of 50 {2} meaning 1 of 10 people who do not have colorectal neoplasia (colorectal cancer or advanced precancerous lesions) will have a false positive test result. - ○ A false negative result occurs when the test does not detect colorectal neoplasia (colorectal cancer or advanced precancerous lesions) although a colonoscopy would identify either of these findings. SimpleScreen CRC has a false negative rate of 21% for colorectal cancer, meaning 21 of 100 people who have colorectal cancer will have a SimpleScreen CRC false negative result. - ● A positive SimpleScreen CRC test result suggests patients may have colorectal neoplasia (colorectal cancer or advanced precancerous lesions). Patients with a positive result should be followed by colonoscopy. - ● A negative SimpleScreen CRC test result does not guarantee absence of colorectal neoplasia (colorectal cancer or advanced precancerous lesions). Patients with a negative result should continue participating in colorectal cancer screening programs, at the appropriate guideline-recommended intervals. - ○ One out of 1600 patients testing negative will be falsely reassured that they are negative for colorectal cancer, given the negative predictive value for CRC of 99.9%. - ○ One out of 10 patients testing negative will be falsely reassured that they are negative for advanced precancerous lesions, given the negative predictive value for advanced precancerous lesions of 91%. - ● CRC screening guideline recommendations vary for persons over the age of 75. The decision to screen patients over the age of 75 should be made on an individualized basis in consultation with a healthcare provider. - ● The performance of SimpleScreen CRC has been established in a prospective, cross-sectional study with consecutively enrolled participants. The benefits and risks of programmatic colorectal screening (i.e., repeated testing over an established period of time) with SimpleScreen CRC have not been studied. - ● The prospective, cross-sectional study cohort may have included participants with one first-degree relative diagnosed with colorectal cancer after age 60. - ● Non-inferiority or superiority of SimpleScreen CRC sensitivity as compared to other recommended screening methods for colorectal cancer or advanced precancerous lesions has not been established. - ● Cross-reactivity was observed in analytical studies using samples from subjects with non-CRC cancers, including gastric, pancreatic, liver, bladder, breast, lung, and prostate cancers. - ● Consult the Freenome Blood Collection Kit (BCK) Instructions for Use for additional precautions and limitations specific to the collection and shipping of blood samples. 3 of 50 {3} # V. DEVICE DESCRIPTION The SimpleScreen CRC is performed at Freenome's CLIA-certified laboratory. SimpleScreen CRC is a qualitative in vitro diagnostic (IVD) test used to detect molecular signals (methylation) associated with colorectal cancer (CRC) from whole blood samples collected from individuals at average risk for CRC. A whole blood sample is collected by licensed healthcare providers using the Freenome blood collection kit (BCK) and is then shipped to Freenome's clinical laboratory for testing. SimpleScreen CRC uses circulating cfDNA, from the plasma portion of peripheral whole blood collected in Streck Cell-Free DNA BCT® blood collection tubes (BCTs) that are included in the BCK, to identify DNA methylation patterns associated with CRC and typically not seen in healthy individuals. The test targets genomic regions that are highly methylated in tumor cells but show only limited methylation in cfDNA from healthy individuals. Once laboratory processing of the blood samples is completed, molecular data are analyzed by a proprietary software platform that includes analytical pre-processing of the input data, transformation of pre-processed inputs into the numeric features used by the trained artificial intelligence/machine learning (AI/ML) classification algorithm, and generation of a binary classification. SimpleScreen CRC yields a result that is "Positive", "Negative", or "No Result". A positive result indicates a signal was detected that may suggest the presence of CRC and should be followed by colonoscopy. A negative result indicates the test did not detect a signal associated with CRC. A negative test result does not guarantee absence of advanced colorectal neoplasia. Healthcare providers should work with individuals to continue recommended colorectal cancer screening programs. In some cases, the SimpleScreen CRC may generate a "No Result." If this happens, the ordering physician will be contacted, and the patient may be asked to provide another blood sample. Reagents, materials, and equipment needed to perform the SimpleScreen CRC test are used exclusively in the Freenome clinical laboratory. The only components distributed outside of Freenome are contained within the Freenome BCK. The following components are required to perform the SimpleScreen CRC test: # Blood Collection Kit: The BCK comprises all components used for the collection, stabilization, packaging, and transportation of whole blood samples. The BCK contains two BCTs and packaging materials with instructions for storage, sample collection, and shipping contents to Freenome's laboratory. - Streck cfDNA BCTs (2) - BCT labels - Patient Brochure - BCK Instructions for Use (IFU) - Biohazard bag - Foil pouch 4 of 50 {4} - Absorbent sleeve - Gel pack - Shipping label and envelope # Assay: The following reagents are required to perform the SimpleScreen CRC test and are qualified by Freenome under the Freenome Quality System: - cfDNA extraction kit - Library preparation kit - DNA quantification kit - Next generation sequencing kit and manufacturer-provided sequencer control # Controls: - Positive control - Negative control - Extraction control - No template control # Instrumentation: The following instruments are required to perform the SimpleScreen CRC test and are qualified by Freenome under the Freenome Quality System: - Hamilton Microlab easyBlood STARlet instrument - Thermo Fisher Qubit® Flourometer - Hamilton Microlab STAR instrument - Molecular Devices SpectraMax® Plate Reader - Agilent Bravo instrument - Illumina NovaSeq™ 6000 # Software: SimpleScreen CRC includes software to process data from the patient's sample and return a test report to the physician. The Freenome Software Platform (FRNM-SP) uses the sequencing data from the samples processed in the laboratory. These sequencing data are transformed and processed by an AI/ML-enabled classification model which produces a score and compares it to a pre-defined threshold in order to generate a binary result that is included in a test report. # Test Procedure: The overall SimpleScreen CRC test is comprised of the following elements: - Blood Collection Kit - Pre-Analytic Process - Genomics Workflow - Freenome Software Platform 5 of 50 {5} # Blood Collection Kit The initial step of the SimpleScreen CRC test is the collection of whole blood in two Streck cfDNA BCTs that are provided as part of the BCK. The BCK enables whole blood collection and preservation of cfDNA for downstream analytical processing. The whole blood specimen is collected from the individual by licensed healthcare providers and is then shipped at ambient temperature to Freenome's clinical laboratory. Samples are received and accessioned at Freenome's clinical laboratory. Prior to blood draw, the Freenome BCK may be stored in conditions consistent with its labeling until the expiration date printed on the BCK label. Complete instructions for sample collection and shipping can be found in the BCK Instructions for Use. # Pre-Analytic Process After accessioning, plasma collected from the BCTs is separated from the whole blood using automated instrumentation. The plasma is then aliquoted and frozen for future analytical processing. Whole blood specimens must be processed within 7 days of blood collection. # Genomics Workflow The genomics workflow begins with 2 mL of plasma and assay controls which are plated together for cfDNA extraction. After the extraction process, the extraction control concentration is quantified as an in-process quality control (QC) checkpoint. Samples then proceed to library preparation. Reagents are prepared prior to the associated library preparation steps. Reagent preparation includes the preparation of reagent master mixes used in library preparation, as well as preparation of the quantification reagent used for in-process QC steps. Library preparation includes end repair, adapter ligation, unmethylated cytosine conversion, and polymerase chain reaction (PCR) steps. After PCR, prepared libraries are quantified for multiplexing as an in-process QC checkpoint. Next, the multiplexing process pools a portion of each of the libraries and controls from a single plate together into an individual plate library pool, concentrates the pool, and adds the prerequisite reagents for target enrichment. Target enrichment is performed next, followed by PCR amplification in order to ensure libraries are of sufficient concentration for sequencing. This is confirmed by a quantification step as another in-process QC checkpoint and for normalization and pooling. After pooling, each individual library pool is quantified as an in-process QC checkpoint. A combined library pool can then be created from up to four individual plate pools using the quantification of each individual plate library pool. If a combined library pool was created, it is then quantified as an additional in-process QC checkpoint to ensure sufficient pooled library mass is present for sequencing. 6 of 50 {6} Sequencing is performed and, when complete, the sequencing data are transferred to a data storage location for downstream processing by the software platform. ## Freenome Software Platform (FRNM-SP) The FRNM-SP includes proprietary cloud-based software used for analytical pre-processing and computational transformation of sequencing data, application of the trained AI/ML classification algorithm, and test report generation. Analytical pre-processing of the sequencing data from laboratory-processed samples includes demultiplexing; sequence alignment; QC assessment of the positive control, negative control, and no template control; and sample QC. After analytical pre-processing, the pre-processed sequencing data are used to analyze cfDNA fragment nucleotide sequence and CpG methylation status, and results are computationally transformed to produce the structured quantitative values used as inputs to the AI/ML-enabled classification model. The AI/ML-enabled classification model uses this information to identify hypermethylated fragments (HMFs) across multiple regions of the genome, then aggregates information from all loci with a non-linear integrative function to generate a score. The score is compared to a threshold, yielding a qualitative result. The learned classification threshold was selected to yield a desired specificity, based on behavior in negative samples during training. If the score is greater than or equal to the threshold, the classifier returns a positive call; if the score is less than the threshold, it returns a negative call. Based on the call from the AI/ML classification model, a test report is generated with a diagnostic result that can be either “Positive”, “Negative”, or in some cases “No Result” for the sample. ## Interpretation of Results A test report will be returned to the ordering healthcare provider with a result of positive or negative. Patients are advised to talk with their healthcare provider about their SimpleScreen CRC results. A positive result indicates a signal was detected that may suggest the presence of CRC or APL and should be followed by colonoscopy. A colonoscopy evaluation is necessary to determine whether CRC is present. A negative result indicates the test did not detect a signal associated with CRC. A negative test result does not guarantee absence of CRC. Healthcare providers should work with individuals to continue guideline-recommended colorectal cancer screening programs. In some cases, SimpleScreen CRC may generate a “No Result”. If this happens, the ordering physician will be contacted, and the patient may be asked to provide another blood sample. A “No Result” test may occur for multiple reasons, some of which include 7 of 50 {7} insufficient specimen volume, compromised specimen or blood collection tube, missing sample information, specimen failed QC. # **Quality Control Measures** SimpleScreen CRC utilizes a number of controls as part of the genomics workflow. - The Positive Control (PC) contains sheared genomic DNA from a cell line with a known (hypermethylated fragments) HMF ratio similar to early-stage CRC samples. - The Negative Control (NC) contains PCR-amplified sheared genomic DNA from a cell line that should yield a negative result. (PCR amplification produces effectively unmethylated DNA.) - The Extraction Control (EC) is used to assess cfDNA extraction success and is then removed from further processing. - The No Template Control (NTC) is used to detect nucleic acid contamination in assay reagents, consumables, or instruments. The controls are treated as individual samples in the workflow. As QC measures, the PC must meet a range criteria while the NC and NTC must fall below respective thresholds for reporting valid patient test results. If there is a QC failure during any of these steps, a “No Result” test report is generated for the sample. # **VI. ALTERNATIVE PRACTICES AND PROCEDURES** There are several other alternatives for colorectal cancer screening. Invasive and non-invasive alternatives are available and described below. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle. Recommended screening for CRC includes both invasive and non-invasive options. Invasive options include colonoscopy, flexible sigmoidoscopy, flexible sigmoidoscopy with fecal immunochemical test (FIT), and CT colonography. Non-invasive screening options include stool DNA- or RNA-based CRC screening tests, guaiac-based fecal occult blood testing (gFOBT), FIT, and blood-based plasma DNA testing. Colonoscopy is considered to be the most accurate screening tool available, which can involve the removal of precancerous lesions to prevent cancer. Patients who have a positive or abnormal test by an invasive or non-invasive screening method, except for colonoscopy, warrant further investigation through conventional colonoscopy. A patient should discuss these alternatives with their healthcare provider to select the method that best meets the patient’s needs, expectations and lifestyle. 8 of 50 {8} ## VII. MARKETING HISTORY SimpleScreen CRC has not been marketed as an IVD in the United States or any foreign country. ## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH Due to the nature of the non-invasive blood collection process, potential adverse events (AEs) caused by or related to testing with SimpleScreen CRC are unlikely. The primary risk associated with the SimpleScreen CRC is a false test result (i.e., a false positive or a false negative result). All positive test results should lead to a colonoscopy. In the instance of a false negative result on SimpleScreen CRC, there is a possibility that a case of CRC could go undetected. ## IX. SUMMARY OF NON-CLINICAL STUDIES Non-clinical studies were conducted at Freenome, LLC to evaluate the analytical performance of SimpleScreen CRC. The studies are described below ### A. Algorithm Development and Cutoff Determination The SimpleScreen CRC AI/ML- enabled classification model was developed and trained using a total of 5,168 clinical samples (negative, CRC and APL) that were collected across numerous geographically diverse sites. Of these, 1,966 negatives were used for establishing the system score cutoff to achieve the desired specificity. ### B. Analytical Sensitivity #### 1. C95 Establishment The C95 (Limit of Detection (LoD)) for SimpleScreen CRC was defined as the system score (S) where the test is expected to declare a sample to be positive 95% of the time (also referred to as the C95). To establish the C95 (LoD), 18 clinical blend samples (3 clinical donors × 6 target hypermethylated fragments (4.00x10-4, 2.00x10-4, 1.00x10-4, 5.00x10-5, 2.50x10-5, and 1.25x10⁻⁵ ( per donor) were tested across 2 reagent lots with 48 replicates per sample for each lot. All batches began with 4 ng cfDNA input into library preparation, an input mass below the fifth percentile of the intended use population. Testing for each reagent lot included three (3) processing runs, four (4) batches per processing run, and four (4) replicates per sample for each batch. In total, 1728 replicates were processed, of which 1698 passed QC and were used to establish the C95 (LoD). 9 of 50 {9} The C95 (LoD) was determined by fitting a probit model to the percentage of replicates positive for each sample (hit rate) as the outcome and the corresponding mean system score (S) as the predictor variable. The C95 (LoD) or each reagent lot was determined as the S value corresponding to 95% positive call rate (Table 1). Table 1. C95 Values per Reagent Lot for System Score S | Lot | Calculated C95 (LoD) | | --- | --- | | 1 | 0.712085 | | 2 | 0.736821 | Note: Lot 2 reported an 6.71E-05 HMF for C95 The C95 (LoD) for the SimpleScreen CRC was determined to be the greater LoD value between the two reagent lots, which was system score (S) = 0.736821 (Lot 2). A subsequent C95 study was conducted as part of the precision study, consisting of unmanipulated clinical samples (12 ACN and 12 non-ACN donors, Table 3) along with three clinical blends prepared from 2 CRC and 1 APL donors (the same unmanipulated donors were also run as part of the precision study). The study was conducted across 6 different days, 3 reagent lots, 2 instrument lines and for a total of 24-36 replicates per sample. The C95 (LoD) was re-established and confirmed across the combined data from unmanipulated clinical samples and clinical blends (S = 0.711697). The C95 value of 0.711697 was used as the C95 across all analytical validation studies. ## 2. Limit of Blank The Limit of Blank (LoB) of SimpleScreen CRC was evaluated by confirming that the false positive rate of the assay was 0% for samples lacking HMFs. Four 0% methylated contrived samples were tested across two reagent lots with 96 replicates per lot (4 samples x 24 reps per sample). Testing for each reagent lot included 3 processing runs, 4 batches per processing run, and 2 replicates per sample for each batch. The false positive rate was 0% for both reagent lots (Table 2). Table 2. False Positive Rate for Two Reagent Lots | Lot | N Positive | N Total | False Positive Rate | | --- | --- | --- | --- | | 1 | 0 | 96 | 0.00% | | 2 | 0 | 95 | 0.00% | 10 of 50 {10} ### C. Precision SimpleScreen CRC precision was evaluated across two combined studies using donors selected that had a system score (S) spanning the assay range and included 8 high positives, 7 low positives, 4 borderline, 6 high negatives and 6 low negative samples. The initial precision study evaluated 24 individual clinical donors (12 ACN and 12 non-ACN) with ACN status confirmed by colonoscopy/pathology. The ACN donors included 9 CRC and 3 APLs. The non-ACN donors included 5 colonoscopy confirmed negatives (NEG) and 7 donors with non-advanced precancerous lesions (NAPLs). Repeated measures for each donor were taken across 12 batches that were processed across 6 different days, 3 reagent lots, 2 instrument lines, and up to 2 runs per condition. Donors were tested with 2-3 replicates per batch for a total of 24-36 replicates across the 12 batches. A supplemental precision study was performed with 7 individual clinical donors (2 CRC and 5 APLs) with ACN status confirmed by colonoscopy/pathology. Repeated measures for each donor were taken across 8 batches that were processed across 2 different days, 1 reagent lot and 1 instrument line. Donors were tested with 3 replicates per batch for a total of 24 replicates across the 8 batches. To evaluate the qualitative output from the SimpleScreen CRC AI/ML-enabled classification model, the majority call for each donor was reported and individual replicates were compared to the majority call for each donor. The majority call agreement for each donor is provided in Table 3. The majority call agreement for ACN donors ranged from 52% - 100% and the aggregate majority call agreement across all non-borderline ACN donors was 92.2%. The majority call agreement for non-ACN donors ranged from 86.1% - 100% and the aggregate majority call agreement across all non-borderline non-ACN donors was 94.3%. Table 3. Detection Rates for Clinical Donors | Sample ID | System Score Bin | Colonoscopy Status | Majority Call | Mean System Score | Total Replicates | % Called Positive Test Result | % Called Negative Test Result | | --- | --- | --- | --- | --- | --- | --- | --- | | CD23 | Low Negative | NEG | Negative | 0.395289 | 36 | 0/36 (0%) | 36/36 (100%) | | CD10 | Low Negative | NAPL | Negative | 0.396721 | 36 | 0/36 (0%) | 36/36 (100%) | | CD19 | Low Negative | NAPL | Negative | 0.397109 | 36 | 1/36 (2.78%) | 35/36 (97.22%) | | CD11 | Low Negative | NEG | Negative | 0.398026 | 34 | 1/34 (2.94%) | 33/34 (97.06%) | | CD20 | Low Negative | NEG | Negative | 0.4024 | 36 | 1/36 (2.78%) | 35/36 (97.22%) | | CD18 | Low Negative | NAPL | Negative | 0.402839 | 36 | 2/36 (5.56%) | 34/36 (94.44%) | | CD12 | High Negative | NEG | Negative | 0.405771 | 34 | 1/34 (2.94%) | 33/34 (97.06%) | 11 of 50 {11} | Sample ID | System Score Bin | Colonoscopy Status | Majority Call | Mean System Score | Total Replicates | % Called Positive Test Result | % Called Negative Test Result | | --- | --- | --- | --- | --- | --- | --- | --- | | CD2 | High Negative | NAPL | Negative | 0.408844 | 36 | 4/36 (11.11%) | 32/36 (88.89%) | | CD3 | High Negative | NAPL | Negative | 0.410831 | 36 | 4/36 (11.11%) | 32/36 (88.89%) | | CD6 | High Negative | NAPL | Negative | 0.410973 | 36 | 5/36 (13.89%) | 31/36 (86.11%) | | CD24 | High Negative | NEG | Negative | 0.411744 | 36 | 3/36 (8.33%) | 33/36 (91.67%) | | CD9 | High Negative | NAPL | Negative | 0.412533 | 35 | 2/35 (5.71%) | 33/35 (94.29%) | | CD8 | Borderline | APL | Negative | 0.425219 | 35 | 7/35 (20%) | 28/35 (80%) | | CD5 | Borderline | APL | Negative | 0.435738 | 33 | 8/33 (24.24%) | 25/33 (75.76%) | | CD1 | Borderline | CRC | Negative | 0.44109 | 23* | 7/23 (30.43%) | 16/23 (69.57%) | | CD29 | Borderline | CRC | Positive | 0.463669 | 24 | 12/24 (50%) | 12/24 (50%) | | CD32 | Low Positive | APL | Positive | 0.49472 | 23** | 12/23 (52.17%) | 11/23 (47.83%) | | CD31 | Low Positive | APL | Positive | 0.556103 | 24 | 22/24 (91.67%) | 2/24 (8.33%) | | CD13 | Low Positive | APL | Positive | 0.576681 | 36 | 27/36 (75%) | 9/36 (25%) | | CD28 | Low Positive | APL | Positive | 0.579151 | 24 | 18/24 (75%) | 6/24 (25%) | | CD33 | Low Positive | APL | Positive | 0.637812 | 24 | 21/24 (87.5%) | 3/24 (12.5%) | | CD22 | Low Positive | CRC | Positive | 0.677421 | 36 | 31/36 (86.11%) | 5/36 (13.89%) | | CD30 | Low Positive | APL | Positive | 0.703253 | 24 | 24/24 (100%) | 0/24 (0%) | | CD34 | High Positive | CRC | Positive | 0.887552 | 24 | 24/24 (100%) | 0/24 (0%) | | CD4 | High Positive | CRC | Positive | 0.98949 | 35 | 35/35 (100%) | 0/35 (0%) | | CD16 | High Positive | CRC | Positive | 0.991532 | 34 | 34/34 (100%) | 0/34 (0%) | | CD14 | High Positive | CRC | Positive | 0.999227 | 36 | 36/36 (100%) | 0/36 (0%) | *One replicate failed the in-process QC metric “Library Concentration (ng/μL). **One replicate failed the in-process QC metric “CHH Conversion Rate (%). 12 of 50 {12} | Sample ID | System Score Bin | Colonoscopy Status | Majority Call | Mean System Score | Total Replicates | % Called Positive Test Result | % Called Negative Test Result | | --- | --- | --- | --- | --- | --- | --- | --- | | CD7 | High Positive | CRC | Positive | 0.999525 | 36 | 36/36 (100%) | 0/36 (0%) | | CD21 | High Positive | CRC | Positive | 1 | 36 | 36/36 (100%) | 0/36 (0%) | | CD15 | High Positive | CRC | Positive | 1 | 35 | 35/35 (100%) | 0/35 (0%) | | CD17 | High Positive | CRC | Positive | 1 | 36 | 36/36 (100%) | 0/36 (0%) | *One replicate failed the in-process QC metric “Library Concentration (ng/μL). **One replicate failed the in-process QC metric “CHH Conversion Rate (%). A variance component analysis was conducted on the system score (S) generated by the SimpleScreen CRC AI/ML enabled classification model. The system score mean, standard deviation (SD), and coefficient of variation (%CV) were calculated for each donor (Table 4). The total variability (within-laboratory) ranged from 0% CV to 27% CV. Table 4. Variance Component Estimates for the SimpleScreen CRC System Score for each Clinical Donor. Results are Presented as: Standard Deviation (Coefficient of Variation) | Donor ID | ACN Status | Majority Call | Mean System Score | Repeatability | Within Laboratory | Between | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | | Days | Lot | Instrument | Run | | CD21 | ACN | Positive | 1 | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD17 | ACN | Positive | 1 | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD15 | ACN | Positive | 1 | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD7 | ACN | Positive | 0.999525 | 0.000842 (0.08%) | 0.000862 (0.09%) | 0 (0%) | 0.000183 (0.02%) | 0 (0%) | 0 (0%) | | CD14 | ACN | Positive | 0.999227 | 0.001347 (0.13%) | 0.001362 (0.14%) | 0 (0%) | 0 (0%) | 0 (0%) | 0.000199 (0.02%) | | CD16 | ACN | Positive | 0.991532 | 0.014968 (1.51%) | 0.015416 (1.55%) | 0 (0%) | 0 (0%) | 0.002014 (0.2%) | 0.003091 (0.31%) | | CD4 | ACN | Positive | 0.98949 | 0.032519 (3.29%) | 0.032604 (3.29%) | 0 (0%) | 0 (0%) | 0 (0%) | 0.002344 (0.24%) | 13 of 50 {13} | Donor ID | ACN Status | Majority Call | Mean System Score | Repeatability | Within Laboratory | Between | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | | Days | Lot | Instrument | Run | | CD22 | ACN | Positive | 0.677421 | 0.178979 (26.42%) | 0.178979 (26.42%) | 0.000004 (0%) | 0 (0%) | 0.000004 (0%) | 0 (0%) | | CD13 | ACN | Positive | 0.576681 | 0.155874 (27.03%) | 0.155874 (27.03%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD1 | ACN | Negative | 0.44109 | 0.077757 (17.63%) | 0.077757 (17.63%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD5 | ACN | Negative | 0.435738 | 0.052502 (12.05%) | 0.055782 (12.8%) | 0 (0%) | 0.011989 (2.75%) | 0.014539 (3.34%) | 0 (0%) | | CD8 | ACN | Negative | 0.425219 | 0.051409 (12.09%) | 0.062409 (14.68%) | 0 (0%) | 0.000001 (0%) | 0.000001 (0%) | 0.035382 (8.32%) | | CD9 | non-ACN | Negative | 0.412533 | 0.037887 (9.18%) | 0.037887 (9.18%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD24 | non-ACN | Negative | 0.411744 | 0.04148 (10.07%) | 0.04148 (10.07%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD6 | non-ACN | Negative | 0.410973 | 0.040975 (9.97%) | 0.041849 (10.18%) | 0.008508 (2.07%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD3 | non-ACN | Negative | 0.410831 | 0.040728 (9.91%) | 0.040728 (9.91%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD2 | non-ACN | Negative | 0.408844 | 0.029132 (7.13%) | 0.039957 (9.77%) | 0 (0%) | 0 (0%) | 0.023109 (5.65%) | 0.014624 (3.58%) | | CD12 | non-ACN | Negative | 0.405771 | 0.027024 (6.66%) | 0.027335 (6.74%) | 0 (0%) | 0.004117 (1.01%) | 0 (0%) | 0 (0%) | | CD18 | non-ACN | Negative | 0.402839 | 0.021657 (5.38%) | 0.022442 (5.57%) | 0.005881 (1.46%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD20 | non-ACN | Negative | 0.4024 | 0.014064 (3.5%) | 0.014343 (3.56%) | 0.002811 (0.7%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD11 | non-ACN | Negative | 0.398026 | 0.010401 (2.61%) | 0.011607 (2.92%) | 0.002076 (0.52%) | 0 (0%) | 0.004715 (1.18%) | 0 (0%) | | CD19 | non-ACN | Negative | 0.397109 | 0.015312 (3.86%) | 0.015312 (3.86%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD10 | non-ACN | Negative | 0.396721 | 0.009417 (2.37%) | 0.009417 (2.37%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | | CD23 | non-ACN | Negative | 0.395289 | 0.007055 (1.78%) | 0.008578 (2.17%) | 0.00488 (1.23%) | 0 (0%) | 0 (0%) | 0 (0%) | Based on the clinical study, 21.6% (5846/27010) of the Prevention of Colorectal Cancer Through Multiomics Blood Testing (PREEMPT CRC) participants generated a system score within the range that will yield less than 95% concordance call. #### **D. Reagent Cross Reactivity (In-silico Analysis of Primers and Probes)** The specificity of the library construct primers and target capture probes used in the SimpleScreen CRC test was evaluated against the genomes of 21 species of fungi, bacteria, and viruses commonly present in human blood or on skin surfaces that may interfere with test performance, using publicly available sequences in an in-silico analysis. The study found no potential cross reactivity to the common bacterial, fungal, and viral sources. 14 of 50 {14} ### E. Non-CRC Cancer and Comorbid Conditions Cross-Reactivity Cross-reactivity of SimpleScreen CRC was evaluated by determining the positivity rate in non-CRC cancers and non-cancer conditions that may share analytes with CRC and APLs. Whole blood samples collected from subjects with non-CRC cancers and non-cancer conditions were selected and processed using the SimpleScreen CRC workflow to generate a classification call, which was used to assess cross-reactivity. These samples were collected from subjects with a known diagnosis of cancer or disease, which are not representative of an asymptomatic intended use population. As a result, the positivity rate for these samples may be overestimated. Sample size and types are listed below in Table 5. Table 5. Non-CRC Cancer and Non-Cancer Cross-Reactivity Sample Size | Sample Type | Cancer or Disease | Sample Size | | --- | --- | --- | | Non-CRC Cancers | Prostate Cancer | 27 | | | Breast Cancer | 27 | | | Lung Cancer | 26 | | | Pancreatic Cancer | 26 | | | Kidney Cancer | 7 | | | Bladder Cancer | 9 | | | Stomach Cancer | 5 | | | Liver Cancer | 11 | | Non-Cancer Conditions | Rheumatoid Arthritis | 244 | | | Chronic Obstructive Pulmonary Disease | 154 | | | Diabetes Type II | 1442 | | | Inflammatory Bowel Disease | 27 | | | Chronic Kidney Disease | 117 | | | Systemic Lupus Erythematosus | 52 | | | Hypertension | 7769 | | | Dyslipidemia | 3392 | | | Diverticulitis | 98 | | | Diverticulosis | 169 | | | Chronic Gastritis | 105 | | Non-Cancer Conditions | Esophagitis | 46 | | | Chronic Liver Disease | 229 | | | Coronary Heart Disease/Coronary Artery Disease/Other Cardiovascular Diseases* | 336 | *Other cardiovascular diseases (CVDs) categories are based on CVD prevalence (conditions involved in heart or blood vessels such as myocardial infarction, peripheral artery disease, etc.) and based on patient population data. 15 of 50 {15} Using an established analytical approach, the positive detection fraction was calculated as the number of SimpleScreen CRC positive samples out of the total number of passing samples tested per non-CRC cancer and non-cancer condition. The positivity rates in previously diagnosed non-CRC cancers and non-cancer conditions when tested with SimpleScreen CRC are listed in **Table 6** and **Table 7**, respectively. The cross-reactivity / positivity rates of non-cancer conditions were generally consistent with the false positive rate of the test. **Table 6. Cross-reactivity (Positivity) Rate for Non-CRC Cancers** | Cancer | Percent Positive Call n/N (%) | Annual Incidence Rate per 10000 Subjects (45-84)* | Number of SimpleScreen CRC Positive Calls per 10000 Subjects^{§} | | --- | --- | --- | --- | | Prostate Cancer^{†} | 7/27 (25.9%) | 18.63 | 4.8 | | Breast Cancer | 9/27 (33.3%) | 17.48 | 5.8 | | Lung Cancer | 16/26 (61.5%) | 13.83 | 8.5 | | Pancreatic Cancer | 18/26 (69.2%) | 3.72 | 2.6 | | Kidney Cancer | 0/7 (0.0%) | 4.77 | 0 | | Bladder Cancer | 3/9 (33.3%) | 4.81 | 1.6 | | Stomach Cancer | 4/5 (80.0%) | 1.89 | 1.5 | | Liver Cancer | 11/11 (100.0%) | 2.84 | 2.8 | | TOTAL | | | 27.7 | * Information was obtained from National Cancer Institute (http://seer.cancer.gov; last accessed 09 July 2025) † Value adjusted to represent sex-specific disease state. §Calculated by multiplying positive call rates with annual incidence. 16 of 50 {16} Table 7. Cross-reactivity (Positivity) Rate for Non-Cancer Conditions | Disease | Percent Positive Call n/N (%) | Prevalence Observed in PREEMPT CRC* | Number of SimpleScreen CRC positive calls per 10000 Subjects§ | | --- | --- | --- | --- | | Rheumatoid Arthritis | 24/244 (9.8%) | 0.9% | 8.8 | | Chronic Obstructive Pulmonary Disease | 19/154 (12.3%) | 0.9% | 11.1 | | Diabetes Type II | 176/1442 (12.2%) | 5.9% | 72.0 | | Inflammatory Bowel Disease†^ | 4/27 (14.8%) | 1.3% | 19.2 | | Chronic Kidney Disease | 12/117 (10.3%) | 0.5% | 5.2 | | Systemic Lupus Erythematosus | 2/52 (3.8%) | 0.2% | 0.8 | | Hypertension | 739/7769 (9.5%) | 29.5% | 280.3 | | Dyslipidemia | 303/3392 (8.9%) | 12.6% | 112.1 | | Diverticulitis | 11/98 (11.2%) | 0.5% | 5.6 | | Diverticulosis | 16/169 (9.5%) | 0.6% | 5.7 | | Chronic Gastritis | 4/105 (3.8%) | 0.4% | 1.5 | | Esophagitis | 2/46 (4.3%) | 0.2% | 0.9 | | Chronic Liver Disease | 29/229 (12.7%) | 0.9% | 11.4 | * Prevalence of non-cancer conditions was observed from PREEMPT CRC all enrolled subjects (N=48,995) with the exception of IBD. † Data obtained from Dahlhamer et al.¹ ^Individuals with diagnosed IBD are contraindicated for SimpleScreen CRC. Prevalence estimates for undiagnosed IBD could not be determined from PREEMPT CRC. §Calculated by multiplying positive call rates with PREEMPT CRC observed prevalence and 10000. 17 of 50 {17} | Disease | Percent Positive Call n/N (%) | Prevalence Observed in PREEMPT CRC* | Number of SimpleScreen CRC positive calls per 10000 Subjects§ | | --- | --- | --- | --- | | Coronary Heart Disease/Coronary Artery Disease/Other Cardiovascular Diseases | 44/336 (13.1%) | 1.4% | 18.3 | *Prevalence of non-cancer conditions was observed from PREEMPT CRC all enrolled subjects (N=48,995) with the exception of IBD. † Data obtained from Dahlhamer et al.¹ ^Individuals with diagnosed IBD are contraindicated for SimpleScreen CRC. Prevalence estimates for undiagnosed IBD could not be determined from PREEMPT CRC. §Calculated by multiplying positive call rates with PREEMPT CRC observed prevalence and 10000. ### F. Carryover and Cross-Contamination To evaluate carryover and cross-contamination effects on the SimpleScreen CRC, alternating unmethylated contrived samples and methylated contrived samples were processed in a checkerboard layout in 6 batches, 92 samples per batch plus controls, performed consecutively on a single line of instruments and a single side of the sequencer. A total of 273 unmethylated and 279 methylated contrived sample replicates were used in this study. Of the methylated contrived samples, 279/279 (100.0%) produced valid positive results. Of the unmethylated contrived samples, 269/272 (98.8%) produced valid negative results. The three false positive results all had a DNA input of 20 ng and a system score below the C95 (Limit of Detection). ### G. Interfering Substances To evaluate the impact of potential endogenous and exogenous interfering substances on the performance of SimpleScreen CRC, 24 replicates each of clinical blend and healthy multi-donor pool plasma samples, which simulate CRC-negative samples, were tested in the absence (control) and presence (test) of nine endogenous substances (Table 8) and four exogenous substances (genomic DNA, magnetic beads, ethanol, and molecular index barcodes). The difference in system score drift was 0.4% - 5.1%, which was considered acceptable. No interference on the SimpleScreen CRC test was observed for any of the substances at the concentration tested. 18 of 50 {18} Table 8. Endogenous Interfering Substances Tested | Interfering Substance | Level | | --- | --- | | Bilirubin, unconjugated | 40 mg/dL | | Bilirubin, conjugated | 40 mg/dL | | Hemoglobin | 1,000 mg/dL | | Triglycerides | 1,500 mg/dL | | Albumin | 6 g/dL | | Cholesterol | 400 mg/dL | | Red Blood Cells | 0.4% v/v | | Uric acid | 23.5 mg/dL | | Glucose | 1,000 mg/dL | ### H. Robustness #### 1. Assay Workflow Guardbanding The objective of the guardbanding study was to evaluate the tolerance of the SimpleScreen CRC test to variations in critical assay workflow parameters such as incubation temperatures, reagent volumes, and reagent concentrations, throughout the entire SimpleScreen CRC workflow. Guardbanding for the SimpleScreen CRC test was performed using a risk-based approach to identify assay components for guardbanding and their respective ranges (guardbands). Three sample types, contrived high methylated, contrived low methylated, and healthy donor pooled plasma, and three controls (positive, negative, and no-template) were used to test 43 factors (guardbanded conditions) across the SimpleScreen CRC test from extraction to target enrichment. Robustness of the test performance with respect to the 43 critical assay workflow parameters was observed. Across all guardbanded conditions, contrived high samples demonstrated 100% majority call agreement, contrived low samples maintained 81.0% - 91.3% majority call agreement, and healthy donor plasma reported majority call agreement of 91.7% - 100.0%. 19 of 50 {19} ## **2. DNA Input Mass** An input guardbanding study was conducted to evaluate the robustness of the SimpleScreen CRC assay at different cfDNA input levels. The robustness of SimpleScreen CRC to the cfDNA input level was assessed using a total of four cfDNA blends at the predicted range of cfDNA input mass of the Intended Use Population (IUP). Three cfDNA levels (3.5 ng, 11 ng, and 70 ng) covering the minimum and maximum limits of the predicted 95% intended use population (IUP) distribution were tested with a minimum of 20 replicates per sample per cfDNA input mass level for a total of 288 replicates over 4 batches. The study results demonstrated that the SimpleScreen CRC test produces a 94.5% (75/79, 95% CI: (87.5% - 98.6%) positivity rate at the minimum and a 100% (44/44, 95% CI: (92.0% - 100%) at the maximum limits of the predicted 95% IUP distribution (3.5 ng and 70 ng, respectively) comparable to the standard input mass of 11 ng. ## **I. Stability** ### **1. Whole Blood Stability** Whole blood stability was assessed using whole blood samples collected in Streck cfDNA blood collection tubes (BCTs) from 25 self-identified healthy donors from the intended use population, 9 APL donors, and 35 CRC donors. Blood samples were stored at room temperature (16°C – 25°) for up to seven days before plasma separation and downstream processing with SimpleScreen CRC. From each donor, blood was collected in BCTs from two to three lots with various ages from the date of sterilization (DOS) or date of manufacture (DOM) of the Streck cfDNA BCTs (Lot A: ≤ 30 days from DOS at study initiation, Lot B: 12-18 months from DOM, Lot C: > 18 months from DOM). From each self-identified healthy donor, 18 BCTs were collected with six BCTs from each of the three lots. From the CRC donors and APL donors, four BCTs were collected. CRC donors and APL donors were only collected with either Lot A and Lot C or Lot A and Lot B due to the limited number of BCTs that could be collected from these donor types. For each lot, half of the BCTs from each donor were processed to plasma on the day of receipt at Freenome (Day 1 post blood draw) and the other half were stored for seven days (Day 8 post blood draw) before being processed to plasma. to support stability of whole blood specimens collected in BCTs for up to seven days (Day 8 post blood draw). The study results support whole blood samples, collected in Streck cfDNA BCTs aged up to 18 months past the DOM, are stable when stored at room temperature (16°C – 25°) for up to seven days. An additional stability study was performed to evaluate whether whole blood processed on Day 1 post blood draw can represent the device performance for samples processed on the date of collection. Whole blood was collected in four BCTs from each of 32 unique donors (20 healthy and 12 CRC donors). For each donor, 2 BCTs were processed to plasma within 4 hours of the blood draw and 2 20 of 50 {20} BCTs were processed to plasma on Day 1. The NPA and PPA were evaluated by comparing the binary SimpleScreen CRC results from the test condition (Day 1) to the control (within 4 hours) majority calls; and a PPA of 100% and NPA of 90% were observed demonstrating whole blood processed on Day 1 post blood draw represents the device performance for samples processed on the data of collection. ## **2. Whole Blood Transportation Stability** Whole blood stability across different shipping conditions was assessed using whole blood samples collected in Streck cfDNA BCTs from 21 self-identified healthy donors, 18 colonoscopy-confirmed negative donors, 1 non-advanced adenoma (NAA) donor, 7 APL donors, and 15 CRC donors. Whole blood samples were either processed to plasma on the day of receipt (Day 1 post-collection, control condition) or packaged in the SimpleScreen CRC blood collection kit and exposed to winter or summer shipping conditions. Samples were then stored at room temperature (15°C – 25°C) until either Day 5 or Day 8 post-collection when plasma was isolated. A total of seven study conditions (BCTs stored inverted Summer Day 8, upright Summer Day 8, inverted Winter Day 8, upright Winter Day 8, upright Summer Day 5, upright Winter Day 5, and Day 1 control). From each self-identified healthy donor, blood was collected into 21 BCTs with three replicates (i.e., three BCTs) for each of the seven conditions. For ACN and colonoscopy-confirmed negative/NAA donors, blood was collected into five BCTs with one BCT for each of the four conditions where plasma was isolated on Day 8 post-collection in addition to the control. The PPA was 100% for all conditions (Summer/Winter, Inverted/Upright) tested and the NPA for Winter Upright, Summer Upright, Winter Inverted, and Summer Inverted, was 95.8%, 95.7%, 93.9%, and 91.8%, respectively. ## **3. Plasma Stability: Freeze-Thaw** A freeze-thaw plasma stability study was conducted to evaluate the impact of plasma freeze-thaws on the performance of the SimpleScreen CRC assay. A total of 20 healthy, 10 CRC, and 4 APL samples were tested across five freeze-thaw cycles. No notable degradation in the system score in the stability study was observed over at the fifth freeze-thaw demonstrating plasma samples are stable for up to four freeze-thaw cycles. 21 of 50 {21} #### 4. Plasma Stability: Long-Term A long-term plasma stability study was designed to evaluate the stability of plasma under long-term storage conditions at -80°C (-70°C to -90°C). A total of 18 healthy, 15 CRC and 13 APL samples to be tested across 5 time-points. This study is ongoing, and results were obtained for the T0, T1 (12 months), and T2 (24 months) time-points. No notable degradation in the system score the stability study was observed at 24 months. Currently, this study has demonstrated stability for at least 22 months when stored at -80°C. #### 5. Plasma Stability: On-board An on-board plasma stability study was conducted to evaluate the stability of plasma when stored up to eight hours at room temperature (16°C – 25°C) prior to plasma plating. Two control batches were thawed and then proceeded immediately to plasma plating and two test condition batches were held at room temperature for a total of nine hours. Each batch contained 10 replicates for each of two clinical blend samples as well as 20 replicates for one healthy multi-donor pool (HMDP), resulting in a total of 40 replicates for clinical blends and 40 replicates for HMDP for both the control and test condition. The percent difference in system scores between the control and treatment conditions was less than 2% for both the clinical blends and the HMDP. The PPA for both clinical blends was 100.0% for both the treatment and control condition. The NPA for the HMDP was 87.5% in the treatment condition and 95.0% in the control condition. #### 6. cfDNA Stability cfDNA stability was evaluated with a total of 18 clinical blended samples (3 clinical blends x 6 hypermethylated fragment [HMF] levels), 4 unmethylated (0% methylated) contrived samples from manufacturing events, and batch controls (PC, NC, and NTC) across four timepoints (e.g., baseline, 6.5 months, 16 months, and 19 months) at -20°C. No notable differences were observed between baseline and later timepoints, demonstrating cfDNA stability for 17 months when stored at -20°C. #### 7. In-Use Reagent Stability An in-use stability study was conducted to evaluate the stability of 4 hold-points in the SimpleScreen CRC workflow. The in-use stability study utilized two clinical blend samples and one healthy multi-donor pool plasma sample. Twenty clinical blend replicates were tested per batch per condition (10 replicates per clinical blend) and 20 healthy multi-donor pool replicates tested per batch per condition. One baseline batch and one batch stored at the corresponding condition were run. The percent change between the stored mean values and baseline mean values was calculated. The following conditions were tested: 22 of 50 {22} - Samples can be stored after cfDNA extraction at -20°C for 34 days - Samples can be stored after library preparation at -20°C for 37 days - Samples can be stored after target capture and DNA quantification at -20°C for 32 days - Samples can be stored after normalization and pooling at -20°C for 35 days Test condition samples were tested at the conditions listed above and compared against the baseline values. The in-use stability study demonstrated a PPA of 100% and NPA of ≥90% for all test conditions listed. There was no notable difference in mean system score S between the control and test samples for the four tested hold conditions, supporting the use of the hold points as part of the assay process. ### 8. Reagent Shelf-Life Stability A real-time reagent stability study was conducted to establish the shelf-life of SimpleScreen CRC reagents and external batch controls. Three SimpleScreen CRC reagent lots were tested across six timepoints (T0: after QC release; T1: 2 months; T2: 4 months; T3: 6 months; T4: 8 months; T5: 10 months) with 3 unique clinical blends samples at approximately 1.5 times the C95, 3 unique clinical blend samples at approximately 3 times the anticipated C95, and three healthy multi-donor pool plasma samples. A minimum of 24 replicates per sample type per reagent lot per timepoint were tested. The shelf-life stability of SimpleScreen CRC reagents and external batch controls is 10 months when stored at the appropriate conditions. ### 9. On Board Reagent Stability The purpose of the on-board stability study was to determine reagent stability after using and holding reagents under different process steps including on deck (stored on the relevant instrument) of their respective automation platforms and while off deck (stored separate to instrument, e.g. on the bench) for the genomics portion of the SimpleScreen CRC test workflow. These processes include: - cfDNA extraction kit - Library preparation kit - DNA quantification kit - Next generation sequencing kit and manufacturer-provided sequencer control A total of 60 clinical blend samples, 60 healthy multi-donor pool samples at their native systems core (correlating with ≤ C10), and three of each batch control were 23 of 50 {23} tested across three batches. A total of 20 replicates per condition were tested to evaluate the tolerance of the SimpleScreen CRC assay to the final hold conditions tested. Clinical blends were blended to target C100. The results demonstrated that the time reagents on board are stable for 2 hours. ### 10. Reagent Freeze-Thaw Stability A reagent freeze-thaw stability study was conducted to evaluate the impact of freeze-thaw cycles on reagents used in the SimpleScreen CRC test that are stored at -20°C. In addition, the study evaluated the freeze-thaw stability of intermediate reagents (aged master mixes). The study tested two clinical blends (10 replicates each) and one healthy multi-donor pool (20 replicates) per batch under each test condition. One control batch and two test batches were evaluated to determine the maximum number of freeze-thaw cycles that could be tolerated without affecting assay performance. The percent change in the mean system score between the control and test conditions was evaluated, and the percent drift in the mean system score for the test condition remained within the acceptable range (<10%). The reagent freeze-thaw stability study demonstrated an overall PPA of 95% and NPA of 90%. The study results showed that the SimpleScreen CRC reagents, master mixes, and sequencing reagents are stable for up to four, three, and three freeze-thaw cycles, respectively. ### 11. Output Plate Freeze-Thaw Stability An output plate freeze-thaw stability study was conducted to evaluate the freeze-thaw stability of seven out of the nine safe stopping points in the SimpleScreen CRC workflow where intermediate samples and one on-board stability condition (extraction plate on-board stability) are stored frozen. Two clinical blend samples and one healthy multi-donor pool (HMDP) were used in this study. Twenty clinical blend replicates were tested per batch per condition (10 replicates per clinical blend), and 20 healthy multi-donor pool replicates were tested per batch per condition. Twenty clinical blend replicates were tested per batch per condition (10 replicates per clinical blend) and 20 healthy multi-donor pool replicates tested per batch per condition. An overall PPA of 100% and NPA of 90% was observed. The results support intermediate samples stored at the seven stopping points are stable for up to three freeze-thaw cycles; and the extraction output plate is stable for up to 2.5 hours when stored on-board at room temperature (16°C – 25°C). 24 of 50 {24} ## 12. Reagent Lot Interchangeability A Reagent Lot Interchangeability study was conducted to evaluate the interchangeability of nine critical reagent groups across three reagent lots using 24 clinical blend replicates (eight replicates per three clinical blends) and 24 HMDP replicates (eight replicates per three HMDP samples). For seven of the nine critical reagent groups overall PPA of 100% and an overall NPA of 92% was observed across the nine interchangeability conditions. The remaining two reagent groups demonstrated 100% pass rate and 100% concordance with the reference condition. The overall percent drift in the mean system score for the interchangeability conditions remained within the acceptable range (<10%) for both clinical blends and healthy multi-donor pools (HMDPs). ### J. Animal Studies Not Applicable ### K. Additional Studie #### 1. Streck cfDNA BCT Lot-to-Lot Reproducibility Study A lot-to-lot reproducibility study was conducted to evaluate performance of the Streck cfDNA BCT across lots when used with SimpleScreen CRC. Lot-to-lot reproducibility was evaluated using whole blood samples collected across five BCTs, each from a different lot, from 21 healthy donors and 16 ACN donors from the intended use population. The PPA and NPA were evaluated by comparing the binary SimpleScreen CRC results for each lot to the majority call across lots. A PPA and NPA of 100% were observed. #### 2. Streck cfDNA BCT Tube-to-Tube Repeatability Study A tube-to-tube repeatability study was conducted to evaluate performance between Streck cfDNA BCTs when used with SimpleScreen CRC. Tube-to-tube repeatability was evaluated using whole blood samples collected across five BCTs, each from the same lot, from 20 healthy donors and 19 ACN donors from the intended use population. The PPA and NPA were evaluated by comparing the binary SimpleScreen CRC results for each BCT to the majority call across all BTCs. A PPA and NPA of 100% were observed. 25 of 50 {25} ### 3. Streck cfDNA BCT Preservative Guardbanding Study A guardbanding study was performed to evaluate the performance of the SimpleScreen CRC test in the presence of excess components from the Streck cfDNA BCT preservatives. Samples were collected into standard Streck cfDNA BCTs. One standard preservative condition and four conditions containing excess amounts of preservatives were assessed. Twenty-one (21) self-identified healthy donors, 6 APL donors and 30 CRC donors were included in the study. Fifteen (15) BCTs were collected from each self-identified healthy donors with three replicates (three BCTs) for each condition. From APL donors, five BCTs were collected with one BCT for each condition. From CRC donors, three BCTs were collected with one BCT for conditions A, B, and C or for conditions A, D, and E due to the limited number of BCTs that were collected. The NPA and PPA were evaluated by comparing the binary SimpleScreen CRC results from the test conditions to the control condition majority call. The result of the guardbanding study showed underfilling the BCTs with less than 10 mL of blood may impact the SimpleScreen CRC test results (Table 9). Table 9. Streck cfDNA BCT Preservative Guardbanding Study Design | Condition (BCT Type) | Streck cfDNA BCT Preservative (Relative to the Standard) | | | Healthy NPA | ACN NPA | ACN PPA | | --- | --- | --- | --- | --- | --- | --- | | | Component A | Component B | Preservative Volume | | | | | A (Standard) | 1x | 1x | 1x | 100% | 100% | 100% | | B (2x Component A) | 2x | 1x | 1x | 91.4% | 66.7% | 90.0% | | C (2x Component B) | 1x | 2x | 1x | 97.2% | 77.8% | 90.0% | | D (8 mL Short Draw) | 1x | 1x | 1.25x | 91.4% | 50.0% | 87.5% | | E (5 mL Short Draw) | 1x | 1x | 2x | 86.1% | 66.7% | 87.5% | 26 of 50 {26} #### 4. Streck cfDNA BCT Incomplete and Overmixing Verification Study The purpose of the Streck cfDNA BCT Incomplete and Overmixing study was to evaluate the effect of incomplete or over mixing of blood with the anticoagulant and cell preservative in the Streck cfDNA BCTs on the SimpleScreen CRC test. Samples were collected in BCTs and subjected to three study conditions: standard mixing (10 inversion), incomplete mixing (5 inversions), and over mixing (15 inversions). Twenty-one (21) healthy donors with three replicates (three BCTs) for each condition and 16 ACN (CRC or APL) donors with one replicate (one BCT) for each condition were included in the study. The NPA and PPA were evaluated by comparing the binary SimpleScreen CRC results from the test conditions to the control condition majority call. The PPA for incomplete and over mixing was 100%; and the NPA for incomplete and over mixing was 78.8% and 90.9%, respectively. The study results demonstrate that incomplete and over mixing may impact the SimpleScreen test results. #### X. SUMMARY OF PIVOTAL CLINICAL STUDY The Prevention of Colorectal Cancer Through Multiomics Blood Testing (PREEMPT CRC) study² was a prospective, multi-center study conducted to generate data to support the safety and effectiveness of SimpleScreen CRC as a blood-based screening test for the detection of markers associated with the presence of colorectal cancer (CRC) from whole blood samples. To evaluate the clinical performance of SimpleScreen CRC blood test, the test result (negative or positive) was compared with the histopathological result of the index lesion (i.e. most clinically significant lesion) identified from screening colonoscopy. The primary study objectives were sensitivity for CRC and specificity for ACN. A secondary objective was sensitivity for APLs. Based on this evaluation, SimpleScreen CRC sensitivity (true positive fraction), was 79.2% (57/72) for subjects with a histopathological diagnosis of CRC, and 12.5% (321/2567) for subjects with a diagnosis of advanced precancerous lesion (APL, Category 2.1 – 2.5, Table 11), respectively. For subjects without a diagnosis of CRC or APL, the SimpleScreen CRC specificity (true negative fraction) was 91.5% (22306/24371). #### A. Study Design PREEMPT CRC (ClinicalTrials.gov, Identifier: NCT04369053) was a prospective study enrolling a total of 48,995 participants at 200 sites in the United States and one site in the United Arab Emirates (at a facility affiliated with a US healthcare system). Eligible participants provided signed informed consent and were enrolled from May 19, 2020, to April 1, 2022. However, to mitigate the then unknown impact of the COVID-19 pandemic on the representiveness of the study population, the pivotal clinical validation population consisted of participants consecutively enrolled on or after April 5, 2021, to April 1, 2022, for a total of 31,594 participants. Enrolled subjects were 45 to 85 years within 30 days of enrollment, asymptomatic, at average risk for CRC, and 27 of 50 {27} were willing to undergo blood collection (site-based and mobile phlebotomy) and a standard-of-care (SOC) screening colonoscopy within 90 days of blood draw. The window to complete colonoscopy was increased to 120 days to account for decreased access to care due to the COVID-19 pandemic. The blood draw was completed prior to colonoscopy bowel preparation. To evaluate the performance of SimpleScreen CRC, the test results were compared to the colonoscopy results and histopathological information. All colonoscopy reports and applicable histopathology reports were reviewed by a central pathologist to assign a histopathological category. When multiple lesions were found, the most clinically significant finding was defined as the index lesion (Table 10). Histopathology categories were used to designate the clinical ground truth result to determine the test sensitivity and specificity, as well as positive and negative predictive values. Table 10. Histopathological Categories and Definitions | Subcategory | Findings | | --- | --- | | CRC 1.0 | All Stages (I-IV) | | APL 2.1 | Adenoma with carcinoma in situ or high-grade dysplasia, any size | | APL 2.2 | Adenoma, villous growth pattern (\( \geq 25\% \)), any size | | APL 2.3 | Adenoma \( \geq 1.0 \) cm in size | | APL 2.4 | Serrated lesion, \( \geq 1.0 \) cm (includes sessile serrated adenoma/polyp) | | APL 2.5 | Traditional serrated adenoma (TSA), any size | | NAPL 3.0 | \( \geq 5 \) adenomas, all \( < 1.0 \) cm in size, non-advanced | | NAPL 4.0 | 3 to 4 adenomas, all \( < 1.0 \) cm in size, non-advanced | | NAPL 5.1 | 1 to 2 adenomas, all \( < 1.0 \) cm in size, non-advanced | | NAPL 5.2 | Hyperplastic polyps (HP) \( \geq 1.0 \) cm | | NAPL 5.3 | All sessile serrated lesions (SSL) \( < 1.0 \) cm and HP \( < 1.0 \) cm not in sigmoid or rectum | | NEG 6.1 | Negative and other findings upon histopathological review | | NEG 6.2 | No findings on colonoscopy, no histopathological review | ### B. Clinical Inclusion and Exclusion Criteria For enrollment in PREEMPT CRC, the subjects met all the following criteria: 1. 45-85 years of age (inclusive) within 30 days of enrollment 2. Willing to undergo a SOC colonoscopy within 90 days of blood collection 28 of 50 {28} 3. Considered by a physician or healthcare provider to be average-risk for CRC 4. Able and willing to provide blood samples per protocol 5. Able to comprehend and willing to sign and date the written informed consent document(s) Subjects were excluded from enrolling in PREEMPT CRC if they met any of the following criteria: 1. Family history of CRC 1.1 At least one (1) first-degree relative diagnosed with CRC before the age of 60 (Note: First degree relatives include parents, siblings, and offspring) 1.2 At least two (2) first-degree relatives diagnosed with CRC at any age 2. Known hereditary gastrointestinal cancer syndrome including but not limited to the following: 2.1. Hereditary non-polyposis CRC syndrome or Lynch syndrome 2.2. Familial adenomatous polyposis (FAP) 2.3. Cowden's syndrome 2.4. Juvenile polyposis syndrome 2.5. MUTYH-associated polyposis (MAP) 2.6. Peutz-Jeghers syndrome 2.7. Serrated polyposis syndrome 3. Personal history 3.1. CRC or colorectal adenoma 3.2. History of malignancy (except for non-melanomatous skin cancer) at the time of enrollment, and for the 5 years preceding enrollment 3.3. IBD, including CUC and Crohn's disease 3.4. Total colonic resection 3.5. Cystic fibrosis 3.6. Colonoscopy in the 9 years preceding enrollment with the exception of an incomplete colonoscopy, or for a poor or inadequate bowel prep 3.7. Sigmoidoscopy or CT colonography in the 4 years preceding enrollment 3.8. Stool DNA testing in the 2 years preceding enrollment 3.9. Fecal occult blood testing or fecal immunochemical testing in the 6 months preceding enrollment 29 of 50 {29} 3.10. Requiring an immediate or emergent colonoscopy for the investigation of symptoms 3.11. Solid organ or bone marrow transplantation 3.12. Blood product transfusion in the 120 days preceding enrollment 3.13. Any trauma or surgery requiring overnight inpatient hospitalization in the 30 days preceding enrollment 4. A medical condition which, in the opinion of the investigator, should preclude enrollment into the study. 5. Participated or currently participating in a clinical research study in which an experimental medication has been administered during the 60 days leading up to and including the date of providing informed consent or may be administered through the time of the colonoscopy. 6. Known to be pregnant. # **C. Follow-Up Schedule** Not Applicable # **D. Clinical Performance Measures** # **1. Primary Objective** The primary objectives of the study were to determine the sensitivity of SimpleScreen CRC for CRC and specificity for ACN using screening colonoscopy with histopathology, as ground truth, for assessment of the target condition, within 90 days of the blood draw. # **2. Secondary Objective** The secondary objective was to evaluate the sensitivity of Freenome's test for advanced precancerous lesions (APL). # **E. Accountability of PMA Cohort** To mitigate the then-unknown impact of the COVID-19 pandemic on the representativeness of the study population, the pivotal clinical validation population consisted of participants consecutively enrolled on or after a cutoff date of April 5, 2021, to April 1, 2022. - The pivotal clinical performance study included 31,594 eligible subjects (subjects who met inclusion and exclusion criteria) 30 of 50 {30} - Of the 31,594 subjects, 2,497 subjects were excluded due to loss to follow-up (182 subjects) or colonoscopy not performed (2315 subjects) - Of the remaining 29,097 subjects, 1,275 subjects were excluded due to colonoscopy/histopathology issues (e.g. unevaluable colonoscopy, unconfirmed diagnosis). - Of remaining 27822 subjects, 812 subjects were excluded due to sample issues. Ultimately, a total of 27,010 subjects who had confirmed clinical findings and valid SimpleScreen CRC blood test results comprised the evaluable dataset, with 72 CRC, 2,567 APLs, 8,936 NAPLs and 15,435NEGs. This population was used for the primary analysis of the clinical validation of the SimpleScreen CRC test. (Figure 1).  Figure 1. Accountability Flowchart ### F. Study Population Demographics and Baseline Parameters The demographic and baseline characteristics for subjects in the primary effectiveness cohort (evaluable cohort of 27,010 participants) are presented in Table 11. The average age was 58.1 years and 55.8% were female. Compared to the U.S. census distribution, \( ^{3} \) there was a higher proportion of participants aged 50 to 54 years (32.9% vs 15.6%), lower proportion of those aged 75 years and older (3.0% vs 12.5%), and a higher proportion of females (55.8% vs 51.8%). Regarding race, 8.8% were Asian, 11.2% Black or African American, 73.0% White, 0.3% Native Hawaiian or Other Pacific Islander, and 0.3% American Indian or Alaska Native. Regarding ethnicity, 11.8% were Hispanic or Latino. The mean BMI was 29.4 kg/m \( ^{2} \) . 31 of 50 {31} Table 11. Demographic and Baseline Characteristics for the Primary Effectiveness Cohort | Characteristic | Evaluable Cohort (N=27010)^{I,II} | CRC (N=72)^{I,II} | APL (N=2567)^{I,II} | ACN (N=2639)^{I,II} | No ACN (N=24371)^{I,II} | | --- | --- | --- | --- | --- | --- | | Age (Years)^{III} | | | | | | | n | 27010 | 72 | 2567 | 2639 | 24371 | | Mean (SD) | 58.1 (8.2) | 59.9 (9.0) | 58.8 (8.2) | 58.8 (8.2) | 58.0 (8.2) | | Median | 57.0 | 59.0 | 59.0 | 59.0 | 57.0 | | Min, Max^{IV} | 45.0, 86.0 | 46.0, 82.0 | 45.0, 84.0 | 45.0, 84.0 | 45.0, 86.0 | | Age Category (Years), n (%) | | | | | | | [45 to 49] | 2968 (11.0%) | 7 (9.7%) | 247 (9.6%) | 254 (9.6%) | 2714 (11.1%) | | [50 to 54] | 8899 (32.9%) | 18 (25.0%) | 769 (30.0%) | 787 (29.8%) | 8112 (33.3%) | | [55 to 64] | 8725 (32.3%) | 24 (33.3%) | 885 (34.5%) | 909 (34.4%) | 7816 (32.1%) | | [65 to 74] | 5604 (20.7%) | 19 (26.4%) | 587 (22.9%) | 606 (23.0%) | 4998 (20.5%) | | [75 and greater] | 814 (3.0%) | 4 (5.6%) | 79 (3.1%) | 83 (3.1%) | 731 (3.0%) | | Sex, n (%) | | | | | | | Male | 11934 (44.2%) | 37 (51.4%) | 1413 (55.0%) | 1450 (54.9%) | 10484 (43.0%) | | Female | 15076 (55.8%) | 35 (48.6%) | 1154 (45.0%) | 1189 (45.1%) | 13887 (57.0%) | | Ethnicity, n (%) | | | | | | | Hispanic or Latino | 3189 (11.8%) | 9 (12.5%) | 221 (8.6%) | 230 (8.7%) | 2959 (12.1%) | | Not Hispanic or Latino | 22421 (83.0%) | 62 (86.1%) | 2208 (86.0%) | 2270 (86.0%) | 20151 (82.7%) | | Unknown | 1400 (5.2%) | 1 (1.4%) | 138 (5.4%) | 139 (5.3%) | 1261 (5.2%) | | Race, n (%) | | | | | | | American Indian or Alaska Native | 78 (0.3%) | 0 (0.0%) | 9 (0.4%) | 9 (0.3%) | 69 (0.3%) | | Asian | 2381 (8.8%) | 14 (19.4%) | 132 (5.1%) | 146 (5.5%) | 2235 (9.2%) | | Black or African American | 3038 (11.2%) | 7 (9.7%) | 251 (9.8%) | 258 (9.8%) | 2780 (11.4%) | | Native Hawaiian or Other Pacific Islander | 72 (0.3%) | 0 (0.0%) | 11 (0.4%) | 11 (0.4%) | 61 (0.3%) | | White | 19707 (73.0%) | 48 (66.7%) | 2023 (78.8%) | 2071 (78.5%) | 17636 (72.4%) | | Other | 543 (2.0%) | 2 (2.8%) | 34 (1.3%) | 36 (1.4%) | 507 (2.1%) | | Unknown | 1055 (3.9%) | 1 (1.4%) | 87 (3.4%) | 88 (3.3%) | 967 (4.0%) | | More Than One Race | 136 (0.5%) | 0 (0.0%) | 20 (0.8%) | 20 (0.8%) | 116 (0.5%) | | n | 26943 | 72 | 2561 | 2633 | 24310 | | Mean (SD) | 29.4 (6.4) | 30.6 (7.1) | 30.6 (6.7) | 30.6 (6.7) | 29.2 (6.4) | | Median | 28.3 | 29.1 | 29.5 | 29.5 | 28.2 | | Min, Max | 10.8, 78.6 | 18.7, 50.0 | 15.6, 75.5 | 15.6, 75.5 | 10.8, 78.6 | | Missing | 67 | 0 | 6 | 6 | 61 | Note: I Percentage based on N in column heading. II Columns are not mutually exclusive. III Age was recorded based on year of birth; fractional age was not calculated. IV Per eligibility, the study population included subjects aged 45 to 85, within one month of enrollment. Subjects outside this age range were confirmed by the respective sites to meet the age eligibility at time of enrollment. 32 of 50 {32} ACN = advanced colorectal neoplasia; APL = advanced precancerous lesion; BMI = body mass index; CRC = colorectal cancer; SD = standard deviation ### G. Safety and Effectiveness Results #### 1. Safety Results Adverse effects that occurred in the PMA clinical study There were no serious device-related adverse events (SAEs) observed among the 32,731 subjects enrolled for participation in the clinical validation cohort. The SimpleScreen CRC test has the risk of a false test result (i.e. a false positive or false negative result). All positive test results should be followed by a colonoscopy. False positive SimpleScreen CRC test results could lead to an increased number of unnecessary colonoscopies and associated adverse events related to the colonoscopy procedure. A false negative SimpleScreen CRC test result could lead to colorectal cancer or precancerous lesions remaining undetected. #### 2. Effectiveness Results ##### Primary Effectiveness Evaluation The primary clinical performance measures included sensitivity of the SimpleScreen CRC for the CRC and specificity for ACN. A secondary performance measure was sensitivity for APLs. Additional performance measures were positive predictive value (PPV) for CRC and APL, and negative predictive value (NPV) for ACN. SimpleScreen CRC clinical performance was evaluated for the 27,010 subjects meeting criteria for inclusion in the primary effectiveness clinical performance study population. The cross-tabulated results are summarized in Table 12. The results are summarized in Table 13 and Table 14. Sensitivity for CRC was 79.2%, specificity for ACN was 91.5%, NPV for ACN was 90.8% and PPV for ACN was 15.5%. 33 of 50 {33} Table 12. Distribution of SimpleScreen CRC Result by Histopathological Category, n=27,010 | SimpleScreen CRC Blood Test Result | Colonoscopy/Histopathology Lesion Category Grouping | | | Total | | --- | --- | --- | --- | --- | | | CRC (Category 1) | APL (Category 2) | Non-ACN - NAPL or NEG (Category 3-5 and 6) | | | Positive | 57 | 321 | 2065 | 2443 | | Negative | 15 | 2246 | 22306 | 24567 | | Total | 72 | 2567 | 24371 | 27010 | APL = advanced precancerous lesion; CRC = colorectal cancer; non-ACN = non advanced colorectal neoplasia; NAPL = non-advanced precancerous lesion; NEG = negative Table 13. below presents measures of clinical performance of sensitivity and specificity along with two-sided 95%CIs using the Wilson score method. Table 13: Measures of Clinical Performance: Sensitivity and Specificity | Primary Endpoint | Estimate (%), (n/N), 95% CI | | --- | --- | | CRC Sensitivity | 79.2%, (57/72) 68.4%-86.9% | | ACN Specificity | 91.5%, (22306/24371) 91.2%-91.9% | | CRC Specificity | 91.1% (24552/26938) 90.8% - 91.5%371) | ACN = advanced colorectal neoplasia; CI = confidence interval; CRC = colorectal cancer Table 14. Secondary Objective | Secondary Endpoint | Estimate (%) (n/N), 2-sided 95% CI1 | | --- | --- | | APL Sensitivity | 12.5%, 11.3%-13.8% (321/2567) | APL = advanced precancerous lesion; CI = confidence interval 34 of 50 {34} **Predictive Values** for patients with SimpleScreen CRC test positive and negative results along with two-sided 95%CIs are presented in **Table 15**. - The positive predictive value (PPV) for CRC is a fraction of patients with CRC among the patients with positive SimpleScreen CRC test results. - The PPV for APL is a fraction of patients with APL among the patients with positive SimpleScreen CRC test results. - A negative predictive value (NPV) for CRC is a fraction of patients without CRC among the patients with negative SimpleScreen CRC test results and 1-NPV is a fraction of patients with CRC among the patients with negative SimpleScreen CRC test results. - A negative predictive value for ACN is a fraction of patients without ACN (CRC or APL) among the patients with negative SimpleScreen CRC results. **Table 15. Measures of Clinical Performance: Positive and Negative Predictive Values** | | CRC | APL | Non-ACN (NAPL or Negative) | | --- | --- | --- | --- | | **Among Patients with Positive SimpleScreen CRC test results (N = 2443)** | PPV for CRC 2.3% (57/2443) | PPV for APL 13.1% (321/2443) | 84.5% (2065/2443) | | **Among Patients with Negative SimpleScreen CRC Test results (N = 24567)** | 1-NPV for CRC 0.06% (15/24567) NPV for CRC 99.94% (24552/24567) | 9.1% (2246/24567) | NPV for ACN 90.8% (22306/24567) | | **Among All Patients in the Clinical Performance Study (N = 27010) (Prevalence)** | 0.27% (72/27010) | 9.5% (567/27010) | 90.2% (24371/27010) | In the clinical performance study, - PPV for CRC was 2.3% (57/2443) with 95%CI: (2.0%-2.6%) and NPV for CRC was 99.94% (24552/24567) with 95%CI: (99.91%-99.96%) [1-NPV= 0.06% (15/24567) with 95%CI: (0.04%-0.09%)]. Prevalence of CRC in the pivotal clinical performance study was 0.27%. 35 of 50 {35} - Among SimpleScreen CRC test positive patients, there were 13.1% (321/2443) patients with APL (PPV for APL=13.1%). Prevalence of APL in the clinical study was 9.5%. - Among SimpleScreen CRC test negative patients, there were 90.8% (22306/24567) patients with non-ACN (NAPL or Negative). ### Performance of SimpleScreen CRC Test Stratified by Age Groups Compared to the U.S. 2020 census age and sex distribution, the evaluable dataset included a higher proportion of participants aged 50 to 54 years, a lower proportion of those aged 75 years and greater, and a higher proportion of females (Table 16). Table 16. Age and Sex Distribution | Sex | Age Group | U.S. Estimate^{1} | Evaluable Cohort (27,010) | | --- | --- | --- | --- | | Male | 45 to 49 | 7.67% | 4.74% | | | 50 to 54 | 7.78% | 14.12% | | | 55 to 64 | 15.83% | 14.53% | | | 65 to 74 | 11.44% | 9.39% | | | 75 and older | 5.50% | 1.41% | | Female | 45 to 49 | 7.66% | 6.25% | | | 50 to 54 | 7.81% | 18.83% | | | 55 to 64 | 16.50% | 17.77% | | | 65 to 74 | 12.81% | 11.36% | | | 75 and older | 7.01% | 1.60% | 1: U.S. census age and sex data were referenced from published data tables$^{3}$ ### Age-Adjusted Performance Measures After the primary effectiveness endpoints were evaluated based on observed results, they were also evaluated by adjusting the results to reflect the U.S. census age and sex distribution (Census-adjusted). To evaluate the population-level accuracy and representativeness of observed test performance, a pre-specified analysis was conducted to adjust clinical performance to the age and sex distribution of the U.S. census population. U.S. census-adjusted sensitivity for CRC was 81.1%, specificity for ACN was 90.4%, NPV for ACN was 90.5%, and PPV for ACN was 15.5% (Table 17). The positive predicative value and negative predicative value by age is shown in Table 18. 36 of 50 {36} Table 17. U.S. Census Adjusted Primary and Secondary Effectiveness Endpoints | Primary Endpoint | U.S. Census adjusted^{1} %, 2-sided 95% CI^{2,3}, (n/N) | | --- | --- | | CRC Sensitivity | 81.1%, 71.3%-88.1% (66.43/81.91) | | ACN Specificity | 90.4%, 90.0%-90.7% (21938.03/24280.65) | | Secondary Endpoint | | | APL Sensitivity | 13.7%, 12.4%-15.0% (362.03/2647.44) | 1: U.S. 2020 Census age and sex data were referenced from published data tables3 2: 2-sided 95% CIs for sensitivity and specificity were calculated using the Wilson score method 3: 2-sided 95% CIs for PPV and NPV were calculated based on 2-sided 95% score-based CIs for likelihood ratios (Nam 1995) with constant prevalence Table 18. CRC and APL PPV and NPV by Age | Age | Prevalence of CRC (%) | Prevalence of APL (%) | Percent Positive SimpleScreen Results | CRC PPV (%) | CRC NPV (%) | APL PPV (%) | ACN NPV (%) | | --- | --- | --- | --- | --- | --- | --- | --- | | 45-49 | 0.24 | 8.32 | 5.86 | 4.02 | 100.00 | 14.37 | 92.05 | | 50-59 | 0.26 | 9.06 | 7.27 | 2.62 | 99.93 | 12.29 | 91.12 | | 60-69 | 0.23 | 10.25 | 10.45 | 1.67 | 99.94 | 13.08 | 90.02 | | 70-79 | 0.45 | 10.33 | 15.30 | 2.46 | 99.91 | 14.99 | 90.42 | | 80-84 | 0.74 | 8.09 | 19.85 | 3.70 | 100.00 | 7.41 | 91.74 | | Age Adjusted | 0.30 | 9.48 | 10.03 | 2.51 | 99.94 | 13.02 | 90.85 | CRC = colorectal cancer; APL = advanced precancerous lesion; NPV = negative predictive value; PPV = positive…