Clonal Chromosome Rearrangements Research Articles

TMOD-01. EGFR AMPLIFICATION ACCOMPANIES ADAPTATION TO MITOGEN-INDUCED DEFECTIVE MITOSIS IN PDGFA

Abstract Using a newly described in vitro murine model of GBM [Bohm et al., Neuro-Oncol 2020 and Omairi et al., Neuro-Oncol 2023] in which P53 null neural progenitor cells (NPCs) divide abnormally and evolve a GBM-like genome during exposure to Platelet-Derived Growth Factor-AA (PDGFA), we asked how a brain-abundant mitogen could transform NPCs. By analyzing gene and protein expression over time, we found that PDGFA fails to induce the transcription of kinetochore and spindle assembly checkpoint genes, while simultaneously driving NPCs to enter mitosis. These dual effects caused chromosome miss-segregation in continuously dividing NPCs; moreover, they occurred in both WT and null NPCs, although only null cells survived defective mitosis. These surviving cells gradually expanded in PDGFA accumulating both random and clonal chromosomal re-arrangements. Transcriptome analysis of NPCs in PDGFA revealed significant under-expression of Foxm1, the major regulator of kinetochore transcription, together with over-expression and phosphorylation of the immediate early response gene, FOS. Analysis of signalling downstream of PDGFRα identified the Ras-MAPK pathway, especially ERK, as responsible for FOS activation. As surviving null cells gradually expanded, they accumulated random and recurrent chromosomal rearrangements. Expansion and subsequent PDGFA-independent proliferation and tumorigenicity were associated with re-expression of Foxm1 and kinetochore proteins, and accompanied by over-expression of Egfr, an RTK-signature that defines human GBM. By stimulating proliferation without setting the stage for error-free mitosis, exposure to PDGFA transforms p53 null NPCs and generates Egfr amplified GBM-like cancer cells.

The Derivation and Comparative Characterization of Mesenchymal Stem Cell Lines, Isolated from Human Pulp of a Deciduous Tooth of Children of Different Sexes

Two new non-immortalized fibroblast-like cell lines isolated from the pulp of Deciduous Tooth of Children of Different Sexes, named MSC-DP-1 and MSC-DP-2, were derived and characterized. In order to confirm the status of mesenchymal stem cells, a comparative analysis of a some characteristics in these lines was carried out at early and late passages. In the process of long-term cultivation, significant interline differences were revealed in the nature of replicative senescence (RS) and in growth characteristics. The MSC-DP-1 line was characterized by a later entry into the active stage of RS and more active proliferation compared to the MSC-DP-2 line. Karyotypic analysis showed that both lines have a normal human diploid karyotype at early passages. At the late (18th passage), in the stage of active PS, the MSC-DP-2 line also has a normal karyotype. And the MSC-DP-1 line, which enters the active stage of RS much later (at passage 42), has an abnormal karyotype with a large number of clonal chromosomal rearrangements. Both lines showed a high proportion of cells carrying antigens characteristic of human MSCs: CD44, CD73, CD90, CD105, HLA-ABC, and a low frequency of cells with CD34, CD45, and HLA-DR antigens. The cells of the derived lines at an early passage have the ability to differentiate in adipogenic, osteogenic and chondrogenic directions. But the MSC-DP-2 line shows weaker differentiation in the adipogenic direction, than the MSC-DP-1 line. In the process of RS, a significant weakening of adipogenic differentiation takes place in the MSC-DP-1 line, and it disappears in the MSC-DP-2 line. The activity of other differentiations does not change during RS. In general, the derived results confirm the status of MSCs for the derived lines and indicate interline differences in the RS process. However, the derived differences between these lines, as well as the comparison with the previously derived line MSC-DP do not indicate their gender nature. Apparently, they are associated with the genetic characteristics of different donors.

Mitogen-induced defective mitosis transforms neural progenitor cells.

Chromosome instability (CIN) with recurrent copy number alterations is a feature of many solid tumors, including glioblastoma (GBM), yet the genes that regulate cell division are rarely mutated in cancers. Here, we show that the brain-abundant mitogen, platelet-derived growth factor-A (PDGFA) fails to induce the expression of kinetochore and spindle assembly checkpoint genes leading to defective mitosis in neural progenitor cells (NPCs). Using a recently reported in vitro model of the initiation of high-grade gliomas from murine NPCs, we investigated the immediate effects of PDGFA exposure on the nuclear and mitotic phenotypes and patterns of gene and protein expression in NPCs, a putative GBM cell of origin. NPCs divided abnormally in defined media containing PDGFA with P53-dependent effects. In wild-type cells, defective mitosis was associated with P53 activation and cell death, but in some null cells, defective mitosis was tolerated. Surviving cells had unstable genomes and proliferated in the presence of PDGFA accumulating random and clonal chromosomal rearrangements. The outcome of this process was a population of tumorigenic NPCs with recurrent gains and losses of chromosomal regions that were syntenic to those recurrently gained and lost in human GBM. By stimulating proliferation without setting the stage for successful mitosis, PDGFA-transformed NPCs lacking P53 function. Our work describes a mechanism of transformation of NPCs by a brain-associated mitogen, raising the possibility that the unique genomic architecture of GBM is an adaptation to defective mitosis that ensures the survival of affected cells.

Chromosomal Instability and Evolution of Transformed Phenotype in Cell Lines Selected from Senescent Rat Embryonic Fibroblasts with Rapamycin

The karyotype and phenotype evolution in cell lines (Rapa) obtained by rapamycin selection from rat embryonic fibroblasts (REF) has been studied. Rapamycin, an inhibitor of mTORC1 kinase, prevents replicative aging of REF cells starting after seven to ten passages in vitro. The karyotype of the established cell lines undergoes an evolution upon their cultivation in vitro. On early passages, clonal chromosome rearrangements, more specifically trisomy of chromosome 7 and translocations t(2;7) or t(4;11), were observed. Chromosome rearrangements (CR) accumulated at late passages. Simultaneously, signs of the transformed phenotype appeared: capacity for clonal growth, a decrease in contact inhibition, and high proliferation rate in the monolayer at saturating density. The evolution of the transformed phenotype was accompanied by the loss of G1/S and/or G2/M checkpoint control. Rapa lines were characterized by accumulation of γH2AX and p53BP foci, which, however, did not colocalize. Thus indicates the accumulation of unrepaired double-strand DNA breaks (DSB). The Rapa lines are shown to express the pluripotency markers Nanog and Oct3/4. The causes of genetic and karyotype instability of Rapa lines during long-term cultivation have been discussed.

Structural and copy number chromosome abnormalities in canine cutaneous mast cell tumours

Mast cell tumours (MCTs) are the most common skin tumours in dogs. Their clinical behaviour is variable and their aetiology remains largely unknown. We performed a metaphase fluorescence in situ hybridisation (FISH) with whole chromosome painting probes, and interphase FISH with BAC probes for 14 cancer-related genes to reveal clonal structural chromosome rearrangements and copy number variants (CNVs) in canine cutaneous MCTs. The metaphase FISH performed in three MCTs revealed several clonal monosomies and trisomies and two different chromosome rearrangements. No centric fusions were detected. The interphase FISH showed a variety of low frequency CNVs for the individual cancer-related genes. The heterogeneous character of the detected abnormalities indicates increased chromosome instability in canine MCTs. The clonal gain of chromosome 11 was detected in 81% (13/16) of the MCTs. Further research is needed to evaluate the significance of this abnormality as prognostic factor for the survival time or recurrence risk assessments in canine cutaneous MCTs.

The Dynamics of Cell Properties during Long-Term Cultivation of Two Lines of Mesenchymal Stem Cells Derived from Wharton’s Jelly of Human Umbilical Cord

The cell properties of two MSC lines (MSCWJ-1 and MSCWJ-2) established from Wharton’s jelly of human umbilical cord were analyzed at the 6th, 13th, and 28th passages. The following were discovered. (1) The cell morphology during long-term cultivation was changed in both lines, with the cells being larger and having more debris in the MSCWJ-2 line than in the MSCWJ-1 line. (2) As shown by analysis of growth characteristics, long-term cultivation was accompanied with a significant increase of the average population-doubling time in both cell lines. This was correlated with morphological changes and a gradual increase in β-galactosidase activity, indicating the occurrence of replicative senescence. The interline changes concerned the level of proliferative activity, the index of proliferation, the time of the entry into the phase of replicative senescence (30 population doublings in the MSCWJ-2 line and 60 population doublings in the MSCWJ-1 line), and the percentage of senescent cells (80% in MSCWJ-2 line compared to 58% in the MSCWJ-1 line). (3) Numerical karyotypic analysis showed that both cell lines at early and late passages had normal karyotype 46, XX (MSCWJ-1 cells), and 46, XY (MSCWJ-2 cells). Structural karyotypic analysis showed a cytogenetic heterogeneity in both lines: low frequency of random clonal and nonclonal chromosomal rearrangements disappeared during long-term cultivation. Unlike in the case of the MSCWJ-1 line, the clonal chromosomal rearrangement of the short arm of chromosome 7 with a frequency of 39% was detected at the sixth passage in the MSCWJ-2 line but disappeared at the 13th passage. (4) Surface marker analysis of these cell lines at the 6th, 13th, and 28th passages revealed that these cells expressed surface antigens characteristic of human MSC (CD44, CD73, CD90, CD105, HLA-ABC), as well as vimentin, and did not express CD34 and HLA-DR. (5) Analysis of nondifferentiated hESC markers in both lines showed a decrease in the level of expression of the surface marker SSEA-4 and a lack of expression of the transcription factor SOX-2 at the 13th passage in the MSCWJ-2 and at the 28th passage in MSCWJ-1 cells. (6) Cell adhesion in both lines was lower in late passages compared to the cells at the sixth passage. The cells retained an osteogenic and adipogenic differentiation potential at late passages. A lack of chondrogenic differentiation in MSCWJ-2 cells, but not in MSCWJ-1 cells, was registered at late passages. The MSCWJ-2 cell line exhibited a frequently occurring clonal chromosomal rearrangement at an early passage, early replicative senescence with a large proportion of senescent cells, a significantly decreased index of proliferation during long-term cultivation, a decrease or disappearance of the expression of nondifferentiated ESCs of markers, and decreased differentiation potential. Given the metabolic cooperation between the cells, we can cautiously suppose that the clonal chromosomal rearrangement of the short arm of chromosome 7 detected at an early passage with high frequency is coupled with activation of cellular processes that proceed in the absence of this chromosomal aberration.

Bone Marrow Stromal Cells May Have Genetic Aberrations and Are Capable to Gain Them in a Culture

Introduction. Stromal microenvironment posses a key role in the regulation of both normal hematopoiesis and its reconstitution after hematopoietic stem cell transplantation (HSCT). Recent data supports the idea that bone marrow stromal cells (BMSC) also have genetic aberrations and may tightly involved in the pathogenesis of HSCT complications. These findings justify the need for more detailed study of genetic aberrations in BMSC.The aim of this study was to evaluate genetic aberrations inBMSC and check the ability to gain them in coculture system.Materials and methods. The interaction of BMSC with hematopoietic tumor cell lines bearing specific genetic aberrations (BCR-ABL fusion transcript for K-562 and JAK2 V617F mutation for Uke-1 cell line) was investigated in stromal cells harvested from 17 patients and 8 healthy donors. We performed cultivation of BMSC monolayer and tumor cells suspension using semipermeable membrane plates inserts with different pore size (0,4 μm and 3,0 μm) in order to exclude direct cell-to-cell contact. We looked also forexisting specific genetic aberrations (point mutations and fusion transcripts) in BMSC of patients with the respective aberration in their leukemic clone. For this purpose we used both karyotyping (7 patients) and RQ-PCR method. BMSC were examined by flow cytometry to evaluate the possible contamination with cells of hematopoietic lineage.Results.We investigated the BMSC karyotype in seven patients and only one case led us to a remarkable finding. The clonal chromosomal rearrangement t(1;7) was detected in 25% of BMSC metaphases. Interestingly, this aberration was never detected in patient's leukemic cells. Moreover, at the moment of this investigation patient had full clinical and hematological remission, full donor chimerism and no signs of minimal residual disease (MRD).We also examined BMSC from leukemia patients bearing recurrent genetic abnormalities and in one case the leukemia-specific marker was detected by RQ-PCR - we observed expression of ETV6-RUNX1 gene (≈0,02%) in BMSC by patient with t(12;21) acute lymphoblastic leukemia. At the moment of BMSC culture initiation ETV6-RUNX1 expression in patient’s bone marrow was detected at high level (ETV6-RUNX1/ABL*100=321%). Before carrying out RNA extraction BMSC were harvested after the second passage and no contamination with CD45+/CD34+ cells by flow cytometry was observed (50,000 events collected from the sample). When BMSCs and Uke-1 cell line were cocultured by using of 3,0 μm pore culture dishes inserts, the BMSC population gained the Jak2V617F mutation (allele burden ≈ 30,39%). We reproduced similar experiments with the K-562 cell line and got similar results - CD45+ cells were also detected in BMSC population (≈ 30%). Moreover we detected CD45+ non-cellular particles by flow cytometry analysis. Implying K-562 cells are not likely to cross the semipermeable membrane (3,0 μm pores versus 20,0 μm cells as measured during microscopy). Besides BCR-ABL gene expression in BMSC was detected by RQ-PCR (BCR-ABL/ABL*100=19%). We repeated same test with 0,4 μm pore inserts and without them in order to check implication of cell-to-cell interaction. We didn’t obtain any similar results with smaller pores, but the fusion transcript was detected in CD45- BMSC population when these two cell populations weren’t devided. Both findings point out at possible horizontal gene transfer mediated by membrane vesicles larger than 0,4 μm and direct whole cell fusion.ConclusionOur data stands for the existence of horizontal gene transfer between leukemic clone and BMSC. This process seems to be mediated by membrane vesicles larger than 0,4 μm in size, though cell fusion can also take place. We also confirmed the fact BMSCs can bear clonal genetic rearrangements which are not specific to tumor cell populations. These findings show tight interaction between tumor and microenvironment cells and can partly explain nature of PCR-based MRD persistence in complete remission. DisclosuresNo relevant conflicts of interest to declare.

Chromosomal rearrangements in myoepithelial carcinoma of the breast that presented as metachronic double cancer with invasive ductal carcinoma in the ipsilateral breast

Myoepithelial carcinoma of the breast is an extremely rare tumor composed entirely of malignant spindle cells with myoepithelial differentiation. The majority of previously reported cases have mainly described the clinicopathological features of the disease, and few have presented cytogenetic data. We herein present the case of a 48-year-old woman who was admitted with a left-sided breast lump in the inner upper quadrant that was initially diagnosed as a myoepithelioma with potentially malignant disorder. At 12 months after resection, she complained about a newly developed solid mass in the subareolar region of the ipsilateral breast that was diagnosed as an invasive ductal carcinoma. In addition, 16 months after the initial admission, a re-growing remnant lesion recurred in the inner upper quadrant and was ultimately diagnosed as a myoepithelial carcinoma. Lymph node metastasis of the myoepithelial carcinoma was also observed in her left axillary region 11 months after local recurrence. A cytogenetic analysis showed recurring specific chromosomal alterations both in the locally recurrent and in the lymph-node metastatic lesion: 48, XX, t(5;18)(q13;q23),del(6)(q?),+14. + mar1. To our knowledge, this is the first published report of clonal chromosomal rearrangements in myoepithelial carcinoma of the breast that presented as metachronic double cancer with invasive ductal carcinoma in the ipsilateral breast.

Identification of Candidate Oncogenes and Chromosomal Breakpoint Sequencing By Targeted Locus Amplification in T-Cell Acute Lymphoblastic Leukemia

Background: T-cell acute lymphoblastic leukemia is characterized by clonal and mutual exclusive chromosomal rearrangements that recurrently activate TAL1, LMO2, TLX1, NKX2-1, TLX3, HOXA or MEF2C oncogenes. Most of these translocations or chromosomal rearrangements occur as erroneous D-J or V-DJ rearrangement attempts of T-cell receptor beta (TCRB) or TCR alpha/delta (TCRAD) genes, mostly positioning oncogenes under the transcriptional control of TCR enhancer elements. Alternatively, oncogenes can also be activated as consequence of BCL11B chromosomal rearrangements. Although many oncogenes are known in T-ALL, the driving oncogenic lesion in particular T-ALL cases remains unknown.Aims: In this study, we aimed to clone reciprocal breakpoint sequences to elucidate cellular mechanisms that lead to recurrent BCL11B -TLX3 chromosomal translocations. Moreover, we want to identify oncogene candidates in various T-ALL patient samples with BCL11B-, TCRB- or TCRAD-translocations for which the candidate oncogene so far has not been identified.Methods: We used Targeted Locus Amplification procedure, a recently developed method that relies on the crosslinking of DNA in live cells, DNA digestion and re-ligation to allow formation of circular DNA ligation fragments and inverted polymerase chain reaction amplification from specific view-point loci. Amplified DNA fragments are sequenced by next generation sequencing, allowing sequence identification in a region covering 2MB around selected regions of interest, including TLX3, TLX1, TAL1, LMO2, BCL11B, TCRAD (TRAJ61), TCRB (TCRBC2)Results: TLA was successfully performed on 10 T-ALL patients having FISH validated TAL1 translocations (2 patients), LMO2 translocations (3 patients), TLX3 translocations (3 patients), TLX1 translocations (2 patients) or an inversion targeting NKX2.1 (1 patient). Analysis of both TAL1 translocated cases revealed a TAL1-TCRAD genomic fusion due to a classical t(1;14)(q32;q11) in 1 patient, but surprisingly reveal a TAL1-TCF7 genomic fusion due to a t(1;5)(q32;q31.1) chromosomal translocation in the second patient. For the LMO2 translocated cases, two patients showed classical LMO2-TCRAD (t(11;14)(p13;q11)) or LMO2-TCRB (t(7;11)(q35;p13) translocations, whereas the third patient presented with an unusual LMO2-BCL11B genomic fusion due to a t(11;14)(p13;q32). Two out of 3 TLX3-translocated patients had classical t(5;14)(q35;q32) translocations, whereas the TLX3 gene in the third patient was rearranged to the calcyphosine-like gene (CAPSL, which flanks the IL7Ra gene) on chromosomal 5p13.2 due to a t(5;5)( p13.2;q35) or an inv(5). One patient had an inversion on chromosome 14, i.e. inv(14)(q11;q13), that brings the NKX2.1 oncogene under the transcriptional control of the TCRAD enhancer. Finally, one TLX1-rearranged patient had a classical TLX1-TCRAD translocation, whereas the other presented with a chromosomal inversion involving the chromosomal band 10q24 (which included TLX1), but also revealed a novel translocation involving the centromere protein P gene (CENPP) on chromosome 9q22.31 with the TCRAD locus.Summary/Conclusions: Targeted Locus Amplification identification of chromosomal rearrangements and genomic breakpoint sequences reveals novel complex translocations in 3 out of 10 T-ALL cases analyzed thus far, indicating higher complexity of chromosomal translocations of known T-ALL oncogenes as thus far anticipated. It further proved a useful tool to identify novel translocation partners from various loci such as the TCR or BCL11B genes that are recurrently involved in these chromosomal rearrangements in T-ALL. Cloning of molecular translocation breakpoints of diagnostic T-ALL patient samples may further provide excellent minimal residual disease markers for disease monitoring during the course of treatment. DisclosuresSplinter:Cergentis BV: Employment. van Min:Cergentis BV: Employment.

Cytogenetic Assessment of the Rat Cell Line CLS-ACI-1: An in vitro Cell Model for Mycn Overexpression

Breast cancer is a complex and heterogeneous disease, and the establishment of cell models in order to properly study the disease at the molecular and cellular level is of utmost importance. Here, we present the cytogenetic characterization and gene expression analysis of the tumoral mammary rat cell line CLS-ACI-1. The use of banding and molecular cytogenetic techniques allowed the description of the complex CLS-ACI-1 karyotype and the identification of breakpoints in clonal chromosome rearrangements. Moreover, a Mycn and Erbb2 comparative expression analysis by RT-qPCR was performed, revealing a high expression level of Mycn in CLS-ACI-1 cells. Moreover, a considerable number of putative mutated genes and chromosome alterations detected through cytogenetic analysis seem to be in the MYCN biological network. Therefore, the CLS-ACI-1 cell line is presented as a promising cell model for the study of the role of MYCN in breast cancer and also as a tool for developing appropriate cancer therapies, namely for Mycn targeting.

A Review of the Literature on Primary Leiomyosarcoma of the Prostate Gland

Primary leiomyosarcoma of the prostate (PLSOP) is rare, with less than 200 cases reported so far. PLSOPs present with lower urinary tract symptoms, haematuria, and perineal pain; may or may not be associated with a history of previous treatment for adenocarcinoma of prostate by means of radiotherapy and or hormonal treatment; may afflict children and adult male. Examination may reveal benign enlarged prostate and hard enlarged mass. PLSOPs may be diagnosed by histological examination findings of spindle-shaped carcinoma cells in prostate specimens. Immunohistochemical staining tends to be positive for vimentin, CD44, smooth muscle actin, and calponin, focally positive for desmin, and at times positive for keratin. They stain negatively for PSA, S-100, CD34, CD117, and cytokeratin. Cytogenetic study on primary leiomyosarcoma of the prostate gland may show clonal chromosomal rearrangement involving Chromosomes 2, 3, 9, 11, and 19. On the whole the prognosis is poor. Surgery with or without chemotherapy would appear to be the mainstay of treatment for PLSOPs that are operable, but generally there is no consensus opinion on the best therapeutic approach. Most cases of PLSOPs are diagnosed in an advanced stage of the disease. A global multicenter trial is required to find therapies that would improve the prognosis.

Lipoblastoma of the Hand and Cleft Palate

We report a case of lipoblastoma of the hand in a 19-month-old female patient with a history of cleft palate. The incidence of lipoblastoma and cleft palate individually is extremely rare. To the best of our knowledge, only 1 other case of a patient with both cleft palate and lipoblastoma exists in the literature. Lipoblastoma is a rare benign neoplasm in adipose tissue almost exclusively found in children younger than 3 years. Cytogenetic testing has shown that lipoblastomas characteristically share a clonal chromosomal rearrangement affecting the long arm of chromosome 8. Furthermore, recent research has shown that the 8q chromosome is an important genetic risk factor for cleft palate development. We describe the second case linking cleft palate with this rare tumor and provide evidence for a potential genetic association.

Lymph node hyperplasia: clonal chromosomal and genomic rearrangements. Report of two new cases and literature review

Cytogenetic analysis is not routinely performed on lymph node hyperplasia (LH). We describe clonal chromosomal rearrangements in two unrelated cases of LH. Lymph nodes of both patients showed typical morphologic features of benign follicular hyperplasia. Cytogenetic analysis revealed clonal chromosomal rearrangements in both cases. Patient 1 showed interstitial 14q and 6q mosaic deletions, whereas patient 2 showed a terminal 14q mosaic deletion. Fluorescence in situ hybridization with IGH break-apart probes identified a partial deletion of IGH in both cases, but the loss of the LSI IGH in patient 2 and loss of the LSI IGHV in patient 1 were observed on the morphologically normal chromosome 14. In the latter case, the finding of two morphologically normal chromosomes 14 with the IGHV deletion in one of the chromosomes suggested that the first mutational event was the IGH deletion and the second event was the interstitial deletion of chromosome 14 with the IGH intact. Array comparative genomic hybridization performed on both biopsies confirmed the IGH deletion at mosaic, but not the chromosomal deletion. Patient 1 was re-biopsied after 9 months and a marginal zone lymphoma was diagnosed. The finding of clonal cytogenetic abnormalities in LH highlighted the difficulties in interpretation of results and clinical follow-up.

Defining the Sister Rat Mammary Tumor Cell Lines HH-16 cl.2/1 and HH-16.cl.4 as an In Vitro Cell Model for Erbb2

Cancer cell lines have been shown to be reliable tools in genetic studies of breast cancer, and the characterization of these lines indicates that they are good models for studying the biological mechanisms underlying this disease. Here, we describe the molecular cytogenetic/genetic characterization of two sister rat mammary tumor cell lines, HH-16 cl.2/1 and HH-16.cl.4, for the first time. Molecular cytogenetic analysis using rat and mouse chromosome paint probes and BAC/PAC clones allowed the characterization of clonal chromosome rearrangements; moreover, this strategy assisted in revealing detected breakpoint regions and complex chromosome rearrangements. This comprehensive cytogenetic analysis revealed an increase in the number of copies of the Mycn and Erbb2 genes in the investigated cell lines. To analyze its possible correlation with expression changes, relative RNA expression was assessed by real-time reverse transcription quantitative PCR and RNA FISH. Erbb2 was found to be overexpressed in HH-16.cl.4, but not in the sister cell line HH-16 cl.2/1, even though these lines share the same initial genetic environment. Moreover, the relative expression of Erbb2 decreased after global genome demethylation in the HH-16.cl.4 cell line. As these cell lines are commercially available and have been used in previous studies, the present detailed characterization improves their value as an in vitro cell model. We believe that the development of appropriate in vitro cell models for breast cancer is of crucial importance for revealing the genetic and cellular pathways underlying this neoplasy and for employing them as experimental tools to assist in the generation of new biotherapies.

ESRRA-C11orf20 Is a Recurrent Gene Fusion in Serous Ovarian Carcinoma

Author SummarySerous ovarian cancer, the most common form of ovarian cancer, is especially lethal because it is usually only detected at a late stage in its progression, after the cancer has spread to other tissues. We searched for molecular markers of this cancer that might provide a better way to detect tumors at a curable stage and that might provide targets for new treatments. Chromosomal rearrangements that fuse two genes to produce a recombinant gene that enhances growth or spread of the cancer are particularly specific biomarkers and have been found in many cancers. By “deep” sequencing of the RNA molecules that carry genetic information in serous ovarian cancers, we discovered a rearrangement that fuses the same two neighboring genes in at least 15% of these tumors. The two fused genes are ESRRA, which encodes a key regulator of gene expression, and an essentially uncharacterized gene, C11orf20, that is normally adjacent to the ESRRA gene. Chromosomal rearrangements that recombine parts of two nearby genes or even parts of a single gene may be a common, important feature of the cancer genome that eludes detection by most approaches to characterizing cancer genomes.

Jumping Translocations of the Long Arms of Chromosome 1 (1qJT) in Myeloproliferative Neoplasms (MPNs) and Myelodysplastic Syndromes (MDS) Are Associated with High Risk of Transformation to Acute Myelogenous Leukemia (AML).

Abstract 1567Poster Board I-591Jumping translocations (JT) are rare cytogenetic events where by a part of one chromosome is translocated to several recipient chromosomes creating multiple related clones within a single patient (pt). Over two-thirds of the already ∼70 reported cases with JTs had multiple myeloma (JR Sawyer et al, 2005). The most frequent donor chromosome involved in JT is the long arm of chromosome 1 (+1q). Although abnormalities of 1q are the 4th most common recurrent rearrangements in both MPN and MDS, 1qJT have been rarely reported in these disorders. Their role in the pathogenesis of MPN and MDS remains unknown. We report a study of 23 pts with myeloid malignancies to determine the role of 1qJT in MPN and MDS. Patient characteristics are shown in Table 1. Of 512 MPN pts (PV=361, PMF=151) cytogenetically evaluated at our institution, we identified 3 pts (PV=2, PMF=1) at diagnosis with 1qJT (0.6% incidence). Additionally 4 of 169 (PV=96, PMF=69) pts with MPN acquired 1qJT during the serial follow up studies over a period of 11 years (the overall incidence was 1.3%, and 4.2% in pts with cytogenetically abnormal karyotypes). [Display omitted] The median follow up time was 78 months (mos) and the average time to acquire 1qJT was 58 mos. usually only one or two cells with +1q were initially identified but over time the number of partner chromosomes increased. In 4 pts who acquired 1qJT, this genetic event occurred an average after 5 years while in 1 pt it occurred after 11 years. However, once 1qJT was acquired, 3 of 4 pts developed AML, within an average time of 8 months. The transformation to AML was associated with an increased 1q translocated copy number, suggesting that 1qJT may be a marker of disease progression. By contrast, the incidence of 1qJT at diagnosis of MDS was 0.04% (4 of 1,000). Each of 4 pts, who presented with 1qJTs, had up to three different related clonal populations, all characterized by +1q translocated to 3 different chromosomes. The presence of 1qJT at diagnosis was associated with transformation to AML (3 of 4 pts) after less than 4 mos. Additionally of the 300 pts who were sequentially studied for over a period of 34 mos, 12 pts acquired 1qJT within 27 months (the overall incidence 4%, or 8% among the cytogenetically abnormal). Once 1qJT was acquired, transformation to AML occurred after 8 months (range 0-32) in 10 of 12 pts (83%). This was accompanied with an increased number of JT partner chromosomes. Overall, trisomy 1q, was “jumping” to as many as 11 different chromosomes, creating 11 related clonal populations. All chromosomes except 2, 10 and 12, were recipients of 1qJT, with a greater frequency affecting acrocentric chromosomes (55%). More importantly, 81% of the breakpoints in recipient chromosomes were in the pericentromeric regions. This study represents the largest series of pts with MPN and MDS characterized by 1qJT. The presence of 1qJT at diagnosis or the subsequent acquisition of 1qJT was associated with transformation to AML in 86% of MPN cases and 83% of MDS cases after an average of 8 months. The prognosis of pts with 1qJT tends to be dependent on the translocated 1q copy number: the higher the number of “jumping” 1q being translocated to different partner chromosomes the shorter the time to transformation to AML. These data strongly indicate that 1qJT in MPN and MDS patients represents clonal chromosomal rearrangements associated with a high risk of imminent transformation to AML. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic analysis of a primary bone angiosarcoma

Primary bone angiosarcomas are rare and aggressive vascular malignancies with a high mortality rate. To our knowledge, there are no reported cytogenetic abnormalities in primary bone angiosarcomas, although several have been reported in soft tissue angiosarcomas. We report a case of primary bone angiosarcoma, arising in the tibia of a 79-year-old woman, with a unique clonal chromosomal rearrangement: t(1;14)(p21;q24), that has not been reported in either soft tissue or primary bone angiosarcoma. The biologic significance of this translocation is not clear; however, the 1p21 locus is in the region of colony stimulating factor (CSF-1), which may play a role in tumorigenesis, as has been described in pigmented villonodular synovitis and tenosynovial giant cell tumor.

Rearrangements of IER3 Represent a Novel and Recurrent Molecular Abnormality in Myelodysplastic Syndromes (MDS)

Background: For most patients with MDS, the disease-associated molecular abnormalities are unknown, which contributes to diagnostic uncertainty and has limited development of effective therapies. Chromosomal deletions such as del(5q) or monosomy 7 are more common than translocations in MDS, but balanced translocations are more likely to be pathobiologically informative. Here we report cloning of an MDS-associated translocation that revealed a novel recurrent molecular abnormality.Methods: We first studied a 71-year old man who presented with MDS and t(6;9)(p21.3;q34) as an isolated chromosomal abnormality in 18 of 20 metaphases. FISH assays for DEK/CAN and ABL rearrangements were unrevealing. We generated somatic hybrid murine/human cell lines from a buffy coat; microdissected and amplified derivative chromosomes; and hybridized to 6p/9q custom comparative genomic hybridization (CGH) arrays. The CGH results considerably narrowed the 6p and 9q breakpoints, which were then amplified by long-range PCR and sequenced to define the rearrangement.Results: The IER3 gene (Immediate Early Response 3; also known as IEX-1) at 6p21.3 was found to be separated from its upstream regulatory elements in this rearrangement and translocated to a transcript-poor region of 9q. RT-PCR confirmed marked downregulation of IER3 expression in the patient compared to healthy controls. IER3 is a plausible candidate for involvement in MDS because of its known role in regulating death receptor-induced apoptosis, interaction with NF-κb pathways, and its importance in response to genotoxic stresses such as ionizing irradiation. We then identified archival bone marrow cell pellets from 204 additional patients with various clonal hematological disorders and chromosomal rearrangements involving 6p21 or 6p22 (i.e., translocations, inversions, deletions, or additions), and designed a splitsignal FISH probe set to assay IER3. FISH results were abnormal in 8 additional patients (9/205 pts total abnormal, 4.4%): 3 split signals and 6 amplifications (one patient had both abnormalities); all 8 patients had MDS and 6p rearrangements as part of a more complex karyotype. FISH studies in MDS patients without 6p rearrangements are ongoing. RTPCR in MDS patients without 6p rearrangements demonstrated down-regulation of IER3 by >2-fold in 38% of patients (67% lower-risk) and up-regulation in 12% of patients (50% higher-risk) when compared to the mean for healthy controls. Direct sequencing of the IER3 coding region and the promoter region in 30 patients without 6p rearrangements revealed no point mutations, but the prevalence of non-synonymous single nucleotide polymorphism rs3094124 (p.A127P) was higher than expected in patients (variant in 28.3 % of MDS alleles vs. 8.6% of 116 HAP-CEU controls, p=0.0015). IER3−/− mice are known to have hypertension, cardiac hypertrophy, and epithelial proliferation, but are not anemic (Sommer SL et al J Appl Physiol 2006). We performed methylcellulose hematopoietic colony growth assays using bone marrow mononuclear cells from IER3−/− mice and wildtype controls, and noted no differences in BFU-E, CFU-E, and CFU-GM growth under unstressed conditions. Studies of IER3 expression induction under stressed conditions in primary MDS cells are ongoing.Conclusion:IER3 rearrangements represent a novel clonally-restricted recurrent genetic abnormality in MDS. Dysregulation of IER3 expression is common in MDS, even in patients without 6p rearrangements. This is the first time IER3 has been linked to human disease. Clarification of the role of IER3 in MDS is likely to yield new insights into MDS pathobiology.

Lipoblastoma: appreciation of an expanded spectrum of disease through cytogenetic analysis.

Abstract Lipoblastomas are rare soft tissue tumors that predominantly affect the pediatric population. We describe a lipoblastoma of the right hand in a 16-month-old boy. Radiographically the tumor appeared large but fairly well circumscribed and composed primarily of fat. Pathologic evaluation revealed variably sized lobules of adipose tissue and myxoid immature mesenchymal tissue separated by prominent fibrous trabeculae. Cytogenetic analysis showed a clonal chromosomal rearrangement with a breakpoint involving chromosome 8q11.2, confirming the diagnosis of lipoblastoma and thus helping to expand the clinicopathologic spectrum of tumors in this diagnostic category.

Lipoblastoma: Appreciation of an Expanded Spectrum of Disease Through Cytogenetic Analysis

Lipoblastomas are rare soft tissue tumors that predominantly affect the pediatric population. We describe a lipoblastoma of the right hand in a 16-month-old boy. Radiographically the tumor appeared large but fairly well circumscribed and composed primarily of fat. Pathologic evaluation revealed variably sized lobules of adipose tissue and myxoid immature mesenchymal tissue separated by prominent fibrous trabeculae. Cytogenetic analysis showed a clonal chromosomal rearrangement with a breakpoint involving chromosome 8q11.2, confirming the diagnosis of lipoblastoma and thus helping to expand the clinicopathologic spectrum of tumors in this diagnostic category.