Original article

Selecting patients with young-onset colorectal cancer formismatch repair gene analysis

M. Walker1, B. O’Sullivan2, B. Perakath3, P. Taniere2, D. Cruger4 and D. Morton1

Departments of 1Surgery and 2Pathology, University Hospital Birmingham NHS Trust, Birmingham, UK, 3Department of Surgery Unit I, ChristianMedical College and Hospital, Vellore, India, and 4Department of Clinical Genetics, Velije Hospital, Velije, DenmarkCorrespondence to: Mr M. Walker, University Department of Surgery, University Hospital Birmingham, Edgbaston, Birmingham B15 2TT, UK (e-mail:m.walker.2@bham.ac.uk)

Background: Young patients with colorectal cancer are at increased risk of carrying a germlinemutation in mismatch repair (MMR) genes. This study investigated the role of clinical criteria andimmunohistochemistry for MMR proteins in selecting young patients for mutation testing.Methods: A cohort of 56 consecutive patients with colorectal cancer aged less than 45 years werestratified into three groups based on clinical criteria: ‘Amsterdam criteria’, ‘high risk’ and ‘young onsetonly’. Immunohistochemistry for four MMR proteins was carried out and the rate of compliance withclinical guidelines determined.Results: Tumours from 11 patients (20 per cent) had abnormal MMR protein expression, of whom eightwere referred for genetic assessment. Of 21 patients (38 per cent) in total referred to the genetics unit,six MMR gene mutations were identified, all associated with abnormal immunohistochemistry.Conclusion: MMR immunohistochemistry should be considered routine in young-onset colorectalcancer.

Presented in part to the Association of Coloproctology of Great Britain and Ireland, Dublin, July 2005

Paper accepted 2 July 2007Published online 30 July 2007 in Wiley InterScience (www.bjs.co.uk). DOI: 10.1002/bjs.5822

Introduction

Hereditary non-polyposis colorectal cancer (HNPCC)is the commonest inherited condition predisposing tocolorectal cancer1. Originally a clinical diagnosis basedon familial clustering of colorectal cancer2, it is nowconsidered a genetic diagnosis following the identificationof germline mutations in the DNA mismatch repair (MMR)genes mutL homologue 1 (MLH1), mutS homologue 2(MSH2), MSH6 and postmeiotic segregation increased 2(PMS2)2. HNPCC is an autosomal dominant conditionand mutation carriers have an 80 per cent lifetime risk ofdeveloping colorectal cancer with a young average ageof onset. It tends to be under-recognized as, unlike thepolyposis syndromes, it has no pathognomonic features.Regular colonoscopic surveillance in HNPCC familiescan reduce colorectal cancer incidence and mortalityrate by 60 per cent at 15 years’ follow-up3, justifyinga concerted approach to diagnosing these families.To realize the benefits of colonoscopic surveillance,probands from HNPCC families need to be identified

from among all patients presenting with colorectalcancer.

Patients with young-onset colorectal cancer represent asmall proportion, but 14–17 per cent of those aged under50 years and 28 per cent aged less than 30 years carry aMMR gene mutation4–6. Routine mutation screening ofpatients with young-onset disease would be costly andlargely negative. The Amsterdam criteria7 were devisedto select patients for immunohistochemical testing andthe Bethesda guidelines8 were devised to select patientsfor investigation of MMR deficiency prior to mutationtesting, but both lack sensitivity and specificity. MMRdeficiency is found not only in over 90 per cent ofHNPCC but also in 15 per cent of sporadic tumours9.It can be tested for by either immunohistochemistry(MMR protein expression loss) or tumour DNA testingfor microsatellite instability (MSI). Immunohistochemistryis at least equivalent10 to MSI testing for detectingMMR mutations, but is more routinely available inhistopathology laboratories and is not as expensive11. This

Copyright 2007 British Journal of Surgery Society Ltd British Journal of Surgery 2007; 94: 1567–1571Published by John Wiley & Sons Ltd

1568 M. Walker, B. O’Sullivan, B. Perakath, P. Taniere, D. Cruger and D. Morton

study examined a small cohort of patients with young-onset colorectal cancer in terms of clinical criteria andMMR immunohistochemistry to assess their contributionto predicting mutations.

Methods

Between 1996 and 2003, 62 patients aged less than45 years were diagnosed with colorectal cancer. Recordsof 60 patients were available and none came fromknown HNPCC families. Four patients (one with familialadenomatous polyposis and three with colitis) wereexcluded from further analysis. Family histories wereobtained from the medical notes along with the outcomeof any referral to geneticists.

Immunohistochemistry

Immunohistochemical staining was carried out on paraffin-embedded sections which were pretreated with citratebuffer pH 6 in a microwave oven (450 W), at full power for12 min and then medium power for 40 min. Endogenousperoxidase activity was blocked with 1 per cent hydrogenperoxide in methanol. Sections were incubated overnightin primary antibody (MLH1: AbCam, Cambridge UK;MSH2, MSH6 and PMS2: BD Biosciences, Oxford,UK), at dilutions of 1 : 25, 1 : 300, 1 : 60 and 1 : 100respectively. Bound antibodies were detected with the ABCDuet streptavidin–biotin system (Dako, Copenhagen,Denmark). Diaminobenzidine was used as a chromogenand Mayer’s haematoxylin as counterstain. Staining wasassessed by two investigators who were blinded toother data. Normal positive tissue (crypt epithelium orinfiltrating lymphocytes) was used as an internal positivecontrol. The reaction in tumour cells was considerednegative if there was no staining in any of the tumourcell nuclei.

Mutation analysis

DNA was extracted from blood lymphocytes. MLH1and MSH2 were screened for mutations by denaturinghigh-performance liquid chromatography. Any variantsidentified were further examined by direct automatedsequencing. When no mutations were identified multiplexligation-dependent probe amplification was carried outto detect any genomic deletions. MSH6 was sequenceddirectly only in patients showing loss of expression ofMSH6.

Statistical analysis

Statistical analysis was performed using the χ2 test.

Results

The median age at diagnosis was 39 (range 16–45) years;other clinical details are shown in Table 1. Two patients hada personal history of HNPCC-associated tumours (bothbrain12) and three had a history of non-HNPCC-associatedtumours (melanoma, Hodgkin’s disease, neuroendocrinetumour).

Family history criteria

Thirty-nine patients (70 per cent) had no family historyof colorectal cancer, and 17 patients had between oneand five relatives diagnosed with colorectal cancer. Eightpatients had an affected first-degree relative and seven hadan affected relative aged less than 50 years. A family historycould not be provided for two patients (because of adoptionand mental illness) and was unrecorded in one patient.

All 56 patients met the first Bethesda guideline (age lessthan 50 years) of whom 30 (54 per cent) met this criteriaonly; this group was classified as ‘young onset only’. Sixpatients (11 per cent) were from families that fulfilled theAmsterdam criteria. The remaining 20 (36 per cent) metup to four of the Bethesda criteria but did not fulfil theAmsterdam criteria; these patients were identified as ‘highrisk’. Patients were further analysed in these three riskgroups.

Genetic referral

Twenty-one patients (38 per cent) had been referred tothe genetics service and this was commonest in thehigher-risk groups (Table 2) but was incomplete evenfor families meeting the Amsterdam criteria. Youngerpatients were more commonly referred (11 of 15 agedless than 35 years versus 11 of 41 aged 35 years or older;P = 0·002). Mutational analysis was carried out in tenpatients, of whom six had a pathogenic mutation (two inMSH2 and four in MLH1). Two patients did not attendtheir appointments and the remaining seven families wereclassified as moderate risk after investigation of extendedpedigrees. Following counselling, 16 at-risk relatives offive patients with mutations underwent predictive testingfor the specific mutation and seven were found to carry themutation.

Mismatch repair protein immunohistochemistry

Immunohistochemistry was carried out on 54 tumours; noblocks were available for two patients. Loss of expressionof one or more MMR proteins was identified in tumoursfrom 11 patients (20 per cent) (Table 3). The six confirmed

Copyright 2007 British Journal of Surgery Society Ltd www.bjs.co.uk British Journal of Surgery 2007; 94: 1567–1571Published by John Wiley & Sons Ltd

Mismatch repair gene analysis in colorectal cancer 1569

Table 1 Patient and colorectal tumour characteristics

No. of patientsn = 56

Sex ratio (M : F) 29 : 27Emergency presentation 13 (23)TNM stage*

1 62 173 204 12

Tumour site†Proximal 16 (29)Distal 37 (66)Both 3 (5)

Synchronous neoplasiaNone 46 (82)Cancer 3 (5)Adenoma 9 (16)

Mucinous tumours 12 (21)

Values in parentheses are percentages. *One rectal cancer was stagedpathological tumour (pT) 0 after preoperative chemotherapy. †Withrespect to splenic flexure. TNM, tumour node metastasis.

Table 2 Outcome by clinical risk stratification group

Amsterdamcriteria(n = 6)

High risk(n = 20)

Youngonset only(n = 30)

Referred for genetic assessment 4 11 6Mismatch repair deficient 4* 4* 3Mutation positive 3 2 1

*No blocks were available for immunohistochemistry in one patient.

mutation carriers showed loss of at least one MMR proteinon immunostaining. Of the other five patients, two remain

under investigation by the geneticists and three were notreferred for genetic analysis.

Predictors of mutation carriers

Comparison of the three risk groups showed that mutationsand MMR-deficient tumours were most common in theAmsterdam criteria group (Table 2). However, only halfof mutation carriers and four of 11 patients with MMR-deficient tumours met the Amsterdam criteria. Young onsetwas the only identifiable risk factor in one of six mutationcarriers and three of 11 patients with MMR-deficienttumours. All six mutation carriers had loss of at least oneMMR protein; the protein loss was concordant with themutated gene except in one patient with a mutation inMLH1 whose tumour retained expression of MLH1 butlost PMS2 expression.

Synchronous neoplasia

Synchronous adenomas were present in nine (16 per cent)of 56 patients, including three of six patients with mutations(Table 2). Adenomas occurred more commonly in patientswith MMR-deficient tumours (four of 11 versus four of43 MMR-proficient tumours; P = 0·024). Most patientshad one or two adenomas; in only two patients were theremultiple adenomas (four and 28) and both of these patientshad MMR-proficient tumours. Synchronous advancedneoplasia (cancer or adenoma larger than 1 cm) was foundin four of six mutation carriers, four of 11 patients withMMR-deficient tumours and two of 43 patients withMMR-proficient tumours.

Table 3 Characteristics of mismatch repair-deficient tumours

Riskstratification

Age(years) Sex Tumour site*

Synchronousneoplasia

MMR proteinsnot expressed Result of genetic testing

Amsterdam criteria 31 F Distal None MLH1 and PMS2 Not referredAmsterdam criteria 38 M Proximal None PMS2 MLH1; c.1668-1G > AAmsterdam criteria 44 F Synchronous† 2 cancers and 2-cm adenoma MLH1 MLH1; c. 291T > G; p.Tyr97XAmsterdam criteria 45 M Proximal 2-cm adenoma MSH2 and MSH6 Deletion MSH2 exons 9–16High risk 27 M Distal None MSH6 No mutation in MSH6High risk 35 F Proximal None MLH1 and PMS2 MLH1; c.735delC; p. Asn245fsXHigh risk 36 F Distal None MSH6 No mutation in MSH6High risk 37 F Synchronous† 2 cancers MSH2 and MSH6 MSH2; c.1566C > G; p. Tyr522XYoung onset only 21 M Distal 2 adenomas, largest 1·8 cm MLH1 and PMS2 Deletion exon 6 MLH1Young onset only 33 F Proximal None PMS2 Not referredYoung onset only 43 F Proximal One adenoma < 1 cm MLH1, MSH2 and MSH6 Not referred

*In relation to splenic flexure. †Tumours in both distal and proximal colon. MMR, mismatch repair; MLH, mutL homologue; PMS, postmeioticsegregation increased; MSH, mutS homologue; c., no. of affected nucleotides and alteration in relation to coding DNA reference sequence; p., no. andalteration of affected amino acid in relation to reference sequence.

Copyright 2007 British Journal of Surgery Society Ltd www.bjs.co.uk British Journal of Surgery 2007; 94: 1567–1571Published by John Wiley & Sons Ltd

1570 M. Walker, B. O’Sullivan, B. Perakath, P. Taniere, D. Cruger and D. Morton

Compliance with clinical guidelines

Compliance with the Bethesda guidelines was defined byeither referral for genetic assessment or testing tumoursfor MMR deficiency. Compliance was 48 per cent overall,but increased from nine of 31 before 2000 to 18 of 25 after2000 (P = 0·001), following the introduction of MMRimmunohistochemistry to the histology laboratory.

Discussion

In this cohort of patients with young-onset colorectalcancer, the presence of clinical criteria was associated withthe risk of carrying a MMR gene mutation. The strongestassociation was noted in patients with an Amsterdamcriteria-positive family history. However, three of the sixmutation-positive families would have been missed if thesepedigree-based criteria had been used alone. In contrast,loss of MMR protein expression correctly identifiedall six HNPCC families. The overall sensitivity of theAmsterdam criteria for detecting mutation carriers is only78 per cent13. In patients aged under 45 years the reportedsensitivity is 50 per cent5, falling to 21 per cent in patientsaged less than 30 years6. These studies also reported that30 per cent of young probands with MMR gene mutationshad no family history of colorectal cancer5,6. The absenceof a family history of colorectal cancer does not excludethe possibility of a MMR gene mutation, particularly inyoung patients. Complementary diagnostic approaches, inaddition to family history, are required for this group.

Strong support for the use of MMR protein immuno-histochemistry is provided by studies demonstrating sensi-tivities approaching 100 per cent for detecting MMR genemutation carriers5,10. Testing for loss of expression of therarer causative genes (MSH6, PMS2) was justified in thiscohort. Loss of expression of MSH6 or PMS2 alone wasseen in four of 11 tumours, and this is consistent withreports that up to a quarter of mutations occur in MSH6and PMS25. Testing for PMS2 also increases the rate ofdetection of MLH1 mutations14, as was demonstrated inthis series. Immunohistochemistry is potentially a morespecific test in young patients as somatic loss of expressionof MLH1 owing to promoter methylation is rare under theage of 50 years, increasing in frequency with age15. Loss ofprotein expression is therefore more likely to be due to aninherited mutation.

Referral for genetic assessment was incomplete evenfor patients whose families met the Amsterdam criteria.Clinicians may not take appropriate action in responseto a high-risk family history16. Compliance with theBethesda guidelines in this series improved following theintroduction of MMR immunohistochemistry, but was still

incomplete. These findings, along with the identificationof three patients with MMR-deficient tumours who hadnot been referred to the genetics service, suggests that asystem less dependent on one clinician could increase therate of appropriate genetics referrals. The central role ofthe surgeon in the referral of potential HNPCC familiesis demonstrated in this cohort, in which 70 per cent ofgenetics referrals came directly from the surgical clinic.

Our findings, and those of others5,10, support the useof immunohistochemistry for MLH1, MSH2, MSH6 andPMS2 in all patients with young-onset colorectal cancer,regardless of family history. It is proposed that this beperformed routinely. Positive findings could be reportedto the multidisciplinary team triggering notification of thegenetics service. The use of immunohistochemistry is likelyto be extended to other selected groups or all patients withcolorectal cancer. This is not just for selection for mutationtesting of MMR genes but also as a marker of prognosisand responsiveness to fluorouracil-based chemotherapy17.Testing tumours for MMR protein expression does notitself constitute a genetic test as its positive predictivevalue is less than 70 per cent5. Nonetheless, best practicesuggests that patients should be informed that tumourtesting may be carried out that could indicate an inheritedcondition.

References

1 Nagy R, Sweet K, Eng C. Highly penetrant heriditary cancersyndromes. Oncogene 2004; 23: 6445–6470.

2 de la Chapelle A. Genetic predisposition to colorectal cancer.Nat Rev Cancer 2004; 4: 769–780.

3 Jarvinen HJ, Aarnio M, Mustonen H, Aktan-Collan K,Aaltonen LA, Peltomaki P et al. Controlled 15-year trial onscreening for colorectal cancer in families with hereditarynonpolyposis colorectal cancer. Gastroenterology 2000; 118:829–834.

4 Katballe N, Christensen M, Wikman FP, Orntoft TF,Laurberg S. Frequency of hereditary non-polyposiscolorectal cancer in Danish colorectal cancer patients. Gut2002; 50: 43–51.

5 Southey MC, Jenkins MA, Mead L, Whitty J, Trivett M,Tesoriero AA et al. Use of molecular tumor characteristics toprioritize mismatch repair gene testing in early-onsetcolorectal cancer. J Clin Oncol 2005; 23: 6524–6532.

6 Farrington SM, Lin-Goerke J, Ling J, Wang Y, Burczak JD,Robbins DJ et al. Systematic analysis of hMSH2 and hMLH1in young colon cancer patients and controls. Am J Hum Genet1998; 63: 749–759.

7 Vasen HFA, Watson P, Mecklin J-P, Lynch HT. Newclinical criteria for hereditary non-polyposis colorectal cancer(HNPCC, Lynch syndrome) proposed by the InternationalCollaborative group on HNPCC. Gastroenterology 1999; 116:1453–1456.

Copyright 2007 British Journal of Surgery Society Ltd www.bjs.co.uk British Journal of Surgery 2007; 94: 1567–1571Published by John Wiley & Sons Ltd

Mismatch repair gene analysis in colorectal cancer 1571

8 Umar A, Boland CR, Terdiman JP, Syngal S, de laChapelle A, Ruschoff J et al. Revised Bethesda guidelines forhereditary nonpolyposis colorectal cancer (Lynch syndrome)and microsatellite instability. J Natl Cancer Inst 2004; 96:261–268.

9 Muta H, Noguchi M, Perucho M, Ushio K, Sugihara K,Ochiai A et al. Clinical implications of microsatelliteinstability in colorectal cancers. Cancer 1996; 77: 265–270.

10 Hampel H, Frankel W, Martin E, Arnold M, Khanduja K,Kuebler P et al. Screening for the Lynch syndrome(hereditary nonpolyposis colorectal cancer). N Engl J Med2005; 352: 1851–1860.

11 Pinol V, Castells A, Andreu M, Castellvi-Bel S, Alenda C,Llor X et al. Accuracy of revised Bethesda guidelines,microsatellite instability, and immunohistochemistry for theidentification of patients with hereditary nonpolyposiscolorectal cancer. JAMA 2005; 293: 1986–1994.

12 Watson P, Lynch HT. Cancer risk in mismatch repair genemutation carriers. Fam Cancer 2001; 1: 57–60.

13 Kievit W, de Bruin JH, Adang EM, Ligtenberg MJ,Nagengast FM, van Krieken JH et al. Current clinical

strategies for identification of hereditary non-polyposiscolorectal cancer families are inadequate: a meta-analysis.Clin Genet 2004; 65: 308–316.

14 de Jong AE, van Puijenbroek M, Hendriks Y, Tops C,Wijnen J, Ausems MG et al. Microsatellite instability,immunohistochemistry, and additional PMS2 staining insuspected hereditary nonpolyposis colorectal cancer. ClinCancer Res 2004; 10: 972–980.

15 Kakar S, Burgart LJ, Thibodeau SN, Rabe KG,Petersen GM, Goldberg RM et al. Frequency of loss ofhMLH1 expression in colorectal carcinoma increases withadvancing age. Cancer 2003; 97: 1421–1427.

16 Grover S, Stoffel EM, Bussone L, Tschoegi E, Syngal S.Physician assessment of family cancer history and referral forgenetic evaluation in colorectal cancer patients. ClinGastroenterol Hepatol 2004; 2: 813–819.

17 Lanza G, Gafa R, Santini A, Maestri I, Guerzoni LCavazzini L. Immunohistochemical test for MLH1 andMSH2 expression predicts clinical outcome in stage II and IIIcolorectal cancer patients. J Clin Oncol 2006; 24:2359–2367.

Copyright 2007 British Journal of Surgery Society Ltd www.bjs.co.uk British Journal of Surgery 2007; 94: 1567–1571Published by John Wiley & Sons Ltd