Detection of methylated BCAT1 and IKZF1 after curative-intent treatment as a prognostic indicator for colorectal cancer recurrence.

BACKGROUND: The risk of recurrence after completion of curative-intent treatment of colorectal cancer (CRC) is hard to predict. Post-treatment assaying for circulating tumor DNA (ctDNA) is an encouraging approach for stratifying patients for therapy, but the prognostic value of this approach is less explored. This study aimed to determine if detection of methylated BCAT1 and IKZF1 following completion of initial treatment identified patients with a poorer recurrence-free survival (RFS). METHODS: 142 CRC stage I-III cases with at least 2 years of follow up (unless recurrence was evident sooner) and a methylated BCAT1/IKZF1 test result between 2 weeks and 12 months after completion of initial treatment were eligible for study inclusion. The association between BCAT1/IKZF1 and RFS was assessed by the log-rank (Mantel-Cox) method. Cox proportional hazard regression analysis was used for multivariable survival analysis. RESULTS: Thirty-three (23.2%) had recurrence at a median 1.6y (interquartile range: 0.8-2.4). Methylated BCAT1/IKZF1 was detected in 19 of the 142 patients (13.4%) and was associated with a significant risk of recurrence (hazard ratio [HR] 5.7, 95%CI: 1.9-17.3, p = 0.002). Three-year RFS for patients with or without detectable methylated BCAT1/IKZF1 was 56.5% and 83.3%, respectively. Multivariable analysis showed that detection of methylated BCAT1/IKZF1 (HR = 2.6, p = 0.049) and site of the primary tumor (HR = 4.2, p = 0.002) were the only significant prognostic indicators of poor RFS. CONCLUSIONS: BCAT1/IKZF1 methylation testing after curative-intent treatment is an independent prognostic indicator for RFS and identifies a subgroup at high risk. Personalized surveillance is warranted for patients with these ctDNA biomarkers detectable after curative-intent treatment.

Detection of recurrent colorectal cancer with high specificity using a reporting threshold for circulating tumor DNA methylated in BCAT1 and IKZF1.

BACKGROUND: A blood assay measuring methylated BCAT1 and IKZF1 can detect recurrent colorectal cancer (CRC) with high sensitivity but suboptimal specificity. This study aimed to establish an upper reference limit (URL) of these biomarkers in a reference population without CRC, apply that threshold to detecting clinical recurrence in patients who had undergone definitive therapy for CRC, and compare the performance of the biomarkers with carcinoembryonic antigen (CEA). METHODS: The level of methylation was reported as the aggregate methylated BCAT1 and IKZF1 expressed as a percentage of total plasma DNA. A reference population of patients confirmed to have no colorectal neoplasia (n = 857) was used to determine the URL. Test accuracy for clinical recurrence was determined in a post-treatment surveillance population (n = 549; 77 recurrence cases). RESULTS: A methylation level of 0.07%, corresponding to the 98th percentile in the reference population, was set as the URL. In the surveillance population, 60 patients had methylation levels above 0.07%, and 81.7% of these had recurrence. In comparison with no minimum threshold being applied, assay sensitivity with a URL of 0.07% yielded similar sensitivity (63.6% [CI, 51.9%-74.3%] vs 64.9% [CI, 53.8%-74.7%]; P = .87) and higher specificity (97.7% [CI, 95.9%-98.8%] vs 91.3% [CI, 88.4%-93.5%]; P < .001). The BCAT1/IKZF1 test was 2.5-fold more sensitive than CEA for detecting recurrences considered amenable to surgery with curative intent (50.0% vs 20.8%; P = .016). CONCLUSIONS: Applying a threshold for positivity to the methylated BCAT1/IKZF1 blood assay improved the specificity for CRC recurrence without compromising sensitivity. Both the sensitivity and the specificity were superior to those of CEA.

Evaluation of a panel of tumor-specific differentially-methylated DNA regions in IRF4, IKZF1 and BCAT1 for blood-based detection of colorectal cancer.

BACKGROUND: Differentially-methylated regions (DMRs) are characteristic of colorectal cancer (CRC) and some occur more frequently than common mutations. This study aimed to evaluate the clinical utility of assaying circulating cell-free DNA for methylation in BCAT1, IKZF1 and IRF4 for detection of CRC. METHODS: A multiplexed real-time PCR assay targeting DMRs in each of the three genes was developed. Assay accuracy was explored in plasma specimens banked from observational cross-sectional trials or from volunteers scheduled for colonoscopy or prior to CRC surgery. RESULTS: 1620 specimens were suitable for study inclusion including 184 and 616 cases with CRC and adenomas, respectively, and 820 cases without neoplasia (overall median age, 63.0 years; 56% males). Combining the PCR signals for all targeted DMRs returned the best sensitivity for CRC (136/184, 73.9%, 95% CI 67.1-79.7), advanced adenomas (53/337, 15.7%, 95% CI 12.0-20.1) and high-grade dysplastic (HGD) adenomas (9/35, 25.7%, 95% CI 14.0-42.3) with a 90.1%, specificity for neoplasia (739/820, 95% CI 87.9-92.0, p < 0.01). Detection of methylation in all three genes were more likely in CRC cases than those without it (OR 28.5, 95% CI 7.3-121.2, p < 0.0001). Of the 81 positive cases without neoplasia, 62 (76.5%) were positive by a single PCR replicate only and predominantly due to detection of methylated BCAT1 (53.2%). Single replicate positivity was significantly higher than that in CRC (26/136, 19.1%, p < 0.0001), and single BCAT1 replicate positivity was more likely in cases without neoplasia than in CRC (OR 17.7, 95% CI 6.6-43.3, p < 0.0001). When a positive result was limited to those with ≥ 1 PCR replicate positive for either IKZF1 or IRF4, or at least two replicates positive for BCAT1, the multi-panel test maintained a high sensitivity for CRC (131/184, 71.2%, 95% CI 64.3-77.3) and HGD adenomas (8/35, 22.9%, 95% CI 11.8-39.3, p = 0.029) but improved specificity significantly (772/820, 94.1%, 95% CI 92.3-95.6, p < 0.0001 vs. any PCR replicate positive). CONCLUSION: The multi-panel methylation assay differentiates cases with CRC from those without it and does so with high specificity when criteria for BCAT1 detection are applied. The marker panel is flexible and studies in those at average risk for CRC are now warranted to determine which panel configuration best suits screening goals. TRIAL REGISTRATION: ACTRN12611000318987. Registered 25 March 2011, https://www.anzctr.org.au/ ACTRN12611000318987.

Evaluation of Circulating Tumor DNA for Methylated BCAT1 and IKZF1 to Detect Recurrence of Stage II/Stage III Colorectal Cancer (CRC).

BACKGROUND: Most recurrences of early-stage colorectal cancer detected with current surveillance measures are widespread and incurable. Circulating tumor DNA (ctDNA) may facilitate earlier diagnosis of recurrent colorectal cancer and improve cancer-related outcomes. METHODS: Plasma from patients undergoing standard surveillance after definitive treatment for stage II/III colorectal cancer was assayed with COLVERA and carcinoembryonic antigen (CEA) at a single time point. Results were correlated with radiographic imaging. Assay performance, including sensitivity and specificity for recurrence, were compared. Impact of potentially confounding variables was also explored. RESULTS: 322 patients were included in the final analysis, and 27 recurrences were documented over a median follow-up period of 15 months. Sensitivity for recurrence was 63% [confidence interval (CI), 42.4-80.6] and 48% (CI, 28.7-68.1) for COLVERA and CEA (≥5 ng/mL), respectively (P = 0.046), while specificity was 91.5% (CI, 87.7-94.4) and 96.3% (CI, 93.4-98.1), respectively (P = 0.016). Smoking and age were independent predictors of CEA but not COLVERA positivity. CONCLUSIONS: COLVERA was more sensitive but less specific than CEA in detecting recurrent colorectal cancer. Short median follow-up may have been responsible for apparent false positives in COLVERA. Studies with serial sampling and longer follow-up are needed to assess whether earlier detection of colorectal cancer recurrence translates into clinical benefit. IMPACT: This prospective study showed that COLVERA (a two-gene ctDNA assay) was more sensitive for detection of recurrence in a cohort of patients undergoing surveillance after definitive therapy for stages II and III colorectal cancer.

Evaluation of Methylation Biomarkers for Detection of Circulating Tumor DNA and Application to Colorectal Cancer.

Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.

A Blood Test for Methylated BCAT1 and IKZF1 vs. a Fecal Immunochemical Test for Detection of Colorectal Neoplasia.

OBJECTIVES: To compare the performance of a new blood test for colorectal cancer (CRC) to an established fecal immunochemical test (FIT) in a study population with the full range of neoplastic and non-neoplastic pathologies encountered in the colon and rectum. METHODS: Volunteers were asked to complete a FIT prior to colonoscopy. Blood was collected after bowel preparation but prior to colonoscopy, and plasma was assayed for the presence of methylated BCAT1 and IKZF1 DNA using a multiplex real-time PCR assay. Sensitivity and specificity estimates for the blood test were calculated from true- and false-positive rates for neoplasia and compared with FIT at a range of fecal hemoglobin (Hb) concentration positivity thresholds. RESULTS: In total, 1,381 volunteers (median age 64 years; 49% male) completed both tests prior to colonoscopy. Estimated sensitivity of the BCAT1/IKZF1 blood test for CRC was 62% (41/66; 95% confidence interval 49-74%) with a specificity of 92% (1207/1315; 90-93%). FIT returned the same specificity at a cutoff of 60 µg Hb/g, at which its corresponding sensitivity for cancer was 64% (42/66; 51-75%). In the range of commonly used FIT cutoffs, respective cancer sensitivity and specificity estimates with FIT were: 59% (46-71%) and 93% (92-95%) at 80 µg Hb/g, and 79% (67-88%) and 81% (78-83%) at 10 µg Hb/g. Although estimated sensitivities were not significantly different between the two tests for any stage of cancer, FIT showed a significantly higher sensitivity for advanced adenoma at the lower cutoffs. Specificity of FIT, but not of the BCAT1/IKZF1 blood test, deteriorated substantially in people with overt blood in the feces. When combining FIT (cutoff 10 µg Hb/g) with the BCAT1/IKZF1 blood test, sensitivity for cancer was 89% (79-96%) at 74% (72-77%) specificity. CONCLUSIONS: A test based on detection of methylated BCAT1/IKZF1 DNA in blood has comparable sensitivity but better specificity for CRC than FIT at the commonly used positivity threshold of 10 µg Hb/g. Further evaluation of the new test relative to FIT in the population screening context is now required to fully understand the potential advantages and disadvantages of these biomarkers in screening.

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

BACKGROUND: Specific genes, such as BCAT1 and IKZF1, are methylated with high frequency in colorectal cancer (CRC) tissue compared to normal colon tissue specimens. Such DNA may leak into blood and be present as cell-free circulating DNA. We have evaluated the accuracy of a novel blood test for these two markers across the spectrum of benign and neoplastic conditions encountered in the colon and rectum. METHODS: Circulating DNA was extracted from plasma obtained from volunteers scheduled for colonoscopy for any reason, or for colonic surgery, at Australian and Dutch hospitals. The extracted DNA was bisulphite converted and analysed by methylation specific real-time quantitative PCR (qPCR). A specimen was deemed positive if one or more qPCR replicates were positive for either methylated BCAT1 or IKZF1 DNA. Sensitivity and specificity for CRC were estimated as the primary outcome measures. RESULTS: Plasma samples were collected from 2105 enrolled volunteers (mean age 62 years, 54 % male), including 26 additional samples taken after surgical removal of cancers. The two-marker blood test was run successfully on 2127 samples. The test identified 85 of 129 CRC cases (sensitivity of 66 %, 95 % CI: 57-74). For CRC stages I-IV, respective positivity rates were 38 % (95 % CI: 21-58), 69 % (95 % CI: 53-82), 73 % (95 % CI: 56-85) and 94 % (95 % CI: 70-100). A positive trend was observed between positivity rate and degree of invasiveness. The colonic location of cancer did not influence assay positivity rates. Gender, age, smoking and family history were not significant predictors of marker positivity. Twelve methylation-positive cancer cases with paired pre- and post-surgery plasma showed reduction in methylation signal after surgery, with complete disappearance of signal in 10 subjects. Sensitivity for advanced adenoma (n = 338) was 6 % (95 % CI: 4-9). Specificity was 94 % (95 % CI: 92-95) in all 838 non-neoplastic pathology cases and 95 % (95 % CI: 92-97) in those with no colonic pathology detected (n = 450). CONCLUSIONS: The sensitivity for cancer of this two-marker blood test justifies prospective evaluation in a true screening population relative to a proven screening test. Given the high rate of marker disappearance after cancer resection, this blood test might also be useful to monitor tumour recurrence. TRIAL REGISTRATION: ACTRN12611000318987 .

A two-gene blood test for methylated DNA sensitive for colorectal cancer.

BACKGROUND: Specific genes are methylated with high frequency in colorectal neoplasia, and may leak into blood. Detection of multiple methylated DNA biomarkers in blood may improve assay sensitivity for colorectal cancer (CRC) relative to a single marker. We undertook a case-control study evaluating the presence of two methylation DNA markers, BCAT1 and IKZF1, in circulation to determine if they were complementary for detection of CRC. METHODS: Methylation-specific PCR assays were developed to measure the level of methylated BCAT1 and IKZF1 in DNA extracted from plasma obtained from colonoscopy-confirmed 144 healthy controls and 74 CRC cases. RESULTS: DNA yields ranged from 2 to 730 ng/mL plasma (mean 18.6ng/mL; 95% CI 11-26 ng/mL) and did not correlate with gender, age or CRC status. Methylated BCAT1 and IKZF1 DNA were detected in respectively 48 (65%) and 50 (68%) of the 74 cancers. In contrast, only 5 (4%) and 7 (5%) controls were positive for BCAT1 and IKZF1 DNA methylation, respectively. A two-gene classifier model ("either or" rule) improved segregation of CRC from controls, with 57 of 74 cancers (77%) compared to only 11 of 144 (7.6%) controls being positive for BCAT1 and/or IKZF1 DNA methylation. Increasing levels of methylated DNA were observed as CRC stage progressed. CONCLUSIONS: Detection of methylated BCAT1 and/or IKZF1 DNA in plasma may have clinical application as a novel blood test for CRC. Combining the results from the two methylation-specific PCR assays improved CRC detection with minimal change in specificity. Further validation of this two-gene blood test with a view to application in screening is now indicated.

CAHM, a long non-coding RNA gene hypermethylated in colorectal neoplasia.

The CAHM gene (Colorectal Adenocarcinoma HyperMethylated), previously LOC100526820, is located on chromosome 6, hg19 chr6:163 834 097-163 834 982. It lacks introns, encodes a long non-coding RNA (lncRNA) and is located adjacent to the gene QKI, which encodes an RNA binding protein. Deep bisulphite sequencing of ten colorectal cancer (CRC) and matched normal tissues demonstrated frequent hypermethylation within the CAHM gene in cancer. A quantitative methylation-specific PCR (qMSP) was used to characterize additional tissue samples. With a threshold of 5% methylation, the CAHM assay was positive in 2/26 normal colorectal tissues (8%), 17/21 adenomas (81%), and 56/79 CRC samples (71%). A reverse transcriptase-qPCR assay showed that CAHM RNA levels correlated negatively with CAHM % methylation, and therefore CAHM gene expression is typically decreased in CRC. The CAHM qMSP assay was applied to DNA isolated from plasma specimens from 220 colonoscopy-examined patients. Using a threshold of 3 pg methylated genomic DNA per mL plasma, methylated CAHM sequences were detected in the plasma DNA of 40/73 (55%) of CRC patients compared with 3/73 (4%) from subjects with adenomas and 5/74 (7%) from subjects without neoplasia. Both the frequency of detection and the amount of methylated CAHM DNA released into plasma increased with increasing cancer stage. Methylated CAHM DNA shows promise as a plasma biomarker for use in screening for CRC.

A panel of genes methylated with high frequency in colorectal cancer.

BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Discovery and validation of molecular biomarkers for colorectal adenomas and cancer with application to blood testing.

BACKGROUND & AIMS: Colorectal cancer incidence and deaths are reduced by the detection and removal of early-stage, treatable neoplasia but we lack proven biomarkers sensitive for both cancer and pre-invasive adenomas. The aims of this study were to determine if adenomas and cancers exhibit characteristic patterns of biomarker expression and to explore whether a tissue-discovered (and validated) biomarker is differentially expressed in the plasma of patients with colorectal adenomas or cancer. METHODS: Candidate RNA biomarkers were identified by oligonucleotide microarray analysis of colorectal specimens (222 normal, 29 adenoma, 161 adenocarcinoma and 50 colitis) and validated in a previously untested cohort of 68 colorectal specimens using a custom-designed oligonucleotide microarray. One validated biomarker, KIAA1199, was assayed using qRT-PCR on plasma extracted RNA from 20 colonoscopy-confirmed healthy controls, 20 patients with adenoma, and 20 with cancer. RESULTS: Genome-wide analysis uncovered reproducible gene expression signatures for both adenomas and cancers compared to controls. 386/489 (79%) of the adenoma and 439/529 (83%) of the adenocarcinoma biomarkers were validated in independent tissues. We also identified genes differentially expressed in adenomas compared to cancer. KIAA1199 was selected for further analysis based on consistent up-regulation in neoplasia, previous studies and its interest as an uncharacterized gene. Plasma KIAA1199 RNA levels were significantly higher in patients with either cancer or adenoma (31/40) compared to neoplasia-free controls (6/20). CONCLUSIONS: Colorectal neoplasia exhibits characteristic patterns of gene expression. KIAA1199 is differentially expressed in neoplastic tissues and KIAA1199 transcripts are more abundant in the plasma of patients with either cancer or adenoma compared to controls.

Colorectal Neoplasia Differentially Expressed (CRNDE), a Novel Gene with Elevated Expression in Colorectal Adenomas and Adenocarcinomas.

An uncharacterized gene locus (Chr16:hCG_1815491), now named colorectal neoplasia differentially expressed (gene symbol CRNDE), is activated early in colorectal neoplasia. The locus is unrelated to any known protein-coding gene. Microarray analysis of 454 tissue specimens (discovery) and 68 previously untested specimens (validation) showed elevated expression of CRNDE in >90% of colorectal adenomas and adenocarcinomas. These findings were confirmed and extended by exon microarray studies and RT-PCR assays. CRNDE transcription start sites were identified in CaCo2 and HCT116 cells by 5'-RACE. The major transcript isoforms in colorectal cancer (CRC) cell lines and colorectal tissue are CRNDE-a, -b, -d, -e, -f, -h, and -j. Except for CRNDE-d, the known CRNDE splice variants are upregulated in neoplastic colorectal tissue; expression levels for CRNDE-h alone demonstrate a sensitivity of 95% and specificity of 96% for adenoma versus normal tissue. A quantitative RT-PCR assay measuring CRNDE-h RNA levels in plasma was (with a threshold of 2(-ΔCt) = 2.8) positive for 13 of 15 CRC patients (87%) but only 1 of 15 healthy individuals (7%). We conclude that individual CRNDE transcripts show promise as tissue and plasma biomarkers, potentially exhibiting high sensitivity and specificity for colorectal adenomas and cancers.

Map of differential transcript expression in the normal human large intestine.

While there is considerable research related to using differential gene expression to predict disease phenotype classification, e.g., neoplastic tissue from nonneoplastic controls, there is little understanding of the range of expression in normal tissues. Understanding patterns of gene expression in nonneoplastic tissue, including regional anatomic expression changes within an organ, is vital to understanding gene expression changes in diseased tissue. To explore the gene expression change along the proximal-distal axis of the large intestine, we analyzed microarray data in 184 normal human specimens using univariate and multivariate techniques. We found 219 probe sets that were differentially expressed between the proximal and distal colorectal regions and 115 probe sets that were differentially expressed between the terminal segments, i.e., the cecum and rectum. We did not observe any probe sets that were statistically different between any two contiguous colorectal segments. The dominant expression pattern (65 probe sets) follows a dichotomous expression pattern consistent with the midgut-hindgut embryonic origins of the gut while a second pattern (50 probe sets) depicts a gradual change in transcript levels from the cecum to the rectum. While the dichotomous pattern includes roughly equal numbers of probe sets that are elevated proximally and distally, nearly all probe sets that show a gradual change demonstrate increasing expression levels moving from proximal to distal segments. These patterns describe an expression map of individual transcript variation as well as multigene expression patterns along the large intestine. This is the first gene expression map of an entire human organ.