Objective:This study evaluated the nationwide trends in care and accompanied postoperative outcomes for patients with distal esophageal and gastro-esophageal junction cancer. Summary of Background... Show moreObjective:This study evaluated the nationwide trends in care and accompanied postoperative outcomes for patients with distal esophageal and gastro-esophageal junction cancer. Summary of Background Data:The introduction of transthoracic esophagectomy, minimally invasive surgery, and neo-adjuvant chemo(radio)therapy changed care for patients with esophageal cancer. Methods:Patients after elective transthoracic and transhiatal esophagectomy for distal esophageal or gastroesophageal junction carcinoma in the Netherlands between 2007-2016 were included. The primary aim was to evaluate trends in both care and postoperative outcomes for the included patients. Additionally, postoperative outcomes after transthoracic and tran-shiatal esophagectomy were compared, stratified by time periods. Results:Among 4712 patients included, 74% had distal esophageal tumors and 87% had adenocarcinomas. Between 2007 and 2016, the proportion of transthoracic esophagectomy increased from 41% to 81%, and neo-adjuvant treatment and minimally invasive esophagectomy increased from 31% to 96%, and from 7% to 80%, respectively. Over this 10-year period, postoperative outcomes improved: postoperative morbidity decreased from 66.6% to 61.8% (P = 0.001), R0 resection rate increased from 90.0% to 96.5% (P <0.001), median lymph node harvest increased from 15 to 19 (P <0.001), and median survival increased from 35 to 41 months (P = 0.027). Conclusion:In this nationwide cohort, a transition towards more neo-adju-vant treatment, transthoracic esophagectomy and minimally invasive surgery was observed over a 10-year period, accompanied by decreased postoperative morbidity, improved surgical radicality and lymph node harvest, and improved survival. Show less
Objective: This study investigated the patterns, predictors, and survival of recurrent disease following esophageal cancer surgery. Background: Survival of recurrent esophageal cancer is usually... Show moreObjective: This study investigated the patterns, predictors, and survival of recurrent disease following esophageal cancer surgery. Background: Survival of recurrent esophageal cancer is usually poor, with limited prospects of remission. Methods: This nationwide cohort study included patients with distal esophageal and gastroesophageal junction adenocarcinoma and squamous cell carcinoma after curatively intended esophagectomy in 2007 to 2016 (follow-up until January 2020). Patients with distant metastases detected during surgery were excluded. Univariable and multivariable logistic regression were used to identify predictors of recurrent disease. Multivariable Cox regression was used to determine the association of recurrence site and treatment intent with postrecurrence survival. Results: Among 4626 patients, 45.1% developed recurrent disease a median of 11 months postoperative, of whom most had solely distant metastases (59.8%). Disease recurrences were most frequently hepatic (26.2%) or pulmonary (25.1%). Factors significantly associated with disease recurrence included young age (<= 65 y), male sex, adenocarcinoma, open surgery, transthoracic esophagectomy, nonradical resection, higher T-stage, and tumor positive lymph nodes. Overall, median postrecurrence survival was 4 months [95% confidence interval (95% CI): 3.6-4.4]. After curatively intended recurrence treatment, median survival was 20 months (95% CI: 16.4-23.7). Survival was more favorable after locoregional compared with distant recurrence (hazard ratio: 0.74, 95% CI: 0.65-0.84). Conclusions: This study provides important prognostic information assisting in the surveillance and counseling of patients after curatively intended esophageal cancer surgery. Nearly half the patients developed recurrent disease, with limited prospects of survival. The risk of recurrence was higher in patients with a higher tumor stage, nonradical resection and positive lymph node harvest. Show less
Rayner, E.; Tiersma, Y.; Fortuno, C.; Hees-Stuivenberg, S. van; Drost, M.; Thompson, B.; ... ; Wind, N. de 2022
The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain... Show moreThe large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis-based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer-predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). Show less
IJsselsteijn, R.; Hees, S. van; Drost, M.; Jansen, J.G.; Wind, N. de 2022
A dietary mutagen induces loss of the wild-type allele in mismatch repair gene-heterozygous cells. This results in impaired DNA damage signaling and in compound hypermutagenesis. These findings... Show moreA dietary mutagen induces loss of the wild-type allele in mismatch repair gene-heterozygous cells. This results in impaired DNA damage signaling and in compound hypermutagenesis. These findings provide insights into the etiology of colorectal carcinogenesis in Lynch syndrome.The prevalent cancer predisposition Lynch syndrome (LS, OMIM #120435) is caused by an inherited heterozygous defect in any of the four core DNA mismatch repair (MMR) genes MSH2, MSH6, MLH1 or PMS2. MMR repairs errors by the replicative DNA polymerases in all proliferating tissues. Its deficiency, following somatic loss of the wild-type copy, results in a spontaneous mutator phenotype that underlies the rapid development of, predominantly, colorectal cancer (CRC) in LS. Here, we have addressed the hypothesis that aberrant responses of intestinal stem cells to diet-derived mutagens may be causally involved in the restricted cancer tropism of LS. To test this we have generated a panel of isogenic mouse embryonic stem (mES) cells with heterozygous or homozygous disruption of multiple MMR genes and investigated their responses to the common dietary mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Our data reveal that PhIP can inactivate the wild-type allele of heterozygous mES cells via the induction of either loss of heterozygosity (LOH) or intragenic mutations. Moreover, while protective DNA damage signaling (DDS) is compromised, PhIP induces more mutations in Msh2, Mlh1, Msh6 or Pms2-deficient mES cells than in wild-type cells. Combined with their spontaneous mutator phenotypes, this results in a compound hypermutator phenotype. Together, these results indicate that dietary mutagens may promote CRC development in LS at multiple levels, providing a rationale for dietary modifications in the management of LS. Show less
Schubert, S.A.; Ruano, D.; Tiersma, Y.; Drost, M.; Wind, N. de; Nielsen, M.; ... ; Wezel, T. van 2020
We describe a family severely affected by colorectal cancer (CRC) where whole-exome sequencing identified the coinheritance of the germline variants encoding MSH6 p.Thr1100Met and MUTYH p.Tyr179Cys... Show moreWe describe a family severely affected by colorectal cancer (CRC) where whole-exome sequencing identified the coinheritance of the germline variants encoding MSH6 p.Thr1100Met and MUTYH p.Tyr179Cys in, at least, three CRC patients diagnosed before 60 years of age. Digenic inheritance of monoallelic MSH6 variants of uncertain significance and MUTYH variants has been suggested to predispose to Lynch syndrome-associated cancers; however, cosegregation with disease in the familial setting has not yet been established. The identification of individuals carrying multiple potential cancer risk variants is expected to rise with the increased application of whole-genome sequencing and large multigene panel testing in clinical genetic counseling of familial cancer patients. Here we demonstrate the coinheritance of monoallelic variants in MSH6 and MUTYH consistent with cosegregation with CRC, further supporting a role for digenic inheritance in cancer predisposition. Show less
Thompson, B.A.; Walters, R.; Parsons, M.T.; Dumenil, T.; Drost, M.; Tiersma, Y.; ... ; InSiGHT Variant Interpretation Com 2020
Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The... Show moreFunctional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants inMLH1,MSH2, andMSH6, including 12 missense variants that were also assessed using a cell-freein vitroMMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants. Show less
Drost, M.; Tiersma, Y.; Glubb, D.; Kathe, S.; Hees, S. van; Calleja, F.; ... ; Wind, N. de 2020
Purpose Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that... Show morePurpose Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that gene. This not only obfuscates personalized health care but also the development of a rapid and reliable classification procedure that does not require clinical data. Methods The complete in vitro MMR activity (CIMRA) assay was calibrated against clinically classified MSH6 variants and, employing Bayes' rule, integrated with computational predictions of pathogenicity. To enable the validation of this two-component classification procedure we have employed a genetic screen to generate a large set of inactivating Msh6 variants, as proxies for pathogenic variants. Results The genetic screen-derived variants established that the two-component classification procedure displays high sensitivities and specificities. Moreover, these inactivating variants enabled the direct reclassification of human variants of uncertain significance (VUS) as (likely) pathogenic. Conclusion The two-component classification procedure and the genetic screens provide complementary approaches to rapidly and cost-effectively classify the large majority of human MSH6 variants. The approach followed here provides a template for the classification of variants in other disease-predisposing genes, facilitating the translation of personalized genomics into personalized health care. Show less
Purpose: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cellfree in vitro... Show morePurpose: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cellfree in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data.Methods: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories.Results: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible.Conclusion: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants. Show less
Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants... Show moreLynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as __variants of uncertain significance__ (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. I describe different approaches for the functional analysis of MMR gene VUS. Show less
