MSH6 germline mutations in early-onset colorectal cancer patients without family history of the disease

Germline MLH1 and MSH2 mutations are scarce in young colorectal cancer patients with negative family history of the disease. To evaluate the contribution of germline MSH6 mutations to early-onset colorectal cancer, we have analysed peripheral blood of 38 patients diagnosed with this disease before 45 years of age and who presented no family history of hereditary nonpolyposis colorectal cancer-related cancers. Blood samples from 108 healthy volunteers were analysed for those genetic alterations suspected to affect the function of MSH6. Of the seven (18.4%) MSH6 alterations found, we have identified three novel germline mutations, one 8 bp deletion leading to a truncated protein and two missense mutations resulting in the substitution of amino acids belonging to different polarity groups. High-frequency microsatellite instability was found in the patient with the MSH6 deletion, but not in the other 27 carcinomas analysed. No MLH1 promoter methylation was detected in tumour tissue. Our findings suggest that germline MSH6 mutations contribute to a subset of early-onset colorectal cancer. Further studies are warranted to understand the genetic and environmental factors responsible for the variable penetration of MSH6 germline mutations, as well as to identify other causes of early-onset colorectal cancer.     

Inhibition of VEGF induces cellular senescence in colorectal cancer cells

Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16.     

PTEN loss of expression predicts cetuximab efficacy in metastatic colorectal cancer patients

To evaluate whether the epidermal growth factor receptor (EGFR), K-Ras and PTEN, all members of the EGFR signalling pathway, may affect the clinical response in cetuximab-treated metastatic colorectal cancer (mCRC) patients. Twenty-seven cetuximab-treated mCRC patients were evaluated for drug response and investigated for EGFR protein expression and gene status, K-Ras mutational status and PTEN protein expression. Ten patients achieved a partial response (PR) to cetuximab-based therapy. All 27 patients showed EGFR protein overexpression. Epidermal growth factor receptor gene amplification was observed in eight out of 27 (30%) and chromosome 7 marked polysomy in 16 (59%) patients. Partial response was observed in six out of eight patients with EGFR gene amplification, four out of 16 with marked polysomy and none out of three with eusomy (P<0.05). The K-Ras wild-type sequence was observed in 17 patients, and nine of them experienced a PR. Conversely, K-Ras was mutated in 10 cases, of which one patient experienced a PR (P<0.05). The PTEN protein was normally expressed in 16 patients, and 10 of them achieved a PR. In contrast, no benefit was documented in 11 patients with loss of PTEN activity (P<0.001). Patients with EGFR gene amplification or chromosome 7 marked polysomy respond to cetuximab. In addition to K-Ras mutations, we demonstrate for the first time that the loss of PTEN protein expression is associated with nonresponsiveness to cetuximab.     

Down-regulation of KRAS-interacting miRNA-143 predicts poor prognosis but not response to EGFR-targeted agents in colorectal cancer

Background:

MicroRNA-143 (miRNA-143) is frequently down-regulated in colorectal cancer (CRC) and may influence CRC cell proliferation, apoptosis and sensitivity to 5-fluorouracil. mRNA encoded by the KRAS oncogene has been identified as a target of miRNA-143. However, the prognostic significance of miRNA-143 expression and the ability to predict patient response to epidermal growth factor receptor (EGFR)-targeted agents have not yet been explored.

Methods:

We examined 77 CRC patients who were identified by pyrosequencing to have wild-type KRAS and were subsequently treated with EGFR-targeted therapy with the monoclonal antibodies cetuximab or panitumumab. MicroRNA-143 expression was measured in CRC tissue and corresponding non-neoplastic colon tissue by RT–PCR and its expression level was correlated with clinico-pathological characteristics. Univariate and multivariate analyses were used to calculate cancer-specific survival (CSS). The progression-free survival (PFS) and objective response rates on EGFR-targeted therapy were also evaluated.

Results:

Down-regulation of miRNA-143 was observed in 47 out of 77 (61%) tumours. Multivariate Cox regression analysis identified low levels of miRNA-143 expression as an independent prognostic factor with respect to CSS (hazard ratio=1.92, confidence interval=1.1–3.4, P=0.024). A significant difference was also observed with regard to PFS on EGFR-targeted therapy (P=0.031), but there were no significant differences with regard to the objective response rates.

Conclusion:

Our data indicate that miRNA-143 expression levels serve as an independent prognostic biomarker for CRC in KRAS wild-type patients. No role for miRNA-143 expression as a predictive biomarker for EGFR-targeted agents could be identified. Given its negative impact on CSS and PFS, miRNA-143 represents a novel prognosticator and a promising drug target for patients with CRC.     

The in situ local immune response,tumour senescence and proliferation in colorectal cancer

Background:

Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16^ink4a) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16^ink4a, Ki-67 and immune infiltrates have similar prognostic value.

Methods:

Immunostaining of p16^inka and Ki-67 was performed within a CRC tissue microarray. Nuclear p16^inka and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs).

Results:

Two hundred and thirty stage I–III cancers were studied. High nuclear p16^ink4a was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16^ink4a expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16^ink4a demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16^ink4a; P=0.002) were independently associated with poorer cancer survival.

Conclusion:

Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16^ink4a-associated senescence is not associated with an upregulation of antitumour T-cell responses.     

No SMAD4 hypermethylation in colorectal cancer

The chromosome region 18q21 is frequently deleted in colorectal cancers. Three candidate tumour suppressor genes, DCC, SMAD4 and SMAD2, map to this region. The SMAD4(DPC4) gene was recently identified as a candidate pancreatic cancer suppressor gene. It is also a gene for juvenile polyposis tumour predisposition syndrome. Somatic SMAD4 mutations have been detected in some colorectal carcinomas. However, the frequency of these mutations is relatively low, and whether SMAD4 plays a key role in colorectal tumorigenesis is still unclear. In addition to loss of chromosomal material and intragenic mutations there is a third mechanism, DNA methylation, which may have an important role in gene inactivation. In the present study, we examined whether promoter hypermethylation could be a mechanism for SMAD4 inactivation. In total, 42 colorectal tumours were selected for the methylation analysis and no evidence of promoter hypermethylation was found. Our result suggests that hypermethylation of the SMAD4 promoter region is not a frequent event in colorectal tumorigenesis.     

Wild-type APC predicts poor prognosis in microsatellite-stable proximal colon cancer

Background:

APC mutations (APC-mt) occur in ∼70% of colorectal cancers (CRCs), but their relationship to prognosis is unclear.

Methods:

APC prognostic value was evaluated in 746 stage I–IV CRC patients, stratifying for tumour location and microsatellite instability (MSI). Microarrays were used to identify a gene signature that could classify APC mutation status, and classifier ability to predict prognosis was examined in an independent cohort.

Results:

Wild-type APC microsatellite stable (APC-wt/MSS) tumours from the proximal colon showed poorer overall and recurrence-free survival (OS, RFS) than APC-mt/MSS proximal, APC-wt/MSS distal and APC-mt/MSS distal tumours (OS HR⩾1.79, P⩽0.015; RFS HR⩾1.88, P⩽0.026). APC was a stronger prognostic indicator than BRAF, KRAS, PIK3CA, TP53, CpG island methylator phenotype or chromosomal instability status (P⩽0.036). Microarray analysis similarly revealed poorer survival in MSS proximal cancers with an APC-wt-like signature (P=0.019). APC status did not affect outcomes in MSI tumours. In a validation on 206 patients with proximal colon cancer, APC-wt-like signature MSS cases showed poorer survival than APC-mt-like signature MSS or MSI cases (OS HR⩾2.50, P⩽0.010; RFS HR⩾2.14, P⩽0.025). Poor prognosis APC-wt/MSS proximal tumours exhibited features of the sessile serrated neoplasia pathway (P⩽0.016).

Conclusions:

APC-wt status is a marker of poor prognosis in MSS proximal colon cancer.     

Adiponectin gene and risk of colorectal cancer

Background:

Genes of the adiponectin pathway are interesting candidates for colorectal cancer risk based on the potential association between colorectal cancer and obesity. However, variants of the adiponectin gene (ADIPOQ) have been demonstrated to be inconsistently associated with risk of colorectal cancer.

Methods:

The current study attempted to evaluate these findings by examining several single nucleotide polymorphisms (SNPs) that were previously genotyped as part of a genome-wide association study in the ADIPOQ gene. Genotyping was also performed for a previously reported risk variant, rs266729, in 1062 individuals with a diagnosis of colorectal cancer and 1062 controls matched on age, gender and ethnicity (Jewish or not Jewish) as part of a population-based case–control study in Israel.

Results:

No evidence was found for an association between ADIPOQ and risk of colorectal cancer. The single nucleotide variant previously associated with decreased risk of colorectal cancer, rs266729, revealed an adjusted odds ratio of 1.04; 95% confidence interval, 0.88–1.23.

Conclusion:

The SNP, rs266729, was not strongly associated with colorectal cancer in patients of Ashkenazi Jewish descent or other ethnic groups in Israel.     

Mutations in Bcl10 are very rare in colorectal cancer.

Bcl10 is a recently identified gene reported to be involved commonly in human malignancy (Willis et al (1999) Cell 96: 1-20). To investigate whether it is frequently mutated in colorectal cancer we have analysed a series of 132 colorectal cancers and eight colorectal cancer cell lines for mutations in Bcl10. One feature of the Bcl10 gene is that it harbours two polyadenine tracts. These repeating elements in genes can be prone to a high rate of mutation if there is defective mismatch repair. To examine the possibility that Bcl10 may be preferentially mutated in mismatch repair-deficient cancers, 49 of the tumours and cell lines were known to be replication error (RER)-positive and, of these, ten were from individuals harbouring germline mutations in hMLH1 or hMSH2. No pathogenic mutations were detected in the tumours and only one mutation was found in the colorectal cancer cell lines. These results indicate that Bcl10 is unlikely to be involved in the pathways of colorectal carcinogenesis.     

Helicobacter pylori infection predicts favorable outcome in patients with gastric cancer

Background

Recent studies have suggested a controversial role of Helicobacter pylori infection in gastric cancer prognosis. The aim of the present study was to investigate the potential impact of H. pylori status on the prognosis of patients with gastric cancer in a Chinese prospective cohort.

Methods

Between 2007 and 2009, 261 patients with curatively resected gastric cancer were enrolled in the study. H. pylori status was defined by means of immunohistochemical staining in tumour and non-neoplastic tissues. Treatment prognosis was measured in terms of cancer-specific survival and disease-free survival (dfs). Univariate and multivariate Cox regression models were used to assess the association between H. pylori status and patient prognosis.

Results

Positivity for H. pylori infection was observed in 188 of the 261 patients (72.0%). In patients positive for H. pylori, mean cancer-specific survival was 55.2 months [95% confidence interval (ci): 53.4 to 56.9 months] and mean dfs was 53.9 months (95% ci: 51.8 to 56.0 months); the same survivals were, respectively, 45.1 months (95% ci: 42.2 to 47.9 months) and 43.7 months (95% ci: 40.4 to 47.0 months) in patients negative for H. pylori. In univariate analysis, positive H. pylori status was associated with better cancer-specific survival [hazard ratio (hr): 0.486; 95% ci: 0.271 to 0.870; p = 0.015] and dfs (hr: 0.540; 95% ci: 0.307 to 0.950; p = 0.033). In multivariate analysis, H. pylori was an independent prognostic factor for cancer-specific survival (hr: 0.485; 95% ci: 0.265 to 0.889; p = 0.019).

Conclusions

Our study demonstrates that positive H. pylori status is a beneficial prognostic indicator in patients with gastric cancer and might suggest possible therapeutic approaches for gastric cancer. Further research is required to better understand inflammation mechanisms and cancer progression.     

Inhibition of protein kinase C delta leads to cellular senescence to induce anti-tumor effects in colorectal cancer

Protein kinase C delta (PKCδ) is a multifunctional serine–threonine kinase implicated in cell proliferation, differentiation, tumorigenesis, and therapeutic resistance. However, the molecular mechanism of PKCδ in colorectal cancer (CRC) remains unclear. In this study, we showed that PKCδ acts as a negative regulator of cellular senescence in p53 wild-type (wt-p53) CRC. Immunohistochemical analysis revealed that PKCδ levels in human CRC tissues were higher than those in the surrounding normal tissues. Deletion studies have shown that cell proliferation and tumorigenesis in wt-p53 CRC is sensitive to PKCδ expression. We found that PKCδ activates p21 via a p53-independent pathway and that PKCδ-kinase activity is essential for p21 activity. In addition, both repression of PKCδ expression and inhibition of PKCδ activity induced cellular senescence-like phenotypes, including increased senescence-associated β-galactosidase (SA-β-gal) staining, low LaminB1 expression, large nucleus size, and senescence-associated secretory phenotype (SASP) detection. Finally, a kinase inhibitor of PKCδ suppressed senescence-dependent tumorigenicity in a dose-dependent manner. These results offer a mechanistic insight into CRC survival and tumorigenesis. In addition, a novel therapeutic strategy for wt-p53 CRC is proposed.     

Characterization of rare transforming KRAS mutations in sporadic colorectal cancer

KRAS mutational status has been shown to be a predictive biomarker of resistance to anti-EGFR monoclonal antibody (mAb) therapy in patients with metastatic colorectal cancer. We report the spectrum of KRAS mutation in 1506 patients with colorectal cancer and the identification and characterization of rare insertion mutations within the functional domain of KRAS. KRAS mutations are found in 44.5% (670/1506) of the patients. Two cases are found to harbor double mutations involving both codons 12 and 13. The frequencies of KRAS mutations at its codons 12, 13, 61, and 146 are 75.1%, 19.3%, 2.5%, and 2.7%, respectively. The most abundant mutation of codon 12 is G12D, followed by G12V and G12C while G13D is the predominant mutation in codon 13. Mutations in other codons are rare. The KRAS mutation rate is significantly higher in women (48%, 296/617) than in men (42.1%, 374/889, P = 0.023). Tumors on the right colon have a higher frequency of KRAS mutations than those on the left (57.3% vs. 40.4%, P < 0.0001). Two in-frame insertion mutations affect the phosphate-binding loop (codon 10–16) of KRAS are identified. One of them has never been reported before. Compared with wild-type protein, the insertion variants enhance the cellular accumulation of active RAS (RAS-GTP) and constitutively activate the downstream signaling pathway. NIH3T3 cells transfected with the insertion variants show enhanced anchorage-independent growth and in vivo tumorigenicity. Potentially these mutations contribute to primary resistance to anti-EGFR mAb therapy but the clinical implication requires further validation.     

Expression of SDF-1 alpha and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer

Although stromal cell-derived factor (SDF)-1 alpha and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1 alpha and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1 alpha and CXCR4. The relationships between clinicopathological features and SDF-1 alpha or CXCR4 expression were then analysed. Stromal cell-derived factor-1 alpha and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1 alpha and nuclear CXCR4 predicts LN metastasis in CRCs.     

Poorer outcome in stromal HIF-2 alpha- and CA9-positive colorectal adenocarcinomas is associated with wild-type TP53 but not with BNIP3 promoter hypermethylation or apoptosis

Stromal expression of hypoxia inducible factor 2 alpha (HIF-2 alpha) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2 alpha- and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5-8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2 alpha, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2 alpha and CA9 stromal-positive CRCs was associated with wild-type TP53 (P=0.001 and P=0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P=0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis. These results suggest that the biology of CRC cells can be modified by alterations in the tumour microenvironment under conditions of tumour hypoxia.     

EGFR gene copy number assessment from areas with highest EGFR expression predicts response to anti-EGFR therapy in colorectal cancer

Background:

Only 40–70% of metastatic colorectal cancers (mCRCs) with wild-type (WT) KRAS oncogene respond to anti-epidermal growth factor receptor (anti-EGFR) antibody treatment. EGFR amplification has been suggested as an additional marker to predict the response. However, improved methods for bringing the EGFR analysis into routine laboratory are needed.

Methods:

The material consisted of 80 patients with mCRC, 54 of them receiving anti-EGFR therapy. EGFR gene copy number (GCN) was analysed by automated silver in situ hybridisation (SISH). Immunohistochemical EGFR protein analysis was used to guide SISH assessment.

Results:

Clinical benefit was seen in 73% of high (⩾4.0) EGFR GCN patients, in comparison with 59% of KRAS WT patients. Only 20% of low EGFR GCN patients responded to therapy. A high EGFR GCN number associated with longer progression-free survival (P<0.0001) and overall survival (P=0.004). Together with KRAS analysis, EGFR GCN identified the responsive patients to anti-EGFR therapy more accurately than either test alone. The clinical benefit rate of KRAS WT/high EGFR GCN tumours was 82%.

Conclusion:

Our results show that automated EGFR SISH, in combination with KRAS mutation analysis, can be a useful and easily applicable technique in routine diagnostic practise for selecting patients for anti-EGFR therapy.     

CDH13 promoter region is specifically methylated in poorly differentiated colorectal cancer

It has recently become clear that CDH13 (H-cadherin, T-cadherin) expression is frequently silenced by aberrant methylation in colorectal cancers and adenomas. In this study, we investigated the methylation status of CDH13 gene and detected aberrant promoter methylation in 27 of 84 (32%) colorectal cancers. We then correlated the results with the clinicopathological features of affected patients. We found a significant difference in histology (P=0.0053) when we compared the CDH13 methylation of poorly differentiated colorectal cancers to that of differentiated ones. This result suggested that poorly differentiated colorectal cancers specifically exhibited CDH13 methylation, and since CDH13 might be responsible for selective cell recognition and adhesion, inactivation of CDH13 could lead to the formation of scattered carcinoma cells in these cancers.     

Extensive molecular screening for hereditary non-polyposis colorectal cancer

The genetic abnormalities underlying hereditary non-polyposis colorectal cancer (HNPCC) are germline mutations in one of five DNA mismatch repair genes or in the TGFbetaRII gene. The aim of our study was to evaluate the significance of simple tests performed on tumours to select appropriate candidates for germline mutational analysis. We studied three groups of patients, HNPCC kindreds fulfilling the International Collaborative Group (ICG) criteria (n = 10), families in which at least one of the criteria was not satisfied (n = 7) and sporadic colorectal cancer (CRC) diagnosed before the age of 50 (n = 17). We searched for microsatellite instability (MSI), presence of hMSH2 and hMLH1 germline mutations, expression of hMSH2, hMLH1 and p53 proteins in tumoural tissue samples by immunostaining. Fifteen out of 17 (88%) of HNPCC and incomplete HNPCC cases were MSI and eight pathogenic germline mutations in hMSH2 or hMLH1 were detected in these two groups (53%). All the 17 early-onset sporadic cases were MSS and no germline mutations were detected among the seven investigated cases. Thirteen out of 15 (81%) familial cases were MSI and p53 protein-negative, whereas 13/14 (93%) sporadic cases were MSS and strongly p53 protein-positive. This extensive molecular investigation shows that simple tests such as MS study combined with hMSH2 and hMLH1 protein immunostaining performed on tumoural tissues may provide valuable information to distinguish between familial, and probably hereditary, and sporadic CRC cases.     

Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer

Background:

Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies.

Methods:

We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR.

Results:

A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1–35 of CAD with exons 20–29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response.

Conclusions:

We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.     

The role of MYH and microsatellite instability in the development of sporadic colorectal cancer

Biallelic germline mutations in MYH are associated with colorectal neoplasms, which develop through a pathway involving somatic inactivation of APC. In this study, we investigated the incidence of the common MYH mutations in an Australian cohort of sporadic colorectal cancers, the clinicopathological features of MYH cancers, and determined whether inactivation of mismatch repair and base excision repair (BER) were mutually exclusive. The MYH gene was sequenced from lymphocyte DNA of 872 colorectal cancer patients and 478 controls. Two compound heterozygotes were identified in the cancer population and all three cancers from these individuals displayed a prominent infiltration of intraepithelial lymphocytes. In total, 11 heterozygotes were found in the cancer group and five in the control group. One tumour from an individual with biallelic germline mutation of MYH also demonstrated microsatellite instability (MSI) as a result of biallelic hypermethylation of the MLH1 promoter. Although MYH-associated cancers are rare in a sporadic colorectal population, this study shows that these tumours can develop through either a chromosomal or MSI pathway. Tumours arising in the setting of BER or mismatch repair deficiency may share a biological characteristic, which promotes lymphocytic infiltration.     

Abnormal expression of TRIB3 in colorectal cancer: a novel marker for prognosis

Background:

TRIB3 is a human homologue of Drosophila tribbles. Previous studies have shown that TRIB3 controls the cell growth through ubiquitination-dependent degradation of other proteins, whereas its significance in the prognosis of colorectal cancer (CRC) is not yet fully understood.

Materials:

This study comprised 202 patients who underwent surgery for CRC, as well as 22 cell lines derived from human gastrointestinal cancer. The correlation of gene expression with clinical parameters in patients was assessed. The biological significance was evaluated by knockdown experiments in seven colorectal cancer cell lines.

Results:

A total of 20 cancer cell lines (90.9%) expressed the TRIB3 gene. The assessment in surgical specimens indicated that the gene expression was significantly higher in the cancerous region than in the marginal non-cancerous region. Patients with high TRIB3 expression were statistically susceptible to a recurrence of the disease, and showed poorer overall survival than those with low expression. The assessment of TRIB3 knockdown in five cell lines showed that small interfering RNA (siRNA) inhibition resulted in a statistically significant reduction in cell growth.

Conclusion:

These data strongly suggest the usefulness of TRIB3 as a marker for predicting the prognosis of CRC patients, showing a basis for the development of effective treatments for CRC.