(41 days)
Not Found
No
The device description details image analysis based on predefined criteria (EpCAM+, CK+, DAPI+, CD45-) and requires manual operator classification of potential CTCs. There is no mention of automated learning or adaptive algorithms.
No.
The device is intended for the enumeration of circulating tumor cells and provides prognostic information (associated with survival), but does not treat or prevent disease.
Yes
The device is intended for the "enumeration of circulating tumor cells (CTC)", and the presence of these cells "is associated with decreased progression free survival and decreased overall survival in patients treated for metastatic breast cancer," indicating its use in diagnosing or monitoring a medical condition.
No
The device description clearly indicates that the CellSearch™ Circulating Tumor Cell Kit is a reagent kit used in conjunction with specific hardware systems (CellTracks® AutoPrep System, CellSpotter® Analyzer or CellTracks® Analyzer II) for sample preparation, image acquisition, and analysis. While software is involved in image analysis and counting, the device itself is a kit containing reagents and is dependent on external hardware.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states that the kit is for the "enumeration of circulating tumor cells (CTC) of epithelial origin... in whole blood". This involves testing a sample taken from the human body (whole blood) to provide information about a medical condition (presence of CTCs associated with metastatic breast cancer).
- Device Description: The description details a process of immunomagnetic labeling and fluorescent staining of cells from a blood sample, followed by analysis using specialized equipment. This is a typical workflow for in vitro diagnostic tests.
- Clinical Trial Results: The inclusion of clinical trial data demonstrating the association of CTC counts with progression-free survival and overall survival in metastatic breast cancer patients further supports its use in a clinical diagnostic context.
- Predicate Device: The mention of a predicate device (K031588; CellSearch™ Epithelial Cell Kit) which is also an IVD, indicates that this device falls within the same regulatory category.
The device performs tests on a biological sample (blood) outside of the body (in vitro) to provide information for the diagnosis, monitoring, or treatment of a medical condition (metastatic breast cancer). This aligns directly with the definition of an In Vitro Diagnostic device.
N/A
Intended Use / 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.
Product codes (comma separated list FDA assigned to the subject device)
NQI
Device Description
Epithelial cells are immunomagnetically labeled by targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen. Anti-EpCAM monoclonal antibodies conjugated to ferrofluid particles are colloidal and, when mixed with a sample containing the target epithelial cells, bind to the EpCAM antigen associated with the epithelial cells. After immunomagnetic selection of epithelial cells from 7.5 mL of blood. Fluorescent reagents are added at this time to discriminate between the immunomagnetically selected cells. Anti-Cytokeratin - Phycoerythrin (CK-PE) stains the intracellular cytoskeleton cytokeratin proteins expressed in cells of epithelial origin, anti-CD45-Allophycocyan (CD45-APC) stains leukocytes and DAPI stains DNA present in the cell nucleus. A strong magnetic field is applied to the processed reagent/sample mixture that causes the labeled target cells to move to the cartridge surface. The cartridge is then placed on the CellTracks® Analyzer II for data acquisition and analysis. The CellTracks® Analyzer II acquires images of PE, APC and DAPI fluorescence staining of the entire viewing surface.
After data acquisition is completed, the images are analyzed for any event where cytokeratin-PE and DAPI are within a specified space in the cartridge, i.e. indicating the possible presence of a cell with a nucleus that expresses cytokeratin. Images from each fluorescent color as well as a composite image of the cytokeratin staining (green) and the nuclear staining (purple) are presented to the user in a gallery for final cell classification. A cell is classified as a tumor cell when it its EpCAM+ (i.e., it is captured), CK+, DAPI+ and CD45-. A check mark placed by the operator next to the composite images classifies the event as a Circulating Tumor Cell (CTC) and the software tallies all the checked boxes to obtain the CTC count.
Our data demonstrate that metastatic breast cancer patients with 5 or more CTC/per 7.5 mL of blood have a significantly greater probability for shorter progression free and overall survival than patients who have fewer than 5 CTC per 7.5 mL of blood.
Mentions image processing
Acquires images of PE, APC and DAPI fluorescence staining of the entire viewing surface. After data acquisition is completed, the images are analyzed for any event where cytokeratin-PE and DAPI are within a specified space in the cartridge, i.e. indicating the possible presence of a cell with a nucleus that expresses cytokeratin. Images from each fluorescent color as well as a composite image of the cytokeratin staining (green) and the nuclear staining (purple) are presented to the user in a gallery for final cell classification.
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
peripheral blood
Indicated Patient Age Range
Not Found
Intended User / Care Setting
Not Found
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
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.
Linearity/Reportable Range: The previous recovery data was re-analyzed as a dilution series to evaluate test linearity. The confounding variable of percent recovery was removed 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.
Limits of Detection: Not applicable, calculated based on recovery rates.
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.
Comparison of New Device to the Predicate System: Fixed and unstained cells from three different cell lines (SK-Br-3, MCF-7, or PC3-9) were spiked into blood from normal donors at three different levels (potential clinical values) for five days. These cell lines were chosen to cover a broad range of EpCAM and Cytokeratin antigen density.
Interfering Substances: SKBr-3 cells spiked into blood samples were exposed to toxic levels (5 times therapeutic index) of various cancer drugs, over-the-counter drugs, and other exogenous substances. Samples spiked with toxic levels of doxorubicin were also tested. Potential interference from lipemia was studied by adding Intralipid to samples to a concentration of 2.6%. Samples were lysed to simulate total hemolysis. Bilirubin at 7.4 mg/dL, HAMA 1/HAMA 2 and hematocrit from 18-60% were studied.
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).
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Non-Clinical Studies:
- Recovery: A linear regression analysis of 30 samples resulted in Y=0.93x + 3.87, R2=0.999. On average, the recovery was 93%. This was compared to the predicate's 85% recovery (slope 0.85, intercept 5.6, r=0.997).
- Linearity/Reportable Range: Regression of observed vs. expected tumor cells yielded a slope of 1.007, an intercept of 3.0, an r=0.99 and r=0.995, over a reportable range of 0 to 1238 tumor cells. This was comparable to the predicate's slope of 0.99, intercept of 5.7, and r2=0.99 over 4 to 1022 CTCs.
- Limits of Detection: The device can detect 1 CTC per 7.5 mL, with approximately 93% recovery. This is comparable to the predicate's 1.28 cell sensitivity calculation.
- Reproducibility (System Reproducibility with CellSearch™ Circulating Tumor Cell Control): For the low control, N=99, Mean cell count=48, Total Precision Standard Deviation (ST) % CV=18%. For the high control, N=99, Mean cell count=969, Total Precision Standard Deviation (ST) % CV=5%. These results are comparable to the predicate's Total % CV of 9.4% for High Control (Mean 258) and 15.8% for Low Control (Mean 47).
- Comparison of New Device to the Predicate System: For MCF-7 cells, slope=1.03, intercept=1.5, r2=0.994. For SK-Br-3 cells, slope=1.01, intercept=2.9, r=0.984. For PC3-9 cells, slope=1.19, intercept=10.5, r=0.963. The performance was found to be substantially equivalent to the predicate, with no difference at the medical decision level of 5 to 50 CTC.
- Interfering Substances: No significant differences in SKBr-3 cell numbers were detected with various cancer drugs, over-the-counter drugs, and other exogenous substances. Lipemia, hemolysis, icterus, and a broad range of hematocrit values (18-60%) do not interfere. HAMA 1 and HAMA 2 also do not interfere. Doxorubicin at toxic levels resulted in aberrant staining, but a distinct staining pattern allows for identification.
- System Reproducibility with Patient Specimens: Regression equation for 163 duplicate samples was Y=0.98x + 0.67, R=0.9978. For CTC 5 CTC=0, Min=0, Max=1.
- Non-malignant breast disease: N=101, Mean # CTC=0.2, SD=1.2, # Patients with >5 CTC=1, Min=0, Max=12.
- Non-malignant other disease: N=99, Mean # CTC=0.1, SD=0.4, # Patients with >5 CTC=0, Min=0, Max=3.
- Metastatic Breast Cancer Patients (Multi-center prospective, longitudinal clinical trial): N=177
- PFS Using Baseline CTC Results:
- Favorable group (5 CTC at 1st follow-up, N=49): Median PFS = 8.9 weeks (~2.1 months).
- Log-rank p80 weeks (>18 months). (19% died)
- Unfavorable group (≥5 CTC, N=87): Median OS = 43.3 weeks (~10.1 months). (56% died)
- Log-rank p80 weeks (>18 months). (20% died)
- Unfavorable group (≥5 CTC, N=49): Median OS = 30 weeks (~7.0 months). (67% died)
- Log-rank p80 weeks (18 months).
- Patients with ≥5 CTC at baseline that decreased to 5 CTC at 1st follow-up, N=49): Median OS = 35.4 weeks (~8.2 months).
- Log-rank p5 vs. 2nd vs. 1st): HR=2.384, p-value=0.001, chi2=10.32.
- Type of Therapy (Chemo/Other vs. Hormonal/Immuno): HR=2.543, p-value=0.015, chi2=5.90.
- ECOG Status (2 vs. 1 vs. 0): HR=1.478, p-value=0.024, chi2=5.10.
- Time to Metastasis (Time in Years): HR=0.922, p-value=0.028, chi2=4.82.
- OS Risk (N=160):
- 1st Follow-Up CTC Number (≥5 vs.
- PFS Using Baseline CTC Results:
§ 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.
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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:
name, address,
contact. | Veridex, LLC
33 Technology Drive
PO Box 4920
Warren, New Jersey 07059
Telephone:
Fax:
Email: | (908) 791-2438
(908) 791-2381
drasmus1@vrxus.jnj.com |
|------------------------------------------|----------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------|
| Contact Person: | Debra J. Rasmussen
Worldwide Executive Director
Regulatory and Quality Affairs | |
| Identification of the
Device: | Trade/Proprietary Name: | CellSearch™ Circulating Tumor
Cell Kit |
| | Common Name: | CellSearch™ Circulating Tumor
Cell Kit |
| | Classification Name: | Immunomagnetic Circulating Cancer
Cell Selection and Enumeration
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 in vitro devices under
sections 513 and 514. | |
| Establishment
Registration Number: | 3004582358 | |
| Predicate Device: | CellSearch™ Epithelial Cell Kit
K031588 | |
1
| Device Description: | Epithelial cells are immunomagnetically labeled by targeting the
Epithelial Cell Adhesion Molecule (EpCAM) antigen. Anti-EpCAM
monoclonal antibodies conjugated to ferrofluid particles are colloidal
and, when mixed with a sample containing the target epithelial cells,
bind to the EpCAM antigen associated with the epithelial cells.
After immunomagnetic selection of epithelial cells from 7.5 mL of
blood. Fluorescent reagents are added at this time to discriminate
between the immunomagnetically selected cells. Anti-Cytokeratin -
Phycoerythrin (CK-PE) stains the intracellular cytoskeleton
cytokeratin proteins expressed in cells of epithelial origin, anti-
CD45-Allophycocyan (CD45-APC) stains leukocytes and DAPI
stains DNA present in the cell nucleus. A strong magnetic field is
applied to the processed reagent/sample mixture that causes the
labeled target cells to move to the cartridge surface. The cartridge is
then placed on the CellTracks® Analyzer II for data acquisition and
analysis. The CellTracks® Analyzer II acquires images of PE, APC
and DAPI fluorescence staining of the entire viewing surface. |
|-------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | After data acquisition is completed, the images are analyzed for any
event where cytokeratin-PE and DAPI are within a specified space in
the cartridge, i.e. indicating the possible presence of a cell with a
nucleus that expresses cytokeratin. Images from each fluorescent
color as well as a composite image of the cytokeratin staining (green)
and the nuclear staining (purple) are presented to the user in a gallery
for final cell classification. A cell is classified as a tumor cell when
it its EpCAM+ (i.e., it is captured), CK+, DAPI+ and CD45-. A
check mark placed by the operator next to the composite images
classifies the event as a Circulating Tumor Cell (CTC) and the
software tallies all the checked boxes to obtain the CTC count. |
| | Our data demonstrate that metastatic breast cancer patients with 5 or
more CTC/per 7.5 mL of blood have a significantly greater
probability for shorter progression free and overall survival than
patients who have fewer than 5 CTC per 7.5 mL of blood. |
| Intended Use for the
CellSearch Assay: | 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. |
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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
Count | Mean Observed
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 | ||
| 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
6
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.
Table 3. Reproducibility of CTC Counts in Duplicate Samples (n=163) with an Average of 5 CTC per 7.5 mL of blood
| | CTC 5 CTC | Min.* | Max.* |
|----------------------------------------|-----|---------------|-----|----------------------------|-------|-------|
| Healthy | 145 | 0.1 | 0.2 | 0 | 0 | 1 |
| Non-
malignant
breast
disease | 101 | 0.2 | 1.2 | 1 | 0 | 12 |
| Non-
malignant
other
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%) |
| 2nd 25 (14%) | |
| ≥ 3rd 67 (38%) | |
| Unk.* 2 ( 1%) | |
| Type of Therapy | Hormone 47 (26%) |
| Chemo 87 (49%) | |
| Immu/C/H 28 (16%) | |
| H / C 10 ( 6%) | |
| No Tx** 4 ( 2%) | |
| 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. . 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 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 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 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 p18 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> CTC.
Figure 4. OS of Patients with 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 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 80 weeks (18 months). The patients represented by the oliv 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 2nd | | | | | |
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| Categories | OS Risk from Baseline | |||||
|---|---|---|---|---|---|---|
| Parameter | Unfavorable | Favorable | HR | p-value | chi2 | # of Patients |
| Baseline CTC Number | >5 | 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 |