Cytogenetics and genetics of human cancer: methods and accomplishments

Cytogenetics and genetics of human cancer: methods and accomplishments

Cancer Genetics and Cytogenetics 203 (2010) 102e126 Review Cytogenetics and genetics of human cancer: methods and accomplishments Avery A. Sandberga...

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Cancer Genetics and Cytogenetics 203 (2010) 102e126


Cytogenetics and genetics of human cancer: methods and accomplishments Avery A. Sandberga, Aurelia M. Meloni-Ehrigb,* b

a 9185 E. Desert Cove, Scottsdale, AZ 85260 Cytogenetics Laboratory, Quest Diagnostics Nichols Institute, 14225 Newbrook Drive, Chantilly, VA 20151

Received 17 April 2010; received in revised form 22 September 2010; accepted 7 October 2010


Cytogenetic and related changes in human cancer constitute part of a constantly developing and enlarging continuum of known genetic alterations associated with cancer development and biology. The cytogenetic component of this continuum has fulfilled much of its pioneering role and now constitutes a small but dynamic segment of the vast literature on cancer genetics, in which it has played an important if not initiating role. The goals of this article are (a) to address historical and methodological aspects of cancer cytogenetics; (b) to present information on diagnostic translocations in leukemias, lymphomas, bone and soft tissue tumors, and carcinomas; (c) to connect some of these chromosomal aberrations with their molecular equivalents; and (d) to describe anomalies in some solid tumors indicative of the complexity of the genomic alterations in cancer. We also look at a few of the more recent genomic developments in cancer and offer an opinion as to what all these findings add up to. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Since its first application to the study of cancer, cytogenetics has taken us from a state of virtually no knowledge of the chromosome changes in human cancer to a point at which a staggering body of information is available. The latter is evidenced by the nearly 55,000 leukemic and tumor karyotypes now included in the Mitelman Database of Chromosome Aberrations in Cancer [1]. Now more than half a century old [2], the field of cancer cytogenetics has more than lived up to its envisioned task of finding recurrent or specific abnormalities associated with cancer, and continues to provide crucial diagnostic and prognostic information. In current practice, cytogenetic data often serve as a guide in other studies, ranging from the exploration of cytogenetic findings with various methodologies, singly or in combination, including fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), or microarray-based technologies such as comparative genomic hybridization to the use of immunohistochemical techniques by the pathologist. Cytogenetic data also provide key background information for the recognition

* Corresponding author. Tel.: (480) 990-9527; fax: (480) 990-2988. E-mail address: [email protected] (A.M. Meloni-Ehrig). 0165-4608/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.10.004

and identification of genes (and their networks) involved in cancer and for their subsequent application in therapeutic development. Progress in understanding the cytogenetic and molecular basis of neoplastic transformation has strengthened the conception of cancer as a genetic disease. Thus, the finding of apparently normal karyotypes in abnormal cells (as is seen in leukemias and various solid tumors) presents an enigma. It can be assumed that cryptic genetic changes are involved in such cases, as has been shown in some tumors and leukemias. These cryptic changes are not discernible with routine cytogenetic methods, but can be studied with special FISH methods (e.g., spectral karyotyping [SKY] and multiprobe FISH [M-FISH]) or, if a specific karyotypic change is suspected, with appropriate cosmid probes or other molecular means. Indeed, newer technologies promise to shed light on the more complicated and perplexing aspects of cancer that have eluded more traditional cytogenetic studies. For example, molecular studies have demonstrated fusion genes associated with prostate cancer and lung cancer that are not discernible cytogenetically. These findings raise the strong possibility that more epithelial carcinomas, which are usually associated with numerous or complex karyotypic alterations, will be shown to have cryptic primary genetic alterations. Given the daunting quantity of cytogenetic and molecular genetic information on cancer gleaned through the past

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Fig. 1. Metaphase of a cultured cell, as shown in the classic 1956 article by Tjio and Levan [6], containing 46 chromosomes and establishing that number as characteristic of the human normal chromosome complement. Reproduced with permission of the publisher.

half-century of research and clinical application, we cannot in an article cover the myriad known chromosome changes associated with cancer or their downstream effects. Rather, our goal is to focus on some particular conditions that bear directly on a few of the more complicated and perplexing aspects of cancer genetics. 2. Cytogenetic methodologies in cancer 2.1. Historical facets of cytogenetic methodologies In 1956, Tjio and Levan [6] confirmed the correct number of human chromosomes as 46 and established their karyotypic constitution in somatic cells (Fig. 1). Within a few years, the first meaningful chromosomal changes in human cancer were reported in leukemias [3e5]. A historical survey of some of the cytogenetic methodologies introduced over the years is presented in Figures 1e10. Each new method widened the recognition of karyotypic changes, increasing the resolution of cytogenetic details until the limits of microscopic visualization were reached. The evolution of cytogenetics presented in Figures 1e10 encompasses also molecular approaches such as FISH and array comparative genomic hybridization (aCGH). These techniques have revealed novel and otherwise cryptic rearrangements, as well as providing chromosome information for cases in which conventional cytogenetic analysis is not possible.

2.2. Special requirements and limitations of cancer cytogenetic studies Cytogenetic techniques require the presence of dividing cells (preferably in the metaphase stage) for the visualization of chromosomes. Thus, fresh specimens are necessary for establishing short-term cultures (in the case of marrow) or long-term cultures (in the case of solid tumors) cultures. Although uncultured marrow often contains sufficient dividing cells for cytogenetic studies, short-term culture allows for more efficient analysis [2,7e9]. The cytogenetic information in hematologic conditions has become so crucial to clinicians that chromosomal analysis is performed in almost all cases of leukemias and lymphomas. This is not yet true of solid tumors, in which the specimens are often fixed before a small portion is obtained for chromosome analysis. Nevertheless, useful genetic information (and including partial cytogenetic information) can be obtained from fixed specimens with appropriate FISH or other molecular techniques [7]. With increasing appreciation of the value of chromosome findings in the clinical and pathologic aspects of epithelial tumors, we can hope that surgeons and pathologists will become accustomed to securing fresh-frozen tumor tissue suitable for cytogenetic analysis. Cytogenetic studies also fail to provide the immediacy of pathologic examinations because of the long time required for culture, assay performance, and interpretation of results.


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Fig. 2. Metaphase (right) and karyogram (left) of a human uncultured bone marrow cell with the normal set of 46 chromosomes, as obtained in the early days of cancer cytogenetics, before banding. Although some chromosomes could not be identified with the certainty later afforded by banding, a large number of studies, particularly of leukemias, led to rapid development of cancer cytogenetics and the description of specific changes such as the Philadelphia chromosome [5], the t(8;21) rearrangement in acute myeloid leukemia [13,14], and loss of chromosome 22 in meningiomas [15], all of which were later confirmed with banding techniques. The presence of numerical chromosome changes and structurally abnormal chromosomes (marker chromosomes) could be readily ascertained, although the exact recognition of the origin of the latter was not often feasible at the time. Reproduced with permission from Sandberg 1990 [2].

Nonetheless, cytogenetics still plays a crucial role in the comprehensive diagnostic spectrum of lesions in toto. 2.3. Importance of performing cytogenetic studies in all forms of cancer The finding of a normal diploid karyotype in affected cells, as is seen in some of the acute leukemias, lymphomas, and solid tumors, is nonetheless diagnostically valuable in that it generally rules out conditions associated with characteristic chromosomal alterations. Furthermore, the apparently normal karyotype may harbor specific genetic changes, which require molecular methodology for detection. Examples include the KIT mutations [10] and gene amplification (ERBB2 in breast cancer and MYCN in neuroblastoma) [11,12]. Even in the absence of diagnostic or recurrent changes, cytogenetic studies are essential for patient follow-up (e.g., during or after therapy in acute leukemias), because the appearance of additional chromosomal alterations is an indicator of disease progression. Cytogenetic studies are important from a basic research viewpoint as well; failure to obtain cytogenetic information on all available tumors, particularly those of epithelial origin, holds back information from the Mitelman database [1], a database indispensable for the fuller understanding and practical application of the genetic changes in these tumors, Thus, ideally and when feasible, all neoplastic conditions should be studied cytogenetically, both at diagnosis and as part of follow-up. This

approach may yield crucial information for the pathologist, oncologist, and surgeon, as well as for researchers. When dividing cells are not available for cytogenetic studies after therapy, FISH, PCR, M-FISH, or microarray approaches can be used to evaluate genetic changes.

3. Recurrent specific chromosome changes in leukemias, lymphomas, and solid tumors Cytogenetic studies have played an essential role in identifying the recurrent chromosomal abnormalities that are characteristic of various cancers, including leukemias, lymphomas, and solid tumors (Tables 1e4). These changes represent genetic mechanisms that are thought to be responsible for the biology of the respective clinical conditions, and have become important components of diagnostic and prognostic criteria. In the following sections we provide an overview of two different classes of genetic alterations associated with cancer: activation of oncogenes and inactivation of tumor suppressor genes. We also offer a description of the types of genetic alterations most commonly associated with different types of cancer. 3.1. Chromosome changes involving oncogenes and tumor suppressor genes 3.1.1. Oncogenes Several types of alterations have been characterized in dominantly acting oncogenes, whose protein products

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Fig. 3. Schematic presentation of the bands characterizing each chromosome, based on methods introduced by Caspersson et al. [16] in 1970. The most widely used technique was and remains G-banding (based on Giemsa stain) [17e19]; Q-banding (based on quinacrine) and R-banding (which reverses the banding pattern seen with G-banding) were also in use. Subsequently, techniques were developed for C-banding (staining the constitutive heterochromatin characteristic of the centromeric region), T-banding (staining the telomeric region), and NOR-staining (staining the nucleolar organizing region in the satellites and stalks of acrocentric chromosomes) [2e8]. Banding methodologies have been most crucial in the development of cancer cytogenetics, because they allow recognition (although with some limitations) not only of each normal chromosome but also of chromosomes involved in translocations and other structural changes.

exhibit altered functions and serve to accelerate cell growth (Tables 1, 3, and 4). Rearrangements are a common source of activating mutations. Examples include amplification, as with MYCN and ERBB2, and aberrant juxtaposition of a promoter or enhancer of one gene with a protooncogene, which causes altered expression of an oncogenic protein (e.g., MYC or CCND1). This aberrant juxtaposition mechanism is often seen in lymphoid neoplasms (Table 2). Another scenario is exemplified by chromosome translocations, inversions, or insertions that lead to formation of fusion oncogenes, such as BCReABL1 in chronic myelogenous leukemia (CML) or EML4eALK in nonesmall cell lung carcinoma. The oncogene fusion mechanism has received increased attention, because many of these fusions lead to activation of protein tyrosine kinases (PTKs) in various types of cancer. Activating point mutations, as in the case of EGFR, are also common, but are not associated with detectable cytogenetic anomalies. Most of the cytogenetic changes summarized in Tables 1e5 involve activation of receptor proteins, especially PTKs. Receptor PTKs are a highly regulated family of proteins in normal cells, but may undergo activating mutations or structural alterations to become oncoproteins in human malignancies. As already noted, oncogenic activation of

PTKs can result from genetic lesions such as point mutations, deletions, or overexpression by gene amplification. Alternatively, chromosomal rearrangements such as translocations, inversions, and insertions that lead to formation of an oncogenic gene fusion can involve receptor PTK or other PTKencoding genes as fusion partners. In such a scenario, the PTK domain becomes fused to an oligomerization domain contributed by the other fusion partner to create a chimeric oncoprotein. For chimeric PTKs such as BCReABL1, oligomerization through protein self-association leads to ligandindependent auto- or cross-phosphorylation of the kinase domain. This causes constitutive activation of the PTK domain, resulting in aberrant stimulation of downstream signaling pathways. In most cases, these translocationderived gene fusions appear to represent very early genetic changes, suggestive of potential biological roles in oncogenesis. The development of therapeutic agents that nullify the oncogenetic effects of the aberrant PTKs (Tables 1 and 5) is an ongoing process, but their potential has been demonstrated by the success of imatinib mesylate, dasatinib, nilotinib, and other tyrosine kinase inhibitors for treatment of CML and most cases of gastrointestinal tumor [44,45]. The discovery and therapeutic exploitation of the BRCeABL1 fusion PTK in CML will be further addressed in section


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Fig. 4. High-resolution G-banded metaphase (A) and karyogram (B) of a normal cell with 46 chromosomes. With this methodology, the G-bands shown in Fig. 3 are further defined structurally by subbands, which can reveal changes not visible with standard banding techniques.

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Fig. 5. A GTG-banded karyogram (G-banding with trypsin and Giemsa stain) of a clear cell chondrosarcoma [20], showing a 46,XY,der(7)t(7;9) (q11.2;q13),der(9)del(9)(p13)t(7;9) karyotype.

3.2.1, which deals with specific alterations characteristic of leukemias and lymphomas. 3.1.2. Tumor suppressor genes Another type of gene rearrangement involves tumor suppressor genes, whose products normally serve as brakes on cell growth and runaway cell proliferation. Inactivation of tumor suppressor genes leads to uncontrolled cell proliferation and downregulation of apoptosis (programmed cell death). Tumor suppressor gene inactivation can be caused by a number of mechanisms [1,2], including deletion of the tumor suppressor gene, inactivating mutations such as del (13)(q14) in retinoblastoma and del(11)(p13) in Wilms tumor (Table 4), and epigenetic modification. In the last-named mechanism, DNA methyltransferases methylate cytosinephosphate-guanine (CpG) sites in the promoter regions of tumor suppressor genes, silencing their expression [46,47]. 3.1.3. Complex translocations involving oncogenes The activation of oncogenes sometimes results from complex genetic rearrangements. As an example, in

Figure 11 are shown fusion genes formed through translocations involving the oncogene ALK (located on 2p23) with a number of other genes in anaplastic large-cell lymphoma [48,49]. Several of these fusion genes have also been described in inflammatory myofibroblastic tumors [50,51]. In each of the fusion genes shown in Figure 11, the kinase domain of the neural-associated receptor tyrosine kinase gene (ALK ) is fused to an activating domain of another gene. Recently a cryptic fusion gene (EML4eALK ) in lung cancer has been described; this is discussed in more detail in section 4.2.2. The scenario for the Ewing sarcoma (EWS) breakpoint region 1 gene (EWSR1) (Fig. 12) is similar to that of ALK (Fig. 11). EWSR1 forms fusion genes through translocations with six other genes in cases of Ewing sarcoma and peripheral primitive neuroectodermal tumor (pPNET) [43]. In addition, EWSR1 is also involved in translocations and fusion genes in six unrelated tumors (and some of those partner genes are also involved in EWS). Within EWS, the nature of the fusion gene has definite prognostic implications [43].


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cancer genetics in the human species universally [57,58]. The perplexing question is why and how so many different cytogenetic changes are seen in cancer. 3.2. Chromosomal abnormalities characteristic of different types of cancer

Fig. 6. A schematic example of a specific translocation, t(2;13) (q35.2~36.1;q14.1), seen in alveolar rhabdomyosarcoma and leading to the fusion gene PAX3eFOXO1 (previously PAX3eFOXO1A) [9]. Arrowheads indicate breakpoints for the normal chromosomes 2 and 13; for the derivative chromosomes, arrowheads indicate fusion points.

Fusions involving the platelet derived growth factor receptor, beta polypeptide gene (PDGFRB) and 14 separate partner genes occur in myeloproliferative neoplasms (MPN), especially in those conditions associated with abnormalities of the eosinophils (Table 5). A similar situation applies to PDGFR genes [52e56]. The recurrent and consistent nature of chromosome changes in these cancers and leukemias (Tables 1e5), which occur in patients regardless of their hereditary, geography, race and background, points to an atavistic genetic background affecting

3.2.1. Leukemias and lymphomas Reciprocal translocations represent the most common chromosomal abnormality in leukemias and lymphomas [1,59]. These translocations (Tables 1, 3, and 4) usually affect oncogenes that have been identified in almost all of the conditions listed and lead to the genesis of abnormal fusion genes, resulting in the mutation or overexpression of components of the fused genes [1,2]. Among these, t(9;22)(q34.1;q11.2), which generates the Philadelphia (Ph) chromosome associated with CML, has been well characterized [60]. The identification of the BCR and ABL1 genes involved in the translocation has led to the discovery of the BCReABL1 fusion protein, a constitutively active PTK. This in turn has been the contributing factor in the development of PTK inhibitors that are effective not only against the BCReABL1 chimeric protein but also against other neoplasms producing PTKs [44,61]. Detection of abnormalities in leukemia is useful not only for diagnostic and treatment purposes, such as the t(15;17) (q24;q21) in acute promyelocytic leukemia (APL), but also for prognostic risk assessment, such as the t(8;21)(q22;q22) in acute myeloid leukemia (AML). There is continuing interest in understanding what causes identical translocations to yield different effects, a phenomenon seen particularly in leukemia [59]. Today, it is clear that mutations of genes such as KITand WT1 can change the prognostic outcome associated with favorable cytogenetic markers such as t(8;21)(q22;q22) and inv(16)(p13.1;q22.1) [62]. Other common abnormalities that may modify prognosis, even in patients with a normal karyotype or intermediate cytogenetic risk, include mutations in the FLT3, CEBPA, and NPM1 genes [62]. Clearly, more work needs to be done at the molecular level to understand and eventually prevent disease progression. Similar to what has been seen in leukemias, establishing the chromosome changes in lymphomas (Table 2) provides diagnostic information and also forms a basis for followup and prognosis [38]. For example, t(11;14)(q13;q32.3), which leads to the [email protected] fusion, is the molecular hallmark of mantle cell lymphoma and is detectable by Southern blotting, PCR, and FISH [38]. Occasionally, t(11;14) is found in B-cell chronic lymphocytic leukemia, prolymphocytic leukemia, and plasma cell myeloma. In particular, plasma cell myeloma with t(11;14) is characterized by CCND1 upregulation and lymphoplasmacytic morphology; these patients are now placed in a better prognostic outcome group than those with other translocations involving chromosome 14. Translocations involving BCL6 located on 3q27 are present in ~50% of diffuse large B-cell lymphomas and in 10% of follicular lymphomas)

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Fig. 7. Fluorescence in situ hybridization (FISH) methodologies were introduced and developed in the 1980s [21,22]. Modifications in subsequent years [23e28] led to a widespread analysis of chromosomal changes in metaphase and interphase cells not afforded by other methodologies. FISH studies have led to a considerable expansion in the recognition of karyotypic anomalies in cancer. (A) Painting probes for whole chromosomes 8 and 21 reveal a t(8;21) (q22;q22) in a metaphase from a case of AML. Whole chromosome painting probes identify chromosomes both in metaphase and interphase cells and are useful when the precise participants in translocations are not clear from the conventional cytogenetic analysis. (B) Centromeric probes for chromosome 8 reveal trisomy 8 (þ8) in an interphase cell from a case of AML. The centromeres of all chromosomes are characterized by highly repetitive sequences unique to each chromosome, and hence repetitive probes are available for each chromosome. These probes are particularly useful for establishing numerical chromosome changes. (C,D) Cosmid probes for a gene or locus can detect unique sequences in both metaphase spreads and interphase nuclei. These include probes for detecting gene amplifications (e.g., MYCN in neuroblastoma and ERBB2 in breast cancer). Specific cosmid probes are particularly useful for detecting fusion events in translocations. Thus, cosmid probes can reveal specific rearrangements, such as the t(9;22) characteristic of CML and the t(15;17) characteristic of APL. (C) A cosmid probe specific for t(9;22) reveals the characteristic Philadelphia chromosome in an interphase cell of a CML case. (D) A cosmid probe specific for t(15;17) reveals that characteristic translocation in an interphase cell of an APL case.

[1]. Alteration of the gene TAL1, either through translocation or deletion, is the most common genetic event in Tcell acute lymphoblastic leukemia (T-ALL) (seen in nearly 30% of cases) [38]. The presence of t(2;5)(p23;q35) and related translocations involving the anaplastic lymphoma

receptor tyrosine kinase gene (ALK ) at 2p23 is seen in ~67% of CD30-positive anaplastic large cell lymphomas with a favorable outcome [39e41]. The t(11;18)(q21;q21) is associated with 30e40% of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue


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Fig. 8. An example of metaphase FISH analyses with the MLL and AF10 probes (for the MLL and MLLT10 genes, respectively) identifies the MLLeMLLT10 rearrangement in a case of acute monocytic leukemia [29]. (A) Signal pattern obtained with the MLL dual-color probe with green and orange signals (fused yellow) appears on both chromosomes 11 and a separate green signal represents the duplicated 50 part of the MLL gene. (B) MLL dual-color probe and a whole-chromosome paint for chromosome 10 (red) confirms two normal (fused yellow) signals on both chromosomes 11 and the 50 segment of MLL (green signal) on the short arm of chromosome 10. (C) FISH with the red-labeled MLL probe and green labeled AF10 bacterial artificial chromosome (BAC) probe confirmed two MLL (orange) signals on both chromosomes 11, one AF10 (green) signal located on the short arm of chromosome 10, and one fused (yellow) signal, suggesting an MLLeMLLT10 fusion gene. (D) A schematic illustration of the MLL rearrangement. Two standard copies of the MLL gene are located on chromosomal regions 11q23 and the duplicated 50 part of the MLL gene contributing to the 50 -MLLeMLLT10-30 fusion is located on the short arm of chromosome 10.

lymphoma type, where it may be the sole karyotypic aberration [1]. Although most of the specific cytogenetic changes shown in Tables 1e4 can be ascertained molecularly, the presence of chromosomal alterations in addition to the specific ones has important clinical and prognostic implications, and such findings are strong indications for cytogenetic analysis, to establish the complete karyotypic picture in each leukemia and cancer. Examples include the recurrent additional changes seen in CML, usually indicative of disease progression: i(17q), additional Ph chromosome, and þ8 [1,60]. In other leukemias and cancers, the additional chromosome changes may or may not be recurrent, may not necessarily

indicate disease progression or a poor prognosis, and may vary from a few to numerous and complex alterations, the latter being particularly common in epithelial cancers [1]. 3.2.2. Solid tumors The occurrence of specific chromosome changes such as translocations in benign tumors (e.g., lipoma, leiomyoma) [1,2,12], as well as nonspecific changes in a number of others, bears witness to the role of genetic events in cellular proliferation without malignant aspects. In fact, the specific chromosomal alterations in benign tumors not only serve diagnostic purposes, but also are a means of differentiating them from benign neoplasms from their malignant

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Fig. 9. Multicolor FISH (M-FISH) analysis of a leukemic myeloid cell showing unique staining of each chromosome and a number of translocations: a der(1)t(1;5), two copied of a complex der(11)t(1;5;11), a der(17)t(11;17), and a der(14) composed of various segments derived from chromosomes 1, 5, 12, and 14 [30]. The last item is an example of the ability of spectral karyotyping and M-FISH to demonstrate complex translocations not feasible with conventional cytogenetics.

counterparts [12,63e66] (e.g., lipoma vs. liposarcomas, leiomyoma vs. leiomyosarcoma). Some of the genes affected in malignant tumors may be involved in the genesis of benign tumors. The same genes can be altered in a number of different tumors, but apparently at varying chronologies in tumor development and associated with different genetic changes and milieus. In many tumors, a specific translocation may be the only alteration present. Many cases, however, display additional structural or numeric karyotypic changes that may be responsible for, or at least are associated with, disease progression [1,2]. The relevance of additional abnormalities is also reflected by alterations in the expression of a number of genes (not evident cytogenetically) apart from those involved in the translocations. The exact cause or causes of these additional alterations is not known, and it remains uncertain whether the primary translocation per se is responsible for the basic genetic process underlying the tumor genesis. These additional changes usually vary from

tumor to tumor, even among tumors with the same diagnosis. With or without additional chromosome changes, tumors with specific translocations may exhibit a variety of anomalies at the molecular level. The genetic and molecular consequences of inversions and insertions, which are quite rare in leukemias and soft tissue and bone tumors, are probably similar to those associated with translocations, in that they can create fusion genes [12]. The translocation t(11;22)(q24;q12) in Ewing sarcoma (EWS) and related tumors (Table 3; Fig. 12) leads to the genesis of an abnormal fusion gene containing elements of FLII and EWSR1 involved in the breakpoint regions [43]. The products of this translocation show variability, because the breaks occur in different exons within the chromosomal bands indicated, and so lead to different fusion transcripts (Fig. 12). The clinical consequences of such variability are exemplified by the relatively unfavorable clinical course of EWS type 2 tumors, compared with EWS type 1 tumors. Although as many as 18 different


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Fig. 10. Array comparative genomic hybridization (array-CGH) approaches have been developed in which hybridization is performed on a matrix or microarray instead of metaphase chromosomes [31,32]. This provides a locus-by-locus measure of DNA copy number alterations that significantly overcomes some of the limitations of conventional CGH and provides a resolution a number of orders better than that of routine chromosomal CGH. A normal array-CGH plot of all the chromosomes is shown in panel (A). The gain of the X chromosome (green) and loss of the Y chromosome (red) indicates a normal female. Compare this with (B), in which the whole chromosome plot shows a pick indicating a gain in a region of chromosome 5. In (C), a detailed view of

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transcripts resulting from the FLIIeEWSR1 fusion have been identified, too few cases with the other fusion products have been examined to indicate the clinical significance of these various transcripts [43]. 3.2.3. Carcinomas Compared with leukemias, carcinomas are diagnosed relatively late in their development, thus allowing for the genesis of chromosomal rearrangements in addition to the primary genetic event. It is unlikely that the additional chromosome rearrangements are merely incidental to the primary karyotypic abnormality, because biologically and clinically these additional changes are associated with tumor progression. Although some of these chromosomal changes have been related to prognosis and tumor biology, relatively few recurrent chromosomal anomalies have been identified as characterizing these tumors [58] (Table 4). To some extent the role of these additional changes has been clarified by the demonstration of cryptic and specific fusion genes not discernible cytogenetically in prostate and lung cancers (see section 4.2). In all probability, other epithelial cancers will be shown to have similar specific changes. Thus, it is possible that the complex chromosomal alterations seen in carcinomas are additional to the basic fusion or other cryptic event and reflect the biology of the tumors involved.

4. Some genetic aspects of epithelial tumors, both benign and malignant In this section we present genetic findings in a few selected tumors that underscore some unique features of epithelial carcinomas. These include the presence of an array of changes in benign and malignant tumors of the salivary glands and the discovery of cryptic fusion genes in cancers of the prostate and lung. 4.1. Salivary gland tumors A comprehensive review of the genetic aspects of and molecular pathways involved in salivary gland tumors has recently been published, including an extensive bibliography [68]. Salivary gland tumors, both benign and malignant, were among the first solid tumors to be characterized cytogenetically and then also defined molecularly [1,68]. Information regarding these tumors is worthy of


emphasis, because they demonstrate a spectrum of the interrelation of histopathologic, cytogenetic, and molecular data (Table 6). 4.1.1. Benign salivary gland tumors Pleomorphic adenomas (mixed tumors) of the salivary glands arise in tissues that are located in an ontogenetic transitional zone, a region in which ectoderm and endoderm meet. The location of these tumors might contribute to the typical difficulty in histopathologic assignment. These tumors have been well characterized cytogenetically, and the karyotypes of hundreds of cases have been reported. Only 2e3% of pleomorphic adenomas become malignant and metastasize [68]. The most common cytogenetic change in benign tumor pleomorphic adenomas is t(3;8)(p21;q12) [69,70], with breakpoints invariably occurring in the 50 noncoding regions in both affected genes. This translocation brings the coding sequences of PLAG1 (located at 8q12) under the control of the 50 regulatory sequences of CTNNB1 (the gene for b-catenin, located at 3p21) and vice versa, resulting in upregulation of PLAG1 and downregulation of CTNNB1. PLAG1 is developmentally regulated and is normally expressed only in fetal tissues, whereas CTNNB1 is normally expressed in all fetal and adult tissues. Thus, the interaction between PLAG1 and CTNNB1 represents an unusual reciprocal exchange of expression control elements in tumors, with the coding sequences of both genes being preserved. In the t(5;8)(p13;q12) rearrangement, fusion of the leukemia inhibitory factor receptor gene (LIFR) and PLAG1 also involves promoter swapping. Although these translocations account for a large proportion of genetic changes in pleomorphic adenomas, some of the neoplasms have no apparent karyotypic abnormalities. With advances in molecular biology, O60% of pleomorphic adenomas with normal karyotypes were shown to contain cryptic intrachromosomal rearrangements of 8q leading to upregulation of PLAG1. Some of these tumors had intrachromosomal rearrangements on 8q11.2~q12 leading to gene fusions of CHCHD7ePLAG1 and TCEA1ePLAG1 [70], with breakpoints telomeric or centromeric to the PLAG1 gene. Taken together, these studies indicate that PLAG1 activation is a crucial pathogenetic event in pleomorphic adenomas, one that is more common than originally suggested by cytogenetics [69]. The gene involved in pleomorphic adenomas with rearrangements of 12q15 is HMGA2 (previously HMGIC ), which

= chromosome 5 plot reveals the specific gain. (D) An earlier study using conventional CGH shows the profiles of 17 ovarian cancers. Losses of chromosomal material (red bars to the left of each chromosome) and gains (green bars, to the right) are shown [33]. Note the large number of chromosomes involved, a finding not uncommon in epithelial cancers. The advantage of CGH, both array and conventional, is that it can provide genome-wide screening of chromosomal deletion or amplification (i.e., copy number alterations), with no need for metaphase spreads in the specimens tested [33e37]. Furthermore, the technique can be applied to both fresh and fixed material. With limited resolution, however, CGH cannot detect chromosomal structural rearrangements that do not lead to genetic imbalance. Furthermore, the data obtained from CGH represent an average value of all cells in a specimen. If the specimen is heterogeneous in genetic composition then inadequate or imprecise genetic imbalance information may be produced. Reproduced from [33] with permission of the publisher.


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belongs to the high-mobility group protein gene family [71]. Like PLAG1, HMGA2 encodes a DNA-binding transcription factor that affects growth factor signaling and cell cycle regulation [71]. HMGA2 is involved in a variety of benign mesenchymal tumors, such as lipomas, uterine leiomyomas, hamartomas of the breast and lung, fibroadenomas of the breast, angiomyxomas, endometrial polyps, and bone and soft-tissue chondromas [72]. Across the various tumor types, more than a dozen different fusion partners with HMGA2 have been identified [71]. It appears that the breakpoints in HMGA2 are a stronger determinant of tumor type than is the fusion partner. Two fusion genes have been identified in pleomorphic adenomas, NFIBeHMGA2 and FHITeHMGA2 [73]. The former and more common is the result of a t(9;12)(p24;q15) or ins(9;12)(p24;q15). As a result, major parts of the C-terminus of HMGA2 are replaced by the last five amino acids of the transcription domain of the NFIB protein. Warthin tumor (cystadenolymphoma) is a benign salivary gland tumor composed of epithelial and lymphoid parts. Cytogenetic examination of the cultured epithelial cells revealed a t(11;19)(q21;p13.1) as the sole chromosomal abnormality [74]. This translocation was shown to generate the fusion gene MAML2eCRTC1 (CRTC1 was previously named MECT1). Subsequent studies showed normal karyotypes in most Warthin tumors, but also revealed t(11;19) and deletions of 6p to be recurrent in some cases; nonrecurrent changes, including other translocations, were also found. The MAML2eCRTC1 fusion gene seen in most cases of mucoepidermoid carcinoma (MEC) has been observed in some Warthin tumors and possibly is indicative of the derivation of some MECs from Warthin tumors [75]. A recent report concluded that Warthin tumors rarely harbor t(11;19) and that these tumors are often classified as infarcted or metaplastic, features known to overlap considerably with MEC on purely morphological grounds; the authors stated that the presence of the t(11;19) favors the diagnosis of MEC [76]. 4.1.2. Malignant salivary gland tumors The malignant salivary gland tumors for which cytogenetic or molecular data are available include MEC, adenoid cystic carcinoma (ACC), polymorphous low-grade adenocarcinoma, adenocarcinoma, and malignant mixed tumors originating from pleomorphic adenomas. The t(11;19) rearrangement and its related MAML2eCRTC1 fusion (described in section 4.1.1) are expressed in 75% of all MECs [78e81]. Nonsalivary gland adenocarcinomas contained evidence of the fusion gene. The biological behavior of MEC with the MAML2eCRTC1 fusion gene is generally more favorable than that of MEC without it [79]. The t(11;19) rearrangement is of interest in that it has been observed in three different tumors: MEC, Warthin tumor (from which malignant salivary gland tumors may originate, but rarely), and clear cell hidradenoma (eccrine spiradenoma) [81]. The last is a benign tumor of the skin originating from the intraepidermal sweat ducts. The

MAML2eCRTC1 fusion transcript found in this tumor has the same fusion points as those in MEC and Warthin tumors. The same translocation and fusion gene have been described in MEC of the lung and thyroid. Thus, available findings indicate that MAML2eCRTC1 is not tumorspecific, but rather that the cells involved in the three tumors share a genetic link involving the development of both benign and malignant epithelial tumors. Variant translocations and alterations in MEC involving chromosomes other than 19 have been reported. These include t(11;17) (q22;p11), t(11;13)(q24;q12), and del(11)(q22). Also, in addition to the t(9;11) in MEC cell lines, other reciprocal and nonreciprocal translocations not involving either chromosome 9 or 11 have been demonstrated with SKY [82]. The available data indicate that the molecular consequences of the MAML2 fusion are very complex, with diverse effects on several different signaling systems. Molecular pathways involved with or affected by the MAML2eCRTC1 fusion gene include those of NOTCH1 [77], and CREB1 [83,84]. A report on the inhibition of the VNT1 pathway in salivary gland tumors through rearrangement of the WIF1 gene has appeared [85]. Observations suggest that fusion-positive and fusion-negative MEC are two different clinical and molecular subtypes, with the former having a more favorable course [79]. Cytogenetic studies have demonstrated that ACC frequently has structural alterations of chromosomes 6 and 9, including 6q deletions and a t(6;9)(q21;p21), as well as loss of chromosome 22 [86]. Polymorphous low-grade adenocarcinoma was shown to have rearrangements of 8q12, 12q13, and t(6;9)(p21;p22). In one study, 12q13 and 6p21 were found to be involved in rearrangements in both types of tumors, including t(6;12)(p21;q13). Alterations (mainly deletions and loss of heterozygosity) at 6q21 were demonstrated in ACC, indicating the possible presence of a tumor suppressor gene responsible for ACC initiation or progression. Comparative genomic hybridization studies showed losses at 12q13 and 19q associated with ACC development. Cytogenetic similarities between ACC and polymorphous low-grade adenocarcinoma have been described, possibly indicating that, although these tumors are in essence pathologically different, the similarities in chromosome changes point to a common pathogenetic background [87]. Similar to MAML2eCRTC1 fusion, fusions involving PLAG1 and HMGA2 may function by activating basic transformation pathways that operate in several cell types and tissues. From a diagnostic viewpoint, however, fusion genes of PLAG1, HMGA2, and MAML2 in salivary glands are tumor type-specific [68]. The cytogenetic and molecular findings in salivary gland tumors discussed here are indicative of their complexity in several aspects: the presence of recurrent and specific translocations and their fusion gene products in benign epithelioid tumors; the presence of t(11;19) and its fusion gene products in MEC of the salivary glands (a decidedly

A.A. Sandberg, A.M. Meloni-Ehrig / Cancer Genetics and Cytogenetics 203 (2010) 102e126 Table 1 Selected chromosomal rearrangements in leukemias and lymphomas that lead to the creation of a fusion gene encoding a chimeric or abnormal protein Chromosomal abnormality [clinical condition] Chronic myeloid leukemia t(9;22)(q34.1;q11.2) Chronic myelomonocytic leukemia t(5;12)(q33.1;p13) Acute myeloid leukemia inv(16)(p13.1q22.1) t(3;3)(q21.3;q26), inv(3)(q21q26) t(3;5)(q25;q35) t(6;9)(p23;q32) t(7;11)(p15.2;p15) t(8;21)(q22;q22.3) t(11;17)(q23;q21.1) t(9;11)(p22;q23) t(11;19)(q23;p13.1) t(11;17)(q23;q21.1) [APL] t(15;17)(q24;q21.1) [APL] t(16;21)(p11.2;q22.3) Acute lymphoblastic leukemia t(4;11)(q21.3;q23) t(1;19)(q23.3;p13.3) t(9;22)(q34.1;q11.2) t(12;21)(p13;q22.3) t(17;19)(q22;p13.3) t(X;11)(q13.1;q23) t(1;11)(p32;q23) t(6;11)(q27;q23) Acute leukemias of ambiguous lineage t(9;22)(q34;q11.2 t(4;11)(q21.3;q23) t(9;11)(p22;q23) t(11;19)(q23;p13.3) Lymphomas inv(2)(p23q35) t(X:2)(q11.1;p23) t(1;2)(q25;p23) t(2;3)(p23;q21) t(2;5)(p23;q35) t(2;17)(p23;q23) t(2;17)(p23;q35) t(2;19)(p23;q13.1) t(11;18)(q21;q21)


Abbreviations: APL, acute promyelocytic leukemia. Genes are listed by HUGO-approved symbols (http://www.genenames. org). Some genes may be referred to in the literature by aliases (e.g., AF1P or AF-1P as alias of EPS15, or ALO17 for RNF213) or by previous approved symbols (e.g., BIRC3, previously API2).

malignant tumor), lungs, and thyroid; the presence of t(11;19) in rare Warthin tumors (usually benign) undergoing transformation to MEC; and the diversity of molecular effects caused by the fusion genes. 4.2. Carcinomas associated with fusion genes but without apparent cytogenetic findings The cytogenetic findings in epithelial tumors (adenocarcinomas) are greatly outnumbered by those of hematologic neoplasias [48]. This has been primarily due to technical aspects (varying success with tumors in culture and limited


Table 2 Nonfusion genes in leukemias and lymphomas Chromosomal abnormality [clinical condition] Burkitt lymphoma t(2;8)(p12;q24) t(8;14)(q24;q32) t(8;22)(q24;q11) B-cell lymphomas t(1;2)(p22;p12) t(1;14)(p22;q32.3) t(1;14)(q22;q32.3) t(2;3)(p12;q27) t(3;14)(q27;q32.3) t(3;22)(q27;q11.2) t(9;14) (p13;q32.3) t(10;14)(q24;q32.3) t(11;14)(q13;q32.3) t(11;14)(q23.3;q32.3) t(12;14)(q24.1;q32.3) t(12;22)(p13;q11.2) t(14;15)(q32;q11) t(14;18)(q32.3;q21) T-cell lineage acute lymphoblastic leukemia t(1;7)(p34;q34) t(1;14)(p32;q11.2) t(7;9)(q34;q34) t(7;9)(q34;q31) t(7;10)(q34;q24) t(7;11)(q34;p13) t(7;19)(q34;p13) t(10;14)(q24;q11.2) t(11;14)(p15;q11.2) t(11;14)(p13;q11.2) Miscellaneous conditions t(1;14)(q21;q32.3) [Pre-B-ALL] t(5;14)(q31;q32.3) [Pre-B-ALL) t(14;19)(q32.3;q13.3) [CLL] t(2;7)(p12;q21.2) [MCL) t(7;14)(q21.2;q32.3) [MCL) t(1;14)(q21;q32.3) [PCM] t(4;14)(p16.3;q32.3) [PCM] t(14;16)(q32.3;q23.1) [PCM] t(X;14)(q28;q11.2) [T-PLL]

Genes juxtaposed [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

Abbreviations: ALL, acute lymphoblastic leukemia; Pre-B-ALL, preB-cell acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; MCL, mantle cell lymphoma; PCM, plasma cell myeloma; T-PLL, T-cell prolymphocytic leukemia. The nonfusion genes result from the juxtaposition of proto-oncogenes to genes of T-cell receptors ([email protected], [email protected], or [email protected]) or to immunoglobulin genes ([email protected], [email protected], or [email protected]), leading to gene overexpression.

availability of material for repeated cytogenetic studies) and, until recently, the relative lack of karyotypic changes of interest to the surgeon, oncologist, and pathologist. Furthermore, in the past, most adenocarcinomas were found to have complex and numerous cytogenetic alterations without recurrent or specific changes. The importance of recurrent fusion oncogenes in carcinomas resides not only in the fact that their demonstration came some years after those observed in leukemias and lymphomas, but also in the generally held conviction that the common carcinomas are generated through a stepwise process of tumor suppressor gene changes. The different types of


A.A. Sandberg, A.M. Meloni-Ehrig / Cancer Genetics and Cytogenetics 203 (2010) 102e126

Table 3 Specific chromosomal translocations established cytogenetically and the genes involved in bone and soft tissue tumors Translocations Aggressive angiomyxoma t(8;12)(p12;q15) t(12;21)(q15;q21.1) Alveolar soft part sarcoma t(X;17)(p11.2;q25) Aneurysmal bone cyst t(16;17)(q22;p13) t(1;17)(p34.3;p13) t(3;17)(q21.3;p13) t(9;17)(p22.3;p13) t(17;17)(p13;q21.3) Angiomatoid fibrous histiocytoma t(2;22)(q31,1;q12) t(12;16)(q13;p11.2) t(12;22)(q13;q12) Chondrosarcoma, extraskeletal myxoid t(9;15)(q22;q21) t(9;17)(q22;q11) t(9;22)(q22;q12) Clear cell sarcoma t(2;22)(q31.3;q12) t(12;22)(q13;q12) Desmoplastic small round cell tumor t(11;22)(p13;q12) Dermatofibrosarcoma protuberans t(17;22)(q21~22;q13) Endometrial stromal sarcoma t(6;7)(p21;p15.2) t(6;10)(p21;p11) t(7;17)(p15.2;q11.2) Ewing sarcoma and pPNET with EWSR1 translocations t(2;22)(q36;q12) t(7;22)(p22;q12) t(9;22)(q22;q12) t(11;22)(q24;q12) t(12;22)(q13;q12) t(17;22)(q21;q12) t(21;22)(q22.3;q12) Congenital fibrosarcoma t(12;15)(p13.1;q25) Congenital mesoblastic nephroma t(12;15)(p13.1;q25) Hemangioendothelioma, epithelioid t(1;3)(p36;q25) Hemangiopericytoma t(12;19)(q13;q13) Inflammatory myofibroblastic tumor t(1;2)(q21;p23) t(2;2)(p23;q13) t(2;17)(p23;q23) t(2;19)(p23;p13.1) Leiomyoma, uterine t(12;14)(q15;q23~24) Lipoblastoma 8q12 rearrangements t(8;8)(q12;q24.1) t(7;8)(q21.3;q12) Lipoma t(3;12)(q27~28;q15)




Table 3 Continued Translocations

Genes involved

t(12;13)(q15;q12) 6p21 rearrangements Liposarcoma, myxoid and round cell t(12;16)(q13;p11.2) t(12;22)(q13;q12) Low-grade fibromyxoid sarcomas t(7;16)(q34;p11.2) t(11;16)(p11.2;p11) Rhabdomyosarcoma, alveolar t(1;13)(p36.1;q14.1) t(2;13)(q36.1;q14.1) Synovial sarcoma t(X;18)(p11.2;q11.2) t(X;18)(p11.2;q11.2) t(X;18)(p11.2;q11.2) t(X;20)(p11.2;q13.3)


Abbreviations: pPNET, peripheral primitive neuroectodermal tumor. Multiple cytogenetic and gene changes seen in some malignant and benign tumors [42,43,63e66].

carcinomas characterized by fusion oncogenes indicate that the pathogenetic mechanisms involved in epithelial carcinogenesis are probably similar to those known in hematological and soft tissue malignancies and that other fusion oncogenes are likely to be identified in epithelial cancers [39]. These fusion genes were in some tumors associated with relevant chromosome changes (Table 4) and in others were discovered by molecular or other means of identifying cryptic chromosomal changes. Molecular studies have come to the aid of cytogenetics in revealing recurrent and specific genomic changes of possible key import in some tumors. When no recurrent cytogenetic change is present in a tumor (or in leukemia), attention is drawn to changes that may be submicroscopic or too subtle to be seen by cytogenetic techniques. 4.2.1. TMPRSS2eETS fusion genes in prostate carcinomas A bioinformatics approach termed cancer outlier profile analysis led to the identification of fusion oncogenes involving (a) the 50 untranslated region of the androgenregulated member of the type II transmembrane serine protease gene (TMPRSS2) located on 21q22.3 and (b) members of the ETS family of transcription factors, ERG (located on 21q22.2) or ETV1 (located on 7p21) [88]. Subsequently, a rare third fusion gene was described, involving the TMPRSS2 locus and another ETS family gene, ETV4 (located on 17q21) [89]. This constitutes an important contribution to establishing genomic changes in a common cancer of variable biology and clinical behavior. The results obtained support the hypothesis that dysregulation of ETS family members through fusion with TMPRSS2 may be a key event in prostate cancer development. The TMPRSS2eERG fusion gene has been detected in ~50% of prostate carcinomas. The elevated expression of

A.A. Sandberg, A.M. Meloni-Ehrig / Cancer Genetics and Cytogenetics 203 (2010) 102e126 Table 4 Specific cytogenetic changes characteristic of carcinomas and other solid tumors Chromosomal abnormality

Genes involved

Germ cell tumors i(12)(p10) Nonesmall cell lung cancer inv(2)(p21p23) EML4eALK Meningiomas 22 or del(22q) Congenital mesoblastic nephroma t(12;15)(p11;q25) ETV6eNTRK3 NUT midline (aggressive) carcinoma t(15;19)(q13;p13) C15orf55eBRD4 (alias NUTeBRD4) Mucoepidermoid carcinoma t(11;19)(p21;p13.1) MAML2eCRTC1 Warthin tumor t(11;19)(q21;p13.1) MAML2eCRTC1 Oligodendroglioma t(1;19)(q10;p10) Pleomorphic adenomas t(5;8)(p13.1;q12) LIFRePLAG1 Salivary gland tumors t(3;8)(p21.3~22;q12) CTNNB1ePLAG1 Prostate cancer del(21)(q22.2~22.3) ERGeTMPRSS2 t(7;22)(q21.2;q22.3) ETV1eTMPRSS2 t(17;22)(q21.3;q22.3) ETV4eTMPRSS2 Renal cell carcinoma, papillary inv(X)(p11q12) NONOeTFE3 t(X;1)(p11.2;q21) TFE3ePRCC t(X;1)(p11.2;p34) PSFeTFE3 t(X;17)(p11.2;q23) CLTCeTFE3 t(X;17)(p11.2;q25) ASPLeTFE3 Retinoblastoma del(13)(q14) RB1 Secretory breast carcinoma t(12;15)(p11;q25) ETV6eNTRK3 Thyroid carcinoma, follicular t(2;3)(q13;p25) PAX8ePPARG Thyroid carcinoma, papillary inv(10)(q11.2q21) RETeCCDC6 RETeNCOA4 (alias RETePTC3) cryptic inv(10)(q11.2q11.23) t(10;17)(q11.2;q23) RETeRIA Wilms tumor del(11)(p13) WT1 Multiple changes are seen in some tumors (e.g., renal cell carcinoma and prostate and thyroid cancers) [1,58].

TMPRSS2eERG fusion transcripts in prostate cancers has been shown to arise by intrachromosomal rearrangements with the 50 untranslated region of TMPRSS2 fusing inframe with the 30 coding domain of ERG [90e97]. Because both genes have the same transcriptional orientation and are located ~2.9 Mb apart on chromosome 21, an interstitial deletion or a more complex alteration of the intervening DNA is required. More than 20 genes are located within the ~2.9 Mb segment, and changes of some genes (ETS2 and HMGN1) have been shown to be associated with prostate cancer progression [89e93]. The ETV1eTMPRSS2 and ETV4eTMPRSS2 fusion genes are relatively uncommon. FISH and 100k oligonucleotide single nucleotide polymorphism array analyses have shown that a microdeletion


between the two genes (at 21q22) is the mechanism leading to the fusion gene in 60% of TMPRSS2eERGepositive prostate carcinomas. Taking into consideration the incidence of prostate cancer, the TMPRSS2eERG fusion gene may be numerically the most common structural genome aberration in human malignancy described to date. The TMPRSS2eERG fusion gene was detected in 21% of lesions in high-grade prostatic intraepithelial neoplasia, thereby confirming that this condition is a precursor of invasive prostate adenocarcinomas [94]. In addition, analysis of the same tumors by CGH allowed the conclusion that TMPRSS2eETS fusions are early events that precede the appearance of additional chromosomal alterations in prostate carcinogenesis. This raises the hypothesis that fusion oncogenes resulting from changes not detectible by conventional cytogenetics are key events in prostate carcinogenesis, with the additional chromosomal aberrations acquired later during tumor progression being responsible for the biological and clinical aggressiveness of the cancer. Heterogeneity of TMPRSS2 gene rearrangements in multifocal prostate cancer has been proposed as molecular evidence for their development as an independent group of tumors [95e99]. Furthermore, different parts of a prostate cancer may harbor different and distinct patterns of hybrid transcripts [100], suggesting that TMPRSS2eERG gene fusions may arise independently in different regions of a single tumor. This variability may be a reflection of the nearly 20 variant products containing different combinations of TMPRSS2 and ERG gene sequences. Other genetic abnormalities have also been associated with prostate cancer. Both FOXO3 (previously FOXO3A), located on 6q21, and WWOX, a tumor suppressor gene located on 16q23.3~24.1 [101], have been proposed as playing a role in prostate cancer development. The deletion of PTEN has been shown to be associated with a poor clinical outcome [102], as is the overexpression of the tumor protein TPD52 [103e106]. How these events relate to the presence or absence of the TMPRSS2eERG fusion gene and the clinical significance of the fusion gene and changes affecting other genes remain to be firmly established. The studies to date on TMPRSS2eERG in prostate cancer have shed light on the heterogeneity of this disease and its prognosis, the diversity and unbalanced genomic rearrangements of the fusion transcripts, and some clinical parameters [107e110]. Other genetic pathways in prostate cancer carcinogenesis and progression have been described [111]. 4.2.2. EML4eALK fusion gene in nonesmall cell lung cancer Based on reverse-transcriptionepolymerase chain reaction (RT-PCR) analysis of lung cancer specimens [112], an EML4eALK fusion gene was identified in 5 of 75 patients with nonesmall cell lung cancer (7%). These cases appear to constitute a distinct subgroup within this disease entityddiffering, for example, from another subgroup of


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Table 5 Chromosome segments involved in translocations with 5q33 (PDGFRB), the genes fused with GFRB, and the clinical conditions in which these abnormalities have been observed Segment Gene

Clinical condition



1q21 2p16.2 4q21.2 7q11.2 10q21.2 12p13.1


12q24.1 14q22.1 14q32.1 15q15.3 17p11.2

Myeloproliferative neoplasm with eosinophilia Chronic eosinophilic leukemia Eosinophilic leukemia Mast cell disease Chronic myelomonocytic leukemia Atypical chronic myeloid leukemia Chronic myelomonocytic leukemia (4 cases); mast cell disease with eosinophilia Myeloproliferative neoplasm Atypical chronic myeloid leukemia Myeloproliferative neoplasm Eosinophilic myeloproliferative neoplasm Juvenile chronic myelomonocytic leukemia

GIT2 NIN CCDC88C TP53BP1 SPECC1 (alias CYTSB) RABEP1 Chronic myelomonocytic leukemia


In these translocations, the 30 end of PDGFRB is juxtaposed to the 50 end of each of the genes indicated.

cases with mutations (deletions or nucleotide substitutions) of the epidermal growth factor receptor gene (EGFR). EML4 is located on 2p21 and ALK is located on 2p23. These genes are separated by ~12 Mb and have opposite

orientations. Thus, inversion of either gene, through an inv (2)(p21p23), is necessary to generate the fusion gene [112]. The EML4eALK fusion gene was not detected in 260 specimens from patients with AML, lymphoma, gastric cancer, or colorectal carcinoma, indicating its possible specificity for lung cancer [113]. The diversity of the breakpoint regions in the genes involved may have a biologic and clinical significance yet to be determined. Testing by RT-PCR for EML4eALK fusion gene in sputum cells has been advocated as a diagnostic approach for establishing the possible presence of nonesmall cell lung cancer [112]. 5. Diverse conditions associated with the same translocation: t(12;15)(p13;q25) Some translocations are not specific and can be associated with multiple types of cancer. For example, five different conditions have been associated with t(12;15) (p13;q25), a translocation that results in an ETV6eNTRK3 fusion gene (Table 7). The resulting fusion protein product has been identified in mesenchymal, epithelial, and hematopoietic malignancies, each of a different anatomic location [113e119]. The ETV6eNTRK3 gene fusion was first identified by cloning of the t(12;15)(p13;q25) rearrangement (Fig. 13) in a congenital (or infantile) fibrosarcoma, a mesenchymal malignancy of very young children. This

Fig. 11. The ALK gene is involved in fusion with a number of other genes [38e41]. Represented here are eight fusion genes associated with anaplastic largecell lymphoma (ALCL); eight fusion genes associated with inflammatory myofibroblastic tumor, four of which are also involved in ALCL; two fusion genes in papillary thyroid carcinoma; and one fusion gene in lung cancer. The puzzle demonstrated in this figure is how identical fusion genes (and the translocations responsible for them) are present in two different tumor typesdhow the same gene, ALK, is causally related to different tumor types and in the same tumor with a variety of genes. Shown in parentheses are fusion genes seen in papillary thyroid carcinoma, in which the genes fused to ALK are fused with other genes in these tumors. The translocations leading to the fusion genes in this figure are described elsewhere [39e41,44,48,49,51,67].

A.A. Sandberg, A.M. Meloni-Ehrig / Cancer Genetics and Cytogenetics 203 (2010) 102e126


Fig. 12. A situation similar to but less complicated than that shown in Fig. 11 involves the EWSR1 gene (alias EWS ). The EWSR1 gene is fused to six different genes in Ewing sarcoma (EWS) and in peripheral primitive neuroectodermal tumors (pPNET) and to five different genes in five other tumors (see abbreviations) [2,42,43]. In acute myeloid leukemia (AML), ERG is involved, as it is also in EWS. Abbreviations: DSRCT, desmoplastic small round-cell tumor; ESCS, endometrial stromal cell sarcoma; MLPS, myxoid liposarcoma; CCS, clear cell sarcoma; AFH, angiomatoid fibrous histiocytoma. The translocations leading to the fusion genes in this figure are described elsewhere [42,43].

rearrangement fuses the N-terminal SAM domain of ETV6 to the C-terminal PTK domain of NTRK3, generating a fusion protein that is similar in structure to other ETV6 chimeric PTKs. The ETV6eNTRK3 fusion protein has potent in vivo and in vitro transforming activity in several cell lineages including fibroblasts, hematopoietic cells, and epithelial cells. A comprehensive review of the ETV6eNTRK3 fusion gene and its role in various molecular pathways and the clinical conditions associated with the fusion gene has been published [113]. More recently, this translocation has been observed in a new entity, a distinctive salivary gland neoplasm with features resembling both salivary acinic cell carcinoma and low-grade cystadenocarcinoma and displaying strong similarities to breast secretory carcinoma [116]. Translocations involving ETV6 are often subtle and may be missed by conventional cytogenetic examination. 6. MPN with mutations of JAK2 and other genes Myeloproliferative neoplasms represent a cytologically and clinically heterogenous group of diseases, with recent updates to their genetic definition [120]. 6.1. Abnormalities involving JAK2 Janus kinase 2 (JAK2), located on 9p24, is a widely expressed gene whose protein, a tyrosine kinase, associates with the intracellular domains in a number of cytokine receptors and is essential to receptor function [121e124]. The description of JAK2 mutations in MPN, a change not

discernible cytogenetically, is noteworthy in that it represents a frequent finding in these neoplasms [121e124]. Involvement of JAK2 in cytogenetically established translocations had been described in hematological neoplasms, such as t (8;9)(p21;p24) with fusion gene PCM1eJAK2 and t(9;12) (p24;p13) with JAK2eETV6 fusion gene (in atypical CML, chronic eosinophilic leukemia, therapy-related AML, preB-ALL, and T-ALL) some years prior to the discovery of the JAK2 mutation [120,125]. However, the JAK2 gene came to the forefront when within a span of 6 weeks in early 2005 four research groups, each using different methodologies, reported mutations of this gene in MPN [121e124]. Confirming reports soon followed as well as hundreds of articles dealing with various aspects of JAK2 in MPN. The most common mutation of JAK2 consists of a GOT point mutation resulting in the substitution of phenylalanine for valine at position 617 (V617F) in the JAK2 gene and, less frequently, at the 1849GOT locus (JAK1849GOT), with involvement of other loci being a possibility. JAK2 mutations are found in nearly 100% of patients with polycythemia vera and in O50% of those with essential thrombocythemia or primary myelofibrosis. Thus, JAK2 mutation can serve as a reliable diagnostic marker for polycythemia vera and for ~50% of cases of essential thrombocythemia and primary myelofibrosis. Infrequent mutations of JAK2 can be seen less frequently in other MPN conditions (3% of chronic myelomonocytic leukemia and 5% of myelodysplastic syndrome), as well as in some cases of systemic mastocytosis and chronic neutrophilic leukemia [126]. The limited biologic effects of JAK2 mutations and the demonstration that the mutated gene is probably not the


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Table 6 Cytogenetic and molecular features of benign and malignant salivary gland tumors

Table 7 Five clinical conditions associated with t(12;15)(p13;q25) and the ETV6eNTRK3 fusion gene [113e119]

Chromosomal abnormality (frequency)



Congenital fibrosarcomaa

Pediatric spindle cell tumor. Good prognosis. The genetic changes differentiates congenital fibrosarcoma from other confusing tumors Pediatric tumor (cellular variant). Excellent prognosis. Trisomy 11 is not uncommon. Elevated levels of IGF2 mRNA. Subtype of infiltrating ductal carcinoma. Prognosis is age-related. Younger patients appear to have a better prognosis. Only two cases reported to date. The fusion gene was somewhat different from that of congenital fibrosarcoma, as were the transcripts. Features resembling both salivary acinic cell carcinoma and low-grade cystadenocarcinoma

Benign tumors Pleomorphic adenoma Rearrangements of 8q12 (40%) t(3;8)(p21.3;q12) t(5;8)(p13.1;q12) Rearrangements of 12q14.3 (8%) t(9;12)(p23;q15) Nonrecurrent clonal changes (23%) Apparently normal karyotype (30%)

Warthin tumor (cystadenolymphoma) Abnormal karyotypes (10%) t(11;19)(q21;p13.1) 6p rearrangements Malignant tumors Mucoepidermoid carcinoma t(11;19)(q21;p13.1) or variants Adenoid cystic carcinoma Alterations of chromosomes 6 and 9 t(6;9)(q21~25;p21~22) Polymorphous low-grade adenocarcinoma Alterations of 8q12, 12q13~15 t(6;9)(p21;p22) 22 Adenocarcinoma 6q Mixed tumors MYC and MDM2 amplified, as well as genes located on 8q12


PLAG1 rearrangements CTNNB1ePLAG1 LIFRePLAG1 HMGA2 rearrangements NFIBeHMGA2 Molecular evidence of PLAG1 rearrangement in most cases

Congenital mesoblastic nephroma Secretory breast carcinoma Acute myeloid leukemia

Salivary gland neoplasm MAML2eCRTC1

cause of MPN [127e129], and also the observation that the leukemias developing in JAK2 mutation-positive cases may involve cells that are negative for the mutation [130e132], raise a number of questions regarding the mutation. However, the clinical and laboratory domains affected (or not affected) by JAK2 mutations have not been clearly defined [133e135]. Much remains to be resolved regarding the role of JAK2 mutations (heterozygous and homozygous) in MPN, with or without other genomic alterations [136e142]. Some forms of MPN, particularly essential thrombocythemia and primary myelofibrosis do not have mutations in JAK2, but they are characterized by MPL mutations and other less frequent gene mutations (e.g., TET2, ASXL1, CBL, IDH1, and IKZF1) [143]. 6.2. Abnormalities involving the PDGFR genes Abnormalities of PDGFRA (located on 4q12) and PDGFRB (located on 5q33) in MPN have been described, often consisting of translocations [57,59e63]. The relationship of these changes to JAK2 status awaits exploration. Because the domains of the genes fused to the tyrosine kinase domain of PDGFRB differ, the question arises of how their actions are reflected in the cytologic and clinical aspects of the conditions shown in Table 5. Also, PDGFRB

a A typical karyogram for congenital fibrosarcoma is presented in Fig. 13.

receptors have differences in signaling, and other potential effects are reflected in the conditions listed on Table 5. Identification of the conditions shown in Table 5 is essential for detecting the patients in whom treatment with tyrosine kinase inhibitors is likely to be successful [58,64,65]. The tyrosine kinase inhibitors such as imatinib mesylate [44], dasatinib, and nilotinib interact with the ATP-binding site of protein tyrosine kinases and have specific inhibitory activity for the ABL1, ARG1, PDGFRA, PDGFRB, and KIT kinases. Although rearrangements in MPN involve the PDGFRA gene less often than PDGFRB, at least two distinct mechanisms have been demonstrated to produce such rearrangements. One results from the reciprocal translocations associated with atypical CML, such as t(4;22)(q12;q11.2), which leads to the PDGFRAeBCR fusion. The other is a unique interstitial deletion of a small chromosomal segment (~800 bp) within band 4q21 containing the CHIC2 gene, which produces the FIP1L1e PDGFRA fusion. In both instances the mechanism for PDGFRA activation affects exon 12. The 4q21 deletions are generally not visible using standard cytogenetic banding techniques, which explains why patients with these syndromes are usually found to have apparently normal karyotypes in the affected cells [125]. An entity known as 8p11 myeloproliferative syndrome, consisting of a number of different neoplasms (B-cell lymphoma, T-cell lymphoma, eosinophilia, myeloid hyperplasia, systemic malignant mast cell disease, and involvement of multilineage precursors), is characterized by constitutive activation of the FGFR1 gene. The location of this gene has been reassigned to 8p12. The 8p11 myeloproliferative syndrome is associated with translocations involving a number of other chromosomes and genes, such

A.A. Sandberg, A.M. Meloni-Ehrig / Cancer Genetics and Cytogenetics 203 (2010) 102e126


Fig. 13. G-banded karyogram with 47 chromosomes in a case of congenital fibrosarcoma associated with a t(12;15)(p13;q25) (see also Table 7). Note the extra copy of the derivative chromosome 15. Arrows point to the abnormal chromosomes. The exact role of additional changes in tumor biology has not been clearly defined, although there are general indications that they are associated with tumor progression.

as FGFR1OP (6q27), CEP110 (9q33.2), and ZNF (13q12) [125,144,145]. Transformation to acute leukemia may occur in patients with 8p11 myeloproliferative syndrome. Involvement of 8p12 in translocations in acute monocytic leukemia is characterized by erythrophagocytosis. Translocations such as t(8;13)(p12;q12), t(8;16)(p12;q13), t(8;19) (p12;q13), and t(8;22)(p12;q13) are noteworthy in relation to 8p11 myeloproliferative syndrome.

7. Salient aspects of cancer cytogenetics The roles played by microRNAs (miRNAs) are receiving much attention and constitute a rapidly developing field of study at many levels [146e156]. The miRNAs are short segments of RNA (~22 bases in length) that affect mRNA functions, most often by suppressing translation of the protein product or by promoting degradation of the mRNA transcript. The genetic revolution [148] raised by the discovery of miRNAs is likely not the last to be witnessed in cancer genetics. One of the values of miRNAs is that they can be detected and quantified in a variety of samples, including plasma and formalin-fixed, paraffin-embedded tissues. This makes them a valuable testing tool, particularly in the clinical arena. In diagnostics, miRNAs already represent an improvement over other approaches currently available; for example, their expression profile has proven helpful in classifying tumors and predicting outcome [146e156].

8. Concluding remarks In summary, the following aspects of cancer genetics are of note: 1. Chromosome changes, especially translocations, can serve as key diagnostic and prognostic elements in cancer. Thus, chromosome changes have been used in the classification of a variety of leukemias, lymphomas, and solid tumors, complementing morphologic criteria for diagnosis and prognosis [59,150]. Translocations may also correctly categorize several sarcomas and lymphomas lacking unequivocal morphologic features (e.g., PNET, alveolar rhabdomyosarcoma, synovial sarcoma, and mantle cell lymphoma). 2. Cytogenetic studies are often necessary for prognostics and follow-up of cancer patients (particularly for leukemias) and for determination of possible additional karyotypic alterations, which generally indicate more aggressive disease. 3. Chromosome changes serve to guide identification of the genes affected by the rearrangements. In cancer, the combination of cytogenetic and molecular studies (FISH, SKY, PCR, CGH, and related methodologies) can more clearly define pathogenetic pathways and the biologic functions of molecular markers than either approach


A.A. Sandberg, A.M. Meloni-Ehrig / Cancer Genetics and Cytogenetics 203 (2010) 102e126

alone. Such a dual approach should lead to less empiric and more biologically oriented approaches to tumor classification and, ultimately, to more efficient clinical use of biomarkers [28,34,37,157e161]. Findings based on the combination of cytogenetic and molecular approaches have already improved the criteria for diagnosis and prognosis of cancer and provide a basis not only for existing therapy, but also for new and more tailored treatments. In terms of tailored treatments, the emphasis is being shifted from molecular markers as such to their molecular pathways, which could provide additional targets for direct and indirect therapeutic interventions. This combination can also be predictive in nature; examples include (a) the more favorable outcome of pPNET with either t(11;22)(q24;q12) (resulting in the FLIIeEWSR1 fusion gene) or t(21;22)(q22;q12) (resulting in the EWSR1eERG fusion gene) [43] and (b) the unfavorable outcome of synovial sarcoma with t(X;18)(p11.2;q11.2), which results in the SSX1eSS18 or SSX2eSS18 fusion genes [9]. The hypothesis that specific clones of spontaneously evolving aneuploidies or karyotypes, rather than specific mutations, generate the individuality of cancers [162,163], may apply to at least some of the conditions discussed in this article. The nature, number, and specificity of chromosome changes in cancer may be determined in part in terms of the following cellular conditions: 1) cell origin and type (function); 2) cell age and maturation (differentiation); and 3) cell molecular pathways and their uniqueness. Obviously, these do not explain every cytogenetic situation or scenario encountered in cancer, but they may offer an avenue for deciphering what underlies the complexity of the chromosome changes. A challenging scenario involves the presence of specific translocations in each of the various subtypes of the acute leukemias. The cells in these conditions are of the same origin (marrow), but of different age and function. For example, AML with differentiation often contains a t(8;21) (q22;q22), a change not present in the somewhat younger myeloblasts characteristic of undifferentiated and minimally differentiated AML [59]. Furthermore, cells that are of identical origin (such as mesenchymal cells in the marrow), but of somewhat different age and possibly function, demonstrate their individuality through the specific chromosomal alterations seen in each subtype of acute leukemia. Cell origin may account for the different cytogenetic changes in tumors of the same organ, such as pleomorphic adenomas of the salivary glands. The effects of maturation on specific chromosome changes is illustrated by the recurrent translocations and other karyotypic anomalies seen in benign tumors such as lipomas, leiomyomas, pleomorphic adenomas, and meningioma, versus those in their malignant counterparts. The presence of the same cytogenetic changes in different types of tumors at different anatomic locations can possibly be explained by the susceptibility of cells of dissimilar origin to changes affecting similar and unique molecular

pathways. Thus, fusion genes and other genetic changes may function by affecting basic transformation pathways that operate in various cell types and tissues, and so may account for the similarity as well as for the wide range of cytogenetic and genetic changes in cancer. Dynamic gene repositioning, as part of the chromosome territories and the functional nuclear architecture, has emerged as an additional level of epigenetic gene regulation and may account for at least some of the cytogenetic and molecular events in cancer [164]. Thus, the maintenance of imprinting and nuclear architecture and functions unique to cell types might explain the panorama of the chromosome changes seen in some cancers. Activation of oncogenes may occur through loss of miRNA-directed repression, an event that should be given consideration when investigating the effects of mutations in cancer [165]. Furthermore, mutations that create miRNA-directed repression of a tumor suppressor gene might also impart a selective advantage to tumor cells. The additional genetic changes seen in conditions with and without specific cytogenetic changes, call for attention. Even though some of these additional changes are recurrent, most appear to have a stochastic nature that will have to be deciphered for a fuller understanding of the biology of tumors. The mountains of information gathered on the genetics of cancer have not answered the basic question of what is really the exact cause of each neoplasm. Although we might be far from fully understanding the genetics of cancer, the rapid and continuing progress made over the past 50 years seems nonetheless to suggest that current technological or conceptual shortcomings can be remedied. ‘‘He has made everything beautiful in its time. He has also set eternity in the hearts of men; yet they cannot fathom what God has done from beginning to end.’’ (Ecclesiastes 3:11, NIV)

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