Clinical, Pathology, Genetic, and Molecular Features of Colorectal Tumors in Adolescents and Adults 25 Years or Younger.

BACKGROUND & AIMS: Colorectal cancers (CRCs) are rare in adolescents and adults ages 25 years or younger. We analyzed clinical, pathology, and molecular features of colorectal tumors from adolescents and young adults in an effort to improve genetic counseling, surveillance, and, ultimately, treatment and outcomes. METHODS: We analyzed clinical data and molecular and genetic features of colorectal tumor tissues from 139 adolescents or young adults (age, ≤25 y; median age, 23 y; 58% male), collected from 2000 through 2017; tumor tissues and clinical data were obtained from the nationwide network and registry of histopathology and cytopathology and The Netherlands Cancer Registry, respectively. DNA samples from tumors were analyzed for microsatellite instability, mutations in 56 genes, and genome-wide somatic copy number aberrations. RESULTS: Mucinous and/or signet ring cell components were observed in 33% of tumor samples. A genetic tumor risk syndrome was confirmed for 39% of cases. Factors associated with shorter survival time included younger age at diagnosis, signet ring cell carcinoma, the absence of a genetic tumor risk syndrome, and diagnosis at an advanced stage of disease. Compared with colorectal tumors from patients ages 60 years or older in the Cancer Genome Atlas, higher proportions of tumors from adolescents or young adults were microsatellite stable with nearly diploid genomes, or contained somatic mutations in TP53 and POLE, whereas lower proportions contained mutations in APC. CONCLUSIONS: We found clinical, molecular, and genetic features of CRCs in adolescents or young adults to differ from those of patients older than age 60 years. In 39% of patients a genetic tumor risk syndrome was identified. These findings provide insight into the pathogenesis of CRC in young patients and suggest new strategies for clinical management. Performing genetic and molecular analyses for every individual diagnosed with CRC at age 25 years or younger would aid in this optimization.

Mutational Signature Analysis Reveals NTHL1 Deficiency to Cause a Multi-tumor Phenotype.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

Role of germline aberrations affecting CTNNA1, MAP3K6 and MYD88 in gastric cancer susceptibility.

BACKGROUND: In approximately 10% of all gastric cancer (GC) cases, a heritable cause is suspected. A subset of these cases have a causative germline CDH1 mutation; however, in most cases the cause remains unknown. Our objective was to assess to what extent these remaining cases may be explained by germline mutations in the novel candidate GC predisposing genes CTNNA1, MAP3K6 or MYD88. METHODS: We sequenced a large cohort of unexplained young and/or familial patients with GC (n=286) without a CDH1germline mutation for germline variants affecting CTNNA1, MAP3K6 and MYD88 using a targeted next-generation sequencing approach based on single-molecule molecular inversion probes. RESULTS: Predicted deleterious germline variants were not encountered in MYD88, but recurrently observed in CTNNA1 (n=2) and MAP3K6 (n=3) in our cohort of patients with GC. In contrast to deleterious variants in CTNNA1, deleterious variants in MAP3K6 also occur frequently in the general population. CONCLUSIONS: Based on our results MAP3K6 should no longer be considered a GC predisposition gene, whereas deleterious CTNNA1 variants are confirmed as an infrequent cause of GC susceptibility. Biallelic MYD88 germline mutations are at most a very rare cause of GC susceptibility as no additional cases were identified.

A molecular inversion probe-based next-generation sequencing panel to detect germline mutations in Chinese early-onset colorectal cancer patients.

The currently known Mendelian colorectal cancer (CRC) predisposition syndromes account for ~5-10% of all CRC cases, and are caused by inherited germline mutations in single CRC predisposing genes. Using molecular inversion probes (MIPs), we designed a targeted next-generation sequencing panel to identify mutations in seven CRC predisposing genes: APC, MLH1, MSH2, MSH6, PMS2, MUTYH and NTHL1. From a consecutive series of 2,371 Chinese CRC patients, 140 familial and non-familial cases were selected that were diagnosed with CRC at or below the age of 35 years. Through MIP-based sequencing we identified pathogenic variants in six genes in 16 out of the 140 (11.4%) patients selected. In 10 patients, known pathogenic mutations in APC (five patients), MLH1 (three patients), or MSH2 (two patients) were identified. Three additional patients were found to carry novel, likely pathogenic truncating (n = 2) and missense (n = 1) mutations in the MSH2 gene and a concomitant loss of expression of both the MSH2 and MSH6 proteins in their respective tumor tissues. From our data, we conclude that targeted MIP-based sequencing is a reliable and cost-efficient approach to identify patients with a Mendelian CRC syndrome.

Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas.

With the recent introduction of Poly(ADP-ribose) polymerase inhibitors, a promising novel therapy has become available for ovarian carcinoma (OC) patients with inactivating BRCA1 or BRCA2 mutations in their tumor. To select patients who may benefit from these treatments, assessment of the mutation status of BRCA1 and BRCA2 in the tumor is required. For reliable evaluation of germline and somatic mutations in these genes in DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissue, we have developed a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach. Our smMIP-based NGS approach provides analysis of both strands of the open reading frame of BRCA1 and BRCA2, enabling the discrimination between real variants and formalin-induced artefacts. The single molecule tag enables compilation of unique reads leading to a high analytical sensitivity and enabling assessment of the reliability of mutation-negative results. Multiplex ligation-dependent probe amplification (MLPA) and Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were used to detect exon deletions of BRCA1 and methylation of the BRCA1 promoter, respectively. Here, we show that this combined approach allows the rapid and reliable detection of both germline and somatic aberrations affecting BRCA1 and BRCA2 in DNA derived from FFPE OCs, enabling improved hereditary cancer risk assessment and clinical treatment of ovarian cancer patients.

Opportunities for immunotherapy in microsatellite instable colorectal cancer.

Microsatellite instability (MSI), the somatic accumulation of length variations in repetitive DNA sequences called microsatellites, is frequently observed in both hereditary and sporadic colorectal cancer (CRC). It has been established that defects in the DNA mismatch repair (MMR) pathway underlie the development of MSI in CRC. After the inactivation of the DNA MMR pathway, misincorporations, insertions and deletions introduced by DNA polymerase slippage are not properly recognized and corrected. Specific genomic regions, including microsatellites, are more prone for DNA polymerase slippage and, therefore, more susceptible for the introduction of these mutations if the DNA MMR capacity is lost. Some of these susceptible genomic regions are located within the coding regions of genes. Insertions and deletions in these regions may alter their reading frame, potentially resulting in the transcription and translation of frameshift peptides with c-terminally altered amino acid sequences. These frameshift peptides are called neoantigens and are highly immunogenic, which explains the enhanced immunogenicity of MSI CRC. Neoantigens contribute to increased infiltration of tumor tissue with activated neoantigen-specific cytotoxic T lymphocytes, a hallmark of MSI tumors. Currently, neoantigen-based vaccination is being studied in a clinical trial for Lynch syndrome and in a trial for sporadic MSI CRC of advanced stage. In this Focussed Research Review, we summarize current knowledge on molecular mechanisms and address immunological features of tumors with MSI. Finally, we describe their implications for immunotherapeutic approaches and provide an outlook on next-generation immunotherapy involving neoantigens and combinatorial therapies in the setting of MSI CRC.

Identification of Novel Candidate Genes for Early-Onset Colorectal Cancer Susceptibility.

Approximately 25-30% of colorectal cancer (CRC) cases are expected to result from a genetic predisposition, but in only 5-10% of these cases highly penetrant germline mutations are found. The remaining CRC heritability is still unexplained, and may be caused by a hitherto-undefined set of rare variants with a moderately penetrant risk. Here we aimed to identify novel risk factors for early-onset CRC using whole-exome sequencing, which was performed on a cohort of CRC individuals (n = 55) with a disease onset before 45 years of age. We searched for genes that were recurrently affected by rare variants (minor allele frequency ≤ 0.001) with potentially damaging effects and, subsequently, re-sequenced the candidate genes in a replication cohort of 174 early-onset or familial CRC individuals. Two functionally relevant genes with low frequency variants with potentially damaging effects, PTPN12 and LRP6, were found in at least three individuals. The protein tyrosine phosphatase PTP-PEST, encoded by PTPN12, is a regulator of cell motility and LRP6 is a component of the WNT-FZD-LRP5-LRP6 complex that triggers WNT signaling. All variants in LRP6 were identified in individuals with an extremely early-onset of the disease (≤30 years of age), and two of the three variants showed increased WNT signaling activity in vitro. In conclusion, we present PTPN12 and LRP6 as novel candidates contributing to the heterogeneous susceptibility to CRC.

A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis and colorectal cancer.

The genetic cause underlying the development of multiple colonic adenomas, the premalignant precursors of colorectal cancer (CRC), frequently remains unresolved in patients with adenomatous polyposis. Here we applied whole-exome sequencing to 51 individuals with multiple colonic adenomas from 48 families. In seven affected individuals from three unrelated families, we identified a homozygous germline nonsense mutation in the base-excision repair (BER) gene NTHL1. This mutation was exclusively found in a heterozygous state in controls (minor allele frequency of 0.0036; n = 2,329). All three families showed recessive inheritance of the adenomatous polyposis phenotype and progression to CRC in at least one member. All three affected women developed an endometrial malignancy or premalignancy. Genetic analysis of three carcinomas and five adenomas from different affected individuals showed a non-hypermutated profile enriched for cytosine-to-thymine transitions. We conclude that a homozygous loss-of-function germline mutation in the NTHL1 gene predisposes to a new subtype of BER-associated adenomatous polyposis and CRC.

Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development.

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.

Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are risk factors for colorectal cancer.

The spindle assembly checkpoint controls proper chromosome segregation during mitosis and prevents aneuploidy-an important feature of cancer cells. We performed genome-wide and targeted copy number and mutation analyses of germline DNA from 208 patients with familial or early-onset (40 years of age or younger) colorectal cancer; we identified haploinsufficiency or heterozygous mutations in the spindle assembly checkpoint genes BUB1 and BUB3 in 2.9% of them. Besides colorectal cancer, these patients had variegated aneuploidies in multiple tissues and variable dysmorphic features. These results indicate that mutations in BUB1 and BUB3 cause mosaic variegated aneuploidy and increase the risk of colorectal cancer at a young age.