Margot G. van Lier

Prospective evaluation of molecular screening for Lynch syndrome in patients with endometrial cancer ≤70 years

OBJECTIVE Lynch syndrome (LS) is a hereditary syndrome that predisposes to multiple malignancies including endometrial cancer (EC). We aimed to evaluate a diagnostic strategy for LS based on routine analysis of microsatellite instability (MSI) and immunohistochemical (IHC) staining for mismatch repair (MMR) proteins in tumour tissue of all newly diagnosed EC patients ≤ 70 years. METHODS Consecutive EC patients ≤ 70 years were included prospectively in eight Dutch centres. EC specimens were analysed for MSI, IHC of four MMR proteins, MMR gene methylation status and BRAF-mutations. tumours were classified as; 1) likely to be caused by LS, 2) sporadic MSI-H, or 3) microsatellite stable (MSS). RESULTS Tumour specimens of 179 patients (median age 61 years, IQR 57-66) were analysed. In our study 92% of included patients were over 50 years of age. Eleven EC patients were found likely to have LS (6%; 95% CI 3-11%), including 1 patient suspected of an MLH1, 2 of an MSH2, 6 of an MSH6 and 2 of a PMS2 gene defect. Germline mutation analyses revealed 7 MMR gene germline mutations. Ten patients likely to have LS (92%) were older than 50 years. In addition, 31 sporadic MSI-H tumours with MLH1 promoter hypermethylation (17%; 95% CI 13-24%) were identified. CONCLUSIONS Molecular screening for LS in patients with EC diagnosed ≤ 70 years, leads to identification of a profile likely to have LS in 6% of cases. New screening guidelines for LS are needed, including recommendations for EC patients older than 50 years of age.

Somatic aberrations of mismatch repair genes as a cause of microsatellite‐unstable cancers

Lynch syndrome (LS) is caused by germline mutations in mismatch repair (MMR) genes, resulting in microsatellite‐unstable tumours. Approximately 35% of suspected LS (sLS) patients test negative for germline MMR gene mutations, hampering conclusive LS diagnosis. The aim of this study was to investigate somatic MMR gene aberrations in microsatellite‐unstable colorectal and endometrial cancers of sLS patients negative for germline MMR gene mutations. Suspected LS cases were selected from a retrospective Clinical Genetics Department diagnostic cohort and from a prospective multicentre population‐based study on LS in The Netherlands. In total, microsatellite‐unstable tumours of 40 sLS patients (male/female 20/20, median age 57 years) were screened for somatic MMR gene mutations by next‐generation sequencing. In addition, loss of heterozygosity (LOH) of the affected MMR genes in these tumours as well as in 68 LS‐associated tumours and 27 microsatellite‐unstable tumours with MLH1 promoter hypermethylation was studied. Of the sLS cases, 5/40 (13%) tumours had two pathogenic somatic mutations and 16/40 (40%) tumours had a (likely) pathogenic mutation and LOH. Overall, LOH of the affected MMR gene locus was observed in 24/39 (62%) tumours with informative LOH markers. Of the LS cases and the tumours with MLH1 promoter hypermethylation, 39/61 (64%) and 2/21 (10%) tumours, respectively, demonstrated LOH. Half of microsatellite‐unstable tumours of sLS patients without germline MMR gene mutations had two (likely) deleterious somatic MMR gene aberrations, indicating their sporadic origin. Therefore, we advocate adding somatic mutation and LOH analysis of the MMR genes to the molecular diagnostic workflow of LS. Copyright

Endoscopic therapy of small-bowel polyps by double-balloon enteroscopy in patients with Peutz-Jeghers syndrome

BACKGROUND Peutz-Jeghers syndrome (PJS) is a hereditary disorder characterized by mucocutaneous pigmentations and hamartomatous polyps mainly in the small bowel. These polyps may cause complications such as intussusception. OBJECTIVE To assess therapeutic efficacy and safety of double-balloon enteroscopy (DBE) for detection and treatment of small-bowel polyps in patients with PJS. DESIGN Prospective cohort study. SETTING Tertiary-care referral center. PATIENTS This study involved 13 patients with PJS, defined as a proven STK11 gene mutation or according to international diagnostic criteria. INTERVENTION DBE with enteroscopic removal of pedunculated polyps of > or =10 mm. MAIN OUTCOME MEASUREMENTS Location, number and size of small-bowel polyps, polypectomy data, and complications and long-term complications associated with development of small-intestine polyps. RESULTS Thirteen patients with PJS (8 male, mean age 31 years) underwent 29 DBE procedures. Ten patients (77%) had a history of partial small-bowel resection because of small-bowel polyps. Small-bowel polyps were found in all 13 patients. The majority of polyps (94%) were located in the proximal jejunum. A total of 82 polyps of > or =10 mm were detected, and 79 (96%) were endoscopically removed without complications. After the introduction of DBE, no small-intestine-polyp-related complications occurred during a follow-up period of 356 person-months. LIMITATIONS Small number of patients. CONCLUSION DBE is clinically useful and safe for diagnosis and therapy of small-bowel polyps in patients with PJS, even in patients with a history of extensive abdominal surgery. DBE may decrease the need for laparotomy in patients with PJS.

Yield of routine molecular analyses in colorectal cancer patients ≤70 years to detect underlying Lynch syndrome†

Although early detection of Lynch syndrome (LS) is important, a considerable proportion of patients with LS remains unrecognized. We aimed to study the yield of LS detection by routine molecular analyses in colorectal cancer (CRC) patients until 70 years of age. We prospectively included consecutive CRC patients ≤70 years. Tumour specimens were analysed for microsatellite instability (MSI), immunohistochemical mismatch‐repair protein expression and MLH1‐promoter methylation. Tumours were classified as either: (a) likely caused by LS; (b) sporadic microsatellite‐unstable (MSI‐H); or (c) microsatellite‐stable (MSS). Predictors of LS were determined by multivariable logistic regression. A total of 1117 CRC patients (57% males, median age 61 years) were included. Fifty patients (4.5%, 95% CI 3.4–5.9) were likely to have LS, and 71 had a sporadic MSI‐H tumour (6.4%, 95% CI 5.1–8.0). Thirty‐five patients likely to have LS (70%) were aged > 50 years. A molecular profile compatible with LS was detected in 10% (15/144) of patients aged ≤50, in 4% (15/377) of those aged 51–60 and in 3% (20/596) of patients > 61 years. Compared to MSS cases, patients likely to have LS were significantly younger (OR 3.9, 95% CI 1.7–8.7) and more often had right‐sided CRCs (OR 14, 95% CI 6.0–34). In conclusion, molecular screening for LS in CRC patients ≤70 years leads to identification of a molecular profile compatible with LS in 4.5% of patients, with most of them not fulfilling the age criterion (≤50 years) routinely used for LS assessment. Routine use of MSI testing may be considered in CRC patients up to the age of 70 years, with a central role for the pathologist in the selection of patients. Copyright

Pancreatic cancer risk in Peutz-Jeghers syndrome patients: a large cohort study and implications for surveillance

Background Although Peutz-Jeghers syndrome (PJS) is known to be associated with pancreatic cancer (PC), estimates of this risk differ widely. This hampers counselling of patients and implementation of surveillance strategies. We therefore aimed to determine the PC risk in a large cohort of Dutch PJS patients. Methods PJS was defined by diagnostic criteria recommended by the WHO, a proven LKB1 mutation, or both. All patients with a presumptive diagnosis of pancreatic, ampullary or distal bile duct cancer were identified. Cases were reviewed clinically, radiologically and immunohistochemically. Cumulative PC risks were calculated by Kaplan-Meier analysis and relative risks by Poisson regression analysis. Results We included 144 PJS patients (49% male) from 61 families (5640 person years follow-up). Seven (5%) patients developed PC at a median age of 54 years. Four patients (3%) were diagnosed with distal bile duct (n=2) or ampullary cancer (n=2) at a median age of 55 years. The cumulative risk for PC was 26% (95% CI 4% to 47%) at age 70 years and relative risk was 76 (95% CI 36 to 160; p<0.001). The cumulative risk for pancreatico-biliary cancer was 32% (95% CI 11% to 52%) at age 70 years, with a relative risk of 96 (95% CI 53 to 174; p<0.001). Conclusions PJS patients have a highly increased risk for pancreatico-biliary cancer. Therefore, patients are eligible for surveillance within well defined research programmes to establish the benefit of such surveillance.

A review on the molecular diagnostics of Lynch syndrome: A central role for the pathology laboratory

•  Lynch syndrome •  Identification of patients at risk for Lynch syndrome •  Molecular basis of Lynch syndrome and sporadic MMR‐deficient tumours •  Molecular diagnostics of Lynch syndrome •  Description of molecular analyses ‐  MSI analysis ‐  Immunohistochemistry ‐  MLH1 promoter hypermethylation assay ‐  BRAF mutation analysis •  Limitations of molecular analyses •  Conclusions

Underutilization of microsatellite instability analysis in colorectal cancer patients at high risk for Lynch syndrome.

Objective. The revised Bethesda Guidelines were published to improve the efficiency of recognizing Lynch syndrome (LS) by identifying LS-related malignancies that should be analyzed for microsatellite instability (MSI). The aim of this study was to evaluate whether MSI analysis was performed in colorectal cancer patients at risk for LS according to the revised Bethesda Guidelines. Material and methods. Patients diagnosed with colorectal cancer in 11 Dutch hospitals in 2005 and 2006 were selected from a regional database. The patients were included in the study if they met any of the following criteria; 1) diagnosed with colorectal cancer <50 years, 2) a second LS-associated tumor prior to the diagnosis of colorectal cancer in 2005/2006, and 3) colorectal cancer <60 years with a tumor displaying mucinous or signet-ring differentiation or medullary growth pattern. Results. Of 1905 colorectal cancer patients, 169 met at least one of the inclusion criteria. MSI analysis had been performed in 23 (14%) of the 169 tumors. MSI status had been determined in 18 of 80 included patients aged <50 years, in 4 of 70 patients with a second LS-related tumor, and in 3 of 41 patients aged <60 years with high-risk pathology features. Conclusions. There is marked underutilization of MSI analysis in patients at risk for LS. As a result LS might be underdiagnosed both in patients with colorectal cancer and in their relatives.

Peutz-Jeghers syndrome and family planning: the attitude towards prenatal diagnosis and pre-implantation genetic diagnosis

Peutz–Jeghers syndrome (PJS) is a hereditary disorder caused by LKB1 gene mutations, and is associated with considerable morbidity and decreased life expectancy. This study was conducted to assess the attitude of PJS patients towards family planning, prenatal diagnosis (PND) and pregnancy termination, and pre-implantation genetic diagnosis (PGD). In a cross-sectional study, 61 adult PJS patients were asked to complete a questionnaire concerning genetic testing, family planning, PND and PGD. The questionnaire was completed by 52 patients (85% response rate, 44% males) with a median age of 44 (range 18–74) years. A total of 37 (71%) respondents had undergone genetic testing. In all, 24 respondents (46%, 75% males) had children. A total of 15 (29%) respondents reported that their diagnosis of PJS had influenced their decisions regarding family planning, including 10 patients (19%, 9/10 females) who did not want to have children because of their disease. Termination of pregnancy after PND in case of a foetus with PJS was considered ‘acceptable’ for 15% of the respondents, whereas 52% considered PGD acceptable. In conclusion, the diagnosis of PJS influences the decisions regarding family planning in one third of PJS patients, especially in women. Most patients have a negative attitude towards pregnancy termination after PND, while PGD in case of PJS is judged more acceptable. These results emphasise the importance of discussing aspects regarding family planning with PJS patients, including PND and PGD.

High cancer risk and increased mortality in patients with Peutz-Jeghers syndrome response

We thank Dr Vande Casteele and colleagues for their interest in our work and commend them for their interesting case. 2 As written in our manuscript, we have had several, and in some cases serial, measurements of infliximab and antidrug antibody (ADA) levels in the patients reported. However, we limited the results to the last sample available before loss of response occurred to better meet the aim of the study, which was to investigate the immunogenic fragment of infliximab and its possible diagnostic role in patients having inflammatory bowel disease with loss of response. We absolutely agree that serial measurements may allow early detection of pharmacodynamic changes which possibly herald the later onset of clinical events such as loss of response or infusion reactions. The case report by Dr Vande Casteele is an interesting example of this principle and is also notable for demonstrating the variable time course of ADA formation which was detected for adalimumab at one point and later disappeared. However, we would caution that much more evidence is needed regarding the predictive power, the time course and the cost-effectiveness of routine serial measurements of the drug and ADA levels before this approach can be advocated as a clinical recommendation in all patients treated with anti-tumour necrosis factor (anti-TNF). We would also beg to differ with the authors’ assertion that ‘the lack of infliximab and adalimumab blood levels contributed to the formation of antibodies to the drug’. In fact, most data indicate that it is the formation of antibodies and subsequent immune complexes with the drug that hasten drug clearance, thereby leading to absent drug levels in the blood. Finally, there is no mention of concomitant immunomodulators used in the particular patient, if any. Data from the authors’ group previously demonstrated the efficacy of concomitant immunomodulators to reduce immunogenicity, at least in episodically treated patients. Moreover, we have shown that prolonged administration of 6-mercaptopurine mediated the deletion of antigen-specific T helper cells, despite repeated antigen exposures. On a conceptual level, this could suggest that addition of thiopurines may incrementally eliminate the drug-specific memory cells, reduce the titres of ADAs and thereafter restore clinical response in patients who already formed antibodies to anti-TNFs. However, it remains to be proven in clinical trials whether these mechanistic observations in murine experiments could indeed translate into an efficacious management approach for immunogenic loss of response to anti-TNFs.