INTRODUCTION: Glioblastoma multiform (GBM) is a neural stem cell (NSC)-derived malignant brain tumor with complex genetic alterations challenging clinical treatments. FAM72 is a NSC-specific protein comprised of four paralogous genes (FAM72 A-D) in the human genome, but its functional tumorigenic significance is unclear.
METHODS: We conducted an in-depth expression and somatic mutation data analysis of FAM72 (A-D) in GBM using the comprehensive human clinical cancer study database cBioPortal [including The Cancer Genome Atlas (TCGA)].
RESULTS: We established a FAM72 transcription profile across TCGA correlated with the expression of the proliferative marker MKI67 and a tissue-specific gene-mutation signature represented by pivotal genes involved in driving the cell cycle. FAM72 paralogs are overexpressed in cancer cells, specifically correlating with the mitotic cell cycle genes ASPM, KIF14, KIF23, CENPE, CENPE, CEP55, SGO1, and BUB1, thereby contributing to centrosome and mitotic spindle formation. FAM72 expression correlation identifies a novel GBM-specific gene set (SCN9A, MXRA5, ADAM29, KDR, LRP1B, and PIK3C2G) in the de novo pathway of primary GBM predestined as viable targets for therapeutics.
CONCLUSION: Our newly identified primary GBM-specific gene-mutation signature, along with FAM72, could thus provide a new basis for prognostic biomarkers for diagnostics of GBM and could serve as potential therapeutic targets.

BACKGROUND: A Disintegrin and Metalloprotease Domain 29 (ADAM29) is involved in many important physiological processes. Recent studies have demonstrated that ADAM29 is a susceptibility locus showing traits as a risk factor for breast cancer under genome-wide significance, however, the clinical relevance and cellular function of ADAM29 in breast cancer have not been reported.
MATERIALS AND METHODS: In this study, we assessed the expression levels of ADAM29 in a cohort of human breast cancer specimens. We also used MDA-MB-231 cells with differing ADAM29 expression and assessed the influence of ADAM29 and its mutations on the MDA-MB-231 cell line.
RESULTS: Increased transcript expression of ADAM29 was observed in breast cancer tissues compared to normal ones. The expression of ADAM29 and its mutations in different domains significantly influenced proliferation, migration and invasion of breast cancer cells in vitro.
CONCLUSION: ADAM29 may represent a prognostic factor in human breast cancer, as well as a novel molecular candidate to be used as a therapeutic target.

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

BACKGROUND: Different DNA aberrations processes can cause colorectal cancer (CRC). Herein, we conducted a comprehensive molecular characterization of 27 CRCs from Iranian patients.
MATERIALS AND METHODS: Array CGH was performed. The MSI phenotype and the methylation status of 15 genes was established using MSP. The CGH data was compared to two established lists of 41 and 68 cancer genes, respectively, and to CGH data from African Americans. A maximum parsimony cladogram based on global aberrations was established.
RESULTS: The number of aberrations seem to depend on the MSI status. MSI-H tumors displayed the lowest number of aberrations. MSP revealed that most markers were methylated, except RNF182 gene. P16 and MLH1 genes were primarily methylated in MSI-H tumors. Seven markers with moderate to high frequency of methylation (SYNE1, MMP2, CD109, EVL, RET, LGR and PTPRD) had very low levels of chromosomal aberrations. All chromosomes were targeted by aberrations with deletions more frequent than amplifications. The most amplified markers were CD248, ERCC6, ERGIC3, GNAS, MMP2, NF1, P2RX7, SFRS6, SLC29A1 and TBX22. Most deletions were noted for ADAM29, CHL1, CSMD3, FBXW7, GALNS, MMP2, NF1, PRKD1, SMAD4 and TP53. Aberrations targeting chromosome X were primarily amplifications in male patients and deletions in female patients. A finding similar to what we reported for African American CRC patients.
CONCLUSION: This first comprehensive analysis of CRC Iranian tumors reveals a high MSI rate. The MSI tumors displayed the lowest level of chromosomal aberrations but high frequency of methylation. The MSI-L were predominantly targeted with chromosomal instability in a way similar to the MSS tumors. The global chromosomal aberration profiles showed many similarities with other populations but also differences that might allow a better understanding of CRC's clinico-pathological specifics in this population.

BACKGROUND: Gastric carcinoma is the second leading cause of cancer-associated death worldwide. The high mortality associated with this disease is in part due to limited knowledge about gastric carcinogenesis and a lack of available therapeutic and prevention strategies. MUC1 is a high molecular weight transmembrane mucin protein expressed at the apical surface of most glandular epithelial cells and a major component of the mucus layer above gastric mucosa. Overexpression of MUC1 is found in approximately 95% of human adenocarcinomas, where it is associated with oncogenic activity. The role of MUC1 in gastric cancer progression remains to be clarified.
METHODOLOGY: We downregulated MUC1 expression in a gastric carcinoma cell line by RNA interference and studied the effects on cellular proliferation (MTT assay), apoptosis (TUNEL assay), migration (migration assay), invasion (invasion assay) and aggregation (aggregation assay). Global gene expression was evaluated by microarray analysis to identify alterations that are regulated by MUC1 expression. In vivo assays were also performed in mice, in order to study the tumorigenicity of cells with and without MUC1 downregulation in MKN45 gastric carcinoma cell line.
RESULTS: Downregulation of MUC1 expression increased proliferation and apoptosis as compared to controls, whereas cell-cell aggregation was decreased. No significant differences were found in terms of migration and invasion between the downregulated clones and the controls. Expression of TCN1, KLK6, ADAM29, LGAL4, TSPAN8 and SHPS-1 was found to be significantly different between MUC1 downregulated clones and the control cells. In vivo assays have shown that mice injected with MUC1 downregulated cells develop smaller tumours when compared to mice injected with the control cells.
CONCLUSIONS: These results indicate that MUC1 downregulation alters the phenotype and tumorigenicity of MKN45 gastric carcinoma cells and also the expression of several molecules that can be involved in tumorigenic events. Therefore, MUC1 should be further studied to better clarify its potential as a novel therapeutic target for gastric cancer.

We performed a mutational analysis of the 19 disintegrin-metalloproteinases (ADAMs) genes in human cutaneous metastatic melanoma and identified eight to be somatically mutated in 79 samples, affecting 34% of the melanoma tumors analyzed. Functional analysis of the two frequently mutated ADAM genes, ADAM29 and ADAM7 demonstrated that the mutations affect adhesion of melanoma cells to specific extracellular matrix proteins and in some cases increase their migration ability. This suggests that mutated ADAM genes could play a role in melanoma progression.

BACKGROUND: Colon cancer (CRC) development often includes chromosomal instability (CIN) leading to amplifications and deletions of large DNA segments. Epidemiological, clinical, and cytogenetic studies showed that there are considerable differences between CRC tumors from African Americans (AAs) and Caucasian patients. In this study, we determined genomic copy number aberrations in sporadic CRC tumors from AAs, in order to investigate possible explanations for the observed disparities.
METHODOLOGY/PRINCIPAL FINDINGS: We applied genome-wide array comparative genome hybridization (aCGH) using a 105k chip to identify copy number aberrations in samples from 15 AAs. In addition, we did a population comparative analysis with aCGH data in Caucasians as well as with a widely publicized list of colon cancer genes (CAN genes). There was an average of 20 aberrations per patient with more amplifications than deletions. Analysis of DNA copy number of frequently altered chromosomes revealed that deletions occurred primarily in chromosomes 4, 8 and 18. Chromosomal duplications occurred in more than 50% of cases on chromosomes 7, 8, 13, 20 and X. The CIN profile showed some differences when compared to Caucasian alterations.
CONCLUSIONS/SIGNIFICANCE: Chromosome X amplification in male patients and chromosomes 4, 8 and 18 deletions were prominent aberrations in AAs. Some CAN genes were altered at high frequencies in AAs with EXOC4, EPHB6, GNAS, MLL3 and TBX22 as the most frequently deleted genes and HAPLN1, ADAM29, SMAD2 and SMAD4 as the most frequently amplified genes. The observed CIN may play a distinctive role in CRC in AAs.

BACKGROUND: A variety of surrogate markers for genetic features and outcome have been described in chronic lymphocytic leukemia based on gene expression analyses. Previous studies mostly focused on individual markers and selected disease characteristics, which makes it difficult to estimate the relative value of the novel markers. Therefore, in the present study a comprehensive approach was chosen investigating 18 promising, partly novel expression markers in a well characterized cohort of patients with long clinical follow-up and full genetic information (IGHV status, genomic abnormalities).
DESIGN AND METHODS: Expression markers were evaluated using real-time quantitative reverse transcriptase polymerase chain reaction in CD19(+)-purified samples from 151 patients. Multivariate analyses were performed to test the markers' ability to identify patients at genetic risk and as prognostic markers in the context of established prognostic factors.
RESULTS: For individual markers, ZAP70 expression provided the highest rate (81%) of correct assignment of patients at genetic risk (IGHV unmutated, V3-21 usage, 11q- or 17p-), followed by LPL and TCF7 (76% both). The assignment rate was improved to 88% by information from a four-gene combination (ZAP70, TCF7, DMD, ATM). In multivariate analysis of treatment-free survival, IGHV mutation status and expression of ADAM29 were of independent prognostic value besides disease stage. With regards to overall survival, expression of ATM, ADAM29, TCL1, and SEPT10 provided prognostic information in addition to that derived from clinical and genetic factors.
CONCLUSIONS: Gene expression markers are suitable for screening but not as surrogates for the information from genetic risk factors. While many individual markers may be associated with outcome, only a few are of independent prognostic significance. With regard to prognosis estimation, the genetic prognostic factors cannot be replaced by the expression markers.

Several prognostic factors can predict the rapid progression in chronic lymphocytic leukaemia (CLL), including IGHV mutational status, cytogenetic abnormalities and, more recently, LPL/ADAM29 expression. In contrast, few studies have been devoted to the influence of these factors on clinical outcome in responding patients after therapy. We here propose to analyse the impact of IGHV gene status, LPL and ADAM29 gene expression on disease-free survival (DFS) and overall survival (OS) in 41 stage B or C CLL patients in remission after oral fludarabine plus cyclophosphamide. The median follow-up was of 64 (16-74) months. Sequencing of IGHV showed mutated (M) VH genes in 16 of 41 cases and unmutated (UM) in 25 cases. Analysis of LPL and ADAM29 expression in 35 of 41 cases showed overexpression of ADAM29 in 17 cases (14 M and three UM) and LPL in 18 cases (all UM). Patients expressing UM IGHV and LPL had shorter DFS and OS when compared to patients expressing M IGHV and/or ADAM29. Furthermore, blood minimal residual disease (MRD) evaluation using four-colour flow cytometry was performed in 33 out the 41 patients. We showed that patients who achieved phenotypic remission displayed longer DFS than those with MRD(+). Our results support the use of LPL and ADAM29 gene expression associated to IGHV mutational status for predicting the clinical outcome of patients treated by oral fludarabine + cyclophosphamide and could be considered for treatment strategies.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course. Recent studies have shown that expression of lipoprotein lipase (LPL) and ADAM29 may serve as novel prognostic markers in B-CLL. To investigate the prognostic value of these genes, we quantified their expression in peripheral blood mononuclear cells using quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) in a cohort of 133 B-CLL patients and correlated the results with clinical outcome, and other known prognostic factors. LPL, ADAM29, LPL and ADAM29 ratios, as well as CD38 and ZAP-70 protein expression determined by multiparameter flow cytometry, were predictive of treatment-free survival. Multivariate Cox regression analysis identified LPL, ADAM29 and CD38 as independent prognostic markers. Evaluation of several disease characteristics in association with the LPL expression status of the patients' B-CLL cells showed highly significant differences for CD38 and ZAP-70 expression, suggesting a correlation of LPL expression with these established adverse prognostic factors. Sequential RQ-PCR analyses in a subset of 22 patients revealed that LPL mRNA expression was relatively stable in the majority of patients, whereas ADAM29 expression levels varied substantially over time. Furthermore, in a subgroup analysis, LPL provided prognostic information in both early stage (Binet A) and patients with more advanced disease (Binet B and C). Conversely, high ADAM29 expression was predictive of a long treatment-free interval in Binet stage A but did not retain its prognostic significance in Binet B and C patients. The LPL/ADAM29 expression ratio was not found to be an independent prognostic factor and did not offer any advantages over the use of LPL alone. Collectively, our data confirm a role for LPL as a novel prognostic indicator in B-CLL.

BACKGROUND AND OBJECTIVES: The mutational status of the immunoglobulin heavy chain variable region genes (IGVH) is a strong indicator of prognosis in B-cell chronic lymphocytic leukaemia (CLL). Since the determination of the IGVH mutation status is very labor-intensive, alternative prognostically relevant markers would facilitate CLL diagnostics.
DESIGN AND METHODS: Ten genes were selected from previously published gene expression profiling studies based on their differential expression in IGVH mutated versus unmutated cases of CLL, and tested with real-time quantitative polymerase chain reaction (RQ-PCR) in unpurified samples from 130 CLL patients. To ascertain potential contaminating effects by normal hematopoietic cells, the expression levels of the selected genes were determined in normal monocytes, B cells, T cells, NK cells and granulocytes.
RESULTS: The selected genes, i.e., ZAP70, LPL, SPG20, ADAM29, NRIP1, AKAP12, DMD, SEPT10, TPM2 and CLECSF2, showed prognostic significance. In multivariate logistic regression analysis expression levels of LPL, ZAP70, ADAM29 and SEPT10 were the most predictive for IGVH mutational status. In univariate analysis the expression of LPL was the best predictor. For survival, expression of LPL was the strongest prognostic factor. In combination with the three cytogenetic markers associated with a poor prognosis, i.e., deletions 17p13, 11q22 and trisomy 12, expression of LPL and IGVH mutational status performed equally well with regard to their predictive value for survival, both being more predictive than ZAP70.
INTERPRETATION AND CONCLUSIONS: This study demonstrates that LPL expression is a predictor for survival in CLL, and for this purpose is as good as IGVH mutational status and more reliable than ZAP70 expression when tested in unpurified CLL samples.

Although the zeta-associated protein of 70 kDa (ZAP-70) is overexpressed in patients with chronic lymphocytic leukemia (CLL) displaying unmutated IGVH genes and poor prognosis, a previous microarray study from our group identified overexpression of LPL and ADAM29 genes among unmutated and mutated CLL, respectively. To assess the prognostic value of these genes, we quantified their expression by real-time quantitative polymerase chain reaction (PCR) in a cohort of 127 patients with CLL and correlated this with clinical outcome, IGVH mutational status, and ZAP-70 protein expression. IGVH mutational status, ZAP-70, and the LPL and ADAM29 mRNA ratios (L/A ratio) were predictive of event-free survival for the whole cohort and for patients with stage A disease. In patients in stage B and C, the L/A ratio was an independent prognostic factor, whereas ZAP-70 did not predict survival. Simultaneous usage of the L/A ratio and ZAP-70 expression allowed an almost perfect (99%) assessment of the IGVH status in the 80% of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). LPL and ADAM29 gene expression could also be determined by a simple competitive multiplex reverse transcription PCR assay. Overall, quantification of LPL and ADAM29 gene expression is a strong prognostic indicator in CLL, providing better prognostic assessment than ZAP-70 in advanced stages of the disease.