CELLSEARCH CIRCULATING TUMOR CELL KIT

K073338 · Veridex, LLC · NQI · Feb 26, 2008 · Immunology

Device Facts

• Record ID: K073338
• Device Name: CELLSEARCH CIRCULATING TUMOR CELL KIT
• Applicant: Veridex, LLC
• Product Code: NQI · Immunology
• Decision Date: Feb 26, 2008
• 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, colorectal or prostate* cancer. The test is to be used as an aid in the monitoring of patients with metastatic breast, colorectal or prostate cancer. Serial testing for CTC should be used in conjunction with other clinical methods for monitoring metastatic breast, colorectal and prostate 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. *Metastatic prostate cancer patients in this study were defined as having two consecutive increases in the serum marker PSA above a reference level, despite standard hormonal management. These patients are commonly described as having androgen-independent, hormone-resistant, or castration-resistant prostate cancer.

Device Story

The CellSearch™ Circulating Tumor Cell Kit is an in vitro diagnostic system for identifying and counting circulating tumor cells (CTC) in whole blood. The process begins with immunomagnetic enrichment: ferrofluid nanoparticles coated with anti-EpCAM antibodies capture epithelial cells from a 7.5 mL blood sample. Following enrichment, the CellTracks® AutoPrep® System adds fluorescent reagents: anti-cytokeratin (CK-PE) for epithelial identification, DAPI for nuclear staining, and anti-CD45 (APC) to exclude leukocytes. A MagNest® device uses a magnetic field to concentrate labeled cells in a cartridge. The CellTracks Analyzer II or CellSpotter® Analyzer automatically scans the cartridge, acquires images, and displays events where CK-PE and DAPI are co-located. A clinician reviews the gallery of images to classify events as tumor cells if they are EpCAM+, CK+, DAPI+, and CD45-. The resulting CTC count provides prognostic information; patients with ≥5 CTCs per 7.5 mL of blood generally exhibit decreased progression-free and overall survival. This information aids clinicians in monitoring disease status and assessing prognosis in metastatic cancer patients.

Clinical Evidence

Prospective multi-center trial (n=231) of metastatic prostate cancer patients. Primary endpoints: progression-free survival (PFS) and overall survival (OS). Patients with ≥5 CTC/7.5 mL blood at baseline or follow-up showed significantly shorter median PFS and OS compared to those with <5 CTC. Multivariate Cox regression confirmed CTC count as a strong independent predictor of survival, often outperforming PSA reduction metrics. Bench testing established 93% recovery and linearity (0-1238 cells).

Technological Characteristics

The system utilizes immunomagnetic enrichment via ferrofluid nanoparticles (magnetic core, polymeric layer, anti-EpCAM antibodies). Identification uses immunofluorescence (anti-CK-PE, DAPI, anti-CD45-APC). The system includes the CellTracks® AutoPrep® System for automated sample preparation and the CellTracks Analyzer II or CellSpotter® Analyzer for automated image acquisition and display. The device is a standalone diagnostic system.

Indications for Use

Indicated for patients with metastatic breast, colorectal, or prostate cancer to aid in monitoring disease status and assessing prognosis via enumeration of circulating tumor cells (CTC) in peripheral 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

• The CellSearch™ Circulating Tumor Cell Kit (for metastatic breast and colorectal cancer) (k071729)

Submission Summary (Full Text)

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CellSearch™ Circulating Tumor Cell Kit Premarket Notification- Expanded Indications for Use-Metastatic Prostate Cancer

Image /page/0/Picture/1 description: The image shows a sequence of handwritten alphanumeric characters. The sequence starts with the letter 'K', followed by the number '0', then the number '7'. The sequence continues with three '3's and ends with the number '8'. The characters are written in a bold, slightly uneven style.

FEB 26 2003

# SECTION 3

# 510(K) SUMMARY

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

VEΓIDEX
LLC

1001 US Highway 202 Raritan, NJ 08869

# 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

| 807.92 (a)(1): Name: | Veridex, LLC |
|----------------------|-----------------------------------------------------------------------|
| Address: | 1001 US Highway 202<br>Raritan, NJ 08869 |
| 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, K071729

#### 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, colorectal or prostate* cancer. The test is to be used as an aid in the monitoring of patients with metastatic breast, colorectal or prostate cancer. Serial testing for CTC should be used in conjunction with other clinical methods for monitoring metastatic breast, colorectal and prostate 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.

*Metastatic prostate cancer patients in this study were defined as having two consecutive increases in the serum marker PSA above a reference level, despite standard hormonal management. These patients are commonly described as having androgen-independent, hormone-resistant, or castration-resistant prostate cancer

#### 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 indication 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-BR-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 Tumor 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 against the results of the expected cell count. The results are summarized in Table 1.

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| 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% |

#### Table 1: Percent Detection Estimates.

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 vielded 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 turnor 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 I 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 (ST) % CV | 18% | 5% |

#### Table 2. Summary of Precision Analyses

#### 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.

Image /page/4/Figure/6 description: This image is titled "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." The figure is focused on the reproducibility of CTC counts. The samples are duplicate MBC samples. The sample size is 163, and the average is less than 5 or greater than or equal to 5 CTC per 7.5 mL of blood.

Image /page/4/Figure/7 description: This image is a scatter plot comparing 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, also ranging from 0 to 999. The plot shows a positive correlation between the two variables, with most data points clustered in the lower-left corner and a visible upward trend as both counts increase. There are also dotted lines at x=5 and y=5.

Figure 1 Note: There may be more than one point superimposed over another. For example, on this plot, there are 50 instances (31%) where both tubes had 0 CTC, 18 instances (11%) where Tube 1 had 0 CTC and Tube 2 had 1 CTC, and another 18 instances (11%) where Tube 1 had 1 CTC and Tube 2 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.

Image /page/5/Figure/3 description: This image is labeled Figure 2 and describes the reproducibility of circulating tumor cell (CTC) counts in duplicate MCRC samples. The study included 1627 samples. The samples had an average of less than 3 or greater than or equal to 3 CTC per 7.5 mL of blood.

Image /page/5/Figure/4 description: The image is a scatter plot comparing 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, also ranging from 0 to 999. The plot shows a positive correlation between the two counts, with data points clustered along a diagonal line. There are also vertical and horizontal dashed lines at the values of 3 on both axes.

Figure 2 Note: There may be more than one point superimposed over another. For example, on this plot, there are 975 instances (60%) where both tubes had 0 CTC, 116 instances (7%) where Tube 1 had 0 CTC and Tube 2 had 1 CTC, and another 109 instances (7%) where Tube 1 had 1 CTC and Tube 2 had 0 CTC.

The tube-to-tube variation of CTC counts in blood samples from metastatic breast and colorectal cancer patients is shown in Figures 1 & 2. The distribution of infrequent events (such as tumor cells) within a given volume is random and independent of cell or disease type. This is best characterized by the Poisson distribution - a mathematical method employed for modeling systems where the probability of an event occurring is very low but the number of opportunities for such an event to occur is large. For tubes with very few prostatic CTC it is reasonable to expect variation in results similar to what is depicted in Figures 1 & 2. Because the two previous studies in MBC and MCRC patients showed almost identical results, a tube-to-tube comparison of CTC counts in blood samples from metastatic prostate cancer patients was not performed during the CellSearch™ CTC prostate clinical trial. However, results of an independent study using CellSearch™ technology conducted at the Memorial Sloan-Kettering Cancer Center demonstrated no systematic site-to-site or tube-to-tube variation in CTC counts across a range of 0 to 1192 CTC per tube in patients with metastatic prostate cancer.

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# 807.92(b)(2): Brief Description of Clinical Data

# Metastatic Prostate Cancer (MPC) Patients

A multi-center prospective, clinical trial was conducted to determine whether the number of CTC predicted disease progression and survival. Metastatic prostate cancer patients in this study were defined as having two consecutive increases in the serum marker prostate-specific antigen (PSA) above a reference level, despite standard hormonal management. These patients are commonly described as having androgen-independent, hormone-resistant, or castration-resistant prostate cancer. A total of 231 metastatic prostate cancer patients with evidence of (PSA) progression despite standard hormonal therapy and starting a new line or type of chemotherapy were enrolled. Clinical data were analyzed on an intentto-treat basis. Patient demographic information is presented in Table 3.

| Category | Mean ± Std. Deviation (Median) | Number of Subjects |
|---------------------------------------|--------------------------------|------------------------------------|
| Age at Baseline (in years) | $70 \pm 9 (70)$ | 231 |
| Pre-Therapy: | | |
| PSA (ng/mL) | $547 \pm 1616 (144)$ | 231 |
| Hemoglobin (g/dL) | $12.3 \pm 1.6 (12.4)$ | 221 |
| Alkaline Phosphatase(AlkPhos) (IU/mL) | $235 \pm 271 (144)$ | 223 |
| Lactate dehydrogenase(LDH) (IU/mL) | $293 \pm 228 (224)$ | 219 |
| Albumin (g/dL) | $3.9 \pm 2.6 (3.8)$ | 214 |
| | Description of Categories | Number of Subjects<br>(% of total) |
| | White | 209 (90%) |
| Race | Black | 17 ( 7%) |
| | Other | 5 ( 3%) |
| | 0 | 101 (44%) |
| Baseline ECOG Score | 1 | 100 (43%) |
| | 2 | 21 ( 9%) |
| | Unknown | 9 ( 4%) |
| | ≥5 | 18 ( 8%) |
| | 6 | 28 (12%) |
| Gleason Score | 7 | 63 (27%) |
| | 8 | 45 (20%) |
| | ≥9 | 54 (23%) |
| | Unknown | 23 (10%) |
| | 1 | 14 ( 6%) |
| Stage at Primary Diagnosis | 2 | 30 (13%) |
| | 3 | 58 (25%) |
| | 4 | 19 ( 8%) |
| | Unknown | 110 (48%) |
| | 1st | 154 (67%) |
| Line of Therapy | 2nd | 38 (16%) |
| | ≥3rd | 39 (17%) |
| | No | 67 (29%) |
| Taxotere in Current Therapy Line? | Yes | 162 (70%) |
| | Unknown | 2 ( 1%) |
| | Negative | 20 ( 8%) |
| Bone Metastasis | Positive | 207 (90%) |
| | Unknown | 4 ( 2%) |
| | No | 142 (62%) |
| Measurable Disease | Yes | 88 (38%) |
| | Unknown | 1 ( 0%) |
| | No | 141 (61%) |
| Visceral Metastasis | Yes | 89 (39%) |
| | Unknown | 1 ( 0%) |

#### Table 3: MPC Patient Demographics

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Baseline CTC count was determined prior to initiation of a new line of chemotherapy. The following timeframes were chosen for evaluation: baseline (prior to the initiation of therapy), 2-5 weeks (14 - 41 days from baseline), 6-8 weeks (42 - 62 days from baseline), 9-12 weeks (63 - 90 days from baseline), and 13-20 weeks (91 - 146 days from baseline) after the initiation of therapy. If more than one blood draw fell within the designated timeframes, the blood draw furthest from the baseline blood draw was used as the result for each timeframe.

### CTC frequencies

All 231 evaluable MPC patients had a baseline blood draw. Two hundred and twenty-one of these MPC patients had one or more follow-up blood draws after the initiation of therapy. Of the ten MPC patients with only a baseline blood draw, three died before a follow-up blood draw could be obtained, one progressed and was sent to hospice, one stopped their chemotherapy due to a broken hip, one patient moved, three refused any further blood draws, and one withdrew their consent for the study. There were a total of 214, 171, 158, and 149 MPC patients with follow-up blood draws 2-5 weeks, 9-12 weeks, and 13-20 weeks after the initiation of therapy, respectively.

In metastatic prostate cancer, disease progression is primarily determined using changes in PSA For this study, disease progression was determined by the sites using PSA, imaging, and/or clinical signs and symptoms. For the baseline analyses, progression free survival (PFS) was determined from the time of the baseline blood draw to the determination of progression or death, and overall survival (OS) was determined from the time of the baseline blood draw to the date of last contact with the patient. For the follow-up analyses, PFS was determined from the time of the follow-up blood draw to diagnosis of progression or death, and OS was determined from the time of the follow-up blood draw to the date of death or the date of last contact with the patients with progression prior to the date of the blood draw being evaluated were excluded from the PFS analyses of that time point and all subsequent follow-up blood draws. Patients with no additional survival follow-up beyond the date of the blood draw being evaluated were excluded from the PFS & OS analyses of that time point. Table 4 shows the numbers of patients at each time point excluded from the PFS or PFS & OS analyses and the reasons for their exclusion.

| Blood Draw<br>Timing | MPC Patients Not Evaluable: | | | | Total # of MPC Patients Evaluable | | | | |
|----------------------|-----------------------------|---|---|----------|-----------------------------------|-------------------------------------------------------------|-------------------------------------------------|----------------------------------------|--------------------------------------------------------|
| | PFS & OS | | | PFS Only | Blood<br>Not<br>Drawn | Blood Drawn 1-<br>7 days after<br>therapy<br>administration | No Follow-up<br>Beyond Date<br>of Blood<br>Draw | No or Non-<br>Evaluable<br>CTC Results | Blood drawn<br>after date of<br>disease<br>progression |
| Baseline | 0 | 6 | 0 | 6 | 0 | 219 | 219 | | |
| 2-5 Weeks | 17 | 0 | 0 | 11 | 4 | 199 | 203 | | |
| 6-8 Weeks | 60 | 0 | 0 | 8 | 22 | 141 | 163 | | |
| 9-12 Weeks | 73 | 1 | 0 | 8 | 15 | 134 | 149 | | |
| 13-20 Weeks | 82 | 0 | 1 | 5 | 27 | 116 | 143 | | |

Table 4: Exclusions from PFS and OS Analyses in MPC Patients

The CTC results obtained from the baseline and follow-up blood draws at 2-5 weeks, 9-12 weeks, and 13-20 weeks after the initiation of therapy were classified as being favorable (<5 CTC) or unfavorable (≥5 CTC). The PSA, Alkaline Phosphatase, and LDH levels summarized in the demographics table and used in the analyses were all measured at a central laboratory in serum samples collected at the same time as the blood samples used for CTC evaluation. The hemoglobin and albumin levels

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summarized in the tables and used in the analyses were values provided by the sites and verified from the patient's medical records that were determined within ± 30 days of the baseline CTC evaluation.

#### Progression Free Survival (PFS) Analysis of MPC Patients

#### PFS Using Baseline CTC Results

Two hundred nineteen of the 231 evaluable patients had a baseline CTC result available. For Kaplan-Meier analysis, patients were segmented into two groups based upon their CTC count at baseline:

- The Favorable group (N=94), represented in green, consisted of patients with <5 CTC. .
- The Unfavorable group (N=125), represented in red, consisted of patients with ≥5 CTC. .

Median PFS was longer in the Favorable group compared to the Unfavorable group (5.8 vs. 4.2 months, respectively.) These results are illustrated in Figure 3 or Table 5.

Image /page/8/Figure/8 description: This image is a survival plot showing the probability of progression-free survival over time in months. There are two survival curves, one for patients with less than 5 CTC and one for patients with greater than 5 CTC. The median progression-free survival is 5.8 months for patients with less than 5 CTC and 4.2 months for patients with greater than 5 CTC. The Cox Hazard Ratio is 1.6, the chi-square is 11.03, and the p-value is 0.0009.

#### Figure 3: PFS of MPC Patients with < 5 or ≥ 5 CTC at Baseline (N = 219).

#### PFS Using Follow-up CTC Results

For Kaplan-Meier analysis, MPC 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 draws after initiation of therapy for PFS are illustrated in Figure illustrates the ability of CTC in MPC patients with <5 and ≥5 CTC to predict time to clinical progression or death at 2-5 weeks (n=199), 6-8 weeks (n=141), 9-12 weeks (n=134) and 13-20 weeks (n=116) after the initiation of therapy.

- The Favorable group represented in olive green, blue, purple, and cyan consisted of patients . with <5 CTC at 2-5, 6-8, 9-12, and 13-20 weeks after the initiation of therapy, respectively.
- The Unfavorable group, represented in brown, black, grey, and orange consisted of patients . with ≥5 CTC at 2-5, 6-8, 9-12, and 13-20 weeks after the initiation of therapy, respectively.

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Image /page/9/Figure/1 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, each representing a different CTC level at different time intervals, such as <5 CTC at 2-5 weeks (n=122) and >5 CTC at 2-5 weeks (n=77).

Figure 4: PFS of MPC Patients with < 5 or ≥ 5 CTC at different times of Follow-Up

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

| Table 5: Progression Free Survival (PFS) for MPC patients with <5 or ≥5 CTC at different time | | | |
|-----------------------------------------------------------------------------------------------|--|--|--|
| points | | | |

| 1 | 2 | 3 | 4 | 5 | 6 |
|--------------------------------------|-----|-----------|-----------------------------------------|-----------------------------------------|---------------------|
| Sampling Time<br>After Tx Initiation | N | ≥5 CTC | Median PFS in Months (95% CI)<br><5 CTC | Median PFS in Months (95% CI)<br>≥5 CTC | Log-rank<br>p-value |
| Baseline | 219 | 125 (57%) | 5.8 (5.0 - 7.9) | 4.2 (3.1 - 4.9) | 0.0008 |
| 2-5 Weeks | 199 | 77 (39%) | 6.5 (4.9 - 7.4) | 2.1 (1.4 - 3.3) | <0.0001 |
| 6-8 Weeks | 141 | 38 (27%) | 5.9 (4.2 - 7.0) | 1.9 (1.3-2.7) | <0.0001 |
| 9-12 Weeks | 134 | 39 (24%) | 4.9 (3.8 - 6.2) | 1.6 (0.9 - 2.6) | <0.0001 |
| 13-20 Weeks | 116 | 27 (23%) | 4.1 (3.3 - 5.8) | 1.2 (0.5 - 1.5) | <0.0001 |

As illustrated in Figure 4 and Table 5, MPC patients with elevated CTC (25 CTC/7.5mL whole blood) at any of the time points had a much higher likelihood of rapid progression than did those with < 5 CTC. Table 5 column 4 shows the median PFS times for those patients with < 5 CTC ranged from 4.1 to 6.5 months and were substantially longer than the median PFS times for those patients with ≥5 CTC, which ranged from 1.2 to 4.2 months (column 5).

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#### Reduction or Increase in CTC Predicts Improved or Decreased PFS

Elapsed PFS times were calculated from the baseline blood draw. For the Kaplan-Meier analysis shown in Figure 5, MPC patients were segmented into four groups based upon their CTC counts at baseline, 2-5 weeks, 6-8 weeks, 9-12 weeks, and 13-20 weeks after the initiation of therapy:

- · Group 1 (green curve), 88 (38%) patients with <5 CTC at all time points. Five (6%) of these patients only had a baseline blood draw while seven (8%) had a single blood draw between their first and last blood draw that had ≥5 CTC;
- · Group 2 (blue curve), 45 (20%) patients with ≥5 CTC prior to the initiation of therapy but who had decreased to <5 CTC at the time of their last blood draw;
- · Group 3 (orange curve), 26 (11%) patients with <5 CTC at an early draw (baseline, 2-5 weeks, and/or 6-8 weeks) but who increased to ≥5 CTC at the time of their last blood draw;
- · Group 4 (red curve), 71 (31%) patients with ≥5 CTC at all draw time points. Eight (11%) of these patients had only a baseline blood draw and two (3%) had a single blood draw between their first and last blood draw that had <5 CTC.

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

Image /page/10/Figure/8 description: This image is a survival plot that shows the probability of progression-free survival over time. The x-axis represents time in months from baseline blood draw, ranging from 0 to 30 months. There are four different groups represented by different lines on the plot, with group 1 having the highest survival probability and group 4 having the lowest. The image also includes a table that provides the median progression-free survival in months for each group, along with the number of patients in each group and the p-values for curve comparisons.

Figure 5 shows that MPC patients with ≥5 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 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).

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#### Overall Survival (OS) Analysis of MPC Patients

#### OS Analysis Using Baseline CTC Results

Death occurred in 119 (52%) of the 231 MPC patients, with a mean follow-up time for the 112 (48%) patients still alive of 16.1 ± 4.9 months (median = 16.5 months, range = 1.9 to 25.7 months). At the time of these analyses, 28 (30%) of 94 patients from the Favorable group (<5 CTC at baseline) compared to 83 (66%) of 125 from the Unfavorable group (≥5 CTC at baseline) had died.

For Kaplan-Meier analysis, the 219 of the 231 evaluable patients that had baseline results were segmented into two groups based upon their CTC count at baseline:

- The Favorable group (N=94), represented in green, consisted of patients with <5 CTC. .
- The Unfavorable group (N=125), represented in red, consisted of patients with ≥5 CTC. .

Median OS was significantly longer in the Favorable group compared to the Unfavorable group (21.7 vs. 11.5 months, respectively). These results are illustrated in Figure 6.

Image /page/11/Figure/8 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 5 CTC and one for patients with greater than 5 CTC. The median overall survival for patients with less than 5 CTC is 21.7 months, while the median overall survival for patients with greater than 5 CTC is 11.5 months. The Cox Hazard Ratio is 3.3 with a p-value of less than 0.0001.

Figure 6: OS of MPC Patients with <5 or ≥5 CTC at Baseline (N = 219).

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#### OS Using Follow-up CTC Results

The Kaplan-Meier analyses of both MPC patient groups at each of the different follow-up blood draw times after initiation of therapy are illustrated in Figure illustrates the ability of CTC in MPC patients with <5 and >5 CTC 2-5 weeks (n=203), 6-8 weeks (n=163), 9-12 weeks (n=149) and 13-20 weeks (n=143) after the initiation of therapy to predict time to death. 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 <5 CTC,
- The Unfavorable group, represented in brown, black, grey, and orange, consisted of patients . with ≥5 CTC.

Image /page/12/Figure/5 description: This image is a survival plot that shows the probability of survival over time from blood draw in months. There are two sets of survival curves, one for patients with less than 5 CTC and another for patients with greater than 5 CTC. Each set of curves is further divided into subgroups based on the time at which the CTC was measured, specifically 2-5 weeks, 6-8 weeks, 9-12 weeks, and 13-20 weeks, with the number of patients in each subgroup indicated in the legend.

Figure 7: OS of MPC Patients with < 5 or ≥ 5 CTC at different times of Follow-Up.

{13}------------------------------------------------

Table 6 summarizes the results of the OS analysis using the CTC levels and a threshold of ≥5 CTC/7.5mL at each of the different blood draw time points.

| 1 | 2 | 3 | 4 | | 5 | 6 |
|-----------------------------------|-----|-----------|------------------------------|-------------------|---------------------|---|
| Sampling Time After Tx Initiation | N | ≥5 CTC | Median OS in Months (95% CI) | | Log-rank<br>p-value | |
| | | | <5 CTCs | ≥5 CTCs | | |
| Baseline | 219 | 125 (57%) | 21.7 (21.3 - NR) | 11.5 (9.3 - 13.7) | <0.0001 | |
| 2-5 Weeks | 203 | 80 (39%) | 20.7 (20.5 - NR) | 9.5 (5.8 - 10.7) | <0.0001 | |
| 6-8 Weeks | 163 | 53 (33%) | 19.9 (17.9 - NR) | 8.5 (5.0 - 10.2) | <0.0001 | |
| 9-12 Weeks | 149 | 49 (33%) | 19.6 (18.5 - NR) | 7.6 (6.2 - 8.6) | <0.0001 | |
| 13-20 Weeks | 143 | 44 (31%) | 19.8 (17.1 - NR) | 6.7 (4.9 - 7.6) | <0.0001 | |

Table 6: Overall Survival (OS) for MPC patients with <5 or >5 CTC at different time points

As illustrated in Figure 7 and Table 6 in columns 4 & 5, MPC patients with ≥5 CTC at any of the time points had a much higher likelihood of dying sooner than did those with < S CTC. The median OS times for those patients with <5 CTC ranged from 19.6 to 21.7 months and were substantially longer than the median OS times for those patients with ≥5 CTC, which ranged from 6.7 to 11.5 months.

#### Reduction or Increase of CTC Predicts Improved or Decreased OS

Elapsed OS times were calculated from the baseline blood draw. For Kaplan-Meier analysis (Figure 8), patients were segmented into four groups based upon their CTC counts at baseline, 2-5 weeks, 6-8 weeks, 9-12 weeks, and 13-20 weeks after the initiation of therapy:

- Group 1 (green curve), 88 (38%) patients with <5 CTC at all time points. Five (6%) of these . patients had only a baseline blood draw while seven (8%) had a single blood draw between their first and last blood draw that had ≥5 CTC;
- Group 2 (blue curve), 45 (20%) patients with ≥5 CTC prior to the initiation of therapy but who . had decreased to <5 CTC at the time of their last blood draw;
- Group 3 (orange curve), 26 (11%) patients with <5 CTC at an early draw but who increased to . ≥5 CTC at the time of their last blood draw;
- Group 4 (red curve), 71 (31%) patients with ≥5 CTC at all draw time points. Eight (11%) of . these patients had only a baseline blood draw and two (3%) had a single blood draw between their first and last blood draw that had <5 CTC.

{14}------------------------------------------------

Image /page/14/Figure/1 description: This image shows a Kaplan-Meier survival curve comparing four groups based on CTC (circulating tumor cell) counts at different blood draws. The y-axis represents the probability of survival, and the x-axis represents the time from baseline blood draw in months. The table provides the number of patients in each group, along with the median overall survival (OS) in months and the corresponding 95% confidence interval. The log-rank p-values for pairwise comparisons between the groups are also presented.

Figure 8: A Reduction in CTC Below 5 After the Initiation of Therapy Predicts Longer OS whereas an Increase in CTC Count to 5 or above Predicts Shorter OS in MPC Patients

Figure 8 shows that those patients with ≥5 CTC at any point after the initiation of therapy had a much higher likelihood of dying sooner. Patients with ≥5 CTC at all time points (Group 4) had the shortest median OS, which was significantly different compared to the median OS of Group 2, and Group 1 but not Group 3. Patients with <5 CTC at all time points (Group 1) had the longest median OS, which was significantly different compared to the median OS of Group 3, but not Group 2. Figure 8 also demonstrated that patients who showed a decrease in CTC (Group 2) improve their survival chances and had a median OS similar to those patients with favorable CTC at all draws (Group 1). The figure also shows that unfavorable CTC levels after the initiation of therapy significantly decreased overall survival (Group 3 and Group 4).

#### Univariate Cox Regression Analysis in MPC Patients

Univariate Cox proportional hazards regression analysis was used to evaluate the association of the following pre-treatment parameters with PFS and OS: stage of disease at diagnosis (1-4), patient age (≥70 or <70 years), ECOG status before initiation of a new line of therapy (0-2), Gleason score (2-10), hemoglobin level within ± 30 days of baseline draw (g/dL, continuous), albumin level within ± 30 days of baseline draw (g/dL, continuous), testosterone level at the time of the baseline draw (ng/mL, continuous), LDH level at the time of the baseline draw (IU/mL, continuous), alkaline phosphatase level at the time of the baseline draw (IU/mL, continuous), PSA level at the time of the baseline draw (ng/mL, continuous),

{15}------------------------------------------------

pre-treatment PSA doubling time (months, continuous), pre-treatment PSA velocity (ng/mL/month, continuous), line of therapy (1), 2", 3", 4", 5", or 6"), type of therapy (taxotere included or not), presence of measurable disease (yes or no), presence of bone metastasis (yes or no), presence of visceral metastasis (yes or no), and baseline CTC level (≥5 CTC/7.5mL or <5 CTC/7.5mL) and follow up CTC counts at 2-5, 6-8, 9-12 and 13-20 weeks.

For these analyses, the elapsed times for both PFS and OS were calculated from the time of the baseline blood draw. The Cox regression results (i.e. the hazards ratio and associated 95% confidence interval, chi-square value, and associated p-values) for the ability of the parameters to independently predict PFS and OS are provided in Table 7 as well as the number of patients in each evaluation.

| Parameter | Categories | | # of MPC<br>Patients | PFS Risk from<br>Baseline | | OS Risk From<br>Baseline | |
|------------------------------------------|---------------------|----------|----------------------|---------------------------|-----------|--------------------------|---------|
| | Positive | Negative | | HR | p-value ¹ | HR | p-value |
| Stage at Primary Diagnosis | 4 vs. 3 vs. 2 vs. 1 | | 121 | 0.88 | 0.206 | 0.83 | 0.174 |
| Age at Baseline Blood Draw | ≥70 | <70 | 231 | 0.96 | 0.764 | 1.28 | 0.178 |
| ECOG Status at Study Entry | 2 vs. 1 vs. 0 | | 222 | 1.34 | 0.011 | 2.36 | <0.001 |
| Gleason Score | 10 to 2 | | 208 | 1.01 | 0.919 | 1.02 | 0.717 |
| Pre-treatment PSA Doubling Time (Months) | Continuous | | 230 | 0.97 | 0.542 | 0.97 | 0.664 |
| Pre-treatment PSA Velocity (ng/mL/Month) | Continuous | | 230 | 1.00 | 0.200 | 1.00 | 0.544 |
| Baseline Hemoglobin (g/dL) | Continuous | | 221 | 0.87 | 0.002 | 0.71 | <0.001 |
| Baseline Albumin (g/dL) | Continuous | | 214 | 0.99 | 0.748 | 1.02 | 0.557 |
| Baseline Testosterone (ng/mL)¹ | Continuous | | 223 | 1.07 | 0.900 | 2.71 | 0.060 |
| Baseline LDH (IU/mL) ¹ | Continuous | | 219 | 1.001 | <0.001 | 1.002 | <0.001 |
| Baseline Alkaline Phosphatase (IU/mL) ¹ | Continuous | | 223 | 1.00 | 0.158 | 1.0008 | 0.001 |
| Line of Therapy | Continuous (1 - 6) | | 231 | 1.23 | 0.003 | 1.28 | 0.003 |
| Type of Therapy (Taxotere: Yes/No) | Yes | No | 229 | 0.57 | <0.001 | 0.59 | 0.006 |
| Measurable Disease? | Yes | No | 230 | 1.00 | 0.993 | 1.28 | 0.181 |
| Bone Metastasis? | Yes | No | 227 | 1.02 | 0.933 | 2.22 | 0.057 |
| Visceral Metastasis? | Yes | No | 230 | 1.01 | 0.918 | 1.26 | 0.216 |
| Baseline PSA (ng/mL) | Continuous | | 231 | 1.00 | 0.746 | 1.00 | 0.907 |
| 2-5 Week PSA (ng/mL) | Continuous | | 207 | 1.00 | 0.819 | 1.00 | 0.794 |
| 6-8 Week PSA (ng/mL) | Continuous | | 167 | 1.00 | 0.426 | 1.00 | 0.654 |
| 9-12 Week PSA (ng/mL) | Continuous | | 155 | 1.00 | 0.684 | 1.00 | 0.324 |
| 13-20 Week PSA (ng/mL) | Continuous | | 143 | 1.00 | 0.639 | 1.00 | 0.205 |
| 2-5 Week PSA Reduction from BL (%) ¹ | <30% | ≥30% | 207 | 1.56 | 0.006 | 1.24 | 0.318 |
| 6-8 Week PSA Reduction from BL (%) ¹ | <30% | ≥30% | 167 | 2.21 | <0.001 | 2.27 | 0.001 |
| 9-12 Week PSA Reduction from BL (%) ¹ | <30% | ≥30% | 155 | 2.76 | <0.001 | 2.30 | <0.001 |
| 13-20 Week PSA Reduction from BL (%) ¹ | <30% | ≥30% | 143 | 2.69 | <0.001 | 3.19 | <0.001 |

#### Table 7: Univariate Cox Regression Analysis in MPC Patients

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| Parameter | Categories | | # of MPC | PFS Risk from<br>Baseline | | OS Risk From<br>Baseline | |
|-------------------------|------------|----------|----------|---------------------------|----------|--------------------------|----------|
| | Positive | Negative | Patients | HR | p-value2 | HR | p-value2 |
| Baseline CTC Number | ≥5 | <5 | 219 | 1.62 | 0.001 | 3.33 | <0.001 |
| 2 - 5 Week CTC Number | ≥5 | <5 | 203 | 2.34 | <0.001 | 4.46 | <0.001 |
| 6 - 8 Week CTC Number | ≥5 | <5 | 163 | 3.29 | <0.001 | 3.66 | <0.001 |
| 9 - 12 Week CTC Number | ≥5 | <5 | 149 | 3.23 | <0.001 | 5.82 | <0.001 |
| 13 - 20 Week CTC Number | ≥5 | <5 | 144 | 4.82 | <0.001 | 7.18 | <0.001 |

" Determined from Serum Drawn on the Same Date as the Blood Drawn for CTC

2 p-value from Wald test of Z statistic

3 p-value from chi-squared test

## Multivariate Cox Regression Analysis in MPC 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 strongest predictors at most time points of PFS and OS (Table 8).

### Table 8: Multivariate Cox Regression Analysis in MPC Patients

| Parameter | # of<br>Patients | PFS Risk from Baseline | | OS Risk from Baseline | |
|---------------------------------------------------------|------------------|------------------------|----------|-----------------------|----------|
| | | HR | p-value2 | HR | p-value2 |
| Baseline CTC (<5 vs. ≥5) | | 1.14 | 0.455 | 1.92 | 0.009 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.00 | 0.982 | 1.46 | 0.032 |
| Baseline Hemoglobin (g/dL)3 | | 0.88 | 0.027 | 0.81 | 0.007 |
| Baseline LDH (IU/mL)1,3 | 188 | 1.0007 | 0.018 | 1.002 | <0.001 |
| Baseline Alkaline Phosphatase (IU/mL)1,3 | | --- | --- | 1.00 | 0.410 |
| Line of Therapy (1st through 6th) | | 1.14 | 0.145 | 1.07 | 0.547 |
| Type of Therapy (Taxotere: Yes/No) | | 0.63 | 0.009 | 0.70 | 0.139 |
| 2 - 5 Week CTC (<5 vs. ≥5) | | 1.48 | 0.041 | 2.91 | <0.001 |
| 2 - 5 Week PSA Reduction from Baseline (≥30% vs. <30%)1 | | 1.40 | 0.077 | 1.13 | 0.637 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 0.97 | 0.836 | 1.46 | 0.054 |
| Baseline Hemoglobin (g/dL)3 | | 0.93 | 0.246 | 0.89 | 0.141 |
| Baseline LDH (IU/mL)1,3 | 173 | 1.002 | 0.002 | 1.003 | <0.001 |
| Baseline Alkaline Phosphatase (IU/mL)1,3 | | --- | --- | 1.00 | 0.622 |
| Line of Therapy (1st through 6th) | | 1.11 | 0.274 | 1.11 | 0.399 |
| Type of Therapy (Taxotere: Yes/No) | | 0.75 | 0.133 | 0.80 | 0.397 |
| 6 - 8 Week CTC (<5 vs. ≥5) | 139 | 2.14 | <0.001 | 2.13 | 0.009 |
| 6 - 8 Week PSA Reduction from Baseline (≥30% vs. <30%)1 | | 1.88 | 0.002 | 2.38 | 0.007 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.04 | 0.810 | 1.52 | 0.088 |
| Baseline Hemoglobin (g/dL)3 | | 0.97 | 0.695 | 0.79 | 0.013 |
| Baseline LDH (IU/mL)1,3 | | 1.002 | 0.003 | 1.004 | <0.001 |

{17}------------------------------------------------

Veridex, LLC CellSearch™ Circulating Tumor Cell Kit Premarket Notification- Expanded Indications for Use-Metastatic Prostate Cancer

| Parameter | # of<br>Patients | PFS Risk from Baseline<br>HR | PFS Risk from Baseline<br>p-value 2 | OS Risk from Baseline<br>HR | OS Risk from Baseline<br>p-value 2 |
|----------------------------------------------------------|------------------|------------------------------|-------------------------------------|-----------------------------|------------------------------------|
| Baseline Alkaline Phosphatase (IU/mL) 1, 3 | | --- | | 1.00 | 0.780 |
| Line of Therapy (1st through 6th) | | 1.37 | 0.001 | 1.35 | 0.035 |
| Type of Therapy (Taxotere: Yes/No) | | 0.80 | 0.278 | 1.45 | 0.276 |
| 9 - 12 Week CTC (<5 vs. ≥5) | | 1.74 | 0.015 | 3.94 | <0.001 |
| 9 - 12 Week PSA Reduction from Baseline (≥30% vs. <30%)1 | | 2.23 | <0.001 | 1.46 | 0.221 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 1.21 | 0.307 | 1.89 | 0.004 |
| Baseline Hemoglobin (g/dL) 3 | 125 | 0.93 | 0.322 | 0.97 | 0.758 |
| Baseline LDH (IU/mL) 1,3 | | 1.00 | 0.190 | 1.003 | <0.001 |
| Baseline Alkaline Phosphatase (IU/mL) 1,3 | | --- | --- | 1.00 | 0.989 |
| Line of Therapy (1st through 6th) | | 1.25 | 0.052 | 1.11 | 0.499 |
| Type of Therapy (Taxotere: Yes/No) | | 0.97 | 0.903 | 1.26 | 0.486 |
| 13 - 20 Week CTC (<5 vs. ≥5) | | 2.95 | <0.001 | 3.75 | 0.001 |
| 13-20 Week PSA Reduction from Baseline (≥30% vs. <30%)1 | | 1.97 | 0.002 | 1.52 | 0.275 |
| Baseline ECOG Status (0 vs. 1 vs. 2) | | 0.98 | 0.919 | 1.98 | 0.002 |
| Baseline Hemoglobin (g/dL) 3 | 123 | 1.03 | 0.723 | 0.87 | 0.232 |
| Baseline LDH (IU/mL) 1,3 | | 1.00 | 0.380 | 1.003 | <0.001 |
| Baseline Alkaline Phosphatase (IU/mL) 1,3 | | --- | --- | 1.00 | 0.078 |
| Line of Therapy (1st through 6th) | | 1.25 | 0.050 | 1.06 | 0.751 |
| Type of Therapy (Taxotere: Yes/No) | | 1.04 | 0.882 | 0.90 | 0.770 |

1 Determined from Serum Drawn on the Same Date as the Blood Drawn for CTC

2 p-value from Wald test of Z statistic

3 Assessed as a continuous parameter

#### Use of CTC to Monitor Clinical Status of Metastatic Prostate Cancer Patients

### Relationship between survival, CTCs and disease assessment by PSA

At present, a reduction in PSA is one of the primary means to determine response to therapy in MPC patients. To establish the relationship of clinical status as determined by a PSA to CTC, reduction of 230% or ≥50% PSA and CTC were measured 2-5 weeks, 9-12 weeks and 13-20 weeks after initiation of therapy and compared to overall survival.

For the Kaplan-Meier analysis the elapsed OS times were calculated from the time of blood draw. Patients were segmented into Favorable groups based upon a CTC of <5 at the time of evaluation and a ≥30% reduction of PSA from baseline to the time of evaluation. Patients were segmented into Unfavorable groups based upon a CTC of ≥5 and <30% reduction of PSA from baseline to the time of evaluation.

Figure 9 shows the results of the analysis 2-5 weeks after initiation of therapy, Figure 10 the analysis 6-8 weeks after initiation of therapy, Figure 11 the analysis 9-12 weeks after initiation of therapy and Figure 12 the analysis 13-20 weeks after initiation of therapy.

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Image /page/18/Figure/1 description: This image is a survival plot that shows the probability of survival over time for two groups of patients based on their CTC levels at 2-5 weeks. The x-axis represents time in months from a 2-5 week blood draw, and the y-axis represents the probability of survival. The top line represents patients with less than 5 CTC, and the bottom line represents patients with greater than 5 CTC. The median overall survival for patients with less than 5 CTC is 20.7 months, while the median overall survival for patients with greater than 5 CTC is 9.5 months.

# Figure 9: OS of MPC Patients 2-5 weeks after the Initiation of Therapy

#### B. ≥30% PSA Reduction at 2-5 Weeks

Image /page/18/Figure/4 description: This image is a survival plot that shows the probability of survival over time in months. The x-axis represents the time from a 2-5 week blood draw in months, and the y-axis represents the probability of survival. There are two survival curves, one for patients with a PSA reduction of greater than 30% at 2-5 weeks, and one for patients with a PSA reduction of less than 30%. The median overall survival for patients with a PSA reduction of greater than 30% is 17.2 months, while the median overall survival for patients with a PSA reduction of less than 30% is 15.2 months.

Figure 10: OS of MPC Patients 6-8 weeks after the Initiation of Therapy

Image /page/18/Figure/6 description: The image is a survival plot that shows the probability of survival over time for two groups of patients based on their CTC levels at 6-8 weeks. The x-axis represents the time from the 6-8 week blood draw in months, and the y-axis represents the probability of survival. The plot shows that patients with less than 5 CTCs at 6-8 weeks had a median overall survival of 19.9 months, while patients with 5 or more CTCs had a median overall survival of 8.5 months. The Cox Hazard Ratio is 3.6, and the p-value is less than 0.0001.

Page 19 of 27

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Veridex, LLC

CellSearch™ Circulating Tumor Cell Kit Premarket Notification- Expanded Indications for Use-Metastatic Prostate Cancer

Image /page/19/Figure/2 description: This image is a survival plot that shows the probability of survival over time in months. The x-axis represents the time from a 6-8 week blood draw in months, and the y-axis represents the probability of survival. There are two survival curves, one for patients with a greater than 30% PSA reduction at 6-8 weeks, and one for patients with less than 30% PSA reduction. The median overall survival for patients with greater than 30% PSA reduction is 22.8 months, while the median overall survival for patients with less than 30% PSA reduction is 11.5 months.

### B. ≥30% PSA Reduction at 6-8 Weeks

Figure 11: OS of MPC Patients 9-12 weeks after the Initiation of Therapy

#### A. 9-12 Week CTC

Image /page/19/Figure/6 description: This image is a survival plot that shows the probability of survival over time in months. The x-axis represents the time from a 9-12 week blood draw in months, and the y-axis represents the probability of survival. There are two survival curves, one for patients with less than 5 CTC (circulating tumor cells) and one for patients with 5 or more CTC. The median overall survival for patients with less than 5 CTC is 19.6 months, while the median overall survival for patients with 5 or more CTC is 7.6 months.

B. ≥30% PSA Reduction at 9-12 Weeks

Image /page/19/Figure/8 description: This image is a survival plot that shows the probability of survival over time for two groups of patients based on their PSA reduction at 9-12 weeks. The first group had a greater than 30% reduction in PSA, while the second group had less than 30% reduction. The median overall survival (OS) for the first group was 18.5 months, while the median OS for the second group was 10.2 months. The log-rank p-value is 0.0007, and the Cox hazard ratio is 2.2.

{20}------------------------------------------------

Image /page/20/Figure/1 description: This image is a survival plot that shows the probability of survival over time for two groups of patients based on their CTC levels at 13-20 weeks. The x-axis represents the time from the 13-20 week blood draw in months, and the y-axis represents the probability of survival. The plot shows that patients with <5 CTC have a higher probability of survival than patients with ≥5 CTC. The median overall survival (OS) for patients with <5 CTC is 19.8 months, while the median OS for patients with ≥5 CTC is 6.7 months.

# Figure 12: OS of MPC Patients 13-20 weeks after the Initiation of Therapy

B. ≥30% PSA Reduction at 13-20 Weeks

A. 13-20 Week CTC

Image /page/20/Figure/4 description: This image is a survival plot that shows the probability of survival over time for two groups of patients based on their PSA reduction at 13-20 weeks. The top line represents patients with a greater than 30% reduction, while the bottom line represents patients with less than 30% reduction. The median overall survival (OS) for the greater than 30% reduction group is 17.7 months, while the median OS for the less than 30% reduction group is 8.7 months. The log-rank p-value is less than 0.0001, indicating a statistically significant difference in survival between the two groups.

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

Table 9 illustrates the comparison of median overall survival at different time points after therapy with CTC, a 30% PSA reduction and a 50% PSA reduction.

| | CTC / 7.5mL | | | | | | 30% PSA Reduction from Baseline | | | | | | 50% PSA Reduction from Baseline | | | | | |
|----------------|-------------|-----------|-----------------|-----------------|--------------------|-----|---------------------------------|-------------|-------------------|-------------------|--------------------|-----|---------------------------------|-------------|-------------------|-------------------|--------------------|-----|
| Time<br>Point | N | ≥5<br>(%) | Median OS<br><5 | Median OS<br>≥5 | logrank<br>p-value | HR | N | <30%<br>(%) | Median OS<br>≥30% | Median OS<br><30% | logrank<br>p-value | HR | N | <50%<br>(%) | Median OS<br>≥50% | Median OS<br><50% | logrank<br>p-value | HR |
| 2-5<br>Weeks | 203 | 39% | 20.7 | 9.5 | <0.0001 | 4.5 | 207 | 71% | 17.2 | 15.2 | 0.3653 | 1.2 | 207 | 83% | 17.5 | 16.2 | 0.5599 | 1.2 |
| 6-8<br>Weeks | 163 | 33% | 19.9 | 8.5 | <0.0001 | 3.6 | 167 | 60% | 22.8 | 11.5 | 0.0011 | 2.2 | 167 | 75% | 22.8 | 14.4 | 0.0117 | 2.1 |
| 9-12<br>Weeks | 149 | 33% | 19.6 | 7.6 | <0.0001 | 5.3 | 155 | 46% | 18.5 | 10.2 | 0.0007 | 2.2 | 155 | 59% | 19.6 | 10.8 | 0.0006 | 2.3 |
| 13-20<br>Weeks | 143 | 31% | 19.8 | 6.7 | <0.0001 | 6.5 | 142 | 41% | 17.7 | 8.7 | <0.0001 | 2.9 | 142 | 46% | 17.7 | 9.9 | 0.0001 | 2.6 |

Table 9: Comparison of Median OS between Favorable CTC and PSA Reduction

#### Concordances between CTC and PSA Changes in MPC Patients

The data in Figure 9 through 12 and in Table 9, illustrate a highly significant difference in overall survival between patients with Unfavorable CTC and Favorable CTC at all time points tested, whereas PSA evaluations were not significant until 6-8 weeks after the initiation of therapy. Although the differences in median OS between the Favorable (≥30% or ≥50% PSA reduction from baseline) and Unfavorable (<30% or <50% PSA reduction from baseline) PSA reduction groups were significant, the separation between the Favorable (<5 CTC) and Unfavorable (≥5 CTC) CTC groups appeared greater and was significant at all time points after the initiation of therapy.

At present, either a ≥30% or ≥50% reduction in PSA is commonly used to evaluate disease progression in metastatic prostate cancer patients. Therefore, to establish the relationship between CTC and changes in PSA two by two tabulations of concordant and discordant observations between CTC and PSA changes for each time point after the initiation of therapy were constructed. Although comparisons of CTC to PSA change at both magnitudes were calculated, only data from the CTC vs. ≥30% PSA change are reported. This decision was based on a recent publication (J Nat Ca Inst. 98 (8):p.516-521, 2006) demonstrating that a 3-month 30% PSA decline showed a stronger association with decrease in risk of death than did a 50% decrease in PSA. Furthermore, a comparison of patient-wise and observation-wise results from the 30% and 50% PSA decline vs. CTC analyses did not demonstrate substantial differences in the Positive % Agreement, Negative % Agreement and Overall Agreement at any of the observed time points.

A total of 197, 159, 146, and 138 patients had serum samples analyzed by the central laboratory and had evaluable CTC results 2-5 weeks, 6-8 weeks, 9-12 weeks after the initiation of therapy, respectively. To determine a patient's response to therapy, the percent change in PSA from the baseline value was calculated for each of the time points after the initiation of therapy. For PSA changes at each time point, the Favorable group was defined as patients with a ≥30% reduction in PSA and the Unfavorable group was defined as patients with a <30% reduction in PSA. For CTC at each time point, the Favorable group was defined as patients having <5 CTC per 7.5mL of blood and the Unfavorable group was defined as patients having ≥5 CTC.

Because CTC vs. PSA results of the patient-wise comparisons between CTC and a ≥30% PSA reduction at 2-5 weeks and 13-20 weeks after the initiation of therapy showed the most significant discordance and concordance, respectively, these data are presented in Table 10 and Table 11, respectively.

{22}------------------------------------------------

| % Reduction in PSA<br>from Baseline at 2-5 Weeks<br>After Initiation of Therapy | CTCs 2-5 Weeks after the<br>Initiation of Therapy | Total | |
|---------------------------------------------------------------------------------|---------------------------------------------------|--------------------|-----|
| | < 5 CTCs/7.5mL | ≥ 5 CTCs/<br>7.5mL | |
| ≥30% Reduction in PSA | 50 | 11 | 61 |
| <30% Reduction in PSA | 69 | 67 | 136 |
| Total | 119 | 78 | 197 |

# Table 10: MPC Patient-Wise Comparison of CTC and 30% PSA Reduction at 2-5 Weeks

| Measurement | Estimate | Lower<br>95% CI | Upper<br>95% CI |
|---------------------------|----------|-----------------|-----------------|
| Positive % Agreement | 49% | 41% | 58% |
| Negative % Agreement | 82% | 70% | 91% |
| Positive Predictive Value | 86% | 76% | 93% |
| Negative Predictive Value | 42% | 33% | 51% |
| Overall Agreement | 59% | 52% | 66% |
| Odds Ratio | 4.4 | 2.1 | 9.2 |

## Table 11: MPC Patient-Wise Comparison of CTC and 30% PSA Reduction at 13-20 Weeks.

| % Reduction in PSA<br>from Baseline at 13-20 Weeks<br>After Initiation of Therapy | CTC 13-20 Weeks after the<br>Initiation of Therapy | | Total |
|-----------------------------------------------------------------------------------|----------------------------------------------------|---------|-------|
| | < 5 CTC | ≥ 5 CTC | |
| ≥30% Reduction in PSA | 72 | 9 | 81 |
| <30% Reduction in PSA | 23 | 34 | 57 |
| Total | 95 | 43 | 138 |

| Measurement | Estimate | Lower<br>95% CI | Upper<br>95% CI |
|---------------------------|----------|-----------------|-----------------|
| Positive % Agreement | 60% | 46% | 72% |
| Negative % Agreement | 89% | 80% | 95% |
| Positive Predictive Value | 79% | 64% | 90% |
| Negative Predictive Value | 76% | 66% | 84% |
| Overall Agreement | 77% | 69% | 84% |
| Odds Ratio | 11.8 | 4.9 | 28.3 |

The results of an "observation-wise" comparison of CTC and PSA changes using a ≥30% reduction threshold at 2-5 weeks, 6-8 weeks, 9-12 weeks, and 13-20 weeks after the initiation of therapy combined are shown in Table 12.

.

:

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

| % Reduction in PSA<br>from Baseline After Initiation of<br>Therapy | CTC after the<br>Initiation of Therapy | | Total |
|--------------------------------------------------------------------|----------------------------------------|---------|-------|
| | < 5 CTC | ≥ 5 CTC | |
| ≥30% Reduction in PSA | 243 | 41 | 284 |
| <30% Reduction in PSA | 175 | 181 | 356 |
| Total | 418 | 222 | 640 |

## Table 12: MPC Observation-Wise Comparison of CTC and 30% PSA Reduction.

| Measurement | Estimate | Lower<br>95% CI | Upper<br>95% CI |
|---------------------------|----------|-----------------|-----------------|
| Positive % Agreement | 51% | 46% | 56% |
| Negative % Agreement | 86% | 81% | 89% |
| Positive Predictive Value | 82% | 76% | 86% |
| Negative Predictive Value | 58% | 53% | 63% |
| Overall Agreement | 66% | 62% | 70% |
| Odds Ratio | 6.1 | 4.1 | 9.1 |

The overall concordance between CTC and PSA changes at the various time points after the initiation of therapy ranged from 59% to 77% when comparing to a ≥30% PSA reduction and from 52% to 75% when comparing to a ≥50% PSA reduction, showing that there was discordance between CTC and PSA changes in ~25% to 40% of the patients.

#### CTC Levels and PSA Reduction Combined to Predict OS in MPC Patients

. .

To determine which of the discordant results better reflected the prognosis of the patient, CTC assessment and changes in PSA 2-5 weeks, 9-12 weeks and 13-20 weeks after initiation of therapy were compared to overall survival. Elapsed OS times were calculated from the blood draw being evaluated. For the Kaplan-Meier analysis Figure 13 (Panels A, B, C and D) patients were segmented into four groups based upon their CTC counts and PSA reduction at 2-5 weeks, 6-8 weeks, 9-12 weeks, and 13-20 weeks after the initiation of therapy, respectively:

- Group 1 (green curve), patients with <5 CTC at the time of evaluation and a ≥30% reduction of . PSA from baseline to the time of evaluation;
- Group 2 (blue curve), patients with <5 CTC at the time of evaluation and a <30% reduction of . PSA from baseline to the time of evaluation;
- Group 3 (orange curve), patients with ≥5 CTC at the time of evaluation and a ≥30% reduction of . PSA from baseline to the time of evaluation
- Group 4 (red curve), patients with ≥5 CTC at the time of evaluation and a <30% reduction of . PSA from baseline to the time of evaluation.

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## Figure 13: CTC Levels and PSA Changes Combined to Predict OS 2-5 Weeks (Panel A), 6-8 Weeks (Panel B), 9-12 Weeks (Panel C), and 13-20 Weeks (Panel D) After the Initiation of Therapy

Image /page/24/Figure/2 description: This image is a survival plot that shows the probability of survival over time for four different groups of patients. The x-axis represents time in months from a 2-5 week blood draw, and the y-axis represents the probability of survival. The four groups are defined by their CTC levels at week 2-5 and their PSA reduction at 2-5 weeks. The median overall survival (OS) in months with 95% confidence intervals is provided for each group, along with the number of patients in each group and the p-values for comparisons between the groups.

A. 2-5 Weeks

B. 6-8 Weeks

Image /page/24/Figure/5 description: This image is a survival plot that shows the probability of survival over time for four different groups of patients. The x-axis represents time in months from a 6-8 week blood draw, and the y-axis represents the probability of survival. The four groups are defined by their CTC levels at week 6-8 and their PSA reduction at 6-8 weeks. The median overall survival in months with 95% confidence intervals is shown for each group, along with the number of patients in each group and the p-values for comparisons between the groups.

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Veridex, LLC CellSearch™ Circulating Tumor Cell Kit Premarket Notification- Expanded Indications for Use-Metastatic Prostate Cancer

#### C. 9-12 Weeks

Image /page/25/Figure/2 description: This image is a survival plot that shows the probability of survival over time for four different groups of patients. The x-axis represents time in months from a 9-12 week blood draw, and the y-axis represents the probability of survival. The four groups are defined by their CTC levels at week 9-12 and their PSA reduction at 9-12 weeks. The median overall survival (OS) in months with 95% confidence intervals is also provided for each group, along with the number of patients in each group and the p-values for curve comparisons.

D. 13-20 Weeks

Image /page/25/Figure/4 description: This image is a survival plot that shows the probability of survival over time for four different groups of patients. The x-axis represents time in months from a 13-20 week blood draw, and the y-axis represents the probability of survival. The four groups are defined by their CTC levels at week 13-20 and their PSA reduction at 13-20 weeks. The median overall survival in months is 19.8 for group 1, 5.3 for group 3, 6.7 for group 4, and the median overall survival is not specified for group 2.

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

Figure 13 shows that patients with ≥5 CTC at any point after the initiation of therapy had a much higher likelihood of dying sooner, irrespective of the changes in PSA levels from baseline. Patients with ≥5 CTC at all time points (Group 3 and Group 4) had the shortest median overall survivals, which were not significantly different…