Molecular Diagnostics of Soft Tissue Tumors Julia A. Bridge, MD; Allison M. Cushman-Vokoun, MD, PhD N Context.—Soft tissue pathology encompasses a remark- ably diverse assortment of benign and malignant soft tissue tumors. Rendering a definitive diagnosis is complicated not only by the large volume of existing histologic subtypes (.100) but also frequently by the presence of overlapping clinical, histologic, immunohistochemical, and/or radio- graphic features. During the past 3 decades, mesenchymal tumor–specific, cytogenetic and molecular genetic abnor- malities have demonstrated an increasingly important, ancillary role in mesenchymal tumor diagnostics. Objectives.—To review molecular diagnostic tools available to the pathologist to further classify specific soft tissue tumor types and recurrent aberrations frequently examined. Advantages and limitations of individual ap- proaches will also be highlighted. Data Sources.—Previously published review articles, peer- reviewed research publications, and the extensive cytogenetic and molecular diagnostic experience of the authors to include case files of The University of Nebraska Medical Center. Conclusions.—Cytogenetic and molecular genetic assays are used routinely for diagnostic purposes in soft tissue pathology and represent a powerful adjunct to comple- ment conventional microscopy and clinicoradiographic evaluation in the formulation of an accurate diagnosis. Care should be taken, however, to recognize the limita- tions of these approaches. Ideally, more than one technical approach should be available to a diagnostic laboratory to compensate for the shortcomings of each approach in the assessment of individual specimens. (Arch Pathol Lab Med. 2011;135:588–601) S oft tissue tumors constitute a rare, heterogeneous group of mesenchymal neoplasms that during the past 3 decades have been shown by cytogenetic analysis to have a remarkably high incidence of specific and primary alterations. These genetic alterations have not only guided molecular studies in establishing the underlying genes involved, thereby yielding important pathogenetic infor- mation, but also have provided clinicians with a valuable tool to add to their diagnostic armamentarium. The addition of molecular cytogenetic (fluorescence in situ hybridization [FISH], array-based comparative genomic hybridization [aCGH], and single nucleotide polymor- phism [SNP] array) and molecular approaches (eg, reverse transcription–polymerase chain reaction [RT-PCR] and sequencing technology) has further enhanced the sensi- tivity and accuracy of detecting nonrandom chromosomal imbalances and/or structural rearrangements (eg, trans- locations), as well as specific gene mutations in soft tissue tumors, including assessment in formalin-fixed, paraffin- embedded (FFPE) tissues. This review is divided into 2 major sections: (1) summary of recurrent or tumor-specific genetic events in soft tissue tumors, and (2) overview of the molecular approaches commonly used in clinical practice to identify them. The advantages and limitations of individual molecular diagnostic assays used in the management of soft tissue tumors are also underscored, with inclusion of select examples that serve as useful paradigms. GENETIC EVENTS IN SOFT TISSUE TUMORS Simplistically, 2 major groups of genetic events can be appreciated in soft tissue tumors. Many soft tissue sarcomas are characterized by tumor-specific anomalies. Most com- monly, these anomalies are translocations that result in the production of chimeric genes encoding for abnormal, oncogenic proteins that are central to the causation of these tumors. Often, these tumors are in the setting of a simple karyotype. Alternatively, some soft tissue tumors feature specific activating or inactivating mutations within onco- genes or tumor suppressor genes, respectively. A second major group of sarcomas lack a tumor-specific abnormality and are associated with multiple and sometimes complex chromosomal changes and nonspecific genetic alterations. Thus, for most sarcomas in this group, the high degree of genomic complexity and instability (including large num- bers of unidentifiable marker chromosomes and intratu- moral heterogeneity) precludes the use of many routine clinical genetic approaches as a discriminating tool. For this reason, there will be no further discussion of this latter group in the current review. Tumor-Specific Abnormalities Translocations.—Approximately one-third of all sarco- mas exhibit a nonrandom chromosomal translocation (exchange of chromosomal material between 2 or more nonhomologous chromosomes), which leads to the juxta- positioning of 2 genes, one from each translocation partner, resulting in the formation of a fusion gene Accepted for publication December 22, 2010. From the Departments of Pathology and Microbiology (Drs Bridge and Cushman-Vokoun), Pediatrics (Dr Bridge), and Orthopedic Surgery (Dr Bridge), University of Nebraska Medical Center, Omaha. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Julia A. Bridge, MD, Departments of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135 (e-mail: jbridge@ unmc.edu). 588 Arch Pathol Lab Med—Vol 135, May 2011 Molecular Diagnostics of Soft Tissue Tumors—Bridge & Cushman-Vokoun
Transcript
Molecular Diagnostics of Soft Tissue Tumors
Julia A. Bridge, MD; Allison M. Cushman-Vokoun, MD, PhD
N Context.—Soft tissue pathology encompasses a remark-ably diverse assortment of benign and malignant soft tissuetumors. Rendering a definitive diagnosis is complicated notonly by the large volume of existing histologic subtypes(.100) but also frequently by the presence of overlappingclinical, histologic, immunohistochemical, and/or radio-graphic features. During the past 3 decades, mesenchymaltumor–specific, cytogenetic and molecular genetic abnor-malities have demonstrated an increasingly important,ancillary role in mesenchymal tumor diagnostics.
Objectives.—To review molecular diagnostic toolsavailable to the pathologist to further classify specific softtissue tumor types and recurrent aberrations frequentlyexamined. Advantages and limitations of individual ap-proaches will also be highlighted.
Data Sources.—Previously published review articles, peer-reviewed research publications, and the extensive cytogeneticand molecular diagnostic experience of the authors to includecase files of The University of Nebraska Medical Center.
Conclusions.—Cytogenetic and molecular genetic assaysare used routinely for diagnostic purposes in soft tissuepathology and represent a powerful adjunct to comple-ment conventional microscopy and clinicoradiographicevaluation in the formulation of an accurate diagnosis.Care should be taken, however, to recognize the limita-tions of these approaches. Ideally, more than one technicalapproach should be available to a diagnostic laboratory tocompensate for the shortcomings of each approach in theassessment of individual specimens.
(Arch Pathol Lab Med. 2011;135:588–601)
Soft tissue tumors constitute a rare, heterogeneous groupof mesenchymal neoplasms that during the past 3
decades have been shown by cytogenetic analysis to havea remarkably high incidence of specific and primaryalterations. These genetic alterations have not only guidedmolecular studies in establishing the underlying genesinvolved, thereby yielding important pathogenetic infor-mation, but also have provided clinicians with a valuabletool to add to their diagnostic armamentarium. Theaddition of molecular cytogenetic (fluorescence in situhybridization [FISH], array-based comparative genomichybridization [aCGH], and single nucleotide polymor-phism [SNP] array) and molecular approaches (eg, reversetranscription–polymerase chain reaction [RT-PCR] andsequencing technology) has further enhanced the sensi-tivity and accuracy of detecting nonrandom chromosomalimbalances and/or structural rearrangements (eg, trans-locations), as well as specific gene mutations in soft tissuetumors, including assessment in formalin-fixed, paraffin-embedded (FFPE) tissues.
This review is divided into 2 major sections: (1)summary of recurrent or tumor-specific genetic events insoft tissue tumors, and (2) overview of the molecularapproaches commonly used in clinical practice to identify
them. The advantages and limitations of individualmolecular diagnostic assays used in the management ofsoft tissue tumors are also underscored, with inclusion ofselect examples that serve as useful paradigms.
GENETIC EVENTS IN SOFT TISSUE TUMORS
Simplistically, 2 major groups of genetic events can beappreciated in soft tissue tumors. Many soft tissue sarcomasare characterized by tumor-specific anomalies. Most com-monly, these anomalies are translocations that result in theproduction of chimeric genes encoding for abnormal,oncogenic proteins that are central to the causation of thesetumors. Often, these tumors are in the setting of a simplekaryotype. Alternatively, some soft tissue tumors featurespecific activating or inactivating mutations within onco-genes or tumor suppressor genes, respectively. A secondmajor group of sarcomas lack a tumor-specific abnormalityand are associated with multiple and sometimes complexchromosomal changes and nonspecific genetic alterations.Thus, for most sarcomas in this group, the high degree ofgenomic complexity and instability (including large num-bers of unidentifiable marker chromosomes and intratu-moral heterogeneity) precludes the use of many routineclinical genetic approaches as a discriminating tool. For thisreason, there will be no further discussion of this lattergroup in the current review.
Tumor-Specific Abnormalities
Translocations.—Approximately one-third of all sarco-mas exhibit a nonrandom chromosomal translocation(exchange of chromosomal material between 2 or morenonhomologous chromosomes), which leads to the juxta-positioning of 2 genes, one from each translocationpartner, resulting in the formation of a fusion gene
Accepted for publication December 22, 2010.From the Departments of Pathology and Microbiology (Drs Bridge
and Cushman-Vokoun), Pediatrics (Dr Bridge), and Orthopedic Surgery(Dr Bridge), University of Nebraska Medical Center, Omaha.
The authors have no relevant financial interest in the products orcompanies described in this article.
Reprints: Julia A. Bridge, MD, Departments of Pathology andMicrobiology, University of Nebraska Medical Center, 983135Nebraska Medical Center, Omaha, NE 68198-3135 (e-mail: [email protected]).
588 Arch Pathol Lab Med—Vol 135, May 2011 Molecular Diagnostics of Soft Tissue Tumors—Bridge & Cushman-Vokoun
(Table 1).1–10 A soft tissue tumor translocation is oftenpresent as the sole karyotypic aberration and is presumedto be the initiating oncogenic event. Moreover, thesetumor-specific translocations are retained throughout theclinical course, even as a given tumor metastasizes orbecomes less differentiated, emphasizing a role insustaining neoplastic transformation and aiding in themanagement of poorly differentiated sarcomas.11 Domi-nantly acting oncoproteins associated with chromosomaltranslocations in soft tissue tumors have multiple func-tions and may or may not require additional mutations forcell transformation.12–14
For soft tissue tumors, chromosomal translocationsmost commonly result in the production of a highly
specific, novel chimeric gene, such as the EWSR1-WT1fusion corresponding to the t(11;22)(p13;q12) of desmo-plastic small round-cell tumor.15 In rare instances, thesame translocation and gene fusion have been demon-strated in 2 or more unrelated neoplasms, for example,ALK-CLTC in inflammatory myofibroblastic tumor andanaplastic large cell lymphoma and ETV6-NTRK3 ininfantile fibrosarcoma, acute myeloid leukemia, secretorycarcinoma of the breast, and the mammary analoguesecretory carcinoma of the salivary glands.16–21 Theseoccurrences suggest that the type of cell the fusiononcogene is targeting may determine whether the neo-plastic transformation is of mesenchymal, hematologic, orepithelial lineage.
Table 1. Characteristic and Variant Chromosomal Translocations and Associated Fusion Genes in Malignant SoftTissue Tumors
Abbreviation: pPNET, peripheral primitive neuroectodermal tumor; ?, the fusion partner gene is not known.a Most frequently presents as an unbalanced translocation in alveolar soft part sarcoma.b Ring chromosome, composed of amplified 3p11–12, is associated with an increased expression of VGLL3, and CHMP2B is also characteristic.
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Functionally, there are 3 types of resultant fusiononcoproteins in soft tissue tumors:
1. Most often, sarcoma-associated fusion oncogenes en-code for chimeric transcription factors that causetranscriptional deregulation. Synovial sarcoma, myx-oid liposarcoma, and clear cell sarcoma represent just afew of the sarcomas characterized by specific, aberranttranscription factors.
2. Inflammatory myofibroblastic tumor and congenital orinfantile fibrosarcoma are examples of soft tissuetumors whose fusion oncogenes encode for chimerictyrosine kinases that elicit deregulation of kinasesignaling pathways.
3. Kinase signaling pathway deregulation is also theoutcome of the rearrangements in dermatofibrosarco-ma protuberans and giant cell tumors of the tendonsheath or diffuse-type giant cell tumors that featurechimeric autocrine growth factors as their characteris-tic fusion gene. Furthermore, dermatofibrosarcomaprotuberans serves as an example of how the identi-fication of sarcoma-specific rearrangements and asso-ciated fusion oncogenes may also be therapeuticallyimportant in addition to diagnostically useful. Activa-tion of the platelet-derived growth factor receptor b(PDGFRB), a transmembrane tyrosine kinase, by fusion
of the gene encoding for its ligand, PDGFB, withCOL1A1 (collagen, type 1, a 1) renders dermatofibrosar-coma protuberans responsive to targeted therapy withtyrosine kinase inhibitors, such as imatinib mesylate.22
An important aspect of familiarizing oneself with themolecular diagnostics of sarcoma translocations is torecognize that both cytogenetic- and molecular genetic–variant translocations exist in these tumors and that newcytogenetic and molecular genetic variants continue to bediscovered and defined. Therefore, depending on the typeof molecular-pathologic approach used, some varianttranslocations and gene fusions may not be identified(false-negative result).
Cytogenetic-variant translocations arise from a rear-rangement of one consistent gene with differing chromo-somal translocation partners (Figure 1, A). For example,approximately 90% to 95% of Ewing sarcomas/peripheralprimitive neuroectodermal tumors (pPNETs) exhibit an11;22 translocation, resulting in the fusion of the EWSR1gene to FLI1 (an ETS gene family member).23 Less-common cytogenetic variants, however, have also beenidentified and are characterized by the fusion of theEWSR1 gene with other members of the ETS family oftranscription factors, including ERG (21q22), ETV1 (7p22),E1AF (17q21), and FEV (2q35–36).24–27 In addition, a fusion
Figure 1. A, Examples of cytogenetic variant translocations where there is consistent involvement of the EWSR1 gene locus at 22q12 but thetranslocation partners vary. B, The genomic breakpoints of EWSR1 and FLI1 are heterogeneous resulting in various types of in-frame EWSR1-FLI1chimeric transcripts. The 2 main types, fusion of EWSR1 exon 7 to FLI1 exon 6 (type 1) and fusion of EWSR1 exon 7 to FLI1 exon 5 (type 2), accountfor 85% to 90% of EWSR1-FLI1 fusions.
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between EWSR1 and a gene from a transcription factorfamily other than ETS, the NFATC2 gene (encodes for amember of the NFAT transcription factor family), has beendescribed.28 Rarely, fusions between EWSR1 and genesencoding for a member of the zinc finger family ofproteins [PATZ1 (22q12) and SP3 (2q31)] have also beenobserved in Ewing sarcoma.29,30 Most recently, we identi-fied a chromatin remodeling gene, SMARCA5 (4q31), as anovel gene partner in an extraskeletal Ewing sarcoma.31
Infrequently, a substitution of the FUS gene for EWSR1 inEwing sarcoma–associated translocations has been ob-served [eg, FUS-ERG t(16;21)(p11;q22) and FUS-FEVt(2;16)(q35;p11)].32,33
In contrast, molecular variants are often the result ofgenomic breakpoint differences that lead to distinct fusionproduct exon combinations. For example, in Ewingsarcoma/pPNET, the chromosomal translocation break-points may arise within EWSR1 introns 7 to 9 and withinintrons 3 to 9 of FLI1, enabling the generation of severalpossible EWSR1-FLI1 chimeric transcripts. In most cases,exon 7 of EWSR1 is fused to either FLI1 exon 6 (type 1;60%) or to exon 5 (type 2; 20%). In contrast to previousreports, recent studies have shown that the fusion subtypeis not a predictor of clinical outcome for patients withEwing sarcoma who receive contemporary therapy.34,35
However, the methodologic sensitivity and specificity ofRT-PCR analysis has shown promise in research studiesfor monitoring micrometastatic disease in the blood orbone marrow of patients with Ewing sarcoma.36 Type Iand type II molecular variants can readily be detected bytheir unique RT-PCR product-band size (Figure 1, B). Theidentity of less-common or unexpected product-bandsizes should be confirmed using additional approaches,such as direct sequencing or digestion with specific
restriction endonucleases. Cytogenetic analysis does notdistinguish between molecular variants. Fluorescence insitu hybridization analysis may be useful for detectingrare cytogenetic variants that primer sets are not com-monly designed to detect.
Many benign soft tissue tumors are also characterizedby specific cytogenetic translocations or other rearrange-ments that may be useful in distinguishing these lesionsfrom malignant neoplasms (Table 2). For example, distin-guishing between the morphologic variants of the mostcommon group of soft tissue tumors, lipomatous neo-plasms, may be challenging in some clinical situations butis imperative for accurate therapy. Identification ofC11orf95-MKL2, the resulting fusion oncogene oft(11;16)(q13;p13), is useful in establishing the diagnosisof chondroid lipoma, a benign soft tissue tumor that canbe mistaken for a sarcoma, especially myxoid liposarcomaor extraskeletal myxoid chondrosarcoma.37 Cytogeneticstudies of conventional lipoma are abundant and haverevealed rearrangements of 12q14–15 and the underlyingHMGA2 gene as most common (Figure 2, A and B).38 Incontrast, atypical lipomatous tumor/well-differentiatedliposarcoma is characterized by the presence of asupernumerary ring or marker chromosomes (often asthe sole anomaly) containing amplification of the 12q14–15 region and corresponding tumor-associated genes (inparticular MDM2 and CDK4) (Figure 2, C through E). Forthe histopathologic differential diagnosis of lipoma andatypical lipomatous tumor/well-differentiated liposarco-ma, complementary molecular testing should especiallybe considered for ‘‘relapsing lipomas,’’ lipomatous tu-mors with questionable cytologic atypia (even if widelyexcised), and for large lipomatous tumors (.15 cm)without diagnostic cytologic atypia.39
Table 2. Recurrent Chromosomal Abnormalities in Benign Soft Tissue Tumors
Neoplasm Abnormality Molecular Consequence
Aggressive angiomyxoma 12q15 rearrangements HMGA2Aneurysmal bone cyst t(16;17)(q22;p13) CDH11-USP6
6p21 rearrangements HMGA1Schwannoma 22q12 loss NF2 lossSpindle cell lipoma/pleomorphic lipoma 16q13-qter rearrangement/loss, monosomy 13 or partial del(13q) Unknown
a Predominantly in patients with familial adenomatous polyposis or Gardner syndrome.
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Soft Tissue Tumors With Recurrent Alterations inIsolated Genes.—Activating Oncogenic Mutations.—Asubset of soft tissue tumors not harboring specifictranslocations or fusion genes is characterized by identi-fiable, activating oncogenic mutations. For example,constitutive activation of specific kinases by oncogenicmutations with stimulation of downstream targets is analternative mechanism for genetic deregulation of kinasesignaling. This mechanism, in the form of KIT or PDGFRAmutations, is the molecular hallmark of gastrointestinalstromal tumor (GIST).40–42
Approximately 85% of all GISTs demonstrate a primarymutation at 1 of 3 sites: KIT exon 11 mutations (approx-imately 65%), KIT exon 9 mutations (10%), and PDGFRAexon 18 mutations (10%).43–45 Mutations in exons 13 or 17 ofKIT or in exons 12 or 14 of PDGFRA collectively compriseabout 5% of the mutations detected in GIST, and roughly10% of GISTs are so-called wild-type GISTs, without any
detectable mutations in the known hot spots.46 The KIT orPDGFRA exon involved and the mutation type oftencorrelate with GIST anatomic location and/or prognosis.For example, KIT exon 11 mutations include in-framedeletions, internal tandem duplications, and substitutions(point mutations) and are observed in all clinicopathologicGIST subtypes; however, patients with GIST and KIT exon11 single-nucleotide substitutions fare better prognosti-cally than those with deletions.47 A less-aggressive clinicalcourse is also observed in patients with GISTs exhibitingKIT exon 11 internal tandem duplications, which tend toarise in the stomach.47,48 KIT exon 9 mutations are moreoften found in small-intestinal GISTs and, in general, thereappears to be a higher mortality for patients with small-intestinal versus gastric GISTs.42,48 Other phenotypic orprognostic correlates include KIT exon 17 mutations,which are more frequently identified in small-intestinalGISTs49; gastric GISTs with exon 13 mutations, which tend
Figure 2. A, G-banded karyotype illustrating a 3;12 translocation (arrows) involving the 12q15 (HMGA2) locus characteristically rearranged inconventional lipoma. B, Fluorescence in situ hybridization (FISH) analysis conducted on a metaphase cell from a conventional lipoma using aUniversity of Nebraska Medical Center custom-designed HMGA2 break-apart FISH probe shows translocation of the green signal just distal to theHMGA2 breakpoint on chromosome 12 to the derivative 1 of this 1;12 translocation. The orange signal just proximal to the HMGA2 breakpointremains on the derivative 12. Also, a centromeric probe specific for chromosome 1, labeled in orange, was included in this analysis. C,Representative G-banded metaphase cell from a well-differentiated liposarcoma demonstrating the characteristic supernumerary ring chromosome.D and E, The FISH studies conducted with a centromeric probe for chromosome 12, labeled in green, and an MDM2 locus-specific probe, labeled inred, confirm the presence of MDM2 amplification within the ring chromosome (red arrow) and in the formalin-fixed, paraffin-embedded tissuesection interphase cells, respectively. Detection of MDM2 amplification may be useful in distinguishing lipoma and ALT/WDL (atypical lipomatoustumor/well-differentiated liposarcoma) (original magnification 3400).
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to be associated with more aggressive behavior49; and, KIThomozygous mutations, which often relate to GISTs with amore aggressive phenotype.50
Mutations in PDGFRA are almost exclusively localizedto gastric GISTs, are frequently associated with epithelioidmorphology, and exhibit more indolent clinical behav-ior.42,51 Substitutions in the second tyrosine kinase domainof PDGFRA exon 18 are most common; however,activating, in-frame deletions and, rarely, duplications,insertions, and complex structural mutations, have alsobeen identified.42,51–53 Immunohistochemical expression ofKIT (CD117) is seen in most GISTs, including PDGFRAmutated GISTs. (A common misconception is that KITexpression is caused by a KIT mutation).
KIT and PDGFRA, localized to 4q12, encode for thesame class (III) of receptor tyrosine kinases. A schematic ofthe most commonly involved KIT and PDGFRA exons inrelation to their coding regions within the receptortyrosine kinase is shown in Figure 3, A. Patients withGIST have been successfully treated with tyrosine kinaseinhibitors, such as imatinib mesylate and sunitinib.Routine tumor genotyping is recommended by many
experts because the kinase genotype has been shown tocorrelate with different imatinib treatment outcomes andprogression-free and overall survival.43,54–56 KIT exon 11mutations appear to be most responsive; however, othermutations in KIT exons and select PDGFRA exon 18mutations also demonstrate sensitivity.42,53,54 The presenceor absence of a KIT exon 9 mutation may affect imatinibdose selection. Tumors wild type for KIT and PDGFRAhave also shown partial response to tyrosine kinaseinhibitors, indicating that other mechanisms involvingtyrosine kinase activation also exist in GISTs.43 PrimaryPDGFRA mutations in exon 18 (primarily involving codon842, especially p.Asp842Val) have shown resistance toimatinib.53 Testing is particularly advocated for GISTs thatare malignant with metastatic risk or that have shown thedevelopment of resistance to imatinib.
A widely used approach for clinically evaluating GISTsfor resistance mutations is by PCR amplification of themost commonly mutated exons, with subsequent directsequencing analysis of the amplified exon (Figure 3, B andC). Alternatively, some laboratories use mutation scan-ning techniques, such as heteroduplex formation analysis
Figure 3. A, Schematic illustration of a dimerized receptor tyrosine kinase family member, such as KIT and PDGFRA. The domains include theextracellular domain (ECD), juxtamembrane domain (JMD), tyrosine kinase domain 1 (TKD1), tyrosine kinase domain 2 (TKD2), and the tyrosinekinase insert (TKI). The triangles represent tyrosines, which become phosphorylated on ligand activation and dimerization of the receptor. The hot-spot exons containing activating gastrointestinal stromal tumor (GIST) mutations are shown relative to the location of the corresponding affecteddomain for each gene. B and C, Examples of KIT and PDGFRA mutations detected by polymerase chain reaction amplification and directsequencing. Horizontal and vertical arrows indicate the beginning of the deletion and single nucleotide substitution, respectively. B, Ac.1690_1695delTGGAAG leading to p.Trp557_Lys558del at the protein level, the most common KIT deletion identified in GISTs. Thep.Trp557_Lys558del mutants are sensitive to imatinib in vitro and in vivo. C, A missense mutation c.2664A.T leading to p.Asp842Val at the proteinlevel, the most common PDGFRA mutation identified in GISTs. The p.Asp842Val mutants are resistant to imatinib in vitro and in vivo. (Sequencingfigures courtesy of Jerzy Lasota, MD, PhD, Armed Forces Institute of Pathology, Washington, DC).
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with denaturing high-performance liquid chromatogra-phy, to first, broadly scan the PCR-amplified exons forpotential mutations. If a profile suggests a possiblemutation within an exon, direct sequencing of that exonis performed to identify the specific mutation. Althoughsuch scanning techniques entail additional technology, theextra time that otherwise would be required for directlysequencing multiple exons is avoided.
Inactivating Oncogenic Mutations.—Inactivation of atumor suppressor gene represents another form ofaberration characterizing a subgroup of soft tissue tumors.Inactivation of tumor suppressor genes requires loss ofboth alleles or loss of one allele with an inactivatingmutation of the other, either epigenetically (ie, methyla-tion with inactivation of the tumor suppressor genepromoter) or genetically (eg, nonsense mutation, splicesite mutation). Alternatively, there may be copy number–neutral loss of heterozygosity (LOH), also known asacquired uniparental disomy, caused by a mitotic segregationerror with loss of one allele and reduplication of theremaining allele or by segmental mitotic recombinationbetween pairs of high-identity, low-copy repeats, amongother mechanisms.57 Inactivation of the tumor suppressorgene SMARCB (previously INI1) typifies extrarenal (andrenal) rhabdoid tumors, proximal-type epithelioid sarco-ma, and a subset of pediatric undifferentiated soft tissuesarcomas.58–61 Deletion and/or mutation of both copies ofthe SMARCB gene results in loss of INI1 proteinexpression demonstrable with an anti-INI1 antibody andimmunohistochemistry.62
Historically, SMARCB molecular alterations have beendetected in approximately 75% of rhabdoid tumors usingconventional karyotyping, FISH, and direct sequencinganalysis. Recently, a comprehensive molecular study63 of51 rhabdoid tumors using a multimodality approach ofFISH, PCR-based sequencing, multiplex ligation-depen-dent probe amplification analysis of SMARCB, and wholegenome SNP-based array analysis revealed that a varietyof events (deletions, mutations, and LOH) led to SMARCBinactivation in 98% of the rhabdoid tumors (50 of 51)examined. Therefore, although karyotyping, FISH, and/orsequencing are useful for identifying SMARCB alterationsin most cases (Figure 4, A and B), a small subset ofdiagnostically suspicious cases may require higher-reso-lution techniques (multiplex ligation-dependent probeamplification or SNP-array analysis) to identify smalldeletions, duplications, or single base pair (bp) mutationswithin the tumors. Assessment by SNP-array analysis isalso useful for revealing copy number–neutral LOH. Thiscan be illustrated by a case recently encountered at ourinstitution, where FISH analysis with a home-brewSMARCB probe was negative, but chromosome 22 copynumber–neutral LOH was detected by SNP-array analysis(Figure 4, C). SMARCB molecular genetic testing can beused as an aid in the diagnosis and in treatment planningfor most patients with rhabdoid tumors.63
MOLECULAR APPROACHES COMMONLY USED ASDIAGNOSTIC AIDS
Genetic approaches commonly used to identify mesen-chymal tumor–specific abnormalities include convention-al cytogenetic, molecular cytogenetic (FISH with noveltechnical variations), RT-PCR, and sequencing analyses.In this review, emphasis is placed on the practical
applications of each of these techniques including theiradvantages and limitations.
Cytogenetic Analysis
Tissue submitted for cytogenetic analysis must be fresh(not frozen or fixed in formalin) because living, dividingcells are required. A soft tissue tumor sample submittedfor cytogenetic analysis should be representative of theneoplastic process and preferably be part of the specimenundergoing pathologic study. Ideally, a 1- to 2-cm3
(approximately 0.5- to 1.0-g) fresh sample is providedfor analysis. Although fine-needle biopsy or aspirationspecimens (,500 mg) can also be analyzed successfully, aprolonged culture may be necessary to produce enoughcells for examination.64 Notably, a limited sample size mayalso be more restrictive for a few neoplasms, such asbenign adipose tissue tumors, which often have a low celldensity per volume unit. Importantly, however, efforts toperform cytogenetic analysis are worthwhile even whenmaterial is limited because the presence of a single cellexhibiting a tumor-specific chromosomal abnormalityprovides strong diagnostic support.
The basic process of cell culturing and karyotypicanalysis is the same for all soft tissue tumors. A short-term culture usually results in sufficient mitoses in 8 to10 days or less. Lengthy culture times should be avoidedbecause undesired overgrowth by common fibroblasts islikely.
An alternative to tissue culture is direct or same-dayharvest. With this technical option, endemic dividing cellsare arrested after a 1- to 12-hour incubation in a mitoticinhibitor, such as colchicine and culture medium. Thismethod is useful for obtaining fast (,24 hours) orpreliminary results but is constrained by the in vivomitotic index. Effusions (eg, ascites and pleural fluids) andfine-needle aspirations of some tumors, such as smallround blue cell neoplasms, can have a high mitotic indexand are often well-suited for direct preparations orovernight culture. Receipt of the tissue sample in thelaboratory within 1 hour after biopsy can significantlystrengthen the probability for a successful direct harvestanalysis.
G-banding, performed with Giemsa or Wright stainspretreated with trypsin or phosphate buffer, respectively,creates a unique pattern of bands for each chromosome, tofacilitate karyotyping. G-banding is the most commonform of banding because of the relative ease of performingthe technique, the reliability of the results, and thepermanence of the preparations. The number of alternat-ing light and dark bands detectable with G-banding in thehaploid genome varies with the level of chromosomalcontraction in each metaphase cell, but preparations frommost soft tissue tumors typically yield 400 to 600 bandsper haploid set. Cytogenetic analysis is not considered ahigh-resolution technique because one band representsapproximately 5 to 10 3106 bp of DNA and, therefore,could potentially contain hundreds of genes.
A global assessment of primary and secondary, numer-ical and structural abnormalities in a single assay isperhaps the greatest strength of cytogenetic analysis.Moreover, in contrast to FISH or RT-PCR, knowledge ofthe anticipated anomaly or histologic diagnosis is notnecessary. Additional advantages and limitations ofconventional cytogenetic analysis are summarized inTable 3.
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Molecular Cytogenetic Analysis
Fluorescence In Situ Hybridization.—Hybridizationrefers to the binding or annealing of complementary DNAor RNA sequences that serve as probes. For this review, thediscussion will be confined to DNA-based probes. Molec-ular cytogenetic assays typically are performed withchromosome-specific probes labeled with fluorescent dyes(FISH). Alternatively, hybridization signals can be detectedwith peroxidase or alkaline phosphatase, but these ap-proaches are generally less sensitive and may be limited bythe numbers of colors that can be confidently distinguishedby standard bright-field microscopy.
A distinct advantage of FISH, in contrast to conven-tional cytogenetic analysis, is that this technique can beperformed on nondividing (interphase) cells from fresh oraged samples (such as blood smears, touch imprintcytologic preparations, or cytospin preparations), FFPEtissue sections, and disaggregated cells retrieved fromfresh, frozen, or FFPE material. Probe detection or labelingwith fluorescent molecules of different excitation and
emission characteristics permits simultaneous analysis ofseveral different probes. Importantly, this procedure canprovide results (such as identification of a tumor-specifictranslocation, amplification of an oncogene locus, or lossof a tumor suppressor gene locus) when the tissue isinsufficient or unsatisfactory for cytogenetic analysis,when conventional cytogenetic analysis has failed to yieldresults, or when cryptic rearrangements are present.65
Cytologic preparations are usually air-dried and sub-sequently fixed in 3:1 ratio of methanol to glacial aceticacid for 5 minutes. To visualize an anomaly within aspecific region of a tumor or within a specific cell type, a 4-to 6-mm-thick, paraffin-embedded tissue section can beused. Analysis of thin sections, however, is limitedbecause portions of most nuclei are lost during sectioning,and this may lead to false-positive results in the evaluationof chromosomal deletions or losses. For the most accurateassessment of subtle aneuploidy changes, the preferredapproach is to obtain whole or intact nuclei by disaggre-gating and releasing cells from a much thicker (50 to
Figure 4. Examples of a few select, molecular diagnostic approaches for evaluating SMARCB (previously INI1) in malignant rhabdoid tumors. A,Conventional G-banded karyotype, demonstrating monosomy 22 in an extrarenal rhabdoid tumor (arrowhead). B, Fluorescence in situ hybridization(FISH) analysis of a soft tissue rhabdoid tumor using a University of Nebraska Medical Center, custom-designed probe specific for the SMARCB locus(22q11.23), labeled in red, which reveals the loss of one copy of this locus in the interphase cells analyzed. The copy number–control probe, labeledin green, BCL2L13 (22q11.21), shows 2 copies and confirms the presence of a focused deletion of the SMARCB (original magnification 3400). C,Single nucleotide polymorphism array analysis of an extrarenal rhabdoid tumor, whole-genome view. Chromosomes are plotted in numeric orderalong the x-axis. A, Relative copy number. B, Heterozygous call bars indicating AB cells in the tumor. C, Allele-specific analysis plot. D, Copynumber–hidden Markov model (yellow, copy number 2). Arrowhead, copy number–neutral loss of heterozygosity [LOH] of chromosome 22 withdivergence of the red/green allele-specific plots, LOH call bars, and an overall copy number of 2. In this case, FISH analysis could not detect adeletion of the SMARCB locus because of the copy number–neutral LOH (array image courtesy of Jill M. Hagenkord, MD, Creighton UniversitySchool of Medicine, iKaryos Diagnostics, Omaha, Nebraska).
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60 mm) section. Fluorescence in situ hybridization is asame-day or overnight procedure, depending on theprobes used or the type of specimen analyzed (or both).
Chromosomal probes frequently used in clinical prac-tice to examine soft tissue tumors can be divided into 3categories: (1) centromere-specific (repetitive-sequence)probes, (2) locus-specific (unique-sequence) probes, and,(3) ‘‘paint’’ or whole chromosome probes. Although thereare a variety of quality-controlled DNA probes intendedfor clinical purposes manufactured commercially and soldas analyte-specific reagents, there are relatively few ofthese probes designed specifically for the study ofmesenchymal neoplasms. Rearrangements of some ofthese loci are seen in more than one soft tissue tumor type.For example, although the t(11;22)(q24;q12) is character-istic of Ewing sarcoma/PNET, rearrangement of EWSR1(22q12) is not confined to Ewing sarcoma but is also seenin most or in smaller subsets of desmoplastic small round-cell tumor, clear cell sarcoma, extraskeletal myxoidchondrosarcoma, and myxoid or round cell liposarcoma,among others. Some laboratories also elect to develop‘‘home-brew’’ probes for the assessment of specificresearch questions or for routine clinical use in analyzingrearrangements for which commercial probes are notavailable (Figure 5, A through D). Home-brew probes aredeveloped and used exclusively in-house and are not soldto other laboratories. Home-brew probes are not currentlyregulated by the US Food and Drug Administration and,therefore, clinical laboratories using such probes mustverify or establish, for each specific use of each probe, theperformance specifications for applicable performancecharacteristics, eg, accuracy, precision, analytical sensitiv-ity and specificity, among others.66
Advantages of FISH is that it can (1) be informative inboth metaphase and interphase cell preparations, thelatter to include FFPE tissue; (2) assist in deciphering theorigin of marker chromosomes, ring chromosomes, andcryptic or complex chromosomal rearrangements; and (3)reveal tumor-specific anomalies that are diagnosticallyuseful, especially in classifying mesenchymal neoplasmswith atypical clinical or histopathologic features. More-over, FISH provides cellular localization of DNA sequenc-es in a heterogeneous population, including detection oflow-level mosaicism. Additional advantages and limita-tions of FISH approaches are provided in Table 4.
Alternative Novel Molecular Cytogenetic Approach-es.—Alternative, novel FISH approaches, such as multi-
color FISH,67 spectral karyotyping,68 combined binaryratio FISH,69 metaphase-based CGH,70 array-basedCGH71,72 and SNP arrays73 are powerful genome-wideapplications that have expanded the diagnostic andprognostic capabilities of molecular cytogenetics in theanalysis of soft tissue tumors. The overall resolution ofinterphase FISH is approximately 50 to 100 kb comparedwith an overall resolution of 10 Mb for routine cytogeneticanalysis, 2 to 3 Mb for multicolor FISH and spectralkaryotyping, 2 to 10 Mb for metaphase-based CGH, andapproximately 10 to 100 kb for aCGH.74 Note, some high-resolution CGH arrays can reportedly detect structuralvariations at a resolution of 200 bp.75 Similar to aCGH,SNP arrays are capable of identifying copy-numberchanges (gains and deletions) at a high resolution andthroughout the whole genome but unlike aCGH, SNParrays also have the means to detect allele-specific copynumbers and LOH. Loss of heterozygosity does notalways refer to copy-number losses. For example, ac-quired uniparental disomy does not cause any genomiccopy-number changes but, instead, reflects allele-basedchanges (in other words, the changes are copy-numberneutral).57 Acquired uniparental disomy is emerging as acommon event in some types of cancer.
Genomic imbalances or copy-number alterations canlead to tumor development through the loss of tumorsuppressor genes or the amplification of oncogenes.Identification of recurrent copy-number imbalances orLOH using cytogenomic array (aCGH and/or SNP array)technology, respectively, provides valuable clues to poten-tial cancer-related genes, points to potential new therapeu-tic targets, and contributes to tumor classification anddiagnosis.76–78 Moreover, DNA copy-number changesidentified with global genomic approaches may aid inpredicting the prognosis of some soft tissue tumors.79–81
Notably, SNP array platforms may be supplanted withadvancing next-generation sequencers which are able togenerate all of the information that SNP arrays can producebut with (theoretically) greater resolution and accuracy.82
A sampling of some of the advantages and limitations ofgenomic and SNP array approaches are listed in Table 5.
Reverse Transcription–Polymerase Chain ReactionAnalysis.—The highly specific gene rearrangements thatresult from chromosomal translocations in soft tissuetumors can be identified with RT-PCR analysis. The PCRtechnique uses specific, synthetic primers to amplify asection of a gene in vitro. The PCR can be carried out on
Table 3. Advantages and Limitations of Conventional Cytogenetic Analysis
Advantages Limitations
Provides global information in a single assay. includes primary and secondary anomalies. knowledge of anticipated anomaly or histologic diagnosis not
necessary
Requires fresh tissue. although direct preparations can be performed, cell culture is
typically required (1–10 d)
Variants undetectable by interphase FISH or RT-PCR may beuncovered
. may encounter complex karyotypes with suboptimal morphology
. submicroscopic or cryptic rearrangements may result in afalse-negative result
Diagnostically useful. sensitive and specific. can be performed on fine-needle aspirates
Normal karyotypes may be observed following therapy-induced tumornecrosis or overgrowth of healthy supporting stromal cells
Provides direction for molecular studies of pathogeneticallyimportant genes
Difficulties encountered with bone tumors include low cell densityand the release of cells from the bone matrix
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RNA following reverse transcription (messenger RNARcomplementary DNA). Snap-frozen tissue is preferred forRNA extraction and RT-PCR analysis, but this procedurecan also be performed on archival (paraffin-embedded)material. To ensure the integrity of the RNA is of sufficientquality for study (RNA degrades relatively quickly), RT-PCR analysis of a housekeeping gene is also conducted(RNA quality control). This prevents the possibility ofreporting a false-negative result due to inadequate RNA.
Reverse transcription–polymerase chain reaction anal-ysis is remarkably sensitive. It may allow for the detectionof abnormalities present in cells too few to be identifiedwith traditional cytogenetic or FISH methods. Reversetranscription-polymerase chain reaction analysis may besuitable for the detection or monitoring of minimalresidual or minimal disseminated disease. For example,some authors using RT-PCR analysis for the detection of
Ewing sarcoma or alveolar rhabdomyosarcoma–associat-ed fusion transcripts have reportedly identified micro-metastatic disease in bone marrow or circulating tumorscells at diagnosis or posttherapy and relate pooreroutcomes for these patients.83–86 However, a study87 ofEwing sarcoma has suggested, at least at diagnosis, thatbone marrow involvement may be more relevant thanperipheral blood to clinical outcome. Additionally, thepresence of tumor cells in the peripheral blood may beattributable to mobilization by tumor biopsies, rather thaninherent tumor metastasis.88
Another advantage of RT-PCR analysis is that it is notdependent on successful cell culture, and, similar to FISH,it is rapid, with a short turnaround. Compared withcytogenetic analysis, the greatest disadvantage of RT-PCRanalysis is the inability to detect chromosomal anomaliesother than those for which the test was designed. With
Figure 5. Examples of home-brew or custom-designed fluorescence in situ hybridization (FISH) probes. A and B, The FISH analysis on metaphasecells and a corresponding formalin-fixed, paraffin-embedded (FFPE) tissue section of a dermatofibrosarcoma protuberans, with a University ofNebraska Medical Center (UNMC) home-brew probe set, demonstrates fusion of the COL1A1 (17q21) spanning probe in red and the PDGFB(22q13) spanning probe in green in the supernumerary ring chromosome and in the interphase nuclei, respectively (arrows) (original magnifications3400). The presence of a COL1A1-PDGFB fusion is characteristic of dermatofibrosarcoma protuberans. C and D, The FISH analysis performed onFFPE tissue sections of 2 cases of alveolar rhabdomyosarcoma (ARMS) with UNMC custom-designed PAX3 (C) and PAX7 (D) break-apart probes(original magnifications 3400). The ARMS in C shows a rearrangement of the PAX3 locus, indicated by the split of the orange and green signals(PAX3+) and the ARMS in D shows a rearrangement of the PAX7 locus (split orange and green signals) with amplification of the orange signal. PAX7rearranged alveolar rhabdomyosarcomas frequently exhibit both rearrangement and amplification of the derivative 13 chromosome of thecharacteristic t(1;13)(p36;q14) translocation as illustrated in this image (orange signal lies just distal to the PAX7 locus and was translocated to 13q14and amplified).
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conventional cytogenetic analysis, all major chromosomalabnormalities, including those not initially anticipated bythe clinician or laboratorian, may be uncovered. Addi-tional advantages and limitations of RT-PCR analysis arelisted in Table 6.
Sequencing Analysis.—As the genetic characterizationof soft tissue tumors is further refined with respect tospecific gene alterations (either as the inciting primarygenetic event or as secondary events in soft tissue tumorsexhibiting tumor-specific translocations), other molecularmethods will be increasingly used for identification ofthese mutations in oncogenes and tumor suppressor genes.DNA sequencing analysis of specific genes is helpful in thedetection of activating or inactivating missense mutations,deletions, and insertions. Sanger sequencing (dideoxynu-cleotide sequencing), one of the originally developedsequencing options, allows for selection of either a fullgene or specific exons and splice sites to sequence. The firststep of Sanger sequencing requires amplification of theDNA fragments of interest by PCR. Sequencing can beperformed on fresh or FFPE tissue if the DNA is of
sufficient quality. Full gene sequencing can be timeconsuming, and therefore, knowledge of recurrent (hotspot) mutations or important functional exon targets (eg,tyrosine kinase domain) is helpful. A limitation of Sangersequencing is that because of its lower analytical sensitiv-ity, specimen areas rich in tumor content ($20%–25%)must be identified and dissected for mutation detection.Assessment of specimens with lower tumor percentagesmay yield false-negative results, and therefore, onlyspecimens with carefully ascertained calculations of thepercentages of neoplastic cellularity should be studied.
Pyrosequencing is a more recently developed sequenc-ing approach that permits the sequencing of short DNAfragments (10–100 bp) within a gene. In clinical practice,pyrosequencing is usually reserved for 10- to 20-bp genehot spots in which a single site or a few codons maycontain single point mutations. Pyrosequencing is moresensitive than Sanger sequencing (with a detection limit ofabout 5%–10% of mutant alleles).
Of great interest is the rapid and widespread growth ofnext-generation sequencing. Next-generation sequencing
Table 4. Advantages and Limitations of Fluorescence In Situ Hybridization
Advantages Limitations
Can be performed on metaphase or interphase cell preparations(fresh, frozen, or paraffin-embedded material)
. can localize anomaly within specific cells or tissue types
More targeted approach; not a screening tool (generally requires priorknowledge of anomaly of interest)
. exceptions would be CGH and SKY
Can provide results when tissue is insufficient or unsatisfactoryfor cytogenetic analysis, when conventional cytogenetics hasfailed to yield results, or when cryptic rearrangements are present
. still a relatively gross approach when contrasting other molecularapproaches capable of detecting single-base mutations
. number of commercially available probes is limited
. interpretation may be challenging when analyzing suboptimalspecimens (ie, background fluorescence or autofluorescence,particularly with FFPE material)
Rapid turnaround FISH nomenclature not consistent among laboratories
Table 5. Advantages and Limitations of Genomic and Single Nucleotide Polymorphism (SNP) Arrays
Advantages Limitations
Provides global information in a single assay. knowledge of anticipated anomaly or histologic diagnosis
not necessary. powerful method of cancer-gene discovery. array technology continues to evolve coinciding with
computational advancements and biologic insight
Cannot detect balanced structural rearrangements, point mutations, orcopy number changes in chromosomal areas that are notrepresented on the array (ie, in areas between probes)
. some copy number imbalances are not clinically significant
. still learning what genomic imbalance patterns may be useful insoft tissue tumors
. standardization of cancer nomenclature/reporting is lacking
Higher resolution than cytogenetic or FISH analysis. a region of interest can be more densely analyzed with SNP
arrays as compared with microsatellite markers. SNP genotyping of tumor tissue DNA can be analyzed in
the absence of healthy DNA from the same individual
Requires relatively high-quality DNA (degraded DNA samples canlead to lower call rates or unsuccessful assays)
. analysis on FFPE tissue is challenging and is often performed onlower-density SNP arrays
. requires sophisticated, expensive equipment, and standards fordata storage have not been established
. relatively labor intensive
Diagnostically useful. sensitive, may provide results when conventional cytogenetics
or FISH have failed to yield results or when crypticrearrangements are present
Copy number information is relative, unless a cell-based assay is usedto confirm the normalization strategy
Copy number information is an average of all cell populations in thesample so subclones cannot be separately assessed
SNP arrays provide information regarding loss of heterozygosity(including copy number–neutral LOH)
. high-level amplification of a single allele can impair the ability todetect the second allele, rendering a false-LOH appearance
. distinguishing longer segments of autozygosity from somatic LOHevents is a challenging problema
Abbreviations: FFPE, formalin-fixed, paraffin-embedded; FISH, fluorescence in situ hybridization; LOH, loss of heterozygosity.a Examination of patient-matched, nonneoplastic DNA (if available) will alleviate this concern.
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includes various high-throughput sequencing technolo-gies that have the ability to rapidly process millions ofsequence reads and generate large data sets that can beanalyzed in different ways to answer a multitude ofquestions about genomic alterations in cancer. Althoughnot currently used in clinical practice, next-generationsequencing is anticipated to represent the next frontier intumor mutation analysis with the promise of an improvedunderstanding of carcinogenesis and a more preciseclassification system with discovery of new therapeutictargets. For now, the expense and bioinformatics burdenof this technology precludes its use in clinical moleculardiagnostic laboratories.
CONCLUSION
Dramatic advances in cytogenetic and molecular bio-logic techniques have furthered our understanding ofsarcomagenesis. Cytogenetic and molecular genetic as-says are used routinely for diagnostic and prognosticpurposes in molecular pathology laboratories and repre-sent a powerful adjunct to complement conventionalmicroscopy and radiographic assessment in the formula-tion of an accurate diagnosis. Care should be taken,however, to recognize the limitations of these approaches.Ideally, more than one technical approach should beavailable to a diagnostic laboratory to compensate for theshortcomings of another approach in different clinicalsituations.
Exciting new technologic innovations in moleculardiagnostics are on the horizon and soon will allow foreven higher-resolution analysis of the genetic anomaliesunderlying the pathogenesis of soft tissue tumors.Moreover, advancements in soft tissue pathology, notonly at the DNA level but also at the RNA level throughtranscriptome analysis and at the epigenetic level throughgenome-wide analysis of promoter methylation or chro-matin acetylation, can be anticipated. These advance-ments will likely not only affect diagnosis and classifica-tion schemes, but perhaps more important, personalizedtherapy based on identified targets within a given tumorcan be employed for improved outcomes.
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Table 6. Advantages and Limitations of Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Analysis
Advantages Limitations
Can be performed on fresh, frozen, or paraffin-embedded material. tissue quantity requirement is small
Not all sarcomas exhibit characteristic fusion-gene transcripts
Can provide results when tissue is insufficient or unsatisfactoryfor cytogenetic analysis, when conventional cytogenetics hasfailed to yield results, or when cryptic rearrangements are present
Targeted approach; not a screening tool. requires prior knowledge of fusion transcript
Diagnostically useful. sensitive and specific. rapid turnaround
. RNA quality may be inadequate because of RNA degradation
. reagent contamination is a potential hazard in small laboratoryspaces
Because of its remarkable sensitivity, RT-PCR may be useful for thedetection of minimal, residual disease or early relapsed disease
. devised primer sets may not detect unusual molecular variants(false-negative)
. identification of some product bands may require validation byadditional approaches, such as direct sequencing, transfer andhybridization with internal oligonucleotide probes, digestion withspecific restriction endonucleases, or reamplification with internalprimers (nested RT-PCR)
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