Volumetric growth rates of sessile serrated adenomas/polyps observed in situ at longitudinal CT colonography.

OBJECTIVE: Sessile serrated adenomas/polyps (SSA/Ps) are now recognized as potential cancer precursors, but little is known about their natural history. We assessed the in vivo growth rates of histologically proven SSA/Ps at longitudinal CT colonography (CTC) and compared results with non-advanced tubular adenomas (TAs). METHODS: We identified a cohort of 53 patients (mean age, 54.8 ± 5.5 years; M:F, 26:27) from one center with a total of 58 SSA/Ps followed longitudinally at CTC (mean follow-up interval, 5.3 ± 1.9 years). Initial and final size measurements were determined using dedicated CTC software. Findings were compared with 141 non-advanced TAs followed at CTC (mean, 4.1 ± 2.3 years) in 113 patients (mean age, 56.8 ± 6.9 years). RESULTS: SSA/Ps were more often flat (62% [36/58] vs. 14% [20/141], p < 0.0001) and right-sided (98% [57/58] vs. 46% [65/141], p < 0.0001) compared with TAs. Initial average diameter was greater for SSA/Ps (9.3 mm vs. 6.3 mm; p < 0.0001). Mean annual volumetric growth was + 12.7%/year for SSA/Ps vs. + 36.4%/year for TAs (p = 0.028). Using a previously defined threshold of + 20% increase in volume/year to define progression, 22% (13/58) of SSA/Ps and 41% (58/141) of TAs progressed (p = 0.014). None of the SSA/Ps had dysplasia or invasive cancer at histopathology. CONCLUSIONS: Sessile serrated adenoma/polyps demonstrate slower growth compared with conventional non-advanced tubular adenomas, despite larger initial linear size. This less aggressive behavior may help explain the more advanced patient age for serrated pathway cancers. Furthermore, these findings could help inform future colonoscopic surveillance strategies, as current guidelines are largely restricted to expert opinion related to the absence of natural history data. KEY POINTS: • Sessile serrated adenoma/polyps (SSA/Ps) tend to be flat, right-sided, and demonstrate slower growth compared with conventional non-advanced tubular adenomas. • This less aggressive behavior of SSA/Ps may help explain the more advanced patient age for serrated pathway cancers.

Association of sporadic and familial Barrett's esophagus with breast cancer.

Barrett's esophagus (BE) is the only known precursor to esophageal adenocarcinoma (EAC). Based on striking aggregation of breast cancer and BE/EAC within families as well as shared risk factors and molecular mechanisms of carcinogenesis, we hypothesized that BE may be associated with breast cancer. Pedigree analysis of families identified prospectively at multiple academic centers as part of the Familial Barrett's Esophagus Consortium (FBEC) was reviewed and families with aggregation of BE/EAC and breast cancer are reported. Additionally, using a matched case-control study design, we compared newly diagnosed BE cases in Caucasian females with breast cancer (cases) to Caucasian females without breast cancer (controls) who had undergone upper endoscopy (EGD). Two familial pedigrees, meeting a stringent inclusion criterion, manifested familial aggregation of BE/EAC and breast cancer in an autosomal dominant inheritance pattern with incomplete penetrance. From January 2008 to October 2016, 2812 breast cancer patient charts were identified, of which 213 were Caucasian females who underwent EGD. Six of 213 (2.82%) patients with breast cancer had pathology-confirmed BE, compared to 1 of 241 (0.41%) controls (P-value < 0.05). Selected families with BE/EAC show segregation of breast cancer. A breast cancer diagnosis is marginally associated with BE. We postulate a common susceptibility between BE/EAC and breast cancer.

TGF-ß signaling alters the pattern of liver tumorigenesis induced by Pten inactivation.

Hepatocarcinogenesis results from the accumulation of genetic and epigenetic changes in liver cells. A common mechanism through which these alterations induce liver cancer is by deregulating signaling pathways. A number of signaling pathways, including the PI3K/PTEN/AKT and transforming growth factor ß (TGF-ß) pathways have been implicated in normal liver development as well as in cancer formation. In this study, we assessed the effect of the TGF-ß signaling pathway on liver tumors induced by phosphatase and tensin homolog (Pten) loss. Inactivation of only the TGF-ß receptor type II, Tgfbr2, in the mouse liver (Tgfbr2(LKO)) had no overt phenotype, while inactivation of Pten alone (Pten(LKO)), resulted in the formation of both hepatocellular carcinomas and cholangiocarcinomas (CC). Interestingly, deletion of both Pten and Tgfbr2 (Pten(LKO);Tgfbr2(LKO)) in the mouse liver resulted in a dramatic shift in tumor type to predominantly CC. Assessment of the PI3K/PTEN/AKT pathway revealed increased phosphorylation of AKT and glycogen synthase kinase 3 beta (GSK-3ß) in both the Pten(LKO) and Pten(LKO);Tgfbr2(LKO) mice, suggesting that this pathway is constitutively active regardless of the status of the TGF-ß signaling pathway. However, phosphorylation of p70 S6 kinase was observed in the liver of all three phenotypes (Tgfbr2(LKO), Pten(LKO), Pten(LKO);Tgfbr2(LKO)) indicating that the loss of Tgfbr2 and/or Pten leads to an increase in this signaling pathway. Analysis of markers of liver progenitor/stem cells revealed that the loss of TGF-ß signaling resulted in increased expression of c-Kit and CD133. Furthermore, in addition to increased c-Kit and CD133, Scf and EpCam expression were also increased in the double knock-out mice. These results suggest that the alteration in tumor types between the Pten(LKO) mice and Pten(LKO);Tgfbr2(LKO) mice is secondary to the altered regulation of stem-cell features induced by the loss of TGF-ß signaling.

Characterisation of familial colorectal cancer Type X, Lynch syndrome, and non-familial colorectal cancer.

BACKGROUND: Familial Colorectal Cancer Type X (FCCTX) is defined as individuals with colorectal cancer (CRC) who families meet Amsterdam Criteria-1 (AC1), but whose tumours are DNA-mismatch-repair-proficient, unlike Lynch syndrome (LS). FCCTX does not have an increased risk of extra-colonic cancers. This analysis compares epidemiologic and clinicopathologic features among FCCTX, LS, and 'non-familial' (non-AC1) CRC cases. METHODS: From the Colon Cancer Family Registry, FCCTX (n=173), LS (n=303), and non-AC1 (n=9603) CRC cases were identified. Questionnaire-based epidemiologic information and CRC pathologic features were compared across case groups using polytomous logistic regression. RESULTS: Compared with LS, FCCTX cases were less likely to be current (vs never) smokers; have a proximal subsite (vs rectal) tumour; or have mucinous histology, poor differentiation, or tumour-infiltrating lymphocytes. There were no observed differences in co-morbidities or medication usage. CONCLUSIONS: FCCTX were less likely to be current tobacco users; other exposures were similar between these groups. Histopathologic differences highly suggestive of LS CRCs do not appear to be shared by FCCTX.

Inactivation of TGF-ß signaling and loss of PTEN cooperate to induce colon cancer in vivo.

The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-ß) signaling pathway, which is often inactivated by mutations of TGF-ß type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in ∼30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite-unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperates to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2(IEKO)) alone is not sufficient for intestinal tumor formation and lack of Pten (Pten(IEKO)) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (Pten(IEKO);Tgfbr2(IEKO)) led to malignant tumors in both the small intestine and colon in 86% of the mice and to metastases in 8% of the tumor-bearing mice. Moreover, these tumors arose via a ß-catenin-independent mechanism. Inactivation of TGF-ß signaling and loss of Pten in the tumors led to increased cell proliferation, decreased apoptosis and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-ß signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.

RET is a potential tumor suppressor gene in colorectal cancer.

Cancer arises as the consequence of mutations and epigenetic alterations that activate oncogenes and inactivate tumor suppressor genes. Through a genome-wide screen for methylated genes in colon neoplasms, we identified aberrantly methylated RET in colorectal cancer. RET, a transmembrane receptor tyrosine kinase and a receptor for the glial cell-derived neurotrophic factor family ligands, was one of the first oncogenes to be identified, and has been shown to be an oncogene in thyroid cancer and pheochromocytoma. However, unexpectedly, we found RET is methylated in 27% of colon adenomas and in 63% of colorectal cancers, and now provide evidence that RET has tumor suppressor activity in colon cancer. The aberrant methylation of RET correlates with decreased RET expression, whereas the restoration of RET in colorectal cancer cell lines results in apoptosis. Furthermore, in support of a tumor suppressor function of RET, mutant RET has also been found in primary colorectal cancer. We now show that these mutations inactivate RET, which is consistent with RET being a tumor suppressor gene in the colon. These findings suggest that the aberrant methylation of RET and the mutational inactivation of RET promote colorectal cancer formation, and that RET can serve as a tumor suppressor gene in the colon. Moreover, the increased frequency of methylated RET in colon cancers compared with adenomas suggests RET inactivation is involved in the progression of colon adenomas to cancer.

Epigenetic silencing of the intronic microRNA hsa-miR-342 and its host gene EVL in colorectal cancer.

MicroRNAs are small, non-coding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA targets. MicroRNA expression is dysregulated in human malignancies, frequently leading to loss of expression of certain microRNAs. We report that expression of hsa-miR-342, a microRNA encoded in an intron of the gene EVL, is commonly suppressed in human colorectal cancer. The expression of hsa-miR-342 is coordinated with that of EVL and our results indicate that the mechanism of silencing is CpG island methylation upstream of EVL. We found methylation at the EVL/hsa-miR-342 locus in 86% of colorectal adenocarcinomas and in 67% of adenomas, indicating that it is an early event in colorectal carcinogenesis. In addition, we observed a higher frequency of methylation (56%) in histologically normal colorectal mucosa from individuals with concurrent cancer compared to mucosa from individuals without colorectal cancer (12%), suggesting the existence of a 'field defect' involving methylated EVL/hsa-miR-342. Furthermore, reconstitution of hsa-miR-342 in the colorectal cancer cell line HT-29 induced apoptosis, suggesting that this microRNA could function as a proapoptotic tumor suppressor. In aggregate, these results support a novel mechanism for silencing intronic microRNAs in cancer by epigenetic alterations of cognate host genes.

Epigenetic events in the colorectum and in colon cancer.

Colon cancers arise from benign neoplasms and evolve into adenocarcinomas through a stepwise histological progression sequence, proceeding from either adenomas or hyperplastic polyps/serrated adenomas. Genetic alterations have been associated with specific steps in this polyp-adenocarcinoma sequence and are believed to drive the histological progression of colon cancer. Recently, epigenetic alterations, which include CGI (CpG island) DNA methylation, have been shown to occur in colon polyps and colon cancer. The aberrant methylation of genes appears to co-operate with the genetic alterations to drive the initiation and progression of colon polyps to colon cancer. CGI DNA methylation is an epigenetic mechanism that represses gene transcription in normal cellular processes, but it becomes excessive and aberrant in many neoplasms. The aberrant DNA methylation affects CpG-rich regions, called CGIs, in the 5' region of genes and results in transcriptional silencing through effects on transcription factor binding and associated changes in chromatin structure. These hypermethylated genes are not only probable pathogenic events affecting colon-cancer formation, but also neoplasm-specific molecular events that may be useful as molecular markers for colon tumours. Furthermore, aberrant DNA methylation of tumour-suppressor genes may occur secondary to a genetic predisposition or to a field-cancerization effect in the colon and may be useful as molecular markers for the risk of developing colon cancer.

Demographic and phenotypic features of 70 families segregating Barrett's oesophagus and oesophageal adenocarcinoma.

BACKGROUND: Based on reported familial patterns, inheritance of a predisposition of developing Barrett's oesesophagus (BO) and oesophageal adenocarcinoma (OAC) likely follows an autosomal dominant model of most inherited cancer syndromes. oesophagus (BO) and oesophageal adenocarcinoma (OAC) likely follows an autosomal dominant model of most inherited cancer syndromes. AIMS: We analysed the phenotypic features of 70 familial BO/OAC families accrued for the purpose of initiating a linkage study to search for genes that contribute to susceptibility for BO/OAC. METHODS: Families with young or familial BO/OAC were recruited from participating institutions and self-referral from advertisement. RESULTS: A total of 70 families (173 affected and 784 unaffected individuals) were recruited into this study. Mean ages of diagnosis of BO and OAC among males were 50.6 and 57.4 years, respectively; among females, 52.1 and 63.5 years, respectively. The standardised incidence ratio (SIR) of cancers other than OAC or oesophagogastric junctional adenocarcinoma (OGJAC), among probands was 0.71. Seventy one percent of the pedigrees have "typical" structures with less than three affected individuals. Power calculations under realistic model assumptions suggest that if genetic heterogeneity is absent or limited, then DNA collection from members of these pedigrees could enable the identification of a novel candidate susceptibility gene for BO/OAC in a genome scan. CONCLUSIONS: This is the largest series of families with BO/OAC yet reported, features of which are consistent with inherited germline predisposition. Further, the SIR of cancers other than OAC/OGJAC was 0.71 among 70 probands, indicating these individuals were not more likely to develop non-OAC cancers.

Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer.

Serological tumor markers have proven valuable in the care of individuals with cancer for the early detection of primary cancers, early detection of cancer relapse, monitoring the response of cancers to therapy, and as predictors of cancer prognosis. Recently, the aberrant hypermethylation of the hMLH1 promoter and its consequent transcriptional silencing has been shown to be a common event in the formation of sporadic microsatellite unstable colon cancer. The silencing of hMLH1 expression appears to be controlled by the hypermethylation of a specific region in the hMLH1 promoter. We developed a methylation-specific PCR assay that assesses this region of the hMLH1 promoter. We found that this assay is able to detect methylated hMLH1 promoter DNA in the serum of some patients with microsatellite unstable colon cancers. In a panel of sera from 19 colon cancer cases, 9 with hMLH1 promoter methylation in the tumor primary, the assay proved 33% sensitive and 100% specific. This assay offers a potential means for the serum-based detection and/or monitoring of microsatellite unstable colon cancers.

Mutation Detection inTGF-ß Receptors.

The transforming growth factor ß (TGF-ß) receptors are an important class of tumor suppressor gene. TGF-ß markedly inhibits the growth of many epithelial cell types; whereas in contrast, cancers of many different tissue types are commonly TGF-ß resistant (10). Many cancer cell lines are resistant to the growth suppressive effects of TGF-ß and display evidence of disruption of TGF-ß signal transduction (10,4,10). To date, this resistance appears to often result from mutations in the TGF-ß receptors or the smad family of TGF-ß signaling proteins (5,11,12,14,16).

Methylation of the CDH1 promoter as the second genetic hit in hereditary diffuse gastric cancer.

Aberrant promoter methylation and the associated loss of gene expression is a common accompaniment of human cancers. Nonetheless, it has been challenging to demonstrate in any given tumour that methylation of a specific gene was causal and not consequent to malignant transformation. In this regard, our attention was drawn to the genesis of gastric cancers in individuals with hereditary diffuse gastric cancer (HDGC). These individuals harbour germline mutations in the gene encoding E-cadherin, CDH1, but their cancers have consistently demonstrated absence of loss of heterozygosity at the CDH1 locus. These findings suggested the hypothesis that CDH1 promoter methylation might function as the 'second genetic hit' in the genesis of these cancers.

Genomic instability and colorectal cancer.

Colon cancer results from the accumulation of genetic alterations. Genomic instability creates a permissive state in which a potential cancer cell is allowed to acquire enough mutations to become a cancer cell. Several forms of genomic instability are common in colon cancer: microsatellite instability (MSI), chromosome instability (CIN), and chromosome translocations. MSI occurs in approximately 15% of colon cancers and results from inactivation of the mutation mismatch repair (MMR) system secondary to either MMR gene mutations or hypermethylation of the hMLH1 promoter. It promotes tumorigenesis by generating mutations in target genes that possess coding microsatellite repeats, such as the transforming growth factor-beta receptor type II gene. CIN occurs in most other colon cancers and leads to a different pattern of gene alterations that culminate in tumor formation. It seems to result from mutations in genes that control mitosis, DNA damage repair, centrosome structure and function, and other fundamental processes in DNA replication. The clinical significance of genomic instability is now under investigation, and it is hoped that this research will soon yield results that have an immediate effect on the treatment of colon cancer.

E-cadherin germline mutations define an inherited cancer syndrome dominated by diffuse gastric cancer.

To extend earlier observations of germline E-cadherin mutations in kindreds with an inherited susceptibility to diffuse gastric cancer, we searched for germline E-cadherin mutations in five further families affected predominantly by diffuse gastric cancer and one family with a history of diffuse gastric cancer and early-onset breast cancer. Heterozygous inactivating mutations were found in the E-cadherin gene in each of these families. No mutation hotspots were identified. These results demonstrate that germline mutation of the E-cadherin gene is a common cause of hereditary diffuse gastric cancer and suggest a role for these mutations in the incidence of breast cancer.

Mutational inactivation of transforming growth factor beta receptor type II in microsatellite stable colon cancers.

We previously demonstrated that mutational inactivation of transforming growth factor beta type II receptors (RIIs) is very common among the 13% of human colon cancers with microsatellite instability. These mutations principally cluster in the BAT-RII polyadenine sequence repeat. Among microsatellite stable (MSS) colon cancers, we now find that non-BAT-RII point mutations inactivate RII in another 15% of cases, thus doubling the known number of colon cancers in which RII mutations are pathogenetic. Functional analysis confirms that these mutations inactivate RII signaling. Moreover, another 55% of MSS colon cancers demonstrate a transforming growth factor beta signaling blockade distal to RII. The transforming growth factor beta pathway and RII in particular are major targets for inactivation in MSS colon cancers as well as in colon cancers with microsatellite instability.

p27 cell-cycle inhibitor is inversely correlated with lymph node metastases in right-sided colon cancer.

p27, a cyclin-dependent kinase inhibitor, suppresses proliferation of normal and neoplastic cells. Expression of p27 is correlated with survival in colon cancer. To some degree, right-sided colon cancers differ biologically and clinically from left-sided colon cancers. We analyzed 41 patients with right-sided colon cancers, including 18 cases with regional lymph node metastases and 23 cases with negative lymph nodes. Immunostaining for p27 was performed on histologic sections of primary cancers and scored. Correlation of p27 protein expression with histologic parameters was performed by t-test and multivariate analysis. Decreased p27 protein expression was associated with large tumor size. As percentages of positively stained tumor cells decreased from 70 to 29%, the mean tumor size increased from 1.9 to 7.3 cm. p27 protein expression significantly decreased in primary cancers with angiolymphatic invasion or with positive lymph nodes in comparison with those without angiolymphatic invasion (26 +/- 6 vs. 44 +/- 5%, P < 0.03) or with negative lymph nodes (23 +/- 4 vs. 47 +/- 6%, P < 0.003). p27 expression was not statistically different in terms of depth of tumor invasion (T1/T2 vs. T3/T4), tumor type or tumor differentiation. Multivariate analysis revealed that low p27 expression in primary cancers was correlated with lymph node metastases (P = 0.01). However, it did not correlate with any other histologic parameters. In summary, decreased p27 expression was associated with an increased likelihood of lymph node metastases in colon cancers, independent of depth of tumor invasion. This implies that p27 is a potentially important predictor for tumor metastasis and patient's prognosis in right-sided colon cancers.

Mutation of the type II transforming growth factor-beta receptor is coincident with the transformation of human colon adenomas to malignant carcinomas.

The transforming growth factor-beta (TGF-beta) type II receptor (RII) is a colon cancer suppressor gene that is inactivated by mutation in 90% of human colon cancers arising via the microsatellite instability (MSI) pathway of carcinogenesis. To determine the pathophysiological consequence of RII mutations, we have determined the timing of their onset among 22 MSI human colon adenomas of varying stages. No RII mutations were detected in any early MSI adenoma, including all those with simple tubular or villous histology. The earliest RII mutation detected was in a region of high-grade dysplasia but was absent from the surrounding simple adenoma. Six additional RII mutations were all found in highly progressed adenomas that contained regions of frankly invasive adenocarcinoma. These RII mutations were detected in both the advanced adenomas and their adjacent regions of carcinoma. RII mutation is a late event in MSI adenomas and correlates tightly with progression of these adenomas to cancer.

Mini-microabscess syndrome in liver transplant recipients.

Cytomegalovirus (CMV) is a significant cause of morbidity in immunosuppressed patients. It is characterized in the liver by parenchymal microabscesses, usually containing CMV-infected cells. However, not all hepatic microabscesses are due to CMV infection. In 1992, we described "mini" microabscess (MMA) syndrome, a distinct clinical syndrome that occurs in transplanted livers. This report analyzes the clinical and laboratory features of 57 cases of MMA syndrome occurring in 52 patients and compares these with 19 biopsy-proven cases of CMV infection. The diagnosis of MMA syndrome can only be made histologically. The microabscesses are smaller and more numerous than in CMV infection, and there are no viral inclusions present. CMV DNA could not be detected in liver biopsy specimens with MMAs by using "nested" polymerase chain reaction (PCR), indicating that MMA syndrome is not caused by CMV infection. The pattern of liver enzyme and bilirubin elevation is predominantly hepatocellular, with transaminase levels elevated, on average, six to eight times the upper limit of normal. The clinical features of MMA syndrome are that it predominantly affects female (40 of 52 patients) orthotopic liver transplant (OLT) recipients of all ages (range, 11 months to 66.9 years). MMA syndrome is unrelated to the indication for initial OLT and tends to occur later after transplantation than CMV infection (median, 91 days post-OLT vs. 32 days for CMV hepatitis). Although the etiology of MMA syndrome is not clear, it does not appear to adversely affect graft or patient survival.