CELLSEARCH CIRCULATING TUMOR CELL KIT

K050245 · Veridex, LLC · NQI · Mar 15, 2005 · Immunology

Device Facts

• Record ID: K050245
• Device Name: CELLSEARCH CIRCULATING TUMOR CELL KIT
• Applicant: Veridex, LLC
• Product Code: NQI · Immunology
• Decision Date: Mar 15, 2005
• 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 cancer. A CTC count of 5 or more per 7.5mL of blood is predictive of shorter progression free survival and overall survival.

Device Story

The CellSearch™ Circulating Tumor Cell Kit uses immunomagnetic selection to isolate epithelial cells from 7.5 mL of whole blood. Anti-EpCAM monoclonal antibodies conjugated to ferrofluid particles bind to target cells; a magnetic field concentrates these cells onto a cartridge surface. Fluorescent reagents (CK-PE, CD45-APC, DAPI) are added to label cytokeratin, leukocytes, and nuclei, respectively. The cartridge is processed on the CellTracks® Analyzer II, which acquires fluorescent images. Software identifies potential tumor cells (EpCAM+, CK+, DAPI+, CD45-) and presents them in a gallery for operator classification. The operator confirms CTCs by checking composite images; the software tallies the count. Used in clinical laboratories by trained technicians. The CTC count (threshold 5 cells/7.5 mL) assists clinicians in monitoring metastatic breast cancer patients, providing prognostic information regarding progression-free and overall survival.

Clinical Evidence

Prospective longitudinal trial (N=177) of metastatic breast cancer patients. Primary endpoints: Progression-Free Survival (PFS) and Overall Survival (OS) based on baseline and 1st follow-up CTC counts. Patients with ≥5 CTC/7.5 mL had significantly shorter median PFS (11.7 vs 30.3 weeks, p=0.0001) and OS (43.3 vs >80 weeks, p<0.0001) compared to those with <5 CTC. Multivariate Cox regression confirmed CTC count as the strongest predictor of PFS and OS.

Technological Characteristics

Immunomagnetic selection using anti-EpCAM ferrofluid particles. Fluorescent labeling (CK-PE, CD45-APC, DAPI). System includes CellTracks® AutoPrep System and CellTracks® Analyzer II. Connectivity: Standalone system. Software: Automated image acquisition and analysis. Sterilization: Not specified.

Indications for Use

Indicated for patients treated for metastatic breast cancer. The presence of CTC in peripheral blood is associated with decreased progression-free and overall survival. A CTC count of 5 or more per 7.5 mL of blood is predictive of shorter progression-free and overall survival.

Regulatory Classification

Identification

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

Special Controls

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

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

Predicate Devices

• CellSearch™ Epithelial Cell Kit (K031588)

Submission Summary (Full Text)

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### 510(k) SUMMARY

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

The assigned 510(k) number is: K050245

Date Prepared: 03/07/2005

| Submitter:<br>name, address,<br>contact. | Veridex, LLC<br>33 Technology Drive<br>PO Box 4920<br>Warren, New Jersey 07059<br>Telephone:<br>Fax:<br>Email: | [phone]<br>[phone]<br>[e-mail] |
|------------------------------------------|----------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------|
| Contact Person: | Debra J. Rasmussen<br>Worldwide Executive Director<br>Regulatory and Quality Affairs | |
| Identification of the<br>Device: | Trade/Proprietary Name: | CellSearch™ Circulating Tumor<br>Cell Kit |
| | Common Name: | CellSearch™ Circulating Tumor<br>Cell Kit |
| | Classification Name: | Immunomagnetic Circulating Cancer<br>Cell Selection and Enumeration<br>System |
| | Device Classification: | II |
| | Regulation Number: | 21 CFR 866.6020 |
| | Product code: | NQI |
| | Classification Panel: | Immunology Devices - 82 |
| Special Controls: | No Special controls have been issued for <i>in vitro</i> devices under<br>sections 513 and 514. | |
| Establishment<br>Registration Number: | 3004582358 | |
| Predicate Device: | CellSearch™ Epithelial Cell Kit<br>K031588 | |

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| Device Description: | Epithelial cells are immunomagnetically labeled by targeting the<br>Epithelial Cell Adhesion Molecule (EpCAM) antigen. Anti-EpCAM<br>monoclonal antibodies conjugated to ferrofluid particles are colloidal<br>and, when mixed with a sample containing the target epithelial cells,<br>bind to the EpCAM antigen associated with the epithelial cells.<br>After immunomagnetic selection of epithelial cells from 7.5 mL of<br>blood. Fluorescent reagents are added at this time to discriminate<br>between the immunomagnetically selected cells. Anti-Cytokeratin -<br>Phycoerythrin (CK-PE) stains the intracellular cytoskeleton<br>cytokeratin proteins expressed in cells of epithelial origin, anti-<br>CD45-Allophycocyan (CD45-APC) stains leukocytes and DAPI<br>stains DNA present in the cell nucleus. A strong magnetic field is<br>applied to the processed reagent/sample mixture that causes the<br>labeled target cells to move to the cartridge surface. The cartridge is<br>then placed on the CellTracks® Analyzer II for data acquisition and<br>analysis. The CellTracks® Analyzer II acquires images of PE, APC<br>and DAPI fluorescence staining of the entire viewing surface. |
|-------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | After data acquisition is completed, the images are analyzed for any<br>event where cytokeratin-PE and DAPI are within a specified space in<br>the cartridge, i.e. indicating the possible presence of a cell with a<br>nucleus that expresses cytokeratin. Images from each fluorescent<br>color as well as a composite image of the cytokeratin staining (green)<br>and the nuclear staining (purple) are presented to the user in a gallery<br>for final cell classification. A cell is classified as a tumor cell when<br>it its EpCAM+ (i.e., it is captured), CK+, DAPI+ and CD45-. A<br>check mark placed by the operator next to the composite images<br>classifies the event as a Circulating Tumor Cell (CTC) and the<br>software tallies all the checked boxes to obtain the CTC count. |
| | Our data demonstrate that metastatic breast cancer patients with 5 or<br>more CTC/per 7.5 mL of blood have a significantly greater<br>probability for shorter progression free and overall survival than<br>patients who have fewer than 5 CTC per 7.5 mL of blood. |
| Intended Use for the<br>CellSearch Assay: | The CellSearch™ Circulating Tumor Cell Kit is intended for the<br>enumeration of circulating tumor cells (CTC) of epithelial origin<br>(CD45-, EpCAM+, and cytokeratins 8, 18+, and/or 19+) in whole<br>blood. |
| | The presence of CTC in the peripheral blood, as detected by the<br>CellSearch™ Circulating Tumor Cell Kit, is associated with<br>decreased progression free survival and decreased overall survival in<br>patients treated for metastatic breast cancer. A CTC count of 5 or<br>more per 7.5mL of blood is predictive of shorter progression free<br>survival and overall survival. |

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## Summary of Technological Characteristics of the CellSearch™ Circulating Tumor Cell Kit:

The CellSearch™ Circulating Tumor Cell Kit has the same technological characteristics (i.e., design, material, chemical composition, energy source) as the CellSearch™ Epithelial Cell Kit. Modifications have been made to the CellSearch™ Epithelial Cell Kit (K031588) to optimize its performance on the CellTracks® AutoPrep System to deliver the same performance as previously established using the CellPrep™ Sample Preparation System. Modifications include:

- Minor formulation and volume changes .
- Minor incubation time changes .
- Rename product: CellSearch TM Circulating Tumor Cell Kit (Epithelial) .

Additionally, the CellSpotter® Analyzer is being modified to replace obsolete parts, place the analyzer inside an enclosure (cosmetic change to make system look more like/compatible to a CellTracks® AutoPrep System), and is being renamed to CellTracks® Analyzer II. These modifications to the CellSearch™ Circulating Tumor Cell Kit do not raise any new issues of safety and effectiveness and the intended use is identical between the two systems.

#### Clinical and Non-Clinical Studies:

#### Non-Clinical Studies

The following non-clinical studies were selected to compare the CellSearch™ Circulating Tumor Cell Kit performance characteristics to the predicate device to show that the two devices are substantially equivalent. After each newly determined performance characteristic, the results are compared to the predicate and followed by the conclusion statement that the CellSearch™ Circulating Turnor Cell Kit meets the performance specification and is thus substantially equivalent.

#### Recovery

Blood samples from a single healthy donor were pooled and five of six 7.5 mL aliquots were spiked with 5, 20, 81, 325 and 1300 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.

| 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.

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To determine the overall, or least squares fit, for the comparison of the observed and expected cell counts across all the data, linear regression analysis was performed. The regression equation for these 30 samples was Y=0.93x + 3.87, R2=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.

These results agree well with those obtained for the predicate (K031588) pre-clinical study where a slope of 0.85, intercept of 5.6 and an r = 0.997 was observed over a range of 4 to 1142 cells. These data indicate that the recovery of the predicate device was 85%. Thus the new assay's 93% recovery appears to be better than the predicate's.

#### 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 original sample 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 turnor cells versus the numbers of expected tumor cells vielded a slope of 1.007, an intercept of 3.0, an r = 0.99 and r = 0.995. Therefore, once the percent recovery (cell loss) was factored out of the CTC values of each of the original samples, this analysis of the data demonstrated that the detection of CTC was linear over the reportable range of 0 to 1238 tumor cells.

These results demonstrate that the CellSearch™ CTC kit/CellTracks® Analyzer II detected the number of tumor cells expected from the known dilution. They also agree with those obtained previously for the predicate system (K031588) with a slope of 0.99, intercept of 5.7 and r2 = 0.99 over a reportable range of 4 to 1022 CTCs. The linearity and reportable range of the new device is very similar to that of the predicate over a greater range of CTCs.

#### Limits of Detection

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

Therefore, a CellSearch™ CTC kit/CellTracks® Analyzer II detection would require that there be at least 1.1 or approximately 1 cell, in the sample chamber prepared from 7.5 mL of whole blood in order to detect at least one cell. This is comparable to the 1.28 cell sensitivity calculation determined for the CellSearch™ Epithelial Cell kit/CellSpotter® Analyzer. This validates that the

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CellSearch™ CTC kit/CellTracks® Analyzer II is capable of delivering sensitivity equal to that of the CellSearch™ Epithelial Cell Kit/CellSpotter® Analyzer for whole blood.

#### Reproducibility

#### 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 EPS-A. 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.

#### Table 2. Summary of Precision Analyses

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

The results of the system reproducibility with CellSearch™ Circulating Tumor Cell Controls for the CellSearch™ Circulating Tumor Cell Kit are comparable to the reproducibility results for the predicate, which has a Total % CV of 9.4% for the High Control Cell (Mean 258) and 15.8% for the Low Control Cell (Mean 47). The reproducibility of the CellSearch™ Circulating Tumor Cell Kit meets the performance specification and is substantially equivalent to that of the predicate system.

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Comparison Studies:

#### Comparison of New Device to the Predicate System

To directly demonstrate comparable performance, a study was performed using different cell lines tested at varying concentrations on both the predicate device, CellSearch™ Epithelial Cell System (K031588), and the new device, CellSearch™ Circulating Tumor Cell System (K050245).

For this comparison, fixed and unstained cells from three different cell lines were spiked into blood from normal donors at three different levels for five days. The three cell lines (SK-Br-3, MCF-7, or PC3-9) were chosen to cover a broad range of EpCAM and Cytokeratin antigen density representing the capture and detection portions of the assay respectively. Three spike levels of each cell line were chosen to cover a range of potential clinical values. Of the three cell lines tested, the PC3-9 cell line has the lowest Cytokeratin antigen density. SK-Br-3 cells demonstrate an uneven bimodal population consisting primarily of moderate level Cytokeratin antigen density cells and a smaller population of higher expressing cells. MCF-7 cells demonstrate the highest level of consistent Cytokeratin expression. The Cytokeratin antigen is the target of the detection reagent for tumor cells in the CellSearch™ Circulating Turmor Cell kits. For MCF-7 cells, the slope of the regression line = 1.03, an intercept of 1.5 and an r2 = 0.994. For SK-Br-3 cells, the slope of the regression line = 1.01 with an intercept of 2.9 and an r = 0.984. For PC3-9 cells, the slope of the regression line = 1.19 with an intercept of 10.5 and an r = 0.963. The slope of 1.19 for PC3-9 cells may be due to an improved dynamic range of the new device resulting in a flattening out of the response curve at higher cell numbers. Innether words, the recovery of CTC by the CellSearch™ Circulating Tumor Cell System at high numbers of cells may be somewhat more sensitive than recovery by the predicate, paricit arly with lower EpCAM antigen density cells as is the case with PC3-9 cells. This difference could also be attributable to increased reliability and/or stability of the CellTracks® AutoPero Systemas compared to the CellPrep™ Sample Preparation System. Regardless of this potential difference. there appears to be no difference between the CellSearch™ Circulating Tumor Cell System and the predicate at the medical decision level of 5 to 50 CTC.

All of the above new studies with the CellSearch™ Circulating Cell Kit/ AutoPrep/CellTracks® Analyzer II system demonstrate that the detection of turnor cells by the CellSearch™ Circulating Cell Kit/ AutoPrep/CellTracks® Analyzer II system is substantially equivalent to the oreaticate system. Therefore, the following interpretation of results, interfering substance analysis, and clinical data generated using the predicate system (K035188) is applicable to the new device (K050245).

#### Interpretation of Results

Results are reported as the number of CTC / 7.5 mL of blood. A CTC count of 5 or more per 7.5 mL of blood is predictive of shorter progression free survival and overall survival.

Precaution: Specimens with more than 5,000 CTC per 7.5 mL of blood were less than 0.03% of those seen in our clinical studies. Sample carryover is of concern when such a high CTC specimen is immediately followed in the CellTracks® AutoPrep System by a specimen yielding a CTC result in the range 5 to 15 CTC per 7.5 mL of blood. In this case, we recommend obtaining a new blood sample from the low CTC patient and

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performing a confirmatory CTC analysis. To identify following samples. refer to the CellTracks® AutoPrep User's Guide section on View Data and obtain the detailed batch data, including sample and patient identification for each tube in the batch.

#### Interfering Substances

SKBr-3 cells spiked into blood samples were exposed to potential interfering substances and compared to untreated controls. Toxic levels (5 times therapeutic index) of the following cancer drugs, over-the-counter drugs, and other exogenous substances were tested: cyclophosphamide, Mitomycin C, Procrit, biotin, 5-fluorouracil, methotrexate, Tamoxifen Citrate, pacitaxel, Arimidex, acetaminophen, acetylsalicylic acid, caffeine, dextromethorphan, Aredia, Human Anti-Mouse Antibody (HAMA) type 1, HAMA type 2, Herceptin, and ibuprofen. No significant differences in SKBr-3 cell numbers were detected, indicating that these substances do not interfere with the CellSearch™ kit.

Samples spiked with toxic levels of doxorubicin resulted in aberrant staining of leukocytes as cytokeratin and CD45 dual positive cells, due to the doxorubicin being a fluorescence compound that is incorporated into nucleated cells. If seen, the staining pattern, of all cells being CD45 positive and cytokeratin positive, is obvious and easily identified by the operator as a known interference staining profile. If blood is drawn outside of the recommended 7 day wash-out period following doxorubicin infusion, this interference is unlikely to be observed in clinical practice, given controlled therapeutic levels and rapid drug clearance.

Potential interference from lipemia was studied by adding Intralipid to samples to a concentration of 2.6%, which is greater than 1000 mg/dl triglyceride. Samples were lysed to simulate total hemolysis. Bilirubin at 7.4 mg/dL, HAMA 1/HAMA 2 and hematocrit from 18-60% were studied. Lipemia, hemolysis, icterus and a broad range of hematocrit values do not interfere with the Cel!Search™ assay. HAMA 1 and HAMA 2 also do not interfere, indicating that individuals receiving mouse Ig by parenteral routes can be tested successfully with the CellSearch™ assay.

#### System Reproducibility with Patient Specimens

A total of 163 duplicate samples were collected from 47 patients over the course of the clinical study. These samples were processed separately on multiple systems at different sites (including different CellPrep™ instruments) 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 =0.9978. Table 3 shows the summary of the data for replicates where the average of the two CTC results was <5 compared to those where the average was >5.

#### Table 3. Reproducibility of CTC Counts in Duplicate Samples (n=163) with an Average of <5 or >5 CTC per 7.5 mL of blood

| | CTC <5 | CTC ≥5 |
|--------------------------------------|--------|--------|
| Number of Duplicates | 123 | 40 |
| Mean CTC Count of Duplicates | 0.7 | 210.5 |
| Avg. Duplicate Standard<br>Deviation | 0.5 | 120 |
| Avg. %CV of Duplicates | 60.0% | 20.0% |

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#### Summary of Clinical Trial Results

#### Expected Values

Healthy volunteers, non-malignant breast disease, non-malignant other disease Single point CTC analyses were performed on control groups of 145 healthy volunteers, 101 women with non-malignant breast disease, and 99 women with other non-malignant diseases.

Epithelial cells are not expected to be in the peripheral blood. Of the 345 total samples from healthy volunteers and women with non-malignant disease, only one subject had more than 5 CTC/7.5 mL. The results are presented in Table 4.

| Category | N | Mean<br># CTC | SD | # Patients with<br>> 5 CTC | Min.* | Max.* |
|----------------------------------------|-----|---------------|-----|----------------------------|-------|-------|
| Healthy | 145 | 0.1 | 0.2 | 0 | 0 | 1 |
| Non-<br>malignant<br>breast<br>disease | 101 | 0.2 | 1.2 | 1 | 0 | 12 |
| Non-<br>malignant<br>other<br>disease | 99 | 0.1 | 0.4 | 0 | 0 | 3 |

Table 4. Control Subjects

* NCCLS Guideline C28-A2-

#### Metastatic Breast Cancer Patients

A multi-center prospective, longitudinal clinical trial was conducted. Results were used to determine whether the number of CTC predict disease progression and survival. Patients with measurable disease and who were starting a new line of therapy were enrolled (N=177). Clinical data were analyzed on an intent-to-treat basis.

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| | Table 5. Patient Demographics | |
|--|-------------------------------|--|
| | | |

| Age at Baseline (Median) | 58.0 ± 13.4 (58) |
|--------------------------|---------------------------------------------------------------------------------------------------------------|
| Race: White | 153 (84%) |
| Black | 14 ( 8%) |
| Hispanic | 7 ( 4%) |
| Unknown | 3 ( 2%) |
| ER/PR + | 121 (68%) |
| ER/PR - | 54 (31%) |
| Unknown | 2 (1%) |
| Her-2/neu - | 91 (52%) |
| Her-2/neu 1+ | 12 ( 7%) |
| Her-2/neu 2+ | 18 (10%) |
| Her-2/neu 3+ | 27 (15%) |
| Unknown | 29 (16%) |
| Line of Therapy | 1st 83 (47%)<br>2nd 25 (14%)<br>≥ 3rd 67 (38%)<br>Unk.* 2 ( 1%) |
| Type of Therapy | Hormone 47 (26%)<br>Chemo 87 (49%)<br>Immu/C/H 28 (16%)<br>H / C 10 ( 6%)<br>No Tx** 4 ( 2%)<br>Unk.* 1 ( 1%) |

*Unk. = Information not available **No Tx. = No treatment information obtained C or Chemo = Chemotherapy, H or Hormone = Hormone Therapy, 1 or Immuno = Immunotherapy

Baseline CTC count was determined prior to initiation of a new line of therapy. A first follow-up CTC count was determined after the initiation of therapy. For the baseline analyses, Progression Free Survival (PFS) was measured from the time of the baseline blood draw to the diagnosis of progression by CT scans and/or clinical signs and symptoms, and Overall Survival (OS) was measured from the time of baseline blood draw to the time of death. For the first follow-up analyses, PFS was measured from the time of 1st follow-up blood draw (mean 4.5 ± 2.4 weeks following enrollment) to diagnosis of progression or death, and OS was measured from the time of 1st follow-up blood draw to the time of death.

#### Progression Free Survival (PFS) Analysis

PFS Using Baseline CTC Results

All 177 patients had a baseline CTC test performed. For Kaplan-Meier analysis, patients were segmented into two groups based upon their CTC count at baseline:

- . The Favorable group (N=90), represented in green, consisted of patients with <5 CTC.
The Unfavorable group (N=87), represented in red, consisted of patients with >5 CTC. . Median PFS was 30.3 weeks (~7.0 months) for the Favorable group and 11.7 weeks (~2.7 months) for the Unfavorable group. The difference in PFS between the two groups is highly significant (Log-rank p=0.0001, Cox Hazards Ratio=1.9547, chi-square=15.33, p = 0.0001). These results are illustrated in Figure 1.

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Image /page/9/Figure/2 description: This image is a graph that shows the probability of progression-free survival over time. The x-axis represents time from baseline in weeks, and the y-axis represents the probability of progression-free survival. There are two lines on the graph, one representing patients with less than 5 CTCs at baseline and the other representing patients with more than 5 CTCs at baseline. The median PFS time for patients with less than 5 CTCs at baseline is 30.3 weeks, while the median PFS time for patients with more than 5 CTCs at baseline is 11.7 weeks.

# Figure 1. PFS of Patients with < 5 or ≥ 5 CTC at Baseline (N=177)

### PFS Using 1st Follow-up CTC Results

I TS Osmg 1 1 oftow ap OF Steems
Of the 177 patients, 23 were not evaluable at first follow-up. Of these 23 patients, ten patients Of the 177 patients, 25 were not evaluad be obtained, nine patients progressed prior to the 14 died before a follow-up blood draw, and to follow-up. Additionally, the ten patients who died had high to extremely high CTC counts at baseline (CTC counts 9, 11, 15, 24, 111, 126, 301, 1143, 4648 and 23618). For Kaplan-Meier analysis, patients were segmented into two groups based upon their CTC count at 1st follow-up:

- The Favorable group (N=111), represented in green, consisted of patients with <5 CTC, .
- The Unfavorable group (N=43), represented in red, consisted of patients with >5 CTC. .

. . The Ontavorable group (11- 15) . 1 months) for the Favorable group and 5.7 weeks (~1.3 months) for the Unfavorable group. The difference in PFS between the two groups is highly significant (Log-rank p<0.0001, Cox Hazards Ratio=2.4842, chi-square=18.83, p< 0.0001). These results are illustrated in Figure 2.

Figure 2. PFS of Patients with < 5 or > 5 CTC at 1st Follow-Up (N=154)

Image /page/9/Figure/10 description: This image is a graph showing the probability of progression-free survival over time. The x-axis represents time from the first follow-up in weeks, with a conversion to months provided. Two lines are plotted, one representing patients with less than 5 CTCs at the first follow-up, and the other representing patients with 5 or more CTCs at the first follow-up. The graph indicates median progression-free survival times of approximately 1.3 months for patients with 5 or more CTCs and 6.1 months for patients with less than 5 CTCs.

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#### Predictive Value of CTC Reduction on PFS

For Kaplan-Meier analysis, patients were segmented into three groups based on their CTC counts at baseline and 1st follow-up:

- The Favorable group (N=81), represented in green, consisted of patients with <5 CTC at both . time points,
- Patients with 5 or more CTC at baseline that decreased to below 5 CTC at 1st follow-up are . represented in alis e green (N=33).
- The Unfavorable group (N=49), represented in red, consisted of patients with >5 CTCs at 15t . follow-up,

Elapsed PFS time was calculated from the baseline blood draw. Three groups were plotted in Figure 3. The Favorable group (N=81, green line) had a median PFS of 30.3 weeks (~7.0 months) and the patients represented by the offece green line (N=33) had a median PFS of 32.9 weeks (~7.6 months). The Unfavorable group (N=49, red line) had a median PFS of 8.9 weeks (~2.1 months). The difference in the PFS of the patients in the Favorable and olive groups compared to the PFS of the patients in the Unfavorable group is highly significant (Log-rank p<0.0006, Cox Hazards Ratio=1.6600, chi-square=22.08, p< 0.0001).

#### Figure 3. A Reduction in CTC Count to Below 5 at the 1st Follow-Up Time Point After the Initiation of Therapy Predicts Improved PFS (N=163)

Image /page/10/Figure/9 description: This image is a survival plot showing the probability of progression-free survival over time. The x-axis represents time from baseline in weeks, with a conversion to months provided. There are two survival curves, one for patients with less than 5 CTC at baseline and at the first follow-up, and another for patients with greater than or equal to 5 CTC at the first follow-up. The median progression-free survival time is 30.3 weeks for the first group and 8.9 weeks for the second group.

#### Overall Survival (OS) Analysis

OS Analysis Using Baseline CTC Results

Death occurred in 66 (37%) of the 177 patients during this study. Seventeen (19%) of 90 patients from Favorable group (<5 CTC at baseline) compared to 49 (56%) of 87 from Unfavorable group (≥5 CTC at baseline) died. Median OS was greater than 80 weeks (>18 months) for the Favorable group and 43.3 weeks (~10.1 months) for the Unfavorable group. The OS difference between the two groups is highly significant (Log-rank p<0.0001, Cox Hazards Ratio=4.3865,

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chi-square=31.73, p< 0.0001). These results are illustrated in Figure 4. For Kaplan-Meier analysis, patients were segmented into two groups based upon their CTC count at baseline:

- The Favorable group (N=90), represented in green, consisted of patients with <5 CTC. .
- . The Unfavorable group (N=87), represented in red, consisted of patients with >> CTC.

Figure 4. OS of Patients with < 5 or > 5 CTC at Baseline (N=177)

Image /page/11/Figure/6 description: This image is a survival plot comparing two groups of patients based on the number of circulating tumor cells (CTCs) at baseline. The x-axis represents time from baseline in weeks, with a conversion to months provided. The y-axis represents the probability of survival. The plot shows that patients with fewer than 5 CTCs at baseline have a higher survival rate compared to those with 5 or more CTCs at baseline, with median survival times of greater than 80 weeks and 43.3 weeks, respectively.

#### OS Using 1st Follow-up CTC Results

For Kaplan-Meier analysis, patients were segmented into two groups based upon their CTC count at 1st follow-up:

- . The Favorable group (N=114), represented in green, consisted of patients with < > CTC,
The Unfavorable group (N=49), represented in red, consisted of patients with >5 CTC. .

Of the 163 evaluable patients at first follow-up, 56 (34%) died during this study; 23 of 114 (20%) from the Favorable group, 33 of the 49 (67%) from the Unfavorable group. Patients in the Favorable group had a median survival of greater than 80 weeks (>18 months), while the Unfavorable group had a median OS of 30 weeks (~ 7.0 months). The difference in OS between the two groups is highly significant (Log-rank p<0.0001, Cox Hazards Ratio=5.4537, chisquare=37.52, p< 0.0001). Results are summarized in Figure 5.

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Image /page/12/Figure/2 description: This image shows a survival analysis graph. The y-axis represents the probability of survival, ranging from 0% to 100%, while the x-axis represents the time from the first follow-up in weeks. There are two survival curves plotted: one for patients with less than 5 circulating tumor cells (CTCs) at the first follow-up, which shows a higher survival rate, and another for patients with 5 or more CTCs, which shows a lower survival rate. The median survival time for patients with less than 5 CTCs is greater than 80 weeks, while for those with 5 or more CTCs, it is 30 weeks.

#### Figure 5. OS of Patients with < 5 or > 5 CTC at 1st Follow-Up (N=163)

#### Predictive Value of CTC Reduction on OS

For Kaplan-Meier analysis, patients were segmented into three groups based upon their CTC counts at baseline and 1st follow-up:

- . The Favorable group (N=81), represented in green, consisted of patients with <> CTC at both time points,
- Patients with 5 or more CTC at baseline that decreased below 5 CTC at 1st follow-up are . represented by the olive green line (N=33),
- The Unfavorable group (N=49), represented in red, consisted of patients with >5 CTC at 18 . follow-up,

Elapsed OS time was calculated from the baseline blood draw. Figure 6 illustrates that a decrease to <5 CTC after the initiation of therapy significantly impacts OS. The Favorable group (green line) had a median OS of >80 weeks (18 months). The patients represented by the oliv < groun line (N=33) had a median OS of 62.6 weeks (~14.6 months). The Unfavorable group (red line) had a median OS of 35.4 weeks (~8.2 months). This difference in the OS of the patients in the Favorable and olive green groups compared to the OS of the patients in the Unfavorable group is highly significant (Log-rank p<0.0007, Cox Hazards Ratio=2.7462, chi-square=40.51, p<0.0001). These data suggest that baseline and 1st follow-up CTC levels are predictive of overall survival.

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Image /page/13/Figure/2 description: The image shows a small, dark, irregularly shaped blob against a white background. The blob appears to be solid and without any discernible internal features. Due to its simplicity and lack of context, it is difficult to determine the nature or origin of the blob. The image is minimalist, focusing solely on the contrast between the dark shape and the bright background.

Image /page/13/Figure/3 description: This image is a figure titled "Figure 6. A Reduction in CTC Count to Below 5 at the 1st Follow-Up Time Point After the Initiation of Therapy Predicts Improved OS (N=163)". The figure discusses the reduction in CTC count to below 5 at the first follow-up time point after the initiation of therapy. The figure also predicts improved overall survival (OS) with a sample size of 163.

Image /page/13/Figure/4 description: The image is a survival plot showing the probability of survival over time in weeks. There are three survival curves, one that is labeled '>18 Months', and another that has a median survival time of ~14.6 months, and another with a median survival time of ~8.2 months. The plot also includes statistical information such as the Logrank p-value, Cox Hazards Ratio, chi-square value, and p-value.

#### Multivariate Cox Regression Analysis

The following parameters were evaluated using multivariate Cox regression analysis, with the The forming paramons of Survival Data Based on the Cox Proportional Hazards Model), stepwise selection process to evaluate association with PFS and OS: patient age (continuous), stage of disease at diagnosis (I-IV), time to metastasis (continuous), ECOG status (continuous), varge of ar an line of therapy (0-2), ER/PR status (+/-), HER2/neu status (0-3), line of therapy (≥20d or 15), type of therapy (chemo/other or hormonal/immuno), baseline CTC count (≥5 or <5 CTC/7.5mL), and 1st follow-up CTC count (≥5 or <5 CTC/7.5mL). A stringency level (pvalue) of 0.05 was used to both include and exclude parameters in the multivariate analyses. Results for each parameter that demonstrated a statistically significant correlation to PFS and OS are summarized in Tables 6 and 7, respectively. CTC number was the strongest predictor of PFS and OS.

| | | Categories | | PFS Risk from Baseline | | | |
|--|-----------|-------------|-----------|------------------------|---------|------|---------------|
| | Parameter | Unfavorable | Favorable | HR | p-value | chi2 | # of Patients |

## Table 6. Multivariate Cox Analysis: Stepwise Cox Regression for Prediction of PFS

| Parameter | Categories | | PFS Risk from Baseline | | | |
|---------------------|-------------|-----------------|------------------------|---------|-------|---------------|
| | Unfavorable | Favorable | HR | p-value | chi² | # of Patients |
| Baseline CTC Number | ≥5 | <5 | 1.761 | 0.001 | 10.58 | |
| Line of Therapy | ≥2nd | 1st | 1.725 | 0.002 | 9.75 | 172 |
| Type of Therapy | Chemo/Other | Hormonal/Immuno | 1.611 | 0.016 | 5.85 | |

| | Categories | | PFS Risk from Baseline | | | |
|---------------------------|-------------|-----------|------------------------|---------|--|---------------|
| Parameter | Unfavorable | Favorable | | o-value | | # of Patients |
| l1st Follow-Up CTC Number | | | | | | |
| Line of Therapy | >2nd | | | | | |

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| | Categories | | OS Risk from Baseline | | | |
|--------------------------|---------------|-----------------|-----------------------|---------|-------|---------------|
| Parameter | Unfavorable | Favorable | HR | p-value | chi2 | # of Patients |
| Baseline CTC Number | >5 | <5 | 4.261 | <0.001 | 22.35 | |
| Line of Therapy | >2nd | 1st | 2.384 | 0.001 | 10.32 | |
| Type of Therapy | Chemo/Other | Hormonal/Immuno | 2.543 | 0.015 | 5.90 | 170 |
| ECOG Status | 2 vs. 1 vs. 0 | | 1.478 | 0.024 | 5.10 | |
| Time to Metastasis | Time in Years | | 0.922 | 0.028 | 4.82 | |
| Parameter | Categories | | OS Risk from Baseline | | | # of Patients |
| | Unfavorable | Favorable | HR | p-value | chi² | |
| 1st Follow-Up CTC Number | ≥5 | <5 | 6.493 | <0.001 | 38.34 | 160 |
| ER/PR Status | Positive | Negative | 0.349 | 0.001 | 11.19 | |
| Line of Therapy | ≥2nd | 1st | 2.291 | 0.006 | 7.67 | |
| ECOG Status | 2 vs. 1 vs. 0 | | 1.530 | 0.025 | 5.05 | |

# Table 7. Multivariate Cox Analysis: Stepwise Cox Regression for Prediction of OS

#### Conclusion:

New performance data demonstrates that the modifications made in the CellSearch™ system did Now performance data coments of the test. The newly determined performance not arrect the sarety and encerrentess or the specifications) such that the clinical data and interfering substance analysis determined using the predicate device are applicable to the new device and may be included in the new package insert.

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# DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/15/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo features a stylized eagle with three lines forming its body and wings. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" is arranged around the eagle in a circular fashion.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Ms. Debra J. Rasmussen World-wide Director of Quality and Regulatory Affairs Veridex, LLC 33 Technology Drive Warren, New Jersey 07059

K050245 Re:

K020245
Trade/Device Name: CellSearch™ Circulating Tumor Cell Kit Regulation Number: 21 CFR § 866.6020 Regulation Name: Immunomagnetic Circulating Cancer Cell Selection and Enumeration System Regulatory Class: II Product Code: NQI Dated: January 30, 2005 Received: February 3, 2005

MAR 1 5 2005

Dear Ms. Rasmussen:

We have reviewed your Section 510(k) premarket notification of intent to market the device indication we have reviewed your Section 910(t) premained is substantially equivalent (for the indications felerenced above and nave december and are and and and and marketed in interstate for use stated in the enclosule for legally manatible povice Americal Device American sonor Ford, Days commerce proof to May 20, 1976, the excordance with the provisions of the Federal Food. Drug, devices that have been recuire approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The You may, therefore, market the device, object on the requirements for annual registration, listing of general controls provisions of are tice, 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 If your device is classifica (Scc above) the exist on a controls. Section your device can
may be subject to such additional controls. Existing major regulations FDA may be subject to such additional controllations (CFR), Parts 800 to 895. In addition, FDA be found in Title 21, Ood of Peacharing your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean Please be advised that I Dri 3 issuation of a complies with other requirements of the Act that I DA has made a determination in administered by other Federal agencies. You must or any Federal statures and regulations and identify, but not limited to: registration and listing (21 comply with an the Ace Stequire.nems 801 and 809); and good manufacturing practice CFR Part 807), labeling (21 OF RT Rans 80% and (21 CFR Part 820). This letter requirents as set form in the quality by the as described in your Section 510(k) premarket will allow you to begin manceing your intral equivalence of your device to a legally marketed nothication. The PDA miding of succeman vipes and thus, permits your device to proceed to the market.

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Page 2 --

If you desire specific information about the application of labeling requirements to your device, If you desire specific into mioritising of your device, please contact the Office of In of questions on the promotion and as retries as [phone]. Also, please note the VITO Diagnostic Device Devilance and bases - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -You may obtain other general information on your responsibilities under the Act from the You may outlif only Manufacturers, International and Consumer Assistance at its toll-free number [phone] or [phone] or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html

Sincerely yours,

Robert Beckerh

Robert L. Becker, Jr., MD, Po Director Division of Immunology and Hematology Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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510(k) Notification

Veridex LLC CellSearch™ Circulating Tumor Cell Kit

## Indications for Use

510(k) Number (if known): Ko 50245

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 cytokeratin 8 & 18+, and/or cytokeratin 19+) in whole blood in conjunction with the CellTracks® AutoPrep System, the CellSpotter® Analyzer or CellTracks® Analyzer II, and the CellSearch™ Circulating Tumor Cell Control Kit.

The presence of CTC in the peripheral blood, as detected by the CellSearch™ Circulating Turnor Cell Kit, is associated with decreased progression free survival and decreased overall survival in patients treated for metastatic breast cancer. A CTC count of 5 or more per 7.5 mL of blood generally is predictive of shorter progression free survival and shorter overall survival.

X Prescription Use (Part 21 CFR 801 Subpart D)

AND/OR

Over-The-Counter Use (21 CFR 807 Subpart C)

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

Concurrence of CDRH, Office of In Vitro Diagnostic Devices(OIVD)

Mana M Chen

**Division Sign-Off**

Office of In Vitro Diagnostic Device Evaluation and Safety

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510(k) K050245

Regulatory Information