Different APC genotypes in proximal and distal sporadic colorectal cancers suggest distinct WNT/ß-catenin signalling thresholds for tumourigenesis.

Biallelic protein-truncating mutations in the adenomatous polyposis coli (APC) gene are prevalent in sporadic colorectal cancer (CRC). Mutations may not be fully inactivating, instead producing WNT/ß-catenin signalling levels 'just-right' for tumourigenesis. However, the spectrum of optimal APC genotypes accounting for both hits, and the influence of clinicopathological features on genotype selection remain undefined. We analysed 630 sporadic CRCs for APC mutations and loss of heterozygosity (LOH) using sequencing and single-nucleotide polymorphism microarrays, respectively. Truncating APC mutations and/or LOH were detected in 75% of CRCs. Most truncating mutations occurred within a mutation cluster region (MCR; codons 1282-1581) leaving 1-3 intact 20 amino-acid repeats (20AARs) and abolishing all Ser-Ala-Met-Pro (SAMP) repeats. Cancers commonly had one MCR mutation plus either LOH or another mutation 5' to the MCR. LOH was associated with mutations leaving 1 intact 20AAR. MCR mutations leaving 1 vs 2-3 intact 20AARs were associated with 5' mutations disrupting or leaving intact the armadillo-repeat domain, respectively. Cancers with three hits had an over-representation of mutations upstream of codon 184, in the alternatively spliced region of exon 9, and 3' to the MCR. Microsatellite unstable cancers showed hyper-mutation at MCR mono- and di-nucleotide repeats, leaving 2-3 intact 20AARs. Proximal and distal cancers exhibited different preferred APC genotypes, leaving a total of 2 or 3 and 0 to 2 intact 20AARs, respectively. In conclusion, APC genotypes in sporadic CRCs demonstrate 'fine-tuned' interdependence of hits by type and location, consistent with selection for particular residual levels of WNT/ß-catenin signalling, with different 'optimal' thresholds for proximal and distal cancers.

Ultrafast and widely tuneable vertical-external-cavity surface-emitting laser, mode-locked by a graphene-integrated distributed Bragg reflector.

We report a versatile way of controlling the unsaturated loss, modulation depth and saturation fluence of graphene-based saturable absorbers (GSAs), by changing the thickness of a spacer between a single layer graphene (SLG) and a high-reflection mirror. This allows us to modulate the electric field intensity enhancement at the GSA from 0 up to 400%, due to the interference of incident and reflected light at the mirror. The unsaturated loss of the SLG-mirror-assembly can be reduced to ∼0. We use this to mode-lock a vertical-external-cavity surface-emitting laser (VECSEL) from 935 to 981 nm. This approach can be applied to integrate SLG into various optical components, such as output coupler mirrors, dispersive mirrors or dielectric coatings on gain materials. Conversely, it can also be used to increase the absorption (up to 10%) in various graphene based photonics and optoelectronics devices, such as photodetectors.

Low repetition rate SESAM modelocked VECSEL using an extendable active multipass-cavity approach.

Ultrafast VECSELs are compact pulsed laser sources with more flexibility in the emission wavelength compared to diode-pumped solid-state lasers. Typically, the reduction of the pulse repetition rate is a straightforward method to increase both pulse energy and peak power. However, the relatively short carrier lifetime of semiconductor gain materials of a few nanoseconds sets a lower limit to the repetition rate of passively modelocked VECSELs. This fast gain recovery combined with low pulse repetition rates leads to the buildup of multiple pulses in the cavity. Therefore, we applied an active multipass approach with which demonstrate fundamental modelocking at a repetition rate of 253 MHz with 400 mW average output power in 11.3 ps pulses.

The TERT variant rs2736100 is associated with colorectal cancer risk.

BACKGROUND: Polymorphic variation at the 5p15.33 (TERT-CLPTM1L) locus is associated with the risk of many cancers but a relationship with colorectal cancer (CRC) risk has yet to be defined. METHODS: We used data from six genome-wide association studies (GWAS) of CRC, linkage disequilibrium mapping and imputation, to examine the relationship between 73 single-nucleotide polymorphisms at 5p15.33 and CRC risk in detail. RESULTS: rs2736100, which localises to intron 2 of TERT, provided the strongest evidence of an association with CRC (P=2.28 × 10⁻4). The association was also shown in an independent series of 10 047 CRC cases and 6918 controls (P=0.02). A meta-analysis of all seven studies (totalling 16 039 cases, 16 430 controls) provided increased evidence of association (P=2.49 × 10⁻5; per allele odds ratio=1.07). The association of rs2736100 on CRC risk was shown to be independent of 15 low-penetrance variants previously identified. CONCLUSION: The rs2736100 association demonstrates an influence of variation at 5p15.33 on CRC risk and further evidence that the 5p15.33 (TERT-CLPTM1L) locus has pleiotropic effects (reflecting generic or lineage-specific effects) on cancer risk.

High-power MIXSEL: an integrated ultrafast semiconductor laser with 6.4 W average power.

High-power ultrafast lasers are important for numerous industrial and scientific applications. Current multi-watt systems, however, are based on relatively complex laser concepts, for example using additional intracavity elements for pulse formation. Moving towards a higher level of integration would reduce complexity, packaging, and manufacturing cost, which are important requirements for mass production. Semiconductor lasers are well established for such applications, and optically-pumped vertical external cavity surface emitting lasers (VECSELs) are most promising for higher power applications, generating the highest power in fundamental transverse mode (>20 W) to date. Ultrashort pulses have been demonstrated using passive modelocking with a semiconductor saturable absorber mirror (SESAM), achieving for example 2.1-W average power, sub-100-fs pulse duration, and 50-GHz pulse repetition rate. Previously the integration of both the gain and absorber elements into a single wafer was demonstrated with the MIXSEL (modelocked integrated external-cavity surface emitting laser) but with limited average output power (<200 mW). We have demonstrated the power scaling concept of the MIXSEL using optimized quantum dot saturable absorbers in an antiresonant structure design combined with an improved thermal management by wafer removal and mounting of the 8-µm thick MIXSEL structure directly onto a CVD-diamond heat spreader. The simple straight cavity with only two components has generated 28-ps pulses at 2.5-GHz repetition rate and an average output power of 6.4 W, which is higher than for any other modelocked semiconductor laser.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

APC and the three-hit hypothesis.

The seminal 'two-hit hypothesis' implicitly assumes that bi-allelic tumour suppressor gene (TSG) mutations cause loss of protein function. All subsequent events in that tumour therefore take place on an essentially null background for that TSG protein. We have shown that the two-hit model requires modification for the APC TSG, because mutant APC proteins probably retain some function and the two hits are co-selected to produce an optimal level of Wnt activation. We wondered whether the optimal Wnt level might change during tumour progression, leading to selection for more than two hits at the APC locus. Comprehensive screening of a panel of colorectal cancer (CRC) cell lines and primary CRCs showed that some had indeed acquired third hits at APC. These third hits were mostly copy number gains or deletions, but could be protein-truncating mutations. Third hits were significantly less common when the second hit at APC had arisen by copy-neutral loss of heterozygosity. Both polyploid and near-diploid CRCs had third hits, and the third hits did not simply arise as a result of acquiring a polyploid karyotype. The third hits affected mRNA and protein levels, with potential functional consequences for Wnt signalling and tumour growth. Although some third hits were probably secondary to genomic instability, others did appear specifically to target APC. Whilst it is generally believed that tumours develop and progress through stepwise accumulation of mutations in different functional pathways, it also seems that repeated targeting of the same pathway and/or gene is selected in some cancers.

Analysis of copy number changes suggests chromosomal instability in a minority of large colorectal adenomas.

We have examined chromosomal-scale mutations in 34 large colorectal adenomas (CRAs). A small number of changes (median = 2, IQR = 0-4) were found by array-comparative genomic hybridization (aCGH) in most tumours. The most common changes were deletions of chromosomes 1p, 9q, 17, 19, and 22, and gains of chromosomes 13 and 21. SNP-LOH analysis and pseudo-digital SNP-PCR analysis detected occasional copy-neutral LOH. Some aCGH changes found frequently in colorectal carcinomas, such as deletions of chromosomes 4q and 18q, were very infrequent in the adenomas. Almost all copy number changes were of small magnitude, far below the predicted levels even for single copy gain/loss; investigation suggested that these changes were either artefactual or occurred in sub-clones within the tumours. In some cases, these sub-clones may have represented progression towards carcinoma, but comparison with aCGH data from carcinomas showed this to be unlikely in most cases. In two adenomas, there was evidence of a large, outlying number of copy number changes, mostly resulting from part-chromosome deletions. Overall, moreover, there was evidence of a tendency towards part-chromosome deletions-consistent with chromosomal instability (CIN)--in about one-sixth of all tumours. However, there was no evidence of CIN in the form of whole-chromosome copy number changes. Our data did not support previous contentions that CRAs tend to show chromosome breakage at fragile sites owing to CIN associated with an elevated DNA damage response. Chromosomal-scale mutations occur in some CRAs; although CIN is not the norm in these lesions, it probably affects a minority of cases.

Investigation of pathogenic mechanisms in multiple colorectal adenoma patients without germline APC or MYH/MUTYH mutations.

Patients with multiple (5-100) colorectal adenomas (MCRAs) often have no germline mutation in known predisposition genes, but probably have a genetic origin. We collected a set of 25 MCRA patients with no detectable germline mutation in APC, MYH/MUTYH or the mismatch repair genes. Extracolonic tumours were absent in these cases. No vertical transmission of the MCRA phenotype was found. Based on the precedent of MYH-associated polyposis (MAP), we searched for a mutational signature in 241 adenomatous polyps from our MCRA cases. Somatic mutation frequencies and spectra at APC, K-ras and BRAF were, however, similar to those in sporadic colorectal adenomas. Our data suggest that the genetic pathway of tumorigenesis in the MCRA patients' tumours is very similar to the classical pathway in sporadic adenomas. In sharp contrast to MAP tumours, we did not find evidence of a specific mutational signature in any individual patient or in the overall set of MCRA cases. These results suggest that hypermutation of APC does not cause our patients' disease and strongly suggests that MAP is not a paradigm for the remaining MCRA patients. Our MCRA patients' colons showed no evidence of microadenomas, unlike in MAP and familial adenomatous polyposis (FAP). However, nuclear beta-catenin expression was significantly greater in MCRA patients' tumours than in sporadic adenomas. We suggest that, at least in some cases, the MCRA phenotype results from germline variation that acts subsequent to tumour initiation, perhaps by causing more rapid or more likely progression from microadenoma to macroadenoma.

Genetic determinants modulate susceptibility to pregnancy-associated tumourigenesis in a recombinant line of Min mice.

Min mice provide a good model of human familial adenomatous polyposis. Recently, we have reported on two recombinant inbred lines (I and V) and the location of a modifier (Mom3) close to Apc, which altered polyp numbers in our mice possibly by modifying the frequency of wild-type (WT) allele loss at Apc; mice with severe disease (line V) showed elevated rates of loss. We now show that in line I only, a single pregnancy caused a significant increase in adenoma multiplicity compared with virgin controls (P<0.001) and that an additional pregnancy conferred a similar risk. Pregnancy was linked to both adenoma initiation and enhanced tumour growth in line I mice, and interline crosses indicated that susceptibility to pregnancy-associated adenomas was under genetic control. We found no evidence for the involvement of oestrodial metabolizing genes or the oestrogen receptors (Esr1 and 2) in tumour multiplicity. Importantly, a significantly elevated frequency of WT allele loss at Apc was observed in adenomas from parous mice (line and backcrossed) carrying the line I Min allele relative to equivalent virgin controls (P=0.015). Our results provide the first experimental evidence for genetic determinants controlling pregnancy-associated tumourigenesis; analogous genetic factors may exist in humans.

Disease severity and genetic pathways in attenuated familial adenomatous polyposis vary greatly but depend on the site of the germline mutation.

BACKGROUND: Attenuated familial adenomatous polyposis (AFAP) is associated with germline mutations in the 5', 3', and exon 9 of the adenomatous polyposis coli (APC) gene. These mutations probably encode a limited amount of functional APC protein. METHODS AND RESULTS: We found that colonic polyp number varied greatly among AFAP patients but members of the same family tended to have more similar disease severity. 5' Mutants generally had more polyps than other patients. We analysed somatic APC mutations/loss of heterozygosity (LOH) in 235 tumours from 35 patients (16 families) with a variety of AFAP associated germline mutations. In common with two previous studies of individual kindreds, we found biallelic changes ("third hits") in some polyps. We found that the "third hit" probably initiated tumorigenesis. Somatic mutation spectra were similar in 5' and 3' mutant patients, often resembling classical FAP. In exon 9 mutants, in contrast, "third hits" were more common. Most "third hits" left three 20 amino acid repeats (20AARs) on the germline mutant APC allele, with LOH (or proximal somatic mutation) of the wild-type allele; but some polyps had loss of the germline mutant with mutation leaving one 20AAR on the wild-type allele. CONCLUSIONS: We propose that mutations, such as nt4661insA, that leave three 20AARs are preferentially selected in cis with some AFAP mutations because the residual protein function is near optimal for tumorigenesis. Not all AFAP polyps appear to need "three hits" however. AFAP is phenotypically and genetically heterogeneous. In addition to effects of different germline mutations, modifier genes may be acting on the AFAP phenotype, perhaps influencing the quantity of functional protein produced by the germline mutant allele.

Analysis of chromosomal instability in human colorectal adenomas with two mutational hits at APC.

In vitro data show that the adenomatous polyposis coli (APC) protein associates with the mitotic spindle and that mouse embryonic stem cells with biallelic Apc mutations are karyotypically unstable. These findings led to suggestions that APC acts in chromosomal segregation and that APC inactivation leads to chromosomal instability (CIN). An alternative hypothesis based on allelic loss studies in colorectal adenomas proposes that CIN precedes and contributes to genetic changes at APC. We determined whether colorectal adenomas with two mutations at APC show features consistent with these models by studying 55 lesions (average size 5 mm; range 1-13 mm) from patients with familial adenomatous polyposis. A variety of methods was used depending on available material, including flow cytometry, comparative genomic hybridization, and loss of heterozygosity (LOH) analysis. Selected adenomas were assessed for proliferative activity by Ki-67 immunocytochemistry. Seventeen of 20 (85%) tumors were diploid, two were near-diploid, and one was hypotetraploid. Just one (near-diploid) tumor showed increased proliferative activity. LOH was found occasionally on chromosome 15q (2 of 49 tumors), but not on chromosome 18q (0 of 48). In 20 adenomas, LOH at APC was associated with loss at 5q but not 5p markers, with the former encompassing a minimum of 20 Mb. However, three of these lesions analyzed by comparative genomic hybridization displayed normal profiles, suggesting, together with other data, that the mechanism of LOH at APC is probably somatic recombination. Our results therefore do not support the hypothesis that CIN precedes APC mutations in tumorigenesis. Regarding the model in which APC mutations lead directly to CIN, if APC mutations do have this effect in vivo, it must be subtle. Alternatively, CIN associated with APC mutations might be essentially an in vitro phenomenon.

Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or "multiple" colorectal adenomas.

Familial adenomatous polyposis (FAP) is a dominantly inherited colorectal tumor predisposition that results from germ-line mutations in the APC gene (chromosome 5q21). FAP shows substantial phenotypic variability: classical polyposis patients develop more than 100 colorectal adenomas, whereas those with attenuated polyposis (AAPC) have fewer than 100 adenomas. A further group of individuals, so-called "multiple" adenoma patients, have a phenotype like AAPC, with 3-99 polyps throughout the colorectum, but mostly have no demonstrable germ-line APC mutation. Routine mutation detection techniques fail to detect a pathogenic APC germ-line mutation in approximately 30% of patients with classical polyposis and 90% of those with AAPC/multiple adenomas. We have developed a real-time quantitative multiplex PCR assay to detect APC exon 14 deletions. When this technique was applied to a set of 60 classical polyposis and 143 AAPC/multiple adenoma patients with no apparent APC germ-line mutation, deletions were found exclusively in individuals with classical polyposis (7 of 60, 12%). Fine-mapping of the region suggested that the majority (6 of 7) of these deletions encompassed the entire APC locus, confirming that haploinsufficiency can result in a classical polyposis phenotype. Screening for germ-line deletions in APC mutation-negative individuals with classical polyposis seems warranted.

The adenomatous polyposis coli (APC) tumour suppressor--genetics, function and disease.

Mutations in the adenomatous polyposis coli (APC) gene are the basis of familial adenomatous polyposis and the majority of sporadic colorectal cancer. APC is expressed in a wide variety of tissues, interacts with the cytoskeleton, is involved in regulating levels of beta-catenin and, most recently, has been shown to bind DNA, suggesting that it may possess a nuclear role. The mutation spectrum implicated in tumorigenesis and its correlation with disease phenotype is well characterized and has contributed to our understanding of important functional domains in APC. Despite these advances, APC continues to provide a fertile subject of research for both colorectal tumorigenesis and cancer in general.

Germline APC variants in patients with multiple colorectal adenomas, with evidence for the particular importance of E1317Q.

Mendelian tumour syndromes are caused by rare mutations, which usually lead to protein inactivation. Few studies have determined whether or not the same genes harbour other, more common variants, which might have a lower penetrance and/or cause mild disease, perhaps indistinguishable from sporadic disease and accounting for a considerable proportion of the unexplained inherited risk of tumours in the general population. Germline variants at the APC locus are excellent candidates for explaining why some individuals are predisposed to colorectal adenomas, but do not have the florid phenotype of familial adenomatous polyposis. We have screened 164 unrelated patients with 'multiple' (3-100) colorectal adenomas for germline variants throughout the APC gene, including promoter mutations. In addition to three Ashkenazi patients with I1307K, we found seven patients with the E1317Q variant. E1317Q is significantly associated with multiple colorectal adenomas (OR = 11. 17, 95% CI = 2.30-54.3, p < 0.001), accounting for approximately 4% of all patients with multiple colorectal adenomas. In addition, four patients with truncating APC variants in exon 9 or in the 3' part of the gene were identified. Germline APC variants account for approximately 10% of patients with multiple adenomas. Unidentified predisposition genes almost certainly exist. We argue that it is worthwhile to screen multiple adenoma patients for a restricted number of germline APC variants, namely the missense changes E1317Q and I1307K (if of Ashkenazi descent), and, if there is a family history of colorectal tumours, for truncating mutations 5' to exon 5, in exon 9 and 3' to codon 1580.

Shigella sepsis.

It is generally believed that bacteremia rarely occurs with enteric Shigella infections. During a five-year period, 110 children were admitted to the University Hospital of the Arizona Health Sciences Center, Tucson, with Shigella enteritis. Eight (7%) had Shigella bacteremia/septicemia, and four of the eight died. An analysis of these patients and their course disclosed clinical characteristics that were of potential prognostic importance. Some of these indicators are identifiable early and hence could be useful in management. The patients at greatest risk were afebrile; 10% or more dehydrated; malnourished, with a low serum albumin level; infected with ampicillin-resistant strains; had leukopenia during the course of the illness; and had persistent watery hemoglobin-positive stools.

Transient protein-losing enteropathy and enlarged gastric rugae in childhood.

Data on two patients with massive but transient protein-losing enteropathy due to gastric disease suggest that antibody-mediated allergic reactions may have been responsible for the clinical picture. The majority of patients described in the literature have had evidence of a preceding or concomitant viral infection, and three of these have had evidence of infection with cytomegalovirus. Recovery occurred relatively rapidly in all patients without sequelae. The syndrome may be a variant of eosinophilic gastroenteritis.