Merged testing for colorectal cancer syndromes and re‐evaluation of genetic variants improve diagnostic yield: Results from a nationwide prospective cohort

Abstract Approximately 5% of patients with colorectal cancer (CRC) have a Mendelian predisposition for the disease. Identification of the disease‐causing genetic variant enables carrier testing and tailored cancer prevention within affected families. To determine the panorama and genetic variation of Mendelian CRC syndromes among referrals at the cancer genetics clinics in Sweden, 850 patients clinically selected for CRC genetic investigation were included in a prospective study that tested for all major hereditary polyposis and nonpolyposis CRC conditions. Genetically defined syndromes were diagnosed in 11% of the patients. Lynch syndrome was predominant (n = 73) followed by familial adenomatous polyposis (n = 12) and MUTYH‐associated polyposis (n = 8); the latter of which two patients presented with CRC before polyposis was evident. One patient with a history of adolescent‐onset CRC and polyposis had biallelic disease‐causing variants diagnostic for constitutional mismatch repair deficiency syndrome. Post‐study review of detected variants of unknown clinical significance (n = 129) resulted in the reclassification of variants as likely benign (n = 59) or as diagnostic for Lynch syndrome (n = 2). Our results reveal the panorama of Mendelian CRC syndromes at the cancer genetics clinics in Sweden and show that unified testing for polyposis and nonpolyposis CRC conditions as well as regular reexamination of sequence data improve the diagnostic yield.


| INTRODUCTION
Approximately 5% of patients with colorectal cancer (CRC) have a constitutional disease-causing genetic variant (DV) that causes autosomal dominant (AD) or autosomal recessive (AR) predisposition to the disease. 1 Lynch syndrome (LS) is by far the most common known Mendelian CRC condition with a prevalence approximately 1:300, followed by familial adenomatous polyposis (FAP) and MUTYHassociated polyposis (MAP; AR inheritance) with estimated prevalences approximately 1:10 000 to 1:40 000, respectively. 2,3 There are several less common Mendelian conditions that predispose to CRC, such as juvenile polyposis syndrome (JPS), PTEN hamartoma tumor syndrome (PHTS), Peutz-Jeghers syndrome (PJS), polymerase proofreading-associated polyposis (PPAP), and a handful additional more recently defined conditions. 3,4 Because DV may have prognostic, therapeutic, and prophylactic implications, screening for DV in selected patients with CRC and/or colorectal polyposis is routine in many health-care systems. In addition, identification of DV permits carrier testing and personalized health care for relatives. Patients with colorectal polyposis with or without CRC have traditionally been genetically screened FAP and MAP. Conversely, patients with nonpolyposis CRC have traditionally been genetically screened for LS. To date, the outcomes of simultaneous testing for Mendelian CRC have mostly been evaluated in retrospective cohorts of selected or unselected patients. 2,[5][6][7] In this work, we have addressed the panorama and genetic variation of Mendelian CRC syndromes in a prospective national cohort of patients clinically selected for CRC genetic diagnostics.

| Patients
From 2014 to 2019, 861 patients with suspected Mendelian predisposition to CRC were invited to participate in the Swedish-extended genetic analysis of colorectal neoplasia (SWEN) study, a prospective study with inclusion from all cancer genetics clinics in Sweden, including the university hospitals in Umeå, Uppsala, Stockholm, Linköping, Gothenburg, and Lund ( Figure 1). Patients obtained oral and written study information and provided written informed consent. This study was approved by The Regional Ethical Review Board in Lund (application no. 2013/468 and no. 2015/211) and by the Swedish Ethical Review Agency (application no. 2019-02312). The inclusion criteria of the study were as follows: (1) age of majority (≥18 years), (2) CRC clinical genetic investigation decided upon at a cancer genetics clinic in Sweden according to national clinical guidelines (Supplementary Material 1), and (3) written informed consent. After exclusion of 11 patients due to patient withdrawal (n = 5), no sample available (n = 2), incomplete molecular genetic analysis (n = 1), previously genetically screened for DV in MMR genes (n = 2), or inclusion merely on the basis of family history (n = 1), 850 patients were successfully enrolled in the study and cataloged regarding gender, neoplasms, colorectal polyps, and age at diagnosis. Records of unspecified numbers of colorectal polyps stating "polyposis", or "many", "numerous" or "massive numbers of" polyps were interpreted as 10 or more polyps and denoted polyposis.

| Molecular genetic analyses
All patients were screened for DV in genes associated with HiSeq 2000 (n = 616) and analyzed as previously described, 8 or using AmpliSeq following sequencing using an IonTorrent S5 (n = 234) as previously described 9 and analyzed using IonReporter.
All variants identified by capture MPS were confirmed using Sanger sequencing using standard protocols. Samples analyzed with IonTorrent sequencing regarding PMS2 were also subjected to nested PCR followed by Sanger sequencing as described. 10  Age at diagnosis (years) a Mendelian syndrome

| Variant classification
Data were analyzed using Gene Marker (Soft Genetics). Variant classification was performed according to the America College of Medical Genetics (ACMG) 11 and ClinVar. 12 Two main categories of variants were reported: DV including variants of class 4 (likely pathogenic variants) and variants of class 5 (pathogenic variants), and variants of unknown significance (VUS; class 3). Likely benign variants (class 2) and benign variants (class 1) were regarded as normal findings and were not reported. DV and VUS were re-evaluated in October 2021, that is, 2-7 years after initial reporting. 3 | RESULTS

| Clinical characteristics of cohort
The cohort contained 527 females and 323 males (

| Detected genetic variants and variant reclassification
A total of 112 DV and 129 VUS were reported (Table 3). Among the MMR genes, MSH2 displayed the greatest number of DV diagnostic for LS (Table 3). Two DV in PMS2 were confirmed as biallelic compound heterozygote variants diagnostic for CMMRD (

| DISCUSSION
In this work, we have investigated a prospective cohort of patients We found one patient with CMMRD, a severe early-onset multiorgan cancer predisposition syndrome caused by biallelic DV in MMR genes, predominantly in PMS2. 16 Molecular genetic diagnosis of CMMRD can be challenging as it may resemble neurofibromatosis type 1 presenting with café-au-lait macules and brain tumors, or FAP or MAP presenting with adenomatous polyposis. 16

| CONCLUSION
In summary, we describe the panorama and genetic variation of Mendelian CRC syndromes in a prospective cohort of patients with a suspected predisposition to CRC at the cancer genetics clinics in Sweden. Our data show that unified testing for the different Mendelian CRC syndromes as well as an intermittent reexamination of sequence data improve the diagnostic yield.

ACKNOWLEDGMENTS
We would like to thank the patients who participated in the study.
The coworkers at the participating molecular diagnostic laboratories are greatly acknowledged. We also thank the health-care regions in Sweden for support and infrastructure.