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Genetic profiling is important for disease evaluation and prediction of prognosis or responsiveness to therapy in neoplasia. Microarray technologies, including array comparative genomic hybridization and single-nucleotide polymorphism-detecting arrays, have in recent years been introduced into the diagnostic setting for specific types of haematological malignancies and solid tumours. It can be used as a complementary test or depending on the neoplasia investigated, also as a standalone test. However, comprehensive and readable presentation of frequently identified complex genomic profiles remains challenging. To assist diagnostic laboratories, standardization and minimum criteria for clinical interpretation and reporting of acquired genomic abnormalities detected through arrays in neoplastic disorders are presented.

Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.

Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.

Genetic profiling is important for disease evaluation and prediction of prognosis or responsiveness to therapy in neoplasia. Microarray technologies, including array comparative genomic hybridization and single-nucleotide polymorphism-detecting arrays, have in recent years been introduced into the diagnostic setting for specific types of haematological malignancies and solid tumours. It can be used as a complementary test or depending on the neoplasia investigated, also as a standalone test. However, comprehensive and readable presentation of frequently identified complex genomic profiles remains challenging. To assist diagnostic laboratories, standardization and minimum criteria for clinical interpretation and reporting of acquired genomic abnormalities detected through arrays in neoplastic disorders are presented.

EVI1 is a transcription factor that is targeted for de-regulation in 10% of patients with acute myeloid leukemia (AML). Overexpression of EVI1 is associated with a particularly poor prognosis. Various karyotypic abnormalities involving chromosomal region 3q26 have been shown to inappropriately activate expression of EVI1, the most frequent alterations being inv(3)(q21q26)and t(3;3)(q21;q26). Rearrangements of 3q are frequently associated with trilineage myelodysplasia, especially dysmegakaryocytosis. So far, translocation t(3;8)(q26;q24) has been reported in one patient with MDS and two cases of AML without further molecular analysis. No other recurrent chromosome aberration with a breakpoint in 8q24 has been shown in AML. We report a case of a 58 year old male patient with a multilineage AML featuring a t(3;8)(q26;q24) as the sole chromosomal aberration in 80% of bone marrow cells analysed by conventional cytogenetics. The normocellular bone marrow aspirate showed 25–50% atypical blasts, dyserythropoiesis, dysplastic myelopoiesis and hyperactivated megakaryopoiesis with many micromegakaryocytes. Immunophenotyping of the blast population by flow cytometry confirmed the myeloid nature of the cells with CD13+, CD33+, CD64+, CD117+, HLA−DR+ phenotype and variable expression of CD14 and CD34. Persisting pancytopenia occurred after two cycles of chemotherapy and a relapse was evident 4 months later. By real time quantitative RT-PCR (RQ-PCR) we showed that EVI1 is highly overexpressed (>100:1) in this patients bone marrow cells as compared with an AML patient without 3q26 involvement. Fluorescence in situ hybridisation (FISH) is currently being performed to characterize the 8q24 and 3q26 breakpoints in more detail. The chromosomal translocation t(3;8)(q26;q24) leads to overexpression of EVI1 and, as in other aberrations involving 3q, shows a poor prognosis in AML.

Phorbol-12-myristate-13-acetate (PMA) acts as a tumor promoter on mouse skin. It induces inflammation and leukocyte-mediated clastogenicity which appears to be related to rapid changes in lipid metabolism. To identify lipids possessing clastogenic and/or tumor-promoting properties, we have characterized the metabolism and release of arachidonic acid (AA) and related lipids during the formation of lipophilic clastogenic factors by PMA-treated human monocytes. In 1 h, [3H]AA-labeled monocytes spontaneously released significant amounts of their total radioactivity (4%) which increased nearly 4-fold (15%) with PMA (30 ng/ml) treatment. Eighty-five per cent of extracellular 3H-label from both control and PMA-treated monocytes was composed of free AA (plus AA-metabolites), while the remaining radioactivity was incorporated in phospholipids and mono- and diacylglycerols. Treated and non-treated cells released essentially the same kind of metabolites but PMA induced a 3- to 4-fold increase in total amounts. The major products consisted of prostaglandins F2 alpha and E2, thromboxane B2, 12-hydroxy-5,8,10-heptadecatrienoic acid and 5-, 11- and 15-hydroxyeicosatetraenoic acids. PMA also induced increases in the levels of three unidentified products. Neither leukotrienes nor 4-hydroxynonenal, a major alkenal degradation product of AA, were found in medium from PMA-treated monocytes. PMA, in contrast to the first-stage tumor promoter calcium ionophore A23187, failed to stimulate the release of platelet activating factor. The increased formation of phorbol ester-induced AA metabolites was proportional to the increase in free extracellular AA. The source of AA from treated and untreated monocytes consisted of cellular phospholipids with phosphatidylcholine and phosphatidylethanolamine accounting for 85%.

The evidence is convincing that oxidants and agents which induce a cellular pro-oxidant state can act as carcinogens, in particular as promoters and progressors. Importantly, infiltrated phagocytes represent a source of oxidants in inflamed tissues. We have studied the mechanism of the promotional action of active oxygen (AO) in mouse epidermal cells JB6 by comparing the non-promotable clone 30 to the promotable clone 41. In order to mimick AO released by phagocytes we used xanthine/xanthine oxidase as a source of extracellular superoxide and hydrogen peroxide. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Reasons for the higher cytostatic effect of AO on the non-promotable clone 30 were discovered when we measured DNA strand breakage and poly ADP-ribosylation of chromosomal proteins. At equal doses AO induced 4-5 times more DNA breaks in clone 30 in reactions which required iron--and probably also calcium--ions. The higher amount of DNA breakage in clone 30 was reflected in a higher extent of poly ADP-ribosylation. Excessive DNA breakage and poly ADP-ribosylation which causes the depletion of NAD and ATP may be responsible for the strong cytostatic effect of AO in clone 30. We conclude that differential resistance to the cytostatic/cytotoxic effect of AO in part determines the promotability of mouse epidermal cells JB6.

The mouse skin tumor promoter phorbol-12-myristate-13-acetate (PMA) is a potent inflammatory agent that not only elicits the infiltration of neutrophils, monocytes and lymphocytes to the site of application (1) but also induces human neutrophils and monocytes, but not lymphocytes, to release clastogenic factor(s) (CF) (2). CF are characterized as lipophilic (ethylacetate extractable) substances of relatively low molecular weight (

Oxidants and agents that induce a cellular prooxidant state can act as carcinogens. We compared the effect of tert -butyl hydroperoxide (Bu-OOH) on DNA strand breakage, poly ADP-ribosylation of chromosomal proteins and the expression of the proto-oncogenes c-fos and c-myc between non-promotable clone 30 and promotable clone 41 of mouse epidermal cells JB6. These pathophysiological effects of oxidants are mechanistically related. Bu-OOH caused more DNA-strand breakage at high concentrations and more extensive poly ADP-ribose accumulation in clone 30 than in clone 41, in reactions which require intracellular free iron. Clone 41 exhibited constitutive c-myc expression while c-fos mRNA was very low in untreated cultures of both clones. Low concentrations of Bu-OOH induced c-myc and more strongly c-fos in clone 41. Both proto-oncogenes were strongly induced in clone 30. Our results allow insights into the mechanisms of action of a typical organic hydroperoxide in JB6 cells. However, they do not uncover the reasons for the differential promotability of the two JB6 clones by oxidants beyond the implication of the constitutive expression of c-myc in promotable clone 41.