A combination of serum markers for the early detection of...
Transcript of A combination of serum markers for the early detection of...
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
A combination of serum markers for the early detection of
colorectal cancer
Running title: Serum markers for early detection of colorectal cancer
Norbert Wild,1 Herbert Andres,1 Wolfgang Rollinger,1 Friedemann Krause,2 Peter
Dilba,2 Michael Tacke,3 and Johann Karl1
1Department of New Technologies, 2Department of Biostatistics, and 3Department of
Antibody Development, Professional Diagnostics, Roche Diagnostics GmbH, Penzberg,
Germany
Grant support: none
Funding: The study was funded by Roche Diagnostics GmbH, Germany.
Corresponding author:
Dr. N. Wild, Department of New Technologies, Professional Diagnostics, Roche Diagnostics
GmbH, Nonnenwald 2, 82377 Penzberg, Germany
phone: 0049-8856-604547
fax: 0049-8856-604513
Published OnlineFirst on August 26, 2010 as 10.1158/1078-0432.CCR-10-0119
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e-mail: [email protected]
Conflict of interest disclosure:
The study was funded by Roche Diagnostics GmbH, Germany. The authors are employees of
Roche Diagnostics GmbH, Germany. Patents by the authors on the results of this study,
including individual markers as well as marker combinations, have been filed.
Keywords: colorectal cancer, serum markers, FOBT, screening, marker combination
Abbreviations: FOBT, fecal occult blood test; CRC, colorectal cancer; CEA,
carcinoembryogenic antigen; FIT, fecal immunochemical test; OPN, osteopontin, Hb-hpt,
hemoglobin-haptoglobin complex; UICC, international union against cancer. CYFRA 21-1
and ELECSYS are trademarks of Roche
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Translational relevance
The current practice of colorectal cancer screening is based on stool assays and imaging
methods e.g. colonoscopy. To pre-select patients, fecal occult blood testing or fecal
immunochemical testing is routinely used prior to colonoscopy and only patients with a
positive test result are referred to a specialist. While these assays are useful screening tools,
patient compliance with stool based assays tends to be low. Serum based assays for the early
detection of colorectal cancer are highly attractive as they could be integrated into any regular
health check-up without the need for additional stool sampling, thereby increasing acceptance
upon patients. The combination of serum markers presented in this study intends to trigger a
follow-up colonoscopy for a final diagnosis. It could be an alternative approach for the early
detection of colorectal cancer.
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Purpose: Fecal occult blood testing (FOBT) is recommended as first line screening to
detect colorectal cancer (CRC). We evaluated markers and marker combinations in serum as
an alternative to improve the detection of CRC.
Experimental Design: Using penalized logistic regression, six markers were selected for
evaluation in 1027 samples (301 CRC, 143 patients with adenoma, 266 controls, 141 disease
controls, 176 other cancer). The diagnostic performance of each marker and of marker
combinations was assessed.
Results: To detect CRC from serum samples we tested 22 biomarkers. Six markers were
selected for a marker combination including the known tumor markers CEA and CYFRA 21-
1 as well as novel markers or markers that are less routinely used for detection of CRC:
ferritin, osteopontin, anti-p53 and seprase. CEA showed the best sensitivity at 95% specificity
with 43.9%, followed by seprase (42.4%), CYFRA 21-1 (35.5%), osteopontin (30.2%),
ferritin (23.9%) and anti-p53 (20.0%). A combination of these markers gave 69.6% sensitivity
at 95% specificity and 58.7% at 98% specificity. Focusing on UICC stages 0–III reduced the
sensitivity slightly to 68.0% and 53.3%, respectively. In a sub-collective, where matched stool
samples were available (75 CRC cases and 234 controls), the sensitivity of the marker
combination was comparable to fecal immunochemical testing (FIT) with 82.4% and 68.9%
vs. 81.8% and 72.7% at 95% and 98% specificity, respectively.
Conclusions: The performance of the serum marker combination is comparable to FIT.
This provides a novel tool for CRC screening to trigger a follow-up colonoscopy for a final
diagnosis.
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The early detection of colorectal cancer (CRC) significantly improves the prognosis of
patients and is a key factor to reduce the mortality from CRC (1). Recently, the American
Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American
College of Radiology have issued joint guidelines for CRC screening to include well known
procedures as guaiac based fecal occult blood testing (FOBT) and fecal immunochemical
testing (FIT), colonoscopy, sigmoidoscopy and double contrast barium enema, but also two
more recent methods, computer tomography colonography (CTC) and fecal DNA testing (2).
The guidelines of the US Preventive Services Task Force have also been up-dated, but do not
include the latter two methods (3).
Serum-based, minimally invasive markers would be highly attractive for CRC screening
as they could easily be integrated in any health check-up without the need of additional stool
sampling. Though numerous biomarkers are under evaluation for the detection of CRC from
serum, none of them has sufficient sensitivity and specificity to be considered in the current
guidelines (4). CEA and carbohydrate antigens e.g. CA19-9 have been assessed more
intensely but with varying results depending on the study design and the study population (4).
Recently, nucleic acid based markers, including RNA, DNA and epigenetic modifications,
have come into focus (5-12). While some of the markers e.g. a panel of 5 RNA markers (7) or
hypomethylated SEPT-9 DNA (11), showed good sensitivities of 88 % and 68 %, the
respective specificities of 69 % and 89 % will burden a high number of patients with a false
positive result if used for screening. In a more recent study the sensitivity and specificity of
SEPT-9 was confirmed with 73% and 91%, respectively (12). GCC RNA (5) and L6 RNA (6)
showed high sensitivity with 74% and 79% at 95% and 100% specificity but have not yet
been extensively validated.
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Using an ELISA format we evaluated new tumor markers together with established
markers. The clinical performance of these markers was assessed as stand alone markers or in
marker combinations with the intention not to replace but to initiate a follow-up colonoscopy
for a definite diagnosis.
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Materials and Methods
Study Design and Sample Collection
Blood and stool samples were collected prospectively in two European multi-center
studies that have previously been described (13). The respective research protocols were
reviewed and approved by the appropriate ethics committees and written informed consent
was obtained from patients prior to sample collection.
All blood samples were collected before surgery or colonoscopy according to standard
operating procedures in standardized collection tubes that were provided to the participating
centers. Whole blood was collected in S-Monovette tubes without gel separator (Sarstedt).
The blood samples were centrifuged, serum was collected, dispensed into cryotubes and
stored at -70°C. After arrival in our laboratory the samples were thawed, aliquoted and stored
again at -70 °C.
Clinical samples from both multi-center studies were compiled for the evaluation (Table
1 and Figure 1). A: Control cohort with 266 patients. Patients with adenoma or inflammatory
bowel diseases were excluded. GI-healthy controls were only pre-selected to be free of any
bowel disease. No other exclusion criteria, e.g. chronic diseases, were applied to controls. B:
Advanced adenoma cohort containing 143 patients with any lesion containing high-grade
dysplasia, villous or tubovillous architecture, or tubular adenoma with at least 1 cm, and C:
CRC cohort with 301 colorectal cancer patients from both studies. Samples from 141 patients
with benign gastrointestinal conditions were collected in both studies (Table 1). Samples from
patients with other cancer diagnosis were collected in surgical units. Patients with co-
morbidities commonly found in a screening population were not excluded from the study. The
co-morbidities of the control and cancer cohort are given in Supplement S1.
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[Insert Table 1 here]
[Insert Fig. 1 here]
Immunoassays
The ELISA tests were measured blinded in duplicate in serum at Roche Diagnostics
GmbH, Penzberg, Germany. Laboratory personnel were unaware of clinical data. To exclude
lot to lot variance a single assay lot was used for all samples wherever feasible and controls
were run. For each assay a sample aliquot was thawed and residual volume was discarded
after testing. The tumor markers of the Elecsys test menu from Roche Diagnostics, Mannheim,
were measured: CEA, CA15-3, CA125, CA19-9, CA72-4, CYFRA 21-1, ferritin, NSE, AFP
and HCG+β. Commercial ELISA kits were obtained for: TIMP-1 and HGF (R&D Systems),
interleukin-6 (Roche Diagnostics GmbH), interleukin-8 (BD Biosciences).
For all other biomarkers prototype ELISA kits were developed using standard assay
procedures as described previously for Nicotinamide N-methyltransferase (NNMT) (14),
PSME3 (15) and S100A12 (16). The technical performance of the prototype ELISA kits was
validated to meet the following specifications (not shown): intra-assay CV and inter-assay CV
(< 15 %), linearity of dilution (+/- 10 % of theoretical value) and analyte recovery in spiked
samples (100 +/- 20 %). While the intra-assay CV was specified to be < 15 %, it was found to
be routinely < 10 % for double determinations of patient samples. The sensitivity of the
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assays was adjusted to detect base levels in healthy subjects, with the exception of anti-p53,
since healthy subjects are expected to be autoantibody negative.
For the seprase ELISA (Swiss-Prot accession: Q12884) serum samples were diluted ad
0.05% in an antibody mix in incubation buffer (40 mM phosphate buffer pH7.4, 10 mM
EDTA, 0.1% Tween 20, 2 mg/ml BSA, 1 mg/ml bovine IgG) containing monoclonal anti-
seprase antibodies D8 and D28 0.375 µg/ml each, labeled with digoxigenin and biotin,
respectively (17). After 2 hours at room temperature aliquots were transferred onto a
strepavidin-coated microtiter plate and incubated for one hour. After washing (TBS, pH7.4,
0.05% Tween 20), 100 µl of anti-digoxigenin peroxidase conjugate (30 mU/ml) were added
for one hour, washed and then followed by tetramethylbenzidine (TMB) substrate for color
development. The reaction was stopped after one hour with 2 N sulfuric acid and the
absorbance was measured at 450 / 620 nm. The seprase concentrations were calculated in
arbitrary units using a serial dilution of a human serum containing a high seprase
concentration.
Osteopontin (OPN) was measured using an ELISA based on rabbit polyclonal antibodies
against peptides covering amino acids 211–228 and 288–304 of human osteopontin (Swiss-
Prot accession: P10451). Biotinylated anti-osteopontin (288–304) antibodies were used to
capture and digoxygenylated anti-osteopontin (211–228) antibodies to detect the analyte.
Samples were diluted 1:100 into an antibody mix in incubation buffer (s. seprase assay)
containing 2.2 µg/ml of each of the anti-osteopontin antibodies. After 1 hour 100 µl aliquots
were transferred to a streptavidin-coated microtiter plate, incubated for another hour and
finally processed as described above. The assay was calibrated in pmol/l using a serial
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dilution in equine serum of an osteopontin fragment, amino acids 211–314, expressed in E.
coli.
ASC (Swiss-Prot accession: Q9ULZ3) and Serpin B5 (Swiss-Prot accession: P36952)
were measured with ELISA assays using rabbit polyclonal antibodies raised against full
length recombinant protein from E.coli. Additionally an antibody against amino acids 331-
347 of Serpin B5 was raised and used as capture antibody. Capture antibodies were
biotinylated, while the detection antibodies were digoxigenylated. The respective sandwich
forming antibodies were diluted in incubation buffer (s. seprase assay) at a concentration of 1
µg/ml each, 100 µl of this mix were added to 20 µl standard or serum and incubated in a
streptavidin-coated microtiter plate. The plates were washed and processed as above. The
assays were calibrated against a serial dilution of recombinant protein from E. coli in equine
serum.
For the detection of anti-p53 auto-antibodies two biotinylated peptides representing
amino acids 11–35 and 41-60 of human p53(Swiss-Prot accession: P04637) in PBS, 0.05 %
Tween 20 at 50 ng/ml were immobilized on a streptavidin-coated microtiter plate with 100
µl/well. After washing 100 µl of serum samples diluted 1:50 in PBS / 0.05 % Tween 20 were
added and incubated for 120 min at 30° C. After washing bound IgG was labeled with 25
mU/ml anti-human-IgG-peroxidaxe-conjugate. Subsequent to washing, bound conjugate was
detected as in the seprase assay. The assay was calibrated using a highly positive serum with
1000 U/ml correspondings to an optical density of 3.0 at 450 nm.
Fecal assays, hemoglobin-haptoglobin complex and S100A12, were performed as
reported (13).
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Statistical Analysis
A rule for serum biomarkers was determined on a training-test-design based on Monte
Carlo Cross Validation (MCCV). A special type of penalized logistic regression, Lasso-
Regression, was used, both as classification algorithm and as selection procedure for a subset
of relevant biomarkers (18,19).
Two different biomarker rules were established for different subsets of the study
population, one including all CRC patients and controls (algorithm I), and a second rule for
the same collective but without CRC stage IV (algorithm II) as classified according to the
staging of the “International union against cancer” (UICC). The software “R” was used for
rule generation.
The results were assessed by receiver operating characteristics (ROC) analysis and by
determining the sensitivity at a preset specificity of 95% or 98%, respectively (20).
Data were first divided randomly into training (67%) and test set (33%) and stratified by
age, gender, survey panel and site. Then a MCCV with 100 runs was executed on the training
set. The medians of the sensitivities and specificities over all 100 runs of MCCV are reported
as training results. They were used to determine an optimized set of parameters for the Lasso-
Regression. The optimized Lasso-Regression was applied to the training-set for generating a
final diagnostic rule and determining the final thresholds at the specificities of 95% and 98%.
This rule was then applied to the independent test-set. By application of the final rule to the
test-set, the test results were obtained (ROC-curves, sensitivities, specificities) for the
assessment of the diagnostic potential of the biomarkers. The final rule was also applied to the
adenoma panel and to the individual CRC stages. Due to sample numbers of the individual
cancer stages the analysis by stages included samples from training and test set.
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Results
Marker candidates
Marker candidates were identified using different methods covering proteomics methods,
Affymetrix studies, literature search and through collaborations (not shown). 60 biomarkers,
including 10 established tumor markers from the Elecsys system (Roche Diagnostics,
Mannheim), were considered as potential biomarkers. Of these 52 were evaluated in 50 CRC
patients and 50 apparently healthy controls, that were not used later on in the enlarged sample
panel for the multivariate analysis (not shown). Finally 22 biomarkers were assessed in an
extensive patient panel that included screening relevant controls (Table 1).
[Insert Table 2 here]
Univariate analysis
When the biomarkers were measured in the patient collectives described in Table 1, the
median concentrations of the markers differed by a factor of < 3. While most markers gave
increased concentrations in CRC patients vs. controls, the concentrations of ferritin and
surprisingly of seprase were decreased (Table 2). When we pre-validated seprase with western
blots of tumor and adjacent healthy tissue from CRC patients, we detected increased
expression in cancer tissue (not shown) as has been described by others (21). However, when
we measured seprase in serum, we found decreased concentrations in cancer patients with a
median of 26.1 vs. 37.6 U/ml.
In a clinical setting the sensitivity of a biomarker is most relevant. Therefore, the cut-off
was set to achieve a specificity of 95% in controls that reflected the screening situation. The
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control cohort was age matched with a median age of 62.0 years to the CRC patients and
included relevant gastrointestinal diseases. As no exclusion criteria besides inflammatory
bowel diseases or adenoma were applied to the control collective, patients with co-morbidities
commonly found in people aged over 50 e.g. hypertension or diabetes, were included
(Supplement S1). When the specificity was set to 95 %, none of the markers was sufficiently
sensitive to be a screening marker on its own. The established tumor marker CEA gave the
highest sensitivity with 43.9%, followed by the novel marker seprase (42.4%), CYFRA 21-1
(35.5%) and OPN (30.2%). Ferritin and anti-p53 were less sensitive with 23.9% and 20.0%,
respectively. The tumor markers AFP, CA15-3 and HCG+β reached sensitivities below 10 %
and were excluded from further evaluation. None of the markers selected for a marker
combination (s. multivariate analysis) detected advanced adenomas with sufficient sensitivity
(Table 3).
[Insert Table 3 here]
When analyzed by tumor stages, seprase was at least 10% more sensitive in early stages
than CEA or CYFRA 21-1 (Table 3), in stage I even 22.8% compared with these two markers.
In the screening relevant stages 0–III seprase reached a sensitivity of 45.4% and a very similar
sensitivity of 42.4% including all stages. CEA and CYFRA 21-1 gave a comparable
sensitivity only when all stages were considered as these markers have an excellent sensitivity
in stage IV patients. However, they were less sensitive in stages 0–III with 30.9% and 22.3%,
respectively.
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Multivariate analysis
To improve the clinical performance, we evaluated marker combinations by application
of Lasso-Regression including either all stages (algorithm I) or only the more screening
relevant stages 0–III (algorithm II). Focusing on stages 0–III (Table 4), the following markers
were selected with Lasso Regression: CEA, ferritin and seprase in each of 100 runs of the
MCCV in the training-set. OPN was selected in 99, CYFRA 21-1 and anti-p53 in 68 runs,
respectively. Though additional markers were selected by the algorithm, a marked drop in the
selection frequency from 68 to 26 runs or less beyond these six markers was evident
(Supplement S2). To estimate the stability of the algorithm, the marker combination selected
in the training-set was validated in an independent test-set (Table 4). The combination of the 6
top markers significantly improved the sensitivity of CRC detection. For the evaluation of
cancer patients with stages 0-III the sensitivity at 95% specificity increased from 45.4% to
68.0 % in the test-set as compared to the best single marker seprase (Tables 3 and 4). When
all tumor stages were included, the sensitivity improved to 69.6% vs. 43.9% for CEA. A
detailed analysis by tumor stages revealed a remarkable increase of the sensitivity at 95%
specificity. The sensitivities for the stages I-III improved from 36.0%, 49.3% and 51.3%,
respectively, with the best single marker, to 44.0%, 75.4% and 71.4% with the marker
combination. Due to sample numbers, patients from the training set were included in this
analysis, which might lead to an increased sensitivity in the individual stages. In patients with
adenomas the sensitivity increased to 22.7%, but was still insufficient.
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[Insert Table 4 here]
For a screening marker, that will trigger a follow-up colonoscopy for the final diagnosis,
a higher specificity would reduce the number of unwarranted colonoscopies, thereby reducing
the burden of false positive results for the patients. Hence, we investigated the sensitivity at
98% specificity. Though algorithm I lost 9.6% and algorithm II 8.4% in the training set, the
sensitivity was still high with 61.2% (all stages) and 56.2% (stages 0–III), that translated in
the test set to a sensitivity of 58.7% and 53.3%, respectively. For the individual cancer stages
the sensitivity of the serum marker combination was at 98% specificity comparable to or
higher than the sensitivities found for the individual markers in the univariate analysis at 95%
specificity (Table 3).
We applied algorithm II (stages 0-III) to CRC patients from the screening situation,
(study I, Figure 1). Based on n=12, where values for all 6 markers were available, the
sensitivity was 66.7% (34.9 - 90.1, 90% CI) and 41,7% (15.2 - 72.3, 90% CI) at 95 % and
98 % specificity, respectively. However, these results are preliminary and have to be taken
with care due to the low sample number. Clearly, the sensitivity of the marker combination
needs to be tested in an independent screening study to fully assess its potential.
The specificity of the CRC marker combination vs. disease controls, including benign
gastrointestinal diseases (Table 5), was assessed by applying the cut-off derived from controls
at 98% specificity. The specificity of the combination was 60.3% against all diseases with
variations against specific subgroups. When the specificity was assessed in patients suffering
from other cancer types, applying again the cut-off derived from controls at 98% specificity,
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the specificity was 75.0% (Table 5). It ranged from excellent 97.0 % in prostate cancer to
46.7 % in lung cancer.
[Insert Table 5 here]
We compared the serum marker combination to fecal hemoglobin-haptoglobin and to a
combination of hemoglobin-haptoglobin with S100A12 (13). In a subset including 75 CRC
patients and 234 controls from the total study collective, where matched serum and stool
samples were available, the respective assays were run in parallel. In these patients the
sensitivity of the serum marker combination was higher than in the complete patient collective
(Table 4). When compared to the fecal methods, the serum marker combination appeared to
be as sensitive as hemoglobin-haptoglobin with 82.4% vs. 81.8% and 68.9% vs. 72.7%, but it
was less sensitive than the fecal marker combination that gave a sensitivity of 90.9% and
79.2% at 95% and 98% specificity, respectively.
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Discussion
Biomarker based CRC screening currently applies the detection of occult fecal blood
using either FOBT or FIT though these methods have limitations in sensitivity and / or
specificity. We had recently evaluated the potential of fecal marker combinations to improve
the sensitivity of CRC screening (13). A combination of Hb-Hpt, S100A12 and, depending on
the specificity level, TIMP-1 resulted in an increased sensitivity. While such a combination of
fecal markers is useful, serum markers could be integrated in a health check-up more easily
having a significant impact on CRC screening due to improved patient compliance. In
contrast to stool samples, microbial contamination or interference by an ill-defined matrix is
not an issue with serum, implicating a more reproducible recovery of analytes. Unfortunately,
current serum markers lack sufficient sensitivity or specificity to be used as standalone
screening markers (4). In consequence marker combinations have been explored to solve this
dilemma. e.g. for CRC two studies showed an increase of the AUC from 0.75 with the best
single marker to 0.80 and 0.81 with combinations of CEA + CA72-4 or CEA + CA72-4 +
CA19-9 (22, 23), respectively.
When we applied the principles from our recent study to serum markers and reduced the
number of potential marker candidates in a stepwise procedure, we identified a combination
of six markers that detects CRC in serum with high sensitivity: Seprase, osteopontin, anti-p53
autoantibodies, ferritin, CEA and CYFRA 21-1.
An increased expression of seprase in cancer including colorectal cancer has been shown
and our western blot results from tissue confirmed this (21, 24-26). However, most
surprisingly the concentrations in serum appeared to be reduced instead, when we used an in-
house ELISA to discriminate CRC from controls. As seprase is a membrane bound protein,
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this might be due to processing that could differentially lead to soluble seprase or to a loss of
an epitope of the specific antibodies used in our assay. Alternatively, isoforms might be
considered. However, only an additional, intracellular isoform has so far been reported, which
does not explain the discrepant results in tissue and serum (27). The mechanism behind the
serum concentrations is therefore currently unclear. Although the concentrations found in
serum are in opposition to the expression in tissue, we found a higher sensitivity in early CRC
stages as has been demonstrated by Henry et al using semiquantitative immunohistochemistry
(25).
Osteopontin has been linked to various cancer types e.g. breast, colon, prostate or lung
cancer (28-31). A special focus has been its contribution to tumor cell invasion and metastasis
making it a promising tumor marker, although serum concentrations reported by Fedarko et al
were not elevated in colon cancer (31). The concentrations found in our study were only
slightly increased but still contributed to the marker combination.
This also applies to ferritin, where a lower serum concentration in CRC is correlated with
tumor size and blood loss in the bowel (32, 33). However, it appears that such small
differences of CRC vs. controls give an insufficient sensitivity for a standalone screening
marker (34).
Anti-p53 autoantibodies in cancer have been investigated in depth (35, 36). They lack
sensitivity but contribute excellent specificity. Our results confirm findings of Müller et al
that a combination of serum anti-p53 with conventional tumor markers increases the
sensitivity of cancer detection (37). For CRC it increased by 13.5% from 59.7% to 73.2%,
when anti-p53 positive status was added to CEA. The high sensitivity in this study might be
attributed to the patient collective lacking early stages almost entirely. In contrast, we
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included a significant number of patients with early stage disease as we wanted to assess our
marker combination in samples closely representing a screening situation.
The markers selected by Lasso-regression are insufficient diagnostic markers when
considered on their own but showed an additive value when combined. CEA is most
commonly used in the follow-up of CRC patients after surgery: CEA is recommended for
follow-up but not for diagnosis due to its low sensitivity in early CRC stages in the guidelines
of the American Society of Clinical Oncology (38). In our study both established tumor
markers CEA and CYRA 21-1 showed a good sensitivity in stage IV but only an intermediate
performance in early stages. When all six markers were combined to analyze stages I-IV, the
sensitivity increased notably as compared to the best single marker CEA. The increase was
also evident when only stages 0–III were considered. Here especially seprase contributed to
the marker combination as it had the best performance of all markers in early stages.
The current benchmark for marker based CRC screening is FIT and any serum marker
should at least be as sensitive. The sensitivity of our marker combination in stages 0–III
(68.0%) is comparable to a Japanese screening study that reported 65.8% sensitivity, both at
95% specificity (39). In a subset of our patients we could confirm this in a side by side
comparison. The sensitivity was slightly higher in these patients than in the total study
collective suggesting that the subset was not strictly representative for all patients. However,
the combination of serum markers and FIT, represented in our study by fecal hemoglobin-
haptoglobin, achieved an almost identical performance.
The benefit of an algorithm based marker combination is an improved sensitivity at high
specificity. Simple addition of a positive status for several assays will immediately introduce
a loss of specificity. The combination of CEA with anti-p53 as done by Müller et al (37) using
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a simple “AND”-rule for the addition of true positives without adding false positives is only
feasible due to the excellent specificity of anti-p53. The more markers are combined with
such a procedure, the more false positives will be included, as the specificity of these markers
is usually lower than 100%. Since different markers will generate false positive results in
different patients, it will significantly reduce the overall specificity.
The specificity of the serum combination was assessed in disease controls that included
benign gastrointestinal conditions and in patients suffering from other cancer types. A
specificity of 60.3% in disease controls might be less problematic as the patients are
symptomatic and can easily by identified by a physician in an otherwise non-symptomatic
screening population. Such patients might be referred for colonoscopy without the need of an
initial screening assay. On the other hand, a specificity of 75.0% against other cancer types
will lead to positive results in colonoscopy negative patients where no symptoms might be
obvious. While a positive rate of 25% in these patients appears high at first glance, it is also a
chance to identify undetected cancer when the follow-up is adjusted to the specific patient
situation, e.g. lung cancer in smokers or breast cancer in women. In patients where no cancer
is detected despite of a positive test result and negative follow-up procedures, monitoring is
an option, to control for changes of marker concentrations over time. This could either reveal
a false positive result, or trigger another follow-up if a significant increase is observed.
However, only longitudinal studies with high patient numbers can address this issue.
Considering the sensitivity of the novel test procedure, the specificity in normal screening
participants and the anticipated increased acceptance upon patients as compared to stool
testing, we believe that the advantages of a serum screening assay outweigh the risks
associated with it.
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The primary focus of our study was to evaluate the feasibility of the detection of CRC
from serum. Though we included a significant number of patients with early CRC stages, the
study population was collected only partially in a screening setting (study I, Figure 1). The
validation of the results in a true screening population with increased patient numbers is
therefore mandatory.
In conclusion, we describe a combination of six serum markers for the early detection of
CRC that will initiate a follow-up colonoscopy for a definite diagnosis. The performance of
the marker combination is comparable to FIT and might improve the acceptance of CRC
screening in the general population as it can easily be integrated in any health check-up
without the need for stool sampling. We are currently validating the algorithm in a multicenter
screening study.
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Acknowledgements
We are indebted to S. Ahmeti, H. Bodenmueller, H. Duefel, A. Engel, S. Freiburghaus, M.
Grünefeld, M.L. Hagmann, E. Hoess, D. Knodel, T. Kott, B. Kanne, U. Kunert, M. Roeßler, P.
Stegmueller, M. Thierolf, W. Uhl and R. Vogel for their contributions to this study.
Participating Clinicians
The following clinical investigators contributed to the prospective sample collection: H.
Bertram, GPR Klinikum Rüsselsheim, Germany; R. Dahmen, Universitätsklinikum
Heidelberg, Germany; A. Eickhoff, Klinikum Ludwigshafen, Germany; H. Friess,
Universitätsklinikum Heidelberg, Germany; F. van de Mierop, AZ Sint Augustinus Wilrijk,
Belgium; H.D. Janisch, Gastroenterology Specialist, Erlangen, Germany; J. Janssen, Sint-
Elisabeth ZH, Turmhout, Belgium; K.W. Jauch, Klinikum Großhadern, Germany; H. Kalthoff,
Conbio GmbH, Hamburg, Germany; M. Khoury, Gastroenterology Specialist, Hof, Germany;
W. Kerzel, Gastroenterology Specialist, Forchheim, Germany; M. Prilutskaya, ProteoGenex,
Los Angeles, US; J.F. Riemann, Klinikum Ludwigshafen, Germany; W. Rösch, Krankenhaus
Nordwest, Frankfurt am Main, Germany; G. Rohr, Hochtaunus Kliniken GmbH, Bad
Homburg v.d.H., Germany; S. Rossol, Krankenhaus Nordwest Frankfurt am Main, Germany;
D. Scheck, Gastroenterology Specialist, St. Ingbert, Germany; W. Tacke, Gastroenterology
Specialist, Königstein, Germany; H. Thiel, Gastroenterology Specialist, Homburg/Saar,
Germany; W. Weber-Guskar, Gastroenterology Specialist, Feldafing, Germany.
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Table 1. Basic characteristics of patient collectives.
Patients, n
Age, mean, median, range, y
Sex female male
CRC collective 301 66.6 / 67 (35-96) 144 157 - UICC 0 / I 6/53 65.9 / 66 (44-83) 25 34 - UICC II 68 71.2 / 72 (45-93) 34 34 - UICC III 76 63.6 / 62 (35-91) 34 42 - UICC IV 68 66.6 / 66 (42-89) 34 34 - w/o staging† 30 65.0 / 64 (43-96) 17 13 Control Collective 266 62.1 / 62 (18-89) 162 104 - GI-healthy* 135 61.6 / 62 (18-79) 83 52 - Hemorrhoids 29 55.9 / 57 (27-74) 15 14 - Diverticulosis 76 64.9 / 65 (37-89) 47 29 - Hyperplastic polyps 14 67.9 / 67 (54-86) 8 6 - Other bowel diseases 12 58.7 / 59 (37-75) 9 3 Advanced adenoma 143 66.6 / 66 (41-85) 59 84 Disease controls 141 59.1 / 62 (23-91) 76 65 - Diverticulitis 30 69.1 / 69 (51-91) 13 17 - Colitis 29 61.9 / 64 (25-90) 15 14 - Morbus Crohn 18 41.3 / 39 (23-67) 13 5 - Colitis ulcerosa 14 56.1 / 55 (24-83) 9 5 - Infection related diarrhea 12 56.2 / 61 (29-79) 8 4 - Inflammatory GI-disease 22 56.6 / 60 (32-87) 9 13 - Ulcer 12 66.7 / 69 (40-89) 8 4 - Other GI-disease 4 55.0 / 61 (31-68) 1 3 Other cancer 176 63.2 / 64 (34-88) 101 75 - Bladder 8 64.4/ 68 (53-75) 3 5 - Breast 44 62.8 / 65 (34-88) 44 - - Endometrium 15 66.3 / 67 (47-84) 15 - - Kidney 23 62.7 / 62 (49-83) 9 14 - Lung 30 65.1 / 65 (47-84) 7 23 - Ovary 23 59.9 / 61 (34-86) 23 - - Prostate 33 62.7 / 62 (51-74) - 33
* no evidence of bowel disease. † staging as given by a pathologist I-III or II-III.
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Table 2. Univariate analysis of marker performance ranked by AUC of CRC vs. controls.
Marker Units Concentration, median, range Cut-off at 95 %
specificity
Sensitivity (%) at 95 % specificity
AUC CRC vs. controls
Controls (n = 266)
Advanced adenoma (n = 143)
Cancer (n = 301)
Seprase U/ml 37.6 (6.99 - 125.0) 33.7 (5.25 - 70.8) 26.1 (1.93 - 81.8) 24.3 42.4 0.79 CYFRA 21-1 ng/ml 0.83 (0.14 - 8.66) 0.99 (0.25 - 6.20) 1.55 (0.17 - 433.5) 2.1 35.5 0.78 CEA ng/ml 1.49 (0.20 - 9.03) 1.80 (0.23 - 10.0) 3.86 (0.20 - 5187) 4.8 43.9 0.77 OPN pmol/ml 405.7 (7.06 - 1264) 480.2.1 (167.4 - 1513) 627.0 (124.3 - 6396) 814.2 30.2 0.77 Il-8 pg/ml 18.6 (5.55 - 1175) 20.1 (7.66 – 243.7) 35.2 (7.84 - 2656) 71.4 27.9 0.76 Il-6 pg/ml 2.52 (0.32 - 164.5) 2.54 (1.07 - 77.5) 4.36 (0.90 - 304.1) 6.7 30.6 0.74 NNMT pg/ml 307.1 (42.7 - 2600) 372.5 (98.1 - 2035) 582.9 ( 39.3 - 2633) 958.8 30.6 0.72 HGF pg/mL 451.3 (2930 - 4207) 490.5 (253.2 - 10438) 633.9 (104.4 - 11856) 812.8 30.4 0.70 ASC pg/mL 312.8 (121.5 - 3052) 322.7 (160.4 - 1981) 434.0 (124.9 - 3163) 828.2 18.3 0.69 S100 A12 ng/mL 14.9 (2.03 - 133.0) 13.7 (0.71 - 133.6) 25.7 (5.08 - 1718) 61.9 20.3 0.69 CA72-4 U/mL 0.94 (0.36 - 24.7) 0.90 (0.37 - 106.6) 1.59 (0.48 - 853.6) 8.0 20.6 0.66 TIMP-1 ng/mL 96.6 (55.6 - 275.2) 95.84 (58.3 - 713.1) 115.9 (51.1 - 1769) 156.7 26.8 0.66 FERR ng/mL 148.6 (7.56 - 2000) 143.2 (7.86 - 1435) 85.5 (2.39 - 1921) 35.2 23.9 0.65 CA19-9 U/mL 6.60 (0.60 - 116.0) 8.14 (0.60 - 100.3) 11.6 (0.60 - 18316) 34.0 22.6 0.64 PSME-3 pg/mL 53.0 (0.00 - 1402) 59.3 (0.00 – 935.9) 80.3 (0.00 - 5263) 189.9 19.7 0.64 Serpin B5 mU/mL 149.4 (0.00 - 872.9) 193.5 (0.00 - 2069) 209.6 (0.00 - 7303) 409.0 23.9 0.62 anti-p53 U/ml 15.4 (0.00 - 327.0) 15.3 (0.00 - 538.4) 15.7 (0.00 - 42144) 43.5 20.0 0.57 NSE ng/mL 9.70 (1.19 - 23.7) 8.61 (0.54 - 30.5) 8.73 (2.11 - 315.5) 16.1 14.0 0.57 AFP IU/mL 2.00 (0.50 - 19.7) 2.23 (0.50 - 10.3) 1.76 (0.5 - 19.2) 5.8 6.6 0.56 CA125 U/mL 11.7 (1.34 - 108.3) 11.8 (2.58 - 134.6) 12.4 (0.60 - 716.3) 28.3 17.6 0.56 CA15-3 U/mL 17.2 (5.37 - 73.1) 16.5 (4.51 - 92.8) 16.3 (1.42 - 48.1) 33.3 4.0 0.53 HCG+ß mIU/mL 0.31 (0.10 - 8.24) 0.1 (0.1 - 10.9) 0.27 (0.10 - 847.7 5.3 3.7 0.51
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Table 3. Sensitivities (%) of marker candidates by tumor stages at 95 % specificity.
N / N* CEA Seprase CYFRA 21-1 OPN Ferritin Anti-p53 Colorectal cancer
Stage 0 6 / 6 16.7 16.7 16.7 16.7 16.7 33.3 Stage I 53 / 50 13.2 36.0 13.2 13.2 13.2 16.0
Stage II 68 / 67 36.8 49.3 26.5 22.1 30.9 17.9 Stage III 76 / 76 34.2 51.3 23.7 27.6 34.2 15.8 Stage IV 68 / 66 88.2 31.8 80.9 54.4 13.2 24.2
Stage 0 – III† 233 / 229 30.9 45.4 22.3 23.2 27.0 18.8 All stages 301 / 295 43.9 42.4 35.5 30.2 23.9 20.0
Adenoma Advanced adenoma 143 / 141 7.7 11.3 8.4 9.1 9.8 5.6
* Reduced sample number for seprase and anti-p53 due to sample volume. † 30 additional samples included with staging as given by a pathologist I-III or II-III.
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Table 4. Sensitivities of the serum marker combination. Comparison of the serum combination vs. fecal markers in a sub-colletive (75 CRC, 234 controls) with matched serum and stool samples.
N Sensitivity test-set (%) at 95% specificity Sensitivity test set (%) at 98% specificity
Training-set* Test-set† Training-set* Test-set† Algorithm I (all stages)
- Stage 0‡ 6 n.a. 50.0 (15.3 - 84.7) n.a. 33.3 (6.3 - 72.9) - Stage I‡ 50 n.a. 44.0 (32.0 - 56.6) n.a. 32.0 (21.2 - 44.5) - Stage II‡ 65 n.a. 75.4 (65.0 - 83.9) n.a. 64.6 (53.7 - 74.5) - Stage III‡ 70 n.a. 71.4 (61.2 - 80.2) n.a. 64.3 (53.8 - 73.8) - Stage IV‡ 65 n.a. 93.8 (86.5 - 97.9) n.a. 87.7 (78.9 - 93.7) - all stages§ 285 70.8 (60.0 - 79.7) 69.6 (60.7-77.4) 61.2 (47.6 - 73.9) 58.7 (49.6-67.4)
Algorithm II (stages 0-III) - Stages 0-III§ 220 64.6(53.2 - 72) 68.0 (58.0 - 76.9) 56.2 (40.8 - 66.7) 53.3 (43.2 - 63.2)
Algorithm II (adenoma) - Advanced adenoma 141 n.a. 22.7 (17.0 - 29.3) n.a. 12.1 (7.8 - 17.5)
Algorithm I: Serum combination vs. fecal markers in 75 CRC and 234 controls with matched serum and stool samples׀׀ - Serum marker combination 75 82.4 (73.5 - 89.3) 68.9 (58.9 - 77.7) - Fecal Hb-Hpt 75 81.8 (73.0 – 88.7) 72.7 (63.2 – 80.9) - Fecal marker combination:
Hb-Hpt + S100A12 75 90.9 (83.6 – 95.7) 79.2 (70.2 – 86.5)
*One hundred fold Monte Carlo Cross Validation, median plus 0.05 and 0.95 quantile. †Apparent sensitivities when the cut-off from the training set was applied, 90 % CI. ‡The cut-off from the training set was applied to all CRC samples with the respective stage, including samples from training and test set. §29 additional samples included with staging as given by a pathologist I-III or II-III. .Samples from the training and test set where matched stool samples were available. Algorithm from the training set was applied׀׀
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N Total number of cancer patients where results were available for all six markers of the combination. Cancer patients and the control collective (n = 266) were split 2/3 (training set) and 1/3 (test set) to optimize and test algorithms I and II. n.a. Not applicable. The cut-off from the training set on CRC and controls was applied. Hb-Hpt: Hemoglobin-haptoglobin complex.
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Table 5. Specificity of the marker combination against disease controls and other cancer
N Specificity (%)* Disease Controls 141 60.3 (53.0 - 67.2) - Chronic bowel diseases† 44 47.7 (34.6 - 61.1) - Colitis 29 62.1 (45.1 - 77.1) - Diverticulitis 30 63.3 (46.7 - 77.9) - Inflammatory GI-disease‡ 22 68.2 (48.5 – 84.0) - Other§ 4 100.0 (47.3 – 100)- Ulcer 12 66.7 (39.1 - 87.7) Other Cancer 176 75.0 (69.1 - 80.3) - Bladder cancer 8 87.5 (52.9 - 99.4) - Breast cancer 44 77.3 (64.5 - 87.1) - Endometrium 15 86.7 (63.7 - 97.6) - Kidney cancer 23 69.6 (50.4 - 84.8) - Lung cancer 30 46.7 (30.8 – 63.0) - Ovary cancer 23 69.6 (50.4 - 84.8) - Prostate cancer 33 97.0 (86.4 - 99.8)
*Apparent specificities when the algorithm was applied using a cut-off derived from the control cohort at 98 % specificity, 90 % CI. †Colitis ulcerativa, infection related diarrhea, Morbus Crohn. ‡Appendicitis, cholangitis, mesenteritis, pancreatitis, proctitis. §Carcinoid, lipoma.
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Figure Legends Fig. 1. Flow chart of the patients selected for the study.
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Published OnlineFirst August 26, 2010.Clin Cancer Res Norbert Wild, Herbert Andres, Wolfgang Rollinger, et al. colorectal cancerA combination of serum markers for the early detection of
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