In this prospective study using colonoscopy as the reference standard, we assessed the ability of a plasma based DNA marker, mSEPT9, to detect preclinical CRC in blood samples collected prospectively from an average risk screening population. Retrospective case control studies had suggested that detectable levels of mSEPT9 in plasma identify individuals with CRC with a sensitivity between 52% and 72% and specificity between 90% and 95%.8–10
Another recent case control study by Warren et al24
obtained estimates of 90% for the sensitivity and 88% for the specificity of mSEPT9. These previous study results motivated the present prospective study, which was performed in a screening setting, in contrast with previously published results derived from cases diagnosed symptomatically (thus likely to be qualitatively different from asymptomatic cases) and convenience sample controls (which may not represent the asymptomatic population) and so had limited generalisability.25
Our study design and analysis avoided those pitfalls; our results demonstrated lower sensitivity than the previous case control studies.
The primary assay used for the study, based on the first commercially available mSEPT9 assay, could detect 48.2% of cases of preclinical CRC with 91.5% specificity in the target population. Most of the CRC identified during the course of examination were stages I–III, and sensitivity was lower for earlier tumour stages. Sensitivity was very low for AA and only slightly higher than the false positive rate for all individuals who did not have cancer, indicating that there is at present no utility in detecting important precancerous lesions.
Screening for CRC not only reduces mortality from the disease26–29
but also incidence.30
However, a national survey in the USA reported that only about 50% of the eligible population had been screened according to guidelines.32
In Germany, a survey among 1808 randomly selected persons revealed that about 40% of those over the age of 50 years had undergone some form of colonoscopy.33
Blood based tests have long been sought in the cancer detection field for their convenience and potential for higher compliance; nevertheless, higher uptake, higher yield of precancerous lesions and finally any effect on mortality would still have to be shown. At present, it can therefore only be speculated whether a blood test with faecal occult blood test (FOBT)-like performance would produce similar reductions in incidence and mortality to those seen in previous randomised FOBT trials.26–29
Our study only assessed performance in a screening setting, and cannot assess other factors such as uptake. In comparison with FOBT, which is still widely used and implemented in some national programmes,34
the mSEPT9 assay yielded a CRC sensitivity of 48%, which is at the lower end of the guaiac FOBT range (37–79%).35
The specificity of mSEPT9 was 92%, compared with a range of 87–98% for the guaiac test.35
PPV for standard rehydrated guaiac FOBT used in the Minnesota study was 5.6%,26
compared with 5.2% for mSEPT9 in this study. However, these comparative performance characteristics would suggest that, even in a programme of repetitive annual screening, use of the mSEPT9 assay would not produce a decline in mortality commensurate to that seen with a sensitive guaiac test.
Furthermore, newer faecal immunochemical stool tests for haemoglobin (FIT) may provide improved performance characteristics36
as well as slightly superior uptake rates37
; therefore, FIT is increasingly being used instead of guaiac FOBT in countries introducing screening programmes (eg, The Netherlands).38
A newer faecal DNA test has reported sensitivities of 85% for CRC and 54% for adenomas ≥1 cm (based on a single case control design using a stool bank).39
The same group40
recently evaluated mSEPT9 in parallel with a stool based DNA test in a small retrospective study in CRC and large adenomas. The study also reported a modest sensitivity and very low AA detection rate for mSEPT9. They estimated the sensitivity of mSEPT9 for large adenomas at 14% and for CRC 60%, somewhat higher than our study, but with larger CI. The source of the samples was the Mayo Tumour Registry, but the exact selection criteria for the samples are not given and the mSEPT9 test was not performed in the same manner. In the same study, comparative sensitivity of faecal DNA was higher for cancer (87%) as well as adenomas ≥1 cm (82%). However, evidence from these latter studies is limited as they were neither performed prospectively nor in a screening setting.
As a large proportion of human CRC tissue contains relatively high amounts of mSEPT9 regardless of tumour stage, improvement of DNA isolation or increased PCR efficiency may result in better marker performance. Unfortunately, tissue samples from the current study were not collected for testing to determine whether the tumours contained significant amounts of mSEPT9. Improvements in the test appear to be possible, as shown by our post hoc analysis using the three PCR replicate emulation which mimics the second generation commercially available mSEPT9 assay and which detected nearly two-thirds of cases, but yielded false positives in 12% of non-cases. The three replicate approach needs to be tested prospectively. The emulation used in this post hoc analysis imprecisely reproduced a simultaneous three replicate assay, for three reasons: (1) the decision to look at a third PCR replicate was based on lower than expected two replicate assay sensitivity, leading to lower estimated three replicate assay sensitivity; (2) the third PCR replicates were not done in the same run on the same machine as the first two, unlike an actual three PCR replicate assay, allowing increased run to run variation to lower the assay discrimination; and (3) criteria for invalid results differ slightly because in practice, all three replicates would have common control samples and be subject to the same probability of invalidity. For these reasons, estimated sensitivity of the three replicate assay is likely to be smaller than true sensitivity; the effect on specificity is less clear but probably small. Thus the three replicate results are strictly exploratory. In addition, because the assay is working with what are small quantities, the assay clearly involves a stochastic component, and hence a particular PCR replicate may lack adequate numbers of mSEPT9 copies to trigger DNA amplification. Additional replicates merely provide more opportunities to yield a positive, thus increasing sensitivity while decreasing specificity. Therefore, it can only be speculated whether this more recent assay version might perform better in the screening setting. Similarly, we do not know whether the accuracy of the three replicate mSEPT9 assay might approximate available immunochemical stool tests. Still, detection of adenomas as precursor lesions would also have to be improved. The choice of a blood based assay, such as mSEPT9, in a programme of CRC screening depends on multiple features not assessed in this study, including the potential for increased compliance, the necessary frequency of testing and features of competing tests, such as cost. The current cost of the mSEPT9 test in Europe is approximately 150 Euros, considerably more than faecal tests but less than a colonoscopy. In any event, the comparative effectiveness and cost of various strategies of screening with mSEPT9 requires further evaluation.41
There are limitations to our study:
- Because the centres enrolling in the study were not randomly sampled and study subjects were sampled from the study centres disproportionately by clinical findings for laboratory testing, standardisation to some target screening population was necessary to obtain relevant performance estimates. Although in any non-random sample there may be unknown factors for which standardisation is impossible and there is always some error in the standardisation variables, our standardisation included the main determinants of CRC incidence, age and sex, and they had negligible measurement errors.
- To increase accrual, the entry criterion regarding prior endoscopy was relaxed soon after the beginning of enrolment. This probably reduced the overall prevalence rate, but we believe it had little impact on the assay parameter estimation.
- Although no split sample quality control was performed to examine consistency between laboratories, there was no statistically significant difference in the frequency of findings between them (data not shown).
- Although subjects with other cancers found on examination were excluded from the primary analysis, sensitivity analyses including these cases only slightly affected the quantitative results (analyses not shown).
It should also be mentioned that we do not believe that the mSEPT9 results are generalisable to other blood based tests for CRC. To the contrary, each marker should be evaluated not only in isolation but also as an adjunct to other markers, much as Ahlquist and colleagues43
have done with stool based markers.
In conclusion, the blood based mSEPT9 assay detected about half of the preclinical CRC with specificity similar to guaiac based FOBT, in this prospective masked study of screening subjects. Sensitivity of mSEPT9 for advanced adenomas was very low. Although this renders the clinical utility very low, there might be ways to achieve possible improvements, as suggested by our post hoc analysis as well as by a recent case control study.24
The utility of the test for population screening for CRC will require improved sensitivity for detection of early cancers and advanced adenomas. Nevertheless, these and all other tests potentially to be used for CRC screening have to be prospectively tested in the screening setting, where they would finally be applied. One implication of our study is that performance estimates may differ substantially in such settings compared with retrospective case control studies.