Genomic temporal heterogeneity of circulating tumour DNA in unresectable metastatic colorectal cancer under first-line treatment.

OBJECTIVE: Circulating tumour DNA (ctDNA) sequencing is increasingly used in the clinical management of patients with colorectal cancer. However, the genomic heterogeneity in ctDNA during treatments and its impact on clinical outcomes remain largely unknown. DESIGN: We conducted a prospective cohort study (NCT04228614) of 171 patients with unresectable metastatic colorectal cancer (mCRC) who underwent first-line treatment and prospectively collected blood samples with or without tumour samples from patients at baseline and sequentially until disease progression or last follow-up. RESULTS: The RAS/BRAF alterations in paired baseline tissue and plasma samples from 63 patients displayed a favourable concordance (81.0%, 51/63). After a period of first-line treatment (median time between baseline and last liquid biopsy, 4.67 months), 42.6% (26/61) of RAS-mutant patients showed RAS clearance and 50.0% (5/10) of BRAF-mutant patients showed BRAF clearance, while 3.6% (3/84) and 0.7% (1/135) of patients showed new RAS or BRAF mutations in ctDNA. Patients with plasma RAS/BRAF clearance showed similar progression-free survival (PFS) and overall survival (OS) with patients who remained RAS/BRAF wild-type, while much better outcomes than those who remained RAS/BRAF mutant. Patients who gained new RAS/BRAF mutations showed similar prognosis as those who maintained RAS/BRAF mutations, and shorter PFS and OS than those who remained RAS/BRAF wild-type. CONCLUSION: This prospective, serial and large-scale ctDNA profiling study reveals the temporal heterogeneity of mCRC-related somatic variants, which should be given special attention in clinical practice, as evidenced by the finding that the shift in plasma RAS/BRAF mutational status can yield a drastic change in survival outcomes.

Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles.

OBJECTIVE: Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. DESIGN: We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. RESULTS: We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time. CONCLUSIONS: These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

MED12 is recurrently mutated in Middle Eastern colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a common cancer and a leading cause of cancer deaths. Previous studies have identified a number of key steps in the evolution of CRC but our knowledge of driver mutations in CRC remains incomplete. Recognising the potential of studying different human populations to reveal novel insights in disease pathogenesis, we conducted genomic analysis of CRC in Saudi patients. DESIGN: In the discovery phase of the study, we conducted whole genome sequencing of tumour and corresponding germline DNA in 27 patients with CRC. In addition to known driver mutations, we identified three MED12 somatic mutations. In the replication phase, we employed a next-generation sequencing approach to capture and sequence MED12 and other candidate genes in a larger sample of 400 patients with CRC and confirmed the enrichment for recurrent MED12 mutations. RESULTS: In order to gain insight into a plausible biological mechanism for the potential role of MED12 mutations in CRC, we studied CRC cell lines that differ substantially in the expression level of MED12, and found the latter to be correlated inversely with transforming growth factor (TGF)-ß signalling and directly with apoptosis in response to chemotherapeutic agents. Importantly, these correlations were replicated when MED12 expression was experimentally manipulated. CONCLUSIONS: Our data expand the recently described role of MED12 as a tumour suppressor in other cancers to include CRC, and suggest TGF-ß signalling as a potential mediator of this effect.

Clinicopathological and molecular features of sessile serrated adenomas with dysplasia or carcinoma.

OBJECTIVE: Sessile serrated adenomas (SSAs) are the precursors of at least 15% of colorectal carcinomas, but their biology is incompletely understood. We performed a clinicopathological and molecular analysis of a large number of the rarely observed SSAs with dysplasia/carcinoma to better define their features and the pathways by which they progress to carcinoma. DESIGN: A cross-sectional analysis of 137 SSAs containing regions of dysplasia/carcinoma prospectively collected at a community GI pathology practice was conducted. Samples were examined for BRAF and KRAS mutations, the CpG island methylator phenotype (CIMP) and immunostained for MLH1, p53, p16, ß-catenin and 0-6-methylguanine DNA methyltransferase (MGMT). RESULTS: The median polyp size was 9 mm and 86.5% were proximal. Most were BRAF mutated (92.7%) and 94.0% showed CIMP. Mismatch repair deficiency, evidenced by loss of MLH1 (74.5%) is associated with older age (76.7 versus 71.0; p<0.0029), female gender (70% versus 36%; p<0.0008), proximal location (91% versus 72%; p<0.02), CIMP (98% versus 80%; p<0.02) and lack of aberrant p53 (7% versus 34%; p<0.001) when compared with the mismatch repair-proficient cases. Loss of p16 (43.1%) and gain of nuclear ß-catenin (55.5%) were common in areas of dysplasia/cancer, irrespective of mismatch repair status. CONCLUSIONS: SSAs containing dysplasia/carcinoma are predominantly small (<10 mm) and proximal. The mismatch repair status separates these lesions into distinct clinicopathological subgroups, although WNT activation and p16 silencing are common to both. Cases with dysplasia occur at a similar age to cases with carcinoma. This, together with the rarity of these 'caught in the act' lesions, suggests a rapid transition to malignancy following a long dwell time as an SSA without dysplasia.

Incidence of and survival after subsequent cancers in carriers of pathogenic MMR variants with previous cancer: a report from the prospective Lynch syndrome database.

OBJECTIVE: Today most patients with Lynch syndrome (LS) survive their first cancer. There is limited information on the incidences and outcome of subsequent cancers. The present study addresses three questions: (i) what is the cumulative incidence of a subsequent cancer; (ii) in which organs do subsequent cancers occur; and (iii) what is the survival following these cancers? DESIGN: Information was collated on prospectively organised surveillance and prospectively observed outcomes in patients with LS who had cancer prior to inclusion and analysed by age, gender and genetic variants. RESULTS: 1273 patients with LS from 10 countries were followed up for 7753 observation years. 318 patients (25.7%) developed 341 first subsequent cancers, including colorectal (n=147, 43%), upper GI, pancreas or bile duct (n=37, 11%) and urinary tract (n=32, 10%). The cumulative incidences for any subsequent cancer from age 40 to age 70 years were 73% for pathogenic MLH1 (path_MLH1), 76% for path_MSH2 carriers and 52% for path_MSH6 carriers, and for colorectal cancer (CRC) the cumulative incidences were 46%, 48% and 23%, respectively. Crude survival after any subsequent cancer was 82% (95% CI 76% to 87%) and 10-year crude survival after CRC was 91% (95% CI 83% to 95%). CONCLUSIONS: Relative incidence of subsequent cancer compared with incidence of first cancer was slightly but insignificantly higher than cancer incidence in patients with LS without previous cancer (range 0.94-1.49). The favourable survival after subsequent cancers validated continued follow-up to prevent death from cancer. The interactive website http://lscarisk.org was expanded to calculate the risks by gender, genetic variant and age for subsequent cancer for any patient with LS with previous cancer.

RNF43 germline and somatic mutation in serrated neoplasia pathway and its association with BRAF mutation.

OBJECTIVE: Serrated polyps (hyperplastic polyps, sessile or traditional serrated adenomas), which can arise in a sporadic or polyposis setting, predispose to colorectal cancer (CRC), especially those with microsatellite instability (MSI) due to MLH1 promoter methylation (MLH1me+). We investigate genetic alterations in the serrated polyposis pathway. DESIGN: We used a combination of exome sequencing and target gene Sanger sequencing to study serrated polyposis families, sporadic serrated polyps and CRCs, with validation by analysis of The Cancer Genome Atlas (TCGA) cohort, followed by organoid-based functional studies. RESULTS: In one out of four serrated polyposis families, we identified a germline RNF43 mutation that displayed autosomal dominant cosegregation with the serrated polyposis phenotype, along with second-hit inactivation through loss of heterozygosity or somatic mutations in all serrated polyps (16), adenomas (5) and cancer (1) examined, as well as coincidental BRAF mutation in 62.5% of the serrated polyps. Concurrently, somatic RNF43 mutations were identified in 34% of sporadic sessile/traditional serrated adenomas, but 0% of hyperplastic polyps (p=0.013). Lastly, in MSI CRCs, we found significantly more frequent RNF43 mutations in the MLH1me+ (85%) versus MLH1me- (33.3%) group (p<0.001). These findings were validated in the TCGA MSI CRCs (p=0.005), which further delineated a significant differential involvement of three Wnt pathway genes between these two groups (RNF43 in MLH1me+; APC and CTNNB1 in MLH1me-); and identified significant co-occurrence of BRAF and RNF43 mutations in the MSI (p<0.001), microsatellite stable (MSS) (p=0.002) and MLH1me+ MSI CRCs (p=0.042). Functionally, organoid culture of serrated adenoma or mouse colon with CRISPR-induced RNF43 mutations had reduced dependency on R-spondin1. CONCLUSIONS: These results illustrate the importance of RNF43, along with BRAF mutation in the serrated neoplasia pathway (both the sporadic and familial forms), inform genetic diagnosis protocol and raise therapeutic opportunities through Wnt inhibition in different stages of evolution of serrated polyps.

Carbonic anhydrase IV inhibits colon cancer development by inhibiting the Wnt signalling pathway through targeting the WTAP-WT1-TBL1 axis.

OBJECTIVE: We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. DESIGN: The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. RESULTS: CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 inhibited cell proliferation, induced apoptosis and cell cycle arrest in the G1 phase. CA4 inhibited the activity of the Wnt signalling pathway and mediated the degradation of ß-catenin. CA4 interacted with Wilms' tumour 1-associating protein (WTAP) and induced WTAP protein degradation through polyubiquitination. Moreover, CA4 promoted the transcriptional activity of Wilms' tumour 1 (WT1), an antagonist of the Wnt pathway, which resulted in the induction of transducin ß-like protein 1 (TBL1) and the degradation of ß-catenin. CONCLUSIONS: CA4 is a novel tumour suppressor in CRC through the inhibition of the Wnt signalling pathway by targeting the WTAP-WT1-TBL1 axis. CA4 methylation may serve as an independent biomarker for the recurrence of CRC.

A plasma microRNA panel for early detection of colorectal cancer.

Colonoscopy remains the standard screening method for detecting colorectal cancer (CRC) at an early stage. However, many people avoid having a colonoscopy because of the fear for its potential complications. Our study aimed to identify plasma microRNAs for preliminarily screening CRC in general population, so that some unnecessary colonoscopies can be avoided. We investigated plasma microRNA expression in three independent cohorts including the discovery (n = 80), training (n = 112), and validation (n = 49) phases recruited at two medical centers. Microarrays were used for screening 723 microRNAs in 80 plasma samples to identify candidate microRNAs. Quantitative reverse-transcriptase PCR was performed on the 161 training and validation plasma samples to evaluate the candidate microRNAs discovered from microarrays. A logistic regression model was constructed based on the training cohort and then verified by using the validation dataset. Area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy. We identified a panel of miR-409-3p, miR-7, and miR-93 that yielded high diagnostic accuracy in discriminating CRC from healthy group (AUC: 0.866 and 0.897 for training and validation dataset, respectively). Moreover, the diagnostic performance of the microRNA panel persisted in nonmetastasis CRC stages (Dukes' A-B, AUC: 0.809 and 0.892 for training and validation dataset, respectively) and in metastasis CRC stages (Dukes' C-D, AUC: 0.917 and 0.865 for training and validation dataset, respectively). In conclusion, our study reveals a plasma microRNA panel that has potential clinical value in early CRC detection and would play a critical role on preliminarily screening CRC in general population.

Claudin-4-targeted optical imaging detects pancreatic cancer and its precursor lesions.

OBJECTIVES: Novel imaging methods based on specific molecular targets to detect both established neoplasms and their precursor lesions are highly desirable in cancer medicine. Previously, we identified claudin-4, an integral constituent of tight junctions, as highly expressed in various gastrointestinal tumours including pancreatic cancer. Here, we investigate the potential of targeting claudin-4 with a naturally occurring ligand to visualise pancreatic cancer and its precursor lesions in vitro and in vivo by near-infrared imaging approaches. DESIGN: A non-toxic C-terminal fragment of the claudin-4 ligand Clostridium perfringens enterotoxin (C-CPE) was labelled with a cyanine dye (Cy5.5). Binding of the optical tracer was analysed on claudin-4 positive and negative cells in vitro, and tumour xenografts in vivo. In addition, two genetically engineered mouse models for pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer were used for in vivo validation. Optical imaging studies were conducted using 2D planar fluorescence reflectance imaging (FRI) technology and 3D fluorescence-mediated tomography (FMT). RESULTS: In vitro, the peptide-dye conjugate showed high binding affinity to claudin-4 positive CAPAN1 cells, while claudin-4 negative HT1080 cells revealed little or no fluorescence. In vivo, claudin-4 positive tumour xenografts, endogenous pancreatic tumours, hepatic metastases, as well as preinvasive PanIN lesions, were visualised by FRI and FMT up to 48 h after injection showing a significantly higher average of fluorochrome concentration as compared with claudin-4 negative xenografts and normal pancreatic tissue. CONCLUSIONS: C-CPE-Cy5.5 combined with novel optical imaging methods enables non-invasive visualisation of claudin-4 positive murine pancreatic tumours and their precursor lesions, representing a promising modality for early diagnostic imaging.

Canonical and uncanonical pathogenic germline variants in colorectal cancer patients by next-generation sequencing in a European referral center.

BACKGROUND: Despite increasing use of next-generation sequencing (NGS), data concerning the gain in germline pathogenic variants (PVs) remain scanty, especially with respect to uncanonical ones. We aimed to verify the impact of different cancer predisposition genes (CPGs) on colorectal cancer (CRC) in patients referred for genetic evaluation. MATERIALS AND METHODS: We enrolled for NGS, by Illumina TruSight Cancer panel comprising 94 CPGs, 190 consecutive subjects referred for microsatellite instability (MSI) CRC, polyposis, and/or family history. RESULTS: Overall, 51 (26.8%) subjects carried 64 PVs; PVs coexisted in 4 (7.8%) carriers. PVs in mismatch repair (MMR) genes accounted for one-third of variant burden (31.3%). Four Lynch syndrome patients (20%) harbored additional PVs (HOXB13, CHEK2, BRCA1, NF1 plus BRIP1); such multiple PVs occurred only in subjects with PVs in mismatch syndrome genes (4/20 versus 0/31; P = 0.02). Five of 22 (22.7%) patients with MSI cancers but wild-type MMR genes harbored PVs in unconventional genes (FANCL, FANCA, ATM, PTCH1, BAP1). In 10/63 patients (15.9%) with microsatellite stable CRC, 6 had MUTYH PVs (2 being homozygous) and 4 exhibited uncanonical PVs (BRCA2, BRIP1, MC1R, ATM). In polyposis, we detected PVs in 13 (25.5%) cases: 5 (9.8%) in APC, 6 (11.8%) with biallelic PVs in MUTYH, and 2 (3.9%) in uncanonical genes (FANCM, XPC). In subjects tested for family history only, we detected two carriers (18.2%) with PVs (ATM, MUTYH). CONCLUSION: Uncanonical variants may account for up to one-third of PVs, underlining the urgent need of consensus on clinical advice for incidental findings in cancer-predisposing genes not related to patient phenotype.

Significance of rare variants in genes involved in the pathogenesis of Lynch syndrome.

The molecular characterization of patients with Lynch syndrome (LS) involves germline testing to detect a deleterious mutation in one of the genes of the mismatch repair (MMR) pathway. To date, however, a large proportion of patients with a clinical suspicion of LS who undergo genetic testing do not show a germline pathogenetic variant in these genes. Germline DNA from 73 patients with a clinical suspicion of LS was examined with next­generation sequencing methods, using a multigene custom panel designed and standardized by our research group, that targets a set of 15 genes. Deleterious variants were identified in 5.6% of index cases, while unclassified variants were identified in 80.3% of probands. To evaluate the pathogenicity of these uncertain variants, the American College of Medical Genetics and Genomics criteria was used, also considering wherever possible the microsatellite instability (MSI) status detected on tumor tissues as pathogenic criterion. In this manner, 8 of these uncertain significance variants were classified as likely pathogenic variants. Notably, some of these likely pathogenetic variants were also identified in the MLH3 gene that is a gene not routinely analyzed for cases with a clinical suspicion of LS. The present study highlighted the importance of verifying the pathogenicity of the numerous variants of unknown significance identified in patients for whom heredity is already clinically confirmed suggesting the importance of considering the MSI­H status on the tumor of patients carrying an uncertain variant to evaluate its pathogenicity. Moreover, the present study also suggested analyzing other MMR genes, such as MLH3, in panels used for the molecular screening of LS.

Gastrointestinal polyposis syndromes for the general gastroenterologist.

The occurrence of colonic polyps is a common phenomenon; however, where there are numerous adenomas or other polyps, and/or the patient is at a relatively young age, an inheritable form of gastrointestinal polyposis should be considered. Patients can present via different referral routes, for example, at colonoscopy where multiple polyps are detected, following a personal diagnosis of colorectal cancer, or by family screening. This article outlines the important considerations in the diagnosis of a polyposis syndrome and key diagnostic features to consider. It will also describe the underlying genetic factors associated with the common polyposis syndromes, including classical familial adenomatous polyposis (FAP), attenuated FAP, MUTYH-associated adenomatous polyposis, Peutz-Jeghers syndrome, juvenile polyposis syndrome, Cowden syndrome and serrated polyposis, and the subsequent management of each condition.

The molecular genetics of colorectal cancer.

Colorectal cancer is a common but heterogeneous disease, which arises through the accumulation of genetic mutations. Knowledge of the molecular basis of colorectal cancer has advanced at a rapid pace in recent years, reflecting progress made in the field of genomic medicine. Targeted therapies have come into mainstream use, and the exciting prospect of treatment regimens tailored to the mutation profile of individual tumours is beginning to emerge. In order to understand the development and application of the next generation of colorectal cancer treatments, it is important that gastroenterologists have a working knowledge of the pathological mechanisms that drive the disease. This review examines our current understanding of the molecular genetics of colorectal carcinogenesis.

UK colorectal cancer patients are inadequately assessed for Lynch syndrome.

OBJECTIVE: To establish whether colorectal cancer patients in two centres in the UK are screened appropriately for Lynch syndrome, in accordance with current international guidance. DESIGN: Patients newly diagnosed with colorectal cancer over an 18-month period were identified from the UK National Bowel Cancer Audit Programme. Their records and management were reviewed retrospectively. SETTING: Two university teaching hospitals, Imperial College Healthcare and Oxford Radcliffe Hospitals NHS Trusts. OUTCOMES MEASURED: Whether patients were screened for Lynch syndrome-and the outcome of that evaluation, if it took place-were assessed from patients' clinical records. The age, tumour location and family history of screened patients were compared to those of unscreened patients. RESULTS: Five hundred and fifty three patients with newly diagnosed colorectal cancer were identified. Of these, 97 (17.5%) satisfied the revised Bethesda criteria, and should have undergone further assessment. There was no evidence that those guidelines had been contemporaneously applied to any patient. In practice, only 22 of the 97 (22.7%) eligible patients underwent evaluation. The results for 14 of those 22 (63.6%) supported a diagnosis of Lynch syndrome, but only nine of the 14 (64.3%) were referred for formal mismatch repair gene testing. No factors reliably predicted whether or not a patient would undergo Lynch syndrome screening. CONCLUSIONS: Colorectal teams in the UK do not follow international guidance identifying the patients who should be screened for Lynch syndrome. Patients and their families are consequently excluded from programmes reducing colorectal cancer incidence and mortality. Multidisciplinary teams should work with their local genetics services to develop reliable algorithms for patient screening and referral.

A national survey of hereditary colorectal cancer services in the UK.

OBJECTIVES: The British Society of Gastroenterology (BSG) Cancer Group designed a survey to determine how we might understand and improve the service for patients at elevated risk of hereditary colorectal cancer (CRC). DESIGN AND SETTING: United Kingdom (UK) gastroenterologists, colorectal surgeons, and oncologists were invited by email to complete a 10 point questionnaire. This was cascaded to 1,793 members of the Royal College of Radiologists (RCR), Association of Cancer Physicians (ACP), the Association of Coloproctology of Great Britain and Ireland (ACPGBI), as well as BSG members. RESULTS: Three hundred and eighty-two members responded to the survey, an overall response rate of 21.3%. Although 69% of respondents felt there was an adequate service for these higher risk patients, 64% believed that another clinician was undertaking this work. There was no apparent formal patient pathway in 52% of centres, and only 33% of centres maintain a registry of these patients. Tumour block testing for Lynch Syndrome is not usual practice. Many appeared to be unaware of the BSG/ACPGBI UK guidelines for the management of these patients. CONCLUSIONS: There is wide variability in local management and in subsequent clinical pathways for hereditary CRC patients. There is a perception that they are being managed by 'another', unspecified clinician. National guidelines are not adhered to. We therefore recommend improved education, well defined pathways and cyclical audit in order to improve care of patients with hereditary CRC risk.