Daniela Falzetti

DNA copy number changes in familial malignant mesothelioma

Malignant mesothelioma (MM) is predominantly a sporadic malignancy linked to exposure to asbestos. Clustering of MM in families suggests genetic susceptibility as a contributing factor. We performed comparative genomic hybridization (CGH) analysis on tumor samples from members of a family with MM of the pleura and a history of parental cancer. Our specific aim was to find a recurrent copy number loss indicating the chromosomal area to which a gene underlying the development of MM could be assigned according to the Knudson two-hit hypothesis. We found losses at 1p, 6q, 9p, 13q, and 14q. The copy number changes were very similar to those reported in sporadic cases. Our findings and results from sporadic cases highlight the importance of cloning the genes in the loss sites at 1p, 6q, 14q, and 22q.

Regions of juxtaposition in unbalanced 1q rearrangements of malignant hemopathies

861 22 showed that the majority of chromosome 22 material remains present on the der(22) implying that a second break in the chromosome 22 has occurred at a site considerably telomeric to the BCR gene. Whole chromosome paint analysis failed to locate the region 9q34-qter, previously thought to be present on der(5). Application of 9q34 BSP produced three hybridisation signals: (1) on the normal chromosome 9 q-terminus; (2) on the der(9) subterminal region; and (3) on the distal half of one homologue of chromosome 22 (Figure 1iv). The intensities of BSP signals seen on the der(9) and der(22) chromosomes were similar, which suggested that approximately equal sized segments of band 9q34 were present on both these abnormal chromosomes, thus confirming that the break in band 9q34 occurred within the q34.1 sub-band. To conclude, LSPs, WCPs and BSPs revealed the involvement of chromosome 22 in a simple variant Philadelphia translocation assessed by G-banding as t(5;9)(q23;q34). Unexpected rearrangements in the structure of the der(22) were revealed, suggesting highly complex involvement of this chromosome in the variant translocation. In this study, whole chromosome paints failed to locate the segment 9q34.1-qter in a patient with variant Ph positive CML. The presence of this 9q material was revealed using a 9q34 band specific probe. Our results clearly demonstrate that rearrangements affecting regions smaller than an average G-band can be visualised using BSPs. This observation suggests that the resolution of WCPs is within 10 Mb while BSPs can identify regions in the range of 5 Mb. The increased sensitivity of BSPs may be due to several factors: (1) Improved representation of sequences from a chromosome band in a BSP; (2) the use of a new and efficient method of labelling involving incorporation of directly labelled primers; or (3) specific features of the chromosome material used for the production of WCP 9. In conclusion BSPs provide a sensitive and reliable tool for the detection of cryptic chromosome rearrangements in malignant karyotypes.

Identification of multiple copies of a 20q-chromosome in a case of myelodysplastic syndrome: a FISH study

In myelodysplastic syndromes (MDS) karyotypic aberrations identify subgroups of patients with distinct clinical-morphological features and can be relevant in risk assessment of developing leukemia. Often conventional cytogenetic analysis is not sufficiently informative due to the presence of partially or completely unrecognizable chromosome markers. By chromosome microdissection (MD) and fluorescence in situ hybridization (FISH) we investigated the nature of a karyotypic marker occurring in multiple copies in one case of MDS arisen in a patient previously treated for breast cancer. Results showed dicentrics derived from telomeric fusion between interstitially deleted 20q-chromosomes. The abnormal karyotype resulted into polysomy for a deleted chromosome 20q.

Microdissection and FISH investigations in acute myeloid leukemia : A step forward to full identification of complex karyotypic changes

Complex chromosomal rearrangements in malignant hemopathies frequently remain unclarified because of paucity of material for further fluorescence in situ hybridization analyses and/or lack of suitable probes. Chromosome microdissection (MD) can be an adequate approach to elucidate chromosome aberrations unrecognizable by conventional karyotyping. We applied MD in two patients with acute myeloid leukemia (AML) and unidentified chromosome changes at karyotype. Microdissection of a ring chromosome in an AML-M5 case revealed 21q polysomy. In an AML-M4 case, MD of an add(15p) disclosed a t(8;15) with over-representation of both 8q22 and 8q24 bands. YAC probes were helpful in showing duplication of the ETO gene at 8q22, and amplification of C-MYC, at 8q24.

14q+ chromosome marker in a T-cell-rich B-cell lymphoma.

Cytogenetic, in situ hybridization, and molecular studies were performed in a case of T‐cell‐rich B‐cell lymphoma. Demonstration of Ig gene rearrangements for both heavy and light chains confirmed the B‐lineage restriction of the neoplastic cell population. Moreover, as expected in B‐cell malignancies, all abnormal karyotypes showed a 14q+, as defined by chromosome painting with a library for chromosome 11, and Southern blotting for c‐myc and bcl‐2 rearrangements, remains unknown

Interpretation of the complex karyotype and identification of a new 6p amplicon by integrated comparative genomic hybridization and fluorescence in situ hybridization on the U937-I cell line

Molecular cytogenetics is helpful to identify complex and cryptic genomic changes in malignancy. Human leukemic cell lines are an important tool for advancements of biological research on malignant cells, one critical step being characterization of genomic changes. We used fluorescence in situ hybridization and comparative genomic hybridization to refine karyotypic interpretation of the diffuse histiocytic lymphoma derived U937-1 cell line. From this integrated approach, chromosome material involved in nine karyotypic markers and in unbalanced translocations could be identified. Moreover, a previously undetected amplicon emerged within band 6p21. The U937-I is a new in vitro model to study genome amplification and unknown recombinations in leukemic cells, such as those involving the centromeric region of chromosome 1.

Multiple small accessory marker chromosomes from different centromeric origin in a moderately mentally retarded male

Abstract. The occurrence of more than two small accessory chromosomes (SACs) in a single individual is extremely rare. Here, we characterize six SACs found in the cells of two different tissues of a moderately mentally retarded male. Microdissection combined with regular FISH demonstrates that the SACs are ring chromosomes derived from the centromeres of different chromosomes. The SACs are often associated with the centromeres of other chromosomes. Immunofluorescence with an anti-CENP-C antibody demonstrates that the SACs contain an active centromere. A possible mechanism by which the SACs originated and their clinical relevance are discussed.

Rearrangement Between the MYH11 Gene at 16p13 and D12S158 at 12p13 in a Case of Acute Myeloid Leukemia M1 (AML-M1)

A case of acute myeloid leukemia (AML) M1 with bone marrow eosinophilia was characterized by cytogenetics and fluorescence in situ hybridization (FISH). A complex karyotype including a der(12)t(12;17)(p12‐13;q11) and a der(16)t(16;20)(p13;p11) was found at diagnosis. FISH studies with probes for chromosome 16 and for the short arm of chromosome 12 showed even more complex rearrangements. Analysis with a panel of probes for 12p showed that D12S158 spanned the breakpoint on the der(12). Unexpectedly, FISH signals were found on the der(12) and on the der(16) at band p13, the site of juxtaposition between the short arm of chromosome 16 and chromosome 20. Moreover, both YAC 854E2, containing the MYH11 gene, and cosmid ZIT133, encompassing the MYH11 breakpoint in inv(16) and t(16;16) of AML‐M4 with eosinophilia, demonstrated fluorescent signals on the normal 16, on the der(16), and on the der(12). These data clearly support a reciprocal exchange between D12S158 at 12p13.3 and the MYH11 gene at 16p13. In addition, experiments with two PAC clones for the CBFB gene at 16q22 excluded the presence of a masked inv(16). An interstitial deletion, independent from the translocation and flanked by VWF and KRAS2, was also detected on the der(12). Genes Chromosomes Cancer 23:10–15, 1998.

Philadelphia-positive acute lymphoblastic leukemia with multiple subclones including duplication of the Philadelphia chromosome and Abelson oncogene

A case of Philadelphia (Ph)-positive acute lymphoblastic leukemia (ALL) with multiple subclones including duplication of the BCR-ABL1 fusion gene and of the Abelson oncogene (ABL1) is reported. Cytogenetically, two different rearrangements of chromosome 9 not involved in the t(9;22) were found in two subclones. In one subclone the normal 9 was lost and replaced by an acrocentric marker, which contained an additional copy of the BCR-ABL1 fusion gene. Reverse transcriptase polymerase chain reaction detected the fusion transcripts p210 (e13a2 junction) and p190 (e1a2 junction), whereas fluorescence in situ hybridization showed the major BCR-ABL1 junction in both Ph chromosomes, strongly suggesting that the presence of the p210 and p190 proteins in this case was due to mechanisms of alternative or mis-splicing at the transcriptional level. The second subclone showed the classic t(9;22) plus an add(9)(p24) containing two copies of the ABL1 gene. Other molecular events involving chromosome 9 were a monoallelic loss of JAK2 in both subclones and an additional loss of P15/P16 in the subclone with the acrocentric marker bearing the extra Ph chromosome.