Evaluation of an assay for methylated BCAT1 and IKZF1 in plasma for detection of colorectal neoplasia

This was a multi-centre predominantly prospective study funded in part by the National Health and Medical Research Council (NHMRC) and Clinical Genomics Technologies Pty Ltd (CGT) to estimate the sensitivity and specificity of a test detecting methylated BCAT1 and/or IKZF1 DNA in blood from people with neoplasia or non-neoplastic pathologies likely to be encountered in the colon and rectum. Findings at colonoscopy were used as the diagnostic standard. The study was approved by the Southern Adelaide Clinical Human Research Ethics Committee (April 4, 2005) and Medical Ethical Board of Academic Medical Centre Amsterdam (July 12, 2011). Written informed consent was obtained from all recruits prior to any procedures. Clinical and research staff at the medical institutions audited clinical data and verified case classification blinded to assay results determined by CGT. The clinical data were only released subsequent to completion of testing of all collected samples. Test results were not disclosed to subjects or their physicians. The trial is registered at Australian and New Zealand Clinical Trials Registry trial registration number 12611000318987.

Subjects aged 33-85 years old and either scheduled for colonoscopy for standard clinical indications (prospective element), or shown at colonoscopy within the prior ten days to have CRC that had not been treated (retrospective element), were approached about volunteering for the study. The participating centres were Repatriation General Hospital (Daw Park, South Australia), Flinders Medical Centre (Bedford Park, South Australia), Academic Medical Centre (Amsterdam, The Netherlands) and Flevo Hospital (Almere, The Netherlands). Following enrolment, cases were excluded if the scheduled colonoscopy was cancelled or if insufficient blood was available.

Venous blood was collected into two 9mL K3EDTA Vacuette tubes (Greiner Bio-One, Frickenhausen, Germany) from subjects either prior to them being sedated for colonoscopy but after consumption of bowel preparation solution, or prior to preparation for surgery but following colonoscopic diagnosis. A second sample was obtained from 26 CRC cases one month or more after surgery. Blood tubes were kept at 4 °C until commencing plasma processing. Plasma was prepared within 4 hours of blood collection by centrifugation at 1,500 g for 10 minutes at 4 °C (no braking), followed by retrieval of the plasma fraction and a repeat centrifugation. The resulting plasma was stored at -80 °C. Frozen plasma samples were shipped on dry ice to CGT and stored at -80 °C until testing.

No study-wide control of colonoscopy or pathology procedures or quality was undertaken as the study aimed to assess marker performance relative to outcomes determined in usual clinical practice. All procedures were performed by hospital-accredited specialists and so met site-specific standards for sedation, monitoring, imaging, and equipment. Histopathology and staging of neoplasia used routine procedures at each clinical site. Cases were excluded if any data crucial to clinical diagnosis was not obtainable, e.g. if colonoscopy was incomplete.

An independent physician assigned diagnosis for all cases used in this study on the basis of colonoscopy, surgical and histopathological findings. CRC was staged according to AJCC 7th Edition [18]. Advanced adenoma was defined as adenoma with any of the following characteristics: (a) ≥ 10 mm in size, (b) >20 % villous change, (c) high grade dysplasia, or (d) serrated pathology. Cases with more than two tubular adenomas or stage 0 cancer were also classified as advanced adenoma. Non-advanced adenoma refers to those not meeting the characteristics of an advanced adenoma. Hyperplastic polyps were classed as non-neoplastic pathologies. Where multiple pathologies were present, the most advanced neoplasm was used as the principal diagnosis. Location of the principal neoplasm was defined as that of the most advanced lesion in a patient with multiple neoplasms. Where multiple non-neoplastic diagnoses were present, the principal diagnosis was allocated in the following hierarchy (descending): inflammatory bowel disease (IBD), hyperplastic polyp, angiodysplasia, haemorrhoids, diverticular disease.

All plasma samples of at least 3.9mL were assayed for the presence of methylated BCAT1 and IKZF1 DNA at CGT’s laboratories by trained and qualified staff blinded to clinical results (see Additional file 1 for details). Samples were analysed in batches of 22 clinical samples and two process controls. Batches were loaded on a QIASymphony SP instrument (Qiagen, Hilden, Germany) and cell-free DNA was extracted using a QIASymphony Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions (Additional file 1). The extracted DNA was bisulphite-converted using the EpiTect Fast Bisulfite Conversion kit (Qiagen) and QIACube instrument (Qiagen) as recommended by manufacturer but with minor modifications (see Additional file 1). The resulting bisulphite-converted DNA was analysed as three replicates in a triplex real-time qPCR assay (ACTB control, methylated BCAT1 and IKZF1) performed on a Roche LightCycler 480 Model II instrument (see Additional file 1). A sample was deemed positive if at least one qPCR replicate was positive for either BCAT1 or IKZF1 DNA methylation; no cycle threshold (Ct) value cut-offs were applied. Each PCR plate included three no-template control samples and a standard curve based on 0-2ng bisulphite converted fully methylated human DNA (Merck-Millipore, MA, United States) prepared in a background of nuclease-free water (Promega, WI, United States). The mass of methylated BCAT1 and IKZF1 DNA in each plasma specimen was determined from the batch specific standard curve. The level of methylation was expressed as the total mass of methylated (BCAT1 plus IKZF1) DNA as a percentage of the total amount of recovered DNA per processed specimen.

Subjects were recruited until at least 100 cancer cases had been identified (keeping 95 % CI of sensitivity estimates to less than 20 %) with at least 25 cases at each of stages I-III (to enable determination of the relationship between positivity rate and stage). The main outcome measure was positivity rate by diagnosis. GraphPad online scientific software tool, http://​graphpad.​com/​scientific-software/​, was used to calculate 95 % confidence intervals (binomial distribution assumed), Chi-square values (using 2x2 contingency tables without Yates’ correction) and McNemar’s test. Linear weighted Kappa statistic and odds ratios were calculated using www.​vassarstats.​net and www.​medcalc.​org/​calc/​odds_​ratio.​php, respectively.

Analysis of potential confounding co-variables was performed using a logistic generalised linear model fitted to a binary positivity variable (R package version 3.1.2) or by using a 2-sample z-test (two-tailed, 95 % significant level, http://​www.​socscistatistics​.​com/​tests/​ztest/​Default2.​aspx) on sample proportions (positive results observed in a given sample size). Continuous variables included age and DNA; dichotomous variables included smoking status, gender, and family CRC history.

An ANOVA Chi-square test (R version 3.1.2) was performed on assay positivity rates corrected for stage distribution in proximal and distal cancers using a generalised linear model with a logistic regression model fitted to two covariate models including stage and lesion, or lesion only.

The log values of the percentages of methylated BCAT1 and IKZF1 DNA measured in amount of DNA retrieved per processed specimens were used to create empirical density plots for three clinical classes: non cancer (all pathologies minus CRC cases), early stage cancer (Stage I + II) and late stage cancer (Stage III + IV). A minus infinity value was assigned to all cases with no methylation signal, whereas a Gaussian distribution was assumed for all non-zero values. By fitting Gaussian distribution curves to the empirical density plots, relative risk was calculated as the ratio of the conditional probability for early or late stage cancer compared to non-cancer based on the equation \( \frac{\mathrm{P}\left(\left.\mathrm{X}=1\right|\left.\mathrm{Y}=1\right)\right.}{\mathrm{P}\left(\left.\mathrm{x}=0\right|\left.\mathrm{Y}=1\right)\right.}=\frac{{\mathrm{P}}_{11}}{{\mathrm{P}}_{01}} \), where X = 1 means cancer, X = 0 means no cancer and Y is the test result (positive (Y = 1) or negative (Y = 0)) at a given threshold value.

Reported p-values are 2-tailed and values <0.05 were considered statistically significant.

Subjects were recruited from the Australian sites during the period September 2011 to May 2014 and from Dutch sites during July 2011 until September 2013 (see Additional file 2 for details). Figure 1 summarises the disposition of volunteers from initial approach through to diagnosis, including the reasons for exclusion or withdrawal. Sufficient plasma was collected prospectively as per protocol, i.e. following ingestion of bowel preparation but prior to colonoscopy, for almost all recruits (2078 of 2105, 99 %). Table 1 shows age and gender relative to principal diagnosis. Diagnoses in the 27 retrospective cases were 21 with cancer, 2 with diverticular disease, 1 with advanced adenoma, 1 with benign polyps and 2 with no evidence of pathologies.

Cancer was the principal diagnosis in 6 % of all enrolled study subjects (129 of 2105 recruits) while adenoma (including stage 0 cancer) was diagnosed in 33 % of the recruits. Non-neoplastic pathologies (including IBD) were diagnosed in 40 % while 21 % recruits (452) showed no evidence of pathology in the colon or rectum. These phenotype frequencies reflect the recruitment strategy, which was designed to capture cases with a broad range of pathologies including all stages of neoplasia. More males (53.7 %) than females were recruited and more cancer patients were male (58.1 %) as would be expected [19].

The two-marker blood test was run successfully (i.e. meeting minimum quality control criteria) on 2127 samples, with 26 of these blood samples obtained after surgical resection of cancers. Table 2 shows the number of cases positive by one or both methylation markers according to diagnosis. Of the 129 cancer cases, 57 % were methylation positive for BCAT1 and 48 % for IKZF1, with 66 % methylation positive by either gene. The true positive rate increased with stage for each marker and for the combined two-marker blood test (either methylation marker positive). Sensitivity estimates for the two-marker blood test for detection of earlier stage cancer (I or II) was 56 % (95 % CI: 44–68) and for later stage cancer (III + IV) was 79 % (95 % CI: 66–88), p = 0.009.

By contrast, sensitivity estimates for adenomas of any type were low, at 6 % (95 % CI: 4–9) for advanced adenoma and 7 % (95 % CI: 4–10) for non-advanced adenoma. These estimates were not significantly different compared to positivity rates in those with a normal colon or benign pathology (Table 2, p > 0.05).

Specificity estimates for the combined two-marker blood test were 94 % (95 % CI: 93–95, 1288 non-neoplastic cases) to 95 % (95 % CI: 92–97, 450 cases with no evidence of disease).

Methylated IKZF1 DNA was typically detected at a lower rate in blood compared to methylated BCAT1 DNA across all diagnostic sub-classes. Concordance between the two markers is shown for selected clinical phenotypes in Table 3. For those with cancer, 51/129 (40 %) were concordant and 34/129 discordant (26 %), with BCAT1 detecting most of the discordant cases (23/34, 68 %) (McNemar’s, p = 0.06). The linear weighted Kappa statistic as a measure of agreement was 0.476 for cancer cases (95 % CI: 0.327–0.625).

In subjects with no evidence of pathologies in colon and rectum, only one case of the 24 positive results showed concordance between the methylation markers with BCAT1 being responsible for most (21/23) of the discordant cases (McNemar’s, p = 0.0002). Linear weighted Kappa measure of agreement was 0.07 (95 % CI: 0–0.213).

The influence of recruitment site, age, gender, smoking status, family history of CRC and amount of cell free DNA on assay positivity was assessed. Recruitment site (see Additional file 2), gender, family history of CRC (see Additional file 3) and age (see Additional file 4) were not significant predictors of assay positivity (p > 0.05).

For 286 cases with known smoking habits, 62 % were current smokers. Excluding the 16 CRC cases that smoked, 11/165 smokers were methylation positive compared to 11/105 non-smokers (Fisher’s p-value = 0.362).

The majority of processed specimens had cell free DNA amounts of 1.6-2.5ng per mL plasma (95 % CI). There was no significant difference in levels of cell-free DNA between all subjects without CRC and cancer cases of stages I to III, however some stage IV cancer cases had a significantly higher amount of DNA (see Additional file 5, p > 0.0001). Excluding cases with cancer, the average amount of cell-free DNA was 2.1ng/mL (95 % CI: 1.9-2.2). Higher DNA amounts (>3ng/mL) were observed in 192 of 1972 non-CRC cases (9.7 %), of which 19 (10 %) were two-marker blood test positive. Increased DNA amounts was associated with an increased chance of a positive result, as the odds ratio for positivity increased 2.7-fold for each increment of one in log (DNA pg/mL), p value <0.0001.

The estimated positivity rates for proximal (60 %) and distal (67 %) cancers were not significantly different (Chi-square test, p value = 0.603). Cancer location, corrected for stage distribution, did not influence detection of markers in blood (Additional file 3, p value = 0.555).

The relationship between detection of methylated BCAT1 and IKZF1 DNA in blood and degree of invasiveness (by pT stage) for cancers is shown in Fig. 2. Although not statistically significant (ANOVA with Tukey post-hoc test), a positive trend was observed between positivity rate and pT stage (degree of invasion) for each marker, and the two-marker blood test.

As per study protocol, the two-marker blood test performance estimates have been qualitatively reported as any detectable signal for methylated BCAT1 and/or IKZF1 DNA. However, positive qPCR methylation results can also be reported quantitatively as the fraction of methylated BCAT1 plus IKZF1 DNA measured in the total yield of DNA isolated per specimen. We modelled the relationship between disease severity (non-cancer, early stage cancer and late stage cancer) and the fraction of methylated BCAT1 and IKZF1 DNA. Figure 3a shows that the fraction of methylated BCAT1 and IKZF1 DNA in blood increased as a function of degree of invasiveness. The generated models were used to calculate the relative risk of disease (early stage or late stage cancer) compared to non-cancer for a given methylation fraction value. The models indicated a low relative risk of having cancer if no methylation was detected. For a specimen containing approximately 5 % methylated BCAT1 and IKZF1 DNA the models estimate a relative risk of 5 for having early stage cancer (Fig. 3b). On the other hand, the relative risk of being late stage cancer given a specimen with approximately 40 % methylation is 125.

Of the 129 cancer cases, a post-resection sample was available for 12 of the 85 cases with a positive two-marker blood result at initial diagnosis, and for 14 of the 44 cases with a negative result at diagnosis. As can be seen in Table 4, ten of twelve initially positive cases became negative after resection. We note that the BCAT1 and IKZF1 methylation levels <5 % are values obtained from extrapolation due to these methylation signals being below the linear range of the qPCR assay. Of the 14 cases that were negative at diagnosis, all but one remained negative after resection (data not shown).