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The clinical course of breast cancer varies from one patient to another. Currently, the choice of therapy relies on clinical parameters and histological and molecular tumor features. Alas, these markers are informative in only a subset of patients. Therefore, additional predictors of disease outcome would be valuable for treatment stratification. Extensive studies showed that the degree of variation of the nuclear DNA content, i.e., aneuploidy, determines prognosis. Our aim was to further elucidate the molecular basis of aneuploidy. We analyzed five diploid and six aneuploid tumors with more than 20 years of follow-up. By performing FISH with a multiplexed panel of 10 probes to enumerate copy numbers in individual cells, and by sequencing 563 cancer-related genes, we analyzed how aneuploidy is linked to intratumor heterogeneity. In our cohort, none of the patients with diploid tumors died of breast cancer during follow-up in contrast to four of six patients with aneuploid tumors (mean survival 86.4 months). The FISH analysis showed markedly increased genomic instability and intratumor heterogeneity in aneuploid tumors. MYC gain was observed in only 20% of the diploid cancers, while all aneuploid cases showed a gain. The mutation burden was similar in diploid and aneuploid tumors, however, TP53 mutations were not observed in diploid tumors, but in all aneuploid tumors in our collective. We conclude that quantitative measurements of intratumor heterogeneity by multiplex FISH, detection of MYC amplification and TP53 mutation could augment prognostication in breast cancer patients., (Published 2018. This article is a US Government work and is in the public domain in the USA.)

Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to the discovery of novel therapeutic opportunities improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics., (© 2017 Wiley Periodicals, Inc.)

During the course of disease, many cancer patients eventually present with metastatic disease including peritoneal or pleural spread. In this context, cytology specimens derived from ascites or pleural effusion may help to differentiate malignant from benign conditions and sometimes yield diagnosis of a malignancy. However, even when supported by immunohistochemistry, cytological interpretation can be challenging, especially if tumor cellularity is low. Here, we investigated whether targeted deep sequencing of formalin-fixed and paraffin embedded (FFPE) cytology specimens of cancer patients is feasible, and has diagnostic and clinical impact. To this end, a cohort of 20 matched pairs was compiled, each comprising a cytology sample (FFPE cell block) and at least one biopsy/surgical resection specimen serving as benchmark. In addition, 5 non-malignant effusions were sequenced serving as negative-controls. All samples yielded sufficient libraries and were successfully subjected to targeted sequencing employing a semiconductor based next-generation sequencing platform. Using gene panels of different size and composition, including the Oncomine Comprehensive Assay, for targeted sequencing, somatic mutations were detected in the tissue of all 20 cases. Of these, 15 (75%) harbored mutations that were also detected in the corresponding cytology samples. In four of these cases (20%), additional private mutations were detected in either cytology or tissue samples, reflecting spatiotemporal tumor evolution. Of the five remaining cases, three (15%) showed wild type alleles in cytology material whereas tumor tissue had mutations in interrogated genes. Two cases were discordant, showing different private mutations in the cytology and in the tissue sample, respectively. In summary, sequencing of cytology specimens (FFPE cell block) reflecting spatiotemporal tumor evolution is feasible and yields adjunct genetic information that may be exploitable for diagnostics and therapy., (© 2017 Wiley Periodicals, Inc.)

Amplification of the MYCN gene in neuroblastoma is associated with a poor prognosis and is considered to remain unchanged in post-treatment specimens and metastases. While heterogeneity of MYCN copy number in tumor cells has been reported, serial samples have only been studied in a limited way, and the biologic relevance of this finding is not well understood. We used in situ hybridization on paraffin sections of 102 specimens from 30 patients with MYCN-amplified neuroblastoma to determine MYCN copy number in the primary tumor, pre- and post-treatment, and in metastatic samples. Nineteen cases (63%) showed diffuse MYCN amplification in all samples tested. Nine cases (30%) showed a reduction in MYCN copy number: five cases with diffuse amplification subsequently showed focal amplification, one case with diffuse MYCN amplification showed MYCN gain after treatment, and three focally amplified cases were non-amplified in later specimens. In two cases (7%), focal amplification became diffuse in subsequent samples. Histology was not predictive of the temporal or spatial pattern of MYCN amplification for a particular tumor. If extent of amplification (focal vs. diffuse) is not considered, 26/30 (87%) of cases were consistently MYCN-amplified. However, our data suggest that MYCN status can be heterogeneous between tumor sites, during tumor progression or following treatment, challenging the notion that MYCN copy number does not change for a particular neuroblastoma. Assessing the biologic significance of MYCN heterogeneity will require larger studies of clinically annotated tumor samples, and will depend on interpreting heterogeneity in MYCN status in combination with other genetic changes. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Primary tumor (PT) heterogeneity can significantly affect the genetic profile of clones at metastatic sites. To understand the mechanisms underlying metastasis, we compared the genetic profile of paired PT and metastatic lymph node (MLN) samples obtained from patients with oral tongue squamous cell carcinoma (OTSCC). Large-scale genetic profiling was performed on paired PT-MLN samples obtained from 10 OTSCC patients using high-density single-nucleotide polymorphism microarrays. We compared the genetic profile of PT and MLN OTSCC samples to identify common and specific copy number alterations and copy-neutral loss-of-heterozygosity (CN-LOH). Unsupervised hierarchical clustering analysis indicated that 8 of the 10 PT-MLN sample pairs formed clusters, indicating that the primary and metastatic tumors were composed of predominantly genetically similar tumor cells. In 6 of the 10 pairs, 8q11.21, 8q12.2-3, and 8q21.3 gains, and 22q11.23 loss were detected in both the PT and MLN. In addition, 16p11.2 CN-LOH was identified in 9 of the 10 pairs. Conversely, 20q11.2 gain was only observed in the MLNs of 5 of the 10 sample pairs, indicating that genes in this chromosomal region may play a significant role in OTSCC lymph node metastasis. To confirm this, we investigated the expression of two candidate 20q11.2 genes in a separate patient cohort. The expression of one of these genes, E2F1, was significantly increased during the process of metastasis. This study indicates that additional genetic changes, such as 20q11.2 gain, which encodes the E2F1 gene, can be acquired through clonal evolution, and may be required for the metastatic process. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Acute lymphoblastic leukaemias (ALL) with 51-67 chromosomes are defined as high hyperdiploid (HHD) and are generally associated with good prognosis. However, several studies show heterogeneity in HHD ALL and suggest that the favourable prognosis is associated rather with higher ploidy defined by DNA index (DNAi) ≥ 1.16 or with a presence of specific single or combined trisomies. HHD ALL with DNAi < 1.16 are only rarely studied separately. Using single nucleotide polymorphism array, we analysed 89 childhood HHD ALL patients divided into groups with lower (<1.16; n = 34) and higher (≥1.16; n = 55) DNAi. We assessed treatment response, presence of secondary aberrations, mutations in RAS pathway genes and CREBBP and also gene expression profile (GEP) to reveal differences between the two subgroups. Cases with 51-54 chromosomes had DNAi 1.1-1.16 and cases with 55-67 chromosomes had DNAi ≥ 1.16. The groups with lower and higher DNAi had distinct response to early treatment and distinct GEP. The better response of the group with higher DNAi was associated with specific trisomies (trisomy of chromosome 10 or combined with trisomies 4 and/or 17). Our results suggest that cytogenetically defined HHD ALL can in fact be divided into two biologically distinguishable subgroups and that DNAi 1.16 is a relevant value to separate between the two. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

We applied a targeted sequencing approach to identify germline mutations conferring a moderately to highly increased risk of cutaneous and uveal melanoma. Ninety-two high-risk melanoma patients were screened for inherited variation in 120 melanoma candidate genes. Observed gene variants were filtered based on frequency in reference populations, cosegregation with melanoma in families and predicted functional effect. Several novel or rare genetic variants in genes involved in DNA damage response, cell-cycle regulation and transcriptional control were identified in melanoma patients. Among identified genetic alterations was an extremely rare variant (minor allele frequency of 0.00008) in the BRIP1 gene that was found to cosegregate with the melanoma phenotype. We also found a rare nonsense variant in the BRCA2 gene (rs11571833), previously associated with cancer susceptibility but not with melanoma, which showed weak association with melanoma susceptibility in the Swedish population. Our results add to the growing knowledge about genetic factors associated with melanoma susceptibility and also emphasize the role of DNA damage response as an important factor in melanoma etiology. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Despite the many efforts already spent to enumerate somatic mutations in diffuse large B-cell lymphoma (DLBCL), previous whole-genome and whole-exome studies conducted on patients of mixed outcomes failed at characterizing the 30% of patients who will relapse or resist current immunochemotherapies. To address this issue, we performed whole-exome sequencing of normal/tumoral DNA pairs in 14 relapsed/refractory (R/R) patients subclassified by full-transcriptome arrays (six activated B-cell like, three germinal center B-cell like, and five primary mediastinal B-cell lymphomas), from the LNH-03 LYSA clinical trial program. Aside from well-known DLBCL features, gene and pathway level recurrence analyses proposed several interesting leads including TBL1XR1 and activating mutations in IRF4 or in the insulin regulation pathway. Sequencing-based copy number analysis defined 23 short recurrently altered regions involving genes such as REL, CDKN2A, HYAL2, and TP53. Moreover, it highlighted mutations in genes such as GNA13, CARD11, MFHAS1, and PCLO as associated with secondary variant allele amplification events. The five primary mediastinal B-cell lymphomas (PMBL), while unexpected in a R/R cohort, showed a significantly higher mutation rate (P = 0.003) and provided many insights on this classical Hodgkin lymphoma related subtype. Novel genes such as XPO1, MFHAS1, and ITPKB were found particularly mutated, along with various cytokine-based signaling pathways. Among these analyses, somatic events in the NF-κB pathway were found preponderant in the three DLBCL subtypes, confirming its major implication in DLBCL aggressiveness and pinpointing several new candidate genes., (© 2015 Wiley Periodicals, Inc.)

Over 100 single nucleotide polymorphisms (SNP) are validated in the TP53 tumor suppressor gene. They define haplotypes, which may differ in their activities. Therefore, mutation in cancer may occur at different rates depending upon haplotypes. However, these associations may be masked by differences in mutations types and causes of mutagenesis. We have analyzed the associations between 19 SNPs spanning the TP53 locus and a single specific aflatoxin-induced TP53 mutation (R249S) in 85 in hepatocellular carcinoma cases and 132 controls from Thailand. An association with R249S mutation (P = 0.007) was observed for a combination of two SNPs (rs17882227 and rs8064946) in a linkage disequilibrium block extending from upstream of exon 1 to the first half of intron 1. This domain contains two coding sequences overlapping with TP53 (WRAP53 and Hp53int1) suggesting that sequences in TP53 intron 1 encode transcripts that may modulate R249S mutation rate in HCC., (Copyright © 2013 Wiley Periodicals, Inc.)

Chronic myeloid leukemia (CML) is a rare disease in children and adolescents and various aspects-from molecular genesis to therapy regimen-have been taken over from studies on the more prevalent adult CML. However, differences have been observed between malignancies with identical underlying chromosomal translocations, but occurring at different age groups, suggesting some diversity in the mechanisms of formation and leukemogenesis. A multiplex long-range PCR-based assay was developed to allow fast and reliable amplification of patient-specific BCR-ABL1 fusion sequences from genomic DNA. The localization of breakpoints was analyzed with respect to distribution within the breakpoint cluster regions, sequence features, and association to repetitive elements or motifs associated with DNA recombination. The genomic fusion sites of 59 pediatric CML patients showed a bimodal breakpoint distribution in BCR that was different from the distribution in adult CML cases, but with similarities to BCR-ABL1-positive, acute lymphoblastic leukemia in adults. BCR breakpoints were found more frequently positioned within, or close to, Alu repeats than would be expected based on their overall sequence proportion. Technical aspects of the highly sensitive DNA-based quantification of residual CML cells by specific fusion sequence during tyrosine kinase inhibitor therapy are exemplified in a subcohort of pediatric CML patients., (Copyright © 2012 Wiley Periodicals, Inc.)

Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region. In addition, transcriptome and computational analyses were performed to search for nonannotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. Our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus., (Copyright © 2012 Wiley Periodicals, Inc.)

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which--owing to extensive interparalog sequence exchange--closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples--all publicly available--and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5' breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations., (Copyright © 2012 Wiley Periodicals, Inc.)

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in Western countries. Chromosomal abnormalities commonly found using conventional cytogenetics and FISH are del(11)(q22-23), trisomy 12, del(13)(q14), and del(17)(p13). Trisomy 12 is the most frequent numerical abnormality in CLL. It can appear isolated or associated with other chromosomal aberrations, including t(14;18)(q32;q21) and trisomy 18. The aim of this study was to determine whether CLL patients with isolated trisomy 12 or associated with other chromosomal alterations have different clinico-pathological features, including a different distribution NOTCH1 mutation. Patients were classified into four groups: Group 1, isolated trisomy 12 (n=14); Group 2, trisomy 12 plus trisomy 18 (n=4); Group 3, trisomy 12 plus t(14;18) (n=8); and Group 4: patients with trisomy 12 plus other abnormalities not involving BCL2 (n=28). The Binet stage and expression of ZAP70 were significantly different among cytogenetic groups. NOTCH1 mutations were detected in 6/12 (50%) patients from Group 1, 4/25 (16%) patients from Group 4, and in no patient from groups 2 and 3 (P=0.020). Patients in Group 2 had a more rapid disease progression (median Treatment-free Survival 2 months) as against patients from Groups 1 (50 months), 3 (69 months), or 4 (68 months; P=0.001). These findings indicate that the distribution of NOTCH1 mutations in CLL with trisomy 12 is heterogeneous and that the presence of additional chromosomal abnormalities such as trisomy 18 could change the prognosis of these patients., (Copyright © 2012 Wiley Periodicals, Inc.)

Formalin-fixed, paraffin-embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH. Aim of this study is to compare the commercially available aCGH platforms suitable for high-resolution copy number analysis using FFPE-derived DNA. Two dual channel aCGH platforms (Agilent and NimbleGen) and a single channel SNP-based platform (Affymetrix) were evaluated using seven FFPE colon cancer samples, and median absolute deviation (MAD), deflection, signal-to-noise ratio, and DNA input requirements were used as quality criteria. Large differences were observed between platforms; Agilent and NimbleGen showed better MAD values (0.13 for both) compared with Affymetrix (0.22). On the contrary, Affymetrix showed a better deflection of 0.94, followed by 0.71 for Agilent and 0.51 for NimbleGen. This resulted in signal-to-nose ratios that were comparable between the three commercially available platforms. Interestingly, DNA input amounts from FFPE material lower than recommended still yielded high quality profiles on all platforms. Copy number analysis using DNA derived from FFPE archival material is feasible using all three high-resolution copy number platforms and shows reproducible results, also with DNA input amounts lower than recommended., (Copyright © 2011 Wiley Periodicals, Inc.)

Somatic mutations in mitochondrial DNA (mtDNA) have been identified in various tumors, including breast cancer. However, their clinicopathological impact on breast cancer still remains unclear. In this study, we re-sequenced the entire mtDNA from breast cancer samples together with paired non-tumorous breast tissues from 58 Taiwanese patients. We identified 19 somatic mutations in the mtDNA coding region of 16 breast cancers. Out of these mutations, 12 of the 19 mutations (63%) are missense or frame-shift mutations that have the potential to cause mitochondrial dysfunction. In combination with our previously study on the D-loop region of mtDNA, we found that 47% (27/58) of the breast cancers harbored somatic mtDNA mutations. Among a total of 40 somatic mutations, 53% (21/40) were located in the D-loop region of the mtDNA, 5% (2/40) were in the ribosomal RNA genes, 5% (2/40) were in the tRNA genes, and 38% (15/40) occurred in mRNA genes. The occurrence of these somatic mtDNA mutations is associated with an older onset age (≥ 50-year old, P = 0.039), a higher TNM stage (P = 0.027), and a higher histological grade (P = 0.012). Multiple logistic regression analysis revealed that an older onset age (P = 0.029) and a higher histological grade (P = 0.006) are significantly correlated with patients having somatic mutations in the mtDNA in their breast cancer sample. In conclusion, our results suggest that somatic mtDNA mutations may play a critical role in the progression of breast cancer., (Copyright © 2011 Wiley-Liss, Inc.)

Disseminated tumor cells (DTC) in the bone marrow (BM) are present in about 35% of ovarian cancers before surgery and after chemotherapy and are associated with worse prognosis. A molecular biomarker in the primary tumor predicting tumor cell spread would be highly desirable. The purpose of the study was to investigate loss of heterozygosity (LOH) in primary ovarian tumors at four ovarian cancer-relevant chromosomal loci involved in apoptosis, platinum sensitivity, or DNA-repair, to assess the prognostic value of LOH and to correlate LOH with DTC occurrence before surgery and after chemotherapy. Primary tumor DNA of 88 patients was analyzed for LOH at four polymorphic microsatellite markers using PCR-based fluorescence microsatellite analysis. BM aspirates were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. LOH at the entire marker set correlated with tumor grading (P = 0.0001) and histology (P = 0.004). LOH at marker D10S1765 correlated with FIGO stage (P = 0.046) and grading (P = 0.05), whereas LOH at D17S855 significantly associated with grading (P = 0.023) and histology (P = 0.012), respectively. DTC were detected in 49% of patients before surgery and in 50% of patients after chemotherapy. Interestingly, LOH proximal to D6S1581 significantly correlated with DTC presence before surgery (P = 0.05) and after chemotherapy (P = 0.022). Conclusively, our data suggest that allelic loss at D6S1581 (proximal to M6P/IGF2R locus) serves as a molecular biomarker for the presence of DTC in the BM before and after chemotherapy., (Copyright © 2011 Wiley-Liss, Inc.)

Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare tumor often difficult to differentiate from fibrosarcoma of bone (FSb), diagnostically. We applied array comparative genomic hybridization (array CGH) to screen for genes with potential importance in the tumor and compared the results with alterations seen in FSb. Twenty-two fresh frozen tissue specimens from 20 patients (18 primary tumors and 4 local recurrences) with UPSb were studied. DNA was isolated and hybridized onto Agilent 244K CGH oligoarrays. The hybridization data were analyzed using Agilent DNA Analytics Software. The number of changes ranged from 2 to 168 (average = 66). Losses were most frequently seen at 8p, 9p, 10, 13q, and 18q, and gains at 4q, 5p, 6p, 7p, 8q, 12p, 14q, 17q, 19p, 20q, 22q, and X. Homozygous deletions of CDKN2A, RB1, TP53, and ING1 were seen in 8/20, 7/20, 3/20, and 2/20 cases, respectively. Hypermethylation of both p16(INK4a) and p14(ARF) was found in two cases with loss at CDKN2A. Inactivation either of CDKN2A, RB1, or TP53 was detected in 18/20 cases. One case showed high level gains of CDK4 and MDM2. Frequent gains were seen at MYC, PDGFRA, KIT, and KDR. Immunohistochemical positivity of KIT, PDGFRA, KDR, and PDGFRB was found in 8/14, 5/14, 4/14, and 4/14 cases, respectively. The regions most significantly discriminating between UPSb and FSb included RB1 and MYC. No homozygous deletions of RB1 were found in FSb. In conclusion, our analysis showed the disruption of G1/S checkpoint regulation to be crucial for the oncogenesis of UPSb., (2011 Wiley-Liss, Inc.)

Oral squamous cell carcinoma (OSCC) has the highest rate of increase among male cancers in Taiwan. An understanding of the molecular pathogenesis of this disease as well as the development of prognostic markers for the clinical management of this disease is very important. Thus, a systematic loss of heterozygosity (LOH) analysis was performed to define minimally deleted regions (MDRs) in 63 male OSCCs using 400 polymorphic microsatellite markers. For increasing reliability, genomic DNA was extracted from >90% tumor cells that had been purified by LCM, and only when a microsatellite marker provided LOH information in >30% of the OSCCs was there considered to be successful allelotyping. A correlation of the various MDRs with clinicopathological parameters and prognosis was carried out. In total, 32 MDRs were identified and ten were noted as novel. In addition, six MDRs were found to be associated with cigarette smoking. Among these markers, a loss of MDR c7r2 (7q32.2-q35) was significantly associated with poor disease-free survival (DFS) and ten MDRs were associated with allelic imbalance (AI) in tumors. Among the latter, a loss of MDR c14r1 (14q24.2-q32.12) and c11r1 (11q13.4-q25) had a synergistic effect on poor DFS and were able to reduce further the DFS rate in patients with MDR c7r2 loss. Taken together, the results generated in this study provide new insights that help with exploring the molecular mechanisms associated with OSCC tumorigenesis and cigarette smoking. They also should aid the development of potential prognostic markers for the clinical management of OSCC., (2011 Wiley-Liss, Inc.)

Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker-assisted laser-capture and laser-beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high-density array-comparative genomic hybridization (array-CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.5 kb to 784.3 kb in size, derived from different portions of chromosome 8. Among these segments, 242 were common in both aberrant chromosomes, but 75 were found to be chromosome-specific. Sequences of 263 junction sites connecting the ends of segments were determined using a PCR/Sanger-sequencing procedure. Overlapping microhomologies were found at 169 junction sites. Junction partners came from various portions of chromosome 8 and no biased pattern in the positional distribution of junction partners was detected. These structural characteristics suggested the occurrence of random fragmentation of the entire chromosome 8 followed by random rejoining of these fragments. Based on that, we proposed a model to explain how these aberrant chromosomes are formed. Through these structural analyses, it was demonstrated that the optimized chromosome isolation method described here can provide high-quality chromosomal DNA for high resolution array-CGH analysis and probably for massively parallel sequencing analysis., (Copyright © 2011 Wiley-Liss, Inc.)

Using a custom CGH-like oligonucleotide array to measure the global microsatellite content in the genomes of 72 cancer, cancer-free, and high risk patient and cell line samples (56 germline DNA and 16 in tumor or tumor cell line DNA) we found a unique, reproducible, and statistically significant pattern of 18 motif-specific microsatellite families (out of 962 possible 1-6 mer repeats) in breast cancer patient germline and tumor DNA, but not in germline DNA of cancer-free volunteer controls or in breast cancer patients with BRCA1/2 mutations. These high-similarity A/T rich repetitive motifs were also more pronounced in the germlines and tumors of colon cancer tumor patients (3/6 samples) and microsatellite unstable colon cancer cell lines; however, germline DNA of sporadic breast cancer patients exhibited the largest global content shift for those motifs with extreme AT/GC ratios. These results indicate that global microsatellite variability is complex, suggest the existence of a previously unknown genomic destabilization mechanism in breast cancer patients' germline DNA, and warrant further testing of such microsatellite variability as a predictor of future breast cancer development., (Copyright © 2011 Wiley-Liss, Inc.)

In B-cell malignancies, genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In this study, we identified the BTB and CNC homology 2 (BACH2) gene as a novel translocation partner of the immunoglobulin heavy chain (IGH) locus in a patient with IGH-MYC-positive, highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). Fluorescence in situ hybridization analysis using an IGH/MYC probe detected an IGH-MYC fusion signal on der(14) and IGH signal on del(6). Genome copy number analysis showed a deletion in the 6q15-25 region and a centromeric breakpoint within the BACH2 gene. cDNA bubble polymerase chain reaction using BACH2 primers revealed that the first exon of Cδ was fused to the 5'-untranslated region of BACH2 exon 2. The Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, encompassing the entire BACH2 coding region, and the BACH2 was highly expressed in this patient. These results indicate that Cδ-BACH2 fusion may cause constitutive activation of BACH2. Although additional screening of 47 samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients and 29 cell lines derived from B-cell malignancies by double-color fluorescence in situ hybridization analysis detected a split signal with deletion of centromeric region of BACH2 only in a patient with follicular lymphoma, BACH2 was highly expressed in lymphoma cells of the patient and B-NHL cell lines with IGH-MYC translocation. These findings suggest that BACH2 plays a critical role in B-cell lymphomagenesis, especially related to IGH-MYC translocation in some way., (Copyright © 2011 Wiley-Liss, Inc.)

Predicting evolution of small node-negative breast carcinoma is a real challenge in clinical practice. The aim of this study was to search whether qualitative or quantitative DNA changes may help to predict metastasis of small node-negative breast carcinoma. Small invasive ductal carcinomas without axillary lymph node involvement (T1T2N0) from 168 patients with either good (111 patients with no event at 5 years after diagnosis) or poor (57 patients with early metastasis) outcome were analyzed with comparative genomic hybridization (CGH) array. A CGH classifier, identifying low- and high-risk groups of metastatic recurrence, was established in a training set of 78 patients, then validated, and compared with clinicopathological parameters in a distinct set of 90 patients. The genomic status of regions located on 2p22.2, 3p23, and 8q21-24 and the number of segmental alterations were defined in the training set to classify tumors into low- or high-risk groups. In the validation set, in addition to estrogen receptors and grade, this CGH classifier provided significant prognostic information in multivariate analysis (odds ratio, 3.34; 95% confidence interval 1.01-11.02; P = 4.78 × 10(-2), Wald test). This study shows that tumor DNA contains important prognostic information that may help to predict metastasis in T1T2N0 tumors of the breast., (© 2010 Wiley-Liss, Inc.)

Conventional osteosarcoma is characterized by rapid growth, high local aggressiveness, and metastasizing potential. Patients developing lung metastases experience poor prognosis despite extensive chemotherapy regimens and surgical interventions. Previously we identified a subgroup of osteosarcoma patients with loss of CDKN2A/p16 protein expression in the primary tumor biopsies which was significantly predictive of a very poor prognosis. Here we aimed to identify the underlying mechanism(s) of this protein loss in relation to osteosarcoma behavior. The CDKN2A locus was analyzed in osteosarcoma cases with total loss of CDKN2A/p16 expression and in cases with high protein expression using melting curve analysis-methylation assay (MCA-Meth), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and mutation analysis. All cases with complete CDKN2A/p16 protein loss showed homozygous deletions at the CDKN2A locus. In none of the cases hyper methylation of the promoter region was seen which was confirmed by sequencing this region. Taken together we show that large or smaller deletions of the CDKN2A locus are evident in patient samples and underlie the CDKN2A/p16 protein expression loss while promoter methylation does not appear to be a mechanism of this expression loss. Genomic loss of CDKN2A instead of promoter methylation might be a plausible explanation for the rapid proliferation and high aggressiveness of osteosarcoma by simultaneous impairment CDKN2A/p14(ARF) function., (© 2010 Wiley-Liss, Inc.)

The chromosome location, 11q21-23, is linked to loss of heterozygosity (LOH) in multiple tumors including those of breast, lung, and head and neck. To examine the process of LOH induction, the H292 cell line (human muco-epidermoid carcinoma) was irradiated or treated with anti-CD95 antibody, and individual clones isolated through two rounds of cloning. Regions of LOH were determined by screening a suite of eight polymorphic microsatellite markers covering 11p15-11q24 using fluorescent primers and genetic analyzer peak discrimination. LOH induction was observed extending through 11q21.1-11q23.3 in 6/49 of clones surviving 4 Gy and 8/50 after 8 Gy. Analysis of selected clones by Affymetrix 6.0 single nucleotide polymorphism (SNP) arrays confirmed the initial assessment indicating a consistent 27.3-27.7 Mbp deletion in multiple clones. The telomeric border of LOH mapped to a 1 Mbp region of elevated recombination. Whole genome analysis of SNP data indicated that site-restricted LOH also occurred across multiple additional genomic locations. These data indicate that 11q21.1-11q23.3, and potentially other regions of this cell line are sites of intrinsic cell-specific instability leading to LOH after irradiation. Such deletions may subsequently be propagated by genetic selection and clonal expansion., ((c) 2010 Wiley-Liss, Inc.)

We compared genomic breakpoints at the PML and RARA loci in 23 patients with therapy-related acute promyelocytic leukemia (t-APL) and 25 de novo APL cases.Eighteen of 23 t-APL cases received the topoisomerase II poison mitoxantrone for their primary disorder. DNA breaks were clustered in a previously reported 8 bp "hot spot" region of PML corresponding to a preferred site of mitoxantrone-induced DNA topoisomerase II-mediated cleavage in 39% of t-APL occurring in patients exposed to this agent and in none of the cases arising de novo (P = 0.007). As to RARA breakpoints, clustering in a 3' region of intron 2 (region B) was found in 65% of t-APL and 28% of de novo APL patients, respectively. Scan statistics revealed significant clustering of RARA breakpoints in region B in t-APL cases (P = 0.001) as compared to de novo APL (P = 1). Furthermore, approximately 300 bp downstream of RARA region B contained a sequence highly homologous to a topoisomerase II consensus sequence. Biased distribution of DNA breakpoints at both PML and RARA loci suggest the existence of different pathogenetic mechanisms in t-APL as compared with de novo APL.

C/EBPalpha plays an essential role as a transcription factor in myeloid cell differentiation. Here, we describe a Japanese family in which two individuals with acute myeloid leukemia (AML) and one healthy individual had an identical 4-base pair insertion in the N-terminal region of CEBPA (350&#95;351insCTAC), resulting in the termination at codon 107 (I68fsX107). The father and a son at diagnosis of AML had different in-frame insertion mutations in the C-terminal region of C/EBPalpha. These C-terminal mutations disappeared upon remission in both patients. Interestingly, the father showed different in-frame insertion mutations in the C-terminal CEBPA at the time of diagnosis and relapse. These data strongly suggest that the N-terminal C/EBPalpha mutation predisposes to the occurrence of a C-terminal C/EBPalpha mutation as a secondary genetic hit, causing AML.

Very little is known about the genetics of fibrosarcoma (FS) of bone. We applied array comparative genomic hybridization (CGH) to identify genes and genomic regions with potential role in the pathogenesis of this tumor. Seventeen patients with FS of bone were included in the study. Array CGH analysis was carried out in 13 fresh frozen tissue specimens from 11 of these patients (nine primary tumors and four local recurrences). DNA was extracted and hybridizations were performed on Agilent 244K CGH oligoarrays. The data were analyzed using Agilent DNA Analytics Software. The number of changes per patient ranged from 0 to 132 (average = 43). Losses were most commonly detected at 6q, 8p, 9p, 10, 13q, and 20p. CDKN2A was homozygously deleted in 7/11 patients. Hypermethylation of both p16(INK4a) and p14(ARF) was found in 1/14 patients. An internal deletion of STARD13 was found in a region with common losses at 13q13.1. The most frequent gains were seen at 1q, 4q, 5p, 8q, 12p, 15q, 16q, 17q, 20q, 22q, and Xp. Single recurrent high level amplification was detected at 4q12, including KIT, PDGFRA, and KDR. No activating mutations were found in any of them. Immunohistochemistry revealed expression of PDGFRA and/or PDGFRB in 12/17 samples. Moreover, small regions of gains pinpointed genes of particular interest, such as IGF1R at 15q26.3 and CHD1L at 1q21.1. In conclusion, our analysis provided novel findings that can be exploited when searching for markers for diagnosis and prognosis, and targets of therapy in this tumor type.

To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4-9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) > MLH1 (46%) > RBSP3 (45%) > ITGA9 (41%), methylation: RBSP3 (25%) > ITGA9 (24%) > STAC (19%) > MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN --> Stage I/II and for ITGA9 from CIN --> Stage I/II and also from Stage I/II --> Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p-RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p-RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (>or=5)/early sexual debut (

Pancreatic cancer is a highly aggressive malignancy and one of the leading causes of cancer deaths, mainly due to the lack of methods for early diagnosis and the lack of effective therapies. Recent CGH microarray studies have revealed several regions that are recurrently amplified in pancreatic cancer; these are thus likely to contain genes that contribute to cancer pathogenesis and thereby could serve as novel diagnostic and therapeutic targets. Here, we performed a detailed characterization of the 7q21-q22 amplicon in pancreatic cancer to identify putative amplification target genes. Fluorescence in situ hybridization analyses in 16 pancreatic cancer cell lines and 29 primary pancreatic tumors revealed an increased copy number in approximately 25% of cases in both sample groups, and the cell line data also allowed us to identify a 0.77 Mb amplicon core region containing ten transcripts. Gene expression analyses by qRT-PCR highlighted the ARPC1A gene as having the statistically most significant correlation between amplification and elevated expression (P = 0.004). Silencing of ARPC1A by RNA interference in AsPC-1 cells having high level amplification and expression resulted in a slight decrease in cell proliferation, but a massive reduction in cell migration and invasion. ARPC1A codes for the p41 subunit of the Arp2/3 protein complex, which is a key player in actin polymerization and thus regulates cell mobility. Taken together, our data implicate ARPC1A as a novel target for the 7q21-q22 amplification and a regulator of cell migration and invasion in pancreatic cancer, thus making it an interesting target for antimetastasis therapy.

Glioblastoma multiforme shows multiple chromosomal aberrations, the impact of which on gene expression remains unclear. To investigate this relationship and to identify putative initiating genomic events, we integrated a paired copy number and gene expression survey in glioblastoma using whole human genome arrays. Loci of recurrent copy number alterations were combined with gene expression profiles obtained on the same tumor samples. We identified a set of 406 "cis-acting DNA targeted genes" corresponding to genomic aberrations with direct copy-number-driving changes in gene expression, defined as genes with either significantly concordant or correlated changes in DNA copy number and expression. Functional annotation revealed that these genes participate in key processes of cancer cell biology, providing insights into the genetic mechanisms driving glioblastoma. The robustness of the gene selection was validated on an external microarray data set including 81 glioblastomas and 23 non-neoplastic brain samples. The integration of array CGH and gene expression data highlights a robust cis-acting DNA targeted genes signature that may be critical for glioblastoma progression, with two tumor suppressor genes PCDH9 and STARD13 that could be involved in tumor invasiveness and resistance to etoposide.

Array comparative genomic hybridization (array CGH) is widely used for studying chromosomal copy number aberrations (CNAs) on a genome-wide and high-resolution scale in heritable disorders and cancers. The aim of this study was to test if the separate channels of dual channel arrays can be interchanged (across array) to either make array CGH more sensitive and cost effective and/or to generate profiles of CNAs and copy number variations (CNVs). Therefore the BT474 breast cancer cell line was compared with a mix of normal reference DNAs hybridized on different arrays and days and DNA copy number profiles were evaluated. Quality was assessed, using regular dual channel array CGH as a standard, using four quality measures, i.e., the median absolute deviation value of chromosome 2, the amplitude of the ERBB2 gene amplification, a deletion on chromosome 9, and the deflection on chromosome 8. The quality of the across array CGH profiles matched or even surpassed the quality of regular dual channel array CGH. In addition, this across array approach was tested for genomic DNA derived from formalin-fixed paraffin-embedded tumors tissue samples, resulting in high-quality copy number profiles, comparable to regular dual channel arrays. Finally, we demonstrated this approach to obtain both CNA and CNV profiles. In summary, across array CGH avoids redundant hybridizations of the same reference material in every experiment either allowing hybridization of two test samples on one array or producing both CNA and CNV profiles simultaneously.

Loss of chromosome 22 and gain of 1q are the most frequent genomic aberrations in ependymomas, indicating that genes mapping to these regions are critical in their pathogenesis. Using real-time quantitative PCR, we measured relative copy numbers of 10 genes mapping to 22q12.3-q13.33 and 10 genes at 1q21-32 in a series of 47 pediatric intracranial ependymomas. Loss of one or more of the genes on 22 was detected in 81% of cases, with RAC2 and C22ORF2 at 22q12-q13.1 being deleted most frequently in 38% and 32% of ependymoma samples, respectively. Combined analysis of quantitative-PCR with methylation-specific PCR and bisulphite sequencing revealed a high rate (>60% ependymoma) of transcriptional inactivation of C22ORF2, indicating its potential importance in the development of pediatric ependymomas. Increase of relative copy numbers of at least one gene on 1q were detected in 61% of cases, with TPR at 1q25 displaying relative copy number gains in 38% of cases. Patient age was identified as a significant adverse prognostic factor, as a significantly shorter overall survival time (P = 0.0056) was observed in patients <2 years of age compared with patients who were >2 years of age. Loss of RAC2 at 22q13 or amplification of TPR at 1q25 was significantly associated with shorter overall survival in these younger patients (P = 0.0492 and P = < 0.0001, respectively). This study identifies candidate target genes within 1q and 22q that are potentially important in the pathogenesis of intracranial pediatric ependymomas.

BRAF and NRAS are commonly mutated in cancer and represent the most frequent genetic events in malignant melanoma. More recently, a subset of melanomas was shown to overexpress KIT and harbor KIT mutations. Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, about 10% of the cases lack mutations in these genes. It is our hypothesis following the melanoma model that mutations in BRAF or NRAS may play a role in wild-type GIST pathogenesis. Alterations in RAS/MEK/ERK pathway may also be involved in development of imatinib resistance in GIST, particularly in tumors lacking secondary KIT or PDGFRA mutations. Imatinib-naive wild-type GISTs from 61 patients, including 15 children and 28 imatinib-resistant tumors without secondary KIT mutations were analyzed. Screening for hot spots mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3) was performed. A BRAF exon 15 V600E was identified in 3 of 61 GIST patients, who shared similar clinical features, being 49- to 55-years-old females and having their tumors located in the small bowel. The tumors were strongly KIT immunoreactive and had a high risk of malignancy. An identical V600E BRAF mutation was also identified in one of 28 imatinib resistant GIST lacking a defined mechanism of drug resistance. In conclusion, we identified a primary BRAF V600E mutations in 7% of adult GIST patients, lacking KIT/PDGFRA mutations. The BRAF-mutated GISTs show predilection for small bowel location and high risk of malignancy. A secondary V600E BRAF mutation could represent an alternative mechanism of imatinib resistance. Kinase inhibitors targeting BRAF may be effective therapeutic options in this molecular GIST subset.

Lynch syndrome is an inherited disease resulting predominantly in colorectal cancer (CRC). The crucial cause is DNA mismatch repair (MMR) malfunction that is associated mostly with MLH1 or MSH2 germline mutations. A significant hallmark of repair defects is a high level of instability in microsatellites (MSI-H). In many sporadic unstable CRCs, the MLH1 gene is inactivated by promoter hypermethylation in addition to extensive promoter methylation in many tumor-suppressor genes known as CpG island methylation phenotype (CIMP). To investigate the possible role of epigenetic alterations in causing MMR deficiency and thereby Lynch syndrome, we evaluated the MLH1 specific and global hypermethylation in hereditary CRCs. Of 22 Lynch-syndrome-related CRCs, 18 (81.8%) demonstrated various levels of DNA methylation; of these, 14 (63.6%) and 4 (18.2%) were methylated in distal and both distal and proximal regions of the MLH1 promoter, respectively. However, only 7/18 (38.9%) of results were confirmed by bisulfite sequencing. Similar methylation patterns in tumors and frequently in matched normal DNA were found in twelve and four patients with MLH1 and MSH2 alterations documented by the absence of protein or presence of germline mutation, respectively. Moreover, the same results were observed in five stable CRCs. None of 22 Lynch-syndrome-related tumors presented CIMP in contrast to 3/10 (30%) stable carcinomas. The rather randomly distributed weak methylation patterns in hereditary CRCs indicate that epigenetic events are redundant in Lynch-syndrome etiology, in contrast to the widespread DNA methylation in sporadic unstable CRCs. These methylation-profile differences can lead to more effective molecular diagnosis of Lynch syndrome.

Somatically acquired chromosomal imbalances are a key feature of neuroblastoma, a heterogeneous pediatric solid tumor. Among these alterations, genomic amplification targeting the MYCN oncogene and observed in about 25-30% of the cases, strongly correlates with advanced stage and poor outcome. In this work, we have used BAC and SNP arrays as well as gene expression arrays to characterize amplifications in neuroblastoma. Eighty-eight distinct BACs defining high-level amplification events were identified in 65 samples, including 43 tumors and 22 cell lines. Although the highest recurrence was observed on chromosome 2, clones on chromosomes 8, 12, 16, and 17 also revealed genomic amplification in several samples. A detailed analysis of the 2p22-2p25 MYCN containing region indicated highly complex patterns in a number of cases. Coamplifications involving MYCN and other regions were explored by FISH in three cell lines. High-resolution arrays then allowed us to further refine the mapping of 25 amplicons in 19 samples, either reducing the size of a single continuous amplicon or increasing the complexity by highlighting multiple noncontiguous regions of amplification. Combined analysis of gene expression profiling and array-CGH data indicated that 12 to 25% of the genes that are targeted by genomic amplification are actually over-expressed in tumor cells, several of them having already been implicated in cancer. Finally, our results suggest that the presence of amplicons localized outside of chromosome 2, in addition to MYCN amplification, may be linked to a particularly severe outcome in neuroblastoma patients.

Approximately 25,000 ovarian cancers are diagnosed in the US annually, and 75% of cases are in the advanced stage when they are largely incurable. There is a critical need for improved early detection tools and development of novel treatments. Because chromosome band 20q13 is a commonly DNA amplified region in ovarian cancer and increase in 20q13 copy number may be an early event, we examined the DNA amplification and RNA expression pattern of 239 microarray probes mapping to this region with the goal of identifying gene(s) associated with ovarian cancer. Using Agilent expression microarray analysis and FISH to tumor tissue arrays, we narrowed the candidates to 19 genes that were consistently overexpressed in a subset of tumors amplified for both ZNF217 and TPD54, although, interestingly the candidates do not include these two amplified genes. Unsupervised clustering of 225 ovarian samples with respect to RNA expression of these 19 genes allowed identification of a 20q-amplified subset of 51 (23%) tumors and this subset was significantly correlated with poor outcome. Of the 19 candidate genes in this subset, ADRM1 overexpression was the most highly correlated with amplification, was amplified in a higher percentage of tumors than ZNF217 and TPD54, and was significantly upregulated with respect to stage, recurrence and metastasis. In addition, overexpression of ADRM1 correlates significantly with shorter time to recurrence and overall survival. Functional analysis is now warranted to determine whether ADRM1 is a target for early screening and/or therapy for ovarian cancer.

The MLH1 -93 G>A promoter polymorphism has been reported to be associated with an increased risk of microsatellite unstable colorectal cancer. Other than microsatellite instability, however, the genetic and most epigenetic changes of tumors associated with this polymorphism have not been studied. We evaluated associations between the -93 G>A polymorphism and CpG island methylator phenotype (CIMP), BRAF V600E mutations, and MLH1 methylation in tumors from a sample of 1,211 individuals with colon cancer and 1,968 controls from Utah, Northern California, and Minnesota. The -93 G>A polymorphism was determined by the five prime nuclease assay. CIMP was determined previously by methylation-specific PCR of CpG islands in MLH1, methylated in tumors (MINT)1, MINT2, MINT31, and CDKN2A (p16). The BRAF V600E mutation was determined by sequencing exon 15. The MLH1 -93 G>A promoter polymorphism was associated with CIMP (odds ratio (OR) 3.44, 95% confidence interval (CI) 1.85, 6.42), MLH1 methylation (OR 4.16, 95%CI 2.20, 7.86), BRAF mutations (OR 4.26, 95%CI 1.83, 9.91), and older age at diagnosis (OR 3.65, 95%CI 2.08, 6.39) in microsatellite unstable tumors. These associations were not observed in stable tumors. Increased age at diagnosis and tumor characteristics of microsatellite unstable tumors associated with MLH1 -93 G>A suggests the polymorphism is acting at a relatively late stage of colorectal carcinogenesis to drive CIMP+ tumors down the microsatellite instability pathway.

Gene amplification is among the most common genetic abnormalities that cause cancer. One of the most clinically important gene amplifications in human cancer causes extensive reduplication of ERBB2. A variety of cancers also occasionally harbor somatic mutations in ERBB2. Gene amplification and activating mutations both have predictive value for clinical response to targeted inhibitors. Since the number of gene copies in an amplicon may exceed 100, and since amplicons may encompass multiple genes, high-resolution analysis of gene amplifications poses considerable technical challenges. We have overcome this obstacle by using emulsion-based resequencing to determine the sequence of many independently-amplified individual DNA molecules in parallel. We used this high throughput sequencing technology to analyze ERBB2 mutational status in five ERBB2 amplified cell lines (four breast, one ovarian) and two breast tumors. Genomic DNA was isolated and the 28 exons of ERBB2 were independently amplified. Amplicons were then pooled at equimolar ratios, subjected to emulsion PCR (emPCR) and finally to picotiter plate pyrosequencing. High-quality sequence data were obtained for all amplicons analyzed and no activating mutations within ERBB2 were identified. Although we did not find activating mutations within the multiple copies of ERBB2 in these samples, the results establish the utility of this technology as a feasible and cost-effective approach for high resolution analysis of amplified genes., ((c) 2008 Wiley-Liss, Inc.)

Triple-negative breast cancer (TNBC) is defined by a lack of expression of estrogen, progesterone, and HER2 receptors, and genetically most of them fall into the basal subgroup of breast cancer. The important issue of TNBC is poorer clinical outcome and absence of effective targeted therapy. In this study, we sought to identify DNA copy number alterations and expression of relevant genes characteristic of TNBC to discover potential therapeutic targets. Frozen tissues from 114 breast cancers were analyzed using high-resolution array comparative genomic hybridization. The classification into subtype was determined by estrogen and progesterone receptor expression, and by the presence or absence of gain on the ERBB2 containing clone. The ACE algorithm was used for calling gain and loss of clones. Twenty-eight cases (25%) were classified as TNBC. Recurrent gains (> or =25%) unique to TNBC were 9p24-p21, 10p15-p13, 12p13, 13q31-q34, 18q12, 18q21-q23, and 21q22. Two published gene expression array data sets comparing basal subtype versus other subtype breast cancers were used for searching candidate genes. Of the genes upregulated in the basal subtype, 45 of 686 genes in one data set and 59 of 1,428 in the second data set were found to be located in the gained regions. Of these candidate genes, gain of NFIB (9p24.1) was specific for TNBC in a validation set by real-time PCR. In conclusion, we have identified recurrently gained regions characteristic of TNBC, and found that NFIB copy number and expression is increased in TNBC across the data sets. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Analysis of recurrent DNA amplification can lead to the identification of cancer driver genes, but this process is often hampered by the low resolution of existing copy number analysis platforms. Fifty-one breast tumors were profiled for copy number alterations (CNAs) with the high-resolution Affymetrix 500K SNP array. These tumors were also expression-profiled and surveyed for mutations in selected genes commonly mutated in breast cancer (TP53, CDKN2A, ERBB2, KRAS, PIK3CA, PTEN). Combined analysis of common CNAs and mutations revealed putative associations between features. Analysis of both the prevalence and amplitude of CNAs defined regions of recurrent alteration. Compared with previous array comparative genomic hybridization studies, our analysis provided boundaries for frequently altered regions that were approximately one-fourth the size, greatly reducing the number of potential alteration-driving genes. Expression data from matched tumor samples were used to further interrogate the functional relevance of genes located in recurrent amplicons. Although our data support the importance of some known driver genes such as ERBB2, refined amplicon boundaries at other locations, such as 8p11-12 and 11q13.5-q14.2, greatly reduce the number of potential driver genes and indicate alternatives to commonly suggested driver genes in some cases. For example, the previously reported recurrent amplification at 17q23.2 is reduced to a 249 kb minimal region containing the putative driver RPS6KB1 as well as the putative oncogenic microRNA mir-21. High-resolution copy number analysis provides refined insight into many breast cancer amplicons and their relationships to gene expression, point mutations and breast cancer subtype classifications. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Neuroblastoma is a heterogeneous neoplasm that has served as a paradigm for the clinical utility of somatically acquired genomic aberrations. DNA copy number alterations (CNA) are currently used to predict prognosis, including MYCN amplification and deletions at chromosome bands 1p36 and 11q23. We predicted that genome-wide assessment of DNA aberrations in neuroblastoma tumors would provide a more precise estimation of clinical phenotype, and could be used to predict outcome. We measured CNAs in a representative set of 82 diagnostic tumors on a customized high-resolution BAC array-based CGH platform supplemented with additional clones across 1p36, 2p24, 3p21-22, 11q14-24, and 16p12-13, and integrated these data with RNA expression data. We used an unbiased statistical method to define a set of minimal common regions (MCRs) of aberration. Unsupervised hierarchical clustering identified four distinct genomic subclasses. First, a subset of tumors with a clinically benign phenotype showed predominantly whole chromosome gains and losses. Second, tumors with MYCN amplification had a unique genomic signature of 1p deletion and 17q gain, but few other rearrangements. Third, tumors with an aggressive clinical phenotype without MYCN amplification, showed multiple structural rearrangements. Most notable were deletions of 3p, 4p, and 11q and gain of 1q, 2p, 12q, and 17q. Lastly, there was a subset of tumors with an aggressive clinical phenotype and no detectable DNA CNAs. The genomic subsets were highly correlated with patient outcome, and individual MCRs remained prognostic in a multivariable model. DNA signature patterns embed important prognostic information in diagnostic neuroblastoma samples, and can identify candidate cancer-related genes., (Copyright (c) 2007 Wiley-Liss, Inc.)

Patients who receive curative treatment for lung cancer can develop additional lung tumors that may or may not be related to the original tumor and thus require different clinical management. If a subsequent tumor has a pattern of allele loss, revealed by allelotype analysis, overlapping that of the first tumor, it is believed to be a local recurrence or metastasis. In this case history, we present loss of heterozygosity analyses of the original primary tumor, and two second primary tumors occurring in the ipsilateral and the contra-lateral lungs. The allelotyping suggests that these tumors are all clonally related but concordance is not complete. Our interpretation is that the original primary tumor and the two new primary tumors have developed to full malignancy independently, but are clonally related, possibly via a clone of motile progenitor cells. Deletion mapping of DNA from biopsies of this patient delineated a region in 4p16 that we had previously shown to be lost in the transition from carcinoma in situ to invasive tumor. We identified a minimally deleted region encompassing six genes including two candidate tumor suppressor genes, CRMP1 a lung cancer metastasis-suppressing gene and PPP2R2C a gene for a regulatory subunit of the PP2 complex known to suppress tumorigenesis, particularly viral induced transformation., ((c) 2007 Wiley-Liss, Inc.)

Somatic interstitial deletions of chromosome segment 7q22-q31 in uterine leiomyomas are a frequent event, thought to be indicative of a tumor suppressor gene in the region. Previous LOH and CGH studies have refined this region to 7q22.3-q31, although the target gene has not been identified. Here, we have used tiling-path resolution microarray CGH to further refine the region and to identify homozygous deletions in fibroids. Furthermore, we have screened all manually annotated genes in the region for mutations. We have refined the minimum deleted region at 7q22.3-q31 to 2.79 Mbp and identified a second region of deletion at 7q34. However, we identified no pathogenic coding variation., ((c) 2007 Wiley-Liss, Inc.)

Multiple osteochondromas (MO), also known as hereditary multiple exostoses (HME), is one of the most common hereditary musculoskeletal diseases in Caucasians (1/50,000) with wide clinical variability and genetic heterogeneity. Two genes have thus far been identified as causing the disease, namely EXT1 and EXT2. Various methods to detect mutations in the EXT genes have been used. Here a cohort of 100 MO patients belonging to unrelated Italian families have been analyzed by single-strand conformation polymorphism (SSCP) analysis or by denaturing high performance liquid chromatography (DHPLC). However, neither of these techniques can detect deletions or duplications of entire exons. Families that were negative at SSCP/DHPLC analysis underwent two-color multiple ligation-dependent probe amplification (MLPA) analysis. By these complementary techniques mutation detection was significantly improved and 26 novel mutations have been revealed as well as 18 previously described mutations to give a total of 44 different mutations. Thus we can conclude that combining MLPA with DHPLC in point-mutations negative MO families, the detection of mutations in EXT genes can significantly improve the identification of both point-mutations and mid-size rearrangements. More important, we were able to characterize all those patients who were negative at the first PCR-based method screening., ((c) 2007 Wiley-Liss, Inc.)

Undifferentiated embryonal sarcoma of the liver is a highly malignant and aggressive tumor that occasionally arises within mesenchymal hamartoma of the liver (MHL), a benign tumor that typically occurs in young children. Undifferentiated embryonal sarcoma arising in MHL, as well as uncomplicated MHL, frequently harbor rearrangements of band 19q13.4, including the translocation t(11;19)(q13;q13.4). In this study we report the cloning and DNA sequence analysis of the translocation breakpoints in an undifferentiated embryonal sarcoma arising in MHL known to harbor t(11;19). In this case, the breakpoint at 11q13 occurred in the MALAT1 gene, also known as ALPHA. MALAT1 is rearranged in renal tumors harboring the t(6;11)(p21;q13) translocation, and noncoding MALAT1 transcripts are overexpressed in a number of human carcinomas. The breakpoint at 19q13.4 occurs at a locus we refer to as MHLB1, for Mesenchymal Hamartoma of the Liver Breakpoint 1. Although the MHLB1 locus does not contain a known gene, several human ESTs map to the region (a subset of which show homology to the nuclear RNA export factor (NXF) gene family), and the region is conserved between many mammalian species., ((c) 2007 Wiley-Liss, Inc.)

Breast cancer is a leading cause of cancer-death among women, where the clinicopathological features of tumors are used to prognosticate and guide therapy. DNA copy number alterations (CNAs), which occur frequently in breast cancer and define key pathogenetic events, are also potentially useful prognostic or predictive factors. Here, we report a genome-wide array-based comparative genomic hybridization (array CGH) survey of CNAs in 89 breast tumors from a patient cohort with locally advanced disease. Statistical analysis links distinct cytoband loci harboring CNAs to specific clinicopathological parameters, including tumor grade, estrogen receptor status, presence of TP53 mutation, and overall survival. Notably, distinct spectra of CNAs also underlie the different subtypes of breast cancer recently defined by expression-profiling, implying these subtypes develop along distinct genetic pathways. In addition, higher numbers of gains/losses are associated with the "basal-like" tumor subtype, while high-level DNA amplification is more frequent in "luminal-B" subtype tumors, suggesting also that distinct mechanisms of genomic instability might underlie their pathogenesis. The identified CNAs may provide a basis for improved patient prognostication, as well as a starting point to define important genes to further our understanding of the pathobiology of breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat, ((c) 2006 Wiley-Liss, Inc.)

Although activated Wnt/beta-catenin pathway is considered to be one of the main driving forces of hepatocarcinogenesis, no somatic mutations of the adenomatous polyposis coli (APC) gene have been found in sporadic hepatocellular carcinoma (HCC) to date. Here we present a case of a sporadic nodule-in-nodule-type HCC that provides the first evidence that biallelic genetic inactivation of the APC gene contributed to the development of the tumor. High-density array-based comparative genomic hybridization (aCGH) was performed to clarify genome-wide chromosomal structural alteration profiles of both early and advanced components of this nodule-in-nodule HCC. aCGH analysis revealed a chromosomal loss of the APC gene locus only in the inner advanced component of this tumor. Direct sequencing of the remaining allele revealed a nonsense mutation at codon 682 in the Armadillo repeats, resulting in a truncated protein that lacked all of the beta-catenin-binding motifs. Nonsense mutations at this location are rare among other types of cancer. In conclusion, combined with an immunohistochemical analysis of the beta-catenin protein, this case provides the first evidence that genetic inactivation of the APC gene can play a significant role in the progression of sporadic HCC, probably by activating the Wnt/beta-catenin pathway., ((c) 2006 Wiley-Liss, Inc.)

Although multiple recurrent chromosomal alterations have been identified in prostate cancer cells, the specific genes driving the apparent selection of these changes remain largely unknown. In part, this uncertainty is due to the limited resolution of the techniques used to detect these alterations. In this study, we applied a high-resolution genome-wide method, Affymetrix 100K SNP mapping array, to screen for somatic DNA copy number (CN) alterations among 22 pairs of samples from primary prostate cancers and matched nonmalignant tissues. We detected 355 recurrent deletions and 223 recurrent gains, many of which were novel. As expected, the sizes of novel alterations tend to be smaller. Importantly, among tumors with increasing grade, Gleason sum 6, 7, and 8, we found a significant trend of larger number of alterations in the tumors with higher grade. Overall, gains are significantly more likely to occur within genes (74%) than are deletions (49%). However, when we looked at the most frequent CN alterations, defined as those in > or =4 subjects, we observed that both gains (85%) and deletions (57%) occur preferentially within genes. An example of a novel, recurrent alteration observed in this study was a deletion between the ERG and TMPRSS2 genes on chromosome 21, presumably related to the recently identified fusion transcripts from these two genes. Results from this study provide a basis for a systematic and comprehensive cataloging of CN alterations associated with grades of prostate cancer, and the subsequent identification of specific genes that associated with initiation and progression of the disease. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat, ((c) 2006 Wiley-Liss, Inc.)

The molecular pathogenesis of mantle cell lymphomas (MCL), a subset of B-cell non-Hodgkin's lymphomas with a poor prognosis, is still poorly understood. In addition to the characteristic primary genetic alteration t(11;14)(q13;q32), several further genetic changes are present in most cases. One of the most frequent genomic imbalances is the deletion of 1p22.1-p31.1 observed in nearly one-third of MCL cases. This might indicate the presence of tumor suppressor gene(s) in this critical region of deletion. Quantitative microsatellite analysis (QuMA) is a real-time PCR-based method to detect DNA copy number changes. Since QuMA has the resolving power to detect subtle genomic alterations, including homozygous deletions, this may help to identify candidate tumor suppressor genes from deleted regions. To gain more insight into the molecular pathogenesis of MCL, QuMA was performed on genomic DNA from 57 MCL cases. Eight microsatellite loci mapping to the chromosomal region 1p22.3 were analyzed. Losses were observed in 51 of the 57 ( approximately 89.5%) samples. Two cases showed a homozygous deletion at the locus containing the gene SH3GLB1, which plays a key role in Bax-mediated apoptosis. Two hotspots with copy number losses were detected at chromosomal localizations 85.4 and 86.6 Mb encompassing BCL10 and CLCA2. Both the genes seem to be attractive candidates to study tumor suppressor function in MCL., ((c) 2006 Wiley-Liss, Inc.)