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Articles |
1
Lab Testing Segment, Business Group Diagnostics, Bayer Corporation, Tarrytown, NY 10591.
2
Business Group Diagnostics, Bayer Corporation, Elkhart,
IN 46514.
3
Oncogene Science Diagnostics, Inc., 80 Rogers St.,
Cambridge, MA 02142-1168.
a Author for correspondence. Fax 914-524-2543; e-mail david.morris.B{at}bayer.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used a monoclonal antibody labeled with fluorescein for capture and a monoclonal Fab' fragment labeled with alkaline phosphatase for detection. Separation of bound and free detection conjugate was performed with magnetizable particles coated with monoclonal antibody to fluorescein. Alkaline phosphatase activity was measured kinetically at 405 or 450 nm.
Results: The assay was linear from 0.1 to 250 µg/L. No hook effect was evident up to 10 000 µg/L. Within-run imprecision (CV) was 0.8–1.2%, and total imprecision was 1.1–1.7%. Cross-reactivity with human epidermal growth factor receptor, which has extensive homology with HER-2/neu extracellular domain, was <0.6%. Human anti-mouse antibodies, heterophilic antibodies, and rheumatoid factor did not interfere, nor did the therapeutic monoclonal antibody Herceptin®. In 51 healthy females, the mean value was 9.3 µg/L with a range of 6.4–14.0 µg/L. No reagent lot-to-lot variability was detected over four lots of reagents tested.
Conclusion: The Bayer Immuno 1TM assay for HER-2/neu was precise and resistant to interferences, characteristics that are essential for longitudinal monitoring of cancer patients.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since 1989, many reports have appeared that evaluated the importance of measuring circulating HER-2/neu ECD of women with breast cancer (4)(5)(6)(7)(8)(9). The shed ECD has been shown to be present in the sera of healthy women and to be increased above the health-related reference range in some women with breast cancer and in particular women with metastatic breast cancer. Many of these studies have shown that patients with increased HER-2/neu have a poor prognosis and shorter overall survival. Moreover, it has been shown that the tumors of these breast cancer patients grow more aggressively than those of patients who do not have increased serum concentrations of HER-2/neu. These studies raise the possibility that measuring HER-2/neu serum concentrations may be useful in monitoring patients for early detection of breast cancer recurrence, in predicting the outcome of hormonal therapy, as an aid to decisionmaking about appropriate treatment, and in monitoring the response of patients to treatment. In addition, there may be applications of the serum test to other cancers. Overexpression of this gene has been shown to have potential prognostic significance in patients with ovarian (10), gastric (11), endometrial (12), salivary gland (13), lung (14), colon (15), pancreatic (16), and prostate (17) cancers.
A new therapeutic approach to treatment of metastatic breast cancer is the use of a humanized monoclonal antibody (Mab) to HER-2/neu marketed as Herceptin® by Genentech. This is a murine Mab (Mab 4D5) that was engineered to give a human IgG1 molecule with the murine sequences necessary for binding HER-2/neu ECD retained (18). Clinical studies have demonstrated that Herceptin has value in the treatment of patients with HER-2/neu-overexpressing metastatic breast cancer (19)(20). Measurement of serum HER-2/neu may therefore have potential value in monitoring patients on Herceptin therapy.
Evaluation of the HER-2/neu status of breast cancer patients has been limited to testing tumor tissue, which is done by immunohistochemical staining techniques for overexpression of the p185 protein or by fluorescent in situ hybridization assays for amplification of the HER-2/neu oncogene. These methods limit testing for HER-2/neu status to the time of diagnosis. Once the tumor has been surgically removed, these methods are no longer applicable and cannot be used for testing for recurrence or monitoring metastatic breast cancer patients. This situation may be remedied by development of a test that can accurately determine the circulating concentrations of HER-2/neu ECD.
We have developed a fully automated random access immunoassay for the quantitative measurement of HER-2/neu ECD in serum on the Bayer Immuno 1TM immunoanalyzer. Here we describe the development and analytical performance of this assay. As a part of this evaluation, we sought to determine whether Herceptin would interfere with this new method with the purpose of establishing the future potential use for this test in monitoring patients on Herceptin therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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calibrators and controls
The Bayer Immuno 1 assay uses six calibrators containing 0, 10,
25, 60, 125, or 250 µg/L HER-2/neu ECD. The calibrator
matrix is delipidated bovine serum supplemented with detergent and
sodium azide. The HER-2/neu ECD used to make the calibrators
is a recombinant protein secreted by transfected mouse NIH 3T3 cells
and was developed by OSDI. The original cloned DNA containing the
sequence for the complete HER-2/neu protein was isolated
from human breast carcinoma cells, SK-BR-3, by OSDI. A further
modification was used to generate a truncated version coding for the
ECD fragment of the HER-2/neu protein, which was inserted in
the mouse cell designated as 3-30 (p105). The cells were grown in
roller bottles using 100 mL/L bovine calf serum in DMEM. The
supernatant was concentrated to give an ECD concentration of ~8000
µg/L and was diluted into the calibrator matrix to give the required
final ECD concentrations.
The formulation of the Bayer controls was the same as those of the calibrators at concentrations of 15, 50, and 100 µg/L.
The Medical Decision Pool (MDP) is a serum-based control material manufactured at Bayer Corporation and used internally for product development and routine quality control. This is a pool of human serum to which recombinant HER-/neu ECD is added to obtain a concentration of ~15 µg/L.
blocker optimization
Human anti-mouse antibody (HAMA) type 1 and type 2 controls were
obtained from Boehringer Mannheim Biochemicals. HAMA type 1 is a pool
of sera from healthy donors who have never been immunized with mouse
proteins and represents a rare interference that has an occurrence of
<0.5%. This control can show severe interference in sandwich assays
that use intact IgG/IgG or IgG/Fab' mouse Mab pairs.
HAMA type 2 interference occurs in donors who have been immunized with mouse proteins. It is frequent in special populations. More than 30% of people who have been treated with monoclonal murine IgG develop HAMAs. This control shows strong interference in all sandwich assays based on mouse Mabs in the absence of appropriate blockers.
Poly MAK 33, obtained from Boehringer Mannheim Biochemicals, is a
chemically polymerized mixture of whole antibody and Fab fragment of
MAK 33 Mab, which is provided as a lyophilized preparation. Mouse
-globulin (MGG) was obtained as a concentrated solution from
Scantibodies Inc.
The Poly MAK 33 and MGG are incorporated into reagent 2. The performance evaluations were all performed with the optimized concentrations of 200 mg/L Poly MAK 33 and 0.5 g/L MGG.
patient samples
Breast cancer patient serum samples with increased
HER-2/neu concentrations were obtained from BioClinical
Partners Inc. Serum samples from patients receiving mouse
monoclonal therapy were obtained from New York Medical College. Female
Red Cross blood donor serum samples were obtained from South Bend
Medical Foundation. The serum samples from healthy females were
collected in-house at Bayer Corporation (Elkhart, IN). Rheumatoid
factor-positive serum samples were obtained from the Foundation for
Blood Research.
interference studies
HAMA interference.
The Immunomedics
ImmuSTRIP® HAMA IgG ELISA assay for human
antibodies to mouse IgG was used to assess the presence of HAMAs in
samples from patients receiving mouse monoclonal therapy. HAMA
interference in the Bayer Immuno 1 HER-2/neu assay was tested before
and after the addition of 5 µL of a stock solution of
HER-2/neu ECD (250 µg/L) into 95 µL of HAMA sample. In
the calculation of the recovery of the amount of HER-2/neu
added to a sample, a correction was made for the dilution of the
endogenous HER-2/neu in the sample. The expected recovery
was determined from a MDP control to which a similar quantity of
HER-2/neu ECD stock solution had been added. The
apparent concentrations of the stock HER-2/neu were
calculated for each sample and then expressed as a percentage of the
value obtained for the MDP.
Herceptin interference.
Herceptin, a therapeutic agent
approved for use in patients with metastatic breast cancer, was
obtained from Genentech. Herceptin is a humanized recombinant
DNA-derived antibody that selectively binds with high affinity to the
ECD of HER-2/neu. Herceptin is provided as a lyophilized
powder that when reconstituted according to the manufacturer’s
instructions gives a solution of 21 g/L Herceptin. The stock solution
of Herceptin was diluted into samples to give concentrations of 0–420
mg/L. The samples were incubated for 30 min at room temperature to
allow antibody-HER-2/neu complexes to form before the
HER-2/neu concentration was determined in the Bayer Immuno 1
assay.
Cross-reactants and endogenous compounds.
Lyophilized
affinity-purified human EGFr from human carcinoma A431 cells was
purchased from Sigma. The material was reconstituted with 100 mL/L
glycerol in water to give a stock solution of 370 mg/L. The
stock solution was diluted into the MDP or bovine serum to give EGFr
concentrations of 0–7500 µg/L for cross-reactivity measurements. The
extent of cross-reactivity was calculated from the slope of the linear
regression analysis obtained by comparing the concentration of EGFr
with the apparent concentration of HER-2/neu obtained in the
Bayer Immuno 1 assay.
Potentially interfering endogenous compounds were tested at
NCCLS-recommended concentrations (21). Human serum from
healthy subjects was supplemented with bilirubin (500 mg/L),
hemoglobin (7.5 g/L), triglycerides (10 g/L), cholesterol (5 g/L), and
human -globulin (50 g/L). Each sample was diluted with MDP to give
final concentrations representing 100%, 75%, 50%, 25%, 10%, and
0% of initial concentration of the compound. The HER-2/neu
results for these samples were compared to that of MDP to which the
appropriate diluent had been added, and the percentage of bias between
the supplemented and unsupplemented MDPs was calculated to determine
interference.
precision
Imprecision of the HER-2/neu assay was evaluated by
analysis of the MDP, three Bayer controls, and Immuno 1 calibrators.
Samples were run in duplicate in 20 independent analyses over
10 days with two lots of reagents and two Bayer Immuno 1 systems at the
same site. Data were analyzed for variance components to give
statistical estimates of total and within-run imprecision.
linearity
To validate linearity over the dynamic range of the assay
(0.1–250.0 µg/L), two human serum samples (245.6 and 193.5 µg/L)
were diluted with the level 1 calibrator to generate a series of
dilutions over the entire calibration range. Final concentrations
representing 100%, 75%, 50%, 25%, 10%, and 0% of each sample were
assayed. Linear regression analysis of the diluted sample results
compared to the expected values was used to assess linearity.
lower limit of detection
The lower limit of detection was evaluated by determination of the
minimum detectable concentration of HER-2/neu that could be
statistically distinguished from the concentration of the level 1
calibrator. Results were collected with two reagent lots, two
calibrator lots, and two magnetic particle reagent lots on two systems
to provide a total of 16 combinations. Measurements of the level 1
calibrator response rates (mA/min) were made on 4 days. The
rates were pooled for each combination. The overall minimum detectable
concentration is defined as the mean HER-2/neu concentration
calculated from the mean of twice the pooled SDs for each of the 16
combinations.
method comparison
The quantitative HER-2/neu microtiter plate ELISA
(product no. OSDI-10) was obtained from OSDI and used according to the
manufacturer’s instructions. The development and performance of this
assay has been described previously (22). For the method
comparison study, 43 serum samples from breast cancer patients were
tested in duplicate using both the Bayer Immuno 1 and OSDI methods.
Assay values generated from the methods were compared by least-squares
regression statistics.
lot-to-lot variability
To evaluate reproducibility in the reagent manufacturing process,
four lots of reagents made in the Bayer Diagnostics manufacturing
facility in Bridgend, Wales were used to assay 17 samples from breast
cancer patients.
sample stability
Fresh serum samples were collected from 10 healthy women. The
HER-2/neu concentrations in these samples ranged from 3.8 to
11.5 µg/L. Aliquots of the samples were stored frozen at -20 °C
and refrigerated at 2–8 °C. The HER-2/neu values were
tested within 5 h after the blood was drawn and on days 1, 8, 15,
and 30. In addition, the stability at room temperature of fresh serum
samples from three women was evaluated by testing immediately after
collection and at 3, 6, and 24 h. The effect on sample stability
of allowing the blood sample to stand for 3 and 6 h at room
temperature before separating the serum was tested for the same three
women.
high-dose hook effect
To evaluate high-dose hook effect, MDP to which recombinant
HER-2/neu ECD concentrate had been added to give a final
concentration of 10 000 µg/L was tested in the assay. We concluded
that there is no high-dose hook at the test sample concentration when
that sample gives a rate greater than that of the level 6 calibrator.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Both HAMA type 1 and HAMA type 2 produced severe interference
when only base buffer was used. It was assumed that the true endogenous
concentrations of these control interference samples were within the
reference range and close to the mean concentration of 9.3
µg/L found for a group of healthy females with the Bayer Immuno 1
assay. Poly MAK 33 was more effective in eliminating interference from
HAMA type 1, whereas MGG was more effective with HAMA type 2. Neither
Poly MAK 33 nor MGG were completely effective alone at the highest
concentrations used. The lowest concentration achieved with HAMA type 1
with MGG alone was 12.9 µg/L compared with 7.0 µg/L with Poly MAK
33 alone. The lowest concentration achieved for HAMA type 2 with Poly
MAK 33 alone was 29.1 µg/L compared with 7.7 µg/L with MGG
alone. The combination of Poly MAK 33 and MGG was the most
effective formulation, with 6.9 µg/L and 7.3 µg/L obtained for HAMA
type 1 and type 2, respectively. Because these concentrations were well
within the expected reference range, we concluded that this
combination had eliminated all significant interference from these HAMA
controls.
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A comparison was made between the resistance to interference obtained with Mab TA-1 alkaline phosphatase conjugate made with whole IgG or Fab' fragment. The conjugates were used in formulations with and without the combination of Poly MAK 33 and MGG present.
Rheumatoid factor-positive serum samples with titers of
51 000–1 330 000 IU/L were used to test the assay as shown in Table 2
. A total of 20 of 42 samples gave significantly
increased results with whole antibody and no blockers. Interference was
frequent with the IgG conjugate in the absence of blockers. In none of
these cases was there any evidence of interference when the Fab'
fragment conjugate was used, even in the absence of blockers. The
presence of blockers made no difference to the results with Fab'
fragment conjugate but produced results with the IgG conjugate that
were equivalent to those with the Fab'. There was only one sample in
which interference was not eliminated, but the apparent
HER-2/neu concentration was reduced from 153 µg/L to 12.5
µg/L compared with the result of 8.7 µg/L with Fab' fragment
conjugate plus blockers. The data in Table 2
show that the Fab'
fragment alkaline phosphatase conjugate used in the assay provides
protection against rheumatoid factor interference.
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interference studies
HAMA samples.
Table 3
shows that recovery of HER-2/neu from individual
HAMA-positive serum samples was 85–111%. There was no correlation
between the HAMA concentration and recovery of HER-2/neu.
The HER-2/neu concentrations of all the samples were within
the reference range.
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Herceptin.
Table 4
shows that Herceptin, when added to
HER-2/neu-positive patient samples at therapeutic
concentrations and higher, had no effect on the measured
HER-2/neu concentration.
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Cross-reactants and endogenous compounds.
The cross-reactivity
with EGFr was estimated as 0.05% when EGFr was added into MDP and
0.06% when it was added into calibrator 1.
Bilirubin (500 mg/L), hemoglobin (7.5 g/L), triglycerides (10 g/L),
cholesterol (5 g/L), and human -globulin (50 g/L) were added into
human serum from healthy subjects up to the concentrations indicated.
All HER-2/neu results were within 98–108% of the expected
value.
precision
Precision data for the MDP, three levels of Bayer controls, and
six calibrators are presented in Table 5
. Pooled within-run CVs were 0.8–1.6%, and total pooled CVs
were 1.1–1.7%.
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linearity
Linearity was examined by measuring HER-2/neu in five
serial dilutions of two positive patient serum samples diluted with
level 1 calibrator. In one case, the range of concentrations obtained
was 0.1–245.6 µg/L; in the other, it was 0.1–193.5 µg/L. The
clinical samples diluted linearly as determined by regression analysis,
which yielded slopes of 1.0022 and 1.0015
(r2 = 0.999).
lower limit of detection
The lower limit of detection, defined as the concentration 2 SD
above the value for the level zero calibrator, was 0.1 µg/L.
high-dose hook effect
No high-dose hook effect was seen when recombinant
HER-2/neu ECD was added to samples up to a concentration of
10 000 µg/L (data not shown).
samples from healthy blood donors
The results obtained with samples from 51 healthy women Red Cross
blood donors are shown in Fig. 1
. The range of results was 6.4–14.0 µg/L with a mean (±
SD) of 9.3 ± 1.9 µg/L.
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method comparison with osdi elisa
A total of 43 serum samples from breast cancer patients were
tested in duplicate in both the Bayer Immuno 1 assay and the OSDI
quantitative HER-2/neu microtiter plate ELISA. Fig. 2
shows the regression plot for the means of all the sample
results. The concentration range for the Bayer Immuno 1
HER-2/neu assay was 10.3–231.9 µg/L. The regression
equation for this range was y = 0.88x + 0.9,
with a Sy|x of 4.3 µg/L and correlation
coefficient of 0.995. For the narrower range of 10.3–72.0 µg/L into
which most of the samples fell (n = 39), the regression equation
was y = 0.96x - 1.8, with a
Sy|x of 3.3 µg/L and a correlation coefficient
of 0.983.
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lot-to-lot variability
The regression analysis statistics for a comparison of results
with a panel of serum samples from 17 breast cancer patients using four
distinct lots of reagents are shown in Table 6
. There was no significant bias among the results for these
reagent lots.
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sample stability
A total of 10 fresh serum samples were collected and stored frozen
at -20 °C and 2–8 °C for 1 month. The samples were assayed at
intervals during this period, and no significant changes in the
HER-2/neu concentrations were found (data not shown). There
were no change in the HER-2/neu concentrations of three
fresh samples stored for 24 h at room temperature. Allowing blood
from three women to stand at room temperature for up to 6 h before
separation of the serum had no effect on assay results.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The resistance to interference conferred on the assay by the use of Fab' fragment instead of whole antibody conjugates was demonstrated by comparing the effect of rheumatoid factor-positive samples on assay results. We concluded that 20 of 47 samples gave significantly increased results with whole antibody and no blockers. The use of Fab' fragment abolished the interference even without the addition of specific blockers. Thus, the combination of Fab' fragment and specific blockers provides a potent resistance to rheumatoid factor interference. The effectiveness of the blockers was also demonstrated by the elimination of interference when they were combined with the whole antibody conjugate. In the analysis of these results, it was assumed that all of the rheumatoid factor samples contained endogenous HER-2/neu concentrations within the reference interval. In all cases, the use of the Fab' fragment conjugate produced concentrations in the expected range. Moreover, the fact that the same results were obtained for the Fab' fragment conjugate with and without blockers is a strong indication that all interference was eliminated.
We concluded that EGFr would have an insignificant effect on HER-2/neu measurements for the following reasons. Partanen et al. (23) measured mean EGFr concentrations of 336 ± 228 pmol/L and 636 ± 299 pmol/L in the serum of populations of healthy subjects and cancer patients, respectively. The concentrations in cancer patients were increased, with the highest serum concentration of EGFr reported to be 4583 pmol/L. In comparison, the concentration of HER-2/neu ECD in healthy subjects was 9.3 ± 1.9 µg/L, or 88.6 ± 18.1 pmol/L. The cross-reactivity of EGFr in the Bayer Immuno 1 HER-2/neu assay was 0.06%. Thus, a serum EGFr concentration of 4583 pmol/L would produce an apparent HER-2/neu concentration of 2.7 pmol/L or 0.3 µg/L in addition to the endogenous HER-2/neu.
The absence of interference by Herceptin is an important result that will allow the value of the assay in monitoring patients being treated with this drug to be assessed. Herceptin binds to the ECD of HER-2/neu as do the Mabs TA-1 and NB-3 used in the Bayer Immuno 1 assay. Our interpretation of the results is that Herceptin binds at a site independent from those of the two Mabs used in the assay with no overlap or steric interference. The Herceptin concentrations tested were present in 5.4- to 34.5-fold molar excess over the measured HER-2/neu in the samples used. According to product information provided by Genentech, Herceptin serum concentrations under steady-state conditions give mean trough and peak concentrations of ~79 and 123 mg/L, respectively.
Because no interference was found at 420 mg/L, we concluded that therapeutic concentrations of Herceptin in serum will not interfere in the accurate measurement of HER-2/neu using the Bayer Immuno 1 assay.
Lot-to-lot variation can be an important source of analytical error in an immunoassay. Van Dalen (24)(25) has stressed the importance of standardization in the manufacture of immunoassay reagents and has recommended that manufacturers report lot-to-lot variation for their tumor marker assays. The results reported here for the Immuno 1 HER-2/neu assay demonstrate that the reagents exhibit insignificant lot-to-lot variation.
The Bayer Immuno 1 HER-2/neu assay has the important attributes needed for monitoring cancer patients. The total imprecision is at most 1.7%. Combined with the resistance to interference and low lot-to-lot variability, this makes the assay very suitable for longitudinal monitoring over extended periods. Accurate results can be expected at widely separated time points. This is the first description of a fully automated assay for HER-2/neu in serum. The Bayer Immuno 1 HER-2/neu assay may be a valuable tool to aid oncologists in the management of cancer patients as more and more studies, particularly for breast cancer patients, show the value of knowledge of HER-2/neu status for prognosis, selection of therapy, and monitoring.
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Acknowledgments |
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Footnotes |
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-globulin.
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References |
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