Need for Sufficient Viable Tumor Cells

Solid tumors may comprise substantial nonviable or hypocellular regions containing few neoplastic cells. Such regions may be extensively necrotic because the tumor cells have died, having outstripped their blood supply. Other regions of a tumor mass may be composed largely of blood (hemorrhage) or scar tissue (fibrosis). Therefore in the case of solid tumor karyotyping, it is crucial that the pathologist select a maximally viable region for analysis.

Unpredictable Tumor Cell Growth in Culture

Even when tumor tissue has been selected from an optimal region, the unpredictable growth of neoplastic cells in culture remains a major consideration. Benign tumors often contain only few mitotic cells and so one generally has to wait several days before such specimens proliferate actively in culture. In the meantime, the culture may become overgrown by nonneoplastic cells, such as fibroblasts. Perhaps surprisingly, even highly malignant solid tumors may grow poorly in tissue culture, despite the fact that they grew well in the patient. Such tumor cultures may occasionally be stimulated by the use of specialized culture media or growth factors, but it is impractical in most clinical cytogenetic laboratories to troubleshoot tissue cultures to optimize the growth of each tumor type. Therefore in practice it may be challenging to culture and karyotype certain types of pediatric tumors in the clinical laboratory. In the case of bone marrow cytogenetics, it is well known that posttherapeutic specimens can be difficult to analyze, being hypocellular and containing cells—reactive and/or neoplastic—that have been temporarily growth-arrested by therapy and therefore fail to give rise to metaphases in culture.

Nonrepresentative Cell Culture

It is also important to recognize that only a subpopulation of cells within a given sample might be capable of growing under a particular set of culture conditions. Therefore one cannot necessarily assume that a clonally abnormal karyotype is representative of the overall neoplastic process. For example, in a given tumor, the final karyotype might be representative of the components of the tumor that were more or less clinically aggressive, depending on which component was best suited for growth under the particular culture conditions used at that time.

Culture Overgrowth by Nonneoplastic Cells

All tumor samples contain mixtures of neoplastic and nonneoplastic cells. The nonneoplastic elements may include hematopoietic cells, fibroblasts, normal epithelial cells, endothelial cells, or glial cells, depending on the type and location of the tumor. Any of these reactive or support cell types might proliferate more successfully than the neoplastic cells in culture. Invariably, when a pediatric cancer submitted for cytogenetic analysis is reported to have a normal karyotype, the normal karyotype is a false-negative result, only signifying that the culture was overgrown by normal cells. Therefore the cytogenetic analysis must always be timed carefully, so that metaphases are analyzed at a point at which the neoplastic population is actively dividing. This technical challenge can, in the case of solid tumors, be met if the cytogeneticist develops familiarity with the distinctive morphologies of the various neoplastic and reactive cell types and then inspects the tissue cultures daily to determine when the neoplastic cells have the proliferative advantage.

Complex Karyotype

Beyond the initial challenges incurred at the culture stage, various additional considerations influence the success of cytogenetic evaluation in different types of pediatric cancer. For example, conventional karyotyping can be challenging in many high-grade solid tumors, in which the karyotypes are often complex compared with those seen in leukemias and lymphomas. A single metaphase cell from such cancers can contain dozens of clonal and nonclonal chromosomal aberrations, making it impractical to characterize the exact mechanisms of rearrangement responsible for each chromosomal aberration or to estimate the relative significance of any of these abnormalities.

Technical Limitations in Detecting Aberrations

There are certain aberrations that cannot be detected by karyotyping, because the resolution is too low. These include smaller deletions or amplifications and point mutations, but also cryptic or masked aberrations, which include, for example, the chromosomal translocation t(12;15) characteristic of congenital or infantile fibrosarcoma, congenital cellular mesoblastic nephroma, secretory breast carcinomas, and a subset of acute myelogenous leukemias ( Fig. 45-2 ). Another translocation that cannot be resolved by conventional karyotype analysis is the t(12;21), which is the most common translocation in pediatric leukemia. It is found in a large fraction of common acute lymphoblastic leukemia (ALL) in children and fuses the ETV6 (TEL) and RUNX1 (AML1) genes.

A, Giemsa banding studies in infantile fibrosarcoma do not reveal the characteristic, but cytogenetically cryptic t(12;15), associated with TEL-NTRK3 oncogenic fusion. Black arrows indicate the normal (top) and rearranged (middle) copies of chromosome 12, as demonstrated by TEL break-apart fluorescence in situ hybridization (FISH). B, The FISH studies reveal yellow probe fusion signal on the normal chromosome 12, whereas the t(12;15) results in split red and green FISH signals on rearranged chromosomes 12 and 15, respectively.

In essence, then, the major advantage of karyotypic evaluation of solid tumors is the breadth of the data provided. Notable challenges include the successful culture of representative tumor cells, particularly because this is essentially a one-shot approach, with no opportunity to return to the specimen, because the window of opportunity for culture is confined to the short period of viability of fresh cells after biopsy. Even when culture is successful, meaningful interpretation of the findings on karyotype may represent a further challenge, depending on the number and nature of the aberrations.

Although solid tumors are less readily accessible for biopsy compared with hematologic neoplasms, minimally invasive sampling by a percutaneous approach is becoming more common. Needle biopsy is often performed under ultrasound or computed tomographic (CT) guidance and can involve fine-needle aspiration or needle core biopsy of the tumor. Solid tumor samples obtained by these methods can be karyotyped successfully, but the small amount of starting material is a constraint in that fewer cultures can be established. Because the successful cytogenetic evaluation of tumors in standard practice is limited by these factors, alternative, more robust methods are needed for the successful routine demonstration of genetic aberrations. Traditionally, these have included fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) techniques, which have the distinct advantage of applicability to fixed material and therefore also to large cohorts of archival cases. Both FISH and molecular analyses can be straightforward also in fine-needle specimens.

Recombinant DNA Technologies

Southern Blot Analysis.

In pediatric solid tumors, leukemias, and lymphomas, recombinant DNA technologies, including Southern blot analyses of candidate genes relevant to particular disease states, can overcome limitations of conventional karyotype analysis for the detection of chromosomal aberrations ( Fig. 45-3 ). Although no longer used extensively in most clinical diagnostic laboratories, Southern blotting has served an important role in gene discovery. That is, genomic Southern blot analyses, coupled with PCR-based molecular cloning and gene sequencing strategies, have identified unknown partner genes fused to various known genes in chromosomal translocations. For example, the demonstration of immunoglobulin (IG) and T-cell receptor (TCR) loci rearrangements in leukemias and lymphomas, as evidenced by altered size of restriction enzyme fragments on genomic Southern blot analyses, has been essential in highlighting crucial regions that could be studied by molecular cloning strategies and that characterize the genomic translocation breakpoint junction sequences that define these tumors ( Fig. 45-4 ). The molecular principles that provide the basis for current clinical molecular cytogenetic diagnostic strategies (e.g., FISH) in pediatric leukemias and solid tumors are not substantially different from the recombinant DNA concepts used in Southern blotting. Some seminal examples of gene rearrangements characterized originally by Southern blotting and now detected routinely by FISH include MYC gene rearrangements, involving the IGH or IGL chain loci in Burkitt lymphoma, and MLL (KMT2A) rearrangements, of which the first characterized type was associated with t(4;11), resulting in MLL-AFF1 (AF4) fusion.

Detection of cryptic MLL bcr rearrangement by Southern blot analysis in treatment-associated acute myelogenous leukemia (AML). The karyotype was normal, but the French-American-British (FAB) M4 morphology and clinical history of dactinomycin exposure informed further analysis of MLL . The dash indicates the germline pattern, and the arrow shows a rearrangement, which panhandle polymerase chain reaction molecular cloning revealed to be an MLL internal tandem duplication.

(Modified from Megonigal MD, Rappaport EF, Jones DH, et al: Panhandle PCR strategy to amplify MLL genomic breakpoints in treatment-related leukemias. Proc Natl Acad Sci U S A 94:11583–11588, 1997.)

A, Southern blot analysis indicating MLL gene amplification in treatment-related acute myelogenous leukemia (AML). Southern blot analysis of HindIII- digested genomic DNA from French-American-British M4 AML cells (RUPN84) of a patient whose AML karyotype showed no evidence of chromosome band 11q23 rearrangement that would suggest MLL gene rearrangement and a cell line established from the same AML (2L1). Cohybridization with a MLL bcr-region probe and a loading control SCL probe demonstrates unrearranged but amplified MLL with signal intensity of 4.3 : 1 in the AML compared with normal peripheral blood mononuclear cell (PBMC) control DNA. B, Fluorescence in situ hybridization confirms MLL genomic amplification in RUPN84.

(Modified from Felix CA, Megonigal MD, Chervinsky DS, et al: Association of germline p53 mutation with MLL s egmental jumping translocation in treatment-related leukemia. Blood 91:4451–4456, 1998.)

Another application of Southern blot analysis has been in discerning gene copy number variation. One example is MLL gene amplification (see Fig. 45-4 ), involving an interlocus DNA rearrangement known as segmental jumping translocation in which the unrearranged MLL DNA segment is amplified and translocated to various regions of the genome.

Fluorescence and Chromogenic in Situ Hybridization.

Whereas conventional cytogenetic analyses are performed using staining techniques that highlight chromosomal banding patterns (1;2), the various molecular cytogenetic methods interrogate chromosomal regions of interest by using nucleic acid probes. Most molecular cytogenetic methods use some variant of in situ hybridization (ISH), in which DNA probes are hybridized and evaluated in the cellular context. These probes are usually cosmids, containing an approximately 40-kb sequence of interest or bacterial artificial chromosomes, containing hundreds to a few thousand kilobases of sequence. ISH assays can be performed with fluorescence or enzymatic detection, referred to as FISH and CISH (chromogenic in situ hybridization), respectively ( Figs. 45-5 and 45-6 ).

A, Giemsa-stained neuroblastoma metaphase cell (and with nucleus from a different cell on right) showing hundreds of variably sized double-minute chromosomes, as indicated by arrows. B, MYCN fluorescence in situ hybridization (FISH) evaluation of neuroblastoma metaphase cell, showing numerous extrachromosomal double minutes containing MYNC (green) whereas the reference chromosome 2 pericentromeric FISH probe (red) shows two copies.

MYCN chromogenic in situ hybridization in a neuroblastoma paraffin section. Peroxidase-DAB detection (brown) of MYCN fluorescence in situ hybridization probe demonstrates high-level MYCN amplification in neuroblastoma cells but not in fibrovascular nonneoplastic cells (arrow).

Flexibility of in Situ Hybridization: Substrate Options.

FISH and CISH analyses are performed routinely in cytogenetic preparations (metaphase spreads) and in paraffin sections and other archival pathologic preparations. Substantial advantages of using paraffin sections include the following: (1) the abundance of source material for each case permits retrospective and repeat analyses; and (2) the well-preserved cell morphology can guide evaluation of the chromosomal events in the relevant cell populations. However, a drawback in the use of paraffin sections is that the nuclei are generally incomplete, having been sliced through during preparation of the sections by microtomy. This limitation can be addressed by performing the ISH assays with nuclei disaggregated from thick (50- to 60-µµ) paraffin sections or from thin cores of the paraffin block.

Resolution.

Most ISH studies have a resolution of one to several megabases on metaphase spreads. Notwithstanding the aforementioned problems of interphase FISH on fixed material, the technique offers the advantage of higher resolution, given the less compact arrangement of DNA in interphase nuclei. Higher-resolution FISH techniques have been developed in which the DNA is essentially stretched out over a slide, as in direct visual hybridization (DIRVISH), resulting in a resolution of between 700 and 5 kb, or fiber FISH, in which the resolution is between 500 kb and just a few kilobases.

Highly Combinatorial Modifications of FISH.

Vari­ous molecular cytogenetic methods have expanded the capabilities of FISH, enabling evaluation of the entire genome or karyotype. Examples include comparative genomic hybridization (CGH) and spectral karyotyping (SKY). CGH is performed by extracting total genomic DNA from a tumor of interest and from a nonneoplastic control cell population. These DNAs are differentially labeled (e.g., tumor DNA with fluorescein and control DNA with rhodamine) and in the earliest applications were cohybridized against normal metaphase cells (metaphase CGH), although these methods have been rapidly supplanted by higher-resolution assays in which the sample DNAs are hybridized against arrayed clones (array CGH) or interrogated for presence of informative single nucleotide polymorphisms (SNPs) as an indicator of copy number and heterozygosity across the genome (discussed later).

An advantage of CGH, compared with conventional karyotyping, is that the tumor DNA can be isolated from frozen or even paraffin specimens, without any need for cell culture, thereby avoiding the selection pressures imposed by culture and instead capturing an overview of the in vivo situation, although diluted by the admixture of support stromal DNA. In addition, the array CGH methods (discussed later) can detect very small deletions, which would be overlooked by traditional cytogenetic banding assays and might even be missed by FISH. However, CGH does not detect the balanced chromosomal rearrangements, particularly the translocations that are the genetic hallmarks of many pediatric cancers. Furthermore, while detecting chromosomal amplifications and deletions reliably, the technique does not provide a functional assay in terms of expression level changes.

Genome-wide molecular cytogenetics can be performed using spectral karyotyping or M-FISH, in which entire panels of DNA probes are simultaneously hybridized against tumor metaphase cells. Whereas conventional FISH techniques involve hybridization of one or two fluorescence-tagged probes, the spectral karyotyping and M-FISH methods use probes for each chromosome or chromosome arm (24 or more probes). Each probe is then detected combinatorially, using different ratios of fluorescence markers, such as fluorescein and rhodamine. By varying the ratio of fluorescence tags, each chromosome can be visualized with a unique color ( Fig. 45-7 ). Thus spectral karyotyping enables a comprehensive ISH screen of the entire tumor cell karyotype. Spectral karyotyping and M-FISH are powerful research tools in tumor cytogenetics and have been useful in identifying rearrangements that are cryptic by conventional cytogenetic banding methods or that are highly complex. However, these techniques have generally been supplanted by array CGH and SNP profiling methods (see later).

Metaphase spectral karyotype in treatment-associated acute myelogenous leukemia, showing typical 5q deletion, among other clonal aberrations.

(Courtesy Hesed, National Institutes of Health, Bethesda, Md.)

Routine Applications of FISH.

Most ISH studies performed in clinical laboratories are focused on investigating deletions, rearrangements, or amplifications of particular gene loci or chromosomes. The probes used for FISH analysis of chromosomal translocations (and other balanced cytogenetic rearrangements) generally fall into two categories. First, there are those that hybridize to a region spanning a breakpoint on one involved chromosome, producing a split signal in the presence of chromosomal rearrangement (see Fig. 45-2 ), Second, there are probes designed to come together in the presence of a fusion, in which a probe labeled with one fluorochrome hybridizes to one partner gene and the other, differentially labeled, to its fusion partner, with these producing a fused fluorescent signal only in the presence of a gene rearrangement.

Maximizing Data Yield from Individual FISH Probes for Rearrangements.

Judicious selection of break-apart FISH probes can allow applicability to a wide variety of tumors, in which the probe interrogates a frequently rearranged gene such as EWSR1 in various solid tumors or MLL in leukemias. The EWS gene undergoes rearrangement with various ETS family genes in Ewing sarcoma and EWSR1 is rearranged in pediatric desmoplastic round cell tumors and clear cell sarcomas of soft parts, among others. Each of these diagnostically useful EWSR1 rearrangements can be demonstrated using a single EWSR1 break-apart FISH probe ( Fig. 45-8 ), with ultimate diagnosis requiring interpretation of the molecular findings in the context of the histology.

EWS break-apart fluorescence in situ hybridization probe, hybridized to Ewing sarcoma metaphase cell, in which probe components centromeric and telomeric to EWSR1 are detected with fluorescein isothiocyanate (green) and rhodamine (red), respectively. EWSR1 rearrangement is indicated by the breaking apart of the normal yellow ( green-red fusion) signal into the red and green components, which localize to different chromosomes as a result of translocation.

Additional gross genetic aberrations routinely accompanying chromosomal translocation may be exploited to refine interpretation of FISH-based analysis in certain contexts. For example, FOXO1 gene FISH can distinguish the PAX7-FOXO1 and PAX3-FOXO1 oncogenic fusions in alveolar rhabdomyosarcoma, given that the PAX7-FOXO1 (but not the PAX3-FOXO1 ) is invariably highly amplified in these tumors. This distinction is warranted because of its prognostic significance. In the typical scenario, in which routine pathologic evaluation by light microscopy of hematoxylin and eosin (H&E)-stained sections with accompanying immunohistochemistry has generated a reasonably narrow differential diagnosis, genetic testing by this type of assay is highly informative and increasingly being used. Generally, this somewhat looser FISH-based assay may be regarded as having optimal attributes, providing greater sensitivity than karyotypic analysis and being capable of detecting cryptic or masked translocations while not being burdened by the restrictive specificity of PCR-based assays, a feature that is especially advantageous for detection of variant translocations.

FISH in the Analysis of Gains and Losses.

Although quantitative assessment of gene amplifications and deletions can be obtained by quantitative PCR (qPCR) or FISH, in practice such assays are more often conducted in clinical laboratories by FISH-based methods. In the example of MYCN amplification in neuroblastoma, FISH detection is performed routinely (see Figs. 45-5 and 45-6 ). Assessment of MYCN amplification may alternatively be done by semiquantitative PCR, using a standard PCR assay with normal (unamplified) and known amplified controls and multiplexing the reaction to include a housekeeping gene along with the gene of interest. However, such assessments can be confounded by intratumoral heterogeneity. In neuroblastoma, this heterogeneity may encompass substantial variations in cellular composition and maturation, and thus careful selection of tumor tissue for analysis is essential. The analysis of poorly selected tissue for MYCN evaluation (e.g., submission of schwannian stroma) will otherwise yield a false-negative result. Moreover, contributions of different components of the neuroblastic tumor in the final DNA isolate can dilute out regional (neuroblast-specific) MYCN amplification, again yielding false-negative results. In this context, there is a distinct advantage to the FISH assays for MYCN amplification, because such methods permit correlation with cytology or morphology for the detection of regional heterogeneity of MYCN amplification, particularly when performed in tissue sections or multifocal touch imprints.

Detection of deletions by FISH is a more complex matter and particularly challenging when using fixed tissue, in which the assay is for an absence of signal and one is dealing with interphase nuclei, by definition incomplete (sliced through by microtomy), with an admixture of normal stromal cells further confounding the picture. Where cytogenetically abnormal metaphase spreads are available, these problems are largely circumvented, but there are other potentially more suitable technical strategies that might be used, including PCR-based assays— again with the caveat of normal cell admixture—to establish loss of heterozygosity (LOH). The immunohistochemical stain may provide a ready assay for such detection, as in the case of the SMARCB1 gene on chromosome 22q, commonly deleted in malignant rhabdoid tumors ( Fig. 45-9 ), and atypical teratoid rhabdoid tumors.

A, Rhabdoid tumor showing loosely cohesive cells, many containing intracytoplasmic inclusions (arrows). Nuclei are vesicular and contain prominent nucleoli (short arrows) (H&E stain). B, INI1 immunohistochemistry, showing entirely negative reaction in the neoplastic cells, reflecting loss of 22q, and with only the endothelial and occasional admixed inflammatory cells staining positively.

General Applications of PCR-Based Methods

Since the initial description of the basic PCR assay, critical modifications of the technique have led to massive expansion of the applicability of this technology. The initial methodology involved the use of a DNA template from which a sequence of interest could selectively be amplified. This methodology provides the basis for testing for LOH, which relies on the natural allelic heterogeneity of repeat sequences in the vicinity of a gene of interest. Where a genomic region is deleted, the concomitant loss of an adjacent allelic repeat sequence results in PCR amplification of only one version of the repeat sequence, thus producing hemizygosity (seeming homozygosity) at that locus.

Reverse-Transcriptase PCR Assay.

When a particular question revolves around the presence of a specific transcript, the starting material is RNA, which is subjected to reverse-transcriptase PCR (RT-PCR), a highly sensitive technique. The principle is that total RNA extracted from fresh, flash-frozen, or fixed material is reverse-transcribed into first-strand complementary DNA (cDNA). This provides the template for testing for the presence of fusion oncogene transcripts using primers complementary to the involved partner genes flanking the fusion point in the chimeric transcript and which cannot therefore generate a product from unrearranged normal transcript. The technique is extraordinarily sensitive, able to detect as few as 1 in 10,000 cells bearing a fusion transcript. The presence of just a small neoplastic component within a tissue sample for genetic testing is therefore sufficient for detection. Thus this PCR-based technology is useful for the detection of minimal residual disease and well suited for the surveillance of hematologic malignancy, in which serial peripheral blood or bone marrow specimens are obtained for routine follow-up of patients. However, this same sensitivity of PCR-based methods carries with it the risk for false-positive results, from cross-contamination of minute amounts of fusion gene templates within assays or detection of very-low-level biologically insignificant fusion transcripts within specimens. Although moot in the context of demonstrating uniquely tumor-associated aberrations (e.g., fusion products), ideally DNAse I treatment of the RNA substrate should be performed to eliminate the possibility of inadvertent genomic amplification during RT-PCR.

Specificity of RT-PCR Reaction.

Breakpoint variability within the genes involved in translocation-generated fusion oncogenes has prognostic implications in some cases. This is reported for Ewing sarcoma and synovial sarcoma, in which a favorable prognosis is associated with a type I fusion of EWS exon 7 to FLI1 exon 6 compared with all other combinatorial variations in Ewing sarcoma ( Fig. 45-10 ). SYT-SSX2 fusion in synovial sarcoma is similarly reportedly associated with better patient outcome. Assays may be designed to address breakpoint variability through choice of primers, selectively amplifying only one variant product, or by choosing consensus primers that will amplify several transcripts and then sequencing the product to establish the precise nature of the transcript. An indication of transcript type might already have been obtained from gel electrophoresis demonstrating the product size ( Fig. 45-11 ; see Fig. 45-10 ). PCR-based assays are best suited to the detection of such fine genetic detail, which cannot be detected by karyotypic analysis and only by specifically designed FISH assays. Currently, distinction among variant transcripts generally remains of academic interest because it provides no basis for therapeutic stratification.

A, EWS and FUS genes encode an N-terminal transcriptional activation domain, RGG-rich regions, RNA recognition motif, and a zinc finger. Arrows indicate various breakpoints reported in EWS or FUS oncogenes. B (top) , Domains encoded by the various ETS-family genes fused with EWS or FUS in Ewing sarcoma. Arrows indicate various breakpoints that have been reported in fusion rearrangements. The most common oncogenic rearrangements in Ewing sarcoma involve the fusion of the EWS exon 7 to FLI1 exon 6 (type 1 transcript) or exon 5 (type 2 transcript). B (bottom) , Domains encoded by the WT1 gene, which is fused with EWS in desmoplastic small round cell tumor. Two WT1 isoforms are produced by an alternative spicing event (±KTS) between zinc fingers 3 and 4. C (top) , Domains encoded by oncogenic fusions between EWS or FUS with an ETS family member in Ewing sarcoma. Dotted lines indicate regions that are variably included, depending on the breakpoint locations. C (bottom) , Domains encoded by the EWS-WT1 fusion oncogene in desmoplastic small round cell tumor. Dotted lines indicate encoded sequences that are variably included, depending on the breakpoint locations (RGG-rich region) and alternative splicing events (KTS). D, Reverse-transcriptase polymerase chain reaction demonstration of type 1 EWS-FLI1 transcript in Ewing sarcoma (lane 1). Positive controls for types 1 and 2 transcripts are in lanes 3 and 4, respectively.

Reverse-transcriptase polymerase chain reaction (RT-PCR) demonstration of various TPM4-ALK fusion oncogene transcripts in inflammatory myofibroblastic tumors, as distinguished by gel electrophoresis of PCR products generated with the same primers (A) , with primer hybridization sites as indicated (B) .

In the case of MLL translocations, not only can one of more than 50 different partner genes be fused to MLL but it has also been suggested that the various partner genes of MLL affect the prognosis. In this case, PCR with gene-specific primers has been one approach to identify fusion transcripts involving some of the more common partner genes of MLL, and primer combinations for different MLL transcripts can be combined in a multiplex PCR reaction. This is most feasible in ALL, where the MLL partner genes are less heterogeneous than in acute myeloid leukemia (AML). Alternatively, approaches such as cDNA panhandle PCR can detect the fusion transcript produced by the 5′- MLL partner gene 3′ rearrangement using primer sequences derived from MLL. This is accomplished by the generation of a stem loop template in which a known MLL sequence has been attached to the unknown partner sequence by reverse transcribing the first-strand cDNA from total RNA using a 5′- MLL -random hexamer adapter-3′ primer for the primer extension.

Additionally, genomic panhandle PCR approaches and long distance inverse PCR approaches are alternatives for characterizing translocations in which the sequence of only one of the involved genes is known. And increasingly, intron-capture methods, coupled with sequencing, are used in high-throughput sequencing screens to identify oncogene fusion partners.

One clinical scenario that requires the ability to detect a broad range of chromosomal translocations, including unknown or rare fusion variants, is in surveillance screening for evidence of a preleukemic state in chemotherapy-exposed patients, before these patients manifest frank secondary leukemia. Advances in sensitive molecular PCR-based screening methods and molecular cytogenetic screening methods such as FISH will better enable the prospective detection of chromosomal translocations, which is crucial in such applications.

Ewing Sarcoma

Ewing sarcomas are highly aggressive tumors, characteristically occurring in bone and soft tissue, but also, less commonly, in viscera, and in which the neoplastic cells are generally of the small, round, blue cell type. Ewing sarcoma and peripheral primitive neuroectodermal tumor (pPNET), although historically considered distinct entities, share molecular and morphologic features and are now regarded as essentially the same entity (herein referred to simply as Ewing sarcoma). Ewing sarcomas typically contain chromosomal translocations involving the Ewing sarcoma gene ( EWS or EWSR1 ), which is located on the long arm of chromosome 22. These translocations involve a number of partner genes (see Table 45-1 ), with the most common translocation being t(11;22)(q24;q12), resulting in oncogenic fusion of 3′ sequences of the FLI1 gene on chromosome 11 with 5′ sequences of EWS. FLI1 belongs to the winged helix-loop-helix transcription factor family that share a conserved 85-amino acid ETS domain that is present in all EWS-ETS fusions. The oncogenic EWSR1-FLI1 fusion gene encodes an activated version of transcription factor in which the DNA binding domain of FLI1 is fused to the more potent trans -activating domain (TAD) of EWSR1, replacing the TAD of FLI1. In variant translocations, EWS is fused with alternative ETS transcription factor family members (see Fig. 45-10 and Table 45-1 ) and, rarely, the EWS -related gene, FUS, can also be fused with an alternative ETS family member in Ewing sarcomas ( Fig. 45-18 ; see Table 45-1 ). The EWS family and ETS family gene translocations are apparently essential for oncogenesis, being found in almost all Ewing sarcomas.

Ewing sarcoma with t(2;16)(q35;p11), resulting in oncogenic fusion of the FEV1 and FUS genes ( arrows indicate translocation breakpoints).

These translocations are readily detected by conventional cytogenetic methods, even using needle biopsy material, because Ewing sarcoma cells grow well in tissue culture. The translocations can also be detected by FISH, typically using dual-color probes flanking the EWSR1 locus (see Fig. 45-8 ). Although this does not identify the partner gene fused to EWSR1, that information is generally unnecessary for diagnostic purposes. Another method for detecting these translocations is RT-PCR. Advantages of PCR include superior sensitivity, identification of the EWS fusion partner gene, and identification of the breakpoint locations within the genes. The most common fusion is between EWS exon 7 and FLI1 exon 6, known as type I fusion, whereas type II fusion involves fusion of EWSR1 exon 7 with FLI1 exon 5, and many further variants have been described (see Fig. 45-10 ). Although several studies have indicated that EWSR1-FLI1 breakpoint locations might be prognostic in Ewing sarcoma, with tumors bearing the type I transcript doing better than all others, more recent studies do not confirm this, and such information is not used routinely to guide therapeutic decisions.

Although histomorphologic and immunohistochemical features are often sufficient to establish the diagnosis of Ewing sarcoma, genetic corroboration of the diagnosis is highly reassuring in distinguishing Ewing sarcoma from other small round blue cell tumors, particularly in situations in which tumor arises at an unusual site, or outside the typical age range, in the context of smaller biopsies, or if tumor cell preservation is compromised and/or immunohistochemistry is suboptimal. Although the cells are essentially undifferentiated, and superficially similar or identical to those in many other small round cell tumors, there are nonetheless characteristic findings, such as subtle cytologic features and crisp cytoplasmic membrane–associated expression of the CD99 (O13-HBA71, pseudoautosomal gene-encoded membrane glycoprotein [ Fig. 45-19 ], or specifically for Erg -rearranged Ewing sarcoma, anti-Erg monoclonal antibody ) that aid the diagnosis.

Ewing sarcoma composed of undifferentiated round cells (A) arranged around thick fibrovascular cores and demonstrating strong and crisp CD99 expression confined to the cell surface (B) , producing a mosaic-like effect.

Thus the combined traditional histologic evaluation of H&E-stained sections, together with immunohistochemical profiling, enables distinction of Ewing sarcoma from, for example, a reasonable mimic in alveolar rhabdomyosarcoma by the demonstration of CD99 expression in Ewing sarcoma versus expression of myogenic markers such as myogenin, MyoD1, and desmin in rhabdomyosarcoma ( Fig. 45-20 ). The characteristic oncogene fusions in Ewing sarcoma (EWS-FLI1) and alveolar rhabdomyosarcoma (PAX-FOXO1) might contribute to the distinct cell differentiation profiles that separate these two round cell tumors immunophenotypically and morphologically. Evidence to this effect has been obtained from in vitro studies in which EWS-ETS oncogenes can induce neuroectodermal differentiation, whereas PAX-FOXO1 induces a myogenic program. Secondary genetic aberrations in Ewing sarcoma include TP53 mutation and CDKN2A deletion, which are each found in approximately 25% of cases and are more frequent in cases that are clinically aggressive and poorly chemoresponsive.

Solid variant of alveolar rhabdomyosarcoma composed of undifferentiated, moderately pleomorphic round cells histologically (A) , but with convincing evidence of myogenic differentiation, as demonstrated by strong and diffuse cytoplasmic expression of desmin (B) and nuclear reactivity for MyoD1 (C) and myogenin (D) . Of note, the percentage of nuclei reacting for both myogenin and MyoD1 is very high, as is typical of the alveolar variant of rhabdomyosarcoma.

Similarly, gain of 1q was strongly associated with relapse and poor outcome, and this is apparently due to overexpression of DTL (CDT2), a ubiquitin ligase gene located on chromosome 1q. Independent loss of 16q was also predictive of worse overall and event-free survival regardless of stage at presentation. In that study, gain of chromosome 12 was, at least in cases of localized disease at presentation, associated with worse event-free survival whereas deletion of 6p has been associated with worse outcome also. Genomic instability portends poorly insofar as tumors with more than three copy number alterations reportedly did worse than those with three of fewer. However, currently these secondary genomic findings are not used in therapeutic decisions.

Rhabdomyosarcoma

Rhabdomyosarcomas are malignant tumors featuring skeletal muscle differentiation, with several histologic subtypes (see Fig. 45-20 ). The major varieties in childhood are the embryonal and alveolar subtypes and, although there are classic morphologies of alveolar and embryonal rhabdomyosarcoma, the distinction is not always clear-cut, because a solid variant alveolar rhabdomyosarcoma exists and composite cases also occur. Therefore especially with decreasing biopsy size, traditional morphologic distinction between embryonal and alveolar subtypes of rhabdomyosarcoma is not always a reliable means of subclassifying the tumors but is nonetheless essential for appropriate therapeutic stratification of patients with these two biologically different tumors. Alveolar rhabdomyosarcoma exhibits a higher proportional nuclear reactivity for both myogenin and MyoD1 than embryonal rhabdomyosarcoma, but even this feature may be unconvincing in small biopsies and thus be of little value in firmly establishing the diagnosis. The biologically distinct nature of embryonal and alveolar subtypes is confirmed by genetic studies, which show translocations targeting the chromosome 13 FOXO1A (forkhead transcription factor) gene in most alveolar rhabdomyosarcomas, and nontranslocation cytogenetic alterations, including 11p deletion, trisomy 8, and trisomy 20, in embryonal rhabdomyosarcomas. In 70% to 80% of alveolar rhabdomyosarcomas there is fusion of the FOXO1 gene with the PAX3 gene on chromosome 2, and approximately 10% have fusions of FOXO1 with the PAX7 gene on chromosome 1. The PAX7-FOXO1 fusion is often highly amplified in the form of double-minute chromosomes (see Fig. 45-13B ), whereas the PAX3-FOXO1 fusions are not usually amplified. This difference appears to reflect the lower intrinsic expression of PAX7-FOXO1, relative to that of PAX3-FOXO1, with genomic amplification therefore required to provide a comparable and sufficient level of oncogenic transcript. The FOXO1, PAX3, and PAX7 genes encode transcription factors, and the PAX3-FOXO1 and PAX7-FOXO1 fusion oncogenes encode activated forms of those transcription factors that function to induce myogenic differentiation programs. Notably, alveolar rhabdomyosarcomas with PAX7-FOXO1 rearrangements were reported in one study to have better prognoses than those with PAX3-FOXO1 when comparing patients presenting with metastatic disease. Furthermore, an argument has been made for movement toward designation of rhabdomyosarcomas based on translocation status rather than on morphology; and in that vein, cases of morphologic alveolar rhabdomyosarcoma have been termed fusion-positive versus fusion-negative. The observation that gene expression profiles of fusion-negative alveolar rhabdomyosarcomas are indistinguishable from those of embryonal rhabdomyosar­comas might lend considerable support to such subclassification. Whether molecular categorization how­ever translates well to clinical prognosis and treatment stratification remains unresolved, and a combined morphomolecular diagnosis is currently considered best practice.

A proportion of cases of rhabdomyosarcoma lacking the canonical PAX-FOXO1 fusions show t(2;2)(p23;q35) or t(2;8)(q35;q13) in which PAX3 is fused with either NCOA1 or NCOA2, which are nuclear receptor transcriptional coactivators, whereas isolated individual cases of rhabdomyosarcoma were reported to show t(2;6)(p23;p21.1), t(4;22)(q35;q12) fusing EWSR1 with DUX4, and a complex t(8;13;9)(p11.2;q14;q32) apparently resulting in amplification of FGFR1-FOXO1 fusion.

Synovial Sarcoma

Synovial sarcomas may be monophasic ( Fig. 45-21 ), in which the tumor is predominantly composed of spindle cells, or biphasic, in which the tumor contains both spindle cell and epithelioid elements, with the latter exhibiting more convincing epithelial immunophenotypic characteristics or, uncommonly, poorly differentiated, with a primitive round cell morphology reminiscent of Ewing sarcoma. Apart from the epithelial markers epithelial membrane antigen (EMA) and cytokeratins, transducer-like enhancer protein 1 (TLE1) also has been validated from gene expression profiles as a reliable marker for synovial sarcoma. Synovial sarcomas feature translocation of chromosomes X and 18, t(X;18)(p11;q11). t(X;18) is found in more than 90% of synovial sarcomas but not generally in histologic mimics such as hemangiopericytoma, mesothelioma, leiomyosarcoma, or malignant peripheral nerve sheath tumor. The molecular underpinnings of t(X;18) are complex in that the oncogene on chromosome 18 ( SYT or SS18 ) can be fused with one of several almost identical genes (generally SSX1 or SSX2 and, rarely, SSX4 ) on chromosome X. SSX1 and SSX2 are adjacent genes and, given their close proximity, it is impossible to distinguish SS18-SSX1 and SS18-SSX2 translocations using conventional chromosomal banding methods. However, the alternative SSX fusions can be demonstrated by FISH or RT-PCR using specific probes or primers, respectively. One study has suggested that synovial sarcomas with the SS18-SSX1 fusion are typically biphasic, whereas those with SS18-SSX2 can be biphasic or monophasic and have better metastasis-free survival compared with those bearing SS18-SSX1 fusions. This morphologic-genotypic correlate is substantiated by studies of SS18-SSX1 and SS18-SSX2 interactions with SNAIL and SLUG, both transcriptional repressors of E-cadherin, which mediates mesenchymal to epithelial transition.

A, Monophasic synovial sarcoma, with interdigitating fascicular arrangement of fusiform spindle cells. B, Weak, patchy immunohistochemical staining for epithelial membrane antigen (EMA) in the spindle cells.

The SS18-SYT fusion protein is both necessary and sufficient for cellular transformation. SS18 and SSX contribute, respectively, transcriptional coactivator and repressor domains to the SS18-SSX fusion proteins, and, despite the lack of any DNA binding domain, exert an effect on gene transcriptional dysregulation apparently through association with chromatin remodeling proteins Trithorax (TRX) and the Polycomb repressor complex (PRC2). Mass spectrometry has revealed that SS18-SSX fusion proteins bind TLE1 and ATF2 to form a complex necessary for tumor cell survival. ATF2 is the DNA binding partner of SS18-SSX, and the epigenetic repression of ATF2 is dependent on TLE1, the recruitment of which can be abrogated by HDAC inhibitors, which have already shown considerable promise in preclinical trials. In addition, SS18-SSX competes physically with wild-type SS18, forming an altered complex lacking SMARCB1. This altered complex transactivates SOX2 , which is a diagnostic marker that is universally overexpressed in synovial sarcoma and essential for synovial sarcoma proliferation.

Adipose Tumors

Most subtypes of adipose tumors, whether benign or malignant, contain distinctive genetic aberrations (see Table 45-1 ). Useful diagnostic markers include 8q rearrangement in lipoblastoma, 12q rearrangement in lipoma, ring chromosomes in well-differentiated and dedifferentiated liposarcoma, and t(12;16) in myxoid or round cell liposarcoma.

Lipoblastomas are pediatric adipose tumors containing variable numbers of primitive cells (lipoblasts) and generally bearing translocations of the chromosome 8 long arm at bands 8q11-12, resulting in rearrangement of the PLAG1 zinc finger oncogene. Reports of individual cases each of lipoblastoma and lipoma bearing translocations involving 8q21.1 region in each case as well as with a t(9;12)(p22;q14) in a pediatric intramuscular lipoma add to the variability in observations in these benign fatty neoplasms. Hibernomas, which contain adipose cells with a brown fat phenotype, generally have rearrangements of the chromosome 11 long arm, although the gene target of that translocation has not been identified.

Another diagnostically useful aberration is the translocation of chromosomes 12 and 16 found in myxoid liposarcomas. This translocation is also found in round cell liposarcomas and is retained in myxoid liposarcomas that acquire round cell features and thereby undergo transition to higher histologic grade. t(12;16) results in fusion of the DDIT3 (CHOP) transcription factor gene on chromosome 12 to the TLS gene on chromosome 16, and the resultant fusion oncoprotein is an activated transcription factor. t(12;16) has not been found in other subtypes of liposarcoma or in other types of myxoid soft tissue tumors. It appears to be relevant therapeutically, in that patients with myxoid or round cell liposarcoma show impressive responses to trabectedin chemotherapy. However, liposarcoma is rare in childhood and virtually unheard of in the first decade of life.

Clear Cell Sarcoma: Malignant Melanoma of Soft Parts

Clear cell sarcomas of the soft tissues resemble cutaneous melanomas histologically, and therefore have been referred to as melanoma of soft parts. Immunohistochemically, the tumors are consistently positive for S-100 protein, usually at least focally for HMB45, microphthalmia transcription factor (MITF), and Melan A. Despite their histologic and immunohistochemical overlaps, clear cell sarcoma and true melanoma are different clinically. Whereas most melanomas are of cutaneous origin, clear cell sarcomas generally present as isolated masses in deep soft tissues, without apparent origin from, or involvement of, skin. Less commonly they arise in the gastrointestinal tract. Most clear cell sarcomas contain a chromosomal translocation, t(12;22)(q13;q12), while those arising in the gastrointestinal tract will more commonly bear the variant t(2;22)(q34;q12). The latter has, however, also been described in a minor subset of soft tissue–based clear cell sarcoma. These translocations have never been reported in cutaneous melanoma and therefore serve as a reliable markers in distinguishing these two tumor types ( Fig. 45-22 ). t(12;22) fuses the ATF1 gene on chromosome 12 with the EWS gene on chromosome 22. ATF1 encodes a transcription factor, and the biologic implications of the translocation are probably similar to those in Ewing sarcoma translocations (see earlier). t(2;22) fuses the CREB1 gene on chromosome 2, a CREB gene family member closely related to ATF1, with the EWSR1 gene.

Clear cell sarcoma karyotype showing balanced translocation, t(12;22), as the only cytogenetic aberration. Arrows indicate translocation breakpoints.

Desmoplastic Small Round Cell Tumor

DRSCTs are aggressive and chemotherapy-resistant neoplasms that arise most commonly from the peritoneum or intraabdominal soft tissues, particularly in adolescent males. They are composed of undifferentiated malignant small round cells in a striking desmoplastic reaction, ensnaring sharply demarcated islands of tumor cells ( Fig. 45-23 ). Almost all cases express an EWSR1-WT1 fusion oncogene resulting from translocation between the chromosome 11 short arm and chromosome 22 long arm. The EWS-WT1 oncoproteins are expressed as several isoforms (see Fig. 45-10 ), each of which is composed of the EWSR1 amino-terminal transactivation region fused to the last three of four zinc fingers at the WT1 carboxyl-terminal end. These oncoproteins result from fusion of EWSR1 exon 7, 8, or 9 to WT1 exon 8. The EWSR1-WT1 fusion typically results in expression of the carboxyl-terminal but not amino-terminal aspect of WT1 in DSRCT, which may be detected by immunohistochemistry using appropriate antibodies (see Fig. 45-23 ). Quantitatively minor secondary transcripts lacking exon 6 of EWSR1 and/or exon 9 of WT1 are also expressed, although the functional relevance of these transcripts is unknown. However, it is worth noting that the usual immunostaining pattern was not observed in an isolated reported case with atypical transcript that lacked WT1 exons 9 and 10. The EWSR1-WT1 oncoprotein retains an alternative splicing site (±KTS) between the third and fourth WT1 zinc fingers (see Fig. 45-10 ). The KTS− isoform regulates genes that are believed to have important biologic roles in DSRCTs, including platelet-derived growth factor-α (PDGFA) and insulin-like growth factor receptor I, whereas the KTS+ isoform regulates a different set of genes. Notably, only the KTS− isoform has shown transforming activity when evaluated in vitro. PDGFA is known to activate potent receptor (platelet-derived growth factor receptor [PDGFR])-mediated mitogenic signaling pathways in fibroblasts, and EWSR1-WT1 might therefore induce desmoplastic proliferation via PDGFA transcriptional upregulation.

A, Desmoplastic small round cell tumor with sharply demarcated islands of blue tumor cells separated by pink densely fibrotic “desmoplastic” stroma. Immunoreactivities with antibodies to N-terminal (B) and C-terminal (C) regions of WT1 are negative and strongly positive, respectively, in the tumor cells, in keeping with EWS-WT1 fusion oncoprotein expression.

Dermatofibrosarcoma Protuberans

DFSP is a low-grade spindle cell tumor, which can occasionally progress to a more aggressive fibrosarcomatous phase. Most DFSPs contain ring chromosomes composed of sequences from chromosomes 17 and 22 (see Fig. 45-13A ). The ring chromosomes contain multiple copies of a fusion gene, COL1A1-PDGFB, in which COL1A1 (a collagen gene) is contributed from chromosome 17 and PDGFB (platelet-derived growth factor-β gene) from chromosome 22. Alternatively, DFSP, particularly in pediatric patients, occasionally has t(17;22) translocations, resulting in a single copy of the COL1A1-PDGFB fusion gene. The COL1A1-PDGFB oncogene results in overexpression of PDGFB, which is a growth factor that activates platelet-derived growth factor receptor-β (PDGFRB) and PDGFRA. This cytogenetic observation has suggested that patients with inoperable DFSP might benefit from treatment with PDGFR inhibitors such as imatinib, and this hypothesis has been confirmed by impressive clinical responses to PDGFRB therapeutic inhibition with imatinib. COL1A1-PDGFB oncogenic fusion is also seen in giant cell fibroblastoma (GCF), a pediatric neoplasm closely related to DFSP. Studies of GCF and composite GCF/DFSP cases have shown the presence of a single translocation t(17;22) and the resultant COL1A1-PDGFB fusion, in the GCF component, with acquisition of additional copies of the oncogene being associated with progression to DFSP.

Desmoid Tumors

Desmoid tumors, also known as deep fibromatoses, contain various genetic aberrations, including the APC (adenomatous polyposis coli) gene or CTNNB1 mutations, and trisomies for chromosomes 8 or 20. Cytogenetic deletions of the chromosome 5 long arm are seen in occasional desmoids, resulting in loss of the APC tumor suppressor gene. However, the most common mutations, particularly in nonfamilial desmoid tumors, are activating CTNNB1 mutations, which are found in at least 50% of cases. Both APC inactivating and β-catenin activating mutations result in stabilization and resultant overexpression, of the β-catenin protein with roles in cell proliferation, motility, and differentiation. This overexpression of β-catenin can be detected immunohistochemically in a nuclear distribution. In a retrospective analysis of primary desmoid tumor, the precise β-catenin mutation type was strongly predictive of recurrence, with S45F being associated in multivariate analysis with decreased time to recurrence and overall percentage recurrence. The cytogenetic aberrations in most des­moid tumors, particularly the trisomies 8 and 20, are mechanisms of progression and are acquired subsequent to the APC or CTNNB1 mutations. Interestingly, an as yet unexplained association of desmoidal fibromatosis in patients with gastrointestinal stromal tumor (GIST) has been reported.

Infantile Fibrosarcoma

Infantile (also congenital/infantile) fibrosarcomas characteristically present within the first year of life. These are tumors composed of plump fibroblast-like cells (see Fig. 45-16 ), which often show high mitotic activity, and feature trisomies of chromosomes 8, 11, 17, and 20. This same group of trisomies occur in the histologically identical pediatric renal tumor, congenital cellular mesoblastic nephroma (see Fig. 45-18 ). Most infantile fibrosarcomas and congenital cellular mesoblastic nephromas also contain a diagnostic chromosomal translocation, t(12;15)(p13;q26), which is cryptic when studied by traditional cytogenetic banding methods (see Fig. 45-2 ). A complex three-way t(12;15;19) translocation has also been reported in infantile fibrosarcoma. t(12;15)(p13;q26) results in fusion of the TEL gene on chromosome 12 to the NTRK3 receptor tyrosine kinase gene on chromosome 15, producing constitutive tyrosine kinase activation. Although challenging to detect by banding methods, the t(12;15) translocation is demonstrated readily by FISH with a dual-color break-apart TEL probe (see Fig. 45-2 ) or RT-PCR assay.

Inflammatory Myofibroblastic Tumor

Inflammatory myofibroblastic tumors, belonging to the group of inflammatory pseudotumors, are relatively rare tumors occurring primarily in children and young adults. They are composed of myofibroblastic cells admixed with an infiltrate of lymphocytes and plasma cells (see Fig. 45-17 ). The inflammatory component of these tumors is nonneoplastic and therefore lacks cytogenetic aberrations, whereas the myofibroblastic cells often contain clonal chromosomal aberrations involving rearrangement and oncogenic activation of the ALK receptor tyrosine kinase gene at chromosome band 2p23. The ALK oncogenes cause constitutive activation of the ALK kinase by enhanced oligomerization resulting from fusion of the ALK kinase domain to the oligomerization domain of the partner gene. ALK oncogenic activation is associated with strong, immunohistochemically detectable ALK expression in the inflammatory myofibroblastic tumor spindle cells in about 50% of cases (see Fig. 45-17 ). The ALK immunohistochemical staining pattern reflects the fusion type, insofar as fusion of ALK with most of the various partners (TPM3, TPM4, CARS, CLTC, SEC31A (SEC31L1), PPFIBP1, ALO17) produces cytoplasmic staining, whereas ALK-RANBP2 fusion produces nuclear membrane–associated reactivity. These immunohistochemical findings can distinguish inflammatory myofibroblastic tumor from its histologic mimics, but, given the considerable rate of nonimmunoreactivity, demonstration of ALK rearrangement is especially helpful in confirming the diagnosis. An aggressive form of IMT characterized by round cell or epithelioid morphology is described that typically arises intraabdominally in male patients and shows in virtually all cases ALK immunoreactivity in a nuclear, or less commonly, cytoplasmic and perinuclear distribution.

ALK-rearranged inflammatory myofibroblastic tumors are effectively treated with ALK inhibitor crizotinib; however, the emergence of secondary resistance can limit efficacy.

Gastrointestinal Stromal Tumor

Most adult GISTs contain activating mutations of the KIT or PDGFRA oncogenes or rarely BRAF. These mutations have been targeted with considerable success using the KIT and PDGFRA inhibitor imatinib (Gleevec) and second-line tyrosine kinase inhibitors in the context of resistant disease. In addition, germline (inherited) KIT mutations are responsible for rare syndromes of familial, multifocal GISTs. However, because most pediatric GISTs do not contain KIT or PDGFRA mutations they have been termed wild-type GISTs, and these predominate as gastric tumors in prepubescent girls. In keeping with this, children with metastatic GISTs show only very limited benefit from the KIT-PDGFRA kinase inhibitor therapies. Moreover, pediatric GISTs lack the typical sequential cytogenetic deletions that seem to drive genetic and clinical progression in adult GISTs. Largely through an appreciation of the occurrence of GIST as a component of Carney-Stratakis syndrome/dyad, which is the dominantly inherited predisposition to paraganglioma and GIST, underpinned by germline inactivation of subunits B, C or D of succinate dehydrogenase (SDH), it has become evident that deficiency in cellular respiration in fact appears to provide the pathologic basis to all pediatric/wild-type GISTs, despite the low incidence of SDH mutation in such tumors. Germline SDHA mutations have been described in isolated cases of paraganglioma and adult wild-type GIST. Inactivation of any of the four subunits A, B, C, or D of SDH leads to enzyme complex destabilization and inactivation. Immunohistochemical staining for the B subunit of succinate dehydrogenase (SDHB) provides a surrogate marker for SDH function and is characteristically negative in all pediatric/wild-type GISTs. On this basis, a proposal has been made that GISTs no longer be classed as “mutant” and “wild-type” based on receptor tyrosine kinase mutation but rather be designated type 1 and type 2 as SDHB-positive and SDHB-negative, respectively. Although a strong association with neurofibromatosis type 1 (NF1) exists, in this particular context GISTs usually arise only in adulthood.

Malignant Peripheral Nerve Sheath Tumors

Benign peripheral nerve sheath tumors and malignant peripheral nerve sheath tumors (MPNSTs) are frequent complications in patients with hereditary neurofibromatosis syndromes. These syndromes are the most common tumor predisposition syndromes, affecting 1 in 3500 individuals worldwide. Neurofibromas and malignant peripheral nerve sheath tumors are common in those NF1, whereas benign schwannomas are common in neurofibromatosis type 2 (NF2, central neurofibromatosis). Characterization of the neurofibromatosis syndrome genes has shed substantial light on the pathogenesis of peripheral nerve sheath tumors. The NF1 and NF2 genes are located on chromosomes 17 and 22, respectively, and both these genes encode tumor suppressor proteins that normally constrain cellular proliferation. NF1 protein tumor suppressor mechanisms involve regulation of RAS GTPase activity, with NF1 gene deletion therefore resulting in constitutive RAS activation, suggesting that therapeutics targeting RAS (or downstream signaling intermediates such as RAF and MEK-MAPK) might be useful for clinical management of MPNSTs and other NF1-associated cancers. MPNSTs often have NF1 gene deletions, which can be demonstrated by FISH assay. The NF1 gene aberrations are accompanied by a generally complex karyotype, suggesting that genetic instability plays a prominent role in the development of MPNSTs. Notably, NF1 gene deletions can be shown in only the Schwann cell component of neurofibromas. This observation supports the view that neurofibromas are clonal Schwann cell neoplasms, whereas the other admixed cell lineages—including fibroblasts, mast cells, and perineural cells—are reactive.

Recent studies demonstrated that the evolution of benign peripheral nerve sheath tumors to MPNST is accompanied predominantly by loss of expression of a large number of genes, including, in a vast majority of cases, inactivation of TP53 with associated downregulation of the pro-apoptotic miR-34a. MiR-10b, which specifically targets NF1, is upregulated in NF1-associated MPNSTs. Homozygous loss of CDKN2A is also associated with malignant transformation, whereas high-resolution copy number profiling has shown, among others, recurrent gains of PDGFRA, MET, HGF, TP73, EGFR, TERT, MYC, and IGF1R and losses of NF1, p16 ^INK4A /CDKN2A, RB1, and TP53, providing potential basis for novel therapeutic agents in this therapeutically challenging sarcoma.

Neuroblastoma

Most neuroblastomas have distinctive cytogenetic features that correlate with their clinical behavior. Favorable prognosis neuroblastomas, which respond well to chemotherapy and can even undergo spontaneous regression, generally feature near-triploid karyotypes without 1p deletion or MYCN amplification. Unfavorable prognosis neuroblastomas ( Fig. 45-24 ), by contrast, feature near-diploid or near-tetraploid karyotypes, often accompanied by 1p deletion, 17q amplification, and/or MYCN amplification, whereas deletions of 3p and 11q are reportedly predictive of metastasis. In essence, gains of whole chromosomes are more typical of low- or intermediate-risk neuroblastoma whereas segmental chromosomal aberrations are more commonly seen in high-risk cases. MYCN amplification, typically manifested as double-minute chromosomes (see Figs. 45-5 and 45-6 ), is arguably the most ominous of the adverse genetic markers. MYCN -amplified neuroblastomas are rarely curable, although complete remission can be achieved through intensive myeloablative chemotherapy. Therefore genetic parameters can be useful adjuncts for determining appropriate intensity of therapy, particularly for those children whose prognoses are not clear based on clinical parameters alone. Cytogenetic analysis of neuroblastoma has been difficult, however, because the tumor cells from most favorable prognosis neuroblastomas fail to divide in culture. MYCN amplification and chromosome 1p or 11q deletion and 17q gain can be demonstrated in interphase cells by FISH or CISH (see Fig. 45-6 ), obviating the need for tumor cell culture. It remains unclear whether one or more conventional tumor suppressor genes are targeted by the 1p deletions found in most poor-prognosis neuroblastomas. However, several microRNAs in the critical 1p deletion region function to maintain neuroblastoma survival and activate MYCN transcription. ALK kinase domain mutations and gene amplifications have been demonstrated in hereditary neuroblastoma that may otherwise be underscored by PHOX2B germline mutation. About 15% of sporadic neuroblastomas also show aberrations of ALK, suggesting that ALK inhibitors might be useful therapeutically in neuroblastoma. SNP variations at 6p22.3, 2q35 (the BARD1 gene), and copy number aberration at 1q21 (the NBPF23 gene) are associated with increased risk for development of neuroblastoma but without further elucidation of the likely mechanisms involved biologically.

Bone marrow involvement by disseminated neuroblastoma, showing a classic rosette of tumor cells, with bluish cytoplasm and neuropil.

(Courtesy Marybeth Helfrich, Children’s Hospital of Philadelphia.)

Rhabdoid Tumor

Malignant rhabdoid tumors generally have dismal outcomes, particularly those presenting in infancy, and most cases, whether arising in soft tissues, kidney, or CNS (where they are known as atypical teratoid/rhabdoid tumors), have deletions of the chromosome 22 long arm, targeting the SMARCB1 ( INI1) tumor suppressor gene. Biallelic SMARCB1 mutation is present in the vast majority of rhabdoid tumors, with frequent truncating mutations occurring. In a small number of cases, instead of SMARCB1, the SMARCA4 (BRG1) gene is inactivated. SMARCB1 encodes a member of the SWI/SNF protein complex involved in chromatin remodeling. Chromosome 22 deletion is often the only detectable cytogenetic aberration, suggesting that SMARCB1 inactivation is an early oncogenic event in these remarkably aggressive tumors, which are classically diploid and genomically stable. Indeed, sequencing studies have revealed a remarkably simple tumor genome otherwise with virtually no oncogenic mutations identifed. Additional evidence of an essential tumorigenic role includes the finding of germline INI1 mutations in some individuals with rhabdoid tumors and the development of rhabdoid tumor models in mice with inactivating SMARCB1 mutations. Notably, germline SMARCB1 mutations are not restricted to rhabdoid tumors but have been implicated also in familial schwannomatosis and in nonrhabdoid CNS tumors, including medulloblastoma. and multiple meningiomas SMARCB1 inhibits cyclin D1 expression by recruiting a histone deacetylase 1 complex to the CCND1 (cyclin D1) promoter, resulting in G1 cell cycle arrest. The relevance of cyclin D1 in SMARCB1 -mediated tumorigenesis is underscored by the observation that rhabdoid tumors develop in SMARCB1+/− mice expressing cyclin D1 whereas SMARCB1+/− mice with cyclin D1 deficiency (cyclin D1−/−) do not develop rhabdoid tumors. These observations suggest that drugs targeting cyclin D1 might be relevant for rhabdoid tumors, and clinical trials testing this are planned.

Recent work has unraveled an epigenetic antagonism between SMARCB1 and Polycombs of the PRC2 complex. The Polycomb proteins are transcriptional repressors that modulate chromatin structure to silence gene expression, and they are key regulators of cell fate transitions. Their function frequently becomes deregulated in cancer. The Polycombs have been shown to be required for the repression of differentiation genes in stem cells so that this repression of differentiation might be facilitated by loss of the PRC2 antagonist SMARCB1.

Rhabdoid tumors are characteristically composed of cells with vesicular nuclei and prominent nucleoli, with the nucleus often being pushed to an eccentric location within the cell by a dense intracytoplasmic inclusion (see Fig. 45-9 ), which, as seen by electron microscopy, consists of a tightly arranged whorl of intermediate filaments. These filaments stain nonspecifically for various markers, giving the tumor a polyphenotypic appearance. Absence of SMARCB1 expression in the tumor cells but not the normal stromal support nuclei, as is readily demonstrable by immunohistochemistry, is useful for diagnosing rhabdoid tumor (see Fig. 45-9 ).

Alveolar Soft Part Sarcoma

Alveolar soft part sarcoma is a rare tumor, occurring mostly in adolescents and young adults, with a predilection for involvement of soft tissues in the head and neck region. However, other soft tissue and even hollow viscera may be involved. Characterized by a distinctive morphology, with nested or alveolar-like growth, the tumor cells are epithelioid, with abundant clear to eosinophilic cytoplasm, sharply defined cellular borders, and diastase-resistant, periodic acid–Schiff (PAS)-positive intracytoplasmic crystals ( Fig. 45-25 ), which on electron microscopic examination are rhomboidal. Alveolar soft part sarcoma generally exhibits indolent behavior but nonetheless has a high risk for late metastases and indeed represents the sarcoma most likely to metastasize to the CNS. There are no histologic features to predict clinical behavior. The classic cytogenetic feature of ASPS is t(X;17)(p11;q25), producing fusion of the ASPSCR1 and TFE3 genes. This same translocation is seen in a subset of pediatric renal cell carcinomas, with the distinct difference that the ASPS -related translocation is un balanced and involves gain of Xp telomeric to TFE3 and loss of 17q sequence telomeric to q25 while the renal carcinoma–associated translocation is balanced. The ASPSCR1-TFE3 fusion protein induces strong expression of MET tyrosine kinase in tumor cells. Antibody raised to the carboxyl terminus of TFE3 produces strong nuclear reactivity in ASPSCR1-TFE3 fusion-positive tumors.

Characteristic histologic features of alveolar soft part sarcoma include an alveolar-like arrangement of cells with abundant deeply eosinophilic cytoplasm (A) and diastase-resistant, periodic acid–Schiff-positive intracytoplasmic crystals (B) .

Renal Tumors

Most pediatric renal tumors contain characteristic cytogenetic aberrations. Deletion of 11p is a well-known aberration in Wilms tumor, but several other aberrations, including extra copies of chromosome 12, are more frequent and have apparent prognostic relevance, as does LOH of 1p and 16q. Another pediatric renal tumor with consistent cytogenetic aberrations is congenital cellular mesoblastic nephroma, in which various chromosomal trisomies and oncogenic fusion of the TEL and NTRK3 proteins are observed reliably. More recently, it has emerged that translocation-associated carcinomas make up a majority of pediatric renal carcinomas. In addition, neuroblastoma patients are at hugely increased risk (329-fold) of developing oncocytoid renal carcinoma ; however, pediatric renal carcinoma may also follow certain sarcomas.

Wilms Tumor.

Wilms tumors are the most common type of renal cancer in children. They typically contain primitive blastemal cells that differentiate into tubular epithelial, glomerular, and/or mesenchymal populations. The classic triphasic Wilms tumor contains an admixture of blastemal, epithelial, and mesenchymal components ( Fig. 45-26 ), and all three cell types may contain the same clonal chromosomal aberrations. Various genetic alterations are found in subsets of Wilms tumors, including trisomies 6, 8, 12, and 18, and deletions of 11p13 (see later). Deletions of 11p15, 1p, and 16q bode ill in patients with low-stage favorable histology tumors and 1q gains are associated with relapse. These can all readily be tested on interphase nuclei by FISH assay (see Table 45-1 ).

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

The Neonatal Erythrocyte and Its Disorders Diagnostic Approach to the Anemic Patient Approach to the Child with a Suspected Bleeding Disorder Disorders of the Red Cell Membrane Pediatric Myeloid Leukemia, Myelodysplasia, and Myeloproliferative Disease Nonrhabdomyosarcoma Soft Tissue Sarcomas and Other Soft Tissue Tumors

Stay updated, free articles. Join our Telegram channel

Join