Breast cancer is the most common and the leading cause of female mortality among South African (SA) women. Several non‑biological and biological risk factors may be attributed to their observed high mortality rate; however, the molecular profiles associated with their breast tumors are poorly characterized. The present study examined the patterns of genome-wide copy number alterations (CNAs) and their potential impact on functional cellular pathways targeted by cancer driver genes in patients with breast cancer from the Western Cape region of SA. Array-comparative genomic hybridization analysis, performed in 28 cases of invasive breast cancer, revealed a mean number of 8.68±6.18 CNAs per case, affecting primarily the Xp22.3 and 6p21-p25 cytobands (57.14% of the cases), followed by 19p13.3-p13.11 (35.7%), 2p25.3-p24.3, 4p16.3-p15.3, 8q11.1-q24.3 and 16 p13.3-p11.2 (32.14%). Functional enrichment analysis of genes and microRNA targets mapped in these affected cytobands revealed critical cancer-associated pathways, including fatty acid biosynthesis and metabolism, extracellular matrix-receptor interaction, hippo and tumor protein p53 signaling pathways, which are regulated by known cancer genes, including CCND1, CDKN1A, MAPK1, MDM2, TP53 and SMAD2. An inverse correlation was observed among the number of CNAs and tumor size and grade; CNAs on the 4p and 6p cytobands were also inversely correlated with tumor grade. No association was observed in the number of CNAs and/or the affected cytobands and the different ethnic groups of the SA patients, indicating that their tumor genome is affected by CNAs, irrespectively of their genetic descent. Additional genomic tumor profiling in SA and other Sub-Saharan African patients with breast cancer is required to determine the associations of the CNAs observed with prognosis and clinical outcome.

Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) is a rare provisionally categorized cutaneous lymphoma characterized by an aggressive course. Its pathogenesis and molecular mechanisms are still unknown, and only two individual cases have so far been molecularly characterized. The aim of this study was to define the pattern of numerical chromosomal alterations in tumor samples taken from 20 patients with pcAECyTCL at the time of diagnosis by means of array-comparative genomic hybridization (a-CGH). a-CGH detected numerous genomic aberrations in all the patients and, putting these together as a whole, they affected all the chromosomes. However, no specific profile of recurrent copy number alterations (CNAs) was found. Most of the gains involved regions previously described in other aggressive cutaneous lymphomas such as 7q, 8q24.3, and 17q, whereas the most significant CNA was the loss of 9p21.3 (CDKN2A-CDKN2B), which has already been found in a variety of malignant tumors and is associated with aggressive cutaneous T-cell lymphomas. In brief, CGH analysis revealed a large number of CNAs with only few recurring regions that probably do not represent driving events. The genomic instability found in this aggressive variant of cutaneous lymphoma may therefore be a secondary event but, at the time of the diagnosis of pcAECyTCL, the genomic integrity of tumor cells is already compromised.

BACKGROUND: The present study was conducted to discover genetic imbalances such as DNA copy number variations (CNVs) associated with gastric cancer (GC) and to examine their association with different genes involved in the process of gastric carcinogenesis in Saudi population.
METHODS: Formalin-fixed paraffin-embedded (FFPE) tissues samples from 33 gastric cancer patients and 15 normal gastric samples were collected. Early and late stages GC samples were genotyped and CNVs were assessed by using Illumina HumanOmni1-Quad v.1.0 BeadChip.
RESULTS: Copy number gains were more frequent than losses throughout all GC samples compared to normal tissue samples. The mean number of the altered chromosome per case was 64 for gains and 40 for losses, and the median aberration length was 679115bp for gains and 375889bp for losses. We identified 7 high copy gain, 52 gains, 14 losses, 32 homozygous losses, and 10 copy neutral LOHs (loss of heterozygosities). Copy number gains were frequently detected at 1p36.32, 1q12, 1q22, 2p11.1, 4q23-q25, 5p12-p11, 6p21.33, 9q12-q21.11, 12q11-q12, 14q32.33, 16p13.3, 17p13.1, 17q25.3, 19q13.32, and losses at 1p36.23, 1p36.32, 1p32.1, 1q44, 3q25.2, 6p22.1, 6p21.33, 8p11.22, 10q22.1, 12p11.22, 14q32.12 and 16q24.2. We also identified 2 monosomy at chromosome 14 and 22, 52 partially trisomy and 22 whole chromosome 4 neutral loss of heterozygosities at 13q14.2-q21.33, 5p15.2-p15.1, 5q11.2-q13.2, 5q33.1-q34 and 3p14.2-q13.12. Furthermore, 11 gains and 2 losses at 1p36.32 were detected for 11 different GC samples and this region has not been reported before in other populations. Statistical analysis confirms significant association of H. pylori infection with T4 stage of GC as compare to control and other stages.
CONCLUSIONS: We found that high frequency of copy number gains and losses at 1p36.23, 1p32.1, 1p36.32, 3q25.2, 6p21.33 and 16q24.2 may be common events in gastric cancer. While novel CNVs at 1p36.32 harbouring PRDM16, TP73 and TP73-AS1 genes showed 11 gains and 2 losses for 11 different GC cases and this region is not reported yet in Database of Genomic Variants may be specific to Saudi population.

BACKGROUND: Neuroblastoma is the most common pediatric malignancy with heterogeneous clinical behaviors, ranging from spontaneous regression to aggressive progression. Many studies have identified aberrations related to the pathogenesis and prognosis, broadly classifying neuroblastoma patients into high- and low-risk groups, but predicting tumor progression and clinical management of high-risk patients remains a big challenge.
RESULTS: We integrate gene-level expression, array-based comparative genomic hybridization and functional gene-interaction network of 145 neuroblastoma patients to detect potential driver genes. The drivers are summarized into a driver-gene score (DGscore) for each patient, and we then validate its clinical relevance in terms of association with patient survival. Focusing on a subset of 48 clinically defined high-risk patients, we identify 193 recurrent regions of copy number alterations (CNAs), resulting in 274 altered genes whose copy-number gain or loss have parallel impact on the gene expression. Using a network enrichment analysis, we detect four common driver genes, ERCC6, HECTD2, KIAA1279, EMX2, and 66 patient-specific driver genes. Patients with high DGscore, thus carrying more copy-number-altered genes with correspondingly up- or down-regulated expression and functional implications, have worse survival than those with low DGscore (P = 0.006). Furthermore, Cox proportional-hazards regression analysis shows that, adjusted for age, tumor stage and MYCN amplification, DGscore is the only significant prognostic factor for high-risk neuroblastoma patients (P = 0.008).
CONCLUSIONS: Integration of genomic copy number alteration, expression and functional interaction-network data reveals clinically relevant and prognostic putative driver genes in high-risk neuroblastoma patients. The identified putative drivers are potential drug targets for individualized therapy.
REVIEWERS: This article was reviewed by Armand Valsesia, Susmita Datta and Aleksandra Gruca.

Reliable interpretation of comparative genomic hybridization array (aCGH) results requires centralization and normalization of the data. We evaluated the reliability of aCGH centralization by comparing aCGH results (with classical centralization-normalization steps) to fluorescence in situ hybridization (FISH) results. In addition, we propose a method to correct centralization bias. Sixty-six pituitary tumors were analyzed (Agilent aCGH + SNP 4 × 180K microarray). For each tumor, the FISH-based log

BACKGROUND: Mortality rates of pancreatic cancer remain high, which is mainly due to advanced disease and metastasis. We hypothesized that genomic copy number alterations are enriched in metastatic cells compared to autologous primary tumors, which may inform on cancer-related pathways possibly serving as potential targets for specific therapies. We investigated 18 pancreatic ductal adenocarcinomas, including 39 lymph node and 5 distant metastases after surgical resection. Analysis was performed with array-based comparative genomic hybridization (aCGH).
RESULTS: Metastases acquire a higher frequency of copy number alterations with the highest in distant metastasis (median = 42, lymph node metastases: median = 23, primary tumors: median = 17). In lymph node metastases, gains were prevalent on chromosome bands 8q11.23-q24.3, 12q14.1, 17p12.1, 21q22.12, and losses on 3p21.31, 4p14, 8p23.3-p11.21,17p12-11.2. Genes on amplified regions are involved in cancer-related pathways such as WNT-signaling, also involved in metastasis.
CONCLUSIONS: Pancreatic cancers show a high degree of intratumor heterogeneity, which could lead to resistance of chemotherapy and worse outcome. ACGH analysis reveals regions preferentially gained or lost in synchronous metastases encoding for genes involved in cancer-related pathways, which could lead to novel therapeutic opportunities.

Application of distinct technologies of cancer genome analysis has provided important information for the molecular characterization of several human neoplasia, including follicular cell-derived thyroid carcinoma. Among them, comparative genomic hybridization (CGH)-based procedures have been extensively applied to evaluate genomic imbalances present in these tumours, obtaining data leading to an increase in the understanding of their complexity and diversity. In this review, after a brief overview of the most commonly used CGH-based technichs, we will describe the major results deriving from the most influential studies in the literature which used this approach to investigate the genomic aberrations of thyroid cancer cells. In most studies a small number of patients have been analyzed. Deletions and duplications at different chromosomal regions were detected in all investigated cohorts. A higher number of genomic imbalances has been detected in anaplastic or poorly differentiated thyroid carcinomas compared to well differentiated ones. Limitations in the interpretation of the results, as well the potential impact in the clinical practice are discussed. Though a quite heterogeneous picture arises from results so far available, CGH array, combined with other methodologies as well as an accurate clinical management, may offer novel opportunities for a better stratification of thyroid cancer patients.

Logistic Regression Model (LRM) and artificial neural networks (ANNs) as two nonlinear models have been used to establish a novel two-stage hybrid modeling procedure for prediction of metastasis in advanced colorectal carcinomas. Two different datasets were used in training and testing procedures. For the first stage of hybrid modeling procedure, LRM was used to evaluate the contribution of DNA sequence copy number aberrations detected by Comparative Genomic Hybridization in advanced colorectal carcinoma and its metastasis. Then, the most effective parameters were selected by the LRM. Selected effective parameters among 565 detected chromosomal gains and losses were as follows: gain of 20q11.2, loss of 1q42, loss of 13q34, gain of 5q12, gain of 17p13, loss of 2q22, loss of 11q24 and gain of 2p11.2. Consequently, neural network models were constructed and fed by the parameters selected by LRM to build hybrid predictors on the two databases during self-consistency and jackknife tests, and performance of the hybrid model was verified. The results showed that our two-stage hybrid model approach is very promising for prediction of metastasis in advanced colorectal carcinomas.

Motivation: Although sequencing-based technologies are becoming the new reference in genome analysis, comparative genomic hybridization arrays (aCGH) still constitute a simple and reliable approach for copy number analysis. The most powerful algorithms to analyze such data have been freely provided by the scientific community for many years, but combining them is a complex scripting task.
Results: The cghRA framework combines a user-friendly graphical interface and a powerful object-oriented command-line interface to handle a full aCGH analysis, as is illustrated in an original series of 107 Diffuse Large B-Cell Lymphomas. New algorithms for copy-number calling, polymorphism detection and minimal common region prioritization were also developed and validated. While their performances will only be demonstrated with aCGH, these algorithms could actually prove useful to any copy-number analysis, whatever the technique used.
Availability and implementation: R package and source for Linux, MS Windows and MacOS are freely available at http://bioinformatics.ovsa.fr/cghRA.
Contact: mareschal@ovsa.fr or fabrice.jardin@chb.unicancer.fr.
Supplementary information: Supplementary data are available at Bioinformatics online.

Chromosomal deletions at 11p13 are a frequent cause of congenital Aniridia, a rare pan-ocular genetic disease, and of WAGR syndrome, accounting up to 30% of cases. First-tier genetic testing for newborn with aniridia, to detect 11p13 rearrangements, includes Multiplex Ligation-dependent Probe Amplification (MLPA) and karyotyping. However, neither of these approaches allow obtaining a complete picture of the high complexity of chromosomal deletions and breakpoints in aniridia. Here, we report the development and validation of a customized targeted array-based comparative genomic hybridization, so called WAGR-array, for comprehensive high-resolution analysis of CNV in the WAGR locus. Our approach increased the detection rate in a Spanish cohort of 38 patients with aniridia, WAGR syndrome and other related ocular malformations, allowing to characterize four undiagnosed aniridia cases, and to confirm MLPA findings in four additional patients. For all patients, breakpoints were accurately established and a contiguous deletion syndrome, involving a large number of genes, was identified in three patients. Moreover, we identified novel microdeletions affecting 3' PAX6 regulatory regions in three families with isolated aniridia. This tool represents a good strategy for the genetic diagnosis of aniridia and associated syndromes, allowing for a more accurate CNVs detection, as well as a better delineation of breakpoints. Our results underline the clinical importance of performing exhaustive and accurate analysis of chromosomal rearrangements for patients with aniridia, especially newborns and those without defects in PAX6 after diagnostic screening.

Previous precision medicine studies have investigated conventional molecular techniques and/or limited sets of gene alterations. The aim of this study was to describe the impact of the next-generation sequencing of the largest panel of genes used to date in tumour tissue and blood in the context of institutional molecular screening programmes. DNA analysis was performed by next-generation sequencing using a panel of 426 cancer-related genes and by comparative genomic hybridization from formalin-fixed and paraffin-embedded archived tumour samples when available or from fresh tumour samples. Five hundred sixty-eight patients were enrolled. The median number of prior lines of treatment was 2 (range 0-9). The most common primary tumour types were lung (16.9%), colorectal (14.4%), breast (10.6%), ovarian (10.2%) and sarcoma (10.2%). The median patient age was 63 years (range 19-88). A total of 292 patients (51.4%) presented with at least one actionable genetic alteration. The 20 genes most frequently altered were TP53, CDKN2A, KRAS, PTEN, PI3KCA, RB1, APC, ERBB2, MYC, EGFR, CDKN2B, ARID1A, SMAD4, FGFR1, MDM2, BRAF, ATM, CCNE1, FGFR3 and FRS2. One hundred fifty-nine patients (28%) were included in early phase trials. The treatment was matched with a tumour profile in 86 cases (15%). The two main reasons for non-inclusion were non-progressive disease (31.5%) and general status deterioration (25%). Twenty-eight percent of patients presented with a growth modulation index (time to progression under the early phase trial treatment/time to progression of the previous line of treatment) >1.3.Extensive molecular profiling using high-throughput techniques allows for the identification of actionable mutations in the majority of cases and is associated with substantial clinical benefit in up to one in four patients.

OBJECTIVES: Lung cancer in Xuanwei (LCXW), China, is known throughout the world for its distinctive characteristics, but little is known about its pathogenesis. The purpose of this study was to screen potential novel "driver genes" in LCXW.
METHODS: Genome-wide DNA copy number alterations (CNAs) were detected by array-based comparative genomic hybridization and differentially expressed genes (DEGs) by gene expression microarrays in 8 paired LCXW and non-cancerous lung tissues. Candidate driver genes were screened by integrated analysis of CNAs and DEGs. The candidate genes were further validated by real-time quantitative polymerase chain reaction.
RESULTS: Large numbers of CNAs and DEGs were detected, respectively. Some of the most frequently occurring CNAs included gains at 5p15.33-p15.32, 5p15.1-p14.3, and 5p14.3-p14.2 and losses at 11q24.3, 21q21.1, 21q22.12-q22.13, and 21q22.2. Integrated analysis of CNAs and DEGs identified 24 candidate genes with frequent copy number gains and concordant upregulation, which were considered potential oncogenes, including CREB3L4, TRIP13, and CCNE2. In addition, the analysis identified 19 candidate genes with a negative association between copy number change and expression change, considered potential tumor suppressor genes, including AHRR, NKD2, and KLF10. One of the most studied oncogenes, MYC, may not play a carcinogenic role in LCXW.
CONCLUSIONS: This integrated analysis of CNAs and DEGs identified several potential novel LCXW-related genes, laying an important foundation for further research on the pathogenesis of LCXW and identification of novel biomarkers or therapeutic targets.

BACKGROUND/AIM: Molecular mechanisms of alterations in protein tyrosine phosphatases (PTPs) genes in cancer have been previously described and include chromosomal aberrations, gene mutations, and epigenetic silencing. However, little is known about small intragenic gains and losses that may lead to either changes in expression or enzyme activity and even loss of protein function.
MATERIALS AND METHODS: The aim of this study was to investigate 25 phosphatase genes using customized array comparative genomic hybridization in 16 sporadic colorectal cancer tissues.
RESULTS: The analysis revealed two unique small alterations: of 2 kb in PTPN14 intron 1 and of 1 kb in PTPRJ intron 1. We also found gains and losses of whole PTPs gene sequences covered by large chromosome aberrations.
CONCLUSION: In our preliminary studies using high-resolution custom microarray we confirmed that PTPs are frequently subjected to whole-gene rearrangements in colorectal cancer, and we revealed that non-polymorphic intragenic changes are rare.

AIM: To investigate whether chromosomal instability (CIN) is associated with tumor phenotypes and/or with global genomic status based on MSI (microsatellite instability) and CIMP (CpG island methylator phenotype) in early-onset colorectal cancer (EOCRC).
METHODS: Taking as a starting point our previous work in which tumors from 60 EOCRC cases (≤45 years at the time of diagnosis) were analyzed by array comparative genomic hybridization (aCGH), in the present study we performed an unsupervised hierarchical clustering analysis of those aCGH data in order to unveil possible associations between the CIN profile and the clinical features of the tumors. In addition, we evaluated the MSI and the CIMP statuses of the samples with the aim of investigating a possible relationship between copy number alterations (CNAs) and the MSI/CIMP condition in EOCRC.
RESULTS: Based on the similarity of the CNAs detected, the unsupervised analysis stratified samples into two main clusters (A, B) and four secondary clusters (A1, A2, B3, B4). The different subgroups showed a certain correspondence with the molecular classification of colorectal cancer (CRC), which enabled us to outline an algorithm to categorize tumors according to their CIMP status. Interestingly, each subcluster showed some distinctive clinicopathological features. But more interestingly, the CIN of each subcluster mainly affected particular chromosomes, allowing us to define chromosomal regions more specifically affected depending on the CIMP/MSI status of the samples.
CONCLUSIONS: Our findings may provide a basis for a new form of classifying EOCRC according to the genomic status of the tumors.

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable- SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.

BACKGROUND: Routine feasibility and clinical impact of genomics-based tumor profiling in advanced breast cancer (aBC) remains to be determined. We conducted a pilot study to evaluate whether precision medicine could be prospectively implemented for aBC patients in a single center and to examine whether patient-derived tumor xenografts (PDX) could be obtained in this population.
RESULTS: Thirty-four aBC patients were included. Actionable targets were found in 28 patients (82%). A targeted therapy could be proposed to 22 patients (64%), either through a clinical trial (n=15) and/or using already registered drugs (n=21). Ten patients (29%) eventually received targeted treatment, 2 of them deriving clinical benefit. Of 22 patients subjected to mouse implantation, 10 had successful xenografting (45%), mostly in triple-negative aBC.
METHODS: aBC patients accessible to tumor biopsy were prospectively enrolled at the Institut Paoli-Calmettes in the BC-BIO study (ClinicalTrials.gov, NCT01521676). Genomic profiling was established by whole-genome array comparative genomic hybridization (aCGH) and targeted next-generation sequencing (NGS) of 365 candidate cancer genes. For a subset of patients, a sample of fresh tumor was orthotopically implanted in humanized cleared fat pads of NSG mice for establishing PDX.
CONCLUSIONS: Precision medicine can be implemented in a single center in the context of clinical practice and may allow genomic-driven treatment in approximately 30% of aBC patients. PDX may be obtained in a significant fraction of cases.

Recently reported genome-wide association studies have identified more than 20 common low-penetrance colorectal cancer (CRC) susceptibility loci. Recent studies have reported that copy number variations (CNVs) are considered important human genomic variants related to cancer, while the contribution of CNVs remains unclear. We performed array comparative genomic hybridization (aCGH) in 36 CRC patients and 47 controls. Using breakpoint PCR, we confirmed the breakpoint of the PKD1L2 deletion region. High frequency of PKD1L2 CNV was observed in CRC cases. We validated the association between PKD1L2 variation and CRC risk in 1,874 cases and 2,088 controls (OR = 1.44, 95% CI = 1.04-1.98, p = 0.028). Additionally, PKD1L2 CNV is associated with increased CRC risk in patients younger than 50 years (OR = 2.14, 95% CI 1.39-3.30, p = 5.8 × 10

PURPOSE: Detection of predisposing copy number variants (CNV) in 330 families affected with hereditary breast and ovarian cancer (HBOC).
METHODS: In order to complement mutation detection with Illumina's TruSight Cancer panel, we designed a customized high-resolution 8 × 60k array for CGH (aCGH) that covers all 94 genes from the panel.
RESULTS: Copy number variants with immediate clinical relevance were detected in 12 families (3.6%). Besides 3 known CNVs in CHEK2, RAD51C, and BRCA1, we identified 3 novel pathogenic CNVs in BRCA1 (deletion of exons 4-13, deletion of exons 12-18) and ATM (deletion exons 57-63) plus an intragenic duplication of BRCA2 (exons 3-11) and an intronic BRCA1 variant with unknown pathogenicity. The precision of high-resolution aCGH enabled straight forward breakpoint amplification of a BRCA1 deletion which subsequently allowed for fast and economic CNV verification in family members of the index patient. Furthermore, we used our aCGH data to validate an algorithm that was able to detect all identified copy number changes from next-generation sequencing (NGS) data.
CONCLUSIONS: Copy number detection is a mandatory analysis in HBOC families at least if no predisposing mutations were found by sequencing. Currently, high-resolution array CGH is our first choice of method of analysis due to unmatched detection precision. Although it seems possible to detect CNV from sequencing data, there currently is no satisfying tool to do so in a routine diagnostic setting.

Genomic alteration in head and neck squamous cell carcinoma (HNSCC) was studied in two cell line pairs (HN30-HN31 and HN4-HN12) using conventional C-banding, multiplex fluorescence in situ hybridization (M-FISH), and array comparative genomic hybridization (array CGH). HN30 and HN4 were derived from primary lesions in the pharynx and base of tongue, respectively, and HN31 and HN12 were derived from lymph-node metastatic lesions belonging to the same patients. Gain of chromosome 1, 7, and 11 were shared in almost all cell lines. Hierarchical clustering revealed that HN31 was closely related to HN4, which shared eight chromosome alteration cases. Large C-positive heterochromatins were found in the centromeric region of chromosome 9 in HN31 and HN4, which suggests complex structural amplification of the repetitive sequence. Array CGH revealed amplification of 7p22.3p11.2, 8q11.23q12.1, and 14q32.33 in all cell lines involved with tumorigenesis and inflammation genes. The amplification of 2p21 (SIX3), 11p15.5 (H19), and 11q21q22.3 (MAML2, PGR, TRPC6, and MMP family) regions, and deletion of 9p23 (PTPRD) and 16q23.1 (WWOX) regions were identified in HN31 and HN12. Interestingly, partial loss of PTPRD (9p23) and WWOX (16q23.1) genes was identified in HN31 and HN12, and the level of gene expression tended to be the down-regulation of PTPRD, with no detectable expression of the WWOX gene. This suggests that the scarcity of PTPRD and WWOX genes might have played an important role in progression of HNSCC, and could be considered as a target for cancer therapy or a biomarker in molecular pathology.

Melanoma remains one of the most aggressive forms of cutaneous malignancies. While its diagnosis based on histologic parameters is usually straight forward in most cases, distinguishing a melanoma from a melanocytic nevus can be challenging in some instances, especially when there are overlapping clinical and histopathologic features. Occasionally, melanomas can histologically mimic other tumors and even demonstration of melanocytic origin can be challenging. Thus, several ancillary tests may be employed to arrive at the correct diagnosis. The objective of this review is to summarize these tests, including the well-established and commonly used ones such as immunohistochemistry, with specific emphasis on emerging techniques such as comparative genomic hybridization, fluorescence in situ hybridization and imaging mass spectrometry.

Secondary chromosomal aberrations are necessary for development of overt leukemia in t(12;21)/ETV6-RUNX1-positive acute lymphoblastic leukemia (ALL). Conventional cytogenetic analysis supplemented with locus-specific FISH analyses is gold standard to detect important clonal aberrations in this disease group. However, adequate chromosome banding analysis may often be hampered by poor chromosome morphology and banding patterns in pediatric ALL cases, which may hinder identification of possible clinical important additional chromosomal aberrations. We used oligo-based high-resolution aCGH (oaCGH) analysis as an adjunct tool to enhance conventional cytogenetic analysis in pediatric acute B-cell lymphoblastic leukemia in a prospective single center study during a 4-year period (2012-2015). In a consecutive series of 45 pediatric B-ALLs, we identified eight patients with t(12;21)/ETV6-RUNX1 fusion by FISH analysis. In three of the patients, oaCGH analysis revealed concurrent Xq duplication and 6q deletion, which was cryptic by G-banded analysis. FISH analyses with whole chromosome painting probes confirmed the imbalances and showed an unbalanced translocation der(6)t(X;6) in all three patients. A search in the literature revealed two additional pediatric patients with cryptic der(6)t(X;6) in t(12;21)-positive ALLs. No common break points on Xq or 6q could be determined between the five patients. This study highlights the importance of oaCGH analysis as an adjunct cytogenetic tool to detect cryptic chromosomal aberrations. Further, the study adds to understanding the full spectrum of secondary chromosomal aberrations in the very common t(12;21)-positive pediatric ALL disease group. We suggest that the unbalanced der(6)t(X;6), which is cryptic to conventional cytogenetics, is a non-random secondary event in this disease group. It might be that the specific combination of concurrent Xq duplication and 6q-deletion results in gain of possible oncogenes on Xq and loss of possible tumor suppressor genes on 6q that are important for the leukemic propagation of t(12;21)-positive hematopoietic cells in a subset of ALLs.

Malignant peritoneal mesotheliomas (MPM) are rare, accounting for approximately 8% of cases of mesothelioma in France. We performed comparative genomic hybridization (CGH) on frozen MPM samples using the Agilent Human Genome CGH 180 K array. Samples were taken from a total of 33 French patients, comprising 20 men and 13 women with a mean (range) age of 58.4 (17-76) years. Asbestos exposure was reported in 8 patients (24.2%). Median (range) overall survival (OS) was 39 (0-119) months. CGH analysis demonstrated the presence of chromosomal instability in patients with MPM, with a genomic pattern that was similar to that described for pleural mesothelioma, including the loss of chromosomal regions 3p21, 9p21, and 22q12. In addition, novel genomic copy number alterations were identified, including the 15q26.2 region and the 8p11.22 region. Median OS was associated with a low peritoneal cancer index (P=.011), epithelioid subtype (P=.038), and a low number of genomic aberrations (P=.015), all of which constitute good prognostic factors for MPM. Our results provide new insights into the genetic and genomic background of MPM. Although pleural and peritoneal mesotheliomas have different risk factors, different therapeutics, and different prognosis; these data provide support to combine pleural and peritoneal mesothelioma in same clinical assays.

BACKGROUND: Although asbestos acts as a potent carcinogen in pleural mesothelial and pulmonary epithelial cells, it still remains unclear whether asbestos causes specific and characteristic gene alterations in these different kinds of target cells, because direct comparison in an identical patient is not feasible. We experienced a rare synchronous collision tumor composed of malignant pleural mesothelioma (MPM) and primary pulmonary adenocarcinoma (PAC) in a 77-year-old man with a history of long-term smoking and asbestos exposure, and compared the DNA copy number alteration (CNA) and somatic mutation in these two independent tumors.
METHODS: Formalin-fixed paraffin-embedded (FFPE) tissues of MPM and PAC lesions from the surgically resected specimen were used. Each of these MPM and PAC lesions exhibited a typical histology and immunophenotype. CNA analysis using SNP array was performed using the Illumina Human Omni Express-12_FFPE (Illumina, San Diego, CA, USA) with DNA extracts from each lesion. Somatic mutation analysis using next-generation sequencing was performed using the TruSeq Amplicon Cancer Panel (Illumina).
RESULTS: The CNA analysis demonstrated a marked difference in the frequency of gain and loss between MPM and PAC. In PAC, copy number (CN) gain was detected more frequently and widely than CN loss, whereas in MPM there was no such obvious difference. PAC did not harbor CNAs that have been identified in asbestos-associated lung cancer, but did harbor some of the CNAs associated with smoking. MPM exhibited CN loss at 9p21.2-3, which is the most common genetic alteration in mesothelioma.
CONCLUSION: In this particular case, asbestos exposure may not have played a primary role in PAC carcinogenesis, but cigarette smoking may have contributed more to the occurrence of CN gains in PAC. This comparative genetic analysis of two different lesions with same amount of asbestos exposure and cigarette smoke exposure has provided information on differences in the cancer genome related to carcinogenesis.

BACKGROUND: The incidence of and mortality from colorectal cancers (CRC) can be reduced by early detection. Currently there is a lack of established markers to detect early neoplastic changes. We aimed to identify the copy number variations (CNVs) and the associated genes which could be potential markers for the detection of neoplasia in both ulcerative colitis-associated neoplasia (UC-CRN) and sporadic colorectal neoplasia (S-CRN).
METHODS: We employed array comparative genome hybridization (aCGH) to identify CNVs in tissue samples of UC nonprogressor, progressor and sporadic CRC. Select genes within these CNV regions as a panel of markers were validated using quantitative real time PCR (qRT-PCR) method along with the microsatellite instability (MSI) in an independent cohort of samples. Immunohistochemistry (IHC) analysis was also performed.
RESULTS: Integrated analysis showed 10 overlapping CNV regions between UC-Progressor and S-CRN, with the 8q and 12p regions showing greater overlap. The qRT-PCR based panel of MYC, MYCN, CCND1, CCND2, EGFR and FNDC3A was successful in detecting neoplasia with an overall accuracy of 54% in S-CRN compared to that of 29% in UC neoplastic samples. IHC study showed that p53 and CCND1 were significantly overexpressed with an increasing frequency from pre-neoplastic to neoplastic stages. EGFR and AMACR were expressed only in the neoplastic conditions.
CONCLUSION: CNVs that are common and unique to both UC-associated and sporadic colorectal neoplasm could be the key players driving carcinogenesis. Comparative analysis of CNVs provides testable driver aberrations but needs further evaluation in larger cohorts of samples. These markers may help in developing more effective neoplasia-detection strategies during screening and surveillance programs.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), are at increased risk for small bowel or colorectal cancers (colitis-associated cancers [CACs]). We compared the spectrum of genomic alterations in CACs with those of sporadic colorectal cancers (CRCs) and investigated differences between CACs from patients with CD vs UC.
METHODS: We studied tumor tissues from patients with CACs treated at Memorial Sloan Kettering Cancer Center or Weill Cornell Medical College from 2003 through 2015. We performed hybrid capture-based next-generation sequencing analysis of >300 cancer-related genes to comprehensively characterize genomic alterations.
RESULTS: We performed genomic analyses of 47 CACs (from 29 patients with UC and 18 with CD; 43 primary tumors and 4 metastases). Primary tumors developed in the ileum (n = 2), right colon (n = 18), left colon (n = 6), and rectosigmoid or rectum (n = 21). We found genomic alterations in TP53, IDH1, and MYC to be significantly more frequent, and mutations in APC to be significantly less frequent, than those reported in sporadic CRCs by The Cancer Genome Atlas or Foundation Medicine. We identified genomic alterations that might be targeted by a therapeutic agent in 17 of 47 (36%) CACs. These included the mutation encoding IDH1 R132; amplification of FGFR1, FGFR2, and ERBB2; and mutations encoding BRAF V600E and an EML4-ALK fusion protein. Alterations in IDH1 and APC were significantly more common in CACs from patients with CD than UC.
CONCLUSIONS: In an analysis of CACs from 47 patients, we found significant differences in the spectrum of genomic alterations in CACs compared with sporadic CRCs. We observed a high frequency of IDH1 R132 mutations in patients with CD but not UC, as well as a high frequency of MYC amplification in CACs. Many genetic alterations observed in CACs could serve as therapeutic targets.

Personalized medicine uses genomic information for selecting therapy in patients with metastatic cancer. An issue is the optimal tissue source (primary tumor or metastasis) for testing. We compared the DNA copy number and mutational profiles of primary breast cancers and paired metastases from 23 patients using whole-genome array-comparative genomic hybridization and next-generation sequencing of 365 "cancer-associated" genes. Primary tumors and metastases harbored copy number alterations (CNAs) and mutations common in breast cancer and showed concordant profiles. The global concordance regarding CNAs was shown by clustering and correlation matrix, which showed that each metastasis correlated more strongly with its paired tumor than with other samples. Genes with recurrent amplifications in breast cancer showed 100% (ERBB2, FGFR1), 96% (CCND1), and 88% (MYC) concordance for the amplified/non-amplified status. Among all samples, 499 mutations were identified, including 39 recurrent (AKT1, ERBB2, PIK3CA, TP53) and 460 non-recurrent variants. The tumors/metastases concordance of variants was 75%, higher for recurrent (92%) than for non-recurrent (73%) variants. Further mutational discordance came from very different variant allele frequencies for some variants. We showed that the chosen targeted therapy in two clinical trials of personalized medicine would be concordant in all but one patient (96%) when based on the molecular profiling of tumor and paired metastasis. Our results suggest that the genotyping of primary tumor may be acceptable to guide systemic treatment if the metastatic sample is not obtainable. However, given the rare but potentially relevant divergences for some actionable driver genes, the profiling of metastatic sample is recommended.

Array Comparative Genomic Hybridization (aCGH) is a rapid screening technique to detect gene deletions and duplications, providing an overview of chromosomal aberrations throughout the entire genome of a tumor, without the need for cell culturing. However, the heterogeneity of aCGH data obfuscates existing methods of data analysis. Analysis of aCGH data from a systems biology perspective or in the context of total aberrations is largely absent in the published literature. We present here a novel alternative to the functional analysis of aCGH data using the phylogenetic paradigm that is well-suited to high dimensional datasets of heterogeneous nature, but has not been widely adapted to aCGH data. Maximum parsimony phylogenetic analysis sorts out genetic data through the simplest presentation of the data on a cladogram, a graphical evolutionary tree, thus providing a powerful and efficient method for aCGH data analysis. For example, the cladogram models the multiphasic changes in the cancer genome and identifies shared early mutations in the disease progression, providing a simple yet powerful means of aCGH data interpretation. As such, applying maximum parsimony phylogenetic analysis to aCGH results allows for the differentiation between drivers and passenger genes aberrations in cancer specimens. In addition to offering a novel methodology to analyze aCGH results, we present here a crucial software suite that we wrote to carry out the analysis. In a broader context, we wish to underscore that phylogenetic analysis of aCGH data is a non-parametric method that circumvents the pitfalls and frustrations of standard analytical techniques that rely on parametric statistics. Organizing the data in a cladogram as explained in this research article provides insights into the disease common aberrations, as well as the disease subtypes and their shared aberrations (the synapomorphies) of each subtype. Hence, we report the method and make the software suite publicly and freely available at http://software.phylomcs.com so that researchers can test alternative and innovative approaches to the analysis of aCGH data.

BACKGROUND: Array CGH analysis of breast tumors has contributed to the identification of different genomic profiles in these tumors. Loss of DNA repair by BRCA1 functional deficiency in breast cancer has been proposed as a relevant contribution to breast cancer progression for tumors with no germline mutation. Identifying the genomic alterations taking place in BRCA1 not expressing tumors will lead us to a better understanding of the cellular functions affected in this heterogeneous disease. Moreover, specific genomic alterations may contribute to the identification of potential therapeutic targets and offer a more personalized treatment to breast cancer patients.
METHODS: Forty seven tumors from hereditary breast cancer cases, previously analyzed for BRCA1 expression, and screened for germline BRCA1 and 2 mutations, were analyzed by Array based Comparative Genomic Hybridization (aCGH) using Agilent 4x44K arrays. Overall survival was established for tumors in different clusters using Log-rank (Mantel-Cox) Test. Gene lists obtained from aCGH analysis were analyzed for Gene Ontology enrichment using GOrilla and DAVID tools.
RESULTS: Genomic profiling of the tumors showed specific alterations associated to BRCA1 or 2 mutation status, and BRCA1 expression in the tumors, affecting relevant cellular processes. Similar cellular functions were found affected in BRCA1 not expressing and BRCA1 or 2 mutated tumors. Hierarchical clustering classified hereditary breast tumors in four major, groups according to the type and amount of genomic alterations, showing one group with a significantly poor overall survival (p = 0.0221). Within this cluster, deletion of PLEKHO1, GDF11, DARC, DAG1 and CD63 may be associated to the worse outcome of the patients.
CONCLUSIONS: These results support the fact that BRCA1 lack of expression in tumors should be used as a marker for BRCAness and to select these patients for synthetic lethality approaches such as treatment with PARP inhibitors. In addition, the identification of specific alterations in breast tumors associated with poor survival, immune response or with a BRCAness phenotype will allow the use of a more personalized treatment in these patients.

BACKGROUND: Molecular tumour profiling technologies have become increasingly important in the era of precision medicine, but their routine use is limited by their accessibility, cost, and tumour material availability. It is therefore crucial to assess their relative added value to optimize the sequence and combination of such technologies.
PATIENTS AND METHODS: Within the MOSCATO-01 trial, we investigated the added value of whole exome sequencing (WES) in patients that did not present any molecular abnormality on array comparative genomic hybridization (aCGH) and targeted gene panel sequencing (TGPS) using cancer specific panels. The pathogenicity potential and actionability of mutations detected on WES was assessed.
RESULTS: Among 420 patients enrolled between December 2011 and December 2013, 283 (67%) patients were analysed for both TGPS and aCGH. The tumour sample of 25 (8.8%) of them presented a flat (or low-dynamic) aCGH profile and no pathogenic mutation on TGPS. We selected the first eligible 10 samples-corresponding to a heterogeneous cohort of different tumour types-to perform WES. This allowed identifying eight mutations of interest in two patients: FGFR3, PDGFRB, and CREBBP missense single-nucleotide variants (SNVs) in an urothelial carcinoma; FGFR2, FBXW7, TP53, and MLH1 missense SNVs as well as an ATM frameshift mutation in a squamous cell carcinoma of the tongue. The FGFR3 alteration had been previously described as an actionable activating mutation and might have resulted in treatment by an FGFR inhibitor. CREBBP and ATM alterations might also have suggested a therapeutic orientation towards epigenetic modifiers and ataxia-telangectasia and Rad3-related inhibitors, respectively.
CONCLUSION: The therapeutic added value of performing WES on tumour samples that do not harbour any genetic abnormality on TGPS and aCGH might be limited and variable according to the histotype. Alternative techniques, including RNASeq and methylome analysis, might be more informative in selected cases.

BACKGROUND: The purpose of the present study was to evaluate the detection rate of genomic aberrations in haematological malignancies using oligobased array-CGH (oaCGH) analysis in combination with karyotyping and fluorescence in situ hybridization (FISH) analyses, and its feasibility in a clinical pragmatic approach.
MATERIALS AND METHODS: The 4x180K Cancer Cytochip array was applied in 96 patients with various haematological malignancies in a prospective setting and in 41 acute myeloid leukemia (AML) patients retrospectively.
RESULTS: Combined use of oaCGH analysis and karyotyping improved the overall detection rate in comparison to karyotyping-alone and vice versa. In cases with normal karyotypes oaCGH analysis detected genomic aberrations in 66% (39/60) of cases. In the group of simple karyotypes oaCGH analysis extended karyotypic findings in 39% (12/31) while oaCGH analysis extended the karyotypic findings in 89% (39/44) of cases with complex karyotypes. In 7% (5/75) of cases oaCGH analysis failed in detecting the observed abnormalities by karyotyping.
CONCLUSION: oaCGH analysis is a valuable asset in routine cytogenetics of haematological malignancies.