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Coelho et al. BMC Cancer (2017) 17:836 DOI 10.1186/s12885-017-3820-5 RESEARCH ARTICLE Open Access A systematic review of test accuracy studies evaluating molecular micro-satellite instability testing for the detection of individuals with lynch syndrome Helen Coelho1* , Tracey Jones-Hughes1, Tristan Snowsill1, Simon Briscoe1, Nicola Huxley1, Ian M. Frayling2 and Chris Hyde1 Abstract Background: A systematic review was conducted to assess the diagnostic test accuracy of polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing for identifying Lynch syndrome in patients with colorectal cancer (CRC). Unlike previous reviews, this was based on assessing MSI testing against best practice for the reference standard, and included CRC populations that were unselected, age-limited or high-risk for Lynch syndrome. Methods: Single- and two-gate diagnostic test accuracy studies, or similar, were identified, assessed for inclusion, data extracted and quality appraised by two reviewers according to a pre-specified protocol. Sensitivity of MSI testing was estimated for all included studies. Specificity, likelihood ratios and predictive values were estimated for studies that were not based on high-risk samples. Narrative synthesis was conducted. Results: Nine study samples were included. When MSI-Low results were considered to be negative, sensitivity estimates ranged from 67% (95% CI 47, 83) to 100% (95% CI 94, 100). Three studies contributed to estimates of both sensitivity and specificity, with specificity ranging from 61% (95% CI 57, 65), to 93% (95% CI 89, 95). Good sensitivity was achieved at the expense of specificity. When MSI-L was considered to be positive (effectively lowering the threshold for a positive index test result) sensitivity increased and specificity decreased. Between-study heterogeneity in both the MSI and reference standard testing, combined with the low number of studies contributing to both sensitivity and specificity estimates, precluded pooling by meta-analysis. Conclusions: MSI testing is an effective screening test for Lynch syndrome. However, there is significant uncertainty surrounding what balance of sensitivity and specificity will be achieved in clinical practice and how this relates to specific characteristics of the test (such as the panel of markers used or the thresholds used to denote a positive test). Keywords: Lynch syndrome, HNPCC, Microsatellite instability, Diagnostic testing, Test accuracy, Systematic review Background Lynch syndrome is caused by heritable constitutional pathogenic mutations in the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) or, rarely, by certain mutations in nearby genes that affect expression of the adjacent MMR gene, (i.e. EPCAM and MSH2, and * Correspondence: h.coelho@exeter.ac.uk 1 Peninsula Technology Assessment Group (PenTAG), University of Exeter Medical School, South Cloisters, St Lukes Campus, Heavitree Road, Exeter, Devon EX1 2LU, UK Full list of author information is available at the end of the article LRRFIP2 and MLH1), due to epigenetic silencing caused by promoter methylation [1, 2]. It is responsible for around 2.8% of colorectal cancer (CRC), [3] conveys a high risk of colorectal and endometrial cancer, and increases the risk of other cancers, such as ovarian and gastric cancer [4, 5]. In people with Lynch syndrome, CRC has an earlier onset than CRC in the general population (44 years, compared with 60–65 years respectively) [5, 6]. Currently, the best method for diagnosing Lynch syndrome is comprehensive screening for constitutional mutations in the MMR genes and EPCAM, using a combination of (i) DNA sequencing © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Coelho et al. BMC Cancer (2017) 17:836 in order to detect point mutations and small insertions and deletions, and (ii) multiplex ligation-dependent probe amplification (MLPA) to detect large structural DNA abnormalities [7]. Patients with CRC can be selected for comprehensive screening for constitutional mutations by first applying other diagnostic tests. Due to the fact that there is a high probability of loss of MMR function in Lynch syndrome cancers, and that tumours which have lost MMR function display microsatellite instability, one such test is microsatellite instability (MSI) testing. This involves polymerase chain reaction (PCR) amplification of DNA markers (using tumour tissue and healthy tissue). The two samples are compared to assess whether abnormal patterns of microsatellite repeats are observed in the tumour DNA. Mono- and dinucleotide markers are the most frequently used with the Bethesda/NCI markers (BAT-25, BAT-26, D2S123, D5S346, D17S250) often being used [8]. However, other markers are in use (e.g. BAT-40, MYCL, MONO-27, NR-21, NR-24), and it has been argued that the panel should contain at least three mononucleotide markers [9], and thus individual laboratories may develop their own panels [10]. Microsatellite instability is categorised trimodally (MSI-High, MSI-Low, or MS-Stable) or bimodally (MSI-positive or negative), according to the proportion of markers demonstrating MSI. When a trimodal categorisation is initially used, a decision must then be taken as to whether MSI-Low (MSI-L) will then be further categorised as a positive or negative test result. A previous Health Technology Assessment in England and Wales evaluated the diagnostic test accuracy of MSI for Lynch syndrome in early-onset (aged under 50 years) CRC patients [7]. However, most of the included studies were at risk of bias because the reference standard was not conducted on all participants. Additionally, because this previous review did not include unselected CRC samples, the results may have been subject to spectrum effects and not generalisable to all CRC patients. Furthermore, this previous review [7], and others before it [4, 9, 11], have been obliged to include a wide range of techniques as their reference standard rather than focusing on the primary standard of comprehensive screening for constitutional mutations using a DNA sequencing method combined with MLPA or another appropriate technique to detect large structural DNA abnormalities. This systematic review was, therefore, conducted to address the need for clearer information regarding the diagnostic test accuracy of PCR-based MSI testing (with or without BRAF V600E mutation testing and with or without MLH1 methylation testing) for identifying Lynch syndrome in patients in the general CRC population. The review was conducted as part of a Diagnostics Assessment Report (DAR) which was commissioned by the National Institute for Health Research (NIHR) Health Technology Page 2 of 13 Assessment Programme in England and Wales to support the National Institute for Health and Care Excellence (NICE) Diagnostics Assessment of molecular testing for Lynch syndrome in people with colorectal cancer [https:// www.nice.org.uk/guidance/dg27] [12]. Methods The systematic review was undertaken in accordance with a predefined protocol. The protocol for the review (and other reviews in the DAR) can be found at http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42016033879. This review departs from the diagnostic test accuracy review described in the original protocol in that it focuses only on PCRbased MSI testing as the index test, whereas the full review also included immunohistochemistry (IHC) as an index test. However, no studies were found that directly compared MSI testing with IHC and the two index tests were, therefore, reviewed in parallel but evaluated separately. Searches The following bibliographic databases were searched using population terms for Lynch syndrome and intervention terms for MSI or IHC: the Cochrane Database of Systematic Reviews, CENTRAL and HTA [all via the Cochrane library]; MEDLINE, MEDLINE In-Process & Other NonIndexed Citations, Embase and the Health Management Information Consortium [all via Ovid]; Web of Science [including conference proceedings, via Thomson Reuters]. Search results were limited by date from 2006 to current and to English language publications. The full search strategies are available from the authors. Four key systematic reviews [4, 7, 9, 11] (and other systematic reviews identified by the bibliographic database searches) were screened in order to source further relevant studies published before 2006 and additional studies published after 2006. These four key systematic reviews [4, 7, 9, 11] were examined prior to the start of this review and were judged to have sufficiently robust searching methods to identify relevant studies published before 2006. Studies which cited the included studies were identified using Scopus (Elsevier). The reference lists of all included studies were screened in order to identify any additional relevant studies. Study selection Titles and abstracts of the studies retrieved from the searches were screened, independently by two reviewers, according to the predefined inclusion criteria specified below. Disagreements between reviewers were resolved by discussion, with arbitration from a third reviewer where necessary. Full texts of included titles/abstracts (from bibliographic database searches, and forward and Coelho et al. BMC Cancer (2017) 17:836 backward citation chasing), and full texts of studies identified from systematic reviews, were obtained. These were screened in the same way as titles and abstracts. Inclusion criteria Studies had to be single-gate (also known as diagnostic cohort studies) or two-gate (also known as diagnostic case–control studies) diagnostic test accuracy studies (or a variation of one of these designs) [13]. They had to recruit individuals with colorectal cancer and investigate the diagnostic test accuracy of molecular MSI testing (with or without BRAF V600E mutation testing and with or without MLH1 methylation testing). MSI must have been compared with the reference standard, which was constitutional MMR mutation testing (including DNA sequencing of MLH1, MSH2 and MSH6 and MLPA or another appropriate technique for detecting large genomic abnormalities as a minimum), by providing sufficient data for at least sensitivity to be estimated. Other outcomes (in addition to sensitivity) were: specificity, likelihood ratios (LR+ and LR-), predictive values (PPV and NPV), concordance (with the reference standard), diagnostic yield, and test failure rates. To be included in the review, studies must have been designed for all participants to receive both the index test and reference standard. However, studies recruiting a representative sample of all patients with CRC (including where an age limit was applied), the reference standard may have been applied to all MSI positive-tumours and to a representative (e.g., random) sample of MSI negative-tumours. Abstracts were included if they reported data from an included study that was published in full. Data extraction and quality appraisal (risk of bias assessment) All included studies were given a study identification label: first-author date. Where needed for clarity, included studies are identified by their study identification label in the results and discussion sections below. Data extraction and quality appraisal were conducted, for all included studies, by one reviewer and checked by another. Discrepancies were resolved by discussion with the involvement of a third reviewer where necessary. Extracted data included details of the study’s design and methods, participants’ characteristics and study results. Risk of bias in individual studies was assessed according to criteria in Phase 3 of the QUADAS-2 tool [14]. Analysis and synthesis The data extracted from the included studies was analysed in STATA 13 (StataCorp LP) using the “diagt” command [15]. For single-gate studies that were not based on high-risk samples (including age-limited population studies), and where data permitted, sensitivity, Page 3 of 13 specificity, LR+, LR-, PPV and NPV, diagnostic yield and concordance with the reference standard (with 95% confidence intervals [CIs]) were calculated. However, for the studies based on high-risk samples, sensitivity (with 95% CI) was calculated (spectrum effects that occur when using a high-risk sample have not been found to lead to significant bias in sensitivity estimates for MSI) [9]. Although not considered an outcome, for illustrative purposes, disease prevalence was also calculated for all included studies, based upon data extracted to 2X2 tables, and representing, therefore, the prevalence in the analysed samples rather than the recruited population. Where extracted data resulted in zero counts in one or more cells, one-sided 95% CIs were calculated. In primary analyses, MSI-Low was considered as a negative index test result. Unclassified variants (mutations of unknown clinical significance with regards Lynch syndrome), where reported, were considered to be negative reference standard results. The main method of synthesis was narrative. Results Ten studies were included in the HTA upon which this review is based (Fig. 1). However, in two of these studies, MSI was not assessed. Therefore, eight studies (Barnetson 2006, Southey 2005, Poynter 2008, Caldes 2004, Mueller 2009, Overbeek 2007, Shia 2005 and Hendriks 2003) [16–23] were eligible for inclusion in this review of MSI testing. It should be noted that Poynter 2008 [21] had two distinct samples (a population-based sample and a highrisk sample) and, therefore, had two distinct sets of includable data. These two samples were treated separately and both included in this review. Study and participant characteristics Of the nine study samples included in this review, three report data from a population-based sample, although only Poynter 2008 recruited an apparently unselected CRC population [21]. The other two studies restricted the population by applying a maximum limit to age of diagnosis: Barnetson 2006 applied an age limit of <55 years and Southey 2005 applied a limit of <45 years [16, 23]. All three of these studies used single-gate designs but varied in size with Barnetson 2006 and Poynter 2008 recruiting 1259 participants and 1061 participants respectively but Southey 2005 recruiting only 131 participants [16, 21, 23]. Disease prevalence in the analysed study samples is provided in Table 1 and was similar in Barnetson 2006 and Poynter 2008 and higher in Southey 2005 [16, 21, 23]. The remaining six studies selected participants with CRC who were at high-risk for Lynch syndrome (Caldes 2004, Hendriks 2003, Mueller 2009, Overbeek 2007, Shia 2005 and the other sample in Poynter 2008) [17–22]. Five of these studies had a single-gate design [17, 19–22]. The Coelho et al. BMC Cancer (2017) 17:836 Page 4 of 13 Fig. 1 Flow-chart detailing selection of studies. 1Abstracts were excluded when they not linked to an included study and did not provide sufficient methodological information to meet the review inclusion criteria or have data extracted. 2These studies evaluated IHS and not MSI. 3One of these studies included two distinct populations, both of which are included in this review. Although there are eight included studies, there are, therefore, nine included datasets remaining study (Hendriks 2003) was a variation on a two-gate study design; although participants with positive reference standard results were recruited, no reference standard negatives were included [18]. We referred to this as a reference standard positive study design. The six high-risk studies varied in size; the largest study was Poynter 2008 with 172 participants and the smallest study was Hendriks 2003 with 45 participants [18, 21]. Further details on study and participant characteristics, including disease prevalence in the analysed samples, are given in Table 1. There was a great deal of between-study variation in both the reference standard and in the MSI testing methods as well as in the reporting of methods. For example, in the studies by Poynter 2008, Mueller 2009, and Overbeek 2007 microdissection techniques were not reported [19–21]. In addition, none of the population-based studies assessed the same panel of markers, with differences existing in both the number and type of markers, see Table 1. Five studies (Barnetson 2006, Southey 2005, Poynter 2008, Mueller 2009, Hendriks 2003) defined tumours tri-modally (i.e. as MSI-H, MSI-L or MSS) [16, 18, 19, 21, 23], two (Overbeek 2007; Participant selection and disease prevalence a Poynter, 2008b Recruitment through population-based cancer registries (population-based sample), selection process unclearc Disease prevalence 9.2% (95% CI 7.1 to 11.8) Barnetson, 2006 Diagnosed <55yrs of age, consecutive recruitment Disease prevalence 8.5% (95% CI 5.8 to 11.9) Population-based and age-limited single-gate studies Study Table 1 Study and population characteristics 870 352 Receiving RS Receiving MSI 46.9% Female 42.7 (±7.7) 38.5 (±8.4) 43.8 (±6.1) 49.0 (±3.9) Carrier MLH1 MSH2 MSH6 64.0% RBG 726 1061 Receiving RS Receiving MSI Clinical criteria: NCR Age (in years): NCR Gender: NCR 1061 Recruited Number: 4.0% AMS II Clinical criteria: 48.2 (±6.0) Noncarrier Age (in years): 53.1% Male Gender: 1259 Recruited Number: Participant characteristics 10μm tumour sections; microdissection performed on purified tumour DNA, and control DNA from blood or normal tissue in the section Microdissection For all MSI-H or MSI-L probands and in NR a random sample of 300 MSS population-based probands: Mutations in MSH2 and MLH1 were detected using a combined approach of dHPLC/ direct sequencing and MLPA. Direct sequencing was used to detect MSH6 mutations in cases with absent IHC staining of MSH6. For all participants: Germ-line DNA obtained from blood leukocytes analysed for MLH1, MSH2, and MSH6 mutations. dHPLC analysis was used for MSH2 and MLH1. Variants noted on chromatography were then sequenced. Mutations were confirmed by reamplification of an independent sample of DNA and resequencing in both directions. MLH1 and MSH2 were assessed for deletions by MLPA, with products separated on a genetic analyser. Reference standard BAT25, BAT26, D5S346, D17S250, BAT40, MYCL, ACTC, Dl 8S55, D1OS197, BAT34C4 Bethesda/NCI panel MSI Panel MSI-H: ≥30% MSI-L: >0% and <30% MSS: 0% MSI-H: >1 marker MSI-L: 1 marker MSS: 0 markers MSI thresholds Coelho et al. BMC Cancer (2017) 17:836 Page 5 of 13 Diagnosed <45yrs of age, random recruitment Disease prevalence 30.5% (95% CI 19.2 to 43.9) Participant selection and disease prevalence a HNPCC families selected through a clinic for familial cancer, selection process unclear Disease prevalence 58.6% (95% CI 44.9 to 71.4) ‘Suspected Lynch syndrome’ participants who met Amsterdam criteria, modified Amsterdam criteria, were ‘HNPCC-like’ or met Bethesda criteria, selection process unclear Disease prevalence 58.3% (95% CI 43.2 to 72.4) Caldes, 2004 Mueller, 2009 High-risk, single-gate studies Southey, 2005 Study 105 Receiving MSI 37.3% Female RGB 58 58 Receiving RS Receiving MSI 48 48 Receiving RS Receiving MSI Gender: NR 48 Recruited Number: Clinical criteria: NR Age (in years): NR Gender: NR 58 Recruited Number: 9.2% NR AMS II Clinical criteria: 37.1 (range 24 to 42) Age (in years): 62.7% Male Gender: 59 Receiving RS For all participants: Sequencing and MLPA. Limited details provided. For all participants: Genomic DNA was isolated from peripheral blood lymphocytes was analysed for MLH1, MSH2 and MSH6. DNA was amplified using PCR and all amplicons were subjected to DGGE or cycle sequencing. The MSI-H cases that were negative for mutations were analysed for genomic deletions in MLH1 and MSH2 by Southern Blotting. For all MSI-H or MSI-L probands, those that lacked expression of at least one MMR protein and a random sample of 23 patients selected from those who had tumours that were MS stable and did not lack expression of any MMR protein: MLH1, MSH2, MSH6, and PMS2 genes were screened for germline mutations using sequencing approaches or dHPLC. Confirmation of putative mutations was sought using an independent polymerase chain reaction for direct automated sequencing. MLPA was used to detect large genomic alterations in MLH1 and MSH2 on samples from 10 patients who had tumours lacking at least one MMR protein expression and for which no previous mutation had been identified by sequencing. 131 Number: Recruited Reference standard Participant characteristics Table 1 Study and population characteristics (Continued) NR 5 and 10 panel markers, no further details provided Bethesda/NCI panel NR MSI-H: >1 marker, or 1 marker if BAT26 MSI-L: Not used MSS: 0 markers MSI-H: >5 markers MSI-L: 2-5 markers MSS: <2 markers BAT25, BAT26, D2S123, D5S346, D17S250, BAT40, MYB, TGFRII, IGFIIR, BAX 5μm tumour sections; microdissection performed on invasive tumour cells from paraffin-embedded archival tumour tissue stained with 1% methylgreen, and normal cells from colonic or lymph node tissue/DNA extracted from peripheral blood lymphocytes 10μm tumour sections; microdissection performed on H&E stained slides with demarked areas containing cancer cells, and corresponding areas on unmarked slides MSI thresholds MSI Panel Microdissection Coelho et al. BMC Cancer (2017) 17:836 Page 6 of 13 Families history that fulfilled one of the following criteria: 1) Amsterdam II criteria 2) Bethesda guidelines 3) a history very close to the Bethesda guidelines, selection process unclear Disease prevalence NC Recruitment through high-risk clinics (clinic-based sample), selection process unclearc Disease prevalence 30.9% (95% CI 23.7 to 38.9) Family history that fulfilled one of the following criteria: I ) Amsterdam I or II criteria 2) a set of relaxed AC three or more colorectal cancers among the first and second-degree relatives of a family that we referred to as ''HNPCC-like,'' and 3) Bethesda criteria, selection process unclear Disease prevalence 49.2 (95% CI 36.1 to 62.3) Poynter, 2008b Shia, 2005 Participant selection and disease prevalence a Overbeek, 2007 Study NR Receiving RS Receiving MSI 172 Receiving RS Receiving MSI 83 Unclear Receiving RS Receiving MSI d 83 Recruited Number: Clinical criteria: NR Age (in years): NR Gender: NR 172 152 Recruited Number: Clinical criteria: NR Age (in years): 40.7 (range 29 to 51) Gender: NR NR NR Recruited Number: Clinical criteria: NR Age (in years): NR Participant characteristics Table 1 Study and population characteristics (Continued) Microdissection NR For all participants: Each of the exons Microdissection performed on DNA of MLH1, MSH2 and MSH6 was from paraffin-embedded tissue blocks. amplified by PCR, and heteroduplex No further details reported analyses were performed using dHPLC. DNA fragments that displayed an abnormal chromatogram were sequenced directly. Cases with tumours that exhibited MSI but in which a point mutation was not For all participants: Mutations in MSH2 and MLH1 were detected using a combined approach of dHPLC/direct sequencing and MLPA. Direct sequencing was used to detect MSH6 mutations in cases with absent IHC staining of MSH6. For all participants: Mutation analysis of NR MLH1, PMS2, MSH2, and MSH6 was performed in DNA from peripheral blood lymphocytes by a combination of either single-strand conformation polymorphism analysis or DGGE and direct sequence analysis. For the detection of large deletions and duplications in MLH1, MSH2, MSH6, and PMS2, MLPA was used. All deletions and duplications were confirmed by Southern blot analysis or with a specific PCR. Reference standard BAT25, BAT26, D2S123, D17S250, BAT40, PAX6, MYCL1 BAT25, BAT26, D5S346, D17S250, BAT40, MYCL, ACTC, Dl 8S55, D1OS197, BAT34C4 BAT25, BAT26, D2S123, D5S346, D17S250 (BAT40 was also added to the standard set of markers but it is unclear for which participants) MSI Panel Tumours categorised as positive (≥30%) or negative MSI-H: ≥30% MSI-L: >0% and <30% MSS: 0% Tumours categorised as positive (>2 Bethesda markers) or negative MSI thresholds Coelho et al. BMC Cancer (2017) 17:836 Page 7 of 13 8.2% Participant selection and disease prevalence a Germline mutation in MLH1, MSH2 or MSH6, selection process unclear Disease prevalence 84.8% (95% CI 68.1 to 94.9)e 45 33 Receiving RS Receiving MSI 40.0% Female Clinical criteria: NR MSH6 62 (range 26 to 84) MSH2 40 (range 23 to 61) MLH1 48 (range 29 to 90) Age (in years): 35.6% Male Gender: 45 38.2% Recruited Number: AMS II Clinical criteria: 50 (range 23 to 78) Age (in years): Female 43.6% 56.3% For all participants: DGGE or Southern blotting. Limited details provided detected were analysed for large deletions in MLH1 and MSH2 using a procedure based on the multiplex PCR of short fluorescent fragments. Gender: Male Reference standard Participant characteristics Microdissection not specifically reported, paired tumour and normal tissue DNA samples were used Microdissection MSI thresholds MSI-H: >1 BAT25, BAT26, D2S123, Bethesda D5S346, D17S250, BAT40, MSH3 and MSH6 markers MSI-L: 1 Bethesda marker MSS: 0 Bethesda markers MSI Panel NC not calculable, total number of participants with CRC not reported, NR Not reported, NCR Not clearly reported; aDisease prevalence calculations were based on participants who received both the index test and the reference standard and for whom data were reported. The disease prevalence may, therefore not be an accurate representation of the prevalence in the recruited population, and this is more likely in studies that did not aim to assess the reference standard in all recruited participants; for example, two study samples (the population based sample in Poynter, 2008, and Southey, 2005) did not perform the reference standard in all participants with an MSS result. bPoynter (2008) reports data from two distinct samples, a population-based sample and a high-risk sample; cAlthough Poynter (2008) reports that ‘some centres recruited all incident cases of CRC while others over sampled cases with a family history or early age of onset’ it is not clear whether this applies to the high-risk sample alone or in part to the high-risk sample and in part to the population based sample; d MSI data are available for 61 participants but it is unclear how many received the test; eAlthough only reference standard positives were recruited, this included those with an unclassified variant, so when those unclassified variants were considered to be reference standard negatives disease prevalence is 84.8% Hendriks, 2003 Reference standard positive study RBG Study Table 1 Study and population characteristics (Continued) Coelho et al. BMC Cancer (2017) 17:836 Page 8 of 13 Coelho et al. BMC Cancer (2017) 17:836 Shia 2005) defined tumours bi-modally (MSI positive or negative) [20, 22], and Caldes 2004 used a bimodal categorisation but defined tumours as either MSI-H or MSS [17]. The thresholds used to categorise the MSI status of tumours also varied across studies (Table 1), with some studies using positivity of only 20% of markers as the cut-off between MSI-H and MSI-L (Barnetson 2006) [16], and others requiring 50% (Southey 2005) [23], although different numbers of markers were also used in these two studies. It should also be mentioned that two of the populationbased or age-limited studies (Poynter 2008, Barnetson 2006) and three of the high-risk studies (Caldes 2004, Shia 2005, Hendriks 2003) report on unclassified variants (i.e. mutations where the association with Lynch syndrome is unclear) [16–18, 21, 22]. Notably, all of the nine studies included in this report predate what is now considered to be the definitive interpretation of MMR gene variants [24]. In this review, therefore, unclassified variants have primarily been considered to be reference standard negative results. Risk of bias in individual studies None of the included studies displayed any evidence to suggest that they were at high-risk of bias (Table 2). It should be noted that an absence of evidence to suggest that the included studies were at high risk of bias does not mean that the studies were free from bias. In fact, none of the studies adequately reported whether MSI was interpreted without knowledge of the reference standard results, or whether the thresholds used to denote a positive MSI result were pre-specified, so it was unclear whether the conduct of the MSI test would have introduced bias. Likewise, for all of the included studies, it was not clear whether the flow and timing of the study would have introduced bias. In all studies except Hendriks 2003 it was unclear whether the conduct of the reference standard would have introduced bias [18]. Additionally, only Barnetson 2006 and Southey 2005 reported sufficient information to determine that participant selection was unlikely to have introduced bias [16, 23]. Sensitivity and specificity Table 3 gives sensitivity and specificity estimates from primary analyses, where unclassified variants were considered to be index test negative results and MSI-L was considered to be an index test negative result (for studies using a tri-modal distribution of MSI). Only three studies contributed estimates of both sensitivity and specificity (Barnetson 2006, Southey 2005, Poynter 2008) [16, 21, 23]. These suggested, in one study (Poynter 2008), that good sensitivity (100.0%, 95% CI 93.9100.0) could be achieved at the expense of specificity (61.1%, 95% CI 57.0, 65.1) [21]. Conversely a second study (Barnetson 2006) achieved good specificity (92.5%, 95% CI 89.1, 95.2) but at the expense of sensitivity (66.7%, 95% CI 47.2, 82.7) [16]. The Page 9 of 13 third study (Southey 2005) had intermediate values of sensitivity (72.2%, 95% CI 46.5, 90.3) and specificity (87.8%, 95% CI 73.8, 95.9) [23]. Although this pattern would be consistent with a threshold effect, it is difficult to establish this because, in addition to using different numbers of unstable markers to denote a positive MSI result, the panel of markers differed between studies (Table 1). The range of sensitivities in the single gate, high risk samples (Caldes 2004, Mueller 2009, Overbeek 2007, Shia 2005 and the other sample in Poynter 2008) [17, 19–22] and the reference standard positive study (Hendriks 2003) [18] all fell within the range of sensitivities identified in the three single-gate population-based sample studies (Barnetson 2006, Poynter 2008, Southey 2005) [16, 21, 23]. In secondary analyses, when MSI-L was considered to be a positive index test result, sensitivity increased (for the six study samples where a tri-modal distribution of MSI was used) and specificity decreased (for the three population-based samples). This is unsurprising; including MSI-L as an index test positive essentially decreases the threshold for a positive test result. Fig. 2 illustrates this effect for the three population based studies. Further analyses were also conducted where unclassified variants were considered to be positive reference standard results. Only Caldes 2004 and Hendriks 2003 provided sufficient data for these analyses [17, 18]. Both of these studies were based on high-risk populations, so only sensitivity estimates were made, and these were largely unchanged from the primary analyses, most likely because of the low absolute numbers of unclassified variants involved. Pooling of sensitivity and specificity data was considered but rejected, primarily because of the marked betweenstudy methodological and clinical heterogeneity (e.g. differences in sequencing methods, which genes were tested, in techniques used to test for large genomic deletions and alterations, whether unclassified variants were tested and in the number and nature of the microsatellite makers assessed). Further, as only three studies provided both sensitivity and specificity estimates, this precluded the application of potentially useful test accuracy meta-analytic models such as the hierarchical summary ROC model [25]. Although nine study samples provided estimates of sensitivity, pooling of sensitivity alone is not recommended because of the interdependence with specificity [25–27]. Secondary outcomes For the population-based sample in Poynter 2008 and for the other two studies that recruited population-based samples (Barnetson 2006 and Southey 2005) [16, 21, 23], LR+, LR−, PPV, NPV, diagnostic yield and concordance with the reference standard were also calculated. These were primarily estimated based on MSI-L being an index test negative result and these results are given in Table 4. None of the studies reported test failure rates. Coelho et al. BMC Cancer (2017) 17:836 Page 10 of 13 Table 2 Risk of bias assessment using Phase 3 of the QUADAS-2 tool Domain Item Population-based, single-gate High-risk,single-gate Barnetson 2006 [16] Southey 2005 [23] Poynter 2008a [21] Caldes 2004 [17] Mueller 2009 [19] Overbeek 2007 [20] Poynter 2008 a [21] Shia 2005 [22] Hendriks 2003 [18] Y Y U U U U U U U Was a case-control design avoided? Y Y Y Y Y Y Y Y Nb Did the study avoid inappropriate exclusions? Y Y U Y U U U U Y Could the selection of patients have introduced bias? L L U U U U U U Uc Is there concern that the included patients do not match the review question? L L L L L L L L L Were the index test results U interpreted without knowledge of the results of the reference standard? U U U U U U U U If a threshold was used, was it prespecified? U U U U U U U U U Could the conduct or interpretation of the index test have introduced bias? U U U U U U U U U Is there concern that the index test, its conduct, or interpretation differ from the review question? L L L L L L L L L Is the reference standard likely to Y correctly classify the target condition? Y Y Y Y Y Y Y Y U Were the reference standard results interpreted without knowledge of the results of the index test? U U U U U U U Y Could the reference standard, its conduct, or its interpretation have introduced bias? U U U U U U U U L L Is there concern that the target condition as defined by the reference standard does not match the review question? L L L L L L L L Was there an appropriate interval between index test(s) and reference standard? U U U U U U U U U Did all patients receive a reference standard? Y N N Y Y Y Y Y Y Did patients receive the same reference standard? Y N N N U U N N U Were all patients included in the analysis? N N N N Y U N U N Could the patient flow have introduced bias? U U U U U U U U U Patient selection Was a consecutive or random sample of patients enrolled? Index test Reference standard Flow and timing Other Notes: aPoynter was assessed twice because data were reported for both a population-based sample and a high-risk sample; bA case-control design was only avoided because there was no control group (half a case control study); cAn unbiased estimate of sensitivity (but not specificity) can be ascertained from this study design, however an unclear rating is given because it is not clear if a consecutive or random sample was recruited Key: L = low, N = no, U = unclear, Y = yes Discussion This systematic review was conducted by an independent, experienced research team working to a prespecified protocol. It is notable that, of the nine study samples included in this review, only one (Poynter 2008) appeared to recruit an unselected CRC population, although even in this study it was not clear how the participants were selected [21]. This paucity of large, unselected, population-based studies is unsurprising; it is costly to provide all participants with both the index test