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 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.

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 -.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Device: CellSearch™ Circulating Tumor Cell Kit (expanded indications for use in Metastatic Prostate Cancer)

1. Table of Acceptance Criteria and Reported Device Performance:

Acceptance Criteria Category	Specific Metric (as applicable)	Predetermined Acceptance Criteria (Implied)	Reported Device Performance
Recovery	Regression analysis (slope, R-squared)	Slope = 1.0 (ideal)	Y = 0.93x + 3.87, R=0.999 (0.999) Average recovery: 93%
Linearity / Reportable Range	Regression analysis (slope, R-squared)	Slope ≈ 1.0 (after factoring out recovery loss)	Slope = 1.007, Intercept = 3.0, R² = 0.990 (R = 0.995) Range: 0 to 1238 tumor cells
Limits of Detection	Minimum detectable CTC count	1 CTC per 7.5 mL	1 CTC per 7.5 mL
System Reproducibility (Control)	% CV (low control)	Not explicitly stated, but generally <20% is acceptable for clinical assays	18%
System Reproducibility (Control)	% CV (high control)	Not explicitly stated, but generally <10% is acceptable for clinical assays	5%
System Reproducibility (Patient Samples MBC)	Regression analysis (slope, R)	Slope ≈ 1.0, R ≈ 1.0	Y = 0.98x + 0.67, R=0.99
System Reproducibility (Patient Samples MCRC)	Regression analysis (slope, R-squared)	Slope ≈ 1.0, R-squared ≈ 1.0	Y = 0.98x + 0.18, R²=0.96
Clinical Efficacy (PFS - Baseline CTC)	Log-rank p-value for <5 vs ≥5 CTC	p < 0.05	p = 0.0008
Clinical Efficacy (PFS - Follow-up CTC)	Log-rank p-value for <5 vs ≥5 CTC	p < 0.05	<0.0001 for all follow-up time points
Clinical Efficacy (OS - Baseline CTC)	Log-rank p-value for <5 vs ≥5 CTC	p < 0.05	p < 0.0001
Clinical Efficacy (OS - Follow-up CTC)	Log-rank p-value for <5 vs ≥5 CTC	p < 0.05	<0.0001 for all follow-up time points
Clinical Efficacy (PFS - CTC Reduction)	Survival difference between groups	Significantly longer PFS for CTC reduction groups	Median PFS for Group 2 significantly longer than Group 3; Group 4 shortest, Group 1 longest.
Clinical Efficacy (OS - CTC Reduction)	Survival difference between groups	Significantly longer OS for CTC reduction groups	Group 4 shortest median OS; Group 1 longest. Group 2 similar to Group 1; Group 3 shorter than Group 1, not significantly different from Group 4.
Multivariate Cox Regression (PFS AUC)	Hazard Ratio (HR) and p-value for CTC	HR > 1 and p < 0.05	HR > 1 and p < 0.05 at most time points
Multivariate Cox Regression (OS AUC)	Hazard Ratio (HR) and p-value for CTC	HR > 1 and p < 0.05	HR > 1 and p < 0.05 at most time points
Concordance with PSA (Overall Agreement)	% Agreement with 30% PSA reduction	Implied to be clinically acceptable for combined assessment	Ranging from 59% to 77% (patient-wise); 66% (observation-wise)

Notes on Acceptance Criteria: The document does not explicitly state numerical acceptance criteria for most performance metrics. Instead, it presents the results of studies and statistical analyses, implying that the observed performance (e.g., strong linearity, good reproducibility, and statistically significant associations with clinical outcomes) met the internal and regulatory expectations for device approval. For clinical effectiveness, the key acceptance criterion is the demonstration of statistically significant associations between CTC counts and clinical outcomes (PFS and OS), and that CTC provides prognostic information, even in cases of discordance with PSA.

2. Sample Sizes Used for the Test Set and Data Provenance:

Recovery: 30 samples (5 different spike levels x 5 donors + unspiked control, processed once). Data provenance: Blood samples from a single healthy donor pooled and spiked with cultured breast cancer cells (SK-BR-3), repeated for four additional donors. Not explicitly stated if prospective or retrospective, but likely prospective lab study.
Linearity/Reportable Range: Same data as Recovery study (30 samples).
Limits of Detection: Not a separate sample set, derived from Recovery data and system specifications.
System Reproducibility (Control): N=99 for both low and high controls. Not explicitly stated if prospective or retrospective or country of origin, but implies controlled laboratory conditions over 30 days.
System Reproducibility (MBC Patient Samples): 163 duplicate blood samples from 47 metastatic breast cancer patients. Data provenance: Not explicitly stated country of origin, appears to be retrospective analysis of samples collected during a clinical study.
System Reproducibility (MCRC Patient Samples): 1,627 duplicate blood samples from 430 MCRC patients. Data provenance: Not explicitly stated country of origin, appears to be retrospective analysis of samples collected during a clinical study.
Clinical Study (Metastatic Prostate Cancer Patients):
- Test set for clinical efficacy: 231 metastatic prostate cancer patients enrolled.
- PFS/OS analyses:
  - Baseline: 219 patients
  - 2-5 Weeks: 199 patients (PFS), 203 patients (OS)
  - 6-8 Weeks: 141 patients (PFS), 163 patients (OS)
  - 9-12 Weeks: 134 patients (PFS), 149 patients (OS)
  - 13-20 Weeks: 116 patients (PFS), 143 patients (OS)
- Concordance with PSA: 197, 159, 146, and 138 patients at 2-5, 6-8, 9-12, and 13-20 weeks respectively had paired CTC and PSA data.
- Data Provenance: Multi-center prospective clinical trial. Country of origin not specified, but typically such trials would involve multiple sites in the US and/or Europe.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications:

The document does not mention the use of experts to establish a "ground truth" for the test set in the traditional sense of image adjudication or pathological review for the clinical study.

For the technical performance studies (Recovery, Linearity, LOD, Reproducibility), the "ground truth" for cell counts was effectively established by the engineered spiking of known numbers of cells (Recovery, Linearity) or by established analytical methods for the control materials. The CellTracks Analyzer II or CellSpotter Analyzer automatically scans and displays events, with a user performing final classification based on EpCAM+, CK+, DAPI+, CD45- phenotype and morphological features. While this involves human review, the expertise and number of individuals are not specified as a "ground truth" panel.
For the clinical study (Metastatic Prostate Cancer), the "ground truth" for clinical outcomes (Progression-Free Survival and Overall Survival) was established through:
- Disease Progression: Determined by the clinical sites using PSA, imaging, and/or clinical signs and symptoms. This implies a standard clinical assessment by the patient's treating physicians and/or study investigators, not a centralized expert panel for ground truth labeling of images.
- Overall Survival: Determined from the date of death or date of last contact (for those still alive) from the study follow-up. This is an objective outcome.

Essentially, the device itself generates the "CTC count," and the study evaluates how these counts correlate with established clinical endpoints, rather than evaluating the accuracy of the CTC count against a separate "expert ground truth" for each specific scan.

4. Adjudication Method for the Test Set:

Clinical Outcomes (PFS/OS): As noted above, disease progression was determined by the clinical sites using a combination of PSA, imaging, and clinical signs/symptoms. It is not described as a formal "adjudication panel" in the sense of multiple independent reviewers resolving discrepancies. Survival is an objective endpoint.
CTC Enumeration: The CellTracks Analyzer II or CellSpotter Analyzer displays images of potential CTC events to a user for "final classification." The document does not detail an adjudication method (e.g., 2+1, 3+1) for these user classifications of CTCs, implying a single user's classification is considered the final count.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study Was Done:

No, a Multi Reader Multi Case (MRMC) comparative effectiveness study was not reported. The study focused on the prognostic value of the device's output (CTC counts) as a standalone marker, not on how the device assists human readers in making a diagnosis or prognosis. The device enumerates CTCs, and a human user reviews the images for final classification, but the study design does not involve comparing human readers with and without AI assistance on a case set.

6. If a Standalone Study Was Done (Algorithm only without human-in-the-loop performance):

The device is described as having a human-in-the-loop component where 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." Therefore, the reported performance throughout the document (e.g., clinical efficacy) represents the performance of the system including the final human classification, not a purely standalone algorithm.

7. The Type of Ground Truth Used:

For Technical Performance (Recovery, Linearity): Known spike concentrations of cultured cancer cells.
For Clinical Efficacy (PFS, OS):
- Progression-Free Survival (PFS): Clinical determination of disease progression (based on PSA, imaging, and clinical signs/symptoms) or death.
- Overall Survival (OS): Date of death or last contact.
  These are real-world clinical outcomes and are considered the definitive "ground truth" for prognostic value.

8. The Sample Size for the Training Set:

The document does not explicitly describe a separate "training set" in the context of developing the CellSearch™ Circulating Tumor Cell Kit. The studies described are primarily performance validation and clinical validation studies. The CellSearch system and its CTC enumeration method likely underwent internal development and optimization using various samples, but a formally documented "training set" for an AI/algorithm component is not presented. It refers to a "long run method of NCCLS guideline EP5-A2" for reproducibility with control samples, which implies standardized internal quality control, not an AI training set.

9. How the Ground Truth for the Training Set Was Established:

Since a formal "training set" for an AI/algorithm in the sense of machine learning is not described, the method for establishing its ground truth is also not detailed. The "ground truth" for the technical aspects of the device's operation (e.g., successful capture and identification of cells) would have been established through microscopy, immunostaining controls, and other laboratory techniques during the device's design and analytical validation.

Summary

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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 202Raritan, NJ 08869
Phone:	(908) 541-5843
FAX:	(908) 575-3010
Contact:	Debra J. RasmussenWorldwide 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 andEnumeration System, Class II, 21 CFR 866.6020, ProductCode 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

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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.

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 CellCount	Mean ObservedTumor 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(% 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 DrawTiming	MPC Patients Not Evaluable:				Total # of MPC Patients Evaluable
	PFS & OS			PFS Only	BloodNotDrawn	Blood Drawn 1-7 days aftertherapyadministration	No Follow-upBeyond Dateof BloodDraw	No or Non-EvaluableCTC Results	Blood drawnafter date ofdiseaseprogression
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 TimeAfter Tx Initiation	N	≥5 CTC	Median PFS in Months (95% CI)<5 CTC	Median PFS in Months (95% CI)≥5 CTC	Log-rankp-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.

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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
Sampling Time After Tx Initiation	N	≥5 CTC	Median OS in Months (95% CI)		Log-rankp-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.

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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),

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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 MPCPatients	PFS Risk fromBaseline		OS Risk FromBaseline
	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 fromBaseline		OS Risk FromBaseline
	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	# ofPatients	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

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

Parameter	# ofPatients	PFS Risk from BaselineHR	PFS Risk from Baselinep-value 2	OS Risk from BaselineHR	OS Risk from Baselinep-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.

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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.

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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
TimePoint	N	≥5(%)	Median OS<5	Median OS≥5	logrankp-value	HR	N	<30%(%)	Median OS≥30%	Median OS<30%	logrankp-value	HR	N	<50%(%)	Median OS≥50%	Median OS<50%	logrankp-value	HR
2-5Weeks	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
6-8Weeks	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
9-12Weeks	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
13-20Weeks	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.

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% Reduction in PSAfrom Baseline at 2-5 WeeksAfter Initiation of Therapy	CTCs 2-5 Weeks after theInitiation of Therapy	Total
	< 5 CTCs/7.5mL	≥ 5 CTCs/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	Lower95% CI	Upper95% 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 PSAfrom Baseline at 13-20 WeeksAfter Initiation of Therapy	CTC 13-20 Weeks after theInitiation 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	Lower95% CI	Upper95% 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.

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% Reduction in PSAfrom Baseline After Initiation ofTherapy	CTC after theInitiation 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	Lower95% CI	Upper95% 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. However, the median OS of these two groups was significantly different compared to the median OS of the patients with <5 CTC at all time points (Group 1 and Group 2). These two groups (Group 1 and Group 2) had the longest median overall survivals, which were not significantly different. The important finding illustrated in Figure 13 is that although a reduction of PSA at some points after initiation of therapy may reach significance for prediction of survival, Favorable CTC at any time point were more accurate than the PSA evaluation. The practical implication is the use of CTC analysis for the evaluation of the probability of survival of MPC patients. In cases where CTC and PSA change were discordant, CTC provided the most accurate assessment of prognosis.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/27/Picture/1 description: The image shows the seal of the Department of Health & Human Services. The seal is circular and contains the words "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" around the perimeter. In the center of the seal is a stylized image of an eagle with its wings spread.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

FEB 2 6 2008

Veridex, LLC c/o Ms. Debra J. Rasmussen Worldwide Executive Director Regulatory Affairs 1001 US Highway 202 Raritan, NJ 08869

Re: K073338 Trade/Device Name: CellSearchTM Circulating Tumor Cell Kit (Epithelial) Regulation Number: 21 CFR 866.6020 Regulation Name: Immunomagnetic Circulating Cancer Cell Selection and Enumeration System Regulatory Class: Class II Product Code: NQI Dated: November 27, 2007 Received: November 28, 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 begin marketing your device as described in your Section 510(k) premarket notification. The

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Page 2 -

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 (240) 276-0450. 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 (800) 638-2041 or (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours.

Robert Becker/

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

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

INDICATIONS FOR USE

510(K) Number (if known): K073338

Device Name: CellSearch™ Circulating Tumor Cell Kit

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

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

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use X (Part 21 CFR 801 Subpart D) AND/OR

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

Maria M Chan
Division Sign-Off

Office of In Vitro Diagno Device Evaluation and Sa

510(k) Ko 73338

Regulation Number and Section

§ 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system.

(a)
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.(b)
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.