Diagnostic – PLOS Currents Evidence on Genomic Tests http://currents.plos.org/genomictests Tue, 21 Aug 2018 20:46:31 +0000 en-US hourly 1 https://wordpress.org/?v=4.5.3 CYLD GeneticTesting for Brooke-Spiegler Syndrome, Familial Cylindromatosis and Multiple Familial Trichoepitheliomas http://currents.plos.org/genomictests/article/cyld-genetic-testing-for-brooke-spiegler-syndrome-familial-cylindromatosis-and-multiple-familial-trichoepitheliomas/ http://currents.plos.org/genomictests/article/cyld-genetic-testing-for-brooke-spiegler-syndrome-familial-cylindromatosis-and-multiple-familial-trichoepitheliomas/#respond Thu, 19 Feb 2015 15:15:49 +0000 http://currents.plos.org/genomictests/?post_type=article&p=22519 CYLD (OMIM 605018). Brooke-Spiegler Syndrome (BSS), familial cylindromatosis (FC) and multiple familial trichoepitheliomas (MFT) (OMIM #605041, #132700, #601606 respectively) differ due to the types of other skin appendage tumour seen together with cylindroma, such as spiradenoma and trichoepithelioma. Previously thought to be separate entities, they are now viewed as allelic variants with overlapping phenotypes, supported by mutation analysis of CYLD . The conditions display autosomal dominant inheritance and affected individuals develop multiple benign skin tumours most commonly on the head and neck. CYLD testing can be performed using PCR and Sanger sequencing for patients with: 1. Multiple cylindromas, spiradenomas or trichoepitheliomas. 2. A single cylindroma, spiradenoma or trichoepithelioma and an affected first-degree relative with any of these tumours. 3. An asymptomatic family member at 50% risk with a known mutation in the family. ]]>

Clinical Phenotype

BSS, FC and MFT arise from heterozygous mutations in the CYLD gene1. Cylindromas are benign appendageal tumours occurring mainly on the scalp, but can occur on any hair bearing skin. The lesions typically present as painless, smooth pink nodules, which may be either solitary or clustered together. They are slow-growing and vary in size from a few millimetres to over six centimetres.

Fig. 1: Cylindroma

Several well-circumscribed, pink, nodular cylindromas with arborizing blood vessels on the surface, occurring on the scalp of a patient with BSS.

Spiradenomas often occur in conjunction with cylindromas in BSS, but tend to be painful rather than painless. They can present as a dermal nodule, often with a blue/black appearance.

Fig. 2: Spiradenoma

Nodular lesion with characteristic blue/black appearance.

Definitive diagnosis of cylindromas and spiradenomas requires histopathological examination of an excised lesion. Histopathologically, cylindromas consist of nests of basaloid cells arranged into nodules that resemble a cylinder in cross section. These form an irregular pattern, often a likened to a jigsaw.

Fig. 3: Histology of a cylindroma

The typical appearance of a cylindroma at low power (10x), consisting of well-defined nests of basaloid cells separated by an eosinophilic basement membrane.

These tumours are rare in the general population, and a histology result should raise the suspicion of a germline CYLD mutation in a young person.

A diagnosis of FC or MFT is prompted by the dominant tumour type being cylindroma or trichoepithelioma respectively. BSS presents with a variety of skin appendage tumours including cylindromas, spiradenomas and trichoepitheliomas. The distinction made between the different conditions hence depends on the combination of tumour types identified. These divisions are not thought to be useful in the clinic in terms of prognostic information or counselling patients, and hence the term CYLD cutaneous syndrome has been proposed2 . The timing of tumour onset is usually in adolescence, but can occur from after adrenarche in childhood, up to the 4th decade. The skin appendage tumours have a predilection for the face and scalp, although also affect the trunk, most commonly in hair-bearing areas2 .

The morbidity associated with skin appendage tumours in these conditions can be substantial. The tumours are disfiguring, can be painful and occur in multiple sites. Affected individuals face repeated episodes of surgery to remove lesions, and in some cases this may culminate in removal of the entire scalp3 . Patients may request surgery when tumours are painful, ulcerated, bleeding, unsightly or causing sexual dysfunction4 . In affected families, genetic testing allows individuals to ascertain their own risk and to use the information for family planning purposes.

Test Description

Blood in EDTA is the preferred tissue but buccal swabs, mouthwash samples or solid tissue can also be tested. The testing protocol involves PCR amplification of exons 4-20 of CYLD in a total of 18 amplicons. In addition to the exonic sequence at least 10bp of flanking intronic sequence is captured at the 5’ and 3’ end of each exon. Following PCR amplification each amplicon is sequenced by bi-directional fluorescent Sanger sequencing and the data analysed using the current version of Mutation Surveyor™ software.

Public Health Importance

The estimated prevalence of CYLD mutations in the UK population is 1:100000, although this is difficult to accurately determine. The penetrance in terms of tumour development in those with a mutation in CYLD is estimated to be close to 100%. Most cases of BSS, FC and MFT result from autosomal dominant inheritance, although sporadic cases can also occur.

A genetic test result allows an individual to make decisions relating to family planning. It can also be useful to inform treatment planning. Patients with a CYLD mutation may be offered the opportunity to enrol into clinical trials. CYLD encodes a deubiquitinating enzyme that negatively regulates the nuclear factor-kappa beta pathway. A clinical trial using topical salicylic acid was not promising5 . More recently gene expression profiling of tumours in patients with germline CYLD mutations have shown dysregulated tropomyosin kinase (TRK) signalling, and treatment with lestaurtinib, a TRK inhibitor reduced growth of cells established from CYLD mutant tumours in vitro6 .

Published reviews, recommendations and guidelines

Systematic evidence reviews: None identified

Recommendations by independent groups: UK GTN – Gene dossier

Guidelines by professional groups: None identified

Evidence Overview

Analytic validity:

The testing methodology involves bi-directional fluorescent DNA sequencing of all coding exons of CYLD (exons 4-20, exonic sequence plus at least 10bp at the 5’ and 3’ of each intron). This method would be expected to detect >99% of mutations within the coding region/splice sites, excluding duplications and large deletions. Large deletions have been reported to affect a minority of patients that are mutation negative following Sanger sequencing.7 cDNA testing is also carried out where appropriate. The generic techniques are well established in the CPA-accredited Northern Genetics Service laboratory.


The testing protocol was validated in confirming the findings of the research study described below.

Analytic sensitivity:

The analytic sensitivity and specificity are close to 100% for the techniques used.

Clinical validity:

A study investigating the mutational spectrum of CYLD and genotype-phenotype correlation8 tested a total of 47 samples from 25 families exhibiting BSS, MFT or FC phenotypes. Of these families, 13 had clinical features of BSS, three of FC and nine of MFT. Mutations in CYLD were present in 11/13 with BSS (85%), 3/3 with FC (100%) and 4/9 families with MFT (44%). This gave an overall mutation detection rate of 18/25 (72%) in these families.

Clinical Utility

The genetic predisposition to these tumours is rare. Although cylindromas carry a significant burden of disease, they are usually not life threatening, which may explain why there are few studies looking at the benefits of genetic testing in affected or at risk individuals. Anecdotal evidence is described in one study of 26 affected patients.2 In this study, issues relating to genetic counselling which are relevant to those undergoing genetic testing for CYLD mutations are explored. When a pathogenic mutation in CYLD is identified in a patient who meets the criteria for testing, they have the advantage of knowing their diagnosis is confirmed, and can use the information for their benefit. Patients who present with multiple skin appendage tumours but are not known to have a family history may not have considered that their tumours could represent an underlying genetic cause. The knowledge that this is so can help them anticipate the fact that further tumours may develop and prepare for necessary treatment. Testing is also facilitated for family members should they develop tumours. Individuals with a family history but no tumours themselves are aware they are at 50% risk of having a CYLD mutation. Having the test on a presymptomatic basis can reassure patients if they receive a negative result, and allow them to make decisions about the future if they know the result is positive. Although prenatal diagnostic testing is not offered for this condition, some patients with a CYLD mutation may choose to use the information to influence family planning decisions. Patients with a mutation may choose to be entered into appropriate clinical trials when the opportunity arises.


UKGTN Homepage: http://ukgtn.nhs.uk/

UKGTN Test Dossier: http://ukgtn.nhs.uk/find-a-test/gene-dossiers/

UKGTN Testing Criteria: http://ukgtn.nhs.uk/resources/testing-criteria/

Competing Interests

The authors have declared that no competing interests exist.

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Genetic Testing Strategies in Newly Diagnosed Endometrial Cancer Patients Aimed at Reducing Morbidity or Mortality from Lynch Syndrome in the Index Case or Her Relatives http://currents.plos.org/genomictests/article/genetic-testing-strategies-in-newly-diagnosed-endometrial-cancer-patients-aimed-at-reducing-morbidity-or-mortality-from-lynch-syndrome-in-the-index-case-or-her-relatives/ http://currents.plos.org/genomictests/article/genetic-testing-strategies-in-newly-diagnosed-endometrial-cancer-patients-aimed-at-reducing-morbidity-or-mortality-from-lynch-syndrome-in-the-index-case-or-her-relatives/#respond Mon, 16 Sep 2013 13:09:59 +0000 http://currents.plos.org/genomictests/?post_type=article&p=21281

Clinical scenario

Approximately 2-4% of endometrial cancer (10% in women diagnosed under the age of 50) is attributable to Lynch syndrome,1,2,3,4 an autosomal dominant cancer-prone syndrome caused by germline mutations in the MLH1, MSH2, MSH6 or PMS2 genes, which encode components of the DNA mismatch repair (MMR) pathway, or, in a small proportion of cases, by deletions in the EPCAM gene that lead to epigenetic silencing of the adjacent MSH2 gene (reviewed in 5). Individuals with Lynch syndrome are at increased risk for cancers of the colon, rectum, endometrium, ovary, small bowel, urothelium, pancreas, biliary tract, stomach, brain, skin and possibly breast (reviewed in 6). In 50-60% of women with Lynch syndrome, endometrial cancer is the first malignancy.7,8 Those who have already been diagnosed with cancer are also at risk of developing a Lynch-syndrome-associated cancer at another site, or a second primary cancer in the same organ.

If Lynch syndrome is suspected in a cancer patient, DNA testing can be used to determine whether the patient has a MMR or EPCAM gene mutation. However, because the prevalence of Lynch syndrome amongst those diagnosed with cancer is low, even for the two most common Lynch Syndrome-associated cancers (colorectal and endometrial), germline DNA testing for all cancer patients is not currently feasible so various clinical triage approaches have been developed to identify the subset of patients most likely to have Lynch syndrome. Triage has typically been based on factors such as family history of Lynch syndrome or Lynch-syndrome-associated cancers, age at diagnosis, and features of the primary tumour (usually assumed to be colorectal).9,10 However, such criteria have been criticised as lacking adequate clinical validity for identifying endometrial cancer patients who should be offered genetic testing.2,11,12,13,14,15 Inadequate clinical validity in unselected endometrial cancer patients with Lynch syndrome has also been found for a variety of clinical prediction rules (PREMM1,2,6, MMRpredict and MMRpro) developed to predict the probability of a Lynch syndrome mutation in cancer patients.16,17,18,19 Young age at diagnosis (usually <50 years) has also been criticised as likely to miss 30-70% of endometrial cancer patients with Lynch syndrome.11,13,14,20

To improve the sensitivity of detecting Lynch syndrome among endometrial cancer patients, tumour testing by microsatellite instability (MSI) and/or immunohistochemistry (IHC) analysis has been suggested for all newly diagnosed patients.2 If this analysis indicates that one or more of the MMR proteins is absent or non-functional in the tumour, the patient would be referred for further investigation and, if sporadic cancer can be excluded, offered germline DNA testing for MMR mutation(s). Those who test positive for a Lynch syndrome mutation can enter surveillance programmes or be offered preventive interventions with the aim of reducing mortality and morbidity from metachronous Lynch syndrome cancers. In addition, their relatives can be offered diagnostic testing for Lynch syndrome, with subsequent risk-reducing surveillance or prevention for those who test positive.2,13 An analogous strategy has been found to have clinical utility for the relatives of newly diagnosed colorectal cancer patients and has been recommended by the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group.21,22,23,24

In some centres in the US, screening of all newly diagnosed colorectal cancer and/or endometrial cancer patients by tumour testing is already being carried out. A recent survey identified 29 insitutions screening all colorectal tumours, and 11 screening all endometrial tumours.25

The aim of this paper is to gather and present evidence on offering molecular tumour testing for Lynch syndrome to all newly diagnosed endometrial cancer patients, with the aim of reducing morbidity or mortality from subsequent cancers in the index case, and/or from Lynch syndrome cancers in her relatives.

Test description

Loss or malfunction of the MMR system leads to numerous unrepaired errors in the genome. Some of these errors are manifested as changes in the lengths of short repeated sequences known as microsatellites. Testing for microsatellite instability (MSI) is done by comparing the distribution of PCR-amplified microsatellite fragment lengths between tumour and normal tissue.26 Tumours in which more than 30% of the microsatellites are unstable are classified as high-frequency instability (MSI-H). A low frequency of instability (MSL-L) is one in which one or up to 30% of the markers show instability, while a microsatellite-stable (MSS) tumour shows no unstable markers.5,26 In 1998 a panel of 5 microsatellite markers, including 2 mononucleotide markers, was recommended in the proceedings of a workshop convened by the National Cancer Institute (NCI), at which the Bethesda guidelines were established.27 More recently, incorporation of additional mononucleotide markers has been found to improve sensitivity.21

In the IHC method, loss of MMR expression is determined by lack of immunohistochemical staining of MMR proteins (MLH1 MSH2, MSH6, PMS2) in the nuclei of tumour cells.5 Because it detects the absence of specific MMR proteins, IHC can also inform subsequent genetic testing for germline mutations. IHC is performed on slices of paraffin-embedded tumour tissue transferred to microscope slides. Nuclei showing any detectable staining (>1%) are scored as positive. The MMR proteins function as heterodimeric complexes: MLH1 with PMS2 (or PMS1) and MSH2 with MSH6 (or MSH3).28 As some of these proteins are unstable when not paired in a complex, a defective MMR system may involve loss of expression of more than one protein: tumours of individuals with germline MLH1 mutations generally lack both MLH1 and PMS2 expression, while those with a germline MSH2 mutation (or, more rarely, a deletion in the EPCAM gene) lack both MSH2 and MSH6 expression. However, germline mutations in MSH6 or PMS2 do not result in loss of MSH2 or MLH1; therefore, tumours with isolated loss of MSH6 or PMS2 expression indicate a possible germline mutation in the respective gene.29

Tumour testing by MSI and/or IHC typically yields abnormal results in 15-25% of unselected endometrial cancer patients.4,19 As the current cost of germline DNA testing for such a large number of patients would be prohibitively high in a population screening programme, further tumour analysis may be undertaken to identify those tumours that are likely to be sporadic.30 Most of these are tumours that show an MSI-H phenotype and loss of MLH1 and PMS2 expression as a result of somatic methylation of the MLH1promoter.31 In colorectal cancer, sporadic cancers can be distinguished from Lynch syndrome cancers by testing for the V600E mutation in the BRAF gene, which is frequently mutated in the former but not the latter, and is strongly associated with MLH1 promoter methylation.32,33 In contrast, the frequency of BRAF gene mutations in endometrial tumours is thought to be low (no higher than 1%), regardless of methylation status, suggesting that BRAF gene analysis is not useful in testing of endometrial cancer patients.4,34,35 If MLH1 expression is absent in endometrial tumours, the methylation status of the MLH1 promoter can be tested directly, for example by bisulphite treatment of the DNA (which converts all unmethylated cytosines to uracil) followed by sequencing or restriction analysis or methylation-specific PCR (MSP).1,2 An alternative method is methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA).4

Germline DNA testing in peripheral blood or normal endometrium is usually carried out by direct gene sequencing of the MLH1, MSH2, MSH6 and (in some laboratories) PMS2 genes, together with a method such as MLPA to detect large genomic rearrangements.2,4 MLPA can also be used to detect deletions in the EPCAM gene.36 MMR gene mutations are classified as deleterious if they are predicted to encode a truncated or unstable protein (e.g. frameshift, nonsense, splice site). Missense mutations may often have uncertain clinical significance.

In some patients with tumour test results suggestive of Lynch syndrome, no mutations are found in the four known MMR genes. There may be additional causative genes, mutations in known MMR genes that are not detected by current sequencing protocols (e.g., deep intronic mutations or promoter mutations) or epigenetic effects that remain to be discovered.37

Availability of tumour testing and DNA testing for Lynch syndrome

The GeneTests Laboratory Directory (http://www.ncbi.nlm.nih.gov/sites/GeneTests/lab) lists 28 laboratories offering MSI analysis for Lynch syndrome. All of these laboratories test at least the 5 Bethesda guidelines markers; according to GeneTests, most use a panel of 10. Nine of the laboratories offer both tumour analysis by IHC and germline mutation testing. Approximately 50 labs are listed as offering DNA testing for MLH1 and MSH2 mutations (generally full sequence analysis plus testing for large deletions or duplications, but a few offering most restricted testing of specific exons). Smaller numbers of laboratories are listed for MSH6 and PMS2 mutation testing: approximately 40 and 25 respectively. 10 listed laboratories offer deletion testing of the EPCAM gene.

The GeneTests Laboratory Directory is not exhaustive and includes mostly US laboratories; Lynch syndrome diagnostic testing is also carried out by many additional commercial and hospital- or university-based diagnostic laboratories worldwide.

Public health importance

If screening of all newly diagnosed endometrial cancer patients were to be undertaken, the incidence of endometrial cancer in the population will determine the number of women who will need to be offered tumour testing, and the expected proportion of these who have Lynch syndrome will determine the numbers who subsequently need surveillance for colorectal cancer (and possibly other Lynch syndrome-associated cancers). In the US, approximately 49,500 new cases of uterine cancer are expected in 2013, and 8,200 deaths from this cause.38 About 95% of uterine cancers are endometrial.37 Lynch syndrome accounts for about 2-4% of those diagnosed with endometrial cancer. Using the American Cancer Society figures for total diagnoses annually,38 the expected number of endometrial cancer diagnoses attributable to Lynch syndrome in 2013 is approximately 940-1880. Estimates of the prevalence of Lynch syndrome in the population range from approximately 1 in 300039 to 1 in 370.40

Colorectal cancer (for which both the index case with Lynch syndrome and her relatives are at risk) is the fourth most common cancer in the US, with 142,820 new diagnoses and 50,830 deaths expected in 2013.38 In both men and women, it is the third-highest cause of cancer related deaths. Approximately 2-4% of colorectal cancer cases are attributable to Lynch syndrome, accounting for 2850-5700 of the expected incident cases in 2013.41

Estimated cancer risks for those with Lynch syndrome vary widely, even among studies that are either population-based or use statistical methods to correct for the ascertainment bias resulting from selection of patients from familial cancer clinics.42,43,44,45,46,47 Reported risks to age 70 for colorectal cancer range from 20-90% for men and 10-55% for women. Endometrial cancer risk estimates range from 15-55%, and risk of any Lynch syndrome cancer from 40-80% for men and 25-80% for women. Cumulative lifetime risks for other Lynch syndrome cancers are even more uncertain, but estimates generally do not exceed 15%.46,48,49,50,51 Some, but not all, of the variation is accounted for by different risk profiles for different MMR gene mutations: in general, risks for MLH1 or MSH2 carriers are higher, and age at onset of disease lower, than for those with mutations in MSH6 or PMS2.52,53

The cancer risks faced by those with Lynch syndrome may be compared with an average population risk to age 75 for colorectal cancer in the US of 3.0% in men and 2.3% in women, while women have a 2.0% risk of developing uterine cancer by age 75.54 Corresponding lifetime risks in the US are 5.3% and 4.9% for colorectal cancer in men and women respectively, and 2.6% for uterine cancer in women.54

Published reviews, recommendations and guidelines

Investigation of index endometrial cancer patients

  • The Society of Gynecologic Oncologists guidelines recommend that women at greater than 20-25% risk of Lynch syndrome should be offered genetic assessment.55 Risk is calculated on the basis of the revised Amsterdam criteria, or a diagnosis of synchronous or metachrononous endometrial or colorectal cancer before the age of 50, or a first or second degree relative with a known MMR mutation, or with evidence of a MMR defect by MSI or IHC analysis. The guidelines state that it is “reasonable” to offer genetic assessment to women whose risk is greater than 5-10%. The guidelines also acknowledge that a lower threshold for genetic referral may be reasonable in some circumstances; for example for women with very few female relatives or where there is adoption in their lineage. The strategy for “genetic risk assessment” is not specified.
  • NCCN guidelines version 2.2012 recommend tumour testing when the patient meets Amsterdam or Revised Bethesda criteria, or has been diagnosed with endometrial cancer at age <50 years, or has known Lynch syndrome in the family.56 A testing strategy is specified based on initial tumour testing by IHC and/or MSI followed by germline DNA testing informed by the combination of tumour testing results.
  • The National Society of Genetic Counselors and the Collaborative Group of the Americas on Inherited Colorectal Cancer joint practice guideline recommends that MSI and IHC analysis should be performed on endometrial tumours of patients meeting Amsterdam I or II or Bethesda criteria.57 Outline recommendations for optimum performance of MSI and IHC are included in the guideline.
  • The Netherlands Association of Comprehensive Cancer Centres (ACCC) Guidelines on hereditary colorectal cancer include a recommendation that women with endometrial cancer diagnosed under the age of 50 should be referred to a clinical geneticist.58 For endometrial tumours, testing by both IHC and MSI is recommended, as well as methylation testing of the MLH1 promoter, to determine which women should be offered germline genetic testing.
  • The “Mallorca group” of European experts recommends that all women diagnosed with endometrial cancer up to the age of 70 years should be offered tumour testing by MSI or IHC.59

Genetic testing, surveillance and preventive options for people with Lynch syndrome

There is general expert consensus that, when mutation testing has identified Lynch syndrome in an individual, germline genetic testing for the pathogenic mutation should be offered to his or her first-degree relatives (e.g. 23,56). Guidelines by the American Cancer Society, US Multi-Society Task Force and American College of Radiology state that genetic testing should be offered to first-degree relatives of those with a confirmed MMR mutation or, when the mutation is not known, if one of the first three of the modified Bethesda criteria is met.60

There is also broad consensus on surveillance and preventive options for colorectal cancer in people with Lynch syndrome. Guidelines published by professional associations and expert groups in the US and Europe (e.g. 59,60,61,62) and a systematic review by an independent expert group63 recommend colonoscopic surveillance every 1-2 years, beginning at age 20-25, or 10 years younger than the age of the youngest person diagnosed in the family (or 2-5 years before the earliest colon cancer if it is diagnosed at age <25 years).56 Lindor et al. suggest that screening may begin later (age 30) in those with MSH6 mutations.63

There is less consensus among professional guidelines on surveillance options for other Lynch syndrome-associated cancers. Some guidelines suggest screening for endometrial cancer and ovarian cancer by transvaginal ultrasound and endometrial biopsy every 1-2 years from age 30-35, while acknowledging a lack of convincing supportive evidence(e.g. 59,61,63,64). NCCN 2012 guidelines recommend that patients should be made aware that abnormal uterine bleeding warrants investigation, and state that surveillance options may be offered at the physician’s discretion.56 There is general consensus that prophylactic hysterectomy and bilateral salpingo-oophorectomy are of demonstrated benefit in reducing risk of endometrial and ovarian cancers and may be suggested as options for women aged 35 or over who have completed childbearing.56

For gastric cancer, small bowel cancer and urothelial cancer, some guidelines suggest that surveillance options should be considered, although clear supportive evidence is lacking.56,61,63

Analytic validity of diagnostic tests

There is little direct evidence on the analytic validity of tests to diagnose Lynch syndrome in endometrial cancer patients. Tumour testing protocols for colorectal cancer specimens may not be optimal for endometrial tumours. Analytic validity may be affected by tumour sampling and preparation, by treatment the patient has undergone before the tumour is sampled (for example, there is some evidence from colorectal tumours that neoadjuvant therapy can affect MSI and IHC testing),65,66 and by variations in assay methods and competency among different laboratories.


Current best practice, developed for testing colorectal tumours, is considered to include sample preparation by laser microdissection, use of a panel of at least 5 microsatellite markers, at least three of which should be mononucleotide markers, and ensuring a minimum proportion of 30% tumour cells in the sample analysed.23 Two highly polymorphic pentanucleotide markers can also be included to check for sample mix-up or contamination.4,67 Commercial test kits are available.

Many laboratories offering MSI testing participate in proficiency testing programmes (for example, the College of American Pathologists programme 68) and performance standards are assumed to be high if best practice is followed. In analyses of 646 tumour samples (of which 88% were colorectal tumours) Bartley et al. identified failure of DNA amplification in only 0.6% of the microsatellites analysed; in no case did the failure to amplify affect the designation of the tumour as MSS, MSI-H or MSI-L.69 A specific issue for MSI testing in endometrial tumours arises from the higher proportion of MSH6 mutations among endometrial cancer patients with Lynch syndrome (see below) and the fact that MSH6-deficient tumours frequently show an MSI-L and occasionally an MSS phenotype.2,70 Therefore an appropriate proficiency test should include performance in identifying the MSI-L phenotype in endometrial tumours.


Technical guidelines and proficiency testing for IHC are available in the US (for example, the College of American Pathologists programme)68 and in Europe (for example through the United Kingdom National External Quality Assessment Service).71 Most, if not all, proficiency testing programmes relate to the testing of colorectal tumours and specific schemes for endometrial tumours are not described. Clarke and Cooper mention an unpublished proficiency test for IHC involving 14 laboratories; they imply that the results showed high standards of performance but no details are given.14 However, some of the few publicly available results of IHC proficiency testing suggest some concerns. Nordic Immunohistochemistry Quality Control (NordiQC; www.nordiqc.org) publishes aggregated results from 80-90 participating laboratories for MMR protein IHC in colorectal tumour samples of known mutation status. For IHC of MLH1, MSH2 and MSH6, the percentage of submitted tests classed as optimal or good was 57%, 73% and 33% respectively; PMS2 is apparently not assessed by this programme. Substantial variation was found for antibody preparations from different commercial suppliers. Problems such as failure to recognise lack of an internal positive control, inter-observer variation (particularly among less-experienced pathologists) and difficulty in interpreting weak staining have been noted in several reports;69,72,73 these studies relate to colorectal tumours. There is little information about technical performance of IHC specifically in endometrial tumours, however Modica et al. claim that more apparent staining inadequacies are observed in endometrial than in colorectal tumours.74

Methylation tests

Insufficient information was found to enable evaluation of the analytical validity of methylation tests. There are several methylation sites in the MLH1 promoter but only those in the proximal C and D regions of the promoter correlate with silencing and loss of expression of MLH1,67 and some publications that report using methylation-specific PCR (MSP) of bisulphite-treated DNA to assay methylation do not specify which promoter region or regions were tested (e.g., 1). The MSP method is reportedly simple, sensitive, inexpensive and can be used on paraffin-embedded samples.75 Hampel et al. assayed the promoter H region by MSP and the D region by combined bisulphite restriction analysis.2 Testing of the D region was unsuccessful in 2 out of 118 tumours. Leenen et al. tested MLH1 promoter hypermethylation by MS-MLPA, a semi-quantitative method that analyses several promoter methylation sites simultaneously and is reportedly sensitive and reproducible.67 The proprietary kit they used, which contains probes specific for methylation sites in the MMR gene promoters, is available for research use only. Moline et al. report methylation testing by real-time PCR/fluorescence energy transfer at a commercial laboratory; no information on test performance is provided.76

MMR gene sequencing

The current clinical standard is direct gene sequencing combined with appropriate analysis (e.g., by MLPA) to detect large rearrangements in the four MMR genes and the EPCAM gene. It is unclear what the appropriate measures are for determining analytic validity. Analysis of the PMS2 gene is problematic because of the large number of pseudogenes. Improved methods of mutation detection in PMS2 have been described and reported to improve the sensitivity of mutation detection but the optimum method is currently unclear.77,78,79,80

Hampel et al. report failure of MLPA deletion analysis in approximately 15% of patients with MSI-positive tumours.2 Weissman et al. note that some MLPA kits for detection of MSH2 deletions also encompass possible EPCAM deletions57 but data on the performance of this or other MLPA tests for EPCAM deletions (including the commercial MS-MLPA kit which can be used to detect methylation of the MSH2 promoter)67 are not available.

New massively parallel “next-generation” sequencing technologies may eventually improve accuracy and throughput and decrease costs for mutation detection. For example, the Col Seq assay reportedly has 100% sensitivity for identifying pathogenic mutations in the 4 MMR genes and EPCAM, with 100% reproducibility between runs.81

Clinical validity of diagnostic tests

Several studies were found that are relevant to the issue of tumour testing of unselected endometrial cancer patients. These studies (discussed below) were:

  • A retrospective study by the OSU group on a large series of unselected endometrial cancer patients diagnosed between 1999 and 2003 (Hampel et al. with follow-up work reported by Mercado et al.);2,3,19
  • A prospective study, reported by Backes et al., of 140 unselected endometrial cancer patients diagnosed at OSU in 2007 and 2008;82
  • A retrospective study of 384 unselected endometrial cancer patients diagnosed at OSU from 2007-2009, reported by Backes et al.;83
  • A prospective study by Leenen et al. of 179 consecutive endometrial cancer patients diagnosed up to the age of 70 in a multi-centre study in the Netherlands;4
  • A prospective study by Lu et al. of 100 endometrial cancer patients diagnosed under the age of 50;1
  • A retrospective study by Berends et al. of 58 endometrial cancer patients diagnosed under the age of 50;84
  • A prospective study by Goodfellow et al. of 441 unselected endometrial cancer patients; molecular analysis, and mutation testing for MSH6 mutations, was carried out on a subset of 100 of these patients.20

Not all of these studies contained sufficient information to contribute to quantitative estimation of the clinical validity of tumour testing, defined as the performance of the test(s) in identifying patients with a pathogenic germline mutation in an MMR gene or the EPCAM gene: in three of the studies, germline mutation testing was carried out only on some or all of those patients with tumour test results suggestive of Lynch syndrome, and not on patients with negative tumour test results.4,82,83 Moline et al. have recently reported the results of implementing a tumour testing programme for 245 endometrial cancer patients at Cleveland Clinic. However, both the inclusion criteria for tumour testing and the tumour testing protocol changed over the period reported, making it difficult to draw quantitative conclusions from the pooled results.76


The ideal study would comprise both MSI tumour testing, and germline DNA testing for mutations in the MLH1, MSH2, MSH6, PMS2 and EPCAM genes, in a large series of consecutive, unselected endometrial cancer patients. No such studies were found. However, the study by the OSU group used broad inclusion criteria for germline testing (see Table 1 footnotes), with test sensitivity and specificity apparently calculated on the assumption that the overall testing strategy identified all mutation carriers.2,3,19 This study also included analysis of a sample of clinic-based cases and their relatives.

Some studies were also identified in which MSI was analysed only in tumours from patients with known MMR gene mutations.11,70,75 Although not ideal, as no mutation-negative individuals were tested, investigators were not blind to mutation status, and the populations are not representative of an unselected population of endometrial cancer patients, these studies were included to contribute to estimates of test sensitivity.

As shown in Table 1, estimates of the sensitivity of MSI analysis range from 77-100%, with specificity 38-81% overall, and 69-81% in population-based studies. The positive predictive value in the population-based OSU study was 9%; in the two studies restricted to patients under the age of 50, it was higher (20% and 32%). The negative predictive value was 97-100%. However, comparisons are problematic, and conclusions difficult to draw, due to heterogeneity among studies (e.g. different populations; different markers and criteria for MSI analysis; different genes included in germline DNA testing; different mutation detection methods). The EGAPP working group noted similar difficulties in evaluating studies on colorectal tumours.21,23

Table 1. Clinical validity of MSI testing
Study Mutation carriers
(total MSI tested)
MMR genes tested MSI panel (no.
mononuc. markers)
MSI criterion Sens. Spec. PPV NPV
Mercadoa19 13 (560) MLH1, MSH2, MSH6, PMS2 5-6 (2) MSI-H + L 0.92 0.78 0.09 1.00
Mercadob 19 16 (24) MLH1, MSH2, MSH6, PMS2 5-6 (2) MSI-H + L 1.0 0.38 0.76 1.00
Luc 1 18 (95) MLH1, MSH2, MSH6 6 (3) MSI-H 1.0 0.81 0.32 1.0
Berendsd 84 5 (57) MLH1, MSH2, MSH6 5 (2) MSI-H 0.80 0.69 0.20 0.97
Goodfellowe 20 7 (100) MSH6 5 (2) MSI-H 1.0
Ryanf 11 20 (20) MLH1, MSH2, MSH6 5-10 (2-3) Unclear 0.90
de Leeuwg 70 31 (31) MLH1, MSH2, MSH6 5 (2)
24-40 (8)
Kuismanenh85 57 (57) MLH1, MSH2 12 (10) MSI-H + L 0.77

Sens., sensitivity; Spec., specificity; PPV, positive predictive value; NPV, negative predictive value

a MSI analysis on tumour samples for 560 of 563 unselected patients. IHC for all MSI tumours, or for women diagnosed at <50 years, or for women with synchronous or metachronous colorectal cancer and endometrial primaries, or with a first degree relative diagnosed with endometrial or colorectal cancer at any age. 223 MSS tumours also evaluated by IHC. Subjects with MSI-H or MSI-L and/or abnormal IHC underwent germline DNA testing. Germline testing by direct sequencing (MLH1, MSH2, MSH6), and MLPA for all 4 genes.

b Patients from family cancer clinics and their affected and unaffected relatives.

c Consecutive endometrial cancer patients diagnosed <50y. All tested by MSI and germline mutation analysis by full sequencing and large deletion analysis.

d Endometrial cancer patients diagnosed <50y. All tested by MSI and germline mutation analysis (DGGE with sequence variants verified by direct sequencing; MLPA for large deletions). Only those with ≥2 unstable markers were classified as MSI-H.

e Those tested for germline mutations were a subset of 441 unselected patients: all those with MSI-H and unmethylated MLH1 promoter (30), all those with MSI-L (10), 30/92 with MSI-H and methylated MLH1 promoter, and 30/304 MSS. Mutation analysis by SSCV to detect variants which were then confirmed by direct sequencing.

f Patients from family cancer clinics. Initial MSI with 5 markers (Bethesda panel) plus additional analysis with 10-marker panel (3 mononucleotide markers) for MSI-L tumours. Germline testing by direct sequencing and MLPA; no details given.

g Retrospective study on known mutation carriers with endometrial tumours (23 carcinomas, 8 hyperplasias), from families fulfilling Amsterdam I criteria

h Retrospective study on 57 tumours from known mutation carriers from Lynch syndrome families with 8 different MLH1 mutations and one MSH2 mutation.

A common finding in several studies of MSI in Lynch syndrome endometrial tumours has been a lower frequency of MSI in tumours from MSH6 mutation carriers. For example, 3 of the mutation carriers identified in the retrospective OSU studies had tumours that were MSI-L or MSS: all 3 of these patients had MSH6 mutations.2,3 De Leeuw et al. found that tumours from 12 MSH6 mutation carriers showed a low level of MSI across a panel of 30-40 markers. Only 4 were classified as MSI-H by Bethesda guidelines panel (5 marker) criteria, and tumours showed instability only with mononucleotide markers.70 Berends et al. found that the one mutation carrier whose tumour was not identified as MSI-H with the Bethesda guidelines panel was an MSH6 carrier.84


Table 2 shows results for clinical validity of IHC analysis in 3 population-based and 4 clinic-based studies, 3 of which were case-only studies.1,11,19,70,84,85 Overall sensitivity for 3 or 4 MMR proteins ranged from 86-100%, and specificity from 48-81% (though the range for population-based studies was narrower: 59-81%). As for MSI analysis, the positive predictive value of IHC in the OSU study was lower (10%) than in the studies restricted to patients under 50 years (20% and 38%). The negative predictive value in all three studies was 100%. As with MSI analysis, it is difficult to draw overall conclusions because of the heterogeneity among studies. Clarke and Cooper state an overall sensitivity of 96-100% for the four MMR protein markers in an MMR-IHC proficiency test “using tissue microarrays of carcinomas of known germline MMR mutation status”.14 The rest of their paper relates to endometrial cancer, but the authors do not specify whether the proficiency testing results (which are described as unpublished) relate to endometrial tumours, or colorectal tumours, or both, or whether the tumours were from unselected cases or cases ascertained through family cancer clinics. Some pathogenic missense variants of MMR proteins escape detection by IHC, lowering the sensitivity of the test;29,84,85,86 this was the case for two pathogenic mutations in the OSU study.2,84 It has been reported that IHC testing of MSH6 and PMS2 alone in Lynch syndrome tumours is as effective as a four-antibody test,87 and this two-antibody test is in routine use in the endometrial tumour screening programme at the Cleveland Clinic;76 however the clinical validity of the test does not appear to have been formally evaluated.

Table 2 Clinical validity of IHC testing
Study Mutation carriers
(total IHC tested)
MMR genes/proteins tested Sens. Spec. PPV NPV
Mercadoa 13 (352) MLH1, MSH2, MSH6, PMS2 0.86 0.67 0.10 1.00
Mercadob 51 (80) MLH1, MSH2, MSH6, PMS2 0.94 0.48 0.80 0.79
Lu 9 (99) MLH1, MSH2, MSH6 1.00 0.81 0.38 1.00
Berendsc 5 (51/36) MLH1, MSH2, MSH6 1.0 0.59 0.21 1.00
Ryand 23 (23) MLH1, MSH2, MSH6 0.91-1.0
de Leeuwe 31 (31) MLH1, MSH2, MSH6 0.97
Kuismanen 18 (18) MLH1, MSH2 1.0

For references for studies, see legend to Table 1.

The senstivity (Sens.), specificity (Spec.), positive predictive value (PPV) and negative predictive value (NPV) relate to the ability of the test to give a result consistent with the germline mutation.

a Population-based study

b Clinic-based cohort

c 51 tumours were tested for MLH1 and MSH2 and 36 were tested for MSH6.

d The sensitivity of 1.0 includes all tumours with results classed as “definite” or “equivocal”. For those classed as “definite”, sensitivity was 0.96. Both “equivocal” tumours were from MSH2 mutation carriers.

e Note that staining for 6/8 MLH1-deficient tumours was described as “weak” but classed as negative in this study.

Selection of patients with abnormal tumour testing results for referral for germline DNA testing

Two of the population-based studies of unselected endometrial cancer patients contained information relevant to the clinical validity of methylation testing for detecting sporadic tumours, but the data were incomplete.1,2,3 Lu et al. tested for MLH1 promoter hypermethylation by the MSP method in 13 patients (from their total series of 99) whose tumours were MSI-H and showed loss of MLH1 by IHC, or had uncertain IHC results. However, it is not clear which MLH1 promoter region was assayed, and methylation analysis was apparently not performed in the one patient with a germline MLH1 mutation.1 Twelve of the 13 tumours were methylation-positive; the one negative result was for a MSI-H tumour for which IHC testing did not work. No known pathogenic mutations were found in any of these tumours, though variants of uncertain significance were found in two patients (one in MLH1 and one in MSH2). If these variants are assumed to be non-pathogenic, the sensitivity of methylation testing for detecting sporadic tumours after IHC and MSI testing in this study was 92%.

The OSU study reported by Hampel et al. is complicated by the fact that they assayed two regions of the MLH1 promoter, only one of which (the D region) is reported in other publications to be associated with MLH1 silencing. They also used a different method for each region. In addition, one MLH1 mutation carrier reported in the later publication by Mercado et al.19 is not included in the earlier publications on the OSU cohort;2,3 the MSI, IHC and MLH1 promoter methylation status for this patient’s tumour are unclear. Methylation at the D region detected 85% of the sporadic tumours that were both MSI-H and lacked MLH1 protein.2

Leenen et al. found MLH1 promoter methylation in 31 of 32 tumours that were MSI-H and lacked MLH1 expression (18% of the total tumours tested), and considered the test to be robust enough to exclude these patients from germline DNA testing.4 The clinical validity of the methylation test cannot be confirmed from this study. Zauber et al. tested MSI and methylation status by MS-MLPA in 101 unselected endometrial cancer patients under 50 years and 112 older than 50 years.88 The combination of MSI-H and unmethylated MLH1 promoter indicated presumptive Lynch syndrome in 13% of the younger women and 5% of those over 50 years, but mutation testing was not carried out in this study.

The OSU group suggest alternative criteria, instead of methylation analysis, to prompt referral for germline DNA testing after initial tumour testing by IHC: all those with loss of MSH2/MSH6, and those with loss of MLH1/PMS2 who are under 60 or have a concerning family history. (In those over 60, loss of MLH1/PMS2 and no concerning family history is considered to indicate likely MLH1 promoter methylation and therefore a sporadic cancer).13,82,83 All 13 mutation carriers identified in the first two publications on the OSU cohort met these criteria; no published information was found on the subsequently identified MLH1 mutation carrier.2,3 11% of the OSU prospective series of 140 patients met the referral criteria (about half of the number who showed loss of at least one MMR protein by IHC); however, no mutations were found in the two patients who accepted germline genetic testing.82 In the subsequent retrospective study reported by Backes et al., the referral criteria were found to predict an MMR gene mutation in 3 out of 8 patients who underwent genetic testing (out of a total of 27 referrals).83 If the same referral criteria had been applied in the study by Leenen et al., one of 11 patients with likely Lynch syndrome on the basis of IHC and MSI testing would not have been referred for genetic counselling; as this patient (whose tumour was negative for MLH1 promoter hypermethylation) declined germline genetic testing, their mutation status is not known.4

For centres where tumour testing for all endometrial cancer patients is not considered feasible, the OSU group suggest restricting tumour testing to patients under the age of 60.13 However, Leenen et al. note that 5 of the 11 patients referred for genetic consultation in their study were over the age of 60; of these, 3 were found to be mutation carriers and one declined testing. None fulfilled Amsterdam or Bethesda criteria.4

Kwon et al. suggest referral of patients with abnormal IHC results and who have one affected first-degree relative.89 However, the clinical sensitivity of this criterion in unselected patients is likely to be low (and see further discussion below).1,4,19

Age at diagnosis and MMR gene mutations in endometrial cancer patients with Lynch syndrome

Table 3 shows the mutation spectrum, and mean or median age at diagnosis, of Lynch syndrome endometrial cancer in 4 population-based studies and a sample of 3 clinic-based studies. In the two population-based studies not restricted to patients under the age of 50, pathogenic mutations were identified in 2.5% and 3.9% of patients respectively.4,19 The most striking difference between these two studies and those either in clinic-based cohorts11,19,46 or young patients1,84 is in the proportion of MSH6 mutation carriers: 64% in the OSU study19 and 87% in the Netherlands study.4 The average age at diagnosis in the OSU and Netherlands studies is similar to that for MSH6 carriers in clinic-based studies and is older than that for MLH1 or MSH2 mutation carriers. The relatively high frequency of MSH6 mutations and older age at onset appears to be characteristic of mutation carriers from unselected endometrial cancer populations not restricted by age at diagnosis (or with cut-off at a late age). For example, Goodfellow et al. found a minimum frequency of 1.6% MSH6 mutations in 441 unselected endometrial cancer patients; 71% of those with MSH6 mutations were over 50.20 Devlin et al. found 6 MSH6 mutations (4 truncating mutations and 2 possibly pathogenic missense mutations) in 105 unselected patients.90 (No analysis was carried out for the other three MMR genes in either study.) In a large study of MSH6 mutation carriers, Baglietto et al. found a mean age of onset for endometrial cancer of 51.53

In the Lynch syndrome tumour screening programme reported by Moline et al., Lynch syndrome was confirmed by mutation testing in 8 patients, of whom 2 had mutations in MLH1, 2 in MSH2, 2 in MSH6 and 2 in PMS2.76 Three additional patients had tumour testing IHC results strongly suggestive of Lynch syndrome (lack of MSH2/MSH6 by IHC) but no mutations could be found. These patients were considered likely to have Lynch syndrome, giving a total of 11 definite/likely cases out of 245 patients tested (4.5%). As mentioned previously, comparisons with reported research studies are difficult due to the pooling of patients selected by different criteria.

In the Netherlands studies, no pathogenic mutation could be found in 30% (3/10) of individuals whose tumours were MSI-H, methylation-negative for the MLH1 promoter, and had IHC results indicating the loss of a MMR protein.

Table 3. Age at diagnosis and MMR gene mutation spectrum in endometrial cancer patients with Lynch syndrome
Study Mutation carriers Age at diagnosis (mean or median) MLH1 No. (%) MSH2 No. (%) MSH6 No. (%) PMS2 No. (%)
Mercadoa19 14 53 2 (14%) 3 (21%) 9 (64%) 0 (0%)
Leenen (patients <70y)b4 7 59 0 (0%) 0 (0%) 6 (87%) 1 (13%)
Lu (patients <50y)c1 9 42 1 (11%) 7 (78%) 1 (11%) n.d.
Berends (patients <50y)84 5 45 1 (20%) 3 (60%) 1 (20%) n.d.
Ryan11 76 47 (overall)
49 (MLH1)
46 (MSH2)
51 (MSH6)
18 (24%) 50 (66%) 8 (10%) n.d.
Mercado19 80 48 31 (39%) 40 (50%) 9 (11%) 0 (0%)
Bonadona46 182 49 (MLH1)
48 (MSH2)
55 (MSH6)
72 (39%) 87 (48%) 23 (13%) n.d.

a 12 mutations of unknown significance (mostly point mutations or small insertions/deletions) reported: 2 in MLH1, 3 in MSH2 and 7 in MSH6.

b No mutation found in 3 patients with IHC results suggesting loss of expression, and 1 patient declined germline testing

EPCAM gene analysis

EPCAM gene analysis may be considered in cases where a tumour shows absent MSH2 expression but no germline MSH2 mutation is found.91,92 Neither the OSU group nor the Netherlands group tested for EPCAM mutations. Further research is needed to clarify the contribution of EPCAM mutations in endometrial cancer. Kempers et al. found that the endometrial cancer risk for EPCAM mutation carriers depends strongly on the location of the deletion in the EPCAM gene: an average cumulative risk of 12% to age 70 falls to almost zero for deletions located far upstream of the MSH2 promoter, but rises to 30% for deletions extending closer to the promoter (estimates not corrected for ascertainment bias).92

Interpretation of DNA test results

Based on experience with colorectal cancer, it has been estimated that in approximately 7% of at-risk individuals who have genetic testing for MMR gene mutations, a variant of uncertain significance is found.40 The interpretation of DNA test results may be aided by databases of Lynch syndrome mutations such as the InSiGHT (International Society of Gastrointestinal Hereditary Tumours) database (www.insight-group.org, which merged several previous databases and includes information on variants of unknown significance (however, this database relates primarily to Lynch syndrome colorectal cancer).93 A multivariate model has been developed to aid classification of missense variants in MLH1 and MSH2.94 Functional assays may also aid in determining pathogenicity.95

Clinical utility

The clinical utility of screening all incident endometrial cancer cases for Lynch syndrome relates to the likelihood that screening will lead to improved health either in the index case or in her relatives.

Index case

In order to demonstrate clinical utility for the index case, there needs to be adequate evidence that tumour testing and (if indicated) germline DNA analysis are acceptable to women and do not themselves cause significant harms, and that a diagnosis of Lynch syndrome (a) indicates specific treatment for Lynch syndrome endometrial cancer that decreases mortality or morbidity from the condition and/or (b) indicates surveillance and/or preventive options that are acceptable and decrease mortality and/or morbidity from other Lynch-syndrome associated cancers.

Acceptability of tumour testing and germline DNA analysis

Limited published information is available on the acceptability of tumour testing, genetic counselling and germline DNA analysis among unselected women diagnosed with endometrial cancer. Arguments for and against reflex tumour testing without explicit consent have been discussed for colorectal cancer patients and similar considerations apply in the case of endometrial cancer patients.21

Backes et al. found a low level of acceptance of germline DNA testing among endometrial cancer patients referred for genetic counselling (13% of those offered referral in a prospective study and 30% in a retrospective study).82,83 Kuppermann et al. also suggest that recognition of the value of genetic testing may not be high among unselected patients.96 In the Cleveland Clinic programme, 32 out of 42 patients with abnormal tumour test results (76%) accepted referral for genetic counselling and 28 of those accepted an offer of genetic testing.76 In the Netherlands study by Leenen et al., 10 0f 11 patients with IHC, MSI and MLH1 promoter methylation results suggestive of Lynch syndrome consented to germline DNA testing.4 Cultural differences between European and US patients, and differences in health insurance arrangements, may affect the acceptability of genetic testing. Backes et al. found that concerns about insurance coverage, and patients’ underestimation of their cancer risk, were important factors in limiting interest in genetic investigation.83 An emphasis on information and explanation, for physicians as well as patients, is important.4,83

Treatment of Lynch syndrome endometrial cancer

Lynch syndrome endometrial cancers display some morphological and histological differences from sporadic cancers (summarised by Clarke and Cooper).14 However, no information was found to suggest that treatment of endometrial cancer for women with Lynch syndrome differs from treatment for sporadic endometrial cancer.

Cancer risks and surveillance

Endometrial cancer is the first malignancy in approximately 50% of women with Lynch syndrome.7,8 Women remain at risk of subsequent colorectal cancer and, to a lesser extent, of other Lynch-associated cancers. Recently, Win et al. have estimated cancer risks after endometrial cancer for women with Lynch syndrome, using data for 127 women from the international Colon Cancer Family Registry.97 The median age of endometrial cancer diagnosis in this population was 46. The major risk was found to be for colorectal cancer, with 10- and 20-year cumulative risks of 20% (95% CI 13%-28%) and 48% (95% CI 35%-62%) respectively, and a standardised incidence ratio (SIR), compared to the general population, of 39.9 (95% CI 27.2-58.3). The median time from endometrial cancer diagnosis to colorectal cancer diagnosis was 11 years. The study had insufficient power to fully stratify cancer risks by MMR gene mutation; however, a significant difference was nevertheless found between the SIR for colorectal cancer in MSH6 mutation carriers (SIR=4.46, 95% CI 0.00-24.2) and either MLH1 (SIR=38.7, 95% CI 19.5-70.2) or MSH2 (SIR 58.5, 95% CI 36.0-98.4) mutation carriers.

The difference in colorectal cancer risk between MSH6 and MLH1/MSH2 mutation carriers is consistent with data from studies on the cumulative risk of colorectal cancer in people with Lynch syndrome.42,43,44,45,46,47 As shown in Figures 1 and 2, in female MLH1 and MSH2 mutation carriers cumulative risk to age 70 ranges from 20-50%, with risk increasingly more sharply between ages 30-60 than at later ages. In female MSH6 and PMS2 mutation carriers, cumulative risk of colorectal cancer rises from 2-3% at age 50 to 20% at age 80. (Note that, for clarity, confidence intervals have not been shown for the studies illustrated in Figures 1-5. In all studies they are broad, particularly at older ages where numbers of cases are small.)

Fig. 1: Cumulative risk of colorectal cancer in women carrying MLH1 or MSH2 mutations

Studies shown: Bonadona,46 Hampel,42 Dunlop,43 Quehenberger,44 Stoffel,47 Jenkins45

Fig. 2: Cumulative risk of colorectal cancer in women and men with MSH6 or PMS2 mutations

Studies shown: Bonadona,46 Baglietto,53 Senter52

As mentioned previously, colonoscopy every 1-2 years has been shown to reduce morbidity and mortality from colorectal cancer in people with Lynch syndrome. Studies in the United States and Germany report approximately 80% compliance with these recommendations in mutation carriers or those fulfilling Amsterdam or Bethesda criteria for risk of Lynch syndrome;98,99 however compliance was only about 60% in two studies in Italy and Spain.100 Psychological evaluations of the effects of risk reducing interventions are few but those that have been carried out suggest that appropriate advice and information help to improve knowledge and reduce anxiety (reviewed in 100)

The risks of non-colorectal cancers in women with Lynch syndrome who have had endometrial cancer are difficult to assess. Win et al. found increased 10- and 20-year cumulative risks for cancers of the urinary tract (kidney, renal pelvis or ureter) [2% (95% CI 0-5%) and 11% (95% CI 3-20%)], urinary bladder [1% (95% CI 0-4%) and 9% (95% CI 2-17%)] and breast [5% (95% CI 1-10%) and 11% (95% CI 4-19%)].97 Increased risks were also observed for small intestine and pancreatic cancer, though numbers of cases were small and confidence intervals for SIRs very large. In studies on cumulative lifetime risks for Lynch syndrome-associated ovarian, stomach, small intestine and biliary tract cancers, estimates vary so widely that few conclusions can be drawn.44,46,48,49,50,51,52,53 For women, the most significant risks from these studies appear to be for ovarian cancer and, in some recent studies, breast cancer. (see Table 3 in the paper by Win et al. at http://jco.ascopubs.org/content/30/9/958/T3.expansion.html);101 however, a recent systematic review from the same research group found that current evidence at the population level for an increased risk of breast cancer in Lynch syndrome was inconclusive.102

Although there are no surveillance interventions of proven benefit for non-colorectal Lynch syndrome cancers, urinalysis with cytology every 1-2 years beginning age 25-35 and upper gastrointestinal endoscopy every 1-2 years beginning age 30-35 are often recommended by physicians as surveillance regimes for upper urinary tract and stomach cancer respectively (reviewed in 6). In view of the recent evidence that breast cancer risk may be elevated in women with Lynch syndrome,97,101 enhanced surveillance for breast cancer may also be warranted, though Win et al. comment that risk may not reach the threshold lifetime risk of 20% recommended by the American Cancer Society for breast screening by MRI.97

Preventive options

Partial or subtotal colectomy is recommended only for individuals with Lynch syndrome who have been diagnosed with colorectal cancer, to reduce the risk of metachronous cancer.59 It is therefore not an appropriate option for women with Lynch syndrome ascertained through diagnosis of endometrial cancer, unless colorectal cancer is also present.

There is some recent evidence that chemoprophylaxis with aspirin can reduce the risk of colorectal cancer in individuals with Lynch syndrome.103,104 Benefit was not observed after 29 months of treatment but was found on longer-term follow-up (56 months). Further research is needed to confirm these findings and determine optimum aspirin dosage, as well as to investigate any adverse effects.105 No information is available with regard to whether the protective effect of aspirin would also apply to women who have had endometrial cancer.

For other Lynch-associated cancers, there are no preventive interventions of proven benefit except for ovarian cancer, where bilateral salpingo-oophorectomy has been shown to reduce risk and to be cost-effective.106,107,108 This may be an appropriate option for some women with Lynch syndrome identified through screening endometrial cancer patients, if they have not already undergone oophorectomy in conjunction with hysterectomy to treat their cancer. A preliminary report suggests that oral progestins may reduce the risk of endometrial cancer in women with Lynch syndrome.109

Overall, the clinical utility of Lynch syndrome testing for the index case depends on her age and the MMR gene mutated: the net benefit is lower for those diagnosed at older ages and with less-penetrant MSH6 mutations. To date, women with these features are the majority of those diagnosed through screening unselected endometrial cancer patients though the number of studies is small. Taken together, these findings suggest that careful age- and mutation-specific genetic counselling is likely to be essential for women with endometrial cancer who are found to have Lynch syndrome, to assist them in understanding their future risk of cancer and in weighing the risks and benefits of surveillance.

Cost-effectiveness (index case)

Resnick et al. carried out a cost-effectiveness analysis comparing triage strategies for identifying endometrial cancer patients with Lynch syndrome.30 They found that, relative to strategies of MMR gene sequence analysis for all women with endometrial cancer, for all diagnosed under age 60, or for all with endometrial cancer who meet Amsterdam criteria, the OSU strategy of IHC tumour analysis for all, followed by genetics referral and single-gene analysis except for those aged over 60 with loss of MLH1/PMS2 and no concerning family history, had an incremental cost-effectiveness ratio (ICER) of $13,812 per additional case detected, and would detect 858 of the estimated 920 incident Lynch syndrome endometrial cancer patients annually. The costs of subsequent surveillance and preventive interventions were not analysed.

Kwon et al. used a Monte Carlo simulation analysis to compare the costs and benefits of six different strategies for testing women with endometrial cancer for Lynch syndrome, assuming that all those identified would undertake risk-reducing colonoscopy every 1-2 years, and that this surveillance would reduce the risk of colorectal cancer from 40% to 15%.89 They concluded that the ICER for testing all women with endometrial cancer by initial IHC triage followed by germline DNA testing in those testing positive by IHC was unfavourable, at approximately $650,000 per life year gained, but that testing all those with at least one first-degree relative with a Lynch-syndrome associated cancer diagnosed at any age was cost-effective, with an ICER of approximately $9,000 per life year gained relative to the most inexpensive strategy (testing all women with endometrial cancer under the age of 50 who have at least one affected first-degree relative). The input data used by Kwon et al. come from a variety of sources, most of which relate to patients ascertained through family cancer clinics. Their assumptions on the extent of risk reduction, and the sensitivity of restricting germline testing to women with at least one affected first-degree relative, may not be valid for women identified by screening all incident endometrial cancer patients. For example, the family history restriction would exclude 9/11 of those referred for germline genetic testing in the study by Leenen et al., and 5/7 mutation carriers.4

Relatives of the index case

In order to demonstrate clinical utility in relatives of the index case, there needs to be sufficient evidence that targeted germline DNA testing is acceptable to these individuals, and that effective and acceptable interventions are available to enable them to reduce their risk of Lynch syndrome-associated cancers.

Risks of colorectal cancer and other non-uterine Lynch syndrome cancers in women have already been discussed (risks are not significantly affected by whether a woman has previously had endometrial cancer).97 For female relatives of the index case, there is the additional consideration that the diagnosis of Lynch syndrome may be made at a younger age; for young women, risks of colorectal cancer may be substantial for MLH1 or MSH2 mutation carriers (Fig 1).

Figures 3 and 4 show cumulative risks of endometrial cancer for MLH1 and MSH2 carriers,42,43,44,46,47 and MSH6 and PMS2 carriers,46,52,53 respectively. For MLH1 and MSH2 carriers, the risk profile is similar to that for colorectal cancer, with a risk to age 70 of 20-50%. For MSH6 or PMS2 carriers, risk is low to about age 50, rising to 15-25% by age 70 and possibly higher by age 80. Win et al. found overall 5- and 10-year cumulative risks for endometrial cancer of 2.84% and 9.84% respectively (see Table 3 at http://jco.ascopubs.org/content/30/9/958/T3.expansion.html).101

Fig. 3: Cumulative risk of endometrial cancer in MLH1 and MSH2 mutation carriers

Studies shown: as for Figure 1

Fig. 4: Cumulative risk of endometrial cancer in MSH6 and PMS2 mutation carriers

Studies shown: as for Figure 2

Figure 5 shows cumulative risks of colorectal cancer in men for MLH1 and MSH2 carriers.42,43,44,45,46,47 The range of estimates to age 70 is very high: from less than 30% to over 90% in different studies. Risks for colorectal cancer in male MSH6 or PMS2 carriers are shown in Figure 2.46,52,53 The risk for PMS2 mutation carriers is about 15%; for MSH6 mutation carriers, the two available studies found similar risks to age 70 (approximately 15-20%) but the large study by Baglietto et al. found that risk increased substantially between age 70 and 80.53

Fig. 5: Cumulative risk of colorectal cancer in men with MLH1 or MSH2 mutations

Studies shown: as for Figure 1

As discussed above, there is adequate evidence that surveillance by regular colonoscopy is effective in reducing risk of colorectal cancer in individuals with Lynch syndrome. The current recommendation is that screening should begin at age 20-25, or 10 years younger than the age at diagnosis of the youngest person diagnosed in the family. For families ascertained by diagnosis of an endometrial cancer patient with an MSH6 mutation, this might suggest delaying the start of screening to age 30-35 unless colorectal cancer cases in the family suggest otherwise.63

For endometrial cancer, there is some evidence that endometrial biopsy every 1-2 years, either alone or in combination with transvaginal ultrasound, is effective in lowering risk by early detection of cancer (reviewed in 6,110), but a recent systematic review concluded that current evidence is insufficient to enable evidence-based decisions, and that surveillance does not appear to lower risk of ovarian cancer.64 Given the very low risks in MSH6 or PMS2 carriers before middle age, if screening is undertaken it may be reasonable to start at the upper end of the suggested age band in these women.63 As a preventive intervention for endometrial and ovarian cancer, total hysterectomy and bilateral salpingo-oophorectomy has been shown to reduce risk,106 and to be acceptable to women diagnosed with Lynch syndrome mutations who have completed child-bearing.111

Jarvinen et al. found that, over an 11-year follow-up period, there was no difference in cancer mortality between individuals with Lynch syndrome undergoing risk-reducing surveillance and preventive interventions, and mutation-negative members of the same families, suggesting that current surveillance strategies are effective.112 In a large prospective study, Win et al. have found that mutation-negative members of Lynch syndrome families have cancer risks similar to those of the overall population and therefore do not need enhanced surveillance.101

Overall, clinical utility to relatives of an index Lynch syndrome case with endometrial cancer is likely to be higher if the family’s mutation is in MLH1 or MSH2 than for MSH6 or PMS2; however, the limited information available to date suggests that families with MLH1 or MSH2 mutations may be in the minority of those identified through screening endometrial cancer cases. It is clear that genetic counselling needs to be sex-, age- and mutation-specific to help mutation-positive relatives understand their risk.

Some studies reported since publication of the EGAPP analysis have added to the evidence regarding clinical utility to the relatives of colorectal cancer patients diagnosed with Lynch syndrome. Two studies have found that screening may be cost-effective but the different mutation spectrum and risk profiles relevant to screening women with endometrial cancer mandate specific health-economic analysis for this scenario.113,114

Other evidence regarding screening of colorectal cancer patients is likely to be applicable to screening endometrial cancer patients. Some commentators have suggested that there is insufficient evidence that encouraging findings from research studies would be replicated if screening were to be rolled out to much larger numbers of patients and centres,115,116 and that advocates of screening have not taken sufficient account of patient preferences, psychosocial harms or inequity of access.117,118 A recent systematic review found that uptake of genetic testing among first degree relatives of people with Lynch syndrome may be only 34%-52%;119 this level may be too low to meet criteria for cost-effectiveness of screening programmes.113 Recent studies of US cancer centres that have taken up reflex testing of all newly diagnosed colorectal cancer patients have found evidence of heterogeneity in approach, difficulties in ensuring adherence to the screening and follow-up protocol by all the clinical teams involved, and concerns about patients’ willingness to comply with follow-up recommendations and/or insurers’ willingness to pay for testing.25,120,121 Factors important for a successful programme include good communication within the multidisciplinary team, early involvement and integration of the genetics team, and clear protocols and responsibilities for contacting and following up patients and their at-risk relatives.121,122

Gaps in current evidence

An important gap in the evidence base is the relative paucity of studies on unselected endometrial cancer patients not restricted by age. The total number of mutation carriers identified in such studies to date is small, and only one of the two prospective studies was able to carry out mutation testing in almost all those with suggestive tumour test results, so it is difficult to estimate the level of confidence in findings such as mutation spectrum and age of onset.2,3,4,19,82,83 There is very little data on non-white population groups.

There are also uncertainties about the performance of IHC and MSI in analysis of endometrial tumours, as most published data relate to colorectal tumours. There is a need for specification of optimal test parameters, and reliable estimates of clinical sensitivity and specificity of these tests in unselected patients. Additional evidence is also needed to guide the choice of MSI and/or IHC in tumour testing of endometrial cancers. Cost considerations almost certainly rule out use of both tests in population screening programmes and some advocate the use of IHC alone,13 but the available information on proficiency testing for IHC suggests some cause for concern about quality (67,74 and www.nordiqc.org). Evaluation is also needed of the clinical validity and cost-effectiveness of criteria (such as methylation testing, or IHC results combined with features such as age at onset and family history) to exclude some women with abnormal tumour test results from germline DNA testing.

For germline DNA testing, optimum protocols for mutation detection should be determined, especially for the PMS2 gene. As massively parallel next-generation sequencing technologies begin to enter the clinical arena, the potential implications for the cost (and therefore availability) of clinical DNA sequencing will also need to be taken into account.

If screening programmes for newly diagnosed endometrial cancer patients become more widely implemented, further evidence is needed on the acceptability of tumour and germline DNA testing to these women, the likelihood that those who test positive will comply with advice on risk reduction, and the counselling of individuals with suggestive tumour testing results, but in whom no Lynch syndrome mutation is found. Recent evidence suggests that the risks associated with MMR mutation-negative “Lynch-like syndrome” may be intermediate between average population risks and risks in those with confirmed Lynch syndrome.123

Finally, there is a need for health-economic analysis that, as well as using appropriate assumptions (as discussed above), takes into account the costs and benefits of genetic counselling, mutation testing, surveillance and preventive interventions in the relatives of the index case.

http://currents.plos.org/genomictests/article/genetic-testing-strategies-in-newly-diagnosed-endometrial-cancer-patients-aimed-at-reducing-morbidity-or-mortality-from-lynch-syndrome-in-the-index-case-or-her-relatives/feed/ 0
Use of the Corus® CAD Gene Expression Test for Assessment of Obstructive Coronary Artery Disease Likelihood in Symptomatic Non-Diabetic Patients http://currents.plos.org/genomictests/article/use-of-the-corus-cad-gene-expression-test-for-assessment-of-obstructive-coronary-artery-disease-likelihood-in-symptomatic-non-diabetic-patients/ http://currents.plos.org/genomictests/article/use-of-the-corus-cad-gene-expression-test-for-assessment-of-obstructive-coronary-artery-disease-likelihood-in-symptomatic-non-diabetic-patients/#respond Mon, 26 Aug 2013 10:10:28 +0000 http://currents.plos.org/genomictests/?post_type=article&p=22041

Clinical Scenarios

Coronary artery disease (CAD) and its clinical sequelae, including myocardial infarction and heart failure, are the leading causes of morbidity and mortality in the developed and developing world. Symptoms consistent with CAD are common, variable and quite diverse with significant gender-specific differences and overlap with other common conditions. Symptomatic patients are often first seen by primary care physicians who determine if a referral to a cardiology service is warranted, prior to investigation of other causes for the symptoms. In cardiology practices, current practice guidelines suggest non-invasive imaging for medium risk patients and invasive coronary angiography for high risk patients 1. Despite the utilization of non-invasive imaging in most patients prior to ICA, the yield for obstructive CAD was <40% in a recent large national study 2. In addition, the prevalence of positive myocardial perfusion imaging studies, has decreased significantly over the last two decades, from 40% to 10% in a recent report 3. Ruling out obstructive CAD in symptomatic women is particularly problematic as currently used diagnostic tests, such as EKG, myocardial perfusion imaging (MPI), and echocardiography, perform less well in women than in men 4. The Agency for Healthcare Research and Quality highlighted the need for a better diagnostic test for women in a recent report by stating that physicians evaluating women at low to intermediate risk of CAD may want to rule-out disease with a non-invasive test with a high negative predictive value 5. In addition, despite the common use of stress testing prior to coronary angiography, practice utilization varies significantly across different regions 6.

Test Description

When a clinician suspects obstructive CAD as a cause of patient symptoms, a standard venous blood draw is performed into a PAXgeneTM RNA preservation tube (Pre-analytix, Valencia, CA). The sample and accompanying test requisition form are sent under temperature controlled conditions to the CLIA and College of American Pathologist’s certified CardioDx laboratory. The sample is then accessioned, and RNA purification, cDNA synthesis, and quantitative real-time polymerase chain reaction (qRT-PCR) are performed. Test results are calculated based on the age and sex of the patient and the expression levels of the 23 genes in the Corus CAD algorithm and reported on a 1-40 scale 7. The test algorithm differs between men and women based on age-dependent risk, specific genes, and relative gene weighting 7. Increasing score is associated with increasing likelihood of obstructive CAD and increasing disease burden. A pre-specified threshold of ≤15 has been prospectively evaluated as a low risk boundary. Approximately 95% of samples received result in a valid test result and of more than 40,000 patients evaluated since launch, 47% have scores below the pre-specified threshold of ≤15.

Public Health Importance/Prevalence

The evaluation of undiagnosed stable but symptomatic chest pain is associated with as many as 2% of all office visits or 2-3 million visits to primary care outpatient clinics each year in the United States 8,9,10. Patients with symptoms suggestive of CAD undergo extensive non-invasive and invasive testing to exclude the presence of CAD as the cause and, in the process are exposed to risks of iatrogenic side effects such as those from ionizing radiation and contrast dye. The annual U.S. cost of non-invasive imaging tests used in the cardiac work-up of stable symptomatic patients is approximately $7 billion 11,12. Despite the significant resources expended only 10% of patients presenting to primary care with chest pain are ultimately diagnosed with stable obstructive CAD 8.

Recommendations by independent groups. As part of the Clinical Laboratory Improvement Act (CLIA) licensure process, the analytical and clinical validation data for the Corus CAD test were independently evaluated by reviewers from the California and New York Departments of Publish Health. In both cases these reviews resulted in positive recommendations and the CardioDx laboratory is licensed in all 50 states. In addition, as of April, 2013, the CardioDx laboratory is now accredited by the College of American Pathologists (CAP).

Guidelines. A recent policy statement from the American College of Cardiology and American Heart Association discussed the role of genetics and cardiovascular disease treatment and diagnosis but did not address gene expression as is measured in the Corus CAD Test 13. An earlier scientific statement suggested: “Gene expression profiling has potential application in clinical practice once specific molecular and clinically meaningful CVD signatures are developed” 14.

Recent Independent Review Articles. A number of recent independent review articles have described the scientific work underpinning the Corus CAD test 15,16. A very recent review article in Nature Reviews Cardiology was solely focused on Corus CAD 17.

Evidence Overview

Analytical Validity

A large study utilizing more than 800 whole blood control samples was performed to assess the intra and inter-batch variability and inherent reproducibility of the Corus CAD test in the CardioDx commercial laboratory as a function of time, reagent batches, operators, and equipment 18. A total of 11 variables were assessed for their contribution to inter-batch variability, including four individual steps in the process (RNA purification, cDNA synthesis, sample addition, and qRT-PCR) across multiple operator, equipment, and reagent lots. Intra-batch variability estimated from 132 samples was 0.092 Cp units, dominated by inherent PCR stochastic variance, and represented approximately 70% of overall variance. Inter-batch variability was estimated across 895 samples over a two-year time frame; the largest sources of variances were reagent lots, and the overall variance was 0.11 Cp units. A comparison of overall process variability to biological dynamic range across 21,000 clinical samples showed that the biological variability was more than 10 times the process variability, demonstrating that the signal to noise was excellent. Overall process variance standard deviation corresponded to 1 unit on the Corus CAD 1-40 scale, corresponding to a clinically insignificant 1.7% change in obstructive CAD likelihood.

Clinical Validity

Two prospective multi-center trials evaluated the performance of the Corus CAD test across populations of different disease prevalence. The PREDICT trial evaluated test performance in a patient population (N=526) referred for invasive coronary angiography, the gold standard for obstructive disease evaluation 19. Disease prevalence was 37%, as measured by core laboratory quantitative coronary angiography, and very similar to that observed in a very large registry study 2. A gender specific analysis of the PREDICT results showed the obstructive CAD prevalence in women was only 22%, indicating a need for better non-invasive diagnostic tools specifically in women 20.

The COMPASS study evaluated test performance in symptomatic patients (N=431) referred for myocardial perfusion imaging, a procedure used prior to angiography 21. The gold standard was a combination of either invasive angiography and CT-angiography, both determined in core laboratories, so that all patients, independent of their MPI results, had gold standard data on their coronary anatomy. Obstructive CAD prevalence was only 15%, lower than seen in PREDICT. Positive MPI scans were seen in 11% of patients, very similar to that seen in a recent study reporting MPI positivity over the last two decades of 10% 3. Results of the two Corus CAD validation studies representing 58 centers in the US were very consistent.

– The primary endpoint of the area under the receiver-operating characteristics curve (AUC for ROC) analysis for discriminating patients with and without obstructive CAD (50% stenosis by quantitative angiography or core-lab CT-angiography) yielded AUCs of 0.70 and 0.79, for PREDICT and COMPASS, respectively (p<0.001 in both cases).

– In both studies Corus CAD demonstrated excellent sensitivity (85 and 89%) and moderate specificity (43 and 52%), respectively, at a threshold of ≤15 which was derived from the PREDICT study and pre-specified for the COMPASS study

– Corus CAD showed high negative predictive values of 83 and 96%, respectively, in the PREDICT and COMPASS studies, consistent with the differences in obstructive CAD prevalence.

– In the subset of PREDICT patients who had MPI and in the entire COMPASS study Corus CAD showed superior diagnostic performance to MPI driven by much greater sensitivity and diagnostic accuracy (ROC curve AUC). In the COMPASS study the AUCs were 0.79, 0.59, and 0.63 for Corus CAD, site-read, and core-lab read MPI, respectively.

– In both studies increasing Corus CAD score was significantly associated with increasing maximum percent stenosis.

– In both studies clinical follow-up for subsequent revascularization and major adverse cardiovascular events was performed. In PREDICT this was for 1 year post-index catheterization and showed a very significant association of Corus CAD score and the composite revascularization and event endpoint 22. In the COMPASS trial 6 month follow-up also showed significantly fewer revascularization and events with low (≤15) Corus CAD scores 21.

Clinical Utility

Three studies of the clinical utility of Corus CAD have been reported: a multi-center retrospective chart review in primary care, a prospective single center study of change in behavior in cardiology, and a prospective multi-center change in behavior study in primary care.

Retrospective Chart Review in Primary Care

To document the impact of Corus CAD in real-world primary care practice, a retrospective chart review study was completed in four primary care practices currently using Corus CAD, located in Arizona, Georgia, Louisiana, and North Carolina 23. A total of 317 patients who presented to four primary care physician sites with signs and symptoms suggestive of obstructive CAD and underwent Corus CAD testing from January 2011 to September 2011 were determined to be evaluable by medical records review and were included in this retrospective study. The objective of this study was to determine if there was a relationship between Corus CAD score and referral decision to the cardiologist: specifically, if patients with low Corus CAD scores were less likely to be referred to a cardiologist than patients with non-low scores.

In this study, 41% (129/317) of the Corus CAD patients had low scores (≤15), a rate consistent with the broader commercial population receiving the test and the COMPASS clinical trial population. Based upon physician self-reported referral rates, the expected referral rate to cardiology was 56.5%. The data show that the average referral rate to a cardiologist following Corus CAD testing was reduced to 30% (p<0.001). In addition, the referral rate was just 9% (12/129) in the Corus CAD low scoring patient population. In multivariate analysis, after controlling for age, gender, type of symptoms, and practice site, patients with low Corus CAD scores had a relative reduction in referral likelihood of 73% (p=0.01).


The IMPACT-Cardiology (Investigation of a Molecular Personalized Coronary Gene Expression Test on Cardiology Practice Pattern)(IMPACT-CARD) trial sought to assess the impact of Corus CAD use on clinical decision-making during the assessment of stable chest pain patients in the cardiology setting. The study included a prospective cohort of 83 patients eligible for analysis. These patients were referred to six cardiologists for evaluation of suspected CAD in the Vanderbilt University health care system and were matched by clinical factors to 83 patients in a historical cohort 24. The IMPACT-Cardiology protocol was designed to evaluate and compare the cardiologists’ diagnostic strategies before and after receiving the Corus CAD results for their patients. Clinicians performed a pre-Corus CAD assessment of patients’ CAD probability and noted their preliminary management decision (no intervention/medical management, referral for non-invasive imaging, or referral for invasive angiography). This pre-Corus CAD assessment was compared to physicians’ assessment of CAD probability after seeing the Corus CAD result (post-Corus CAD assessment) and determining a final management decision.

In this study following communication of Corus CAD results, a change in diagnostic testing (e.g. myocardial perfusion imaging, CTA and cardiac catheterization) was noted in 48 patients [58%, 95% CI (46%, 69%)]. More patients had a decreased versus increased level of testing (n=32 (39%) vs n=16 (19%), p=0.03). In particular, 91% (29 of 32) of patients with decreased testing had low Corus CAD (≤ 15), while 100% (16 of 16) of patients with increased testing had elevated Corus CAD (p<0.001). The most common change was among patients considered for referral to non-invasive imaging or invasive angiography prior to the Corus CAD test who were then referred to either no intervention or medical management after receiving a low Corus CAD score. Furthermore, none of the patients with low scores (≤15) saw an increase in testing.

The IMPACT-CARD trial demonstrated that among patients with a low Corus CAD score, the management decisions of cardiologists change, leading to a decrease in non-invasive cardiac imaging and invasive angiography.


The IMPACT-PCP (Investigation of a Molecular Personalized Coronary Gene Expression Test on Primary Care Practice Pattern) trial assessed the impact of Corus CAD use on clinical decision-making around the assessment of patients with symptoms of obstructive CAD in the primary care setting. The study included a prospective cohort of 251 patients, eligible for analysis, assessed by 8 community based practitioners at four sites. Clinicians performed a pre-Corus CAD assessment of patients’ CAD probability and noted their preliminary management decisions (no intervention/medical management, referral for non-invasive imaging, or referral for invasive angiography). This pre-Corus CAD assessment was compared to the clinician’s assessment of CAD probability after seeing the Corus CAD results (post-Corus CAD assessment) and determining a final management decision.

In this study, a change in diagnostic testing (e.g. myocardial perfusion imaging, CTA and cardiac catheterization) was noted in 145 patients following Corus CAD testing (58% observed vs 10% expected change, p<0.001). More patients had decreased (n=93, 37%) versus increased (n=52, 21%) intensity of testing (p<0.001). In particular, among the 127 low score Corus CAD patients (51% of study patients), 60% (76/127) had decreased testing, and only 2% (3/127) had increased testing. After more than 30 days of follow-up of 247 (98%) patients, there has been one MACE event (hemorrhagic stroke in a low score Corus CAD patient) reported.

In summary, Corus CAD was associated with a statistically significant and clinically relevant change in clinical decision-making among patients evaluated for suspected symptomatic CAD. In addition, the utilization of Corus CAD showed clinical utility above and beyond conventional decision-making by optimizing the patient’s diagnostic evaluation, particularly around the reduction in the intensity of diagnostic testing among low Corus CAD patients.

Systematic Evidence Reviews

Palmetto Government Benefits Administrators (Palmetto, GBA), the CMS Medicare Administrative Contractor with oversight for Corus CAD, has published its assessment of the test. This review determined that the test meets standards for analytical and clinical validity, and clinical utility and is a reasonable and necessary Medicare benefit, effective January 1, 2012 25.

Overall Analysis of Evidence. The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) working group has published a framework for evaluation of evidence of genomic testing, comprising analytical and clinical validity and clinical utility 26. For Corus CAD with respect to analytic validity, an extensive study demonstrating very good score reproducibility and the ability to result >95% of samples, suggests a Level 1 evidence category in our judgement, for the performance of the test in the CardioDx clinical laboratory. For clinical validity it is our assessment that the two prospective multi-center trials with core laboratory definition of disease status, representing almost 1000 patients, also correspond to Level 1 evidence according to EGAPP criteria 26. The clinical utility data from the IMPACT and retrospective chart review studies are of relatively small size and limited follow-up suggesting a level 2-3 evidence determination.


Although the results of the evaluation of the Corus CAD test are very promising, its results should be interpreted carefully as patients with diabetes mellitus and chronic inflammatory or autoimmune disorders were excluded from test development and validation. Furthermore, this test was derived and tested in predominantly Caucasian patient populations. Given the known variations in the prevalence of CAD in different ethnic/racial backgrounds 27, results of this test in non-Caucasian populations should be interpreted with caution.


The Corus CAD test has been extensively evaluated since it was first derived, including with two prospective multi-center trials. Given the scope of the deleterious effects of CAD and the considerable costs involved in diagnosing obstructive CAD, a blood test that can help in this determination is certainly valuable. The Corus CAD test promises to have an important role in this regard particularly if it continues to perform this well in larger, more diverse cohorts.

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SCN1A Genetic Test for Dravet Syndrome (Severe Myoclonic Epilepsy of Infancy and its Clinical Subtypes) for use in the Diagnosis, Prognosis, Treatment and Management of Dravet Syndrome http://currents.plos.org/genomictests/article/scn1a-genetic-test-for-dravet-syndrome-severe-myoclonic-epilepsy-of-infancy-and-its-clinical-subtypes/ http://currents.plos.org/genomictests/article/scn1a-genetic-test-for-dravet-syndrome-severe-myoclonic-epilepsy-of-infancy-and-its-clinical-subtypes/#respond Thu, 25 Apr 2013 10:24:13 +0000 http://currents.plos.org/genomictests/?post_type=article&p=21183

Clinical Scenario

Test for epilepsy syndromes associated with mutations in the SCN1A gene including the severe infantile onset epilepsies- typical Dravet syndrome (severe myoclonic epilepsy in infancy) and its borderline subtypes. Dravet syndrome typically presents in the first year of life with prolonged febrile and non-febrile, generalised clonic or hemiclonic epileptic seizures in children with no pre-existing developmental problems. Other seizure types including myoclonic, focal and atypical absence seizures appear between the ages of one and four years. The epilepsy is usually refractory to standard anti-epileptic medication and from the second year of life affected children develop an epileptic encephalopathy resulting in cognitive, behavioural and motor impairment. Seizure types within Dravet syndrome such as status epilepticus may be life threatening and sudden unexpected death in epilepsy can occur. Despite the phenotypic variability within the typical and borderline forms they are now all classified together as Dravet syndrome.

  • Referrals made by paediatric neurologists, neurologists, epileptologists, paediatricians, clinical geneticists.
  • Sample processed for SCN1A mutation screening.
  • Target population includes those with electroclinical phenotype of Dravet Syndrome or clinical sub-types – several seizure types in one individual with onset in infancy, refractory to medication and with generalised spike and wave on EEG or infants less than 1 year with 2 or more prolonged hemiclonic febrile seizures in early infancy.
  • The estimated likelihood of detecting an SCN1A mutation in a typical Dravet case is 80-90%.
  • This test is for use in the diagnosis, prognosis, treatment and management of Dravet Syndrome.
  • OMIM number for disease #607208, #182389, #604403
  • Gene – name and description – SCN1A, Sodium channel, neuronal type 1, alpha subunit
  • OMIM number for Gene *182389
  • Technical Method (s) Bi-directional DNA sequencing
  • Multiplex Ligation-dependent Probe Amplification (MLPA)

Test Description

Peripheral blood sample required.

Diagnostic testing methodologies:(DNA sequencing) Mutation scanning in single direction confirmed in opposite direction and again in an exon specific separate assay. All primers are SNP and BLAST alignment checked. All mutations identified in a previous preliminary project were confirmed using this methodology. This methodology is well established in the laboratory for many disorders.

(MLPA) Use of an MLPA kit designed specifically to pick up deletions and duplications in the SCN1Agene. Exon 21 deletion control identified amongst 10 normal control samples, assay repeated to validate results. This methodology is well established in the laboratory for other disorders.

Public Health Importance

The estimated incidence of the disease in the UK population is difficult to ascertain as historically this group of epilepsy syndromes have been excluded from epidemiological studies as they have been difficult to diagnose in electro-clinical terms 1 . A recent study based on a UK birth cohort suggested an incidence of at least 1 in 40,000 live births for SCN1A positive Dravet syndrome and 1 in 29.000 for Dravet syndrome as a whole 2 . Dravet syndrome has been misdiagnosed as whooping cough vaccine damage or pertussis encephalopathy3 .

Where the mutation is inherited the inheritance pattern is autosomal dominant but most cases are found to be de novo. Familial cases most commonly arise in Genetic Epilepsy with Febrile Seizures plus (GEFS+). The majority of cases are sporadic and the great value of this test is providing an early diagnosis and allowing appropriate treatment. Penetrance is difficult to estimate.

A confirmed diagnosis has implications for treatment stragegies and genetic counseling. It can save many additional costly and invasive investigations. When a diagnosis confirms or supports a clinical suspicion, medication changes may result 4. Anti-epileptic medications such as carbamazepine, lamtrigine and pheytoin can worsen seizures in Dravets syndrome whereas there is evidence from placebo controlled trials that a medication called stirpentol in combination with valproate and clobazam may reduce seizures. Infants with Dravet syndrome suffer from developmental regression and there is good evidence that some of this is due to uncontrolled seizures and abnormal EEG activity (an epileptic encephalopathy). There is clinical justification and evidence from recent research on adults with the syndrome to hope that controlling seizures will reduce the cognitive impairment associated with the syndrome 5.

The clinical features of Dravet Syndrome develop over several years so without the support of molecular genetic testing the diagnosis may not be made until 2-4 years of age. By this time the child may have suffered years of uncontrolled seizures and already have significant cognitive impairment 2 .

Published Reviews, Recommendations and Guidelines

Systematic evidence reviews: None identified

Recommendations by independent group: UKGTN – Gene Dossier

Guidelines by professional groups: None identified

Evidence overview

Analytic Validity: Test accuracy and reliability in measuring analytes or other entities measured (analytic sensitivity and specificity).

Diagnostic testing methodologies:(DNA sequencing) Mutation scanning in single direction confirmed in opposite direction and again in an exon specific separate assay. All primers are SNP and BLAST alignment checked. All mutations identified in a previous preliminary project were confirmed using this methodology. This methodology is well established in the laboratory for many disorders.

(MLPA) Use of an MLPA kit designed specifically for the SCN1A gene. Exon 21 deletion control identified amongst 10 normal control samples, assay repeated to validate results. This methodology is well established in the laboratory for other disorders.

Validation: Clinical Molecular Genetic Society (CMGS) Trainee project: 6/6 mutations were identified in “blind” analysis using conformation sensitive capillary electrophoresis (CSCE). A further panel of 20 patients with varied infantile epileptic encephalopathies, referred from a consultant paediatric neurologist, were screened using CSCE and DNA sequencing. Results were confirmed by bi-directional sequencing.

Analytical Sensitivity is estimated at >98% for bi-directional sequencing, 99.5% when MLPA included based upon our own laboratory test performance experience.

Clinical Validity: Test accuracy and reliability in supporting clinical or public health assessment.

In all Dravet syndrome cases the clinical sensitivity is around 80%, rising to 90% in typical Dravet syndrome cases. In our series about 10% of individuals classified as typical Dravet syndrome were not found to have an SCN1A mutation.

The negative predictive value is estimated to be low. Approximately 1/100 of our patients thought to have SMEI/related syndrome (based on clinical, and electro-clinical data) were found not to have an SCN1A mutation. This is most likely to be due to allelic heterogeneity particularly for the related syndromes.

The negative predictive value is estimated to be low. Approximately 1/100 of our patients thought to have SMEI/related syndrome (based on clinical, and electro-clinical data) were found not to have an SCN1A mutation. this is most likely to be due to allelic heterogeneity particularly for the related syndromes.

Clinical Utility: Net benefit of test in improving health outcomes

When a pathogenic mutation is identified the diagnosis can be made and/or confirmed (i.e. some patients are so young that their epilepsy phenotype has not fully evolved enough for a clinical diagnosis to be made). A confirmed diagnosis has implications for treatment strategies and genetic counselling. It can save many additional costly and invasive investigations. When a genetic diagnosis confirms or supports a clinical suspicion, medication changes may result. Anti-epileptic medications such as carbamazepine, lamotrigine and phenytoin can worsen seizures in Dravet Syndrome whereas there is evidence from placebo controlled trials that a medication called stiripentol in combination with valproate and clobazam may reduce seizures. Infants with Dravet syndrome suffer from developmental regression and there is good evidence that some of this is due to the uncontrolled seizures and abnormal EEG activity (an epileptic encephalopathy). There is clinical justification and evidence from recent research on adults with the syndrome to hope that controlling seizures will reduce the cognitive impairment associated with the syndrome 5 .

We undertook a review of our service using questionnaires to ask carers and physicians their views on genetic testing. 187 carers and 163 physicians responded.

In the carers of the mutation positive group, 87% reported genetic testing helpful, 55% said it led to a change in treatment resulting in fewer seizures. 41% described other changes including improved access to therapies and respite care. In 48%, physicians reported that testing facilitated diagnosis earlier than with clinical and EEG data alone. Molecular testing prevented additional investigations in 67% of cases, altered treatment approach in 69%, helped medication choice in 74% and through medication change improved seizure control in 42%. Carer and physician views correlated significantly with regard to the clinical utility of genetic testing. In addition to confirming a clinical diagnosis, SCN1A genetic testing enabled early diagnosis, influenced treatment-choice and facilitated access to additional therapies in a significant proportion of cases4.

UKGTN Testing Criteria: Minimum criteria required for testing to be appropriate as stated in the Gene Dossier.

Electroclinical Phenotype of Dravet Syndrome or clinical subtypes – several seizure types in one individual with onset in infancy, refractory to medication and with generalised spike and wave on EEG OR Infants less than 1 year with 2 or more prolonged hemiclonic febrile seizures in early infancy


  • UKGTN Homepage: http://www.ukgtn.nhs.uk/gtn/Home
  • UKGTN Gene Dossier: http://www.ukgtn.nhs.uk/gtn/Information/Services/Gene+Dossiers
  • UKGTN Testing criteria: http://www.ukgtn.nhs.uk/gtn/Information/Services/Testing_Criteria;
  • GeneReviews: http://www.ukgtn.nhs.uk/gtn/Search+for+a+Test/Search+by+Disease+or+Gene

Competing interests

The authors have declared that no competing interests exist.

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Use of the Afirma® Gene Expression Classifier for Preoperative Identification of Benign Thyroid Nodules with Indeterminate Fine Needle Aspiration Cytopathology http://currents.plos.org/genomictests/article/use-of-the-afirma-gene-expression-classifier-for-preoperative-identification-of-benign-thyroid-nodules-with-indeterminate-fine-needle-aspiration-cytopathology/ http://currents.plos.org/genomictests/article/use-of-the-afirma-gene-expression-classifier-for-preoperative-identification-of-benign-thyroid-nodules-with-indeterminate-fine-needle-aspiration-cytopathology/#respond Mon, 11 Feb 2013 16:50:06 +0000 http://currents.plos.org/genomictests/?post_type=article&p=21197

Clinical Scenario

Thyroid nodules are common and typically benign. However, given that 5-10% of nodules are malignant, current practice guidelines recommend evaluation with ultrasound followed by fine needle aspiration (FNA) biopsy for most clinically significant thyroid nodules.1,2,3 Most diagnostic FNA biopsies are read as cytopathologically benign or malignant, but 15%-30% remain indeterminate.2 Most patients with indeterminate lesions (defined in the Bethesda System as Atypia of Undetermined Significance or Follicular Lesion of Undetermined Significance, suspicious for Follicular or Hürthle Cell Neoplasm and suspicious for malignancy) are referred for a diagnostic thyroid surgery.4 Approximately three-quarters of these nodules are ultimately found to be benign on final surgical pathology.5,6

In 2011, it is estimated that more than 450,000 thyroid FNAs were performed. In that same year, approximately 48,020 primary thyroid malignancies were diagnosed.7 In order to avoid diagnostic surgery on benign thyroid nodules with indeterminate FNA cytopathology, pre-operative FNA-based genomics tests should predict a risk of malignancy comparable to the risk of malignancy in a cytologically benign nodule that is resected (“approximately 5% or less”).8 At this level of risk, physicians can confidently recommend clinical and sonographic monitoring in lieu of thyroid resection as they do for cytologically benign nodules under current clinical management schemas.9 Recent reviews have evaluated known gene mutation marker panels associated with thyroid malignancy and the Afirma GEC towards this end.10,11,12 A recent meta-review of a panel of somatic mutation markers associated with malignancy such as BRAF, RAS, RET/PTC, and PAX8/PPARgamma found sensitivity to be too low (63.7%) to achieve a high enough negative predictive value (NPV) to recommend monitoring when these mutations are absent.13 The Afirma GEC employs a different approach analyzing the mRNA expression of 167 genes with high enough sensitivity (92%) in indeterminate cytology lesions to identify the signature of a benign thyroid nodule with 95% NPV: that is, similar to the risk of malignancy in a resected thyroid nodule with a preoperatively benign FNA cytopathology diagnosis.14,15

Test Description

When needle passes are made for cytologic analysis of sonographically suspicious thyroid nodules, two dedicated passes also are made for Afirma GEC analysis and immediately stored in nucleic acid preservative solution. If the FNA cytopathology is nondiagnostic, benign, or malignant, the sample collected for the Afirma GEC is discarded. If the FNA cytopathology is indeterminate, the Afirma GEC sample undergoes RNA extraction and nucleic acid amplification. Processed Afirma GEC samples are hybridized to a custom Afirma Thyroid microarray and analyzed with a classification algorithm using linear support vector machine logic to produce either a “Benign” or “Suspicious” test result. About 10% of FNA samples have inadequate RNA yield or quality and are reported by the Afirma GEC as “No Result”.16

Public Health Importance

The incidence of thyroid cancer in the U.S. has risen dramatically. In 2009, there were 37,200 new cases of thyroid cancer, while in 2013, it is anticipated there will be 60,220 new cases.17,18 At the same time, there has been an increase in the utilization of thyroid FNA and subsequent thyroid surgery.19 The prevalence of thyroid nodules increases with age and is more common in females. Approximately 50% of women ≥50 years have at least one thyroid nodule based on published ultrasound and autopsy series.20 Two thirds of thyroid nodules have benign FNA cytopathology and monitoring is implemented, whereas those with indeterminate or malignant cytology are generally referred for surgery. Because thyroid nodules with indeterminate FNA cytopathology have a 25% risk of malignancy when resected, 75% of these operations will likely be on nodules determined to be benign post-operatively.5,6 Thyroid surgery is associated with potential complications, including temporary and permanent hypocalcemia, recurrent laryngeal nerve injury (with voice change, dysphagia, and potentially airway compromise if bilateral), and bleeding, with an incidence as high as 2-10%.21,22,23 While there is strong evidence that high volume thyroid surgeons on average have fewer complications than low volume counterparts, 50% of thyroid operations in the U.S. are still performed by surgeons who perform ≤5 thyroidectomies/year.24 Hypothyroidism is an expected sequelae of thyroid surgery, with patients requiring life-long thyroid hormone supplementation or replacement therapy.

Published Reviews, Recommendations and Guidelines

Systematic evidence reviews. Palmetto Government Benefits Administrators (Palmetto GBA), the CMS Medicare Administrative Contractor with oversight for the Afirma GEC, has published its assessment of the test as an update to its local coverage article on molecular diagnostics. This review determined that the test meets criteria for analytical and clinical validity, and clinical utility as a reasonable and necessary Medicare benefit, effective January 1, 2012.25

Recommendations by independent groups. As part of the CLIA Laboratory licensure process, the analytical and clinical validation data for the Afirma GEC were independently assessed by reviewers from the California Department of Public Health and the New York State Department of Health.26,27 Both of these reviews resulted in a favorable licensure outcome.

Guidelines by professional groups. The National Comprehensive Cancer Network (NCCN) thyroid carcinoma guidelines were updated in December, 2012 to state “Molecular diagnostics may be useful to allow reclassification of follicular lesions (follicular neoplasm or follicular lesion of undetermined significance) as more likely to be benign or more likely to be malignant…If molecular testing predicts a risk of malignancy comparable to the risk of malignancy seen with a benign FNA cytology (approximately 5% or less), consider observation.” The NCCN guidelines for abnormal gene/gene expression profile testing are associated with Level of Evidence 2A (lower level evidence, uniform NCCN consensus that the intervention is appropriate).8,28

Evidence Overview

Analytical Validity: test accuracy, reliability in measuring differences in expression of relevant genes (analytic sensitivity and specificity), and robustness.

• Building on an earlier study by Chudova et al.14, a large collaborative study by Walsh et al. reviewed over 30 sub-studies on the Afirma GEC, finding high analytic sensitivity and specificity after dilution of thyroid neoplasm FNA samples with adjacent normal tissue or benign neoplasms (such as nodular hyperplasia and lymphocytic thyroiditis), as well as dilution with blood and genomic DNA, respectively.14,29

• High reproducibility was found in studies of interlaboratory concordance (R2 0.98), as well as intra-assay, inter-assay, and intra-nodule concordance (R2 0.99, 0.98, and 0.95, respectively).29

• The assay was robust to a wide range of temperature, storage and stressed shipping conditions and was reproducible across different operators, runs, and reagent lots with routine use of control reagents/samples for in-process Quality Control monitoring.29

Clinical Validity: test accuracy in correctly determining which indeterminate cytology FNA biopsies are benign compared to expert surgical histopathology.

• Two prospective multicenter studies evaluated the negative predictive value (NPV) for the Afirma GEC, which is the key diagnostic performance metric used to make a decision to monitor patients in lieu of referral for diagnostic thyroid surgery.14,15 Both studies utilized diagnosis of the surgical pathology specimen by a central panel of blinded academic endocrine histopathologists as the reference standard for clinical validation. Approximately one quarter of study sites were academic and three quarters were community-based (total sites n=49).

• In both studies, NPV was >94% for cytologically indeterminate thyroid FNAs with atypia/follicular lesions of undetermined significance and follicular/Hürthle cell neoplasm diagnoses, but lower when the cytology was suspicious for malignancy. Overall, the NPV for the Afirma GEC was similar to the NPV for a resected thyroid nodule with benign cytopathology.5,30

• In the second, larger study15, although the NPV was 95% for all indeterminate cytology nodules (at the 24% risk of malignancy expected in clinical practice), the NPV was 85% for FNAs with a cytopathology diagnosis suspicious for malignancy. Sensitivity was 92% for indeterminate nodules overall. Clinical specificity for indeterminate nodules rose from 0% for cytopathology alone to 52% with the Afirma GEC, meaning that half of the benign nodules with indeterminate cytopathology could be identified with this pre-operative test.

Clinical Utility: net benefit of test in improving health outcomes by allowing recommendation for monitoring instead of a diagnostic surgery on benign thyroid nodules.

• Duick et al. was a retrospective analysis based on chart review of 368 patients (395 indeterminate thyroid nodules) cared for by 51 physicians at 21 practice locations in 11 states. When compared to historical controls, the relative rate of operation on cytologically indeterminate nodules fell 90%, from 74% historically to 7.6% with Afirma GEC benign results (p < 0.001).16 The comparisons between historical controls and operative rates based on the binomial test achieved more than 99% power in detecting a less than 25% change.31 With Afirma GEC benign results, in 92.4% of the cases, physicians recommended watchful waiting in lieu of a diagnostic thyroid resection. The residual rate of surgery was similar to the historic 9% resection rate on cytologically benign thyroid nodules and may relate to other clinical parameters and issues of patient and physician preference, including issues related to nodule size.5

• These results were consistent with a previously conducted web- and mail-based opinion survey of 84 physician practices, with a mean of 89% of physicians reporting that they recommended watchful waiting for patients with cytologically indeterminate FNAs but benign Afirma GEC results.32

Limitations. In the clinical validity studies, thyroid FNAs with indeterminate cytology diagnoses that were cytologically suspicious for malignancy did not have sufficiently low NPV to generally recommend monitoring.10 Secondly, the Duick et al clinical utility study used historical rather than contemporary controls.16 Although historical controls were used, these were appropriately validated based on a meta-review of 11 recent thyroid pathology studies where thyroid nodule evaluation was similar to current community practice.5


The clinical validity studies reviewed would fall into the Level 1 category in the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) hierarchy of data sources and study designs, based on two prospective, multicenter, double blinded cohort studies.33 A validated clinical decision rule was based on classification concordance of Afirma GEC benign results with blinded expert surgical pathology benign diagnosis. 11 Sensitivity in most indeterminate FNAs was high enough (92%) to achieve a NPV of 94-95%, which is comparable to a thyroid nodule that is benign on cytopathology but undergoes surgical resection (93-94%).5,30 However, the Afirma GEC NPV for FNAs where the cytopathology was suspicious for malignancy was only 85%. While this lowers the residual risk of malignancy from 62% to 15%, surgical consultation still should be planned in these patients. The test utilized interlaboratory comparisons in a large collaborative study, an EGAPP criterion for Level 1 hierarchy of analytic validity study design.34 Improvement in the net health outcome is based on avoidance of surgery in patients with indeterminate thyroid FNA cytology that would have been found to be benign on surgical pathology. Clinical utility of the Afirma GEC was evaluated in clinical practice, outside of the investigational setting, in an opinion survey and a controlled study consistent with EGAPP Level 3 criteria for clinical utility study design. The Afirma GEC potentially can improve the net health outcome by providing an alternative to diagnostic thyroid surgery, and therein the risk of downstream complications of surgery, in patients with benign thyroid nodules but indeterminate FNA cytopathology. In summary, the studies reviewed here regarding clinical and analytic validity, and clinical utility support recommendation for offering patients the alternative of using the Afirma GEC to monitor patients in lieu of thyroid resection when applied in the specific case of thyroid FNAs where there is indeterminate cytopathology: atypia/follicular lesion of undetermined significance, and follicular/Hürthle cell neoplasm.

Competing interests

Dr. Lanman is an employee of Veracyte, Inc. The other co-authors have no conflict of interest.

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Genetic testing for long QT syndrome and the category of cardiac ion channelopathies http://currents.plos.org/genomictests/article/genetic-testing-for-long-qt-syndrome-and-the-category-of-cardiac-ion-channelopathies/ http://currents.plos.org/genomictests/article/genetic-testing-for-long-qt-syndrome-and-the-category-of-cardiac-ion-channelopathies/#respond Thu, 03 May 2012 18:12:53 +0000 http://currents.plos.org/genomictests/?post_type=article&p=21021

Clinical Scenario

In patients with fainting (especially occurring during physical exertion or emotional/auditory arousal), seizures, a history of aborted cardiac arrest, a family history of sudden death, or who have themselves succumbed to sudden cardiac death (SCD), cardiac ion channel mutation testing, typically in conjunction with electrocardiography, can provide important information. Performed with cardiac evaluation, genetic testing may be used to determine the status of long QT syndrome (LQTS), short QT syndrome (SQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and Brugada syndrome (BrS) genes from blood, saliva, or tissue specimens, including postmortem samples123. While these conditions demonstrate characteristic electrocardiographic patterns (LQTS – a prolonged QT interval on resting ECG (part of the Schwartz-Moss diagnostic score)4; CPVT – ventricular ectopy5; BrS – ST segment elevation2), these findings may be absent or inconclusive on a single clinical test. Similarly, post-mortem samples may be accompanied by insufficient clinical data for diagnostic classification. Genetic testing has been used to exclude one condition over another and to provide definitive diagnosis in many such situations167. For the patient whose symptoms, ECG, and/or other adjunctive tests58 have led to a clinical diagnosis, the test can identify pertinent risks and assist management. In addition, relatives of affected patients, who may have no symptoms but are capable of passing on an abnormal gene, can be identified through techniques like cascade screening1369. Therapies for patients displaying mutations include avoidance of physical triggers and aggravating drugs, placement on anti-arrhythmic drug or beta blocker therapy10111213, use of mechanical devices (pacemakers, ICDs)2, and sympathectomy surgery4.

Test Description

Cardiac ion channelopathies result from adverse alterations in genes that code for protein subunits of cardiac ion channels1. The literature differentiates these channelopathies in terms of their subtypes (e.g., for long QT syndrome, LQT1, LQT2, LQT3, LQT4, LQT5, LQT6, …, LQT13) and the name of the gene affected (KCNQ1 for LQT1; KCNH2 for LQT2; SCN5A for LQT3; ANK2 for LQT4; KCNE1 for LQT5; KCNE2 for LQT6, …, KCNJ5 for LQT13). Genetic testing is available through a number of commercial and university-based genetic diagnostics laboratories1141516. Transgenomic (formerly PGx Health and Genaissance), GeneDx, and Correlagen all offer genetic assays allowing genotyping of LQTS, SQTS, CPVT, and BrS. For example, panels offered by Transgenomic17 and GeneDx18 test for 13 and 12 LQTS genes, respectively, KCNQ1, KCNH2, andSCN5A (LQT1-3) mutations being most frequent; 3 SQTS genes – mutations in the KCNH2, KCNQ1, andKCNJ2 genes; 4 (Transgenomic) and 2 (GeneDx) CPVT genes, RYR2 mutations being most frequent; and 7 BrS genes, SCN5A mutations being most frequent. The Masonic Medical Research Laboratory covers these conditions for research purposes, but specializes in acquired as opposed to congenital long QT syndrome19. Samples are collected in an EDTA-containing tube; the DNA is isolated from fresh whole blood. DNA amplification by polymerase chain reaction (PCR) is then used to generate templates for direct sequencing. DNA, frozen blood, saliva, and other tissue samples such as buccal specimens have recently been accommodated by some labs1718. Detailed results are provided for each test panel, typically with a description of known literature on any identified mutation and its likelihood to cause disease. Focused testing for a single mutation relevant to a patient’s family, rather than a panel of potentially involved genes, is available at a lower cost.

Public Health Importance

The public health importance of these inherited arrhythmia syndromes is highlighted by their potential lethality, mostly due to ventricular tachyarrhythmias20. Long QT syndrome may be responsible for as many as 3,000 unexpected deaths in children and young adults annually in the United States21. Catecholaminergic polymorphic ventricular tachycardia carries a high mortality in untreated cases57. The current state of technology, however, does not enable arrhythmia screening of broad, potentially at-risk populations – athletes, all children, all patients exposed to QT-prolonging drugs, or all patients with a history of syncope616. Subpopulations of sudden death victims are of great interest. It is estimated that approximately 25-35% of autopsy-negative or unexplained sudden cardiac death in the young, and 10% of sudden infant death (SIDS) may be attributable to mutations in either LQTS or CPVT susceptibility genes22. Brugada syndrome may be responsible for 4-15% of unexpected sudden deaths, particularly in individuals with an apparently normal heart232425.

Table of Cardiac Ion Channelopathies
Long QT syndrome (LQTS) 1726272875 Short QT syndrome (SQTS) 171829 Catecholaminergic polymorphic ventricular tachycardia (CPVT) 5173031 Brugada syndrome (BrS) 1732333435
Prevalence 1:5,000 to 1:2,000 Rare – fewer than 30 cases published 1:7,000 to 1:10,000 1:800 (Japan); 1:6,000 (U.S. & Europe) type 1 ECG pattern
Annual mortality rate 0.3% (LQT1)
0.6% (LQT2)
0.56% (LQT3)
Unidentified 3.1% 4% (pts. with type 1 ECG pattern)
Mean age of first event 14 ± 12 yrs. 40 ± 24 yrs. 15 ± 10 yrs. 42 ± 16 yrs. (pts. with type 1 ECG)
Diagnostic Yield – Genetic Testing 75-80% 15-20% 65-75% 11-40% (see Clinical Validity)

In the case of LQTS, the specific genotype has prognostic value16. In studies ranging from 250 to 300,000 genotyped individuals through age 60 drawn from the International LQTS Registry, the LQT3 genotype has demonstrated a 5- to 8-fold higher risk for life-threatening events compared to the LQT1 and 2 genotypes36. The LQT2 genotype displays a risk intermediate between LQT1 and LQT336, though the statistical strength and ordering of this relationship depends on age, sex, and source of the cohort1037. Subgroup analyses have more precisely defined the impact of QTc duration and genotype in children and adolescents with LQT1 and 238.

Published Reviews, Recommendations and Guidelines

Systematic evidence reviews

The Hayes Inc. Genetic Test Evaluation (GTE) Program has prepared three evaluations of ion channelopathies: long QT syndrome, CPVT, and Brugada syndrome. These reviews were based on studies of primary literature retrieved from PUBMED and Embase in the following date ranges: LQTS – January 1, 1996 to June 16, 2009; CPVT – January 1, 1996 to February 10, 2010; and Brugada syndrome – January 1, 1996 to August 3, 201039. DNA Direct, Inc. has published two systematic reviews of LQTS genetic testing through its Genomic Medicine Institute. The reviews, which the company makes available with interactive tools and decision support, are based on studies of primary patient data published between 2001 and 200840. BlueCross BlueShield Association and Kaiser Permanente published a systematic review of genetic testing for long QT syndrome through the BlueCross BlueShield Technology Evaluation Center (BCBS Tec)41. The review was based on studies of primary patient data appearing in Medline and PUBMED published between 1990 and October 2007. The Australia and New Zealand Horizon Scanning Network released a Horizon Scanning Report on genetic testing for congenital long QT syndrome based principally on nine peer-reviewed articles, and detailing safety (e.g., false negatives), effectiveness, clinical utility (cost-effectiveness), and ethical (consent and privacy, harms from testing, access) considerations42. Investigative teams have published comprehensive clinical-epidemiologic reviews of both long QT syndrome as a family and several of the other channelopathies under Human Genome Epidemiology (HuGE) reviews (e.g., long QT syndrome21) and GeneReviews (e.g., Brugada syndrome43).

Recommendations by independent groups

Two independent reviews have concluded that genetic testing has diagnostic value, including for the identification of asymptomatic heterozygotes, for all four syndromes; definitive prognostic value for LQTS and weak or contingent prognostic value (depending on the nature of the findings) for CPVT and BrS; and practical value in the determination of therapy for LQTS alone130. One of the teams also published an “evidence-based” genetic testing scoring system to compare the relative clinical value of genetic testing over these conditions (LQTS > CPVT > BrS > SQTS)44. SQTS’s low score is associated with paucity of data, this condition having first been described in 200015. Criteria on when to screen for each channelopathy, based on consensus documents and primary literature from 1995 onward, are laid out by Tzou and Gerstenfeld15. This review concluded that genetic testing can lead to genotype-specific therapy in the case of LQTS, and decisions about ICD placement for malignant arrhythmias associated with CPVT and SQTS15.

Guidelines by independent groups

A 2007 consensus report by the U.S. National Heart, Lung, and Blood Institute and the Office of Rare Diseases on gene mutations affecting ion channel function concluded that genetic testing for LQTS must be combined with clinical evaluation, and noted lack of clarity in the proportion of SQTS cases that might be explained by the corresponding KCNH2, KCNJ2, and KCNQ1 genes45. A 2011 Heart Rhythm Society (HRS) / European Heart Rhythm Association (EHRA) consensus statement further states that LQTS genetic testing is recommended for any asymptomatic patient with idiopathic (not attributable to QT prolonging disease states or conditions) QTc values > .48 s. (prepuberty) or > .50 s. (adult), and may be considered for QTc values >= .46 and .48, respectively6. (QTc = “heart rate-corrected QT interval,” as per the Bazett formula4.) The Heart Rhythm UK Familial Sudden Death Syndromes Statement Development Group published in 2008 a position statement on genetic testing for sudden cardiac death syndromes based on a comprehensive review of English language publications, grading of the evidence, and secondary review of the evidence by an external committee3. The Group followed with a position statement on ICD placement for these conditions based on risk of SCD46. The first position statement and the more recent HRS/EHRA report recommend genetic testing for all patients with a firm diagnosis of congenital LQTS and those with clinical features of CPVT (due to its severity, despite an acknowledged lower clinical sensitivity), but that expert clinical and family history assessment are needed when genetic testing is undertaken for borderline LQTS cases and known or suspected cases of BrS. Practice guidelines from the American College of Cardiology / American Heart Association / European Society of Cardiology47 have noted an evolving role for genetic testing of LQTS in risk stratification and clinical decision making. Both independent reviews and professional society guidelines agree that genetic testing by itself is not recommended in making a diagnosis or prognosis for BrS, though it may be used to support clinical diagnosis, and early detection of at-risk relatives62548. Several HRS / EHRA consensus statements clarify that genetic testing can be useful in patients clinically suspected of having BrS with a type 1 (“coved” ST segment elevation) ECG pattern, but that it is not indicated in the setting of an isolated type 2 (less specific, “saddleback” ST elevation) or 3 (either shape but less pronounced elevation) pattern62025.

Evidence Overview

Analytic Validity:

Commercially available channelopathy genetic testing (CGT) for LQTS, SQTS, CPVT, and BrS involves direct sequencing of protein-coding portions and flanking regions of targeted exons following PCR amplification. Sequencing is performed in both forward and reverse directions. Sequences are analyzed for heterozygous and homozygous variants using public reference sequences1741. For the FAMILION® test, variants detected in the initial analysis are confirmed by repeating the sequencing twice in the forward and three times in the backward direction. Transgenomic reports a CGT error rate of < 1% 17. GeneDx indicates a 98% “technical sensitivity” for assessment of each of the four conditions18. The John Welsh Cardiovascular Laboratory at Baylor College of Medicine makes available genetic diagnostic testing using DNA sequencing analysis for KCNJ2 (LQT7) and CAV3 (LQT9) mutations. This facility reports approximately 99% detection of the exons that are sequenced49. Failure to detect variants in the laboratory setting is attributed to refractoriness of the amplicon to analysis by direct DNA sequencing or real-time PCR detection, sample mishandling, sample tracking errors, errors in data analysis, and other gene specific issues (see Clinical Validity)17. The FAMILION® analytic specificity approaches 100% for “Class I” (deleterious or probably deleterious) mutations, and approximates 95% for “Class II” mutations (of uncertain clinical significance – possibly deleterious)41.

Clinical Validity:

The near perfect analytic sensitivities of these assays must be contrasted with the ability to detect genetic variants in the clinical setting. The yield for the first 2,500 consecutive unrelated cases referred by physicians for commercially available long QT syndrome genetic testing (low-, intermediate-, and high-pretest probability) was 36%; values in the literature range between 33 and 39%50. Tester et al. reported greater yields of 72 to 78% using the 5 major LQTS genes for patients with the highest clinical probability for LQTS (Schwartz-Moss score >= 4 4)37. Transgenomic reports a 75 to 80% yield for such patients based upon multiple data sets1751. False negatives may be explained by a number of factors, including the existence of private mutations37, the presence of non-targeted exons17 and relevant introns outside tested splice sites16, and the existence of large deletion and duplication mutations (generally on the scale of a whole exon or more)717. LQTS is in the upper range among cardiac conditions in terms of number of affected genes (13) and allelic mutations (>800) while BrS lies in the midrange (8 genes and >400 allelic mutations). By comparison, current compendia of arrhythmogenic right ventricular dysplasia/cardiomyopathy list 9 affected genes and >400 allelic mutations, while Marfan syndrome has ~600 allelic mutations in the FBN1 fibrillin gene.

Authors have reported various mutational hotspots in the case of LQTS arising either independently or due to founder effects, which because of their severity may aid risk stratification21. Interpretation of results for probands and family members is complicated by variable penetrance in family members with the same genotype52, and the possibility of compound mutations, observed in 4 to 10% of mutation positive individuals37505354. Estimated positive predictive values (EPV – percent of mutations found in definite cases that would cause the condition) for LQTS genetic testing based on an assessment of > 1300 unrelated index cases with Schwartz score >= 4 or QTc >= .48 s. are: 96% (95% C.I., 94-98) for KCNQ1; 93% (89-95) for KCNH2; and 63% (40-77) for SCN5A55. Nonmissense mutations have an EPV > 99% regardless of location55. EPVs for missense mutations range from 0% in the interdomain linker of SCN5A to 100% in the transmembrane/linker/pore regions of KCNH2. Similar figures are lacking for the other channelopathies.

Several groups have investigated ECG genotyping (to be distinguished from use in initial diagnosis) through T-wave morphology as a means of reducing the cost of LQTS mutational genotyping565758. Sensitivity is highly dependent on the gene being assessed, and varies between 92% (LQT2) and 47% (LQT3)5758. QTc interval alone lacks predictive value for genotyping5657.

For channelopathy genetic testing (CGT) of the other inherited arrhythmia syndromes, Transgenomic reports a clinical sensitivity of 65-75% (CPVT), 25-40% (BrS), and 15-20% (SQTS)17. GeneDx18 and the Correlagen CardioGeneScan®59 also test for these conditions. The GeneDx and Correlagen test information / FAQ sheets provide rough estimates of clinical sensitivity; figures from Correlagen are also reported with the assays. Some commercial laboratories only provide CPVT screening of a limited number of select exons encompassing critical RYR2 regions, which can contribute to false negatives30. Disagreement exists on the impact of combining other variables with mutational results in order to increase clinical sensitivity; results vary by arrhythmia syndrome, genotype, and size of the study population102760.

Mutational analysis of 27 SCN5A exons on cases from BrS databases at 9 international centers resulted in yields of 11-28%3543, lower than the 25-40% figure Transgenomic reports174360. The literature suggests that for BrS about a quarter of the patients or fewer carry an SCN5A mutation (commercial assays also include other lower frequency mutations)1225. The low clinical sensitivity of genetic testing for Brugada syndrome, due to the low gene frequency for the most prevalent type of mutation (involving SCN5A), incompleteness of known allelic variants, and the role of an organic substrate in many cases, limits its diagnostic capability156162. Genetic testing for BrS is more often used for confirmatory purposes1. Higher yields for the various channelopathies accrue in families with at least one recorded case of sudden unexpected death, permitting detection of potentially affected relatives24.

Clinical Utility:

Genetic analysis has been found useful in risk stratification of LQTS patients and detection of at-risk family members, though current knowledge of CPVT genetic variants does not in itself does not yield prognostic information15222730364148. “Intragenic risk stratification” based on mutation type and location, and cellular function, particularly for the LQT1 and 2 genotypes, has been an increasing part of large scale studies but is not yet in widespread practice1611414763. A meta-analysis of 30 Brugada syndrome prospective studies by Gehi et al.64 concluded family history of SCD and presence of an SCN5A mutation by themselves are insufficient to predict risk for cardiac events in BrS. Instead, debates about risk stratification for patients with this condition have taken place largely on the electrocardiographic front65. The presence of spontaneous ST-segment elevation, particularly with a type-1 pattern, in conjunction with a history of syncope remains the strongest predictor of BrS risk2.

Mutational information can serve as an adjunct to clinical and phenotypic assessment of genotype in therapeutic decisions for LQTS303648, though this role is not without controversy1012164166. Clear evidence exists for genotype-specific therapy in management of the LQT1, 2, and 3 genotypes, but it is less substantiated for the other LQTS genotypes due to their rarity111213366768. Disagreement exists on the use of intragenic, site-specific information to predict actual clinical phenotype or response to therapy for the LQTS genotypes1137. In analyzing retrospective data on 27 of 128 LQTS patients in the 3 to 13 year-old age range who received either pacemakers or ICDs as therapy, investigators in a 3-area study (Utah, British Columbia, and Arizona) found no association between device placement decisions and implementation of genetic testing. However, the authors also noted that all patients with SCN5A (LQT3) mutations had therapeutic devices, though only 1 of 30 with a KCNQ1 (LQT1) mutation had one69. The use of genetic testing in therapeutic decision making for the other inherited arrhythmia syndromes is not yet substantiated but appears promising16304870. ECG remains the principal tool for BrS diagnosis and therapeutic follow-up21625.

Several teams have evaluated step-wise or tiered strategies also including phenotypic assessment to increase the efficiency of LQTS and CPVT genetic testing17537172. Targeted screening based on phenotypic information can lower the cost of more comprehensive LQTS genetic testing by ~60% 71. Phillips et al. found LQTS genetic testing more cost-effective than not testing for symptomatic index cases at an estimated cost of $2,500 per year of life saved ($50,000 per year of life saved is often used as a standard threshold)7374. Bai et al., in looking at 546 patients referred to a large consortium of LQTS research laboratories, found the highest yield (64%) and lowest cost ($8,418 per positive genotyping) for patients with ECG-confirmed LQTS, but diminishing yields and increasing costs for patients with borderline QTc intervals and those with normal intervals but a positive family history for SCD60. They recommended genetic testing be prioritized to those with a “conclusive diagnosis” of LQTS. Genotyping of individuals with a conclusive diagnosis of CPVT, and of patients with type 1 BrS ECG with atrioventricular block was also found to be cost-effective. Apart from this report, the host of cost-effectiveness analyses for Brugada syndrome deal with the issue of implantation of ICDs.

Perez et al. used a Markov model to assess the cost-effectiveness of different strategies for testing then treating an asymptomatic 10 year-old first degree relative of a patient with clinically evident LQTS75. They concluded that genetic testing is moderately expensive, at $67,400 per quality adjusted life year saved, but improves with higher clinical suspicion of the proband, number of relatives tested, and stronger family history of sudden death.

CGT is covered to different extents by insurance providers, ranging from denial to 100% coverage, with most covering at least 50 to 75% of the cost22. Some health plan policies explicitly limit genetic test reimbursement to LQTS while excluding the other channelopathies76.

Public Health Ethical, Legal, and Social (PHELSI) Considerations:

Genetic testing for cardiac channelopathies is laden with ethical issues, including the search for a balance between individual privacy vs. alerting at-risk family members, as well as psychosocial issues inherent in informing individuals of their risk17778. A review by Wren provides general recommendations for genetic testing of children in families with a history of SCD, and offers ethical considerations in childhood genetic testing for Brugada syndrome79. Use of pacemakers and ICDs in children, while often done under situations of medical necessity, can involve trade-offs between benefit and adverse effects456980. Screening for LQTS mutations in particular racial-ethnic groups deserves further ethical analysis21. Personalization of drug treatment through determination of individually specific genotype remains a hoped-for future direction in the field175. Risk assessment for inherited heart arrhythmias is also becoming a part of direct-to-consumer genetic testing, an area subject to increasing attention by policy makers.


The commercial and university-based laboratories cited above are approved under the Clinical Laboratory Improvement Amendments (CLIA). Several, but not all, are accredited by the College of American Pathologists (CAP). These assays are developed and validated in-house, thus do not require FDA approval.

Relevant web sites:

  • IRCCS Fondazione Salvatore Maugeri Molecular Cardiology Laboratories. Gene Connection for the Heart Inherited Arrhythmias Database. www.fsm.it/cardmoc. Up-to-date compendium of inherited arrhythmia syndrome (cardiac ion channelopathy, arrhythmogenic right ventricular cardiomyopathy, and others) mutations and polymorphisms, and condition synopses.
  • Heart Rhythm Society. www.hrsonline.org. International society concerned with education and advocacy for cardiac arrhythmia professionals and patients. Web site describes and provides access to professional educational programs, clinical guidelines and consensus statements relating to diagnosis and management, and relevant legislation.
  • Cardiac Arrhythmias Research and Education (CARE) Foundation. www.longqt.com. Advocacy and awareness-raising organization aimed at preventing sudden cardiac death due to acquired and heritable heart rhythm disorders. Web site reports professional educational resources, and details emerging advocacy issues, support groups, and genetic testing laboratories.
  • Sudden Arrhythmia Death Syndromes Foundation. www.sads.org. Aimed at preventing sudden and unexpected cardiac death in children and young adults, the Foundation’s web site describes public awareness-raising activities, advocacy initiatives impacting patients and professionals, and patient and family support services.
  • Drugs that Prolong the QT Interval and/or Induce Torsades de Pointes Ventricular Arrhythmia. www.qtdrugs.org. Web site provides a comprehensive list of arrhythmogenic drugs to be avoided by LQTS patients, lists drugs by risk group, provides consumer education tools, and describes the drug-induced arrhythmias case registry.
  • Raymond Brugada Senior Foundation, www.brugada.org. Foundation web site provides a full description of Brugada syndrome, cites relevant literature and professional policies, and offers avenues for partnering in research and joining a support group.
  • BrugadaDrugs.org. www.brugadadrugs.org. Web site provides a comprehensive list of arrhythmogenic drugs to be avoided by Brudaga syndrome patients, lists drugs by risk group, cites drugs diagnostic for Brs, and offers a patient letter listing drugs to be avoided.

Competing Interests

The authors have declared that no competing interests exist.

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