Mechanisms of immunogenicity in colorectal cancer.

BACKGROUND: The immune response in cancer is increasingly understood to be important in determining clinical outcomes, including responses to cancer therapies. New insights into the mechanisms underpinning the immune microenvironment in colorectal cancer are helping to develop the role of immunotherapy and suggest targeted approaches to the management of colorectal cancer at all disease stages. METHOD: A literature search was performed in PubMed, MEDLINE and Cochrane Library databases to identify relevant articles. This narrative review discusses the current understanding of the contributors to immunogenicity in colorectal cancer and potential applications for targeted therapies. RESULTS: Responsiveness to immunotherapy in colorectal cancer is non-uniform. Several factors, both germline and tumour-related, are potential determinants of immunogenicity in colorectal cancer. Current approaches target tumours with high immunogenicity driven by mutations in DNA mismatch repair genes. Recent work suggests a role for therapies that boost the immune response in tumours with low immunogenicity. CONCLUSION: With the development of promising therapies to boost the innate immune response, there is significant potential for the expansion of the role of immunotherapy as an adjuvant to surgical treatment in colorectal cancer.

ANTECEDENTES: La respuesta inmune en el cáncer se considera cada vez más importante por su influencia sobre los resultados clínicos, incluidas las respuestas a las diferentes modalidades de tratamiento. Los nuevos conocimientos sobre los mecanismos implicados en el microambiente inmunitario en el cáncer colorrectal están ayudando a definir el papel de la inmunoterapia y el desarrollo de terapias dirigidas para el tratamiento del cáncer colorrectal en todos los estadios de la enfermedad. MÉTODOS: Se realizó una búsqueda bibliográfica en las bases de datos PubMed, Medline y Cochrane para identificar artículos relevantes. Esta revisión descriptiva discute la comprensión actual de los factores que contribuyen a la inmunogenicidad en el cáncer colorrectal y las posibles aplicaciones en terapias dirigidas. RESULTADOS: La capacidad de respuesta a la inmunoterapia en el cáncer colorrectal no es uniforme. Varios factores, tanto relacionados con la línea germinal, como con el tumor son determinantes potenciales de la inmunogenicidad en el cáncer colorrectal. Los estudios actuales están dirigidos a tumores con alta inmunogenicidad provocada por mutaciones en los genes de reparación de apareamientos erróneos en el ADN. Trabajos recientes sugieren un papel para los tratamientos que estimulan la respuesta inmune en tumores con baja inmunogenicidad. CONCLUSIÓN: Con el desarrollo de tratamientos prometedores para estimular la respuesta inmune innata, existe un potencial significativo para la expansión del papel de la inmunoterapia como adyuvante del tratamiento quirúrgico en el cáncer colorrectal.

Consensus and controversies regarding follow-up after treatment with curative intent of nonmetastatic colorectal cancer: a synopsis of guidelines used in countries represented in the European Society of Coloproctology.

AIM: It is common clinical practice to follow patients for a period of years after treatment with curative intent of nonmetastatic colorectal cancer, but follow-up strategies vary widely. The aim of this systematic review was to provide an overview of recommendations on this topic in guidelines from member countries of the European Society of Coloproctology, with supporting evidence. METHOD: A systematic search of Medline, Embase and the guideline databases Trip database, BMJ Best Practice and Guidelines International Network was performed. Quality assessment included use of the AGREE-II tool. All topics with recommendations from included guidelines were identified and categorized. For each subtopic, a conclusion was made followed by the degree of consensus and the highest level of evidence. RESULTS: Twenty-one guidelines were included. The majority recommended that structured follow-up should be offered, except for patients in whom treatment of recurrence would be inappropriate. It was generally agreed that clinical visits, measurement of carcinoembryoinc antigen and liver imaging should be part of follow-up, based on a high level of evidence, although the frequency is controversial. There was also consensus on imaging of the chest and pelvis in rectal cancer, as well as endoscopy, based on lower levels of evidence and with a level of intensity that was contradictory. CONCLUSION: In available guidelines, multimodal follow-up after treatment with curative intent of colorectal cancer is widely recommended, but the exact content and intensity are highly controversial. International agreement on the optimal follow-up schedule is unlikely to be achieved on current evidence, and further research should refocus on individualized 'patient-driven' follow-up and new biomarkers.

Recommendations and consensus on the treatment of peritoneal metastases of colorectal origin: a systematic review of national and international guidelines.

AIM: This systematic review aimed to provide an overview of (inter)national guidelines on the treatment of peritoneal metastases of colorectal cancer origin (PMCRC) and to determine the degree of consensus and available evidence with identification of topics for future research. METHOD: A systematic search of MEDLINE, Embase, PubMed as well as Tripdatabase, National Guideline Clearinghouse, BMJ Best Practice and Guidelines International Network was performed to identify (inter)national guidelines and consensus statements from oncological or surgical societies on PMCRC. The quality of guidelines was assessed using the AGREE-II score. Topics followed by recommendations were extracted from the guidelines. The recommendations, highest level of supporting evidence and the degree of consensus were determined for each topic. RESULTS: Twenty-one guidelines were included, in most (15) of which cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) was recommended in selected patients based on level 1b evidence. Substantial consensus was also reached on the benefit of multidisciplinary team discussion and the achievability of a (near) complete cytoreduction (CC0-1) without supporting evidence. Both evidence and consensus were lacking regarding other aspects including preoperative positron emission tomography/CT, second look surgery in high risk patients, the optimal patient selection for CRS/HIPEC, procedural aspects of HIPEC and (perioperative) systemic therapy. CONCLUSION: In currently available guidelines, evidence and consensus on the treatment strategy for PMCRC are lacking. Updates of guidelines are ongoing and future (randomized) clinical trials should contribute to multidisciplinary and international consensus on treatment strategies for PMCRC.

Systematic review of evidence and consensus on perianal fistula: an analysis of national and international guidelines.

AIM: Treatment of perianal fistula has evolved with the introduction of new techniques and biologicals in Crohn's disease (CD). Several guidelines are available worldwide, but many recommendations are controversial or lack high-quality evidence. The aim of this work was to provide an overview of the current available national and international guidelines for perianal fistula and to analyse areas of consensus and areas of conflicting recommendations, thereby identifying topics and questions for future research. METHOD: MEDLINE, EMBASE and PubMed were systematically searched for guidelines on perianal fistula. Inclusion was limited to papers in English less than 10 years old. The included topics were classified as having consensus (unanimous recommendations in at least two-thirds of the guidelines) or controversy (fewer than three guidelines commenting on the topic or no consensus) between guidelines. The highest level of evidence was scored as sufficient (level 3a or higher of the Oxford Centre for Evidence-based Medicine Levels of Evidence 2009, http://www.cebm.net/oxford-centre-evidence-based-medicine-levels-evidence-march-2009/) or insufficient. RESULTS: Twelve guidelines were included and topics with recommendations were compared. Overall, consensus was present in 15 topics, whereas six topics were rated as controversial. Evidence levels varied from strong to lack of evidence. CONCLUSION: Evidence on the diagnosis and treatment of perianal fistulae (cryptoglandular or related to CD) ranged from nonexistent to strong, regardless of consensus. The most relevant research questions were identified and proposed as topics for future research.

Influence of day of surgery on mortality following elective colorectal resections.

BACKGROUND: The aim of this study was to investigate whether the increased mortality previously identified for surgery performed on Fridays was apparent following major elective colorectal resections and how this might be affected by case mix. METHODS: Patients undergoing elective colorectal resections in England from 2001 to 2011 were identified using Hospital Episode Statistics. Propensity scores were used to match patients having operations on a Friday in a 1 : 1 ratio with those undergoing surgery on other weekdays. Multivariable analyses were used to investigate overall deaths within 1 year of operation. RESULTS: A total of 204,669 records were extracted for patients undergoing major elective colorectal resections. Patients who had surgery on Fridays were more deprived (4780 (17.1 per cent) of 27,920 versus 28,317 (16.0 per cent) of 176,749; P < 0.001), a greater proportion had had an emergency admission in the 3 previous months (7870 (28.2 per cent) of 27,920 versus 48,623 (27.5 per cent) of 176,749; P = 0.019), underwent minimal access surgery (4565 (16.4 per cent) of 27,920 versus 23,783 (13.5 per cent) of 176,749; P < 0.001) and had surgery for benign diagnoses (6502 (23.3 per cent) of 27,920 versus 38,725 (21.9 per cent) of 176,749; P < 0.001) than those who had surgery on Mondays to Thursdays. In a matched analysis the odds ratio for 30-day mortality after colorectal resections performed on Fridays compared with other weekdays was 1.25 (95 per cent c.i. 1.13 to 1.37); odds ratios for 90-day and 1-year mortality were 1.16 (1.07 to 1.25) and 1.10 (1.04 to 1.16) respectively. CONCLUSION: Patients selected for colorectal resections on Fridays had a higher mortality rate than patients operated on from Monday to Thursday and had different characteristics, suggesting that increased mortality may reflect patient factors rather than hospital variables alone.

Biomarker-based treatment selection in early-stage rectal cancer to promote organ preservation.

BACKGROUND: Total mesorectal excision (TME) remains commonplace for T1-2 rectal cancer owing to fear of undertreating a small proportion of patients with node-positive disease. Molecular stratification may predict cancer progression. It could be used to select patients for organ-preserving surgery if specific biomarkers were validated. METHODS: Gene methylation was quantified using bisulphite pyrosequencing in 133 unirradiated rectal cancer TME specimens. KRAS mutation and microsatellite instability status were also defined. Molecular parameters were correlated with histopathological indices of disease progression. Predictive models for nodal metastasis, lymphovascular invasion (LVI) and distant metastasis were constructed using a multilevel reverse logistic regression model. RESULTS: Methylation of the retinoic acid receptor ß gene, RARB, and that of the checkpoint with forkhead and ring finger gene, CHFR, was associated with tumour stage (RARB: 51·9 per cent for T1-2 versus 33·9 per cent for T3-4, P < 0·001; CHFR: 5·5 per cent for T1-2 versus 12·6 per cent for T3-4, P = 0·005). Gene methylation associated with nodal metastasis included RARB (47·1 per cent for N- versus 31·7 per cent for N+; P = 0·008), chemokine ligand 12, CXCL12 (12·3 per cent for N- versus 8·9 per cent for N+; P = 0·021), and death-associated protein kinase 1, DAPK1 (19·3 per cent for N- versus 12·3 per cent for N+; P = 0·022). RARB methylation was also associated with LVI (45·1 per cent for LVI- versus 31·7 per cent for LVI+; P = 0·038). Predictive models for nodal metastasis and LVI achieved sensitivities of 91·1 and 85·0 per cent, and specificities of 55·3 and 45·3 per cent, respectively. CONCLUSION: This methylation biomarker panel provides a step towards accurate discrimination of indolent and aggressive rectal cancer subtypes. This could offer an improvement over the current standard of care, whereby fit patients are offered radical surgery.

Systematic review of evidence and consensus on diverticulitis: an analysis of national and international guidelines.

AIM: The study aimed to analyse the currently available national and international guidelines for areas of consensus and contrasting recommendations in the treatment of diverticulitis and thereby to design questions for future research. METHOD: MEDLINE, EMBASE and PubMed were systematically searched for guidelines on diverticular disease and diverticulitis. Inclusion was confined to papers in English and those < 10 years old. The included topics were classified as consensus or controversy between guidelines, and the highest level of evidence was scored as sufficient (Oxford Centre of Evidence-Based Medicine Level of Evidence of 3a or higher) or insufficient. RESULTS: Six guidelines were included and all topics with recommendations were compared. Overall, in 13 topics consensus was reached and 10 topics were regarded as controversial. In five topics, consensus was reached without sufficient evidence and in three topics there was no evidence and no consensus. Clinical staging, the need for intraluminal imaging, dietary restriction, duration of antibiotic treatment, the protocol for abscess treatment, the need for elective surgery in subgroups of patients, the need for surgery after abscess treatment and the level of the proximal resection margin all lack consensus or evidence. CONCLUSION: Evidence on the diagnosis and treatment of diverticular disease and diverticulitis ranged from nonexistent to strong, regardless of consensus. The most relevant research questions were identified and proposed as topics for future research.

The costs of surgical site infection after abdominal surgery in middle-income countries: Key resource use In Wound Infection (KIWI) study.

BACKGROUND: Surgical site infection (SSI) is the most common complication of abdominal surgery, with substantial costs to patients and health systems. Heterogeneity in costing methods in existing SSI studies makes multi-country comparison challenging. The objective of the study was to assess the costs of SSI across middle-income countries. METHODS: Centres from a randomized controlled trial assessing interventions to reduce SSI (FALCON, ClinicalTrials.gov, NCT03700749NCT) were sampled from two upper-middle- (India, Mexico) and two lower-middle- (Ghana, Nigeria) income countries. The Key resource use In Wound Infection (KIWI) study collected data on postoperative resource use and costs from consecutive patients undergoing abdominal surgery with an incision >5 cm (including caesarean section) that were recruited to FALCON between April and October 2020. The overall costs faced by patients with and without SSI were compared by operative field contamination (clean-contaminated vs contaminated-dirty), country and timing (inpatient vs outpatient). FINDINGS: A total of 335 patients were included in KIWI; SSI occurred in 7% of clean-contaminated cases and 27% of contaminated-dirty cases. Overall, SSI was associated with an increase in postoperative healthcare costs by 75.3% (€412 international Euros) after clean-contaminated surgery and 66.6% (€331) after contaminated-dirty surgery. The highest and lowest cost increases were in India for clean-contaminated cases (€517) and contaminated-dirty cases (€223), respectively. Overall, inpatient costs accounted for 96.4% of the total healthcare costs after clean-contaminated surgery and 92.5% after contaminated-dirty surgery. CONCLUSION: SSI was associated with substantial additional postoperative costs across a range of settings. Investment in health technologies to reduce SSI may mitigate the financial burden to patients and low-resource health systems.

Methylation profiling of rectal cancer identifies novel markers of early-stage disease.

BACKGROUND: Radical surgery is the de facto treatment for early rectal cancer. Conservative surgery with transanal endoscopic microsurgery can achieve high rates of cure but the histopathological measures of outcome used to select local treatment lack precision. Biomarkers associated with disease progression, particularly mesorectal nodal metastasis, are urgently required. The aim was to compare patterns of gene-specific hypermethylation in radically excised rectal cancers with histopathological stage. METHODS: Locus-specific hypermethylation of 24 tumour suppressor genes was measured in 105 rectal specimens (51 radically excised adenocarcinomas, 35 tissues adjacent to tumour and 19 normal controls) using the methylation-specific multiplex ligation-dependent probe assay (MS-MLPA). Methylation values were correlated with histopathological indices of disease progression and validated using bisulphite pyrosequencing. RESULTS: Five sites (ESR1, CDH13, CHFR, APC and RARB) were significantly hypermethylated in cancer compared with adjacent tissue and normal controls (P < 0·050). Methylation at these sites was higher in Dukes' A than Dukes' 'D' cancers (P = 0·013). Methylation at two sites (GSTP1 and RARB) was individually associated with localized disease (N0 and M0 respectively; P = 0·006 and P = 0·008). Hypermethylation of at least two of APC, RARB, TIMP3, CASP8 and GSTP1 was associated with early (N0 M0) disease (N0, P = 0·002; M0, P = 0·044). Methylation levels detected by MS-MLPA and pyrosequencing were concordant. CONCLUSION: Locus-specific hypermethylation was more prevalent in early- than late-stage disease. Hypermethylation of two or more of a panel of five tumour suppressor genes was associated with localized disease.

Wnt signalling in adenomas of familial adenomatous polyposis patients.

BACKGROUND: Epigenetic silencing of Wnt antagonists and expression changes in genes associated with Wnt response pathways occur in early sporadic colorectal tumourigenesis, indicating that tumour cells are more sensitive to Wnt growth factors and respond differently. In this study, we have investigated whether similar changes occur in key markers of the Wnt response pathways in the genetic form of the disease, familial adenomatous polyposis (FAP). METHODS: We investigated epigenetic and expression changes using pyrosequencing and real-time RT-PCR in samples from seven patients without neoplasia, and matched normal and tumour tissues from 22 sporadic adenoma and 14 FAP patients. RESULTS: We found that 17 out of 24 (71%) FAP adenomas were hypermethylated at sFRP1, compared with 20 out of 22 (91%) of sporadic cases. This was reflected at the level of sFRP1 transcription, where 73% of FAP and 100% of sporadic cases were down-regulated. Increased expression levels of c-myc and FZD3 were less common in FAP (35 and 46% respectively) than sporadic tumours (78 and 67% respectively). CONCLUSION: Overall, the changes in expression and methylation were comparable, although the degree of change was generally lower in the FAP adenomas. Molecular heterogeneity between multiple adenomas from individual FAP patients may reflect different developmental fates for these premalignant tumours.

Reorganisation of Wnt-response pathways in colorectal tumorigenesis.

In most colorectal tumours, APC mutation stabilises beta-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of beta-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca(2+) receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

Hereditary colorectal cancer: screen the parents!

Hereditary nonpolyposis colorectal cancer is the most common form of hereditary colorectal cancer. Occasionally, the presentation of colorectal cancer may be at an early age when parents may be unidentified obligate carriers. The risk of colorectal cancer increases with increasing age, even in inherited disease. Therefore, it is important to screen parents of patients presenting at a young age and to obtain a complete pedigree to identify and screen those who are at risk. Two such families where the index case presented at a young age were encountered in our practice and both their mothers were found to have colorectal cancer on surveillance colonoscopy. We recommend that parents of the index cases should be screened and more detailed family pedigree obtained when patients present at a young age with colorectal cancer.

Silkworm 5S RNA and alanine tRNA genes share highly conserved 5' flanking and coding sequences.

A fragment of Bombyx mori genomic DNA containing one tRNA2Ala gene and one 5S RNA gene has been used to compare the structural features of silkworm 5S RNA and tRNA genes. The nucleotide sequences of both genes and of the primary transcripts produced from them in homologous in vitro transcription systems have been determined. Comparison of the sequences of these two genes with that of another previously analyzed B. mori tRNA2Ala gene reveals common oligonucleotides which may be important transcriptional signals. The oligonucleotides TA(C)TAT, AATTTT, and TTC are located approximately (+/- 1 nucleotide) 29, 19, and 3 nucleotides, respectively, before the transcription initiation sites of the two tRNA2Ala genes and the one 5S RNA gene we have analyzed. The sequence GGGCGTAG(C)TCAG lies within the coding regions of all three genes. The functional significance of these sequences is suggested by their location within regions required for the transcription of silkworm alanine tRNA genes in vitro.

Cellular and subcellular localization of PDE10A, a striatum-enriched phosphodiesterase.

PDE10A is a recently identified phosphodiesterase that is highly expressed by the GABAergic medium spiny projection neurons of the mammalian striatum. Inhibition of PDE10A results in striatal activation and behavioral suppression, suggesting that PDE10A inhibitors represent a novel class of antipsychotic agents. In the present studies we further elucidate the localization of this enzyme in striatum of rat and cynomolgus monkey. We find by confocal microscopy that PDE10A-like immunoreactivity is excluded from each class of striatal interneuron. Thus, the enzyme is restricted to the medium spiny neurons. Subcellular fractionation indicates that PDE10A is primarily membrane bound. The protein is present in the synaptosomal fraction but is separated from the postsynaptic density upon solubilization with 0.4% Triton X-100. Immuno-electron microscopy of striatum confirms that PDE10A is most often associated with membranes in dendrites and spines. Immuno-gold particles are observed on the edge of the postsynaptic density but not within this structure. Our studies indicate that PDE10A is associated with post-synaptic membranes of the medium spiny neurons, suggesting that the specialized compartmentation of PDE10A enables the regulation of intracellular signaling from glutamatergic and dopaminergic inputs to these neurons.

Two novel regions of interstitial deletion on chromosome 8p in colorectal cancer.

We have investigated interstitial deletions of chromosome 8 in 70 colorectal carcinomas and 11 colonic adenomas using 11 microsatellite markers, including eight spanning the centromeric region of chromosome 8p (p11.2-p12). Allelic loss or imbalance was observed in 38 (54%) cancers and four (36%) adenomas. Twenty-eight (40%) of the cancers had deletions of 8p11.2-p12. Two distinct and independent regions of interstitial loss were found within this region. Fluorescent in situ hybridization, using an alpha satellite repeat probe to the centromere of 8p and two probes to the P1 region, was performed in four tumours that demonstrated allelic imbalance. Localized heterozygous deletions were confirmed in all four tumours. Eleven (16%) cancers had localized deletion in the region ANK-1 to D8S255 (P1) and a further eleven (16%) cancers had a less well localized deletion in the region defined by the markers D8S87 to D8S259 (P2). Loss of both centromeric loci was identified in a further six (9%) tumours. A functional significance for these two deletion regions was sought by correlation with primary and secondary tumour characteristics. Isolated P2 deletion was associated with 'early' T1 cancers (2p=0.0002), and were also identified in 3/11 adenomas. Conversely, interstitial deletions of the P1 locus were more frequently seen in 'locally invasive' T3/4 cancers (2p=0.015), and isolated P1 deletions were also associated with the presence of liver metastases (2p=0.016). Our data provide evidence of at least two genes within the 8p11.2-p12 region, mutations in which may confer different and independent roles in the pathogenesis of colorectal cancer.

par-4, a gene required for cytoplasmic localization and determination of specific cell types in Caenorhabditis elegans embryogenesis.

Specification of some cell fates in the early Caenorhabditis elegans embryo is mediated by cytoplasmic localization under control of the maternal genome. Using nine newly isolated mutations, and two existing mutations, we have analyzed the role of the maternally expressed gene par-4 in cytoplasmic localization. We recovered seven new par-4 alleles in screens for maternal effect lethal mutations that result in failure to differentiate intestinal cells. Two additional par-4 mutations were identified in noncomplementation screens using strains with a high frequency of transposon mobility. All 11 mutations cause defects early in development of embryos produced by homozygous mutant mothers. Analysis with a deficiency in the region indicates that it33 is a strong loss-of-function mutation. par-4(it33) terminal stage embryos contain many cells, but show no morphogenesis, and are lacking intestinal cells. Temperature shifts with the it57ts allele suggest that the critical period for both intestinal differentiation and embryo viability begins during oogenesis, about 1.5 hr before fertilization, and ends before the four-cell stage. We propose that the primary function of the par-4 gene is to act as part of a maternally encoded system for cytoplasmic localization in the first cell cycle, with par-4 playing a particularly important role in the determination of intestine. Analysis of a par-4; par-2 double mutant suggests that par-4 and par-2 gene products interact in this system.

Analysis of the Birt-Hogg-Dubé (BHD) tumour suppressor gene in sporadic renal cell carcinoma and colorectal cancer.

Germline mutations in the BHD gene cause the dominantly inherited cancer susceptibility disorder, Birt-Hogg-Dubé (BHD) syndrome. Individuals with BHD are reported to have an increased risk of renal cell carcinoma (RCC) and of colorectal polyps and cancer. The BHD gene maps to 17p11.2, and to investigate whether somatic inactivation of the BHD gene region is implicated in the pathogenesis of sporadic RCC and colorectal cancer (CRC), we performed mutation analysis in 30 RCC primary tumours and cell lines, and 35 CRCs and cell lines. A somatic missense mutation (Ala444Ser) with loss of the wild type allele (consistent with a two hit mechanism of tumorigenesis) was detected in a primary clear cell RCC, and a further missense mutation (Ala238Val) was identified in a clear cell RCC cell line for which matched normal DNA was not available. A somatic missense substitution (Arg392Gly) was identified in a primary CRC, and the same change was detected in three RCCs (all oncocytomas) for which matched normal DNA was not available. A germline Arg320Gln missense variant detected in a primary CRC was not detected in 40 control individuals or in a further 159 familial and sporadic CRC cases. However, AA homozygotes for an intronic single nucleotide polymorphism (c.1517+6 G-->A) were under-represented in familial cases compared with controls (p = 0.03). For some tumour suppressor genes, epigenetic silencing is a more common mechanism of inactivation than somatic mutations. However, we did not detect evidence of epigenetic silencing of BHD in 19 CRC and RCC cell lines, and BHD promoter region hypermethylation was not detected in 20 primary RCCs. These findings suggest that BHD inactivation occurs in a subset of clear cell RCC and CRC.