Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features.

BACKGROUND: Differences exist between the proximal and distal colon in terms of developmental origin, exposure to patterning genes, environmental mutagens, and gut flora. Little is known on how these differences may affect mechanisms of tumorigenesis, side-specific therapy response or prognosis. We explored systematic differences in pathway activation and their clinical implications. MATERIALS AND METHODS: Detailed clinicopathological data for 3045 colon carcinoma patients enrolled in the PETACC3 adjuvant chemotherapy trial were available for analysis. A subset of 1404 samples had molecular data, including gene expression and DNA copy number profiles for 589 and 199 samples, respectively. In addition, 413 colon adenocarcinoma from TCGA collection were also analyzed. Tumor side-effect on anti-epidermal growth factor receptor (EGFR) therapy was assessed in a cohort of 325 metastatic patients. Outcome variables considered were relapse-free survival and survival after relapse (SAR). RESULTS: Proximal carcinomas were more often mucinous, microsatellite instable (MSI)-high, mutated in key tumorigenic pathways, expressed a B-Raf proto-oncogene, serine/threonine kinase (BRAF)-like and a serrated pathway signature, regardless of histological type. Distal carcinomas were more often chromosome instable and EGFR or human epidermal growth factor receptor 2 (HER2) amplified, and more frequently overexpressed epiregulin. While risk of relapse was not different per side, SAR was much poorer for proximal than for distal stage III carcinomas in a multivariable model including BRAF mutation status [N = 285; HR 1.95, 95% CI (1.6-2.4), P < 0.001]. Only patients with metastases from a distal carcinoma responded to anti-EGFR therapy, in line with the predictions of our pathway enrichment analysis. CONCLUSIONS: Colorectal carcinoma side is associated with differences in key molecular features, some immediately druggable, with important prognostic effects which are maintained in metastatic lesions. Although within side significant molecular heterogeneity remains, our findings justify stratification of patients by side for retrospective and prospective analyses of drug efficacy and prognosis.

JARID2 is a direct target of the PAX3-FOXO1 fusion protein and inhibits myogenic differentiation of rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS (t-test; P < 0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n = 120; P = 0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin (MYOG) and Myosin Light Chain (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.

MFG-E8/lactadherin regulates cyclins D1/D3 expression and enhances the tumorigenic potential of mammary epithelial cells.

Milk fat globule-EGF factor 8 (MFG-E8) is a glycoprotein highly expressed in breast cancer that contributes to tumor progression through largely undefined mechanisms. By analyzing publicly available gene expression profiles of breast carcinomas, we found that MFG-E8 is highly expressed in primary and metastatic breast carcinomas, associated with absent estrogen receptor expression. Immunohistochemistry analysis of breast cancer biopsies revealed that MFG-E8 is expressed on the cell membrane as well as in the cytoplasm and nucleus. We also show that increased expression of MFG-E8 in mammary carcinoma cells increases their tumorigenicity in immunodeficient mice, and conversely, its downregulation reduces their in vivo growth. Moreover, expression of MFG-E8 in immortalized mammary epithelial cells promotes their growth and branching in three-dimensional collagen matrices and induces the expression of cyclins D1/D3 and N-cadherin. A mutant protein unable to bind integrins can in part exert these effects, indicating that MFG-E8 function is only partially dependent on integrin activation. We conclude that MFG-E8-dependent signaling stimulates cell proliferation and the acquisition of mesenchymal properties and contributes to mammary carcinoma development.

Genomic gain and over expression of CCL2 correlate with vascular invasion in stage I non-seminomatous testicular germ-cell tumours.

Testicular germ-cell tumours (TGCT) are the most frequent solid tumour to affect young Caucasian adult males and have increased in incidence over recent decades. In clinical stage I non-seminomas, (NSGCT) histological vascular invasion (VI) is a prognostic factor for metastatic relapse. Using array comparative genomic hybridization, we have previously shown that the presence of VI is associated with gain of a region at 17q12, containing a cluster of genes encoding inflammatory cytokines. We here confirm this finding using fluorescence in situ hybridization (FISH) demonstrating gain in 12 out of 42 (29%) assessable samples. Interrogation of previously published expression microarray data suggests that of the genes contained within this region, CCL2 [monocyte chemoattractant protein 1 (MCP1)] is frequently overexpressed in TGCT. Immunohistochemistry confirms this finding in a collection of 67 clinical stage I NSGCT, demonstrating an association with the presence of VI (p=0.049) that was not seen with VEGF-A, MMP2 or MMP9, although all were frequently expressed. This work gives further insight into the mechanisms involved in invasion in this tumour type, which may ultimately have implications for the management of patients with stage I disease.

Activation of the hedgehog pathway confers a poor prognosis in embryonal and fusion gene-negative alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Seventy-five percent of ARMS harbor reciprocal chromosomal translocations leading to fusion genes of the forkhead transcription factor FOXO1 and PAX3 or PAX7. The hedgehog (Hh) pathway has been implied in tumor formation and progression of various cancers including RMS. However, whether Hh pathway activation presents a general feature of RMS or whether it is restricted to specific subgroups has not yet been addressed. Here, we report that marker genes of active Hh signaling, that is, Patched1 (Ptch1), Gli1, Gli3 and Myf5, are expressed at significantly higher levels in ERMS and fusion gene-negative ARMS compared with fusion gene-positive ARMS in two distinct cohorts of RMS patients. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene-negative ARMS, but not in fusion gene-positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene-negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation. Moreover, Myf5 is identified as a novel excellent class predictor for RMS by receiver operating characteristic analysis. Importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene-negative RMS underscoring the clinical relevance of these findings. By showing that Hh signaling is preferentially activated in specific subgroups of RMS, our study has important implications for molecular targeted therapies, such as small molecule Hh inhibitors, in RMS.

MicroRNA-206 expression levels correlate with clinical behaviour of rhabdomyosarcomas.

BACKGROUND: Rhabdomyosarcomas (RMSs) are primarily paediatric sarcomas that resemble developing skeletal muscle. Our aim was to determine the effects of microRNAs (miRNA) that have been implicated in muscle development on the clinical behaviour of RMSs. METHODS: Expression levels of miR-1, miR-206, miR-133a and miR-133b were quantified by RT-PCR in 163 primary paediatric RMSs, plus control tissues, and correlated with clinico-pathological features. Correlations with parallel gene expression profiling data for 84 samples were used to identify pathways associated with miR-206. Synthetic miR-206 was transfected into RMS cell lines and phenotypic responses assessed. RESULTS: Muscle-specific miRNAs levels were lower in RMSs compared with skeletal muscle but generally higher than in other normal tissues. Low miR-206 expression correlated with poor overall survival and was an independent predictor of shorter survival in metastatic embryonal and alveolar cases without PAX3/7-FOXO1 fusion genes. Low miR-206 expression also significantly correlated with high SIOP stage and the presence of metastases at diagnosis. High miR-206 expression strongly correlated with genes linked to muscle differentiation and low expression was associated with genes linked to MAPkinase and NFKappaB pathway activation. Increasing miR-206 expression in cell lines inhibited cell growth and migration and induced apoptosis that was associated with myogenic differentiation in some, but not all, cell lines. CONCLUSION: miR-206 contributes to the clinical behaviour of RMSs and the pleiotropic effects of miR-206 supports therapeutic potential.

Genomic copy number and expression patterns in testicular germ cell tumours.

Testicular germ cell tumours of adults and adolescents (TGCT) include seminomas (SE) and nonseminomas (NS), with spermatocytic seminomas (SSE) representing a distinct entity in older men. SE and NS have gain of 12p material in all cases, whereas SSE are associated with overrepresentation of chromosome 9. Here, we compare at the chromosomal level, copy number imbalances with global expression changes, identified by comparative expressed sequence hybridisation analyses, in seven SE, one combined tumour, seven NS and seven cell lines. Positive correlations were found consistent with copy number as a main driver of expression change, despite reported differences in methylation status in SE and NS. Analysis of chromosomal copy number and expression data could not distinguish between SE and NS, in-keeping with a similar genetic pathogenesis. However, increased expression from 4q22, 5q23.2 and 9p21 distinguished SSE from SE and NS and decreased copy number and expression from 2q36-q37 and 6q24 was a specific feature of NS-derived cell lines. Our analysis also highlights 19 regions with both copy number and expression imbalances in greater than 40% of cases. Mining available expression array data identified genes from these regions as candidates for involvement in TGCT development. Supplementary data is available at http://www.crukdmf.icr.ac.uk/array/array.html.

Predictive factors of efficacy of the somatostatin analogue octreotide as first line therapy for advanced pancreatic endocrine carcinoma.

About 40% of nonfunctioning pancreatic endocrine carcinomas (NF-PEC) cannot be cured by surgery due to advanced stage disease. Somatostatin analogues have been proposed as first line therapy in these cases. We performed a prospective phase IV study to assess the efficacy of octreotide in advanced NF-PEC and identify factors predictive of response to therapy. Twenty-one consecutive patients with octreoscan-positive advanced-stage well-differentiated NF-PEC were treated with long-acting release octreotide 20 mg i.m. at diagnosis. The immunohistochemical expression of somatostatin receptor 2 (SSTR2) and the quantitative mRNA analysis of SSTR2 and SSTR5 were assessed in 12 tumours. The tumour proliferative fraction was assessed by immunohistochemistry for Ki-67. Eight patients (38%) had stable disease (SD) after a median follow-up of 49.5 months. Thirteen patients (62%) developed progression after a median of 18 months. Tumour progression correlated with a proliferative index>or=5% (P=0.016), weight loss (P=0.006) and absence of abdominal pain (P=0.003) at diagnosis. Other clinical (age, gender and primary tumour resection) or pathological parameters (site, size and liver metastasis) lacked significant correlation with tumour progression. No difference in the amount of SSTR2 mRNA and protein or SSTR5 mRNA was found between tumours that were stable (n=5) and seven tumours that progressed (n=7). Treatment with long-acting release octreotide was associated with stabilization of disease and a good quality of life in 38% of patients. A Ki-67 index>or=5% and/or the presence of weight loss may justify more aggressive therapy without waiting for radiologically proven progression of disease.

Prime-boost vaccines encoding an intracellular idiotype/GM-CSF fusion protein induce protective cell-mediated immunity in murine pre-B cell leukemia.

Two vaccines against an intracellularly expressed B cell idiotype were assessed for their ability to induce protective immunity in mice against challenge with a pre-B cell leukemia. One vaccine was based on a plasmid expression vector and the other was a recombinant vaccinia virus; both vaccines expressed a polypeptide derived from the complementarity-determining regions (CDR(2)-CDR(3)) of the leukemic clone-specific immunoglobulin heavy chain (IgH), as a fusion product with mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF). Mice inoculated with either vaccine showed significantly higher survival rates than controls after challenge with leukemia cells. However, protection from tumor challenge was optimal when the DNA vaccine was used for priming, followed by a booster immunization with the vaccinia virus recombinant. This vaccination protocol induced resistance not only to the first tumor challenge given shortly afterwards, but also to a second challenge given months later. Both CD4(+) and CD8(+) T cells contributed to protection in vaccinated mice. These data suggest that such a vaccine regimen might reduce the incidence of recurrence in patients with minimal residual disease after conventional therapy.

Role of disease-causing genes in sporadic pancreatic endocrine tumors: MEN1 and VHL.

Pancreatic endocrine tumors (PETs) occur in association with multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau (VHL) syndromes caused by germline alterations in MEN1 and VHL, respectively. It is thus expected that these genes will also be altered in a proportion of sporadic PETs. Indeed, MEN1 is altered in about 25% of nonfamilial PETs, although no mutations have been found in VHL. For all clinical subtypes, the frequency of allelic loss on chromosome arm 11q mirrors observed mutational frequencies, with the exception of nonfunctional tumors (NF-PETs), in which mutations have been reported in only 8% of cases. As allelic loss on 11q is the most frequent event found in these neoplasms, this low frequency is somewhat puzzling, particularly in light of the fact that most MEN1-associated PETs are nonfunctioning. To clarify the role of these genes in sporadic PETs, we analyzed 31 sporadic NF-PETs, nine insulinomas, and one VIPoma for alterations in MEN1 and VHL. As somatic mutations were observed in eight (26%) of the NF tumors and in one insulinoma, it would therefore appear unlikely that an additional tumor suppressor gene related to sporadic PET pathogenesis is located on 11q. One insulinoma also had a somatic mutation in VHL, and thus this gene may also be altered in these neoplasms, albeit in a small proportion of cases.

High resolution allelotype of nonfunctional pancreatic endocrine tumors: identification of two molecular subgroups with clinical implications.

A high resolution allelotype for nonfunctional pancreatic endocrine tumors (NF-PETs) has been generated by microsatellite analysis of DNA from 16 frozen cases, each probed with 394 markers. Two subgroups of NF-PETs were found. Seven cases showed frequent, large allelic deletions [loss of heterozygosity (LOH)] with an average fractional allelic loss (FAL) of 0.55, whereas nine cases showed a small number of random losses with a FAL of 0.15. Designated high or low FAL, respectively, these genetic phenotypes showed correlation with the ploidy status: high-FAL tumors were aneuploid, low-FAL were diploid. Chromosomes 6q and 11q showed LOH in >60% of cases. About 50% of cases had losses on 11p, 20q, and 21. Selected LOH analysis on an additional 16 paraffin-embedded NF-PETs confirmed the high frequency of 6q and 11q LOH. The allelotype of NF-PET is markedly different from that of either ductal or acinar tumors of the pancreas as well as from that of functional-PETs. Moreover, whereas deletions involving chromosome 11 also are a feature of functional-PETs, the involvement of chromosome 6q is characteristic of NF-PETs. Survival analysis showed that none of the single chromosomal alterations was associated with outcome, whereas ploidy status is an independent factor adding prognostic information to that furnished by the proliferative index measured by Ki-67 immunohistochemistry.