Health Policy Advisory Committee on Technology

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1 Health Policy Advisory Committee on Technology Technology Brief Update Blood and stool biomarker testing for colorectal cancer screening August 2016

2 State of Queensland (Queensland Department of Health) 2016 This work is licensed under a Creative Commons Attribution Non-Commercial No Derivatives 3.0 Australia licence. In essence, you are free to copy and communicate the work in its current form for non-commercial purposes, as long as you attribute the authors and abide by the licence terms. You may not alter or adapt the work in any way. To view a copy of this licence, visit For further information, contact the HealthPACT Secretariat at: HealthPACT Secretariat c/o Clinical Access and Redesign Unit, Health Service and Clinical Innovation Division Department of Health, Queensland Level 2, 15 Butterfield St HERSTON QLD 4029 Postal Address: GPO Box 48, Brisbane QLD HealthPACT@health.qld.gov.au Telephone: For permissions beyond the scope of this licence contact: Intellectual Property Officer, Department of Health, GPO Box 48, Brisbane QLD 4001, ip_officer@health.qld.gov.au, phone (07) Electronic copies can be obtained from: DISCLAIMER: This Brief is published with the intention of providing information of interest. It is based on information available at the time of research and cannot be expected to cover any developments arising from subsequent improvements to health technologies. This Brief is based on a limited literature search and is not a definitive statement on the safety, effectiveness or costeffectiveness of the health technology covered. The State of Queensland acting through Queensland Health ( Queensland Health ) does not guarantee the accuracy, currency or completeness of the information in this Brief. Information may contain or summarise the views of others, and not necessarily reflect the views of Queensland Health. This Brief is not intended to be used as medical advice and it is not intended to be used to diagnose, treat, cure or prevent any disease, nor should it be used for therapeutic purposes or as a substitute for a health professional's advice. It must not be relied upon without verification from authoritative sources. Queensland Health does not accept any liability, including for any injury, loss or damage, incurred by use of or reliance on the information. This Update was commissioned by Queensland Health, in its role as the Secretariat of the Health Policy Advisory Committee on Technology (HealthPACT). The production of this Update was overseen by HealthPACT. HealthPACT comprises representatives from health departments in all States and Territories, the Australian and New Zealand governments and MSAC. It is a subcommittee of the Australian Health Ministers Advisory Council (AHMAC), reporting to AHMAC s Hospitals Principal Committee (HPC). AHMAC supports HealthPACT through funding. This Update was prepared by Linda Mundy from the HealthPACT Secretariat..

3 2016 Summary of findings The national bowel cancer screening program (NBCSP) aims to provide by 2020 biennial screening for asymptomatic individuals aged years, using an immunological faecal occult blood test (FOBT) to detect colorectal cancer (CRC). Since the literature search was conducted for the original Brief, the ColoVantage DNA stool test has been made available in Australia on a user-pays basis. In addition, the USA s Food and Drug Administration (FDA) has approved the use of ColoGuard, a DNA stool-based CRC screening test, and the Epi procolon a plasma-based CRC screening test used to detect methylated DNA. Few new studies have been published in the peer-reviewed literature since the publication of the original brief. The pivotal trial of Epi procolon included in the 2016 application for FDA approval used a patient cohort enriched with CRC positive patients. It is important to note that the performance of a diagnostic test is reliant on the prevalence of the condition in question. A diagnostic test will perform differently in a sample enriched with the condition than when tested in a true screening population of asymptomatic individuals. The positive predictive value (PPV) was poor at 2.4 per cent, however the negative predictive value (NPV) of 99.7 per cent indicates that the Epi procolon is good at reassuring patients with a negative test result that they don t have CRC, which is an important consideration in a screening programme. A further validation study requested by the FDA was conducted, comparing Epi procolon to FOBT. Although participants were considered to be at average risk of CRC, the rate of CRC detection was 34 per cent, compared to an average lifetime risk in Australia of eight per cent. The PPV and NPV for Epi procolon were similar to those obtained in the pivotal study, compared to a PPV and NPV of 15.6 and per cent for FOBT, respectively. No new studies could be identified describing the use of the ColoVantage Plasma DNA test in asymptomatic screening populations. However, an abstract described the use of the ColoVantage Plasma test in conjunction with FOBT, compared to ifobt alone. The addition of the ColoVantage Plasma test detected an additional seven cancers and eight high-risk adenomas and the combined test sensitivity and specificity for CRC (for either test positive) was 89.4 and 77.8 per cent, respectively. A systematic review of the Cologuard stool-based DNA test in asymptomatic adults who underwent CRC screening reported a sensitivity for detecting CRC >90 per cent, but only 40 per cent for detecting advanced adenomas. As with the Epi procolon, the Cologuard test could reliably rule out CRC, which is important in a screening programme, with a negative likelihood ratio between 0.02 and 0.09, however it was poor ruling out advanced adenoma, with a likelihood ratio between 0.5 and 0.7. Cost-effectiveness information in the Australian screening setting of the new commercial tests compared to the established FOBT is still lacking. Blood and stool biomarker testing for CRC screening: Update August 2016 i

4 Other areas of great interest and research, that may provide commercial CRC tests in the future, include the detection of micrornas or specific tumour-associated proteins in the stool or blood. In addition, research into methods to increase participation in the national bowel cancer screening program and to engage non-responders should be given priority to maximise the potential of the NBCSP HealthPACT Advice No new evidence has been provided in this Update of the evidence of new blood-based biomarker and stool-based tests for the detection of colorectal cancer in terms of performance characteristics of the tests assessed that would suggest the ifobt, used in the National Bowel Cancer Screening Program, would be superseded. No evidence has been provided to support the introduction of blood-based biomarker tests for use in population screening in Australia at this stage, and the ifobt used by the NBCSP remains the best performing test for use in the Australian population. The need to increase participation rates, and to target individuals at higher risk, especially in the Indigenous population, may involve greater general practitioner participation in the delivery of screening and education. Increased participation rates in the NBCSP may result in an increase in the number of false positives and in so doing, impact on jurisdictional waiting lists for colonoscopy. It was noted that the use of calprotectin testing to identify patients with intestinal inflammation may provide a potential means to reduce waitlists for colonoscopy and unnecessary colonoscopies. Calprotectin testing has not been approved for public subsidy by the Medical Services Advisory Committee. There is insufficient evidence to support the introduction of blood-based biomarker tests for the use in population screening in Australia at this time. However, given the ongoing research and rapidly evolving evidence base on tests for use in population screening programs for CRC, HealthPACT recommends that this technology be reassessed in 24 months. Blood and stool biomarker testing for CRC screening: Update August 2016 ii

5 Technology, Company and Licensing Register ID Technology name Patient indication Reason for assessment WP151 Blood and stool biomarker tests for colorectal cancer screening People at average risk of colorectal cancer In 2014 a Technology Brief was commissioned to investigate new and emerging blood-based biomarker and stool-based tests for the detection of colorectal cancer (CRC). It is of concern that these new tests for CRC are currently being marketed to consumers in Australia as a more convenient and acceptable alternative to faecal occult blood testing (FOBT) without a rigorous assessment of the safety and effectiveness of these tests in a non-symptomatic population. HealthPACT considered the evidence for these tests and found that, in comparison to the FOBT currently in use in the Australian National Bowel Cancer Screening Program, these tests were expensive and as such would be prohibitive for population screening and may result in an increased number of unnecessary colonoscopies being performed. In 2014 there was insufficient evidence to support the introduction of bloodbased biomarker tests for the use in population screening in Australia, however, due to the rapidly evolving evidence base around a number of these tests, HealthPACT recommended that this technology be reassessed in 24 months. Description of the technology Bowel cancer, which includes cancers of the colon, recto-sigmoid junction and rectum, is a major cause of morbidity and mortality in Australia. Although colonoscopy is the gold standard for the detection of colorectal cancer (CRC), it is expensive, and by virtue of its invasive nature, associated with risks such as perforation of the gut, and is therefore not recommended for use as a routine screening tool. 1, 2 Evidence of several randomised controlled trials (RCTs) demonstrated that bowel cancer mortality could be reduced by per cent by regular bowel screening using the non-invasive guaiac faecal occult blood test (gfobt). As such, in 2006, Australia introduced the national bowel cancer screening program (NBCSP), a population-based screening program. 3 The NBCSP now uses a 4 th generation FOBT - an automated immunochemical faecal occult blood test (ifobt). 4 Faecal occult blood testing looks for minute traces of haemoglobin in stool samples, which may be a result of upper or lower gastrointestinal bleeding potentially caused by not only benign or malignant growths or polyps of the colon, but many conditions including: inflammatory bowel disease, haemorrhoids, ulcerative colitis or Crohn s disease. The gfobt uses hydrogen peroxide, which in the presence of blood results in a colour change, whereas the newer ifobt uses antibodies directed against human haemoglobin to detect blood in the stool sample. 5 Blood and stool biomarker testing for CRC screening: Update August

6 Of the 1.4 million individuals invited to participate in the NBCSP during the year , the overall rate of participation increased slightly when compared to the previous year (36.0 vs 33.4%). Of those who returned a valid screening test, approximately 37,700 (7.5%) returned a positive screening result, and were recommended to seek a follow-up with their general practitioner for a follow-up referral for colonoscopy. 6 Participants may elect to have their colonoscopy through the private a or public health -care system, depending on their individual circumstances and choice. 4 During , sixty-eight per cent of those with a positive screening result were reported as having had a follow-up colonoscopy. Of those participants with a positive screen and who underwent a colonoscopy, 149 and 599 were diagnosed with a confirmed or suspected cancer, respectively. In addition, advanced adenomas, which although benign have the potential to become cancerous, were found in a further 1,691 participants. 6 Since the introduction of the NBCSP, several new colorectal cancer (CRC) diagnostic tests have been brought to market or are in development (Table 1 in the original Brief). The aim of the 2015 Brief was to provide a preliminary assessment of the safety and diagnostic accuracy of these new blood and stool-based biomarker tests in comparison to the established FOBT, and in particular how they performed in the detection of early colorectal cancer and adenomas. As described above, haemoglobin enters the faeces as a result of upper or lower gastrointestinal bleeding. DNA and RNA b biomarkers, such as those described below, continuously enter into the faeces via exfoliation from all stages of CRCs and polyps at higher rates than normal tissue, probably due to greater cell proliferation taking place in tumours. Reduced rates of biomarker release into the bloodstream occurs in the early stages of CRC but increases with advancing stages (Figure 1). 7 The 2016 Update aims to report any new evidence on those diagnostic tests described in the original 2015 Brief, in addition to describing any new and emerging tests used for the detection of bowel cancer. As with the original brief, this update is not a systematic appraisal of all available evidence, nor is it placed to definitively report on the effectiveness of these new technologies in clinical and / or population screening settings. a Using Medicare Benefits Schedule (MBS) item numbers and : Fibreoptic colonoscopy examination of colon beyond the hepatic flexure with our without biopsy 32093: Endoscopic examination of the colon beyond the hepatic flexure by fibreoptic colonoscopy for the removal of 1 or more polyps, or the treatment of radiation proctitis, angiodysplasia or post-polypectomy bleeding by argon plasma coagulation. b RNA = ribonucleic acid Blood and stool biomarker testing for CRC screening: Update August

7 Figure 1 Rates of marker release into the bloodstream via vascular invasion and into the stool via exfoliation 7 DNA stool-based assays Whereas the established FOBT is used to detect minute traces of haemoglobin in stool samples, new tests on the market may be used to detect DNA fragments in stool samples. These tests rely on the fact that the colorectal epithelium sloughs cells into the large bowel, and, if tumours or polyps are present, the exfoliated cells will contain genes or fragments of DNA which can then be detected. Stool samples can be evaluated for the presence of DNA deletions or mutations in the K-RAS, p53 and adenomatous polyposis (APC) genes, all of which are associated with, or present in colorectal cancer. 1 Due to the heterogeneous nature of CRC and the large number of gene mutations associated with CRC, the detection of a single mutation, or a combination of several mutations is not specific or sensitive enough to use as a screening test. However, mutations in these key genes may be used in combination with other biomarkers to test for the presence of CRC. 8 A good example of this is the ColoGuard DNA stool test, which combines assays for the detection of mutations in KRAS mutations in addition to aberrant NDRG4 and BMP3 methylation, and a haemoglobin immunoassay. It is thought that by combining several approaches (DNA mutational analysis and immunological detection) may improve the accuracy of early CRC detection tests. 9 DNA blood-based assays Another area of interest is epigenetics, where the actual sequence of DNA remains unchanged but modifications such as DNA methylation, changes in micro-rna and histone modification, result in changes in gene expression, therefore altering phenotype. Epigenetic modifications may affect the way cells terminally differentiate, leading to the progression of cancer. 1 Blood and stool biomarker testing for CRC screening: Update August

8 DNA methylation is a normal part of the cell differentiation process, however hypermethylation of tumour suppressor genes results in gene silencing, preventing transcription of these genes. Changes in methylation occurs early in carcinogenesis and therefore may be a good biomarker to be used in the early detection of cancer in both stool and blood-based samples. 1, 8, 10 During cell differentiation and embryonic development, cytosine is modified by the addition of a methane group (Figure 2). This occurs in regions of high guaninecytosine c content, which are located in the 5' untranslated region of genes. A number of genes have been identified as targets for methylation during the progression of CRC from early adenoma to adenocarcinoma (Figure 3). 11 Figure 2 The process of DNA methylation 11 Figure 3 Commonly methylated genes that have been identified in the pathogenesis of CRC 11 The Epi procolon and ms9 test for aberrant levels of DNA methylation of the SEPT9 gene in blood samples, and the ColoVantage Plasma test is designed to detect aberrant DNA methylation in two genes, BCAT1 and IKZF1, where hyper-methylation has frequently been observed in benign colorectal polyps and malignant cancers (colorectal neoplasia). 12 The ColoSure faecal-based assay for the detection of methylation of the vimentin gene was mentioned by recent reviews as being the only commercially available test in the USA, however it appears that this test is no longer offered and has been replaced by Exact Sciences ColoGuard test Messenger RNA (mrna) blood-based assays Differences in gene expression, either under or over-expression, between normal and tumour tissue form the basis of mrna tests such as the ColonSentry. The ColonSentry test measures levels of mrna transcripts from whole blood using a seven gene panel d using c Guanine and cytosine are two of the four bases of DNA d ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, and VN1 (over-expressed in CRC), and IL2RB (under-expressed in CRC relative to bowel tissue) Blood and stool biomarker testing for CRC screening: Update August

9 8, 13 real-time PCR. ColonSentry the USA. 13 micrornas is in clinical use in China and Malaysia, and in limited use in One area of intense interest and research is that of micrornas (mirnas), which are short, non-coding RNAs that regulate gene, and therefore protein expression, by binding to the 3' untranslated region of messenger RNA (mrna). One mirna can target several mrnas but one mrna can be the target of multiple mirnas. mirnas are involved in cellular processes including differentiation, proliferation and apoptosis (cell death), and as such are implicated in the occurrence and development of cancer. It has been proposed that differential expression of mirnas in individuals with CRC compared to normal individuals may make mirnas ideal biomarkers for the screening, early diagnosis and prognosis of some cancers, in particular CRC. 1, 2, 14 MicroRNAs can be isolated from stool samples 15, however stable levels of circulating mirnas have been detected and isolated from small samples of whole blood (2ml), serum and plasma samples using commercially available RNA extraction kits, with levels of mirna quantified using quantitative real-time PCR e. 16 A number of single mirnas have been identified as potential biomarkers for the presence of CRC, however it is more likely that a panel of mirnas would give greater discriminatory power. These have been summarised in Table 1. Table 1 Individual and panel mirnas identified as potential biomarkers for CRC 16 Single mirna mirna panels Up-regulated in CRC mirna-21, mirna-92a, mirna-29a, mirna-141, mirna-135b, mirna95, mirna-222, mirna-17-3p mirna-18a, mirna-19a, mirna-19b, mirna- 15b, mirna-29a, mirna-335 mirna-21, mirna-31, mirna-92a, let-7g, mirna-181b, mirna-203 Up-regulated in adenomas mirna-135b, mirna cluster, mirna-18a Down regulated in CRC mirna-24, mirna-320a, mirna-423-5p Can distinguish adenomas from controls Can distinguish advanced colorectal adenoma from CRC CRC = colorectal cancer mirna-532-3p, mirna-331, mirna-195, mirna-17, mirna-142-3p, mirna-15b, mirna-532, mirna-652 mirna-331, mirna-15b, mirna-21, mirna-142-3p, mirna-339-3p e PCR = polymerase chain reaction Blood and stool biomarker testing for CRC screening: Update August

10 Commercial tests for mirnas are not yet available; however it would be possible to test for either a single mirna or, more realistically, a panel of mirnas using next generation sequencers already in use in the health system. More RCTs, comparing the ability of mirnas to discriminate between normal and CRC individuals and assessing mirnas performance against FOBT, are required to demonstrate the clinical utility of mirna before their use can be translated into clinical settings. Other markers Other markers that have been suggested as potential screening tools include areas of microsatellite instability (tandem repeats of short nucleotide sequences), which are frequently detected in tumours and long form DNA, both of which are in the early stages of investigation. 1 The detection of certain proteins in the circulation has also been proposed as a potential tool in the detection of CRC. Glycoproteins such as CA125, CA15-3, prostate-specific antigen and carcinoembryonic antigen (CEA) are blood based biomarkers associated with ovarian, breast, prostate and colon cancer. However, these markers have poor specificity and tend to be more useful in the monitoring of patients already diagnosed with cancer, rather than for screening asymptomatic individuals. 17 Several other proteins have been nominated as candidate protein biomarkers associated with CRC, and tests are currently underway to validate assays. Breath testing of volatile organic compounds analysed gas chromatography coupled with mass spectrometry has also been suggested as a potential non-invasive detection tool of the future, with specific profile patterns associated with CRC. This research is only in the early 9, 18, 19 stages of development Stage of development in Australia The ColoVantage Plasma test is still currently available to a limited population of Australians under trial conditions, however the ColoVantage DNA stool test is available online, from pharmacies and GPs on a user-pays basis ($39.95), some of which may be claimed back via private health insurance. 20 Yet to emerge Experimental Investigational Nearly established Established Established but changed indication or modification of technique Should be taken out of use 2016 Licensing, reimbursement and other approval As stated in the original 2015 Brief, the Therapeutic Goods Administration (TGA) regulatory framework for in vitro diagnostic tests (IVDs) changed in July 2010, and in-house laboratory tests now receive a similar level of regulatory scrutiny as commercial kits. The ColoVantage Blood and stool biomarker testing for CRC screening: Update August

11 Plasma test is a DNA test and is currently provided in Australia as an in-house IVD, and as such is classified as a Class 3 in-house IVD. In August 2014 the USA s Food and Drug Administration (FDA) approved the use of ColoGuard, the first stool-based colorectal screening test. 21 In April 2016, the FDA approved the Epi procolon (Epigenomics AG, Germany), a qualitative IVD for detecting methylated Septin9 DNA, which is associated with the occurrence of CRC, in plasma obtained from whole-blood specimens. It is indicated for use in average-risk patients who have chosen not to undergo other screening methods, including stool-based tests or colonoscopy. This test also has approval for use in Europe Australian Therapeutic Goods Administration approval Yes ARTG number (s) No Not applicable 2016 Diffusion of technology in Australia The ColoVantage Plasma test is still being trialled in the Blue Mountains study, and as mentioned above, the ColoVantage stool based test is currently available on a user-pays basis; however the uptake of this test could not be ascertained Cost infrastructure and economic consequences The ColoVantage DNA stool test is available in Australia online, from pharmacies and GPs on a user-pays basis ($39.95), some of which may be claimed back via private health insurance. 20 Prices identified for other tests described in this brief are as follows: ColonSentry will be reimbursed in the range of US$350 per test in the USA 23, the maximum out-of-pocket cost of Cologuard in the USA is $649, depending on the level of health insurance 24 and Epigenomics estimate that the Centers for Medicare & Medicaid Services would pay US$141 per Epi procolon test Evidence and Policy Safety and effectiveness DNA methylation blood and stool-based tests Xue et al (2015) conducted a systematic review of studies describing DNA methylation profiles in individuals with CRC compared to healthy controls. A total of 74 studies were identified for inclusion, 35 (47%) of which were blinded to CRC histological status. Sampling method in the studies varied, with most analyses using stool samples (50%), blood (40.5%) and a small proportion that used both blood and stool samples (0.03%). 26 Blood and stool biomarker testing for CRC screening: Update August

12 For blood-based assays, there was little consistency in the methylated genes targeted by these studies, with 29 candidate genes described, mostly by single research groups. Four of the five candidate genes described by multiple research groups (hmlh1, CDKN2A, HLTF and ALX4) were reported before 2010 and not reported on after. The target most described was methylation of the SEPT9 gene in plasma samples, with 10 studies reporting results from 2008 onwards. Three of the most recent of these studies reported on the use of the Epi procolon assay, two 27, 28 of which were summarised in the original brief. In addition, nine methylated gene panels were described, with the more recent studies reporting significant levels in CRC individuals compared to healthy controls. 26 Similar results were reported for stool-based assays, with 35 candidate genes described, mostly by single research groups. However, two genes, SFRP2 and VIM, were described by eight different studies, with significant levels of the methylated genes being detected in the CRC group compared to healthy controls. The VIM gene was the basis of the ColoSure assay, which appears to have been superseded. Seventeen stool-based gene panels were described, all of which improved the diagnostic sensitivity compared to single genes, reporting significant levels in CRC individuals compared to healthy controls. 26 Overall, nine methylated single gene candidates and two gene panels were identified as those likely to be useful in discriminating early CRC, with significantly higher levels detected in patients with CRC compared to healthy controls (Table 2). Note that the overall sample sizes in the CRC arms were larger; however this table is only presenting those patients diagnosed with early CRC. The authors note that larger, prospective studies are required before the clinical utility of candidate biomarkers for CRC screening can be confirmed. 26 Table 2 Methylation markers in early CRC 26 Gene Specimen Healthy controls % (n/n) Early CRC* (TNM1) % (n/n) P value CDKN2A Serum 0 (0/10) 100 (2/2) HLTF Serum 0 (0/20) 20 (3/15) hmlh1 Serum 0 (0/20) 26.7 (4/15) SEPT9 Plasma Plasma Plasma Plasma Plasma Plasma 14 (25/179) 8.8 (25/285) 13.8 (45/327) 11.7 (11/94) 15.2 (14/92) 8.6 (80/934) 30 (6/20) 41.2 (35/85) 48.8 (20/41) (21/25) 36.4 (8/22) NA VIM Plasma Stool 7.3 (8/110) 17.1 (62/363) 50 (11/22) 84.2 (16/19) < Blood and stool biomarker testing for CRC screening: Update August

13 Gene Specimen Healthy controls % (n/n) Early CRC* (TNM1) % (n/n) P value NEUROG1 Serum 20 (9/45) 51.9 (14/27) SDC2 Serum 4.8 (6/125) 92.3 (24/26) FBN1 Stool 6.7 (2/30) 91.7 (11/12) CAHM Plasma 6.8 (5/74) 41.7 (5/12) VIM+NDRG4+BMP3+ TFP12+muKRAS+ Hb Stool NDRG4+BMP3+ mukras+ Hb Stool Stool 10.2 (455/4,457) (26/29) 95.2 (20/21) NA Early CRC defined as TNM1 (Tumour, Node, Metastases), Hb = haemoglobin, NA = not applicable Epi procolon The pivotal trial of the Epi procolon included in the 2016 application for FDA approval was conducted by Potter et al (2014) and was not included in the original HealthPACT brief. 29 This multicentre study, conducted in the USA and Germany, used archived plasma samples collected prospectively during the previous PRESEPT study reported by Church et al (reported in the original brief). 27 Participants in the PRESEPT study were asymptomatic individuals, aged >50 years (mean age 60 years) who were at average risk for CRC and scheduled to undergo screening colonoscopy. In the validation study, a total of 1,544 samples from the 6,857 samples from the PRESEPT study were analysed independently using the Epi procolon test, with technicians blinded to the SEPT9 status of the samples. However, samples were selected according to the patient s known CRC status following colonoscopy: invasive adenocarcinoma (n=44), advanced adenoma (polyps 10mm, n=621) small polyps (<10mm, n=435) and no evidence of disease (n=444) (level IV diagnostic evidence). 11, 29 The results are summarised in Table 3. Of interest is the positive and negative predictive values (PPV and NPV), with the high NPV indicating that a negative result obtained with the Epi procolon test is good at reassuring patients that they don t have CRC; however the low PPV indicates that a high proportion of patients who receive a positive result do not, in fact, have CRC. The authors of the study raised the question of health economics, in that the previous PRESEPT study by Church et al (2014) found that testing with Epi procolon was cost effective when compared to no screening; however the poor PPV would raise uncertainty regarding the health economic benefit. A health economic evaluation of Epi procolon is currently underway. 29 Blood and stool biomarker testing for CRC screening: Update August

14 Table 3 Diagnostic outcomes of the FDA pivotal study11, 29 CRC stage Sensitivity 95% CI Stage I 41% (7/17) [22, 64] Stage II 83% (10/12) [55, 95] Stage III 80% (8/10) [49, 94] Stage IV 100% (5/5) [57, 100] Stage I-III 64% (25/39) [48, 77] Total 68% (30/44) [53, 80] No evidence of disease 22% (97/444) [18, 26] Small polyps 20% (87/435) [16,24] Advanced adenomas 22% (134/621) [18, 24] Total non-crc 21% (318/1500) [19, 23] Adjusted specificity of individuals with negative colonoscopy 80% [78, 82] Positive diagnostic likelihood ratio 3.41% [2.72, 4.27] Negative diagnostic likelihood ratio 0.40% [0.26, 0.61] Positive predictive value 2.4% [2.0, 3.0] Negative predictive value 99.7% [99.4, 99.9] See Appendix for definitions of diagnostic accuracy terms The FDA recommended that a second clinical validation study be conducted, comparing the performance of the Epi procolon to a commercial ifobt (OC- FIT-CHEK). A total of 301 individuals, the majority of whom were male (68%), aged who were considered to be at average risk of CRC were enrolled prior to a screening colonoscopy. Blood and faecal sampling took place prior to bowel preparation for colonoscopy (level III-1 diagnostic evidence). Paired Epi procolon and FOBT results were available for 290 individuals. 21 The results of the second validation study are summarised in Table 4. Although the overall sensitivity of Epi procolon was improved slightly when compared to FOBT (72% vs 68%), this was countered by a marked reduction in the specificity (81% vs 97%). There was no difference between the NPV of both tests; however the PPV was markedly improved when the FOBT was used (no statistical analysis reported). Of concern, is that the colonoscopy results revealed that of 301 individuals considered to be at average risk of CRC, 101 (34%) were diagnosed with CRC. This reflects an enriched CRC population, which then impacts on diagnostic outcomes such as the PPV, which is highly dependent on prevalence. 21 In a true screening population, the positivity of ifobt is approximately five per cent, with an eight Blood and stool biomarker testing for CRC screening: Update August

15 per cent average lifetime risk of developing CRC. 30 In addition, the confidence intervals for both the Epi procolon and FOBT for detecting all stages of CRC were wide, indicating a great deal of variation. 21 Table 4 Results of the Epi procolon validation study 21 CRC stage Epi procolon [95% CI] Positivity FOBT [95% CI] Positivity Stage 0 100% (2/2) [34.2, 100] 0% (0/2) [0, 65.8] Stage I 61.5% (16/26) [42.5, 77.6] 65.4% (17/26) [46.2, 80.6] Stage II 80.0% (16/20) [58.4, 91.9] 80% (16/20) [58.4, 91.9] Stage III 65.2% (15/23) [44.9, 81.2] 82.6% (19/23) [62.9, 93.0] Stage IV 92.3% (12/13) [66.7, 99.6] 58.3% (7/12) [32.0, 80.7] Unknown 76.5% (13/17) [52.7, 90.4] 50% (7/14) [26.8, 73.2] Total 73.3% (74/101) [63.9, 80.9] 68% (66/97) [58.2, 76.5] Sensitivity 72.2% (70/97) [62.5, 80.1] 68% (66/97) [58.2, 76.5] Specificity 80.8% (156/193) [74.7, 85.8] 97.4% (188/193) [94.1, 98.9] Positive diagnostic likelihood ratio 3.96% [2.89, 5.42] 26.3% [10.94, 63.05] Negative diagnostic likelihood ratio 0.33% [0.24, 0.46] 0.33% [0.25,.044] Positive predictive value 2.72% [2.0, 3.68] 15.62% [7.16, 30.77] Negative predictive value 99.77% [99.68, 99.83] 99.69% [99.69, 99.83] Of interest is the case control study conducted by Ørntoft et al (2015) that assessed the performance of Epi procolon when stratified by age and underlying comorbidities, including diabetes, hypertension and arthritis (level III-3 diagnostic evidence). Cases (n=150) and controls (n=150) were matched by age and gender and selected from a large cohort (n=4,698) of patients who had been referred for a diagnostic colonoscopy. Overall sensitivity and specificity was 73 and 82 per cent, respectively. The assay had low sensitivity for the detection of early stage tumours (adenomas and stage I carcinoma), which would be of great importance if the test is to be used in a population screening scenario. Further analysis found that there was an association between age and the performance of the test, with a significant increase in the rate of false positives for those aged >65 years (p<0.05). In addition, arthritis was associated with an increased rate of false negatives (p=0.005) and arteriosclerosis was associated with an increased rate of false positives (p=0.007). When these factors, in addition to diabetes, were adjusted for in a multivariate analysis, the odds ratio for a positive Epi procolon test increased from 8.25 (95%CI 4.83, 14.09) to Blood and stool biomarker testing for CRC screening: Update August

16 (95%CI 12.58, 69.02). This analysis should be replicated in larger prospective controlled studies if the Epi procolon is considered as an alternative test in a population screening program. 31 ColoVantage Plasma No new studies could be identified describing the use of the ColoVantage Plasma test in asymptomatic screening populations. However, an abstract was identified that reported the use of the ColoVantage Plasma test in conjunction with FOBT, compared to ifobt alone. This approach was determined as a consequence of a survey aimed at addressing issues of low compliance and in CRC screening programmes. The survey found that participation would increase if combined testing, resulting in higher accuracy, was used. A total of 1,381 individuals were enrolled in the study with ColoVantage Plasma testing conducted just prior to colonoscopy, and FOBT completed within two weeks of colonoscopy (level III-1 diagnostic evidence). The results of colonoscopy were as follows: 4.8 per cent diagnosed with CRC, 13.7 per cent with high-risk adenoma, 18.8 per cent with low-risk adenoma and no evidence of disease in the remaining 62.7 per cent of participants. The FOBT was positive in 309 patients, detecting 78.8 per cent of cancers (52/66) and 42.3 per cent of high-risk adenomas (80/189). FOBT specificity in the cases with no evidence of disease on colonoscopy was 84 per cent. Of the cases found to be negative by FOBT, the ColoVantage Plasma test detected an additional seven cancers and eight high-risk adenomas. These additional cancers were more likely to have lymphovascular invasion than cancers positive for FOBT alone (50% vs 11%, p=0.04). The combined test sensitivity and specificity for CRC (for either test positive) was 89.4 and 77.8 per cent, respectively. Importantly, cases diagnosed with CRC by colonoscopy who were positive for both tests (34/61) had a relative risk of 23.0 for cancer when compared to those cases without a double positive result. The authors concluded that by combining FOBT and the ColoVantage Plasma test increased sensitivity over either test alone using an either or rule. In addition, cases with CRC who identified as positive by ColoVantage Plasma test alone appeared to be phenotypically different to FOBT positive cancers, which may have implications for therapy. 32 ColoGuard stool-based DNA test A systematic review of the Cologuard test in asymptomatic adults who underwent CRC screening was conducted in 2015, however the journal could not be accessed from any sources. The abstract reported that a total of one synthesis report and five diagnostic test studies were identified by the search strategy. Three of the five diagnostic studies were case-control studies of moderate quality, with the remaining two being prospective, high quality studies. The pooled results found the sensitivity for detecting CRC was greater than 90 per cent, but only 40 per cent for detecting advanced adenomas. The Cologuard test could reliably rule out CRC, which is important in a screening programme, with a negative likelihood ratio between 0.02 and 0.09, however it was poor ruling out advanced adenoma, Blood and stool biomarker testing for CRC screening: Update August

17 with a likelihood ratio between 0.5 and 0.7. There was no evidence in terms of mortality, survival or cost-effectiveness. 33 ms9 No new studies describing the ms9 assay were identified. mirnas Yan et al (2016) recently conducted a meta-analysis of studies that reported on the diagnostic value of mirnas for CRC compared to a gold standard. A total of 103 studies were included, with a total of 3,124 CRC patients compared to 2,579 healthy controls. The average numbers of participants in the CRC and control arms were 73 and 61, respectively; however sample size varied greatly with the smallest study have three participants in each arm, and the largest study comparing 200 cases to 80 controls. The majority of studies evaluated mirna levels in blood, serum or plasma, with a smaller number of studies evaluating levels in tissue (20.4%) or faeces (7.8%). The results of this meta-analysis are summarised in Table Table 5 Pooled results of diagnostic accuracy of mirnas for the diagnosis of CRC 14 Sensitivity [95% CI] Specificity [95% CI] AUC Regression analysis P(Sens) P(Spec) Ethnicity Asian Caucasian [0.768, 0.82] [0.554, 0.77] [0.791, 0.844] [0.701, 0.812] Sample size Small Large [0.761, 0.824] [0.691, 0.798] 0.77 [0.734, 0.802] [0.848, 0.786] mirna Single Multiple [0.669, 0.763] [0.822, 0.88] [0.74, 0.801] 0.86 [0.825, 0.889] <0.001 <0.001 Specimen Circulating Non-circulating [0.724, 0.811] [0.705, 0.801] [0.786, 0.843] 0.76 [0.719, 0.798] Specimen Plasma Serum Blood Tissue Faeces [0.623, 0.773] [0.767, 0.854] [0.788, 0.932] [0.749, 0.823] [0.550, 0.766] [0.737, 0.826] [0.855, 0.898] [0.606, 0.786] [0.703, 0.784] [0.709, 0.905] Overall [0.733, 0.802] [0.781, 0.829] AUC = area under the summary receiver operating characteristic (SROC) curve There was significant heterogeneity between the included studies, which was expected due to the inclusion of different populations and types of sample (circulating, tissue and faecal). The overall pooled sensitivity and specificity was and 0.806, respectively, with an area Blood and stool biomarker testing for CRC screening: Update August

18 under the curve (AUC) of indicating a relatively good diagnostic accuracy for the detection of CRC. Of interest is the sub group analysis, which revealed a higher diagnostic accuracy in the Asian population compared to Caucasian (AUC = vs 0.786). More importantly, the use of multiple RNAs resulted in a greater diagnostic accuracy compared to the use of single RNAs (AUC = vs 0.813, respectively). In addition, mirnas isolated from circulating samples, in particular serum, had the best diagnostic accuracy (AUC = 0.915). 14 Protein biomarkers A recent development is the detection of proteins associated with cancer using microarray and biochip technology. A Russian research group have immobilised an array of seven f CRCassociated glycans in addition to the human immunoglobulins IgA, IgM and IgG on hydrogel biochips, resulting in a CRC diagnostic signature. Simultaneous measurement of oncomarkers including CEA, CA125 and CA15-3 was conducted to strengthen the diagnostic signature. When a patient s serum is added to the biochip, antibodies directed against the CRC-associated glycans will bind, if present, to the immobilised glycans, with the degree of binding measured by immunofluorescence. Serum from 69 healthy donors, 33 patients with colorectal cancer and 27 patients with inflammatory bowel disease were analysed using the hydrogel biochip. Combining both diagnostic approaches resulted in CRC being diagnosed in 95 per cent of cases, with a sensitivity and specificity of 88 and 98 per cent for the detection of CRC (in patients with Stage II-IV CRC), respectively. 34 A similar approach was used by Villar-Va zquez et al (2016). A total of 16 candidate tumour associated proteins were identified by screening serum samples from CRC patients using microarrays. Of the 16 candidates, eight g were selected, in addition to p53 (a protein associated with many tumour types) and covalently immobilised onto magnetic beads that act as an array. As with the study above, antibodies if present in the patient s serum will bind to the immobilised proteins with the degree of binding measured by ELISA. A total of 307 serum samples from healthy controls (n=93), CRC patients (n=135, all stages), other types of cancer (n=65) and inflammatory bowel disease (n=14) were analysed, yielding a sensitivity and specificity of 66 and 90 per cent for the diagnosis of CRC, respectively. 35 Although in the initial stages of development, these methods, along with others, may be a promising tool in the future Economic evaluation The only new economic analysis identified for inclusion in this update was reported only in abstract form. This US cost-effectiveness study reported on simulations of CRC screening strategies in the Medicare beneficiary population. Modelling was performed comparing the f SiaTn, Tn, TF, LeC, LeY, SiaLeA, and Manβ1-4GlcNAcβ g AKT1, EDIL3, GTF2B, HCK, IRAK4, MAPKAPK3, PIM1, and STK4 Blood and stool biomarker testing for CRC screening: Update August

19 following screening strategies: 3-yearly screening using the Cologuard stool-based DNA test; 10-yearly colonoscopy; 5-yearly sigmoidoscopy; 10-yearly sigmoidoscopy combined with annual FOBT and annual FOBT (either ifobt or gfobt). Annual testing with the Cologuard was also considered using a sensitivity analysis. 36 In the absence of any CRC screening programme the model predicted that 24.9 (range ) out of 1,000 individuals aged >65 years in the Medicare population died from CRC at the undiscounted cost of US$4.4 million (range US$ million). Three yearly screening with Cologuard prevented 15.5 (range ) CRC deaths at an additional cost of US$410,000 (range US$ ,000), based on the reimbursement cost of the test of US$493. Three-yearly screening with the Cologuard test was more costly than all other screening strategies and less effective than all others with the exception of sigmoidoscopy screening (Figure 4). The sensitivity analysis revealed that 3-yearly screening with the Cologuard test would not be cost-effective at any test cost, however annual screening would be cost-effective if purchased/reimbursed at US$60 (range $56-66) per test. However, 3-yearly screening with the Cologuard test would become cost-effective given higher compliance rates than those gained with other screening modalities (31 to 53% higher) (Figure 5). 36 Figure 4 Costs and life years gained (LYG) of simulated screening strategies (FOBT = gfobt, FIT = ifobt, SIG = sigmoidoscopy, COL = colonoscopy, mtsdna = Cologuard test). Costs in US$ 36 Blood and stool biomarker testing for CRC screening: Update August

20 Figure 5 Threshold cost of 3-yearly or annual screening with the Cologuard test A new cost analysis was identified, however this analysis compared the performance of both ifobt and gfobt to a hypothetical new non-invasive test (with a similar specificity and sensitivity to ifobt), all of which were provided on an annual screening basis. Due to the hypothetical nature of this study, results are not included in this assessment Ongoing research A South Australian study, being conducted at Flinders Medical Centre (ACTRN ) commenced in 2011 and expects to enrol its last participant in late This large prospective cross-sectional study (n=4,000) aims to evaluate the accuracy of several candidate biomarkers for detection of screen-relevant colorectal neoplasia, relative to a proven immunochemical faecal occult blood test (ifobt, FIT) and the diagnostic test colonoscopy. The Netherlands Cancer Institute is currently recruiting patients (n=4,000) to participate in a cohort study that will compare the accuracy of the molecular stool test, Cologuard and FOBT to colonoscopy for detection of advanced adenomas or CRC in a surveillance population (NCT ). In addition the cost-effectiveness and the life time health effects of stool-based surveillance strategies will be evaluated. The study is expected t be completed by July A prospective, non-comparative longitudinal study is currently recruiting patients (n= 2,173) to assess the use of the Cologuard DNA stool collection test for repeat testing (3-years) in average risk patients (NCT ). Several studies were identified that were concerned with identifying factors to increase the uptake of FOBT (NCT , NCT , NCT ). Blood and stool biomarker testing for CRC screening: Update August

21 2016 Other issues Waiting lists and calprotectin testing As discussed in the original brief, a positive result after FOBT or a new and emerging CRC screening test will require clinical management, usually in the form of a colonoscopy. Given that the screening population is healthy, a false positive test result may lead to unnecessary colonoscopies and biopsies. Therefore an issue that should be considered is the number of individuals on the waiting list for colonoscopy following a positive FOBT, and the impact this waiting time may have on patients. Although studies have indicated that there is no evidence to support a link between prolonged colonoscopy waiting time and stage of carcinoma at diagnosis, wait time will likely have a considerable psychological impact on patients and their relatives. 38, 39 Overall data on colonoscopy wait times are difficult to quantify as patients may opt to have their colonoscopy in the public or private system. All jurisdictions have strategies in place to reduce the time between referral from the general practitioner (GP) to public hospital outpatient appointment, and from outpatient appointment to colonoscopy procedure. Generally it is recommended that urgent cases (Category 1) are seen by a specialist within 30 days of referral, with semi-urgent cases (Category 2) being seen within 90 days and nonurgent cases (Category 3) within 365 days. 40 A recent retrospective review of colonoscopy wait times in the public hospital system for patients participating in the NBCSP was conducted in South Australia. A total of 433 patients, representing approximately 65 per cent of those who tested positive by screening as part of the NBCSP between January 2006 and December 2009 in South Australia, underwent a colonoscopy. The average wait time between GP consultation and hospital appointment was 31 days, with an average wait of 52 days between GP consult and colonoscopy. The majority of patients had their colonoscopy within a day timeframe (146/333, 44%) and only 23 per cent had their colonoscopy within the 30-day benchmark (Table 6). It should be noted that these figures are likely to vary greatly depending on the hospital and region. 4 Table 6 Clinic and colonoscopy wait times for South Australian NBCSP participants Number Waiting time mean + SD From initial GP referral to hospital appointment days (+ 27 days) From hospital appointment to colonoscopy days ( days) From initial GP referral to colonoscopy days ( days) In addition to suspected symptoms of CRC, colonoscopy is indicated for a number of gastrointestinal symptoms including inflammatory bowel diseases (IBD), such as Crohn s Blood and stool biomarker testing for CRC screening: Update August

22 disease and ulcerative colitis, and non-inflammatory bowel diseases, such as irritable bowel syndrome (IBS), both of which share many similar symptoms including chronic abdominal 41, 42 pain or discomfort, bloating, excessive flatus or a change in bowel habit. Faecal calprotectin is a marker for intestinal inflammation, with concentrations correlating with the severity of inflammation. Levels of calprotectin are elevated in patients with IBD but not in patients with IBS, and as such a calprotectin test may be used as a first line screen or triage to differentiate between organic IBD and functional disorders such as IBS. 43 Calprotectin has a high negative predictive value, therefore patients with a negative result would be reassured that bowel inflammation could be excluded, thus avoiding the need for an invasive colonoscopy. 41 The identification of low risk patients may reduce the number of unnecessary invasive colonoscopies being performed, and in so doing, reduce colonoscopy waiting lists for those in need. In addition, calprotectin could identify those with a high likelihood of inflammatory bowel disease who would require a colonoscopy. 43 The metaanalysis by van Rheenen et al (2010) reported that screening with faecal calprotectin would result in a 67 per cent reduction in the number of adults requiring colonoscopy, however false negatives would result in six per cent of adults receiving a delayed diagnosis. Similarly, three of 33 adults would undergo colonoscopy despite not having IBD but may have a different condition for which colonoscopy is required. When the population of children and teenagers were considered, 65 instead of 100 would undergo colonoscopy, with nine of them not having IBD, and eight per cent experiencing a delayed diagnosis. 43 In 2013 the UK s National Institute for Health and Care Excellence issued a guidance recommending calprotectin testing as an option to help doctors distinguish between IBD and IBS. 44 In addition, the MSAC have been considering the protocol of an application, sponsored by Taylor Bio-Medical Pty Ltd, for the public subsidy of the quantitative measurement of calprotectin in human stool, to distinguish IBD from IBS. 42 This application could not provide data on the number of potential colonoscopies avoided if calprotectin was used as a screening tool, however it reported a high rate of colonoscopies being performed for chronic diarrhoea and IBS symptoms, with per cent of colonoscopies are performed for inappropriate indications. 42 The use of calprotectin testing may reduce the time patients spent on the colonoscopy waiting list. Calprotectin testing is currently available in Australia on a user-pays basis and cost approximately $ Participation rates Participation in the NBCSP has been an issue since its inception. The AIHW reported that approximately 36 per cent of the 1.4 million individuals invited to participate in the programme during the period from July 2013 to June 2014 returned a completed bowel cancer screening kit for analysis. Participation rates were higher for women than men and increased with age (Figure 6), and although 66 per cent of all participants resided in major cities, participation rates were higher in inner regional (38.8%) and outer regional (37.5%) areas (Figure 7). In addition, invitees from the lowest socioeconomic areas (the areas with Blood and stool biomarker testing for CRC screening: Update August

23 the most disadvantage) had the lowest participation rates than those living in all other socioeconomic areas (Figure 8). 46 Figure 6 Crude participation rates in the NBSCP, by age and sex, Figure 7 Crude participation rates in the NBCSP, by remoteness area, Figure 8 Crude participation rates in the NBCSP, by socioeconomic status area, Blood and stool biomarker testing for CRC screening: Update August