CELLSEARCH CIRCULATING TUMOR CELL KIT

K071729 · Veridex, LLC · NQI · Nov 20, 2007 · Immunology

Device Facts

• Record ID: K071729
• Device Name: CELLSEARCH CIRCULATING TUMOR CELL KIT
• Applicant: Veridex, LLC
• Product Code: NQI · Immunology
• Decision Date: Nov 20, 2007
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6020
• Device Class: Class 2

Intended Use

The CellSearch™ Circulating Tumor Cell Kit is intended for the enumeration of circulating tumor cells (CTC) of epithelial origin (CD45-, EpCAM+, and cytokeratins 8, 18+, and/or 19+) in whole blood. The presence of CTC in the peripheral blood, as detected by the CellSearch™ Circulating Tumor Cell Kit, is associated with decreased progression free survival and decreased overall survival in patients treated for metastatic breast or metastatic colorectal cancer. The test is to be used as an aid in the monitoring of patients with metastatic breast or metastatic colorectal cancer. Serial testing for CTC should be used in conjunction with other clinical methods for monitoring breast and colorectal cancer. Evaluation of CTC at any time during the course of disease allows assessment of patient prognosis and is predictive of progression free survival and overall survival.

Device Story

System captures/enumerates circulating tumor cells (CTC) from 7.5mL whole blood. Process: immunomagnetic enrichment using ferrofluid nanoparticles coated with anti-EpCAM antibodies; fluorescent labeling with anti-CK-PE (epithelial), DAPI (nucleus), and anti-CD45-APC (leukocyte exclusion). CellTracks AutoPrep System automates reagent/sample handling; MagNest device uses magnetic field to concentrate labeled cells in cartridge. CellTracks Analyzer II or CellSpotter Analyzer performs automated scanning/imaging. System displays events with co-located CK-PE and DAPI fluorescence in gallery format for clinician review. Clinician classifies events as tumor cells if phenotype is EpCAM+, CK+, DAPI+, CD45-. CTC counts aid monitoring of metastatic breast/colorectal cancer; serial testing provides prognostic information on progression-free and overall survival, used alongside standard clinical methods like radiological imaging.

Clinical Evidence

Prospective multi-center trial (N=430) of metastatic colorectal cancer patients. Primary endpoints: progression-free survival (PFS) and overall survival (OS). Patients with ≥3 CTC/7.5mL had significantly shorter median PFS (4.5 vs 7.9 months) and OS (9.4 vs 18.5 months) compared to <3 CTC. Multivariate Cox regression confirmed CTC count as an independent predictor of survival. Concordance with radiological imaging (RECIST) was 78%.

Technological Characteristics

Immunomagnetic selection and immunofluorescent enumeration system. Components: ferrofluid nanoparticles (magnetic core, polymeric coating, anti-EpCAM antibodies), fluorescent reagents (anti-CK-PE, DAPI, anti-CD45-APC). Automated sample preparation (CellTracks AutoPrep) and analysis (CellTracks Analyzer II/CellSpotter). Connectivity: standalone system. Software: automated image acquisition and gallery display for user classification.

Indications for Use

Indicated for monitoring patients with metastatic breast or metastatic colorectal cancer to assess prognosis and predict progression-free and overall survival via enumeration of circulating tumor cells (CD45-, EpCAM+, CK 8, 18+, and/or 19+) in whole blood.

Regulatory Classification

Identification

An immunomagnetic circulating cancer cell selection and enumeration system is a device that consists of biological probes, fluorochromes, and other reagents; preservation and preparation devices; and a semiautomated analytical instrument to select and count circulating cancer cells in a prepared sample of whole blood. This device is intended for adjunctive use in monitoring or predicting cancer disease progression, response to therapy, and for the detection of recurrent disease.

Special Controls

The device is classified as Class II under regulation 21 CFR 866.6020 with special controls. The special control guidance document "Immunomagnetic Circulating Cancer Cell Selection and Enumeration System" is available at https://www.fda.gov/cdrh/oivd/guidance/1531.pdf

*Classification.* Class II (special controls). The special control for this device is FDA's guidance document entitled “Class II Special Controls Guidance Document: Immunomagnetic Circulating Cancer Cell Selection and Enumeration System.” See § 866.1(e) for availability of this guidance document.

Predicate Devices

• CellSearch™ Circulating Tumor Cell Kit (k062013)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the word "VERIDEX" in all caps. The letters are stylized with a geometric, sans-serif font. Below the word "VERIDEX" is the text "LLC" in a smaller font size.

33 Technology Drive Warren, NJ 07059

# 510(k) SUMMARY

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is K071729

| 807.92 (a)(1): | Name: | Veridex, LLC | |
|----------------|----------|-----------------------------------------------------------------------|-------------|
| | Address: | 33 Technology Drive<br>PO Box 4920<br>Warren, NJ 07059 | NOV 20 2007 |
| | Phone: | [phone] | |
| | FAX: | [phone] | |
| | Contact: | Debra J. Rasmussen<br>Worldwide Executive Director Regulatory Affairs | |

# 807.92 (a)(2): Device Name - trade name and common name, and classification

| Trade name: | CellSearch™ Circulating Tumor Cell Kit | | |
|-----------------|----------------------------------------------------------------------------------------------------------------------------------------------------|--|--|
| Common name: | CellSearch™ Circulating Tumor Cell Kit | | |
| Classification: | Immunomagnetic Circulating Cancer Cell Selection and<br>Enumeration System, Class II, 21 CFR 866.6020, Product<br>Code NQI, Immunology Devices- 82 | | |

# 807.92 (a)(3): Identification of the legally marketed predicate device CellSearch™ Circulating Tumor Cell Kit, K062013

### 807.92 (a)(4): Device Description

The CellSearch" Circulating Tumor Cell Kit contains a ferrofluid-based capture reagent and immunofluorescent reagents. The ferrofluid reagent consists of nanoparticles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting the EpCAM antigen for capturing CTC. After immunomagnetic capture and enrichment, fluorescent reagents are added for identification and enumeration of CTC. The fluorescent reagents include the following: anti-CK-Phycoerythrin (PE) specific for the intracellular protein cytokeratin (characteristic of epithelial cells), DAPI which stains the cell nucleus, and anti-CD45-Allophycocyanin (APC) specific for leukocytes.

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The reagent/sample mixture is dispensed by the CellTracks® AutoPrep® System into a cartridge that is inserted into a MagNest® cell presentation device. The strong magnetic field of the MagNest® device attracts the magnetically labeled epithelial cells to the surface of the cartridge. The CellTracks® Analyzer II or CellSpotter® Analyzer automatically scans the entire surface of the cartridge, acquires images and displays any event to the user where CK-PE and DAPI fluorescence are co-located. Images are presented to the user in a gallery format for final classification. An event is classified as a tumor cell when its morphological features are consistent with that of a tumor cell and it exhibits the phenotype EpCAM+, CK+, DAPI+ and CD45 -.

## 807.92 (a)(5): Intended use

The CellSearch™ Circulating Tumor Cell Kit is intended for the enumeration of circulating tumor cells (CTC) of epithelial origin (CD45-, EpCAM+, and cytokeratins 8, 18+, and/or 19+) in whole blood.

The presence of CTC in the peripheral blood, as detected by the CellSearch™ Circulating Tumor Cell Kit, is associated with decreased progression free survival and decreased overall survival in patients treated for metastatic breast or metastatic colorectal cancer. The test is to be used as an aid in the monitoring of patients with metastatic breast or metastatic colorectal cancer. Serial testing for CTC should be used in conjunction with other clinical methods for monitoring breast and colorectal cancer. Evaluation of CTC at any time during the course of disease allows assessment of patient prognosis and is predictive of progression free survival and overall survival.

# 807.92 (a)(6): Technological Similarities and Differences to Predicate

There have been no material changes to the CellSearch™ Circulating Tumor Cell Kit, this 510(k) is being submitted for an expanded indications for use.

# 807.92 (b)(1): Brief Description of Non-clinical data

### Recovery

Blood samples from a single healthy donor were pooled and five of six 7.5 mL aliquots were spiked with approximately 1300, 325, 81, 20, and 5 cultured breast cancer cells (SK-RR-3). The sixth tube was unspiked pooled blood and served as a zero point. These samples were processed on the CellTracks® AutoPrep® System with the CellSearch™ Circulating Turnor Cell Kit and CTC counts were determined on the CellTracks® Analyzer II. The experiment was repeated for four additional donors. The observed cell counts were plotted aspanst the results of the expected cell count. The results are summarized in Table 1.

| Table 1. Percent Detection Estimates. | | |
|---------------------------------------|-----------------------------------|---------------------------|
| Expected Tumor Cell<br>Count | Mean Observed<br>Tumor Cell Count | Range of Percent Recovery |
| 1300 | 1215 | 91 to 95% |
| 325 | 308 | 82 to 101% |
| 81 | 85 | 80 to 136% |
| 20 | 22 | 95 to 140% |
| 5 | 7 | 120 to 200% |

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To determine the overall, or least squares fit, for the comparison of the observed and expected cell counts across all the data, linear regression analysis was performed. The regression equation for these 30 samples was Y=0.93x + 3.87 with an R=0.999 (R=0.999). The results of this study indicate that on average, over the tested CTC range, the recovery, as derived from regression analysis, is 93%.

Given the linear response of the tumor cell counts, one would expect the slope of the observed versus expected plot to be 1.0. However, the slope was 0.93. This is because the CellTracks® AutoPrep® System with CellSearch™ CTC Kit involves the capture and fluorescent labeling of cells followed by their detection and enumeration by the CellTracks® Analyzer II. The loss of cells could therefore be attributed to one of the following possibilities; 1) the recovery of only 93% of the tumor cells spiked into 7.5mL of blood by the CellTracks® AutoPrep® System, 2) the detection of only 93% of the tumor cells present in the sample chamber by the CellTracks® Analyzer II or 3) a combination of both of these sources of error.

## Linearity / Reportable Range

Another way to examine the previous data is to analyze it as a dilution series to evaluate test linearity. We removed the confounding variable of percent recovery by using the observed value of the initial sample in the dilution series (i.e. the first tube) divided by the dilution factors to determine the expected values for the dilution series for each patient sample. Regression of all of these numbers of observed tumor cells versus the numbers of expected tumor cells yielded a slope of 1.007, an intercept of 3.0, and an R2 = 0.990 (R = 0.995). Therefore, once the percent recovery (cell loss) was factored out of the CTC values of each of the initial samples, the analysis of the data demonstrated that the detection of CTC was linear over the reportable range of 0 to 1238 tumor cells.

### Limits of Detection

One CTC per 7.5 mL can be detected by the CellTracks® Analyzer II resulting in a limit of detection of 1 CTC in a cartridge. Linear regression shows that on average, 93% of CTC present in a 7.5 mL blood sample are recovered using the CellTracks® AutoPrep® System (see Recovery section). The loss of approximately 7% of the CTC in the sample is not sufficient to reduce the limit of detection of 1 CTC.

# Reproducibility:

# a. System Reproducibility with CellSearch™ Circulating Tumor Cell Control

Three separate CellSearch" Circulating Tumor Cell Control samples were prepared and processed each day for over 30 days, per the long run method of NCCLS guideline EP5-A2. Each single-use sample bottle contains a low and a high concentration of cells from a fixed cell line that have been pre-stained with two different fluorochromes. Summary statistics for the high and low control cells is presented below.

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| | Low | High |
|-------------------------------------------------|-----|------|
| N | 99 | 99 |
| Mean cell count | 48 | 969 |
| Total Precision Standard Deviation<br>(ST) % CV | 18% | 5% |

# b. System Reproducibility with Patient Samples

# Metastatic Breast Cancer (MBC)

A total of 163 duplicate blood samples were collected from 47 metastatic breast cancer patients over the course of the clinical study. These samples were processed at multiple sites to determine the reproducibility of CTC measurements. The regression equation for the comparison of these 163 duplicate samples was Y=0.98x + 0.67, R=0.99. Figure 1 shows a scatter plot of the duplicate CTC results in blood from MBC patients plotted on a logarithmic scale, with the threshold of 5 CTC indicated by the dashed lines.

Figure 1. Reproducibility of CTC Counts in Duplicate MBC Samples (n=163) with Average of <5 or ≥5 CTC per 7.5 mL of blood.

Image /page/3/Figure/7 description: The image is a scatter plot comparing Tube 1 CTC Count and Tube 2 CTC Count. The x-axis represents the CTC count for Tube 1, ranging from 0 to 999, while the y-axis represents the CTC count for Tube 2, also ranging from 0 to 999. The plot includes a note indicating that some points may be superimposed, and it provides examples of instance counts, such as 50 instances (31%) where both tubes had 0 CTC.

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# Metastatic Colorectal Cancer (MCRC)

A total 1,627 duplicate blood samples were collected from 430 MCRC patients over the course of the clinical study. These samples were processed at multiple sites to determine the reproducibility of CTC measurements. The regression equation for the comparison of these 1,627 duplicate samples was Y=0.98x + 0.18, R2=0.96. Figure 2 shows a scatter plot of the duplicate CTC results in blood from MCRC patients plotted on a logarithmic scale, with the threshold of 3 CTC indicated by the dashed lines.

Figure 2. Reproducibility of CTC Counts in Duplicate MCRC Samples (n=1627) with Average of <3 or ≥3 CTC per 7.5 mL of blood.

Image /page/4/Figure/4 description: This image is a scatter plot that compares Tube 1 CTC Count and Tube 2 CTC Count. The x-axis represents Tube 1 CTC Count, ranging from 0 to 999, while the y-axis represents Tube 2 CTC Count, ranging from 0 to 999. The plot shows a positive correlation between the two variables, with most of the data points clustered around the lower values. The figure note mentions that there may be more than one point superimposed over another.

there are 975 instances (60%) where both tubes had 0 CTC, 116 instances (7%) where Tube 1 CTC and Tube 2 had 1 CTC, and another 109 instances (7%) where Tube 1 had 1 CTC and Tube 2 had 0 CTC.

# 1 Metastatic Colorectal Cancer (MCRC) Patients

A multi-center prospective, clinical trial was conducted to determine whether the number of CTC predicted disease progression and survival. Metastatic colorectal cancer patients with measurable (N=430) disease starting a new line of therapy were enrolled. Clinical data wrie analyzed on an intent-to-treat basis. Patient demographic information is presented in Table 3.

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#### Veridex, LLC CellSearch™ Circulating Tumor Cell Kit Premarket Notification- Expanded Indications for Use- Colorectal

Baseline CTC count was determined prior to initiation of a new line of therapy. Follow-up CTC counts were determined after the initiation of therapy at approximately 3 to 4 week intervals. For the baseline analyses, Progression Free Survival (PFS) was measured from the time of the baseline blood draw to the diagnosis of progression by CT scans and/or clinical signs and symptoms, and Overall Survival (OS) was measured from the time of baseline blood draw to the time of death. For the follow-up analyses, PFS was measured from the time of the follow-up blood draw to diagnosis of progression or death, and OS was measured from the time of the follow-up blood draw to the time of death.

| Category | Description of Categories | N=430 Patients |
|---------------------------------------|-----------------------------------------------------|------------------------------------------------------------|
| Age at Baseline (in<br>years) | Mean ± Std. Deviation<br>(Median) | $63.0 \pm 12.6$ (64) |
| Years to Metastasis | Mean ± Std. Deviation<br>(Median) | $0.9 \pm 1.4$ (0.1) |
| | | Number of Subjects<br>(% of total) |
| Gender | Female<br>Male | 192 (45%)<br>238 (55%) |
| Race | White<br>Black<br>Other<br>Unknown | 305 (71%)<br>44 (10%)<br>12 ( 3%)<br>69 (16%) |
| Baseline ECOG Score | 0<br>1<br>2<br>Unknown | 196 (46%)<br>187 (43%)<br>31 ( 7%)<br>16 ( 4%) |
| Tumor Type<br>at<br>Primary Diagnosis | Colon<br>Rectal<br>Colorectal<br>Unknown | 292 (68%)<br>71 (17%)<br>66 (15%)<br>1 ( 0%) |
| Stage<br>at<br>Primary Diagnosis | 1<br>2<br>3<br>4<br>Unknown | 12 ( 3%)<br>45 (11%)<br>118 (27%)<br>232 (54%)<br>23 ( 5%) |
| Liver Metastasis | No<br>Yes | 117 (27%)<br>313 (73%) |
| Line of Therapy | 1st Line<br>2nd Line<br>3rd Line | 309 (72%)<br>95 (22%)<br>26 ( 6%) |
| Type of Therapy | Bevacizumab<br>Irinotecan<br>Oxaliplatin<br>Unknown | 243 (56%)<br>103 (24%)<br>253 (59%)<br>26 ( 6%) |

| | | Table 3: MCRC Patient Demographics | |
|--|--|------------------------------------|--|
| | | | |

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#### 1.1 CTC frequencies

Of the total number of 430 MCRC patients, 9 had a baseline blood draw and no follow-up blood draws. Of these 9 patients, four died before a follow-up blood draw could be obtained, two were taken off their therapy due to treatment related toxicity, one patient had surgery to remove their measurable disease, one patient refused further treatment, and one patient refused any further blood draws. Of the remaining patients, 362, 342, 321, and 211 had follow-up blood daily 18the weeks, 3-5 weeks, 6-12 weeks, and 13-20 weeks after the initiation of therapy, respectively. The difference in the number of patients evaluable for PFS and OS at each time point is due to the progression of some patients prior to the blood draw, while the difference in the number of patients at each time point is due to the number of patients with blood draws and evaluablee 'CT results.

Table 4 shows the numbers of patients at each time point excluded from the PFS, OS, or PFS & OS analyses and the reasons for their exclusion.

| Blood<br>Draw<br>Timing | Reasons for Exclusion of MCRC Patients from Analyses:<br>PFS & OS | | | | PFS Only | | OS Only | | Total # of<br>MCRC<br>Patients<br>Evaluable: | |
|-------------------------|-------------------------------------------------------------------|----------------------------------------------------------------|--------------------------------------------------|-------------------------------------|-----------------------------------------------------------|--------------------------------------------------|---------|-----|----------------------------------------------|--|
| | Blood Not<br>Drawn | Blood Drawn 1-<br>7 days after<br>administration<br>of therapy | No Follow-<br>up Beyond<br>Date of<br>Blood Draw | Non-<br>Evaluable<br>CTC<br>Results | Blood<br>drawn after<br>date of<br>disease<br>progression | No Follow-<br>up Beyond<br>Date of<br>Blood Draw | PFS | OS | | |
| Baseline | 1 | 11 | 0 | 5 | 0 | 0 | 413 | 413 | | |
| 1-2<br>Weeks | 68 | 0 | 0 | 5 | 1 | 0 | 356 | 357 | | |
| 3-5<br>Weeks | 88 | 0 | 1 | 8 | 4 | 0 | 329 | 333 | | |
| 6-12<br>Weeks | 109 | 0 | 4 | 7 | 26 | 0 | 284 | 310 | | |
| 13-20<br>Weeks | 219 | 0 | 9 | 8 | 14 | 1 | 180 | 193 | | |

Table 4: Exclusions from Progression Free and Overall Survival Analyses

The CTC results obtained from the follow-up blood draws at 1-2 weeks, 3-5 weeks, 6-12 weeks, and 13-20 weeks after the initiation of therapy were classified as being favorable (< 1 C Wests, unfavorable (≥3 CTC). If more than one CTC result was obtained within any of the designated follow-up timepoints, the CTC result from the blood draw furthest from the baseline blood draw was used.

Table 6 summarizes the total number of MCRC patients and percentage of patients with unfavorable CTC in the clinical trial that differs from the numbers and nercentages of patents with for Progression Free Survival shown in Table 5.

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#### 1.2 Progression Free Survival (PFS) Analysis of MCRC Patients

### PFS Using Baseline CTC Results

413 of the 430 MCRC patients had a baseline CTC result available. For Kaplan-Mejer analysis. patients were segmented into two groups based upon their CTC count at baseline:

- The Favorable group (N=305), represented in green, consisted of patients with < 3 CTC. .
- The Unfavorable group (N=108), represented in red, consisted of patients with ≥3 CTC. .

Median PFS was significantly longer in the Favorable group compared to the Unfavorable group (7.9 vs 4.5 months, respectively). These results are illustrated in Figure 3 or Table 5.

Image /page/7/Figure/7 description: This image is a survival plot that shows the probability of progression-free survival over time. The x-axis represents the time from baseline blood draw in months, and the y-axis represents the probability of progression-free survival. There are two curves on the plot, one for patients with less than 3 CTC and one for patients with greater than or equal to 3 CTC. The median progression-free survival for patients with less than 3 CTC is 7.9 months, while the median progression-free survival for patients with greater than or equal to 3 CTC is 4.5 months.

Figure 3: PFS of MCRC Patients with < 3 or > 3 CTC at Baseline (N=413).

## PFS Using Follow-up CTC Results

For Kaplan-Meier analysis, patients were segmented into two groups based upon their CTC count at each of the various follow-up blood draws. Both patient groups at each of the different follow-up blood draw times after initiation of therapy for PFS are illustrated in Figure 4. PFS times were calculated from the time of each blood draw, and any patient showing evidence of progression prior to a particular blood draw was excluded from the analysis of that and all subsequent follow-up blood draws. Figure 4 illustrates the ability of CTC in MCRC patients

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with < 3 and ≥3 CTC 1-2 weeks, 6-12 weeks, and 13-20 weeks after the initiation of therapy to predict PFS.

- The Favorable group, represented in olive green, blue, purple, and cyan, consisted of . patients with <3 CTC.
- The Unfavorable group, represented in brown, black, grey, and would, consisted of . patients with >3 CTC.

Figure 4: PFS of MCRC Patients with < 3 or > 3 CTC at different times of Follow-Up

Image /page/8/Figure/5 description: This image is a graph that shows the probability of progression-free survival over time. The x-axis represents time from blood draw in months, ranging from 0 to 30. The y-axis represents the probability of progression-free survival, ranging from 0% to 100%. There are multiple lines on the graph, representing different groups of patients based on CTC levels at different time intervals, such as 1-2 weeks, 3-5 weeks, 6-12 weeks, and 13-20 weeks, with sample sizes indicated for each group.

Table 5 summarizes the results of the PFS analysis using the CTC levels and a threshold of >3 CTC/7.5mL at each of the different blood draw time points.

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| 1 | 2 | 3 | 4 | 5 | 6 |
|--------------------------------------|-----|--------------|-----------------------------------------|-----------------------------------------|---------------------|
| Sampling Time<br>After Tx Initiation | N | ≥3<br>CTC | Median PFS in Months (95% CI)<br><3 CTC | Median PFS in Months (95% CI)<br>≥3 CTC | Log-rank<br>p-value |
| Baseline | 413 | 108<br>(26%) | 7.9 (7.0 - 8.6) | 4.5 (3.7 - 6.3) | 0.0002 |
| 1-2 Weeks | 356 | 41<br>(12%) | 7.3 (6.5 - 8.1) | 3.8 (1.9 - 5.1) | <0.0001 |
| 3-5 Weeks | 329 | 39<br>(12%) | 6.8 (6.1 - 7.6) | 1.9 (1.2 - 4.4) | <0.0001 |
| 6-12 Weeks | 284 | 18<br>(6%) | 6.5 (5.8 - 7.7) | 2.0 (0.5 - 2.5) | <0.0001 |
| 13-20 Weeks | 180 | 16<br>(9%) | 6.3 (4.9 - 7.4) | 1.2 (0.1 - 2.3) | <0.0001 |

Table 5: Progression Free Survival (PFS) for MCRC patients with <3 or > 3 CTC at different time points

As illustrated in Figure 4 and Table 5, MCRC patients with elevated CTC (≥3 CTC/7.5mL whole blood) at any of the time points had a much higher likelihood of rapid progression than did those with < 3 CTC. Table 5 column 4 shows the median PFS times for those patients with < < > CTC ranged from 6.3 to 7.9 months and were substantially longer than the median PFS times for those patients with ≥3 CTC, which ranged from 1.2 to 4.5 months (column 5).

# Reduction or Increase in CTC Predicts Improved or Decreased PFS

Elapsed PFS times were calculated from the baseline blood draw. For Kaplan-Meier analysis (Figure 5), MCRC patients were segmented into four groups based upon their CTC counts at baseline, 1-2 weeks, 3-5 weeks, 6-12 weeks, and 13-20 weeks:

- Group 1 (green curve), 303 (70%) patients with <3 CTC at all time points. Seven (2%) of . these patients only had a baseline blood draw while eight (3%) had a single blood draw between their first and last blood draw that had ≥3 CTC;
- . Group 2 (blue curve), 74 (17%) patients with ≥3 CTC prior to the initiation of therapy but who had decreased to <3 CTC at the time of their last blood draw;
- Group 3 (orange curve), 29 (7%) patients with <3 CTC at an early draw (baseline, 1-2 . weeks, and/or 3-5 weeks) but who increased to ≥3 CTC at the time of their last blood draw;
- Group 4 (red curve), 24 (6%) patients with ≥3 CTC at all time points. Three (13%) of these . patients had only a baseline blood draw, one (4%) had only a 3-5 week blood draw, and one (4%) had a single blood draw between their first and last blood draw that had <3 CTC.

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Image /page/10/Figure/1 description: This image is a survival plot that shows the probability of progression-free survival over time, measured in months from a baseline blood draw. The plot compares four groups based on their circulating tumor cell (CTC) counts at different time points. Group 1, with less than 3 CTC at all draws, has the highest median progression-free survival at 8.1 months, while Group 4, with greater than or equal to 3 CTC at all draws, has the lowest at 2.2 months. The log-rank p-values indicate significant differences in survival between the groups, with some comparisons showing p-values less than 0.0001.

Figure 5: A Reduction in CTC Below 3 After the Initiation of Therapy Predicts Longer PFS in MCRC Patients

Figure 5 shows that MCRC patients with >3 CTC at all time points (Group 4) had the shortest median PFS, which was significantly different compared to the median PFS of Group 3, Group 2 and Group 1. The difference in the median PFS between those patients who showed a CTC reduction after the initiation of therapy (Group 2) was significantly longer compared to those patients who showed a CTC increase (Group 3).

#### 1.3 Overall Survival (OS) Analysis of MCRC Patients

### OS Analysis Using Baseline CTC Results

Death occurred in 202 (47%) of the 430 MCRC patients, with a mean follow-up time for the 228 (53%) patients still alive of 12.6 ± 6.5 months (median = 11.0, range = 0.8 - 30.0). At the time of these analyses, 124 (41%) of 305 patients from Favorable group {<3 CTC at baseline) compared to 68 (63%) of 108 from Unfavorable group (>3 CTC at baseline) had died.

For Kaplan-Meier analysis, patients were segmented into two groups based upon their CTC count at baseline:

- . The Favorable group (N=305), represented in green, consisted of patients with <3 CTC.
- The Unfavorable group (N=108), represented in red, consisted of patients with >3 CTC. .

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Median OS was significantly longer in the Favorable group compared to the Unfavorable group (18.5 vs. 9.4 months, respectively). These results are illustrated in Figure 6.

Image /page/11/Figure/2 description: This image is a survival plot that shows the probability of survival over time, measured in months from a baseline blood draw. There are two survival curves, one for patients with less than 3 CTC and one for patients with greater than or equal to 3 CTC. The median overall survival for patients with less than 3 CTC is 18.5 months, while the median overall survival for patients with greater than or equal to 3 CTC is 9.4 months. The Cox Hazard Ratio is 2.5, and the p-value is less than 0.0001.

Figure 6: OS of MCRC Patients with < 3 or > 3 CTC at Baseline (N=413).

# OS Using Follow-up CTC Results

The Kaplan-Meier analyses of both MCRC patient groups at each of the different follow-up blood draw times after initiation of therapy are illustrated in Figure 7. This figure illustrates the ability of CTC in patients with <3 and >> CTC 1-2 weeks, 3-5 weeks, 6-12 weeks and 13-20 weeks after the initiation of therapy to predict time to death in 421 patients with metastatic colorectal cancer. OS times were calculated from the time of each blood draw.

- The Favorable group, represented in olive green, blue, purple, and cyan, consisted of . patients with < 3 CTC.
- The Unfavorable group, represented in brown, black, grey, and mange, consisted of . patients with >3 CTC.

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Image /page/12/Figure/1 description: This image is a survival plot that shows the probability of survival over time in months from a blood draw. There are two groups of patients, one with less than 3 CTC and one with greater than 3 CTC. The survival curves are stratified by the time it took to clear CTCs, with the groups being 1-2 weeks, 3-5 weeks, 6-12 weeks, and 13-20 weeks. The sample sizes for each group are also provided.

Figure 7: OS of MCRC Patients with < 3 or > 3 CTC at different times of Follow-Up.

Table 6 summarizes the results of the OS analysis using the CTC levels and a threshold of ≥3 CTC/7.5mL at each of the different blood draw time points.

| Table 6: Overall Survival (OS) for MCRC patients with <3 or ≥ 3 CTC at different time |
|---------------------------------------------------------------------------------------|
| points |

| | 2 | 3 | 4 | | 6 |
|-----------------------------------------|-----|--------------|------------------------------|------------------|---------------------|
| Sampling Time<br>After Tx<br>Initiation | N | ≥3 CTC | Median OS in Months (95% CI) | | Log-rank<br>p-value |
| | | | <3 CTC | ≥3 CTC | |
| Baseline | 413 | 108<br>(26%) | 18.5 (15.5 - 21.2) | 9.4 (7.5 - 11.6) | <0.0001 |
| 1-2 Weeks | 357 | 41<br>(11%) | 15.7 (14.3 - 18.4) | 6.1 (4.9 - 8.9) | <0.0001 |
| 3-5 Weeks | 333 | 41<br>(12%) | 16.4 (14.1 - 18.3) | 4.4 (2.6 - 8.7) | <0.0001 |
| 6-12 Weeks | 310 | 25<br>(8%) | 15.8 (13.8 - 19.2) | 3.3 (1.8 - 5.6) | <0.0001 |
| 13-20 Weeks | 193 | 21<br>(11%) | 14.6 (12.0 - 21.5) | 3.3 (2.4 - 8.5) | <0.0001 |

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As illustrated in Figure 7 and Table 6 in columns 4 & 5, MCRC patients with ≥3 CTC at any of the time points had a much higher likelihood of dying sooner than did those with <3 CTC. The median OS times for those patients with < 3 CTC ranged from 14.6 to 18.5 months and were substantially longer than the median OS times for those patients with >3 CTC, which ranged from 3.3 to 9.4 months.

# Reduction or Increase in CTC Predicts Improved or Decreased OS

Elapsed OS times were calculated from the baseline blood draw. For Kaplan-Meier analysis (Figure 8), MCRC patients were segmented into four groups based on their CTC counts at baseline, 1-2 weeks, 3-5 weeks, 6-12 weeks, and 13-20 weeks:

- Group 1 (green curve), 303 (70%) patients with <3 CTC at all time points. Seven (2%) of . these patients only had a baseline blood draw while eight (3%) had a single blood draw between their first and last blood draw that had >3 CTC:
- Group 2 (blue curve), 74 (17%) patients with >3 CTC prior to the initiation of therapy but . who had decreased to <3 CTC at the time of their last blood draw;
- Group 3 (arange curve), 29 (7%) patients with <3 CTC at an early draw (baseline, 1-2 ● weeks, and/or 3-5 weeks) but who increased to ≥3 CTC at the time of their last blood draw;
- Group 4 (red curve), 24 (6%) patients with ≥3 CTC at all draw time points. Three (13%) of . these patients had only a baseline blood draw, one (4%) had only a 3-5 week blood draw, and one (4%) had a single blood draw between their first and last blood draw that had <3 CTC.

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Image /page/14/Figure/1 description: This image is a Kaplan-Meier survival curve, showing the probability of survival over time for four different groups. The x-axis represents time from baseline blood draw in months, and the y-axis represents the probability of survival. The four groups are defined by their CTC (circulating tumor cell) counts at different time points, with group 1 having <3 CTC at all draws, group 2 having >3 CTC at baseline and <3 CTC at the last draw, group 3 having <3 CTC at early draw and >3 CTC at the last draw, and group 4 having >3 CTC at all draws. The median overall survival (OS) in months is also provided for each group, along with the p-values for curve comparisons.

Figure 8: A Reduction in CTC Below 3 After the Initiation of Therapy Predicts Longer OS whereas an Increase in CTC Count to 3 or above Predicts Shorter OS in MCRC Patients

Figure 8 shows that MCRC patients who exceed the threshold of 3 CTC at any point after the initiation of therapy had a much higher likelihood of dying sooner. Patients with >3 CTC at all time points (Group 4) had the shortest median OS, which was significantly different compared to the median OS of Group 2, Group 2 and Group 1. Patients with <3 CTC at all time points (Group 1) had the longest median OS, which was significantly different compared to the median OS of Group 4, Group 3 and Group 2. Figure 8 also shows that patients who show a decrease in CTC (Group 2) have a significantly lower risk of death compared to those patients with an increase in CTC (Croup 3).

### Univariate Cox Regression Analysis in MCRC Patients

The following parameters were analyzed using Univariate Cox regression analysis to evaluate association with PFS and OS: gender, stage of disease at diagnosis (1-4), time to metastasis (continuous), patient age (≥65 or <65), site of primary disease (colorectal or rectal or colon), ECOG status before initiation of a new line of therapy (0-2), line of therapy (100 or 200 or 3th, presence of liver metastasis (yes or no), type of therapy (bevacizumab, irinotecan, and/or oxaliplatin included or not), baseline CTC counts (>3 or <3 CTC/7.5mL), and follow-up CTC counts 1-2 weeks, 3-5 weeks, 6-12 weeks and 13-20 weeks after the initiation of therapy (>3 or <3 CTC/7.5mL). Table 7 shows the results of this analysis and presents the Cox hazard ratio (HR) and associated p-value (Wald test of Z statistic) as well as the number of patients in each evaluation.

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| Parameter | | Categories | # of<br>MCRC | PFS Risk from<br>Baseline | | OS Risk from<br>Baseline | |
|-------------------------------|-----------|------------------------------------------------|--------------|---------------------------|-------------|--------------------------|-------------|
| | Positive | Negative | Patients | HR | p-<br>value | HR | p-<br>value |
| Gender | Male (1) | Female (0) | 430 | 1.01 | 0.944 | 1.23 | 0.156 |
| Stage at Primary<br>Diagnosis | | 4 vs. 3 vs. 2 vs. 1 | 407 | 0.98 | 0.734 | 1.09 | 0.330 |
| Time to Metastasis | | Time in Years | 428 | 1.00 | 0.901 | 0.92 | 0.121 |
| Age at Baseline Blood<br>Draw | ≥65 Years | <65 Years | 430 | 1.65 | <0.001 | 1.82 | <0.001 |
| Site of Primary Disease | | Colorectal (2) vs. Rectal<br>(1) vs. Colon (0) | 429 | 1.03 | 0.733 | 1.02 | 0.866 |
| Baseline ECOG Status | | 2 vs. 1 vs. 0 | 414 | 1.32 | 0.002 | 1.65 | <0.001 |
| Line of Therapy | | 3 vs. 2 vs. 1 | 430 | 2.04 | <0.001 | 1.63 | <0.001 |
| Liver Mets | Yes | No | 430 | 0.86 | 0.225 | 1.23 | 0.198 |
| Bevacizumab | Yes | No | 405 | 0.54 | <0.001 | 0.62 | 0.001 |
| Irinotecan | Yes | No | 405 | 1.51 | 0.001 | 1.39 | 0.029 |
| Oxaliplatin | Yes | No | 405 | 0.53 | <0.001 | 0.69 | 0.008 |
| Baseline CTC Number | >3 | <3 | 413 | 1.59 | <0.001 | 2.48 | <0.001 |
| 1 - 2 Week CTC Number | >3 | <3 | 357 | 2.02 | <0.001 | 3.23 | <0.001 |
| 3 - 5 Week CTC Number | >3 | <3 | 334 | 2.19 | <0.001 | 4.23 | <0.001 |
| 6 - 12 Week CTC Number | >3 | <3 | 314 | 4.59 | <0.001 | 10.88 | <0.001 |
| 13 – 20 Week CTC<br>Number | >3 | <3 | 203 | 5.07 | <0.001 | 4.88 | <0.001 |

# Table 7: Univariate Cox Regression Analysis

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# Multivariate Cox Regression Analysis in MCRC Patients

Multivariate Cox regression analyses were conducted to evaluate the independent predictive power of CTC count by adjusting for the effects of the known important clinical factors that are statistically significant in the univariate analyses. CTC were found to be strong predictors of PFS and OS (Table 8).

| Variable | N | PFS Risk from Baseline<br>Hazard Ratio | PFS Risk from Baseline<br>p-value | OS Risk from Baseline<br>Hazard Ratio | OS Risk from Baseline<br>p-value |
|---------------------------------------|-----|----------------------------------------|-----------------------------------|---------------------------------------|----------------------------------|
| Baseline CTC (<3 vs. ≥3) | 373 | 1.76 | <0.001 | 2.46 | <0.001 |
| Age at Baseline (<65 vs. ≥65) | | 1.47 | 0.002 | 1.84 | <0.001 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.16 | 0.107 | 1.48 | 0.001 |
| Line of Therapy (1st vs. 2nd vs. 3rd) | | 1.59 | <0.001 | 1.41 | 0.009 |
| Bevacizumab (No vs. Yes) | | 0.65 | 0.001 | 0.68 | 0.021 |
| Irinotecan (No vs. Yes) | | 0.76 | 0.156 | 1.25 | 0.363 |
| Oxaliplatin (No vs. Yes) | | 0.57 | 0.002 | 1.00 | 0.984 |
| 1 - 2 Week CTC (<3 vs. ≥3) | 321 | 1.76 | 0.003 | 2.77 | <0.001 |
| Age at Baseline (<65 vs. ≥65) | | 1.53 | 0.001 | 1.85 | <0.001 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.26 | 0.025 | 1.54 | 0.001 |
| Line of Therapy (1st vs. 2nd vs. 3rd) | | 1.76 | <0.001 | 1.62 | 0.001 |
| Bevacizumab (No vs. Yes) | | 0.66 | 0.003 | 0.77 | 0.156 |
| Irinotecan (No vs. Yes) | | 0.67 | 0.066 | 1.25 | 0.402 |
| Oxaliplatin (No vs. Yes) | | 0.53 | 0.002 | 0.97 | 0.904 |
| 3 - 5 Week CTC (<3 vs. ≥3) | 302 | 2.35 | <0.001 | 4.54 | <0.001 |
| Age at Baseline (<65 vs. ≥65) | | 1.58 | 0.001 | 2.06 | <0.001 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.16 | 0.149 | 1.33 | 0.032 |
| Line of Therapy (1st vs. 2nd vs. 3rd) | | 1.74 | <0.001 | 1.65 | 0.001 |
| Bevacizumab (No vs. Yes) | | 0.68 | 0.007 | 0.86 | 0.410 |
| Irinotecan (No vs. Yes) | | 0.58 | 0.012 | 0.99 | 0.966 |
| Oxaliplatin (No vs. Yes) | | 0.47 | <0.001 | 0.88 | 0.594 |
| 6 - 12 Week CTC (<3 vs. ≥3) | 279 | 3.04 | <0.001 | 9.43 | <0.001 |
| Age at Baseline (<65 vs. ≥65) | | 1.43 | 0.013 | 1.73 | 0.005 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.30 | 0.027 | 1.53 | 0.004 |
| Line of Therapy (1st vs. 2nd vs. 3rd) | | 1.73 | <0.001 | 1.20 | 0.282 |
| Bevacizumab (No vs. Yes) | | 0.61 | 0.001 | 0.82 | 0.337 |
| Irinotecan (No vs. Yes) | | 0.78 | 0.258 | 1.47 | 0.181 |
| Oxaliplatin (No vs. Yes) | | 0.62 | 0.020 | 1.35 | 0.278 |
| 13 - 20 Week CTC (<3 vs. ≥3) | 186 | 4.50 | <0.001 | 4.97 | <0.001 |
| Age at Baseline (<65 vs. ≥65) | | 1.26 | 0.218 | 1.55 | 0.061 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.13 | 0.417 | 1.13 | 0.526 |
| Line of Therapy (1st vs. 2nd vs. 3rd) | | 1.68 | 0.004 | 1.12 | 0.628 |
| Bevacizumab (No vs. Yes) | | 0.68 | 0.058 | 0.89 | 0.655 |
| Irinotecan (No vs. Yes) | | 0.73 | 0.311 | 1.20 | 0.636 |
| Oxaliplatin (No vs. Yes) | | 0.65 | 0.135 | 1.31 | 0.477 |

Table 8: Multivariate Cox Regression Analysis

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#### 1.4 Use of CTC to Monitor Clinical Status of Metastatic Colorectal Cancer

# Relationship between survival, CTC, and disease assessment by imaging

Radiological imaging is one of the primary means used to determine disease status and response to therapy in metastatic colorectal cancer patients. To establish the relationship of clinical status as determined by imaging to CTC, CTC measured at two different timepoints and imaging results were compared 1) to the true clinical endpoint overall survival and 2) to each other.

# CTC

Previous data has shown that metastatic colorectal cancer patients with ≥3 CTC / 7.5mL of blood at any succeeding follow-up visit after the initiation of therapy had a higher likelihood of progressive disease and decreased overall survival compared to patients with < 3 CTC / 7.5mL of blood. The CTC results obtained 3-5 weeks after the initiation of therapy as well as the CTC results obtained within ± one month of the imaging study were classified as Favorable (<3 CTC) and Unfavorable (≥3 CTC). If more than one CTC value was obtained within + one month of the imaging study, the CTC result obtained closest to the imaging study was used.

# Imaging

Each MCRC patient had to have measurable disease, i.e. a minimum of one 2cm lesion up to and including a maximum of 10 such lesions. The method of imaging for each patient was determined by the treating oncologist in keeping with the current standard of care. Either CT or MRI of the chest, abdomen and pelvis were performed with the requirement that all lesions seen at baseline were followed using the same method for all subsequent imaging studies. Image interpretation was performed by a certified radiologist at the participating site using RECIST uni-dimensional criteria to classify each follow-up disease assessment as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD).

Each patient was imaged at a minimum of two time points up to 8 different time points. These studies included a baseline image, imaging at subsequent intervals of 2-3 months (6-12 weeks), and a final image study when the patient went off study. Copies of all patients' imaging studies were forwarded to the study coordinator at each clinical site for filing with the patient clinical data.

Out of the total of 430 evaluable MCRC patients enrolled into the study, 28 (7%) did not have a follow-up imaging study performed, 18 (4%) died before a follow-up imaging study could be performed, and 384 (89%) had one or more follow-up imaging studies performed that were assessed using RECIST criteria. At the time of the 1st follow-up in the 384 patients with a follow-up imaging study, 4 (1%) showed a complete response, 117 (31%) showed a partial response, 186 (48%) had stable disease, and 77 (20%) showed progressive disease. For the purposes of these analyses, patients who died before a follow-up imaging study were considered to have progressive disease.

For response to therapy at the first follow-up disease assessment, the Favorable group was defined as those having stable disease (S), partial response (PR) or a complete response (CR) by RECIST criteria (non-progressive disease, NPD) and the Unfavorable group as those with progressive disease or death (PD).

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# Relationship between survival to imaging and CTC

Separate Kaplan-Meier analyses were performed to compare the overall survival of MCRC patients in the Favorable (<3 CTC) and Unfavorable (>3 CTC) groups using CTC results at two different time points and the first follow-up imaging study. Using results from the first followup imaging studies performed 9.1 + 2.9 weeks (median = 8.6 weeks) after initiation of therapy (i.e. the baseline blood draw), the median survival of the 307 (76%) patients determined by imaging to have NPD was 19.1 months (95% CI = 17.0 to 23.1) (Figure 9, Table 9). For the 95 (24%) patients determined by imaging to have PD, the median survival was 5.8 months (95% CI= 4.4 to 7.7).

A total of 320 MCRC patients had imaging studies performed before and after initiation of therapy or they died prior to a follow-up imaging study being performed and they had CTC assessed 3-5 weeks after initiation of therapy (average = 3.8 + 0.7 weeks from the time of the baseline blood draw, median = 4.0 weeks). The median survival of 282 (88%) patients with Favorable CTC results (<3 CTC) was 17.3 months (95% Cl = 15.0 to 19.5 months) (Figure 10. Table 9). The 38 patients (12%) with Unfavorable CTC results (≥3 CTC) had a median survival of 5.4 months (95% CI = 3.6 to 9.4 months).

To determine if CTC assessments performed closer to the imaging resulted in similar survival prospects compared to CTC assessments performed approximately 4 weeks after the initiation of therapy, only those patients with CTC assessments performed within + one month of the first follow-up imaging study were analyzed (Figure 11, Table 9). Three hundred and sixtyfour (364) of the 402 patients (91%) had CTC assessments within one month of the first followup imaging study, which was performed 9.0 + 2.9 weeks (median = 8.5 weeks) after the initiation of therapy. The median survival of 335 (92%) patients with Favorable CTC results was 17.2 months (95% CI = 15.0 to 19.2 months). For the 29 (8%) patients with Unfavorable CTC results, the median survival was 5.4 months (95% CI = 3.2 to 7.5 months). These data show that CTC assessments at both time points provide similar results to imaging conducted approximately nine weeks after the initiation of therapy.

Applying multivariate Cox regression analysis to adjust for imaging indicates that both CTC and imaging at 6-12 weeks are independently associated with overall survival but that CTC [adjusted hazard ratio: 7.9 (4.6-13.6)] are a stronger predictor than imaging [adjusted hazard ratio: 3.1 (2.1-4.6)].

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| | N | Median Survival & (95% CI) in Months |
|------------------------------|-----------|--------------------------------------|
| A. Imaging | 402 | |
| Favorable (NPD) | 307 (76%) | 19.1 (17.0-23.1) |
| Unfavorable (PD) | 95 (24%) | 5.8 (4.4 - 7.7) |
| B. 3-5 week CTC | 320 | |
| Favorable (<3 CTC) | 282 (88%) | 17.3 (15.0 - 19.5) |
| Unfavorable (≥3 CTC) | 38 (12%) | 5.4 (3.6 - 9.4) |
| C. CTC (±1 month of Imaging) | 364 | |
| Favorable (<3 CTC) | 335 (92%) | 17.2 (15.0 - 19.2) |
| Unfavorable (≥3 CTC) | 29 (8%) | 5.4 (3.2 - 7.5) |

Table 9: OS of MCRC Patients with CTC assessment approximately one month after the initiation of therapy and within one month of the radiological assessment

Figure 9: Correlation of Radiological and CTC Assessment with OS: OS of MCRC Patients with NPD or PD at 1st Follow-Up Imaging Study (N=402)

Image /page/19/Figure/4 description: This image is a survival plot showing the probability of survival over time. The x-axis represents the time from baseline blood draw in months, ranging from 0 to 30. The y-axis represents the probability of survival, ranging from 0% to 100%. There are two survival curves, one for NPD (No Progressive Disease) and one for PD (Progressive Disease), with median overall survival (OS) of 19.1 months and 5.8 months, respectively.

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100% CTC / 7.5mL Median OS in at 3-5 Weeks N (%) Months (95% C.I.) 90% <3 СТС 282 (88%) 17.3 (15.0 to 19.5) 5.4 ( 3.6 to 9.4) >3 CTC 38 (12%) 80% Cox Hazard Ratio = 4.1 70% chi-square = 34.85 robability of Surv (p-value < 0.0001) 60% 17.3 Months 50% 5.4 Logrank p < 0.0001 Months ! 40% 30% 20% 10% 0% 2 12 10 14 0 6 8 16 18 20 22 24 28 30 4 26 Time from Baseline Blood Draw (Months)

Figure 10: Correlation of Radiological and CTC Assessment with OS: OS of MCRC Patients with <3 or >3 CTC at 1st Follow-Up after Initiation of Therapy (N=320)

{21}------------------------------------------------

Image /page/21/Figure/1 description: The image is a title for a figure. The title is "Figure 11: Correlation of Radiological and CTC Assessment with OS: OS of MCRC Patients with <3 or ≥3 CTC within +1 Month of 1st Follow-Up Imaging Study or Death (N=364)". The title describes the figure as showing the correlation of radiological and CTC assessment with OS of MCRC patients. The patients have less than 3 or greater than or equal to 3 CTC within +1 month of the 1st follow-up imaging study or death. The sample size is 364.

Image /page/21/Figure/2 description: This image is a survival plot that shows the probability of survival over time in months. There are two survival curves, one for patients with less than 3 CTC and one for patients with greater than or equal to 3 CTC. The median overall survival for patients with less than 3 CTC is 17.2 months, while the median overall survival for patients with greater than or equal to 3 CTC is 5.4 months. The Cox Hazard Ratio is 7.3, the chi-square is 48.34, and the p-value is less than 0.0001.

Concordances between CTC and Radiological Monitoring in MCRC Patients

As noted above, imaging studies are a major component of the current standard of care for determining disease progression and response to treatment in the metastatic colorectal cancer setting. To further support the effectiveness of CTC in making these clinical assessments, twoby-two tabulations of concordant and discordant observations between CTC and radiological imaging were constructed.

For response to therapy, the Favorable group was defined as those having stable disease (S), partial response (PR) or a complete response (CR) by RECIST criteria (non-progressive disease, NPD) and the Unfavorable group as those with progressive disease (PD). Out of the 18 patients who died prior to a follow-up imaging study, 10 had a follow-up blood draw within 30 days of death and these 10 patients were classified as having progressive disease (PD) for the purposes of these comparisons.

The CTC results obtained within + one month of the imaging study were classified as Favorable (<3 CTC) and Unfavorable (≥3 CTC). If more than one CTC value was obtained within + one month of the imaging study, the CTC result obtained closest to the date of the imaging study was used. This analysis used all evaluable blood draws from the patients to match up CTC with the imaging studies, not just the ones that were selected for the designated timepoints as described in 1.1 above.

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A total of 366 MCRC patients had CTC results within one month of the imaging study or death. The result of this "patient-wise" comparison between CTC and imaging (or death) is shown in Table 10.

| Response at 1st Follow-Up<br>Imaging Study | CTC within +/- 1 Month of<br>Imaging Study or Death | | Total |
|--------------------------------------------|-----------------------------------------------------|--------------|-------|
| | <3 CTC/7.5mL | ≥3 CTC/7.5mL | |
| Non-Progressive Disease | 272 | 13 | 285 |
| Progressive Disease | 65 | 16 | 81 |
| Total | 337 | 29 | 366 |

# Table 10: MCRC Patient-Wise Comparison of CTC and Imaging

| Measurement | Estimate | Lower<br>95% CI | Upper<br>95% CI |
|---------------------------|----------|-----------------|-----------------|
| Positive % Agreement | 20% | 12% | 30% |
| Negative % Agreement | 95% | 92% | 98% |
| Positive Predictive Value | 55% | 36% | 74% |
| Negative Predictive Value | 81% | 76% | 85% |
| Overall Agreement | 79% | 74% | 83% |
| Odds Ratio | 5.2 | 2.4 | 11.2 |

Of the 384 MCRC patients with one or more follow-up imaging studies, a total of 911 imaging studies that rendered a useable radiological response were performed. A total of 805 of the 911 (88%) imaging studies had CTC results obtained within ± one month of the imaging study. Of the 18 patients who died prior to a follow-up imaging study, 10 had a follow-up blood draw within 30 days of death and these 10 patients were classified as having progressive disease (PD) for the purposes of these comparisons. The result of this "observation-wise" comparison between CTC and imaging (or death) in the 815 observations is shown in Table 11.

Table 11: Observation-Wise Comparison of CTC and Imaging

| Response at All Follow-Up<br>Imaging Studies | CTC within +/- 1 Month of<br>Imaging Study or Death | | Total |
|----------------------------------------------|-----------------------------------------------------|----------------|-------|
| | <3 CTC / 7.5mL | ≥3 CTC / 7.5mL | |
| Non-Progressive Disease | 597 | 33 | 630 |
| Progressive Disease | 147 | 38 | 185 |
| Total | 744 | 71 | 815 |

| Measurement | Estimate | Lower<br>95% CI | Upper<br>95% CI |
|---------------------------|----------|-----------------|-----------------|
| Positive % Agreement | 21% | 15% | 27% |
| Negative % Agreement | 95% | 93% | 96% |
| Positive Predictive Value | 54% | 41% | 65% |
| Negative Predictive Value | 80% | 77% | 83% |
| Overall Agreement | 78% | 75% | 81% |
| Odds Ratio | 4.7 | 2.8 | 7.7 |

{23}------------------------------------------------

In serial observations, only a minority of the transitions for imaging results between non-progressive disease and progressive disease coincided with a matching transition of CTC counts between <3 and >3 CTC / 7.5 mL.

Because the prognostic value of the CTC results at an earlier time-point were equivalent to that of the CTC results at the time of imaging (Figure 10 & Figure 11), a patient-wise comparison using results from only the 1st follow-up imaging study, performed approximately 9 weeks after the initiation of therapy, and the CTC results obtained approximately 4 weeks after initiation of therapy was constructed. A total of 320 (80%) of the 402 patients had CTC results 3-5 weeks after the initiation of therapy. The result of this "patient-wise" comparison between CTC at an earlier time point and imaging (or death) is shown in Table 12.

| | | | | | Table 12: MCRC Patient-Wise Comparison of CTC and Imaging | | | | on of the comments of the comments of the comments of the comments of the contribution of the contribution of the contribution of the contribution of the contribution of the |
|--|--|--|--|--|-----------------------------------------------------------|--|--|--|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
|--|--|--|--|--|-----------------------------------------------------------|--|--|--|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|

| Response at 1st Follow-Up<br>Imaging Study | CTC 3-5 Weeks<br>After Initiation of Therapy | | Total |
|--------------------------------------------|----------------------------------------------|-------------------|-------|
| | <3 CTC /<br>7.5mL | ≥3 CTC /<br>7.5mL | |
| Non-Progressive Disease | 228 | 18 | 246 |
| Progressive Disease | 54 | 20 | 74 |
| Total | 282 | 38 | 320 |

| Measurement | Estimate | Lower<br>95% CI | Upper<br>95% CI |
|---------------------------|----------|-----------------|-----------------|
| Positive % Agreement | 27% | 17% | 39% |
| Negative % Agreement | 93% | 89% | 96% |
| Positive Predictive Value | 53% | 36% | 69% |
| Negative Predictive Value | 81% | 76% | 85% |
| Overall Agreement | 78% | 73% | 82% |
| Odds Ratio | 4.7 | 2.3 | 9.5 |

# CTC as an Adjunct to Imaging

While the overall agreement between CTC and imaging was good (approximately 78%), there was disagreement in approximately 22% of the MCRC patients. As the information from CTC assessments is intended to be used in conjunction with other diagnostic modalities to make treatment decisions, CTC assessment 3-5 weeks after the initiation of therapy and imaging in the following groups were compared to OS to determine which of the discordant results hetter reflected the prognosis of the patient:

- Group 1 (green curve), 228 (71%) patients with <3 CTC at 3-5 weeks and NPD; .
- Group 2 (blue curve), 54 (17%) patients with <3 CTC at 3-5 weeks and PD; ●
- Group 3 (news curve), 18 (6%) patients with ≥3 CTC at 3-5 weeks and NPD; .
- Group 4 (red curve), 20 (6%) patients with ≥3 CTC at 3-5 weeks and PD. .

{24}------------------------------------------------

Figure 12 suggests that CTC determination is a strong independent predictor of overall survival. This figure also suggests that the combination of CTC and radiological assessments provides the most accurate assessment of prognosis.

Figure 12: OS of MCRC Patients in Groups 1, 2, and 4 using CTC 3-5 Weeks after Initiation of Therapy (n=320) and the Disease Status Determined at the 1st Follow-Up Imaging Study

Image /page/24/Figure/3 description: This image is a survival plot that shows the probability of survival over time for different groups of patients. The x-axis represents time from baseline blood draw in months, and the y-axis represents the probability of survival. There are three groups shown on the plot: Group 1 with less than 3 CTC and NPD, Group 2 with less than 3 CTC and PD, and Group 4 with greater than 3 CTC and PD. The median overall survival in months is also shown for each group, with Group 1 having 19.1 months, Group 2 having 9.1 months, and Group 4 having 3.6 months.

{25}------------------------------------------------

Image /page/25/Picture/1 description: The image shows the logo for the Department of Health & Human Services. The logo is a circular seal with the words "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" around the perimeter. Inside the circle is an abstract image of an eagle or bird-like figure. The image is black and white.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

# NOV 2 0 2007

Veridex, LLC A Johnson and Johnson Company c/o Ms. Debra J. Rasmussen Worldwide Executive Director Regulatory Affairs 33 Technology Drive Warren. NJ 07059

Re: k071729

Trade/Device Name: CellSearch™ Circulating Tumor Cell Kit Regulation Number: 21 CFR 866.6020 Regulation Name: Immunomagnetic circulating cancer cell selection and enumeration system Regulatory Class: Class II Product Code: NQI Dated: October 29, 2007 Received: October 30, 2007

Dear Ms. Rasmussen:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to

{26}------------------------------------------------

Page 2 -

begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at [phone]. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at 240-276-3474. For questions regarding the reporting of device adverse events (Medical Device Reporting (MDR)), please contact the Division of Surveillance Systems at 240-276-3464. You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number [phone] or [phone] or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely vours.

Josephine Bautista
Robert L. Becker, Jr., M.D., Ph.D.

Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

{27}------------------------------------------------

Veridex. LLC CellSearch™ Circulating Tumor Cell Kit Premarket Notification- Expanded Indications for Use- Colorectal

### INDICATIONS FOR USE

#### 510(K) Number (if known): K071729

Device Name: CellSearchTM Circulating Tumor Cell Kit

#### Indications for Use:

The CellSearch™ Circulating Tumor Cell Kit is intended for the enumeration of circulating turnor cells (CTC) of epithelial origin (CD45-, EpCAM+, and cytokeratins 8, 18+, and/or 19+) in whole blood.

The presence of CTC in the peripheral blood, as detected by the CellSearch™ Circulating Tumor Cell Kit, is associated with decreased progression free survival and decreased overall survival in patients treated for metastatic breast or metastatic colorectal cancer. The test is to be used as an aid in the monitoring of patients with metastatic breast or metastatic colorectal cancer. Serial testing for CTC should be used in conjunction with other clinical methods for monitoring breast and colorectal cancer. Evaluation of CTC at any time during the course of disease allows assessment of patient prognosis and is predictive of progression free survival and overall survival.

The CellSearch system includes: CellSave Preservative Tubes, the CellTracks® AutoPrep® System, the CellTracks® Analyzer II or the CellSpotter® Analyzer, and the CellSearch™ Circulating Tumor Cell Control Kit.

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Concurrence of CDRH, Office of Device Evaluation (ODE)

| Prescription Use <u>X</u><br>(Part 21 CFR 801 Subpart D) | AND/OR<br><u>maria in chan</u><br>Division Sign-Off | Over-the-Counter Use <u> </u><br>(21 CFR 801 Subpart C) |
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Office of In Vitro Diagnostic
Device Evaluation and Safety

| 510(k) | <u>K071729</u><br>Page 4 of 33 |
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CONFIDENTIAL

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