Anna V. Roschke

Secondary genomic rearrangements involving immunoglobulin or MYC loci show similar prevalences in hyperdiploid and nonhyperdiploid myeloma tumors.

The pathogenesis of multiple myeloma (MM) is thought to involve at least two pathways, which generate hyperdiploid (HRD) or nonhyperdiploid (NHRD) tumors, respectively. Apart from chromosome content, the two pathways are distinguished by five primary immunoglobulin heavy chain (IGH) rearrangements (4p16, FGFR3, and MMSET; 6p21, CCND3; 11q13, CCND1; 16q23, MAF; 20q12, MAFB) that are present mainly in NHRD tumors. To determine the prevalence and structures of IGH, immunoglobulin (IG) light chain, and MYC genomic rearrangements in MM, we have done comprehensive metaphase fluorescent in situ hybridization analyses on 48 advanced MM tumors and 47 MM cell lines. As expected, the prevalence of the five primary IGH rearrangements was nearly 70% in NHRD tumors, but only 12% in HRD tumors. However, IGH rearrangements not involving one of the five primary partners, and IG light chain rearrangements, have a similar prevalence in HRD and NHRD tumors. In addition, MYC rearrangements, which are thought to be late progression events that sometimes do not involve an IG heavy or light chain locus, also have a similar prevalence in HRD and NHRD tumors. In contrast to the primary IGH rearrangements, which usually are simple balanced translocations, these other IG rearrangements usually have complex structures, as previously described for MYC rearrangements in MM. We conclude that IG light chain and MYC rearrangements, as well as secondary IGH rearrangements, make similar contributions to the progression of both HRD and NHRD MM tumors. Published 2008 Wiley‐Liss, Inc.

Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations.

Genes that play roles in malignant transformation have often been found proximate to cancer‐associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide‐bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Genes Chromosomes Cancer 27:418–423, 2000. Published 2000 Wiley‐Liss, Inc.

Targeting cancer cells by exploiting karyotypic complexity and chromosomal instability.

Multiple karyotypic abnormalities and chromosomal instability are particular hallmarks of many cancers that are relatively resistant to long term control by current chemotherapeutic agents. We have asked whether these same hallmarks, karyotypic complexity and instability, can be used as determinants for the screening of potential anticancer compounds. Using a panel of well characterized cancer cell lines we have been able to identify specific groups of chemical compounds that are more cytotoxic toward the relatively more karyotypically complex and unstable panel members. Thus, we delineate an approach for the identification of “lead compounds” for anticancer drug discovery complementary to approaches that are focused at the outset on a given gene or pathway.

Multi-Layered Cancer Chromosomal Instability Phenotype

Whole-chromosomal instability (W-CIN) – unequal chromosome distribution during cell division – is a characteristic feature of a majority of cancer cells distinguishing them from their normal counterparts. The precise molecular mechanisms that may cause mis-segregation of chromosomes in tumor cells just recently became more evident. The consequences of W-CIN are numerous and play a critical role in carcinogenesis. W-CIN mediates evolution of cancer cell population under selective pressure and can facilitate the accumulation of genetic changes that promote malignancy. It has both tumor-promoting and tumor-suppressive effects, and their balance could be beneficial or detrimental for carcinogenesis. The characterization of W-CIN as a complex multi-layered adaptive phenotype highlights the intra- and extracellular adaptations to the consequences of genome reshuffling. It also provides a framework for targeting aggressive chromosomally unstable cancers.

Drugs aimed at targeting characteristic karyotypic phenotypes of cancer cells

The karyotypic features of cancer cells have not been a particular focus of anticancer drug targeting either as guidance for treatment or as specific drug targets themselves. Cancer cell lines typically have considerable, characteristic, and variable chromosomal aberrations. Here, we consider small-molecule screening data across the National Cancer Institutes 60 tumor cell line drug screening panel (NCI-60) analyzed for specific association with karyotypic variables (numerical and structural complexity and heterogeneity) determined for these same cell lines. This analysis is carried out with the aid of a self-organizing map allowing for a simultaneous assessment of all screened compounds, revealing an association between karyotypic variables and a unique part of the cytotoxic response space. Thirteen groups of compounds based on related specific chemical structural motifs are identified as possible leads for anticancer drug discovery. These compounds form distinct groups of molecules associated with relatively unexplored regions of the NCI-60 self-organizing map where anticancer agents currently standard in the clinic are not present. We suggest that compounds identified in this study may represent new classes of potential anticancer agents.

Integrated high-resolution array CGH and SKY analysis of homozygous deletions and other genomic alterations present in malignant mesothelioma cell lines

High-resolution oligonucleotide array comparative genomic hybridization (aCGH) and spectral karyotyping (SKY) were applied to a panel of malignant mesothelioma (MMt) cell lines. SKY has not been applied to MMt before, and complete karyotypes are reported based on the integration of SKY and aCGH results. A whole genome search for homozygous deletions (HDs) produced the largest set of recurrent and non-recurrent HDs for MMt (52 recurrent HDs in 10 genomic regions; 36 non-recurrent HDs). For the first time, LINGO2, RBFOX1/A2BP1, RPL29, DUSP7, and CCSER1/FAM190A were found to be homozygously deleted in MMt, and some of these genes could be new tumor suppressor genes for MMt. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements that led to the formation of HDs. Our data imply that only with acquisition of structural and/or numerical karyotypic instability can MMt cells attain a complete loss of tumor suppressor genes located in 9p21.3, which is the most frequently homozygously deleted region. Tetraploidization is a late event in the karyotypic progression of MMt cells, after HDs in the 9p21.3 region have already been acquired.

Complex IGH rearrangements in multiple myeloma: Frequent detection discrepancies among three different probe sets.

Primary IGH translocations involving seven recurrent partner loci and oncogenes are present in about 40% of multiple myeloma tumors. Secondary IGH rearrangements, which occur in a smaller fraction of tumors, usually are complex structures, including insertions or translocations that can involve three chromosomes, and often with involvement of MYC. The main approach to detect IGH rearrangements is interphase—but sometimes metaphase—FISH strategies that use a telomeric variable region probe and a centromeric constant region/ Eα enhancer or 3′ flanking probe to detect a separation of these two probes, or a fusion of these probes with probes located at nonrandom partner sites in the genome. We analyzed 18 myeloma cell lines for detection discrepancies among Vysis, Cytocell, and in‐house IGH probe sets that hybridize with differing sequences in the IGH locus. There were no detection discrepancies for the three telomeric IGH probes, or for unrearranged IGH loci or primary IGH translocations using the centromeric IGH probes. However, the majority of complex IGH rearrangements had detection discrepancies among the three centromeric IGH probes.

Novel near-diploid ovarian cancer cell line derived from a highly aneuploid metastatic ovarian tumor

A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a 47-year-old Ashkenazi Jewish patient three years after the first surgery removed the primary tumor, both ovaries, and the remaining reproductive organs. OVDM1 was characterized by cell morphology, genotyping, tumorigenic assay, mycoplasma testing, spectral karyotyping (SKY), and molecular profiling of the whole genome by aCGH and gene expression microarray. Targeted sequencing of a panel of cancer-related genes was also performed. Hierarchical clustering of gene expression data clearly confirmed the ovarian origin of the cell line. OVDM1 has a near-diploid karyotype with a low-level aneuploidy, but samples of the original metastatic tumor were grossly aneuploid. A number of single nucleotide variations (SNVs)/mutations were detected in OVDM1 and the metastatic tumor samples. Some of them were cancer-related according to COSMIC and HGMD databases (no founder mutations in BRCA1 and BRCA2 have been found). A large number of focal copy number alterations (FCNAs) were detected, including homozygous deletions (HDs) targeting WWOX and GATA4. Progression of OVDM1 from early to late passages was accompanied by preservation of the near-diploid status, acquisition of only few additional large chromosomal rearrangements and more than 100 new small FCNAs. Most of newly acquired FCNAs seem to be related to localized but massive DNA fragmentation (chromothripsis-like rearrangements). Newly developed near-diploid OVDM1 cell line offers an opportunity to evaluate tumorigenesis pathways/events in a minor clone of metastatic ovarian adenocarcinoma as well as mechanisms of chromothripsis.

Frequent occurrence of large duplications at reciprocal genomic rearrangement breakpoints in multiple myeloma and other tumors

Using a combination of array comparative genomic hybridization, mate pair and cloned sequences, and FISH analyses, we have identified in multiple myeloma cell lines and tumors a novel and recurrent type of genomic rearrangement, i.e. interchromosomal rearrangements (translocations or insertions) and intrachromosomal inversions that contain long (1–4000 kb; median ∼100 kb) identical sequences adjacent to both reciprocal breakpoint junctions. These duplicated sequences were generated from sequences immediately adjacent to the breakpoint from at least one—but sometimes both—chromosomal donor site(s). Tandem duplications had a similar size distribution suggesting the possibility of a shared mechanism for generating duplicated sequences at breakpoints. Although about 25% of apparent secondary rearrangements contained these duplications, primary IGH translocations rarely, if ever, had large duplications at breakpoint junctions. Significantly, these duplications often contain super-enhancers and/or oncogenes (e.g. MYC) that are dysregulated by rearrangements during tumor progression. We also found that long identical sequences often were identified at both reciprocal breakpoint junctions in six of eight other tumor types. Finally, we have been unable to find reports of similar kinds of rearrangements in wild-type or mutant prokaryotes or lower eukaryotes such as yeast.

Abstract 4208: Integrated oligo aCGH and SKY analysis of genomic alterations in malignant mesothelioma cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Malignant mesothelioma (MMt) is a rare and very aggressive tumor of the mesothelium. Its most prevalent subtype is malignant pleural mesothelioma, eighty percent of which are associated with asbestos exposure. MMt incidence is 0.9 new cases per 100,000 persons per year. The five year relative survival is 7% overall, which positions MMt immediately above pancreatic cancer (6%), at the bottom of the survival list by cancer site. We applied a comprehensive molecular cytogenetic analysis using spectral karyotyping (SKY) and oligo aCGH to a panel of MMt cell lines. We found the largest set of recurrent and non-recurrent homozygous deletions (HD) for MMt (88 HDs in 17 cell lines; 52 recurrent HDs that fall into ten genomic areas). Detailed analysis of the most frequently homozygously deleted area located at 9p21.3 revealed that the main targets are CDKN2A/p14ARF (in 100% of the MM cell lines), CDKN2A/p16INK4A and CDKN2B /p15 (94% of cell lines). p14ARF seems to be the main target of HDs in the 9p21.3 area (deleted in all 17 MMt cell lines studied), followed by p16INK4A (deleted in 16 cell lines) indicating the inactivation of two major tumor suppressing pathways, p53 and Rb. Accordingly, mutations or HDs of p53 and Rb are infrequent events in MMt tumors. Other recurrent HDs were located at 9p21.2 (targeting LINGO2) and at16p13.3 (RBFOX1) in 41% of cell lines, 22q11.23 (GSTT1) in 29%, 22q12.2 (NF2) and 3q26 (no known genes) in 23% each, 8p11.22 -23 (ADAM5P/ADAM3A) in 18%, 3p21.3-p21.2 (RPL29), 3p21 (DUSP7), 4q22.1 (FAM190A) and 13q11-q12 (LATS2) in 12% each of the 17 MM cell lines While p16INK4A, p14ARF, p15, LATS2 and NF2 have been shown to behave as tumor suppressors in MMts, other genes in the HD areas presented here could be new tumor suppressor candidates. Structural chromosomal rearrangements were mainly non-reciprocal translocations (74% of aberrations detected by SKY) and deletions (23%). No recurrent balanced or unbalanced translocations have been found. Structural chromosomal rearrangements resulted in recurrent losses of fragments or whole chromosomal arms of 1p, 3p, 8p, 9p, 13q, 14q, 15q, 18q, 22q and gains of 1q, 5p, 7p, 8q, 17q, 20q. Small HDs were frequently embedded in the areas of focal losses. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements leading to the formation of HDs. Our data imply that only with acquisition of structural or numerical karyotypic abnormalities can MMt cells attain a complete loss of the 9p21.3 genomic area and loss of tumor suppressor genes located there. Tetraploidization seems to be a late event in the karyotypic progression of MMt cells, after HD in the 9p21.3 area has already been acquired. Citation Format: Ester Rozenblum, Geula Klorin, Oleg Glebov, Robert L. Walker, Yoonsoo Park, Paul S. Meltzer, Ilan R. Kirsch, Frederic J. Kaye, Anna V. Roschke. Integrated oligo aCGH and SKY analysis of genomic alterations in malignant mesothelioma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4208. doi:10.1158/1538-7445.AM2013-4208