Molecular Basis of Disease

Chromosomal Translocations and Gene Rearrangements in Sarcomas

Chromosomal abnormalities (translocations, inversions, deletions, and insertions) are associated with DNA recombination and structural rearrangement of the genes located at the DNA breakpoints. If the breakpoint occurs within the involved gene, an altered gene structure arises. If the breakpoint occurs outside the gene, it may involve control elements critical for gene expression. In either case, the result can be a dramatic change in the gene structure, expression levels, or both.

Molecular analysis has identified two general mechanisms through which chromosomal translocations result in altered gene function. The first mechanism is gene fusion, in which chimeric or fusion genes are the result of joining of two parent genes (one upstream, or 5', the other downstream, or 3', to the breakpoint). Both genes are truncated by the translocation involving the coding portions of the parent genes. In general, translocation breakpoints are located in noncoding introns and the normal splicing mechanism removes the chimeric intron sequence. The exons are spliced "in frame" for the translational reading frame and can be translated into a novel fusion protein. In rare instances, the breakpoints are located in the exons of the parent genes. This may result in a novel chimeric product if the translational reading frame is maintained, or it may produce a truncated protein (encoded by the 5' gene sequence) if the reading frame is lost. Transcription of chimeric genes is usually under the control of the upstream parent gene promoter but may be influenced by DNA sequences in or close to the downstream gene.

The second mechanism through which chromosomal translocations result in altered gene function is promoter swapping or exchange. The breakpoint occurs at the 5' end of the coding region of the involved gene. This results in the replacement of its promoter region with enhancer elements or the promoter from the translocation partner. Promoter swapping leads to transcriptional activation, but the protein is wild type.

Numerous fusion genes have been identified in malignant tumors of the soft tissues and in mesenchymal tumors in general (see Table 26-1). The majority of sarcoma translocations result in in-frame fusion genes, resulting

Table 26-1. Chromosomal Alterations and Aberrant Gene Products in Sarcomas

Transcript

Variability

Chromosomal

Fusion

Prevalence

(number of fusion

Aberrant

Tumor Type

Changes

Gene

(%)

transcript variants)

Function

Reference

Ewing's sarcoma

t(11;22)(q24;q12)

EWS-FLI1

95

Large (18)

Transcription

Delattre. Nature.

(ES)/peripheral

factor

1992;359:162.

neuroectodermal

t(21 ;22)(q22 ;q12)

EWS-ERG

5

Large (4)

Transcription

Sorensen. Nat Genet.

tumor

factor

1994;6:146. Zucman.

(PNET)

EMBO J. 1993;12:4481.

t(7;22)(p22;q12)

EWS-ETV1

<1

Unknown

Transcription

Jeon. Oncogene.

factor

1995;10:1229.

t(17;22)(q12;q12)

EWS-E1AF

<1

Transcription

Urano. Biochem Biophys

factor

Res Commun. 1996;

219:608.

t(2;22)(q33;q12)

EWS-FEV

<1

Unknown

Transcription

Peter. Oncogene. 1997;

factor

14:1159.

Desmoplastic small

t(11;22)(p13;q12)

EWS-WT1

100

Small (3)

Transcription

Ladanyi. Cancer Res.

round cell tumor

factor

1994;54:2837.

(DSRCT)

Alveolar

t(2;13)(q35;p14)

PAX3-FKHR

95

None (1)

Transcription

Galili. Nat Genet. 1993;

rhabdomyosarc

factor

5:230. Shapiro. Cancer

oma (ARMS)

Res. 1993;53:5108.

t(1;13)(p36;p14)

PAX7-FKHR

10

None (1)

Transcription

Davis. Cancer Res.

factor

1994;54:2869.

Alveolar soft part

der(17)

ASPL-TFE3

100

Small (2)

Transcription

Ladanyi. Oncogene.

sarcoma

t(X;17)(p11;q25)

factor

2001;20:48-57.

DFSP/giant cell

t(17 ;22)(q22 ;q13)

COL1A1-PDGFB

100

Large (>8)

Autocrine

Simon. Nat Genet. 1997;

fibroblastoma

growth

15:95.

factor

Synovial sarcoma

t(X;18)(p11;q11)

SYT-SSX1

65

None (1)

Transcription

Clark. Nat Genet. 1994;

factor

7:502.

t(X;18)(p11;q11)

SYT-SSX2

35

None (1)

Transcription

Crew. EMBO J. 1995;14:

factor

2333. De Leeuw. Hum

Mol Genet. 1995;4:1097.

t(X;18)(p11;q11)

SYT-SSX4

<1

None (1)

Transcription

Skytting. J Natl Cancer

factor

Inst. 1999;91:974.

Clear cell sarcoma

t(12;22)(q13;q12)

EWS-ATF1

90

Small (2)

Transcription

Zucman. Nat Genet.

(malignant

factor

1993;4:341.

melanoma of

soft parts)

Congenital

t(12;15)(p13;q25)

ETV6-NTRK3

100

None (1)

Receptor

Knezevich. Nat Genet.

fibrosarcoma

tyrosine

1998;18:184.

kinase

Inflammatory

t(1;2)(q25;p23)

TPM3-ALK

20

None (1)

Receptor

Griffin. Cancer Res.

myofibroblastic

tyrosine

1999;59:2776.

tumor*

kinase

t(2;19)(p23;p13)

TPM4-ALK

10

None (1)

Receptor

Lawrence. Am J Pathol.

tyrosine

2000;157:377.

kinase

t(2;17)(p23;23)

CLTC-ALK

Uncertain

None (1)

Receptor

Bridge, Am J Pathol.

tyrosine

2001;159:41.

kinase

12q15

HMGIA2

Uncertain

Unknown

Transcription

Kazmierczac. Cancer

rearrangement

rearrangement

cofactor

Genet Cytogenet. 1999;

112:156.

Table 26-1. Chromosomal Alterations and Aberrant Gene Products in Sarcomas (Continued)

Transcript

Variability

Chromosomal

Fusion

Prevalence

(number of fusion

Aberrant

Tumor Type

Changes

Gene

(%)

transcript variants)

Function

Reference

Extraskeletal myxoid

t(9;22)(q22;q12)

EWS-CHN

75

Small (2)

Transcription

Labelle. Hum Mol Genet.

chondrosarcoma

factor

1995;4:2219. Clark.

Oncogene. 1996;12:229.

t(9;17)(q22;q11)

TAF2N-CHN

25

Unknown

Transcription

Sjogren. Cancer Res.

factor

1999;59:5064.

Panagopoulos.

Oncogene.

1999;18:7594. Attwooll.

Oncogene. 1999;18:7599.

t(9;15)(q22;q21)

TCS12-CHN

Uncertain

Unknown

Transcription

Sjogren. Cancer Res.

factor

2000;60:6832.

Lipoma

12q15

HMGIA2-LPP

50t

Unknown

Transcription

Ashar. Cell. 1995;82:57.

rearrangement

Other

cofactor

Schoenmakers. Nat

Genet. 1995;10:436.

Petit, Genomics.

HMGIC

1996;36:118.

rearrangement

6p21

HMGA1

10t

Unknown

Transcription

Tkachenko. Cancer Res.

rearrangement

cofactor

1997;57:2276.

Lipoblastoma

8q11-13

HAS2-PLAG1

>90

None (1)

Transcription

Hibbard. Cancer Res.

rearrangement

factor

2000;60:4869.

t(7;8)(q22;q12)

COL1A2-PLAGI

Uncertain

Unknown

Transcription

Hibbard. Cancer Res.

factor

2000;60:4869.

Myxoid/round cell

t(12;16)(q13;p11)

TLS-CHOP

>95

Small (3)

Transcription

Crozat. Nature. 1993;363:

liposarcoma

factor

640. Rabbitts. Nat

Genet. 1993;4:175.

t(12;22)(q13;q12)

EWS-CHOP

2-5

Unknown

Transcription

Panagopoulos. Oncogene.

factor

1996;12:489.

Well differentiated

12q13-15

HMGIC,

>60

Unknown

Gene

Meza-Zepeda. Cancer.

liposarcoma

amplification

MDM2,

amplification

2001;31:264. Pedeutour.

SAS, CDK4

resulting

Genes Chromosomes

in cell growth

Cancer. 1994;10:85.

dysregulation

Endometrial stromal

t(7;17) (p15;q21)

JAZF1-JJAZ1

40-100

None (1)

Transcription

Koontz. Proc Natl Acad

tumors

factor

Sci USA. 2001;98:6348.

Aggressive

12q13-15 rearr

HMGIC

Uncertain

Unknown

Transcription

Nucci. Genes

angiomyxoma

rearrangement

cofactor

Chromosomes

Cancer. 2001;32:172.

Angiomatoid fibrous

t(12;16)(q13;p11)

TLS-ATF1

Uncertain

Unknown

Transcription

Waters. Cancer Genet

histiocytoma

factor

Cytogenet. 2000;121:109.

Extrarenal rhabdoid

del22q11.2 or

INI1 deletion!

>90

Unknown

Loss of tumor

Newsham. Genomics.

tumor

22q11.2

suppressor

1994;19:433.

rearrangement

gene

Low grade

t(7;16)(q33;p11)

FUS-CREB3L2

>95 %

Large

Transcription

Mertens. Lab Invest.

fibromyxoid

(BBF2H7)

factor

2005;85,408. Reid, Am

sarcoma

J Surg Pathol. 2003;27,

1229.

t(11;16)(q11;p11)

FUS-CREB3L1

<5 %

Unknown

Transcription

Mertens. Lab Invest.

factor

2005;85,408.

*The proportion of inflammatory myofibroblasts tumors with ALK gene rearrangement is 35%, overall.

^Percentages indicate the proportion of tumors with abnormal karyotypes which have 12q13-15 or 6p21 chromosomal alterations. (Sources:

Heim S, Mitelman F. Cancer Cytogenetics, 2nd ed. New York:Wiley-Liss, 1995;Fletcher CD,Akerman M, Dal Cin P, et al. Correlation between

clinicopathological features and karyotype in lipomatous tumors. A report of 178 cases from the Chromosomes and Morphology (CHAMP)

Collaborative Study Group. Am J Pathol. 1996;148:623-630.

t No fusion gene is present.

in abnormal chimeric transcription factors.3 In a few cases, the gene fusion results in an aberrant tyrosine kinase or an autocrine growth factor.4-6 A recent investigation identified the der(17) associated with the nonreciprocal t(X;17)(p11.2;q25) of human alveolar soft part sarcoma.7 The translocation produces a chimeric transcript between the TFE3 transcription factor gene and ASPL, a novel gene at 17q25. ASPL encodes a UBX-like domain at the C-terminus of the encoded protein. In alveolar soft part sarcoma, the 5' end of ASPL is fused to exon 3 or 4 of TFE3, resulting in a fusion protein retaining the C-terminal TFE3 DNA-binding domain, a possible aberrant transcriptional regulator.

A recurrent t(7;17)(p15;q21) has been identified in endometrial stromal tumors.8 Two new zinc finger genes are fused as a result of the translocation: JAZF1 and JJAZ1. Protein products of the zinc finger genes usually function as transcriptional regulators via specific DNA binding through the zinc finger motif. The chimeric protein in endometrial stromal tumors has a tumor-specific mRNA transcript containing 5' JAZF1 and 3' JJAZ1 sequences including the zinc finger encoding regions from both parent genes. Since gene expression of wild-type JAZF1 is present in normal endometrial stromal cells, the JAZF1-JJAZ1 fusion gene present in endometrial stromal tumors likely results in aberrant transcriptional regulation in a lineage-specific manner.

Specificity and Oncogenic Nature of Sarcoma Fusion Transcripts

Unlike many epithelial neoplasms, where diverse genetic alterations usually underlie the stepwise progression of precursor lesions leading ultimately to the emergence of malignant clones, soft tissue malignancies have no identifiable precursor lesions, and clear, stepwise progressive mutational events have not been described. Rather, more often than not, a single genetic alteration is found in a particular type of sarcoma. In addition, chromosomal fusions in soft tissue sarcomas do not seem to represent a form of generalized genomic instability, as occurs with germline TP53 mutations1 or with microsatellite instability associated with colon carcinoma.9 Benign tumor counterparts of soft tissue sarcomas usually carry quite different genetic or chromosomal abnormalities, or both. For example, the specific sets of chromosomal alterations found in soft tissue lipomas are not among those consistently observed in liposarcoma.10,11 Similar to leukemoge-nesis and lymphomagenesis, the fusion gene in a given sarcoma is speculated to be oncogenic only in a specific cell type at a specific differentiation stage.12

In general, the genes involved in sarcoma translocation are transcription factors or cofactors. Many of the chimeric proteins include a strong transcriptional activator N-terminal domain encoded by one partner gene fused with a DNA-binding domain encoded by the other partner gene.

In fact, fusion of domains capable of activating transcription with other domains featuring specific DNA-binding function appears to be a common theme shared among neoplasms of mesenchymal derivation, such as soft tissue tumors and leukemia. EWS (and its homologous TLS/FUS) is a powerful transcription activator13 and provides a paradigm for this type of oncogenic mechanism, as also indicated by its "promiscuity" as a fusion partner (Table 26-1).

Available data indicate that the fusion genes produced in translocation-specific sarcomas are the initiating events that either are necessary or sufficient for the genesis of these malignant soft tissue tumors. In vitro and in vivo experiments have shown evidence of tumorigenesis with the expression of these fusion genes. The most common chromosomal translocation in myxoid liposarcomas, t(12;16)(q13;p11), creates a TLS/FUS-CHOP fusion gene. Transgenic mice expressing the altered form of FUS-CHOP created by an in-frame fusion of the FUS domain to the carboxy end of CHOP develop liposarcomas. No tumors of other tissues were found in these transgenic mice despite widespread activity of the transgene. The results provided evidence that the FUS domain of FUS-CHOP plays a specific and critical role in the pathogenesis of liposarcoma.14 Alveolar rhabdomyosarcoma (ARMS) is consistently associated with the characteristic translocation t(2;13)(q35;q14) or t(1;13)(p36;q14), which encode the PAX3-FKHR or PAX7-FKHR fusion oncoproteins, respectively. PAX3-FKHR fusion protein contributes to oncogen-esis through abnormal control of growth, apoptosis, differentiation, or motility.15

Available Assays and Interpretation

A significant number of sarcomas have consistent chromosomal abnormalities that are detectable by standard cytogenetics or molecular genetic approaches (Figure 26-1).2,3,16

Target

Method

Tissue Requirement

Chromosome

Fusion gene

Karyotyping FISH

Southern blot Genomic PCR

RT-PCR/sequencing Real-time RT-PCR

Fusion transcript

Viable tissue for culture Touch preparation Unfixed (fresh of frozen) Paraffin fixed

Unfixed (fresh or frozen) Unfixed (fresh or frozen)

Unfixed (fresh or frozen) Unfixed (fresh or frozen) Paraffin fixed

Figure 26-1. Molecular and cytogenetic methods for sarcoma diagnosis.

Sarcomas in this group should be defined by their specific molecular and cytogenetic alterations, although it will continue to be important to determine the sensitivity and specificity of these translocations for specific sarcoma types and the relative roles of molecular and histological classification of each sarcoma.

Karyotyping

Karyotyping is the classic cytogenetic approach for identifying chromosomal alterations including translocations in sarcomas.17 Optimally, the procedure requires a substantial volume of viable, sterile tumor tissue, usually 1 cm3 to 2 cm3. The specimen should be harvested, placed in culture medium as soon as possible, and transported to the laboratory. Specimens also can be sent over a long distance at either room temperature or refrigerated for up to 48 hours. Small samples (limited incisional or needle biopsies) can be successfully cultured and karyotyped, although they may require a longer incubation time (1 to 2 weeks) to obtain enough dividing tumor cells for analysis. Characteristic and diagnostic chromosomal alterations seen in human soft tissue tumors are listed in Table 26-1.

An obvious drawback of conventional cytogenetic analysis is the requirement of adequate tumor cell growth to obtain metaphase spreads. If tumor cells do not grow, a false-negative result of a normal karyotype may occur due to the presence of normal fibroblasts in the specimen. Another pitfall of karyotyping is its limited resolution for identification of cryptic alterations, which may occur in sarcomas. Furthermore, translocations involving specific chromosomal regions may not necessarily represent the characteristic gene fusion for a specific sarcoma. Confirmation by fluorescence in situ hybridization (FISH) or molecular testing, therefore, is recommended for questionable cases or to reach a definitive diagnosis.

Fluorescence In Situ Hybridization

FISH provides a powerful diagnostic modality to demonstrate a specific gene fusion or chromosomal alteration. Metaphase chromosomal preparation from the tumor can be used to demonstrate translocations by chromosomal painting using chromosome-specific probes or gene-specific probes. However, this is not always possible since cell culture of sarcoma tissue to obtain metaphase chromosomes may be unsuccessful. In these cases, interphase FISH provides an excellent alternative using touch preparations from fresh or frozen tumor specimens without the requirement for tissue culture. One major advantage of interphase FISH over traditional karyotyping is the ability to detect cryptic gene rearrangements. In fact, the chromosomes of interphase nuclei are much more extended than metaphase or prometaphase chromosomes. As a result, FISH analysis performed on interphase nuclei permits higher resolution and can help to determine the

Figure 26-2. Dual-color interphase FISH is used to detect gene fusions in soft tissue sarcomas. Breakpoint flanking probes (one red and one green) are hybridized to nuclei preparation from paraffin-embedded tumor samples. The juxtaposed red and green signals, which can appear yellow in some nuclei, represent the chromosomal translocation (present in all four nuclei shown).

Figure 26-2. Dual-color interphase FISH is used to detect gene fusions in soft tissue sarcomas. Breakpoint flanking probes (one red and one green) are hybridized to nuclei preparation from paraffin-embedded tumor samples. The juxtaposed red and green signals, which can appear yellow in some nuclei, represent the chromosomal translocation (present in all four nuclei shown).

physical mapping order of large DNA probes. Dual-colored DNA probes from the two rearranged genes involved in the translocation are most commonly used for the diagnosis of sarcomas with a specific translocation (Figure 26-2). Chromosomal centromeric probes and DNA probes spanning the translocation breakpoint also can be used. The interpretation of the FISH result depends on the types of hybridization probes.

The possibility of performing interphase FISH on fixed paraffin-embedded tissue, either on histology sections or on nuclei preparations from paraffin blocks, has recently increased its potential diagnostic applications.18,19 For interphase FISH of paraffin-embedded specimens, analysis of a nuclei preparation offers several advantages over the analysis of histological sections. The nuclei are more individualized on the slide, offering easy access to probe hybridization and interpretation. The complete genome of each nucleus is available for FISH, because there is no trimming effect as seen with histologic sections, in which some nuclei are only partially present on a slide due to the sectioning of the specimen (4-5 |im thick). On the other hand, direct examiniation of histological sections allows for correlation of FISH results with the architectural features of the tumor.

Southern Blot

Southern blot analysis detects sarcoma-specific translocations using labeled DNA probes specific to the fusion genes. The procedure is highly specific and particularly useful in detecting translocations with frequently variable translocation breakpoints, as for Ewing's sarcoma (ES). The main limitations of the Southern blot method are that it is labor intensive and requires fresh or frozen tissue to obtain high-molecular-weight genomic DNA. False-negative results may arise when DNA fragments surpass the upper limits of DNA length (>15-20 kilobases [kb]), or with fusion genes encompassing large intron(s). Increasing the number of restriction enzymes usually resolves this problem. False-positive results may be seen due to incomplete restriction enzyme digestion, contamination by a cloning vector or bacteria, structural polymorphism of the genes involved in the translocation, and restriction recognition site variation.

Genomic Polymerase Chain Reaction

Polymerase chain reaction (PCR) amplification of the region encompassing the breakpoint of a fusion gene is often problematic. First, the breakpoint positions within the introns of the two rearranged genes are often variable, requiring selection of PCR primers within the adjacent exon and resulting in a variable size of the PCR product from case to case. Also, the introns can be very large, resulting in very large PCR products that may not be completely amplified and reducing the sensitivity of the test. Therefore, genomic PCR of sarcoma-specific translocations is not used for clinical testing.

Reverse Transcription-PCR

Chromosomal translocations in sarcomas give rise to aberrant fusion transcripts that are highly specific to a given tumor type (Table 26-1). More important, the structures of these fusion transcripts are highly consistent. Although the translocation breakpoint may involve various nucleotide positions of an intron at the DNA level, the resulting fusion transcript structure is the same due to RNA splicing. In a given sarcoma type, the gene fusion point at the messenger RNA (mRNA) level is highly precise to the single ribonucleotide. It is the tumor specificity and structural consistency of the fusion transcripts that make reverse transcription-PCR (RT-PCR) the preferred method for molecular detection of specific fusion transcripts.20

After reverse transcription of tumor mRNA into complementary DNA (cDNA), PCR primers complementary to the exons that flank the translocation breakpoint are used to amplify fusion transcript-specific RT-PCR products. Variations of RT-PCR methods have been used for the detection of sarcoma-specific fusion transcripts. Since the fusion joining point may be far away from the poly(A) tail of the aberrant transcript, random hexamers or gene-specific downstream primers should be used instead of oligo d(T) for the reverse transcription of RNA into cDNA. Instead of the traditional two-step RT-PCR method, reverse transcription and PCR can be performed in a single reaction, a procedure called one-step RT-PCR. The reverse tran-scriptase works first at low temperature to convert mRNA into cDNA, while the Taq DNA polymerase is inactive. The temperature then is raised to inactivate the reverse tran-

scriptase, to activate the Taq DNA polymerase, and to initiate the amplification reaction at the same time. Reactions with multiple primer sets are difficult to optimize but are very convenient for molecular testing of sarcomas with numerous fusion gene variants such as ES/peripheral neuroectodermal tumor (PNET).21 Nested PCR using an additional pair of primers to further amplify the first-round RT-PCR product can greatly increase the sensitivity, although it also increases the likelihood of cross contamination.

Identification of a positive RT-PCR result usually relies on detection of the expected-sized product on a common DNA-separating gel. When fresh or frozen tissue is the starting material, the target size of the RT-PCR product is designed carefully to be less than 500 base pairs (bp) and preferably around 200 bp. For fusion genes that have little size variations, such as SYT-SSX fusion of synovial sarcoma, the presence of a single RT-PCR product of the expected size generally is considered adequate for positive identification, although post-PCR confirmatory analysis of the product sequence can be performed. For fusion genes with molecular size variability, such as TLS-CHOP in myxoid liposarcoma, additional analysis of the PCR products is mandatory to confirm the specificity of the PCR products. Confirmatory methods include blot transfer with subsequent hybridization with a fusion gene-specific probe, DNA sequencing of the PCR product, restriction endonuclease digestion analysis, or an additional nested PCR step. Inclusion of positive and negative control reactions is necessary because the high sensitivity of the amplification process and cross-contaminations are major problems with RT-PCR detection of fusion transcripts for clinical diagnosis.

Paraffin-embedded blocks of soft tissue tumors may be used as the source of RNA,22 although with some caution.10 As a result of the fixation and embedding processes, the RNA from tissue blocks is substantially degraded. RNA degradation requires that the size of the amplification product be designed to be considerably smaller (around 100 bp) compared to assays designed for use of fresh or frozen tissue. RT-PCR from tissue blocks usually requires more amplification cycles, and a nested approach may be necessary for the detection of the aberrant transcript. These assay-altered conditions account for an increased risk of false-positive results due to carryover contamination. Very stringent conditions and additional control reactions are necessary to ensure test specificity.

Consideration must be given to the variability of fusion gene transcripts for each type of sarcoma (see Table 26-1) when interpreting RT-PCR test results. For instance, in ES/PNET, there are considerable variations among the different exons involved in the gene fusion.21 Although the tumors may be cytogenetically indistinguishable from one another, they differ in the exon composition of the final fusion transcript. Variant chromosomal translocations involving different chromosomal partners also give rise to variability in approximately 5% of ES/PNET cases. In fact, the t(21;22), instead of the far more common t(11;22), results in the fusion of EWS with ERG.23 Very rarely, EWS is fused with other loci including ETV1 or E1AF in ES/PNET.21 Awareness of these variants is very important when a negative RT-PCR for EWS-FLI1 occurs.

Real-Time RT-PCR

Real-time RT-PCR is a recent advancement in analysis of mRNA.24 Although designed for quantitative measurement, real-time RT-PCR has become a reliable detection method for fusion gene transcripts.25,26 It has three major advantages over the conventional endpoint RT-PCR. First, realtime RT-PCR greatly enhances the specificity of detection by incorporating a specific probe, complementary to the region of the gene internal to the PCR primers. Second, real-time PCR measures the geometric (exponential) phase of a PCR reaction (cycles 20-25), offering an accurate quantification of the fusion transcript. Third, the PCR product accumulation is measured in real time by fluorescence detection through the semitransparent plastic cap of the reaction tube and, therefore, post-PCR manipulation and possible PCR contamination are reduced. Since its introduction for molecular analysis, real-time RT-PCR has shown conclusively high specificity and sensitivity, which are important when analyzing very small samples or target genes with very low expression levels.25 Simultaneous amplification of multiple targets by multiplex real-time RT-PCR is possible by using target-specific probes labeled with different fluorescent labels.27

With careful optimization, real-time RT-PCR can be highly reliable for detection of fusion transcripts using all types of tissue sources.26 The early geometric phase detection with enhanced specificity and elimination of post-PCR manipulation makes real-time RT-PCR ideal for detection of sarcoma fusion transcripts, even if the RNA source is paraffin-embedded tissue.28 The specific and quantitative detection power of real-time RT-PCR using paraffin-embedded tissue is illustrated in Figure 26-3, in which amplification of the SYT-SSX fusion transcript of synovial sarcoma is shown. Positive identification of fusion transcripts can be achieved using as little as 20 ng of RNA extracted from one paraffin block, making small specimens, such as core needle biopsies, suitable for definitive molecular diagnosis. Overall, real-time RT-PCR offers significant advantages over conventional RT-PCR. It is technically easier, more rapid, and more sensitive. Because a specific probe is included in the reaction, secondary confirmation is unnecessary, and the problem of carryover cross-contaminations is minimized. The operatingcost profile and time to reporting also are in favor of real-time RT-PCR over conventional RT-PCR.25 The routine clinical applications of real-time RT-PCR for sarcoma diagnosis and minimal disease detection are highly promising.

Delta Rn vs cycle

1.0e+001

1.0e+000

1.0e-001

1.0e-002

1.0e-003

1.0e-004

Delta Rn vs cycle

1.0e+000

1.0e-001

1.0e-002

1.0e-003

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 Cycle number

Standard curve

Standard curve

d

\

b

\

\ r

2.2 Log C0

Figure 26-3. Real-time RT-PCR detection of gene fusion in a sarcoma. Top panel: Realtime RT-PCR plot of PCR cycle number versus fluorescence signal for SYT-SSX fusion transcript amplification in a synovial sarcoma with (a) 50ng, (b) 100ng, (c) 200 ng, and (d) 500ng of total input RNA, each amplified in triplicate. The RNA was extracted from formalin-fixed, paraffin-embedded tissue. Bottom panel: Log gradient plot of the data from the top panel, demonstrating linear quantitative amplification consistent with the amount of input RNA template. The y-axis represents the threshold cycle number, and the x-axis is the log of the RNA concentration. The quantity of the specific RNA measured by real-time RT-PCR is inversely proportional to the threshold cycle number.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment