A breakpoint map of recurrent chromosomal rearrangements in human neoplasia.

Cytogenetic studies over the past few decades have revealed clonal chromosomal aberrations in almost 27,000 human neoplasms. Many of these neoplasia-associated chromosomal abnormalities have been characterised at the molecular level, revealing previously unknown genes that are closely associated with the tumorigenic process. Information on chromosome changes in neoplasia is growing rapidly, making it difficult to identify all recurrent chromosomal aberrations. We have developed a computer program to ascertain, for the first time, all recurrent structural abnormalities in all haematological malignancies and solid tumours published up to June 1996. Out of 26,523 cases, a total of 215 balanced and 1,588 unbalanced recurrent aberrations were identified among 75 different neoplastic disorders. Our compilation of all recurrent balanced and unbalanced neoplasia-associated rearrangements should help in directing future efforts aimed at identifying the molecular mechanisms involved in tumorigenesis.

The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein.

The recurrent translocation t(8;16)(p11;p13) is a cytogenetic hallmark for the M4/M5 subtype of acute myeloid leukaemia. Here we identify the breakpoint-associated genes. Positional cloning on chromosome 16 implicates the CREB-binding protein (CBP), a transcriptional adaptor/coactivator protein. At the chromosome 8 breakpoint we identify a novel gene, MOZ, which encodes a 2,004-amino-acid protein characterized by two C4HC3 zinc fingers and a single C2HC zinc finger in conjunction with a putative acetyltransferase signature. In-frame MOZ-CBP fusion transcripts combine the MOZ finger motifs and putative acetyltransferase domain with a largely intact CBP. We suggest that MOZ may represent a chromatin-associated acetyltransferase, and raise the possibility that a dominant MOZ-CBP fusion protein could mediate leukaemogenesis via aberrant chromatin acetylation.

Tumor etiology and chromosome pattern.

Fibrosarcomas induced in Chinese hamsters and rats by Rous sarcomla virus and 7,12-dimethylbenz(a)anthracene are associated with nonrandom chromosome variation. Although histologically indistinguishable, the tumors induced by the virus or chemical in each host species are characterized by completely different karyotypic patterns.

A gene fusion network in human neoplasia.

Cancer is, at the cellular level, a genetic disease and acquired gene fusions play a causal role in the initiation of the neoplastic process either by activating proto-oncogenes or creating hybrid genes. We constructed a network by combining the 5' and 3' parts of all presently known gene fusions in human neoplasia and here we show that the observed network is fragmented and that the organization of the genes demonstrates a scale-free network topology with a power law degree distribution meeting the requirements of P(k) approximately k(-gamma), that is, conforming to the distributions found in naturally occurring networks such as the Internet and social or ecological networks. The results hence indicate that the complex system of pairwise interacting genes leading to neoplasia is governed by a universal principle.

A novel and cytogenetically cryptic t(7;21)(p22;q22) in acute myeloid leukemia results in fusion of RUNX1 with the ubiquitin-specific protease gene USP42.

Although many of the chromosomal abnormalities in hematologic malignancies are identifiable cytogenetically, some are only detectable using molecular methods. We describe a novel cryptic t(7;21)(p22;q22) in acute myeloid leukemia (AML). FISH, 3'RACE, and RT-PCR revealed a fusion involving RUNX1 and the ubiquitin-specific protease (USP) gene USP42. The genomic breakpoint was in intron 7 of RUNX1 and intron 1 of USP42. The reciprocal chimera was not detected - neither on the transcriptional nor on the genomic level - and FISH showed that the 5' part of USP42 was deleted. USP42 maps to a 7p22 region characterized by segmental duplications. Notably, 17 kb duplicons are present 1 Mb proximal to USP42 and 3 Mb proximal to RUNX1; these may be important in the genesis of t(7;21). This is the second cryptic RUNX1 translocation in hematologic malignancies and the first in AML. The USPs have not previously been reported to be rearranged in leukemias. The cellular context in which USP42 is active is unknown, but we here show that it is expressed in normal bone marrow, in primary AMLs, and in cancer cell lines. Its involvement in the t(7;21) suggests that deregulation of ubiquitin-associated pathways may be pathogenetically important in AML.

Gene expression profiling of leukemic cell lines reveals conserved molecular signatures among subtypes with specific genetic aberrations.

Hematologic malignancies are characterized by fusion genes of biological/clinical importance. Immortalized cell lines with such aberrations are today widely used to model different aspects of leukemogenesis. Using cDNA microarrays, we determined the gene expression profiles of 40 cell lines as well as of primary leukemias harboring 11q23/MLL rearrangements, t(1;19)[TCF3/PBX1], t(12;21)[ETV6/RUNX1], t(8;21)[RUNX1/CBFA2T1], t(8;14)[IGH@/MYC], t(8;14)[TRA@/MYC], t(9;22)[BCR/ABL1], t(10;11)[PICALM/MLLT10], t(15;17)[PML/RARA], or inv(16)[CBFB/MYH11]. Unsupervised classification revealed that hematopoietic cell lines of diverse origin, but with the same primary genetic changes, segregated together, suggesting that pathogenetically important regulatory networks remain conserved despite numerous passages. Moreover, primary leukemias cosegregated with cell lines carrying identical genetic rearrangements, further supporting that critical regulatory pathways remain intact in hematopoietic cell lines. Transcriptional signatures correlating with clinical subtypes/primary genetic changes were identified and annotated based on their biological/molecular properties and chromosomal localization. Furthermore, the expression profile of tyrosine kinase-encoding genes was investigated, identifying several differentially expressed members, segregating with primary genetic changes, which may be targeted with tyrosine kinase inhibitors. The identified conserved signatures are likely to reflect regulatory networks of importance for the transforming abilities of the primary genetic changes and offer important pathogenetic insights as well as a number of targets for future rational drug design.

Absence of late-replicating X-chromosome in a female patient with acute myeloid leukemia and the 8;21 translocation.

Autoradiography was used to demonstrate that the x-chromosome of the 45,X,-X,t(8;21) stemline of a female patient with acute myeloid leukemia (AML) was the active X-chromosome. This suggested that in patients housing AML with the 8;21 translocation, the loss of the inactive X-chromosome in females and of the Y in males (which is known to occur in nearly half of the patients) entails selective advantage to the stemline.

Consistent involvement of only 71 of the 329 chromosomal bands of the human genome in primary neoplasia-associated rearrangements.

In an attempt to quantify the nonrandomness of primary neoplasia-associated acquired chromosomal aberrations in humans, we have retrieved information from a computerized data base on the chromosomal abnormalities of 9069 human neoplasms. By restricting the survey to the 1985 cases with a solitary structural rearrangement, we attempted to limit the analysis to only those aberrations that were most likely to represent pathogenetically important, primary changes. The breakpoints of the primary abnormalities thus identified clustered to 71 bands. It furthermore turned out that 27 of the 41 oncogene sites known with reasonable precision (i.e., localized within one or two bands) coincide with bands consistently involved in neoplasia-associated rearrangements. These comparisons add to the evidence that acquired, cancer-associated chromosomal aberrations are nonrandom in distribution, that only a limited number of genomic sites are consistently involved in primary neoplasia-associated aberrations, and that the concordance between the breakpoints of primary aberrations and the location of cellular oncogenes is greater than predicted by chance.

Chromosomal imbalance maps of malignant solid tumors: a cytogenetic survey of 3185 neoplasms.

To assess the distribution of gains and losses of genetic material in malignant solid neoplasms, 11 tumor types for which at least 100 short-term cultured cases with clonal chromosome aberrations had been reported in the literature were selected. The study was based on cytogenetic information from 508 breast carcinomas, 447 malignant neuroglial tumors, 435 kidney carcinomas, 333 colon carcinomas, 304 ovarian carcinomas, 303 lung carcinomas, 209 testicular germ cell tumors, 206 head and neck carcinomas, 172 malignant melanomas, 142 Wilms' tumors, and 126 neuroblastomas. In each case, the net imbalances were calculated for each chromosome band. The profiles of gains and losses revealed that all tumor types display unique combinations of imbalances. However, there is also considerable overlap among the profiles of the different diagnostic entities, indicating that similar molecular mechanisms may be operative in the development of many types of neoplasia. Deletions were more common than gains in all tumor types, with chromosomes X, Y, 4, 10, 13-15, 18, and 22 and chromosome segments 1p22-pter, 3p13-pter, 6q14-qter, 8p, 9p, and 11p being particularly often deleted in the majority of tumors. To better delineate critical lost segments, deletion profiles based only on structural rearrangements were made for chromosomes 1, 3-12, and 17, which all had on average at least four registered deletions per band. The relative distribution of losses indicated that different bands/regions are affected in different tumor types and that, often, several distinct candidate tumor suppressor gene loci can be discerned within the same chromosome arm, e.g., 1p12-13, 1p22, 1p34, and 1p36 on the short arm of chromosome 1 and 7q22, 7q32, and 7q36 on the long arm of chromosome 7. The only chromosomes or chromosome segments more often gained than deleted were chromosomes 7 and 20 and the long arms of chromosomes 1 and 12, suggesting the presence there of dominantly acting growth-regulatory genes. The data presented in this study should be valuable as a guide for molecular genetic studies on allelic imbalances and for the interpretation of results from studies using comparative genomic hybridization.

Characterization of the CHOP breakpoints and fusion transcripts in myxoid liposarcomas with the 12;16 translocation.

Myxoid liposarcomas are cytogenetically characterized by t(12;16)(q13;p11). The translocation results in rearrangements of the CHOP gene in 12q13 and the FUS gene in 16p11, creating a fusion gene where the RNA-binding domain of FUS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP. In the present study, we have mapped 16 genomic breakpoints in the region of the CHOP gene and isolated and sequenced a new variant (type II) of the chimeric FUS/CHOP transcript. The genomic breakpoints were dispersed along a 7.50-kilobase pair region from a SstI cleavage site upstream of the promoter of CHOP to a PstI cleavage site within intron 1. Reverse transcriptase-polymerase chain reaction analysis of tumor samples demonstrated the presence of two variant fragments, 654 base pairs (type I) and 378 base pairs (type II) in size. Of the 13 samples analyzed, 7 showed the smaller, 3 showed the larger, and 3 showed both types of transcripts. We cloned and sequenced the two fragments and found in type II a novel fusion point in the FUS mRNA 275 base pairs upstream of that present in the type I transcript. In both types of transcripts the interrupted FUS is followed by the entire exon 2 of CHOP. As a consequence the normally nontranslated exon 2 is translated and in both types there is in the junction between FUS and CHOP a shift from a FUS glycine codon to a valine codon in the chimeric mRNA.

Chromosome abnormalities in eighty-three head and neck squamous cell carcinomas: influence of culture conditions on karyotypic pattern.

Short-term cultures from 115 squamous cell carcinomas (SCC) of the head and neck were cytogenetically investigated. Thirty-six of the tumors have been reported previously, whereas 79 are new cases. The material was divided into two series based on the medium used. The 80 tumors of series I were cultured in RPMI 1640 supplemented with fetal calf serum, glutamine, antibiotics, insulin, cholera toxin, and epidermal growth factor. The 35 tumors of series II were cultured in a chemically defined, serum-free medium with a low calcium concentration, MCDB 153, which stimulates epithelial growth while inhibiting fibroblasts. A total of 83 tumors with clonal karyotypic abnormalities were detected in the two series. Series II had a higher proportion of tumors with complex karyotypic changes than series I (43% versus 15%), a lower proportion of tumors with pseudo- or neardiploid clones characterized by simple rearrangements (3% versus 34%), and a lower frequency of unrelated clones (3% versus 24%), indicating that the different culture conditions favored growth of different cell populations. Except for rearrangements of 1p22, which were mainly found in series I, the distribution of breakpoints in structural aberrations was similar in the two series and clustered to several chromosomal bands or regions, in particular 11q13, 1p22, 1p11-12, 3p11-q11, 5q13, 1q25, 15q10, and 8q10. Unbalanced structural aberrations were more common in series II, frequently leading to loss of segments from chromosome arms 3p, 7q, 8p, 11q, 13p, 14p, and 15p, whereas gain of genetic material often involved chromosome arms 1q, 3q, 8q, and 15q.

Identification of cytogenetic subgroups and karyotypic pathways in transitional cell carcinoma.

The clinical course in urinary bladder cancer is difficult or impossible to predict based on conventional disease parameters. It is a reasonable hypothesis that the genetic aberrations acquired by the tumor cells, being instrumental in bringing about the disease in the first place, may also hold the key to more reliable prognostication. However, though 200 transitional cell carcinomas (TCC), the most common bladder cancer in the Western world, with clonal chromosomal abnormalities have been reported, our knowledge about the karyotypic characteristics of these tumors remains insufficient. The aberration pattern is clearly nonrandom, but no completely specific primary or secondary karyotypic abnormality has been identified, and the chronological order in which the aberrations appear during disease progression is not well known. The high degree of karyotypic complexity in epithelial tumors like TCC is one reason why our picture of the sequential order of cytogenetic evolution is unclear. To overcome some of these difficulties we have used several statistical methods that allow analysis and interpretation of the relationship between cytogenetic aberrations in TCC. We show that there exists a temporal order with respect to the appearance of chromosomal imbalances and that this order is highly correlated with tumor stage and grade. Analyzing changes in the distribution of imbalances per tumor in G1, G2, and G3 tumors, we suggest that progression involves the acquisition of cytogenetically detectable and submicroscopic genetic changes at comparable frequencies. By means of computer simulations, we show that the imbalances -9, +7, and 1q+ appear earlier than expected from random events and that -6q, -5q, -18, +5p, -22p, and -15 appear later than expected. Using principal component analysis, we identify two cytogenetic pathways in TCC, one initiated by -9 and followed by -11p and 1q+, the other initiated by +7 and followed by 8p- and +8q. The -9 pathway was correlated with stage Ta-T2 tumors, whereas the +7 pathway was correlated with stage T1-T3 tumors, i.e., +7 tumors appeared to be more aggressive. Although these pathways are well separated at earlier stages, they later converge to contain a common set of imbalances.

Cytogenetic analysis of 33 basal cell carcinomas.

Cytogenetic analysis of short-term cultures from 33 basal cell carcinomas (BCC), a type of neoplasm for which no previous karyological data exist, revealed clonal chromosome aberrations, all of them different, in 8 tumors. In 2 cases, 2 cytogenetically unrelated clones were detected, suggesting a multicellular origin in at least a subset of BCC. A remarkably high level of nonclonal structural rearrangements, mostly in the form of seemingly balanced translocations, was found in 23 tumors; namely, in 6 of 8 BCC with clonal karyotypic abnormalities and in 17 of 25 without. It is possible that some of these aberrations represent additional neoplastic clones, thus indicating an even higher level of cytogenetic heterogeneity in BCC. We think that the most likely interpretation of the results is that BCC may have a multicellular origin, reflecting field cancerization of the skin. During subsequent tumor development, the selection pressure narrows down the number of clones that infiltrate the surrounding tissue. The finding by karyotypic analysis of some apparently monoclonal, some polyclonal BCC, may reflect that different tumors have been examined at different points in the clonal evolution of the neoplastic cells.

Nonrandom chromosome abnormalities in short-term cultured primary squamous cell carcinomas of the head and neck.

We report the finding of clonal chromosome abnormalities in short-term cultures from 44 squamous cell carcinomas of the head and neck region. Eleven tumors had gain or loss of the Y chromosome, sometimes one clone with +Y and another with -Y, as the sole anomaly, whereas the remaining 33 all carried structural rearrangements and usually were cytogenetically complex with multiple aberrations. The chromosomal bands most frequently involved were, in decreasing order of frequency, 8p11-q11, 1p11-q11, 3p11-q11, 11q13, 13p11-q11, 1p13, 5p11-q11, 7p11-q11, 15p11-q11, and 14p11-q11. Almost one-half of the breakpoints were located in centromeric or juxtacentromeric bands. Recurrent aberrations included i(8q), i(5p), i(1q), del(3)(p11-12), del(5)(p11), t(1;1)(p13;q25), and der(14;15)(q10;q10). To see whether the karyotypic features of head and neck squamous cell carcinoma differ depending on exact tumor site, we added to the present series our previously published 23 karyotypically abnormal head and neck squamous cell carcinomas that had been cultured in the same way as the tumors of the present series. In the ensuing correlation analysis, tumors of the oral cavity and oropharynx and hypopharynx were found to share many features: highly complex karyotypes were frequent, often containing isochromosomes such as i(8q) and i(5p), and also rearrangements of 11q13 (often as homogeneously staining regions) and loss of genetic material from the short arms of chromosomes 3, 13, 14, and 15 were repeatedly seen. Laryngeal carcinomas, on the other hand, often had simple karyotypic changes.

Cancer risk in humans predicted by increased levels of chromosomal aberrations in lymphocytes: Nordic study group on the health risk of chromosome damage.

Cytogenetic assays in peripheral blood lymphocytes (PBL) have been used extensively to survey the exposure of humans to genotoxic agents. The conceptual basis for this has been the hypothesis that the extent of genetic damage in PBL reflects critical events for carcinogenic processes in target tissues. Until now, no follow-up studies have been performed to assess the predictive value of these methods for subsequent cancer risk. In an ongoing Nordic cohort study of cancer incidence, 3182 subjects were examined between 1970 and 1988 for chromosomal aberrations (CA), sister chromatid exchange or micronuclei in PBL. In order to standardize for the interlaboratory variation, the results were trichotomized for each laboratory into three strata: low (1-33 percentile), medium (34-66 percentile), or high (67-100 percentile). In this second follow-up, a total of 85 cancers were diagnosed during the observation period (1970-1991). There was no significant trend in the standardized incidence ratio with the frequencies of sister chromatid exchange or micronuclei, but the data for these parameters are still too limited to allow firm conclusions. There was a statistically significant linear trend (P = 0.0009) in CA strata with regard to subsequent cancer risk. The point estimates of the standardized incidence ratio in the three CA strata were 0.9, 0.7, and 2.1, respectively. Thus, an increased level of chromosome breakage appears to be a relevant biomarker of future cancer risk.

The FHIT and PTPRG genes are deleted in benign proliferative breast disease associated with familial breast cancer and cytogenetic rearrangements of chromosome band 3p14.

We have used nested reverse transcription-PCR (RT-PCR) and PCR on genomic DNA to search for aberrations in the FHIT and PTPRG genes, both located in chromosomal band 3p14.2, in specimens from cytogenetically analyzed benign breast lesions (three samples with atypical hyperplasia and one with fibroadenosis) from two women belonging to breast cancer families. The transcription analysis showed that the FHIT gene was either not expressed or that its expression was dramatically reduced to a level not detectable by nested RT-PCR in the samples with atypical hyperplasia. Genomic analysis of exons 3 and 5 of FHIT and exon 12 of PTPRG provided evidence that these DNA segments were homozygously deleted in the majority of the cells. These data are in line with the histopathological features and cytogenetic findings in the three samples; none contained normal parenchyma, and all had chromosomal aberrations involving band 3p14. RT-PCR analysis of the fibroadenosis specimen, which had a normal karyotype, detected the expected 856-bp fragment as well as an additional alternative transcript variant of FHIT with 1014 bp. The additional 158-bp sequence, which may add 38 amino acids to the NH2-terminal part of the previously described FHIT protein, was inserted between exons 4 and 5 and seems to be a new exon located in intron 4 of FHIT.

The reciprocal translocation t(9;16)(q22;p13) is a primary chromosome abnormality in basal cell carcinomas.

The reciprocal translocation t(9;16)(q22;p13) was identified in three short-term cultured basal cell carcinomas (BCCs). The t(9;16) was the sole anomaly in one clone in two tumors and was accompanied by a second change that also affected the long arm of chromosome 9 in the third. In addition, other cytogenetically unrelated abnormal clones were also found in all three BCCs. The identification of t(9;16)(q22;p13) as a primary chromosomal abnormality in a subset of BCCs (we found it in 3 of 22 tumors) is especially intriguing against the background that the PTCH gene, which when mutated in the germ line presumably gives rise to the autosomal dominant basal cell nevus or Gorlin's syndrome, maps to chromosome band 9q22. None of the genes rearranged in the BCC-specific t(9;16)(q22;p13) translocation have been identified, but we hypothesize that the translocation represents the cytogenetic corollary of a tumorigenic recombination of PTCH with an as yet unknown gene in 16p13. If so, this would be the first time that a tumor suppressor gene causally involved in a hereditary cancer is shown to be frequently rearranged through a specific translocation in sporadic carcinomas of the same type.

Two distinct FUS breakpoint clusters in myxoid liposarcoma and acute myeloid leukemia with the translocations t(12;16) and t(16;21).

The FUS gene, which maps to 16p11, is fused to the CHOP gene in the t(12;16) (q13;p11) that characterizes myxoid liposarcomas (MLS) and to the ERG gene in acute myeloid leukemia (AML) with t(16;21) (p11;q22). In the present study we have mapped the breakpoints within FUS in 13 MLS with t(12;16) and in one AML with t(16;21). This region of FUS is about 3.9 kb and contains four exons. The breakpoints clustered to two zones (1 and 2). A strong association was found between the two known types of FUS/CHOP transcripts and the genomic localization of the breakpoints. In all cases expressing only type I or both type I and II FUS/CHOP transcript the genomic breakpoints mapped to zone 1. In all cases expressing only the type II transcript the breakpoints occurred in zone 2. The breakpoint in the AML case was in zone 1, suggesting that in-frame fusion transcripts are selected by similar mechanisms in both MLS and AML.

Fusion of the EWS and CHOP genes in myxoid liposarcoma.

The translocation t(12;16)(q13;p11), which cytogenetically characterizes myxoid liposarcomas (MLS), results in a fusion of the CHOP gene in 12q13 and the FUS gene in 16p11, creating a chimeric FUS/CHOP gene. We have identified two cases of MLS with translocations giving rise to recombination between 12q13 and 22q12. The result was a fusion of the N-terminal part of the EWS gene in 22q12, involved in a number of mesenchymal tumor types, with the CHOP gene and the creation of an EWS/CHOP chimeric gene. The presence of the EWS/CHOP chimeric gene in MLS shows that (i) the N-terminal part of FUS may be replaced by the N-terminal part of EWS in a CHOP fusion oncoprotein (ii) the two N-terminal parts, when fused to certain transcription factors, have a common or very similar oncogenic potential and (iii) the tumorigenic process in MLS and the morphogenetically distinctly different EWS-associated tumor types may be related.

Characteristic sequence motifs at the breakpoints of the hybrid genes FUS/CHOP, EWS/CHOP and FUS/ERG in myxoid liposarcoma and acute myeloid leukemia.

We have sequenced the breakpoint regions in one acute myeloid leukemia (AML) with t(16;21)(p11;q22) resulting in the formation of a FUS/ERG hybrid gene and in four myxoid liposarcomas (MLS), three of which had the translocation t(12;16) (q13;p11) and a FUS/CHOP fusion gene and one with t(12;22;20)(q13;q12;q11) and an EWS/CHOP hybrid gene. The breakpoints were localized to intron 7 of FUS, intron 1 of CHOP, an intronic sequence of ERG and intron 7 of EWS. In two MLS cases with t(12;16) and in the AML, the breaks in intron 7 of FUS had occurred close to each other, a few nucleotides downstream from a TG dinucleotide repeat region. The break in the two MLS had occurred in the same ATGGTG hexamer and in the AML 40 nucleotides upstream from the hexamer. The third case of t(12;16) MLS had a break upstream and near a TC-dinucleotide repeat region and a sequence similar to the chi bacterial recombination element was found to flank the breakpoint. In the MLS with the EWS/ CHOP hybrid gene, the break in intron 7 of EWS had occurred close to an Alu sequence. Similarly, in all 4 MLS, the breaks in intron 1 of CHOP were near an Alu sequence. No Alu or other repetitive sequences were found 250 bp upstream or downstream from the break in the ERG intron involved in the AML case. In the AML, the MLS with ESW/CHOP and in one MLS with FUS/CHOP there were one, two and six, respectively, nucleotide identity between the contributing germline sequences in the breakpoint. In the other two MLS cases, two and three extra nucleotides of unknown origin were inserted between the FUS and CHOP sequences. At the junction and/or in its close vicinity, identical oligomers, frequently containing a trinucleotide TGG, were found in both partner genes. Our data thus show that all four genes-FUS, EWS, CHOP and ERG-contain characteristic motifs in the breakpoint regions which may serve as specific recognition sites for DNA-binding proteins and have functional importance in the recombination events taking place between the chromosomes. Different sequence motifs may, however, play a role in each individual case.