Eva Maria Murga Penas

Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung

BackgroundPancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.MethodsThe newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay.ResultsPaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis.ConclusionThe established PaCa 5061 cell line and its injection into pfp-/-/rag2-/- mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.

Cytogenetics of extramedullary manifestations in multiple myeloma

Extramedullary disease in patients with multiple myeloma is a rare event, occurring mostly in advanced disease or relapse. Outcome is poor and prognostic factors predicting the development of extramedullary disease have not been defined. We investigated cytogenetic alterations of myeloma cells in different extramedullary manifestations by adapting the fluorescence in situ hybridization (FISH) technique in combination with cytoplasmic immunoglobulin staining to study the cytogenetics of plasma cell tumours on paraffin embedded material. Thirty six patients were investigated: 19 with extramedullary disease, 11 with skeletal extramedullary disease and six with solitary extramedullary plasmacytoma. The first two groups showed the following results: del(17p13) 32% vs. 27%, del(13q14) 35% vs. 27%, MYC‐overrepresentation 28% vs. 18% and t(4;14) 37% vs. 18%. We detected an overall higher incidence of del(17p13) in both groups compared to data from bone marrow samples of multiple myeloma reported to date (range 7–16%). The solitary extramedullary plasmacytomas presented overall less cytogenetic aberrations than the other groups. Most important, three patients with extramedullary disease and one with skeletal extramedullary disease presented different FISH findings in the extramedullary tumour compared to their bone marrow plasma cells. del(17p13), occurring additional in three of four cases, seems a strong marker for extramedullary progression of myeloma.

A novel cryptic translocation t(12;17)(p13;p12-p13) in a secondary acute myeloid leukemia results in a fusion of the ETV6 gene and the antisense strand of the PER1 gene

The ETV6 gene is a member of the ETS family of transcription factors and the main target of chromosomal rearrangements affecting chromosome band 12p13. To date, more than 15 fusion partners of ETV6 have been characterized at the molecular level. Most of these fusions encode chimeric proteins with oncogenic properties. However, some of the translocations do not produce a functional fusion protein, but may induce ectopic expression of oncogenes located close to the breakpoint. We herein report the characterization and cloning of a novel cryptic translocation, t(12;17)(p13;p12–p13), occurring in a patient with an acute myeloid leukemia evolving from a chronic myelomonocytic leukemia. Cytogenetic analysis suggested the presence of a deletion of the short arm of chromosome 12, del(12)(p13), in three of the five metaphase cells analyzed. However, fluorescence in situ hybridization (FISH) with the ETV6‐specific cosmid clones 179A6, 50F4, 163E7, and 148B6 as well as probes hybridizing to the TP53 gene on 17p13 and the subtelomeric region of 17p revealed the presence of a translocation between 12p and 17p. By FISH, the breakpoints could be localized in intron 1 of ETV6 and centromeric to TP53. By 3′ rapid amplification of cDNA ends–polymerase chain reaction (3′ RACE‐PCR), a fusion transcript between exon 1 of ETV6 and the antisense strand of PER1 (period homolog 1, Drosophila), a circadian clock gene, could be identified. This ETV6‐PER1 (antisense PER1 strand) fusion transcript does not produce a fusion protein, and no other fusion transcripts could be detected. We hypothesize that in the absence of a fusion protein, the inactivation of PER1 or deregulation of a gene in the neighborhood of PER1 may contribute to the pathogenesis of leukemias with a t(12;17)(p13;p12–p13).

The t(14;18)(q32;q21)/IGH-MALT1 translocation in MALT lymphomas contains templated nucleotide insertions and a major breakpoint region similar to follicular and mantle cell lymphoma

The t(14;18)(q32;q21) involving the immunoglobulin heavy chain locus (IGH) and the MALT1 gene is a recurrent abnormality in mucosa-associated lymphoid tissue (MALT) lymphomas. However, the nucleotide sequence of only one t(14;18)-positive MALT lymphoma has been reported so far. We here report the molecular characterization of the IGH-MALT1 fusion products in 5 new cases of t(14;18)-positive MALT lymphomas. Similar to the IGH-associated translocations in follicular and mantle cell lymphomas, the IGH-MALT1 junctions in MALT lymphoma showed all features of a recombination signal sequence-guided V(D)J-mediated translocation at the IGH locus. Furthermore, analogous to follicular and mantle cell lymphoma, templated nucleotides (T-nucleotides) were identified at the t(14;18)/IGH-MALT1 breakpoint junctions. On chromosome 18, we identified a novel major breakpoint region in MALT1 upstream of its coding region. Moreover, the presence of duplications of MALT1 nucleotides in one case suggests an underlying staggered DNA-break process not consistent with V(D)J-mediated recombination. The molecular characteristics of the t(14;18)/IGH-MALT1 resemble those found in the t(14;18)/IGH-BCL2 in follicular lymphoma and t(11;14)/CCND1-IGH in mantle cell lymphoma, suggesting that these translocations could be generated by common pathomechanisms involving illegitimate V(D)J-mediated recombination on IGH as well as new synthesis of T-nucleotides and nonhomologous end joining (NHEJ) or alternative NHEJ repair pathways on the IGH-translocation partner.

A novel fusion of the MALT1 gene and the microtubule-associated protein 4 (MAP4) gene occurs in diffuse large B-cell lymphoma

Rearrangements of the MALT1 gene by the t(11;18)(q21;q21) and t(14;18)(q32;q21) are the most frequent structural chromosomal abnormalities in MALT lymphomas. These translocations lead to fusions of BIRC3–MALT1 and IGH–MALT1 respectively, and activate the NF‐κB pathway. Among 122 diffuse large B‐cell lymphomas and 28 Burkitts lymphomas screened by interphase FISH, we found two cases with a break within MALT1, but without a t(11;18) or a t(14;18). Molecular genetic analyses in one of these cases revealed a novel “in frame” fusion of exon 9 of MALT1 and exon 9 of the microtubule‐associated protein 4 (MAP4) gene. The translocation was accompanied by a deletion of MALT1 sequences distal to the breakpoint including the caspase‐like domain, which is essential for activation of NF‐κB. As a result of the deletion, the reciprocal 5′MAP4‐3′MALT1 transcript was not present, demonstrating that the 5′MALT1‐3′MAP4 fusion represents the pathogenetically relevant transcript. Immunohistochemistry with amino‐terminal and carboxy‐terminal MALT1 antibodies, indicated a strong expression of the chimeric MALT1–MAP4 protein. Moreover, NF‐κB activation was not increased in this case as shown by the levels of IκBα phosphorylation and NEMO ubiquitination. Our data demonstrate that the pathogenetic consequences of the novel MALT1–MAP4 fusion are different from those of the known MALT1‐associated chromosomal rearrangements and do not involve NF‐κB activation.

Primary Hepatic Lymphoma of Mucosa-Associated Lymphoid Tissue Type: A Case Report With Cytogenetic Study:

Primary hepatic lymphoma of mucosa-associated lymphoid tissue type is extremely rare. Only 38 cases have been reported to date. A case of a 59-year-old man with Helicobacter pylori—resistant gastric ulcers and Buerger disease who was followed up since 1999 is reported. A 2-cm hepatic nodule was incidentally found during partial gastrectomy and corresponded to mucosa-associated lymphoid tissue—type lymphoma without underlying liver disease. Molecular studies showed a clonal immunoglobulin heavy-chain gene rearrangement. Investigations for the mucosa-associated lymphoid tissue lymphoma-associated translocations t(11;18) and t(14;18), as well as the t(3;14)(q27;q32), were negative, whereas trisomy 3 and trisomy 18 were detected.

Deletion of chromosome 15 represents a rare but recurrent chromosomal abnormality in myelocytic malignancies

Abstract We report on three cases with myelocytic malignancies cytogenetically characterized by a deletion of chromosome 15 occurring as the sole cytogenetic aberration. The deletions were defined as del(15) (q12q21) (two cases) and del(15)(q11q21) (one case). Cytogenetic analysis was supplemented by fluorescence in situ hybridization (FISH) using a chromosome 15 specific whole chromosome painting probe and probes hybridizing to the UBE3A gene on 15q11~q13, the PML gene on 15q22, and the telomeric region of 15q. Hereby, an interstitial deletion of 15q including UBE3A , but not PML and the telomeric region of 15q could be demonstrated. Two of our patients were diagnosed as acute myelocytic leukemia (AML) with bone marrow dysplasia classified as AML-M6 and AML-M4, respectively, according to the French–American–British classification; the third patient suffered from a chronic myelomonocytic leukemia (CMMoL). In two cases, the aberration was found at the time of primary diagnosis, whereas the third case showed the del(15) only during relapse of leukemia. Both cases with acute leukemia did not adequately respond to intensive chemotherapeutic treatment and died 13 and 11 months, respectively, after primary diagnosis. Our findings and the data of five previously published cases with an isolated del(15) indicate that: 1) del(15) represents a rare but recurrent abnormality in myelocytic hemopathies; 2) in our cases, del(15) was interstitial and included the region 15q11~q13/ UBE3A , but not 15q22/ PML and the telomeric region of 15q as shown by FISH; 3) del(15) occurs frequently in disorders with myelodysplastic or myeloproliferative features and may therefore affect early hematopoietic progenitor cells; and 4) del(15) may occur during disease progression and is often associated with an unfavorable prognosis.

Molecular characterization of chromosomal band 5p15.33: A recurrent breakpoint region in mantle cell lymphoma involving the TERT-CLPTM1L locus

Secondary chromosomal aberrations may contribute to the development of a malignant phenotype in mantle cell lymphoma. Chromosomal band 5p15.33 represents a new recurrent breakpoint in B-cell malignancies. We present a molecular cytogenetic study of 8 mantle cell lymphoma (MCL) cell lines and 23 patients with MCL to determine and characterize novel secondary aberrations. We detected new secondary recurrent rearrangements in all cell lines and in 7 patients and confirmed 5p15.33 as a recurrent breakpoint in 4 cell lines and one patient. Further molecular characterization by flow-FISH and quantitative RT-PCR suggest TERT and CLPTM1L as target genes of 5p15.33 rearrangements.

Comprehensive cytogenetic and molecular cytogenetic analysis of 44 Burkitt lymphoma cell lines: Secondary chromosomal changes characterization, karyotypic evolution, and comparison with primary samples

Burkitt lymphoma cell lines (BL‐CL) are used extensively as in vitro models in genetic studies; however, cytogenetic information is not always available or updated. We provide a comprehensive cytogenetic resource of 44 BL‐CL, assessed by G‐banding, multicolor‐FISH, and FISH with 1q, 3p, 7q, and 13q region‐specific probes, including the first cytogenetic characterization of 22 BL‐CL and the revision of further 22 commonly used BL‐CL. Based on these data, we determined a consensus karyotype, evaluated in detail the secondary chromosomal changes (SCC), and the karyotypic stability of these cell lines. An individual karyotype was identified in all investigated BL‐CL, confirming their unique origin. Most of the BL‐CL remained cytogenetically relative stable after years of intensive cultivation. The most frequent structural SCC were dup(1q), del(13q) and the most frequent numerical SCC were +7, +13. Common breakpoints were located on 1q12, 7q11, and 13q31. The most common gains were in 1q and 7q and the most common losses were in 11q and 13q. Interestingly, the frequency of 1q gains and 13q losses was significantly higher in the EBV‐negative than in the EBV‐positive BL‐CL. Furthermore, by reviewing karyotypes of 221 primary BL listed in the Mitelman database, we observed similarities between BL‐CL and primary BL regarding the frequency of numerical and structural SCC and breakpoint distribution. In BL‐CL and in primary BL two SCC, dup(1q), and +12, always occurred mutually exclusive of each other. These findings validate BL‐CL as appropriate model for in vitro studies on the significance of SCC in the pathogenesis of BL.

MALT1 sequencing analyses in marginal zone B-cell lymphomas reveal mutations in the translocated MALT1 allele in an IGH-MALT1-positive MALT lymphoma

MALT1, a paracaspase, forms together with CARD11 and BCL10 a trimolecular complex, and, both as a scaffold protein constitutively associated with BCL10 and as a protease, is essential in B- and T-c...