Identification of a commonly amplified 4.3 Mb region with overexpression of C8FW, but not MYC in MYC-containing double minutes in myeloid malignancies

Double minutes (dmin), the cytogenetic hallmark of genomic amplification, are found in approximately 1% of karyotypically abnormal acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). The MYC gene at 8q24 has been reported to be amplified in the majority of the cases, and generally it has been assumed that MYC is the target gene. However, only a few studies have focused on the extent of the amplicon or on the expression patterns of the amplified genes. We have studied six cases (five AML and one MDS) with MYC-containing dmin. Detailed fluorescence in situ hybridization analyses identified a common 4.3 Mb amplicon, with clustered proximal and distal breakpoints, harboring eight known genes (C8FW, NSE2, POU5FLC20, MYC, PVT1, AK093424, MGC27434 and MLZE). The corresponding region was deleted in one of the chromosome 8 homologues in five of the six cases, suggesting that the dmin originated through extra replication (or loop-formation)--excision--amplification. Northern blot analysis revealed that MYC was not overexpressed. Instead, the C8FW gene, encoding a phosphoprotein regulated by mitogenic pathways, displayed increased expression. These results exclude MYC as the target gene and indicate that overexpression of C8FW may be the functionally important consequence of 8q24 amplicons in AML and MDS.     

Distinct sequences on 11q13.5 and 11q23-24 are frequently coamplified with MLL in complexly organized 11q amplicons in AML/MDS patients

Amplification within chromosome arm 11q involving the mixed-lineage leukemia gene (MLL) locus is a rare but recurrent aberration in acute myeloid leukemia and myelodysplastic syndrome (AML/MDS). We and others have observed that 11q amplifications in most AML/MDS cases have not been restricted to the chromosomal region surrounding the MLL gene. Therefore, we implemented a strategy to characterize comprehensively 11q amplicons in a series of 13 AML/MDS patients with MLL amplification. Analysis of 4 of the 13 cases by restriction landmark genomic scanning in combination with virtual genome scan and by matrix-based comparative genomic hybridization demonstrated that the 11q amplicon in these four cases consisted of at least three discontinuous sequences derived from different regions of the long arm of chromosome 11. We defined a maximally 700-kb sequence around the MLL gene that was amplified in all cases. Apart from the core MLL amplicon, we detected two additional 11q regions that were coamplified. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that sequences in 11q13.5 and 11q23-24 were amplified in 8 of 13 and 10 of 12 AML/MDS cases, respectively. Both regions harbor a number of potentially oncogenic genes. In all 13 cases, either one or both of these regions were coamplified with the MLL amplicon. Thus, we demonstrated that 11q amplicons in AML/MDS patients display a complex organization and have provided evidence for coamplification of two additional regions on the long arm of chromosome 11 that may harbor candidate target genes.     

MYC amplification in two further cases of acute myeloid leukemia with trisomy 4 and double minute chromosomes

We report two cases of trisomy 4 with double minute chromosomes (dmin): one in a woman with acute myeloid leukemia (AML), French-American-British subtype M2, the other in a man with chronic myelomonocytic leukemia. In the former case, many cells without trisomy 4 but with dmin were present, a finding not observed in previously reported cases. In both cases, fluorescence in situ hybridization studies demonstrated the double minutes to be MYC amplicons. Ten cases of AML with trisomy 4 and dmin have now been described; in the five cases investigated, the dmin have been shown to be amplified MYC gene sequences.     

Double minutes and c-MYC amplification in acute myelogenous leukemia: Are they prognostic factors?

A case of acute myelogenous leukemia (AML) with double minutes (dmin) and X chromosome loss is presented. Using comparative genomic hybridization (CGH), a region of high-level DNA amplification was detected at 8q24, the locus of the c-MYC proto-oncogene. Fluorescence in situ hybridization (FISH) with a DNA probe specific for the human c-MYC gene confirmed the extrachromosomal amplification of this proto-oncogene in the dmin of the leukemic cells. During the course of the disease, three relapses occurred; two complete remissions could be achieved by treatment with various chemotherapy regimens. The patient's survival time of 25 months was considerably longer than in most reported cases of AML with extrachromosomal c-MYC amplification. Therefore, the present case challenges the view that the occurrence of dmin in AML is generally an indication of poor prognosis.     

Deletion of MYC and presence of double minutes with MYC amplification in a morphologic acute promyelocytic leukemia-like case lacking RARA rearrangement: could early exclusion of double-minute chromosomes be a prognostic factor?

Gene amplification on double minutes is rarely found in acute myeloid leukemia (AML) and is often linked to poor prognosis. It is often associated with acute myeloid leukemia with differentiation (AML-M2) and is rarely reported in acute promyelocytic leukemia (APL), which is characterized in the vast majority of cases by the reciprocal t(15;17)(q22;q21) with resultant translation of an abnormal PML-RARA fusion protein. Most of the rare cases of APL that lack this translocation have a demonstrable RARA breakpoint. We report on a morphologic APL-like case lacking t(15;17) and the RARA breakpoint and also has the deletion MYC of 8q24 associated with the occurrence of MYC amplification on double-minute chromosomes (dmin). Excessive exclusion of dmin was observed at the initial diagnosis. These findings are compared to the few cases previously reported in the literature.     

The paradox of 20q11.21 amplification in a subset of cases of myeloid malignancy with chromosome 20 deletion

Deletion of the long arm of one chromosome 20 (del(20q)) is a well‐recognized abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and is presumed to cause loss of a tumor suppressor gene at 20q12. In a previously published series of MDS and AML cases, which had lost this region via unbalanced translocation, around 40% of cases were shown to have additional copies of the chromosome 20 abnormalities, with resulting gain or amplification of the retained parts of chromosome 20, most often 20q11.2. We have used FISH and array comparative genomic hybridization, to define further the retained and amplified regions. We now report targeted amplification of 20q11.21 in four of the 22 cases selected for further study and in one new case. The shortest amplified region of 250 kb in a series of five patients with three to ten copies of the 20q11.21 region contained the complete HCK, TM9SF4, PLAGL2, and POFUT1 genes. By RT‐PCR we have shown that there is correlation between amplification and increased expression of these four genes in most cases. Localized and high level amplification of the common 250 kb region is evidence for activation of an oncogene in this region in these MDS and AML cases. Cases with 20q11.21 amplification tended to have a high proportion of erythroblasts in the marrow, with two cases diagnosed as erythroleukemia (AML‐M6). Chromosome sub‐band 20q11.21 amplification may therefore prove to be a marker of a specific subset of AML/MDS with a significant erythroid component. © 2010 Wiley‐Liss, Inc.     

Identification of amplified DNA sequences on double minute chromosomes in a leukemic cell line KY821 by means of spectral karyotyping and comparative genomic hybridization

Double minute chromosomes (dmin) are cytogenetic hallmarks of amplified genes. Using spectral karyotyping (SKY) and comparative genomic hybridization (CGH), we identified the origin of amplified DNA in a leukemic cell line, KY821, that harbors numerous dmin. The SKY revealed that the DNA sequences of dmin are derived from materials of chromosome 8, and CGH showed a high degree of overrepresentation only at 8q22–24, indicating that in KY821 only chromosomal material of 8q22–24, containing MYC, is amplified in dmin. An approach combining SKY with CGH should facilitate efforts to identify novel chromosomal regions of gene amplification and contribute information about genetic lesions that underly neoplastic tumors. Received: March 11, 1998 / Accepted April 9, 1998     

AML/MDS with 11q/MLL amplification show characteristic gene expression signature and interplay of DNA copy number changes

AML/MDS patients carrying 11q amplifications involving the mixed lineage leukemia gene (MLL) locus are characterized by a complex aberrant karyotype (CAK) frequently including deletions within 5q, 17p, and 7q, older age and fast progression of the disease with extremely poor prognosis. MLL has been shown to be overexpressed in cases with 11q amplification. However, in most of the cases, the amplified region is not restricted to the MLL locus. In this study, we investigated 19 patients with AML/MDS and MLL gain/amplification. By means of array CGH performed in 12 patients, we were able to delineate the minimal deleted regions within 5q and 17p and identified three independent regions 11q/I‐III that were amplified in all cases. Gene expression profiles established in 15 cases were used to identify candidate genes within these regions. Notably, analysis of our data suggests a correlation of loss of 5q and 17p and expression of genes present in 11q23‐25. Furthermore, we demonstrate that the gene expression signature can be used to discriminate AML/MDS with MLL amplification from several other types of AML. © 2009 Wiley‐Liss, Inc.     

Amplified, lost, and fused genes in 11q23-25 amplicon in acute myeloid leukemia, an array-CGH study

Gene amplifications occur rarely in hematologic neoplasms. We characterized two cases of acute myeloid leukemia (AML) with marker chromosomes and 11q23-25 amplicons. Case 1 was a 14-year-old male with an additional ring of chromosome 11 material as the sole karyotypic abnormality, as determined by G-banding and multicolor fluorescence in situ hybridization. Standard comparative genomic hybridization (CGH) showed amplification in 11q23-qter. However, the MLL gene, in 11q23, was not amplified by FISH. Case 2 was a 38-year-old male with the G-banding karyotype 51,XY,+8,+19,+3mar and with 11q22-qter amplification by standard CGH. This patient also had the MLL-LARG fusion gene. We used microarray-based CGH (array-CGH) to characterize the amplicons. In case 1, the amplified region in 11q24.3-25 (5.5 Mb) was continuous, and MLL was not amplified, as expected. In case 2, the amplicon was divided into two distinct parts, in 11q23.3 (1.2 Mb) and in 11q23.3-25 (13.3 Mb). It contained a loss ( approximately 1 Mb) in 11q23.3, and the amplicon breakpoint was in the middle of MLL. Although the amplicon size varied, the patients had a common amplified region in 11q24-25 that comprised 14 genes. Expression microarray of case 1 revealed that three of these genes, FLI1, NFRKB, and SNX19, were also overexpressed. The results indicate that the 11q24-q25 region may harbor new candidate oncogenes. In addition, the complex amplicon of case 2 suggests some intriguing chromosomal mechanisms.     

Mutations of the PTPN11 gene in therapy-related MDS and AML with rare balanced chromosome translocations

Activating mutations of the PTPN11 gene encoding the SHP2 tyrosine phosphatase is the most common genetic abnormality in juvenile myelomonocytic leukemia and is sporadically observed in myelodysplasia (MDS) and acute myeloid leukemia (AML). An unselected series of 140 patients with therapy-related MDS or AML were investigated for mutations of PTPN11 in Exons 3, 4, 8, and 13. Four cases had mutations of the gene; three of these had deletions or loss of chromosome arm 7q. Two cases had rare balanced translocations to chromosome band 21q22 with rearrangement of the RUNX1 gene and the other two patients had rare balanced translocations to chromosome band 3q26 with rearrangement of the EVI1 gene. The findings support cooperation between so called Class I and Class II mutations in leukemogenesis.     

Extrachromosomal gene amplification in acute myeloid leukemia; Characterization by metaphase analysis,comparative genomic hybridization,and semi-quantitative PCR

A case of acute myeloid leukemia (M-3) with complex karyotypic aberrations and double minute (dmin) chromosomes is presented. The patient had no history of prior exposure to mutagenic or carcinogenic agents or of other malignancies. She died from CNS involvement six weeks after the initial diagnosis. We used comparative genomic hybridization to identify the amplified sequences presumed to represent the dmin of the leukemic cells; the tumor/normal ratios indicated increased signal intensity at 8q24. This localization prompted investigation by semi-quantitative PCR that revealed amplification of the MYC oncogene. The extent of chromosome aberrations and the oncogene amplification, both linked with poor prognosis, may relate to the rapid course of this patient's disease. © 1993 Wiley-Liss, Inc.     

DNA copy number changes and evaluation of MYC, IGF1R, and FES amplification in xenografts of pancreatic adenocarcinoma

We analyzed eight samples of xenografted human pancreatic tumors and two metastases developed in mice by comparative genomic hybridization (CGH). The most recurrent changes were: gains on chromosomes 8 (8q24-qter; 7/8 cases), 15 (15q25-q26; 6/8 cases), 16 (16p in 6/8 cases; 16q in 5/8 cases), 20 (20q; 6/8 cases), and 19 (19q; 5/8 cases); and losses on chromosomes 18 (18q21; 6/8 cases), 6 (6q16-q21 and 6q24-qter; 5/8 cases each), and 9 (9p23-pter; 5/8 cases). The two metastases maintained the aberrations of the original pancreatic tumor plus gain of 11q12-q13 and 22q. Loss of heterozygosity analysis was carried out for 10p14-pter, a region that was lost in 3/8 samples. All of them presented allelic imbalance for all the informative loci. Fluorescence in situ hybridization and Southern analysis were performed to test some candidate oncogenes in 8q24 (MYC) and 15q25-qter (IGF1R and FES). Two of seven tumors showed high-level amplification of MYC relative to the centromere (> 3-fold), another two tumors had low-level amplification (1.5- to 3.0-fold), and one displayed 5.5 MYC signals/cell. In relation to the FES gene, low-level amplification was found in three tumors. Southern analysis showed five cases with a low-level amplification of IGF1R. Our data suggest that either few extra gene copies may be enough for cancer progression or other genes located in these regions are responsible for the amplifications found by CGH.     

Amplification of MGC2177, PLAG1, PSMC6P, and LYN in a malignant mixed tumor of salivary gland detected by cDNA microarray with tyramide signal amplification

Gene amplifications have been observed in many different tumor cells, and many of these changes are related to tumor pathogenesis. Comparative genomic hybridization (CGH) using metaphase chromosomes can detect changes in chromosome copy number with a resolution of 10-20 Mb. Current advances in CGH analysis in a microarray format allow us to refine such changes down to the gene level. We applied microarray technology to detect novel gene amplification in a malignant mixed tumor of salivary gland. Besides detecting previously known gene amplifications (MDM2 and MYC), we identified four other highly amplified genes located at 8q11.2 approximately q13: MGC2177, PLAG1, PSMC6P, and LYN. The amplification was further validated with real-time quantitative polymerase chain reaction.     

Coamplification of prostate stem cell antigen (PSCA) and MYC in locally advanced prostate cancer

Gain of sequences on chromosome arm 8q is a common feature of prostate cancer that may correlate with metastatic and androgen-independent progression. The target gene(s) for this gain is not known, although MYC is amplified in a subset of advanced tumors and is one potential candidate. Prostate stem cell antigen (PSCA) is a prostate-specific cell surface protein that maps to chromosome region 8q24.2 and is overexpressed in prostate cancer. Our aim in this study was to test the hypothesis that PSCA overexpression may result from overrepresentation of chromosome arm 8q. Twenty locally advanced prostate cancers were analyzed by dual-probe fluorescence in situ hybridization (FISH) for alterations of MYC and PSCA. Extra copies of MYC were found in 12/20 (60%) tumors, including 5 (25%) with simple gain (no increase in MYC copy number relative to the chromosome 8 centromere) and 7 (35%) with an additional increase (AI or overrepresentation) in MYC copy number relative to the centromere. In the five cases with simple gain of MYC, there was a concomitant gain of PSCA. PSCA was overrepresented in 5/7 (71%) cases with AI of MYC. Immunohistochemical staining of the 20 tumors with monoclonal antibodies specific for PSCA showed a high degree of correlation between PSCA gene overrepresentation and protein overexpression. Four of 5 tumors with AI of PSCA overexpressed PSCA protein, compared with only 2/15 tumors with a normal PSCA copy number or simple gain of PSCA (P = 0.014). These results demonstrate that PSCA is co-overrepresented with MYC in a majority of cases, but may not be a necessary part of the 8q amplicon. PSCA protein overexpression can result from AI of PSCA and might be useful as a cell surface marker on prostate cancer cells with 8q overrepresentation. Genes Chromosomes Cancer 27:95-103, 2000.