Colon and Endometrial Cancers With Mismatch Repair Deficiency Can Arise From Somatic, Rather Than Germline, Mutations

doi:10.1053/j.gastro.2014.08.041

Gastroenterology

Volume 147, Issue 6, December 2014, Pages 1308-1316.e1

https://doi.org/10.1053/j.gastro.2014.08.041 Get rights and content

Background & Aims

Patients with Lynch syndrome carry germline mutations in single alleles of genes encoding the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2; when the second allele becomes mutated, cancer can develop. Increased screening for Lynch syndrome has identified patients with tumors that have deficiency in MMR, but no germline mutations in genes encoding MMR proteins. We investigated whether tumors with deficient MMR had acquired somatic mutations in patients without germline mutations in MMR genes using next-generation sequencing.

Methods

We analyzed blood and tumor samples from 32 patients with colorectal or endometrial cancer who participated in Lynch syndrome screening studies in Ohio and were found to have tumors with MMR deficiency (based on microsatellite instability and/or absence of MMR proteins in immunohistochemical analysis, without hypermethylation of MLH1), but no germline mutations in MMR genes. Tumor DNA was sequenced for MLH1, MSH2, MSH6, PMS2, EPCAM, POLE, and POLD1 with ColoSeq and mutation frequencies were established.

Results

Twenty-two of 32 patients (69%) were found to have 2 somatic (tumor) mutations in MMR genes encoding proteins that were lost from tumor samples, based on immunohistochemistry. Of the 10 remaining tumors 3 had one somatic mutation in a MMR gene, with possible loss of heterozygosity that could lead to MMR deficiency, 6 were found to be false-positive results (19%), and 1 had only one mutation in a MMR gene and remained unexplained. All of the tumors found to have somatic MMR mutations were of the hypermutated phenotype (>12 mutations/megabase); 6 had mutation frequencies >200/megabase, and 5 of these had somatic mutations in POLE, which encodes a DNA polymerase.

Conclusions

Some patients are found to have tumors with MMR defects during screening for Lynch syndrome, yet have no identifiable germline mutations in MMR genes. We found that almost 70% of these patients acquire somatic mutations in MMR genes, leading to a hypermutated phenotype of tumor cells. Patients with colon or endometrial cancers with MMR deficiency not explained by germline mutations might undergo analysis for tumor mutations in MMR genes to guide future surveillance guidelines.

Section snippets

Patients

Patients from the previously published Columbus Lynch syndrome study1, 3 and the ongoing state-wide prospective Lynch syndrome screening study in Ohio (OCCPI)¹⁴ were included. Both studies included patients with newly diagnosed colorectal and endometrial cancer, regardless of age at diagnosis or family history. The research protocol and consent form were approved by the Institutional Review Board at each participating hospital, and all patients provided written informed consent. Patients with

Patient Characteristics

A total of 32 patients with either colorectal or endometrial cancer (see Table 1 for patient characteristics) were screened for somatic mutations using ColoSeq, 22 from the Columbus Lynch syndrome study (21% of unexplained colorectal cancer cases and 38% of unexplained endometrial cancer cases) and 10 from the OCCPI (100% of unexplained cases on the study; see Supplementary Figures 1 and 2 for screening outcomes). All of these patients had previously tested negative for MMR germline mutations

Discussion

In one of the largest population-based screening studies for Lynch syndrome in colorectal¹ and endometrial cancer,³ 3.9% of all screened patients had MMR-deficient tumors that were not explained by germline mutations, leaving the treating physician and the patient unsure of the implications of an abnormal screening test. This study confirms that most of these cases can be explained by 2 somatic tumor mutations. The abnormal screening tests were explained by tumor MMR gene mutations in 69% of

Acknowledgments

The authors thank Christina Smith, Karen Koehler, Mallory Beightol, Shelby Flowers, and Tatyana Marushchak for performing genomic library preparation and sequencing, Angela Jacobson and Dr Robert Livingston for help with research coordination, Dr Brian Shirts and Dr Tom Walsh for assistance with variant interpretation, and Dr Stephen Salipante, Dr Emily Turner, and Sheena Scroggins for help with bioinformatics. The authors would also like to thank Dr C. Richard Boland and Dr Jennifer Rhees at

References (31)

C.C. Pritchard et al.
ColoSeq provides comprehensive Lynch and polyposis syndrome mutational analysis using massively parallel sequencing
J Mol Diagn
(2012)
C.C. Pritchard et al.
A mosaic PTEN mutation causing Cowden syndrome identified by deep sequencing
Genet Med
(2013)
C.C. Pritchard et al.
Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and gene rearrangement in clinical cancer specimens
J Mol Diag
(2014)
J.M. Carethers
Differentiating Lynch-like from Lynch syndrome
Gastroenterology
(2014)
C.R. Boland
The mystery of mismatch repair deficiency: Lynch or Lynch-like?
Gastroenterology
(2013)
H. Hampel et al.
Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer)
N Engl J Med
(2005)
H. Hampel et al.
Feasibility of screening for Lynch syndrome among patients with colorectal cancer
J Clin Oncol
(2008)
H. Hampel et al.
Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients
Cancer Res
(2006)
H. Hampel et al.
Comment on: screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients
Cancer Res
(2007)
Comprehensive molecular characterization of human colon and rectal cancer
Nature
(2012)

Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives

Genet Med

(2009)

L.C. Beamer et al.

Reflex immunohistochemistry and microsatellite instability testing of colorectal tumors for lynch syndrome among us cancer programs and follow-up of abnormal results

J Clin Oncol

(2012)

M.J.L. Ligtenberg et al.

Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3[prime] exons of TACSTD1

Nat Genet

(2009)

J. Rhees et al.

Inversion of exons 1–7 of the MSH2 gene is a frequent cause of unexplained Lynch syndrome in one local population

Fam Cancer

(2013)

M. Rodríguez-Soler et al.

Risk of cancer in cases of suspected Lynch syndrome without germline mutation

Gastroenterology

(2013)

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Molecular and Clinicopathologic Characterization of Mismatch Repair-Deficient Endometrial Carcinoma Not Related to MLH1 Promoter Hypermethylation
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Universal tumor screening in endometrial carcinoma (EC) is increasingly adopted to identify individuals at risk of Lynch syndrome (LS). These cases involve mismatch repair-deficient (MMRd) EC without MLH1 promoter hypermethylation (PHM). LS is confirmed through the identification of germline MMR pathogenic variants (PV). In cases where these are not detected, emerging evidence highlights the significance of double-somatic MMR gene alterations as a sporadic cause of MMRd, alongside POLE/POLD1 exonuclease domain (EDM) PV leading to secondary MMR PV. Our understanding of the incidence of different MMRd EC origins not related to MLH1-PHM, their associations with clinicopathologic characteristics, and the prognostic implications remains limited.
In a combined analysis of the PORTEC-1, -2, and -3 trials (n = 1254), 84 MMRd EC not related to MLH1-PHM were identified that successfully underwent paired tumor–normal tissue next-generation sequencing of the MMR and POLE/POLD1 genes. Among these, 37% were LS associated (LS-MMRd EC), 38% were due to double-somatic hits (DS-MMRd EC), and 25% remained unexplained. LS-MMRd EC exhibited higher rates of MSH6 (52% vs 19%) or PMS2 loss (29% vs 3%) than DS-MMRd EC, and exclusively showed MMR-deficient gland foci. DS-MMRd EC had higher rates of combined MSH2/MSH6 loss (47% vs 16%), loss of >2 MMR proteins (16% vs 3%), and somatic POLE-EDM PV (25% vs 3%) than LS-MMRd EC. Clinicopathologic characteristics, including age at tumor onset and prognosis, did not differ among the various groups.
Our study validates the use of paired tumor–normal next-generation sequencing to identify definitive sporadic causes in MMRd EC unrelated to MLH1-PHM. MMR immunohistochemistry and POLE-EDM mutation status can aid in the differentiation between LS-MMRd EC and DS-MMRd EC. These findings emphasize the need for integrating tumor sequencing into LS diagnostics, along with clear interpretation guidelines, to improve clinical management. Although not impacting prognosis, confirmation of DS-MMRd EC may release patients and relatives from burdensome LS surveillance.
Use of multi-gene panels in patients at high risk of hereditary digestive cancer: position statement of AEG, SEOM, AEGH and IMPaCT-GENÓMICA consortium
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Este documento de posicionamiento, auspiciado por la Asociación Española de Gastroenterología, la Sociedad Española de Oncología Médica, la Asociación Española de Genética Humana y el consorcio IMPaCT-Genómica, tiene como objetivo realizar recomendaciones para el uso de paneles de genes en la evaluación de individuos con alto riesgo de cáncer digestivo hereditario. Para medir la calidad de la evidencia y los niveles de recomendación se ha utilizado la metodología basada en el sistema Grading of Recommendations Assessment, Development and Evaluation (GRADE). Se obtuvo el consenso entre expertos mediante un método Delphi. El documento incluye recomendaciones sobre escenarios clínicos en los que se recomienda el uso de paneles de genes en cáncer colorrectal, síndromes polipósicos, cáncer gástrico y pancreático, así como los genes de los paneles a ser considerados en cada una de estas situaciones clínicas. También se establecen recomendaciones sobre la evaluación de mosaicismos, las estrategias de asesoramiento ante la ausencia de sujeto índice y, finalmente, el análisis constitucional tras identificación de variantes patogénicas tumorales.
This position statement, sponsored by the Asociación Española de Gastroenterología, the Sociedad Española de Oncología Médica, the Asociación Española de Genética Humana and the IMPaCT-Genómica Consortium aims to establish recommendations for use of multi-gene panel testing in patients at high risk of hereditary gastrointestinal and pancreatic cancer. To rate the quality of the evidence and the levels of recommendation, we used the methodology based on the GRADE system (Grading of Recommendations Assessment, Development and Evaluation). We reached a consensus among experts using a Delphi method. The document includes recommendations on clinical scenarios where multi-gene panel testing is recommended in colorectal cancer, polyposis syndromes, gastric and pancreatic cancer, as well as the genes to be considered in each clinical scenario. Recommendations on the evaluation of mosaicisms, counseling strategies in the absence of an index subject and, finally, constitutional analysis after identification of pathogenic tumor variants are also made.
Sebaceous carcinoma epidemiology, associated malignancies and Lynch/Muir-Torre syndrome screening in England from 2008 to 2018
2023, Journal of the American Academy of Dermatology
Sebaceous carcinomas (SC) may be associated with the cancer predisposition syndrome Muir-Torre/Lynch syndrome (MTS/LS), identifiable by SC mismatch repair (MMR) screening; however, there is limited data on MMR status of SC.
To describe the epidemiology of SC, copresentation of other cancers, and population level frequency of MMR screening in SC.
A population-based retrospective cohort study of SC patients in the National Cancer Registration and Analysis Service in England.
This study included 1077 SC cases (739 extraocular, 338 periocular). Age-standardized incidence rates (ASIR) were higher in men compared with women, 2.74 (95% CI, 2.52-9.69) per 1,000,000 person-years for men versus 1.47 person-years (95% CI, 1.4-1.62) for women. Of the patients, 19% (210/1077) developed at least one MTS/LS-associated malignancy. MMR immunohistochemical screening was performed in only 20% (220/1077) of SC tumors; of these, 32% (70/219) of tumors were MMR deficient.
Retrospective design.
Incorporation of MMR screening into clinical practice guidelines for the management of SC will increase the opportunity for MTS/LS diagnoses, with implications for cancer surveillance, chemoprevention with aspirin, and immunotherapy treatment targeted to MTS/LS cancers.
A Highly Sensitive Pan-Cancer Test for Microsatellite Instability
2023, Journal of Molecular Diagnostics
Microsatellite instability (MSI) is an evolving biomarker for cancer detection and treatment. MSI was first used to identify patients with Lynch syndrome, a hereditary form of colorectal cancer (CRC), but has recently become indispensable in predicting patient response to immunotherapy. To address the need for pan-cancer MSI detection, a new multiplex assay was developed that uses novel long mononucleotide repeat (LMR) markers to improve sensitivity. A total of 469 tumor samples from 20 different cancer types, including 319 from patients with Lynch syndrome, were tested for MSI using the new LMR MSI Analysis System. Results were validated by using deficient mismatch repair (dMMR) status according to immunohistochemistry as the reference standard and compared versus the Promega pentaplex MSI panel. The sensitivity of the LMR panel for detection of dMMR status by immunohistochemistry was 99% for CRC and 96% for non-CRC. The overall percent agreement between the LMR and Promega pentaplex panels was 99% for CRC and 89% for non-CRC tumors. An increased number of unstable markers and the larger size shifts observed in dMMR tumors using the LMR panel increased confidence in MSI determinations. The LMR MSI Analysis System expands the spectrum of cancer types in which MSI can be accurately detected.
Discordant Staining Patterns and Microsatellite Results in Tumors of MSH6 Pathogenic Variant Carriers
2023, Modern Pathology
Diagnosis of Lynch syndrome (LS) caused by a pathogenic germline MSH6 variant may be complicated by discordant immunohistochemistry (IHC) and/or by a microsatellite stable (MSS) phenotype. This study aimed to identify the various causes of the discordant phenotypes of colorectal cancer (CRC) and endometrial cancer (EC) in MSH6-associated LS. Data were collected from Dutch family cancer clinics. Carriers of a (likely) pathogenic MSH6 variant diagnosed with CRC or EC were categorized based on an microsatellite instability (MSI)/IHC test outcome that might fail to result in a diagnosis of LS (eg, retained staining of all 4 mismatch repair proteins, with or without an MSS phenotype, and other staining patterns). When tumor tissue was available, MSI and/or IHC were repeated. Next-generation sequencing (NGS) was performed in cases with discordant staining patterns. Data were obtained from 360 families with 1763 (obligate) carriers. MSH6 variant carriers with CRC or EC (n = 590) were included, consisting of 418 CRCs and 232 ECs. Discordant staining was reported in 77 cases (36% of MSI/IHC results). Twelve patients gave informed consent for further analysis of tumor material. Upon revision, 2 out of 3 MSI/IHC cases were found to be concordant with the MSH6 variant, and NGS showed that 4 discordant IHC results were sporadic rather than LS-associated tumors. In 1 case, somatic events explained the discordant phenotype. The use of reflex IHC mismatch repair testing, the current standard in most Western countries, may lead to the misdiagnosis of germline MSH6 variant carriers. The pathologist should point out that further diagnostics for inheritable colon cancer, including LS, should be considered in case of a strong positive family history. Germline DNA analysis of the mismatch repair genes, preferably as part of a larger gene panel, should therefore be considered in potential LS patients.
Heterogeneous expression of mismatch repair proteins and interpretation of immunohistochemical results in colorectal cancer and endometrial cancer
2023, Pathology Research and Practice
To investigate the heterogeneous expression patterns of four mismatch repair (MMR) proteins in colorectal cancer (CRC) and endometrial cancer (EC), and their effects on the interpretation of immunohistochemical (IHC) results. A total of 1636 CRC and EC specimens were collected from two hospitals. Seventy-eight cases had heterogeneous expression of MMR proteins, including 49 CRC and 29 EC cases. Polymerase chain reaction-capillary electrophoresis (PCR-CE) was then performed to detect the microsatellite instability (MSI) status, and 44 cases were further verified by targeted next-generation sequencing (NGS). Heterogeneous expression of MMR proteins was observed in 66 cases (66/78, 84.6%) of proficient MMR (pMMR) and 12 cases (12/78, 15.4%) of deficient MMR (dMMR). The proportion of heterogeneous MMR protein expression in EC (12.0%) was higher than that in CRC (3.5%). The heterogeneous expression patterns were divided into focal clonal heterogeneity (6/78, 7.7%) and glandular mosaic heterogeneity (72/78, 92.3%). Surprisingly, three pMMR CRC cases showed isolated small focal clonal heterogeneity of mutS homologue 6 (MSH6), with < 15% positive tumour cells, which was validated as high MSI (MSI-H) with PCR-CE and NGS. However, the other three EC pMMR cases with > 50% focal clonal heterogeneity of MMR proteins were verified as microsatellite stable (MSS) or low MSI (MSI-L). Fifteen EC cases with glandular mosaic heterogeneous expression of MMR proteins included two MSI-H cases, which were validated using PCR-CE and NGS. Among the dMMR cases, only two EC cases with mutL homologue 1 (MLH1)/PMS1 homologue 2, mismatch repair system component (PMS2) loss and MSH2/MSH6 mosaic heterogeneous expression were confirmed as MSS using PCR-CE and NGS, which may be related to the mechanism of MLH1 promoter methylation. Thus, in CRC, only cases with small focal clonal heterogeneous expression of MSH6 have a high likelihood of MSI-H, and further PCR-CE or NGS testing is recommended. The possibility of MSI-H cannot be ruled out in EC cases with glandular mosaic heterogeneous expression of MMR proteins; PCR-CE or NGS detection is therefore necessary.

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: Conflicts of interest This author discloses the following: Heather Hampel has received research funding from Myriad Genetic Laboratories. The remaining authors disclose no conflicts.

: Funding This study was supported by grants from Pelotonia and the National Cancer Institute (CA16058 and CA67941).

: Author names in bold designate shared co-first authorship.

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Original ResearchFull Report: Clinical—Alimentary TractColon and Endometrial Cancers With Mismatch Repair Deficiency Can Arise From Somatic, Rather Than Germline, Mutations

Background & Aims

Methods

Results

Conclusions

Section snippets

Patients

Patient Characteristics

Discussion

Acknowledgments

J Mol Diagn

Genet Med

J Mol Diag

Gastroenterology

Gastroenterology

Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer)

N Engl J Med

Feasibility of screening for Lynch syndrome among patients with colorectal cancer

J Clin Oncol

Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients

Cancer Res

Comment on: screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients

Cancer Res

Comprehensive molecular characterization of human colon and rectal cancer

Nature

Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives

Genet Med

Reflex immunohistochemistry and microsatellite instability testing of colorectal tumors for lynch syndrome among us cancer programs and follow-up of abnormal results

J Clin Oncol

Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3[prime] exons of TACSTD1

Nat Genet

Inversion of exons 1–7 of the MSH2 gene is a frequent cause of unexplained Lynch syndrome in one local population

Fam Cancer

Risk of cancer in cases of suspected Lynch syndrome without germline mutation

Gastroenterology

Original Research
Full Report: Clinical—Alimentary Tract
Colon and Endometrial Cancers With Mismatch Repair Deficiency Can Arise From Somatic, Rather Than Germline, Mutations