Chromosome Arms 19q Research Articles

Copy number profiling by array comparative genomic hybridization identifies frequently occurring BRCA2‐like male breast cancer

Genomic aberrations can be used to subtype breast cancer. In this study, we investigated DNA copy number (CN) profiles of 69 cases of male breast cancer (MBC) by array comparative genomic hybridization (aCGH) to detect recurrent gains and losses in comparison with female breast cancers (FBC). Further, we classified these profiles as BRCA1-like, BRCA2-like or non-BRCA-like profiles using previous classifiers derived from FBC, and correlated these profiles with pathological characteristics. We observed large CN gains on chromosome arms 1q, 5p, 8q, 10p, 16p, 17q, and chromosomes 20 and X. Large losses were seen on chromosomes/chromosome arms 1p, 6p, 8p, 9, 11q, 13, 14q, 16q, 17p, and 22. The pattern of gains and losses in estrogen receptor positive (ER+) MBC was largely similar to ER+ FBC, except for gains on chromosome X in MBC, which were uncommon in FBC. Out of 69 MBC patients, 15 patients (22%) had a BRCA2-like profile, of which 2 (3%) were also BRCA1-like. One patient (1%) was only BRCA1-like; the remaining 53 (77%) patients were classified as non-BRCA-like. BRCA2-like cases were more often p53 accumulated than non-BRCA-like cases (P = 0.014). In conclusion, the pattern of gains and losses in ER+ MBC was largely similar to that of its ER+ FBC counterpart, except for gains on chromosome X in MBC, which are uncommon in FBC. A significant proportion of MBC has a BRCA2-like aCGH profile, pointing to a potentially hereditary nature, and indicating that they could benefit from a drug regimen targeting BRCA defects as in FBC.

Role of microRNAs Located on Chromosome Arm 10q in Malignant Gliomas.

Deletions of chromosome arm 10q are found in most glioblastomas and subsets of lower grade gliomas. Mutations in the PTEN gene at 10q23.3 are restricted to less than half of the 10q-deleted gliomas, suggesting additional glioma-associated tumor suppressors on 10q. We investigated 64 astrocytic gliomas of different malignancy grades for aberrant expression of 16 microRNAs (miRNAs) on 10q. Thereby, we identified four miRNAs (miR-107, miR-146b-5p, miR-346, miR-1287-5p) whose expression was frequently down-regulated in anaplastic astrocytomas and/or glioblastomas. DNA methylation analyses revealed 5'-CpG site hypermethylation of miR-346 in more than two-thirds of primary glioblastomas, while aberrant 5'-CpG site methylation of miR-146b-5p was frequent in IDH1-mutant astrocytomas and secondary glioblastomas. Overexpression of either of the four miRNAs in glioma cell lines reduced cell proliferation and/or increased caspase-3/7 activity. Expression analyses of miRNA overexpressing glioma cells and 3'-untranslated region luciferase reporter gene assays revealed evidence that these miRNAs post-transcriptionally regulate expression of glioma-relevant genes, including CDK6 (miR-107), EGFR (miR-146b-5p, miR-1287-5p), TERT and SEMA6A (miR-346), all of which are overexpressed in malignant gliomas in situ. In summary, we show that the 10q-located miRNAs miR-107, miR-146b-5p, miR-346 and miR-1287-5p are frequently down-regulated in malignant gliomas and thereby may support overexpression of important glioma growth-promoting genes.

Molecular dissection of ependymomas.

Molecular dissection of ependymomas.

Identification of Copy Number Aberrations in Breast Cancer Subtypes Using Persistence Topology.

DNA copy number aberrations (CNAs) are of biological and medical interest because they help identify regulatory mechanisms underlying tumor initiation and evolution. Identification of tumor-driving CNAs (driver CNAs) however remains a challenging task, because they are frequently hidden by CNAs that are the product of random events that take place during tumor evolution. Experimental detection of CNAs is commonly accomplished through array comparative genomic hybridization (aCGH) assays followed by supervised and/or unsupervised statistical methods that combine the segmented profiles of all patients to identify driver CNAs. Here, we extend a previously-presented supervised algorithm for the identification of CNAs that is based on a topological representation of the data. Our method associates a two-dimensional (2D) point cloud with each aCGH profile and generates a sequence of simplicial complexes, mathematical objects that generalize the concept of a graph. This representation of the data permits segmenting the data at different resolutions and identifying CNAs by interrogating the topological properties of these simplicial complexes. We tested our approach on a published dataset with the goal of identifying specific breast cancer CNAs associated with specific molecular subtypes. Identification of CNAs associated with each subtype was performed by analyzing each subtype separately from the others and by taking the rest of the subtypes as the control. Our results found a new amplification in 11q at the location of the progesterone receptor in the Luminal A subtype. Aberrations in the Luminal B subtype were found only upon removal of the basal-like subtype from the control set. Under those conditions, all regions found in the original publication, except for 17q, were confirmed; all aberrations, except those in chromosome arms 8q and 12q were confirmed in the basal-like subtype. These two chromosome arms, however, were detected only upon removal of three patients with exceedingly large copy number values. More importantly, we detected 10 and 21 additional regions in the Luminal B and basal-like subtypes, respectively. Most of the additional regions were either validated on an independent dataset and/or using GISTIC. Furthermore, we found three new CNAs in the basal-like subtype: a combination of gains and losses in 1p, a gain in 2p and a loss in 14q. Based on these results, we suggest that topological approaches that incorporate multiresolution analyses and that interrogate topological properties of the data can help in the identification of copy number changes in cancer.

Patterns of somatic uniparental disomy identify novel tumor suppressor genes in colorectal cancer.

Colorectal cancer (CRC) is characterized by specific patterns of copy number alterations (CNAs), which helped with the identification of driver oncogenes and tumor suppressor genes (TSGs). More recently, the usage of single nucleotide polymorphism arrays provided information of copy number neutral loss of heterozygosity, thus suggesting the occurrence of somatic uniparental disomy (UPD) and uniparental polysomy (UPP) events. The aim of this study is to establish an integrative profiling of recurrent UPDs/UPPs and CNAs in sporadic CRC. Our results indicate that regions showing high frequencies of UPD/UPP mostly coincide with regions typically involved in genomic losses. Among them, chromosome arms 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q and 22q preferentially showed UPDs/UPPs over genomic losses suggesting that tumor cells must maintain the disomic state of certain genes to favor cellular fitness. A meta-analysis using over 300 samples from The Cancer Genome Atlas confirmed our findings. Several regions affected by recurrent UPDs/UPPs contain well-known TSGs, as well as novel candidates such as ARID1A, DLC1, TCF7L2 and DMBT1. In addition, VCAN, FLT4, SFRP1 and GAS7 were also frequently involved in regions of UPD/UPP and displayed high levels of methylation. Finally, sequencing and fluorescence in situ hybridization analysis of the gene APC underlined that a somatic UPD event might represent the second hit to achieve biallelic inactivation of this TSG in colorectal tumors. In summary, our data define a profile of somatic UPDs/UPPs in sporadic CRC and highlights the importance of these events as a mechanism to achieve the inactivation of TSGs.

Abstract 2746: Significant evidence for linkage for lethal prostate cancer on chromosome arm 4q in Utah high-risk pedigrees

Abstract Background: Using the unique Utah genealogical resource that links 12 generations of genealogy data to a statewide cancer registry from 1966 and statewide death certificates from 1904 we have identified a set of high-risk lethal prostate cancer pedigrees with a significant excess of prostate cancer cases having died from prostate cancer. Methods: Clusters of prostate cancer cases descended from a common ancestor, among whose descendants is observed a statistical excess of prostate cancer (high-risk prostate cancer pedigrees) were recruited and sampled over the past 25 years. Pedigrees including 4 or more cases who have subsequently been reported to have died from prostate cancer (as evidenced by inclusion as a cause of death on a Utah death certificate) were genotyped with 720k high-density SNPs across the genome. Linkage analysis with markers not in linkage disequilibrium was performed to identify regions hypothesized to contain a lethal prostate cancer predisposition gene. Results: A single extended Utah high-risk prostate cancer pedigree (p<0.05 for significant excess of deceased prostate cancer cases) including 6 distantly related sampled prostate cancer cases who went on to die as a result of their prostate cancer showed significant evidence for linkage at chromosome arm 4q24 (LOD score = +3.28), with 5 of the 6 cases having inherited the same region of chromosome 4 from a common ancestor. Conclusions: The inability of association studies to identify prostate cancer predisposition genes has returned focus to linkage studies of highly informative high-risk pedigrees, which provide power for identifying and localizing regions of interest for predisposition genes. A focus on only those prostate cancer cases who are members of high-risk lethal prostate cancer pedigrees, who also went on to die as a result of their prostate cancer, has resulted in the identification of evidence for a prostate cancer predisposition gene on chromosome arm 4q24, confirming a previous GWAS that reported association with prostate-cancer-specific-survival (Pomerantz MM et al., 2011) in this region harboring TET2 (a tumor suppressor gene involved in pathogenesis of acute myeloid leukemia, myelodysplastic syndrome, and myeloproliferative neoplasms), and PP2A (implicated in androgen receptor regulation in prostate cancer cell lines). Citation Format: Lisa A. Cannon-Albright, craig C. TEERLINK, Robert A. Stephenson, Neeraj Agarwal. Significant evidence for linkage for lethal prostate cancer on chromosome arm 4q in Utah high-risk pedigrees. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2015-2746

Assessment of 1P/19Q Deletionby Flourescenceinsitu Hybridization (FISH) in Glioma Patientsfrom Asingle Institute Experience

The discovery of 1p and 19q chromosomal arms deletion in glial tumors influences both more objective diagnosis and more accurate prediction of chemotherapy response. As a result an attempt has been made to detect deletion using fluorescence in-situ hybridization (FISH) and analyzed its prognostic value in a cohort of glial tumor patients from Amrita Institute of Medical Sciences and Research Center Kochi. FISH was performed on 66 FFPE tissue sections by using Vyis LSI 1p36/LSI 1q25 and LSI 19p13/LSI 19q13dual coloured FISH probe sets. Signals were scored from at least 150-250 non-overlapping, intact nuclei. 163 cases were analyzed. Both 1p and 19q deletions was observed only in 28/163 (17.17%), - 1p/+ 19q deletion 80/163 (49.07%) and+1p/-19q deletion 55/163(33.74%) . In this work presented the FISH was successfully applied to identify deletion 1p/19q. Its incidence depends on the type of diagnosed gliomas. In contrast to reported data, the present study reveals 49.07% deletion - 1p/ + 19q. Deletions also have prognostic significance in the test group what constitutes the basis for inclusion of determining deletion 1 p/19q into diagnostic and treatment algorithm.

Concordant chromosome 3 results in paired choroidal melanoma biopsies and subsequent tumour resection specimens

Background/aimThe study's aim was to compare chromosome 3 aberrations of choroidal melanoma (CM) as determined by multiplex ligation dependent probe amplification (MLPA) or microsatellite analysis (MSA) in intraocular tumour biopsies...

Beginning at the end: DNA replication within the telomere.

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.

MYCN overexpression is associated with unbalanced copy number gain, altered nuclear location, and overexpression of chromosome arm 17q genes in neuroblastoma tumors and cell lines.

MYCN amplification and MYCN overexpression are poor prognostic factors in neuroblastoma. Tumors with unbalanced chromosome arm 17q gain are often associated with MYCN amplification; however, the relationship between chromosome 17 copy number status and MYCN expression is not known. We investigated the relationship between MYCN expression and chromosome 17 copy number, nuclear location, and gene expression. By performing dual-colored fluorescence in situ hybridization on 16 primary neuroblastomas, we found that those with unbalanced gain of 17q have high MYCN expression, those with no gain have medium expression, and those with numerical gain have low expression (P < 0.0001). We also found that the nuclear location of 17q correlates with chromosome 17 copy number status: copies in tumors with unbalanced gain and no gain of chromosome 17 occupy a more central location than those in tumors with balanced gain (P < 0.0001). We show that a more central nuclear location of 17q coincides with increased expression of genes found within this chromosome arm. To further understand the association between MYCN expression and chromosome 17, we overexpressed MYCN in two low-expressing MYCN cell lines, SHEP and GIMEN. We found that both cell lines had an unbalanced gain of chromosome 17q, a more central nuclear location of the region and increased expression of the 17q genes. Therefore, this study indicates, for the first time, a functional relationship between MYCN overexpression and the gain of 17q in neuroblastoma.

Cryptic insertion of 3′FOXO1 into inverted chromosome arm 2q in the presence of two normal chromosome 13s and 13 small interstitial duplications in a patient with alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) is a pediatric soft tissue neoplasm with a characteristic translocation, t(2;13)(q35;q14), which is detected in 70-80% of cases. This well-described translocation produces the gene fusion product PAX3-FOXO1. Cryptic rearrangements of this fusion have never before been reported in ARMS. Here we describe a patient with ARMS that showed, by fluorescence in situ hybridization and G-banded chromosomes, a cryptic insertion of 3'FOXO1 into inverted chromosome 2q. The inversion breakpoints were depicted by array comparative genomic hybridization as two small interstitial duplications, one of which involved the PAX3 gene. In addition, the array comparative genomic hybridization results revealed 1q gain, 16q loss, and 11 more small duplications, with one of them involving the FOXO1 gene. Although the pathogenesis in classic ARMS cases is thought to be driven by the 5'PAX3-3'FOXO1 fusion on derivative chromosome 13, here we report a novel cryptic insertion of 3'FOXO1 resulting in a pathogenic fusion with 5'PAX3 on inverted chromosome 2q.

Evidence-Based Diagnostic Algorithm for Glioma: Analysis of the Results of Pathology Panel Review and Molecular Parameters of EORTC 26951 and 26882 Trials.

With the rapid discovery of prognostic and predictive molecular parameters for glioma, the status of histopathology in the diagnostic process should be scrutinized. Our project aimed to construct a diagnostic algorithm for gliomas based on molecular and histologic parameters with independent prognostic values. The pathology slides of 636 patients with gliomas who had been included in EORTC 26951 and 26882 trials were reviewed using virtual microscopy by a panel of six neuropathologists who independently scored 18 histologic features and provided an overall diagnosis. The molecular data for IDH1, 1p/19q loss, EGFR amplification, loss of chromosome 10 and chromosome arm 10q, gain of chromosome 7, and hypermethylation of the promoter of MGMT were available for some of the cases. The slides were divided in discovery (n = 426) and validation sets (n = 210). The diagnostic algorithm resulting from analysis of the discovery set was validated in the latter. In 66% of cases, consensus of overall diagnosis was present. A diagnostic algorithm consisting of two molecular markers and one consensus histologic feature was created by conditional inference tree analysis. The order of prognostic significance was: 1p/19q loss, EGFR amplification, and astrocytic morphology, which resulted in the identification of four diagnostic nodes. Validation of the nodes in the validation set confirmed the prognostic value (P < .001). We succeeded in the creation of a timely diagnostic algorithm for anaplastic glioma based on multivariable analysis of consensus histopathology and molecular parameters.

MB-28 * GENOME-WIDE COPY NUMBER ANALYSIS, NEUROPATHOLOGICAL CLASSIFICATION AND ANALYSIS OF HEDGEHOG ACTIVATION: HISTOLOGY REFLECTS GENETICS AND BIOLOGY IN MEDULLOBLASTOMAS OF YOUNG CHILDREN

The identification of desmoplastic/nodular histology as an independent favourable prognostic marker in medulloblastoma (MB) of infancy has allowed the reduction of therapy in these patients to decrease longterm side-effects. Although we were able to demonstrate different origins and pathway activation of desmoplastic versus non-desmoplastic MB, the genomics and pathway activation has not been exactly related to the different histological MB subtypes. 168 MBs of children under 5 years of age were diagnosed according to the WHO 2007 classification, and FFPE material was analysed in detail by immunohistochemical techniques (including staining of hedgehog targets YAP-1 and p75-NGFR) as well as by molecular inversion profiling (MIP) to assess the genomic landscape of infant MB. GISTIC analysis was performed to identify significant copy number alterations in MB subtypes. MB with extensive nodularity (MBEN, n = 22) and desmoplastic MB (DMB, n = 42) were both characterized by LOH of chromosomal arms 9q or 10q (MBEN, 77% of cases; DMB, 76%), whereas classic MB (CMB, n = 90) and large cell or anaplastic MBs (LCA-MB, n = 14) frequently showed specific gains of chromosomal arm 17q (CMB, 67%; LCA-MB, 79%),partially occurring as isochromosome 17q (CMB, 30%; LCA-MB, 57%), and gains of OTX2 (CMB, 73%; LCA-MB, 79%). Moreover 10/14 (71%) of LCA-MB carried MYCC (n = 9) or MYCN (n = 1) amplifications. The overall frequency of chromosomal copy number aberrations was significantly lower in the desmoplastic/nodular subtypes compared to non-desmoplastic subtypes. Importantly, there was a high correlation between desmoplastic/nodular histology (MBEN and DMB) versus non-desmoplastic subtypes with hedgehog pathway activation shown by the expression of Yap-1 and p75-NGFR (concordance rate, >98%). In summary, histology of infant MB is associated with specific recurrent chromosomal alterations and reflects their biology determined by their cellular origin and developmental pathway activation. Immunohistological assessment of pathway markers and genomic profiling by MIP can help to classify infant MBs.

Molecular classification of diffuse cerebral WHO grade II/III gliomas using genome- and transcriptome-wide profiling improves stratification of prognostically distinct patient groups.

Cerebral gliomas of World Health Organization (WHO) grade II and III represent a major challenge in terms of histological classification and clinical management. Here, we asked whether large-scale genomic and transcriptomic profiling improves the definition of prognostically distinct entities. We performed microarray-based genome- and transcriptome-wide analyses of primary tumor samples from a prospective German Glioma Network cohort of 137 patients with cerebral gliomas, including 61 WHO grade II and 76 WHO grade III tumors. Integrative bioinformatic analyses were employed to define molecular subgroups, which were then related to histology, molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q co-deletion and telomerase reverse transcriptase (TERT) promoter mutations, and patient outcome. Genomic profiling identified five distinct glioma groups, including three IDH1/2 mutant and two IDH1/2 wild-type groups. Expression profiling revealed evidence for eight transcriptionally different groups (five IDH1/2 mutant, three IDH1/2 wild type), which were only partially linked to the genomic groups. Correlation of DNA-based molecular stratification with clinical outcome allowed to define three major prognostic groups with characteristic genomic aberrations. The best prognosis was found in patients with IDH1/2 mutant and 1p/19q co-deleted tumors. Patients with IDH1/2 wild-type gliomas and glioblastoma-like genomic alterations, including gain on chromosome arm 7q (+7q), loss on chromosome arm 10q (-10q), TERT promoter mutation and oncogene amplification, displayed the worst outcome. Intermediate survival was seen in patients with IDH1/2 mutant, but 1p/19q intact, mostly astrocytic gliomas, and in patients with IDH1/2 wild-type gliomas lacking the +7q/-10q genotype and TERT promoter mutation. This molecular subgrouping stratified patients into prognostically distinct groups better than histological classification. Addition of gene expression data to this genomic classifier did not further improve prognostic stratification. In summary, DNA-based molecular profiling of WHO grade II and III gliomas distinguishes biologically distinct tumor groups and provides prognostically relevant information beyond histological classification as well as IDH1/2 mutation and 1p/19q co-deletion status.

Examination of genomic copy number alterations in renal cell carcinoma with sarcomatoid features.

478 Background: Sarcomatoid differentiation is an uncommon histological finding in renal cell carcinoma (RCC) that may develop from any RCC subtype and is associated with a very poor prognosis. The identification of genetic alterations that drive this aggressive phenotype could aid in the development of more effective targeted therapies. In this study, we aimed to identify unique copy number alterations (CNAs) in patients with sarcomatoid RCC when compared to those with other RCC subtypes. Methods: Genomic copy number analysis was performed using single nucleotide polymorphism (SNP)-based microarrays on tissue extracted from the tumors of 80 patients (9 with sarcomatoid features (sRCC), 39 clear cell (ccRCC), 26 papillary (pRCC) and 6 chromophobe RCC (chRCC)) who underwent renal mass excision between 2010 - 2014. Statistical analysis was performed using Kaplan Meier (KM) survival analysis, t-tests and Fisher exact tests where appropriate. Results: sRCC tumors exhibited significantly higher numbers of CNAs when compared to ccRCC, pRCC and chRCC (mean 20.1 vs. 6.6 vs. 7.0 vs. 6.3, respectively; p &lt;0.0001). The most common copy number losses occurred in chromosome arms 1p, 3p, 9q, 15q, 18q, 21q, and 22q, with losses of 9q (88%), 15q (77%), 18q (66%), and 22 (77%) being unique among sRCC tumors when compared to the other 3 histologies. The most common copy number gains were in chromosome arms 1q, 8q, 17q, and 20p/q, with 1q (55%) and 8q (66%) gains unique when compared to the other 3 histologies. Of the sRCC tumors, 3 arose from ccRCC, 2 from pRCC and 4 from unclassified RCC. sRCC was associated with worse survival compared to ccRCC, pRCC and chRCC on KM analysis (p=0.0006), and higher rates of lymph node positivity (77% vs. 3% vs. 12% vs. 0%, respectively; p&lt;0.0001) and metastases (100% vs. 13% vs. 4% vs. 0%, respectively; p&lt;0.0001) on presentation were observed with sRCC. Conclusions: Sarcomatoid differentiation in RCC is associated with a high rate of chromosomal changes with unique copy number alterations including losses of 9q, 15q, 18q and 22q and gains of 1q and 8q. Identification and validation of candidate driver genes or tumor suppressor loci within these chromosomal regions may help identify targets for future therapies.

A population-based single nucleotide polymorphism array analysis of genomic aberrations in younger adult acute lymphoblastic leukemia patients.

Adult acute lymphoblastic leukemia (ALL) is characterized by a high frequency of abnormal karyotypes some of which are related to outcome. Single nucleotide polymorphism (SNP) array analysis provides a highly sensitive platform to detect large and small genomic aberrations. SNP array profiling data in adult ALL are limited and further systematic studies of this patient group are needed. We performed a population-based SNP array analysis of genomic aberrations and their influence on survival in 66 Lithuanian 18-65 year old ALL patients diagnosed between 2007 and 2013. Most aberrations were detected in chromosome arm 9p, chromosome arm 6q, chromosome arm 13q, and chromosome 17. The recurrently targeted copy number abnormalities involved several leukemia-related genes-CDKN2A/B, MLL, IKZF1, PAX5, RB1, TP53, and ETV6. We identified several new recurrent aberrations with possible new target genes: SMARCA4 in 19p13.2, RNASEL in 1q25.3, ARHGEF12 in 11q23.3, and LYL1 in 19p13.2. Aberrations in chromosome 13 and the RB1 gene as well as CDKN2A/B gene status were related to the outcome.

Quantitative multigene FISH on breast carcinomas identifies der(1;16)(q10;p10) as an early event in luminal A tumors.

In situ detection of genomic alterations in cancer provides information at the single cell level, making it possible to investigate genomic changes in cells in a tissue context. Such topological information is important when studying intratumor heterogeneity as well as alterations related to different steps in tumor progression. We developed a quantitative multigene fluorescence in situ hybridization (QM FISH) method to detect multiple genomic regions in single cells in complex tissues. As a “proof of principle” we applied the method to breast cancer samples to identify partners in whole arm (WA) translocations. WA gain of chromosome arm 1q and loss of chromosome arm 16q are among the most frequent genomic events in breast cancer. By designing five specific FISH probes based on breakpoint information from comparative genomic hybridization array (aCGH) profiles, we visualized chromosomal translocations in clinical samples at the single cell level. By analyzing aCGH data from 295 patients with breast carcinoma with known molecular subtype, we found concurrent WA gain of 1q and loss of 16q to be more frequent in luminal A tumors compared to other molecular subtypes. QM FISH applied to a subset of samples (n = 26) identified a derivative chromosome der(1;16)(q10;p10), a result of a centromere-close translocation between chromosome arms 1q and 16p. In addition, we observed that the distribution of cells with the translocation varied from sample to sample, some had a homogenous cell population while others displayed intratumor heterogeneity with cell-to-cell variation. Finally, for one tumor with both preinvasive and invasive components, the fraction of cells with translocation was lower and more heterogeneous in the preinvasive tumor cells compared to the cells in the invasive component. © 2014 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

Evidence of an Epigenetic Origin for High-Risk 1q21 Copy Number Aberrations in Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy stratified in part by cytogenetic aberrations including the high-risk copy number alterations (CNAs) of 17p- and +1q21. CNAs of 1q21 are known to occur as a result of jumping translocations of 1q (JT1q12) which increase the copy number (CN) of 1q21, while at the same time causing collateral genomic damage including deletions and amplifications in some receptor chromosomes (RC). We have previously reported the decondensation of the 1q12 region and triradial chromosomes in the bone marrow specimens of patients with aggressive disease, and have speculated that hypomethylation of the pericentromeric region of 1q12 may play a role in the origin of 1q21 CNAs. To test the hypothesis that 1q12 hypomethylation induces CN gains of 1q21, we used the hypomethylating agent 5-azacytidine to treat blood lymphocyte cultures of five myeloma patients with balanced constitutional 1q12 aberrations as well as five normal control subjects. The patients with constitutional aberrations of 1q12 were selected based on their potential to show either novel CN gains of regions translocated distal to 1q12 or CN gains of 1q21 when 1q12 was translocated to a non-homologous chromosome. The constitutional aberrations included two patients with pericentromeric inversions of chromosome 1 inv(1)(p11q12), and one patient each with a balanced translocation of t(1;2)(q12;p11), t(1;2)(q12;q34), or t(1;9)(q12;q11). These particular aberrations should provide a unique model for testing the association between the hypomethylation of 1q12 and the origin of CN gains of 1q21. In vitro blood lymphocyte cultures of patients and controls were treated for 72 hours with 5-azacytidine at a final concentration of 10uM. 5-azacytidine is known to cause site-specific hypomethylation and chromatin decondensation in the 1q12 region. Utilizing FISH probes for 1q12 (satII/III) and 1q21(CKS1B), we analyzed 100 metaphase cells from each patient and each control for CN gains for 1q21 and any novel region translocated distal to 1q12. To fully characterize the induced rearrangements of 1q12, we also applied inverse G-banding and spectral karyotyping in selected cells. Remarkably, all patients with the balanced constitutional 1q12 rearrangements showed CN gains of chromosome regions on the derivative chromosomes distal to the inverted or translocated 1q12 regions. Recurring aberrations of 1q12 included pericentromeric decondensation and triradials of chromosome arms 1q, 1p, and 2p, in addition to localized pulverizations distal to 1q12 of these same arms. Specifically, in both patients with inv(1), whole-arm CN gains of 1p were identified in 2-3% of cells, while in the patient with t(1;2)(q12;p11), whole-arm gains of 2p (MYCN) were identified in 3% of cells. In these same three patients, on the derivative chromosomes where the 1q12 region was separated from 1q21 by an inversion or translocation, no CN gains of the 1q21 occurred. In the patients with 1q12 translocated to a non-homologous chromosome, 1q21 was amplified on the RC in 4% of the cells in the patient with t(1;2)(q12;q34), and in 3% in the patient with t(1;9)(q12;q11). Control subjects showed CN gains of 1q21on both chromosomes 1 in the range of 3-13%. Whole-arm JT1q12s to RC16q were identified in three patients and three controls. This JT1q12 results in a CN gain of 1q21, and a whole-arm CN loss in RC16q, identical to those found in the bone marrow specimens of MM patients. Interestingly, one control showed JT1q12s to two different RCs, 9q and 12q, causing whole-arm deletions in the long arms of both. The key structural aberration responsible for the generation of CN gains of 1q21 and subsequent JT1q12s is the formation of a triradial involving the 1q12 region. The novel finding in this study is the induction of triradials and CN gains for chromosome arms of 1p and 2p, which demonstrates that epigenetic modifications to 1q12 can amplify non-homologous chromosome regions juxtaposed to it. Furthermore, the CN gains induced here occurred only distal to 1q12 on all normal and derivative chromosomes and mimic the triradials and JT1q12s reported in the bone marrow of patients with aggressive disease. These findings demonstrate for the first time that the hypomethylation of the 1q12 region can potentially amplify any genomic region distal to it, and provide evidence for an epigenetic origin of the high-risk 1q21 CNAs found in the progression of MM. DisclosuresNo relevant conflicts of interest to declare.

Analysis for loss of heterozygosity on chromosome arm 13q by STR analysis or SNP sequencing can replace analysis of FLT3-ITD to detect patients with prognostically adverse AML.

We aimed at developing novel assays for Loss of Heterozygosity (LOH) detection on 13q as a substitute for FLT3-ITD analysis to identify acute myeloid leukemia (AML) patients with high risk of shorter survival. To this aim, we first analyzed a selected cohort of 185 patients with (n = 138) or without (n = 47) FLT3-ITD by short tandem repeat (STR) analysis for 13q LOH. In 46 of 138 FLT3-ITD positive cases, a FLT3-ITD/FLT3wt ratio of ≥ 1 was measured indicating LOH. Applying analysis with a combination of five different STR markers, a threshold of an STR allelic ratio of >65% allows the identification of LOH in 40/46 (87%) samples. Survival analysis revealed significantly inferior outcome in patients with LOH as detected by STR analysis (event free survival (EFS): no LOH: 13.3 months versus LOH: 4.5 months, p < 0.001; overall survival (OS): no LOH: 35.8 months versus LOH: 9.7 months, p = 0.001). In multivariate Cox regression analysis, 13q LOH was found to be an independent adverse parameter for EFS and OS (p = 0.003 and p < 0.001, respectively) besides age and white blood cell count. Thus, the prognostic impact of 13q LOH is nearly identical to the one of FLT3-ITD/FLT3wt load of ≥ 1 and thus is a feasible alternative to identify respective patients with high risk AML. In a second approach, a cohort of 91 patients was subjected to a proof-of-principle study of applying single nucleotide polymorphism analysis by next generation amplicon sequencing for detection of LOH. 53/91 cases had LOH and 50 were identified with this new method resulting in a positive detection rate of 94.3%.

P06.02 * ACTIVATION OF MULTIPLE GROWTH FACTOR SIGNALLING PATHWAYS IS FREQUENT IN MENINGIOMAS

Meningiomas are the most common type of primary intracranial tumour, which in a significant minority of cases are either histologically aggressive or surgically unresectable. Treatment of these latter tumours can be difficult, and currently there are very limited chemotherapeutic options available. We have investigated the expression of a number of growth factors, their receptors incl. activated forms and signalling pathways in 30 meningiomas and control tissue using immunohistochemical markers, and correlated expression with presumed NF2 gene status, by using fluorescent in situ hybridisation to look for chromosome 22q loss. The 8 meningiomas which showed evidence of loss from chromosome arm 22q by in situ hybridisation, had very similar patterns of expression to the remaining tumours. Our findings suggest that the majority of meningiomas show activation of multiple growth factor receptor pathways, irrespective of chromosome 22q loss, and may be susceptible to specific targeted therapeutic options directed against these pathways.