MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/Deletions.

Acute myelogeneous leukemia (AML) with inv(3)/t(3;3)(q13q25) is associated with aberrant expression of the stem-cell regulator MECOM (aka EVI1). Two bone marrow samples received in the OHSU Knight Diagnostic Laboratories (KDL) Cytogenetics Laboratory for chromosomes and FISH for a question of progression of myelodysplastic syndrome (MDS) to AML showed complex abnormalities including a deletion of chromosome 3q, one with del(3)(q13q25) and the other with del(3)(q22q25). In light of the prognostic importance of the activation of the MECOM oncogene and the concurrent inactivation of the GATA2 tumor suppressor that occurs with the classic inversion of chromosome 3q, fluorescence in situ hybridization (FISH) was performed using two different probe designs to better define the 3q deletions in the two cases. Using the Abbott Molecular Laboratories dual fusion MECOM/RPN1 probe, interphase and metaphase cells in both patients showed a variant single fusion (orange/green/fusion) signal pattern consistent with fusion and deletion. Using the three-color (red/green/aqua) Cytocell EVI1 probe, interphase cells in both cases showed a split red/green signal with the aqua signal remaining with the green signal. The distance between the split signals was generally less than is usually seen in the commonly described inverted chromosome 3. These findings are therefore consistent with a complex inversion and concurrent deletion/deletions of chromosome 3q. Thus, the deletion 3q seen in G-banded chromosomes from bone marrow from these two patients is most consistent with the activation of MECOM and the inactivation of GATA2.

Cytogenetic instability in ovarian epithelial cells from women at risk of ovarian cancer.

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

In vivo genetic selection of renal proximal tubules.

Repopulation by transplanted cells can result in effective therapy for several regenerative organs including blood, liver, and skin. In contrast, cell therapies for renal diseases are not currently available. Here we developed an animal model in which cells genetically resistant to a toxic intermediate of tyrosine metabolism, homogentisic acid (HGA), were able to repopulate the damaged proximal tubule epithelium of mice with fumarylacetoacetate hydrolase (Fah) deficiency. HGA resistance was achieved by two independent mechanisms. First, Fah+ transplanted bone marrow cells produced significant replacement of damaged proximal tubular epithelium (up to 50%). The majority of bone marrow-derived epithelial cells were generated by cell fusion, not transdifferentiation. In addition to regeneration by fusion-derived epithelial cells, proximal tubular repopulation was also observed by host epithelial cells, which had lost the homogentisic acid dioxygenase gene. These data demonstrate that extensive regeneration of the renal proximal tubule compartment can be achieved through genetic selection of functional cells.

Heterozygosity for p53 (Trp53+/-) accelerates epithelial tumor formation in fanconi anemia complementation group D2 (Fancd2) knockout mice.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure and an increased susceptibility to cancer. FA is genetically heterogeneous, consisting of at least 11 complementation groups, FA-A through L, including FA-D1 (BRCA2) and D2. We have previously reported an increased incidence of epithelial tumors in Fancd2 knockout mice. To further investigate the role of the FA pathway in tumor prevention, Fancd2 mutant mice were crossed to mice with a null mutation in the tumor suppressor gene, Trp53. The tumor spectrum in Fancd2(-/-)/Trp53(+/-) mice included sarcomas expected in Trp53 heterozygotes, as well as mammary and lung adenocarcinomas that occur rarely in Trp53 heterozygotes. These tumors occurred earlier than in Fancd2(-/-) control mice. Therefore, the Fancd2(-/-)/Trp53(+/-) mice represent an improved model for the study of adenocarcinoma in FA. In addition, it was found that Fancd2(-/-) mouse embryonic fibroblasts but not Fancd2(-/-)/Trp53(-/-) mouse embryonic fibroblasts arrest following DNA damage. Therefore, Trp53 is required for the S phase checkpoint activation observed in Fancd2 mutant cells. Fancd2(-/-)/Trp53(-/-) cells showed an increase in aneuploidy and had multiple gross chromosomal rearrangements.

Interstrand crosslink-induced radials form between non-homologous chromosomes, but are absent in sex chromosomes.

Fanconi anemia (FA) and cells lacking functional BRCA1 and BRCA2 proteins are hypersensitive to interstrand crosslinking (ICL) agents and show increased numbers of chromosomal breaks and radials. Although radial formation has been used to diagnose FA for more than 30 years, there has been little analysis of these characteristic formations. In this study, radials were analyzed from FA-A and FA-G fibroblasts as well as normal and retrovirally-corrected FA-A fibroblasts treated with high doses of ICLs. Radials were found to only involve non-homologous chromosome interactions and to be distributed nearly randomly along the length of chromosomes. Sites on chromosomes that did show increased frequency of radial involvement did not correlate with known fragile sites or pericentric regions. Hybrid radials were observed between mouse and human chromosomes in human-mouse hybrid cells produced by microcell-mediated chromosome transfer of mouse chromosomes into human FA-A fibroblasts. Both X and Y chromosomes were notably not involved in radials. These observations suggest that ICL repair may involve short stretches of homology, resulting in aberrant radial formation in the absence of FA proteins.

Cell fusion is the principal source of bone-marrow-derived hepatocytes.

Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.