Archer® FusionPlex® Sarcoma Kit

 

The Archer® FusionPlex® Sarcoma kit is a targeted sequencing assay to simultaneously detect and identify fusions of 26 genes associated with soft tissue cancers. Using Archer’s proprietary Anchored Multiplex PCR™-based enrichment, fusions of all genes in this kit can be identified in a single sequencing assay, even without prior knowledge of fusion partners or breakpoints.

For Research Use Only. Not for use in diagnostic procedures.

Highlights

  • Comprehensive - identify sarcoma-related fusions in a single assay
  • Detailed - characterize fusion partners at single-nucleotide resolution
  • Innovative - first comprehensive NGS-based sarcoma fusion test on the market
  • Streamlined – reduce turn-around time and eliminate reflex testing

Assay Targets

ALK CAMTA1 CCNB3 CIC
EPC EWSR1 FKHR FUS
GLI1 HMGA2 JAZF1 MEAF6
MKL2 NCOA2 NTRK3 PDGFB
PLAG1 ROS1 SS18 STAT6
TAF15 TCF12 TFE3 TFG
USP6 YWHAE    

Sarcoma Gene Fusion Map

Archer FusionPlex Sarcoma Kit Gene Fusions This schematic shows known soft tissue sarcoma gene fusions. Everything shown is detected by this assay.

Assay Targets

Gene Accession Target Exon Direction Assay Type
ALK NM_004304 19,20,21,22 5' Fusion
CAMTA1 NM_015215 8,9,10 5' Fusion
CCNB3 NM_033031 2,3,4,5,6 5' Fusion
CIC NM_015125 19,20 3' Fusion
EPC1 NM_025209 9,10,11 3' Fusion
EWSR1 NM_005243 4,5,6,7,8,9,
10,11,12,13
3' Fusion
FOXO1 NM_002015 1,2,3 5' Fusion
FOXO1 NM_002015 1,2,3 3' Fusion
FUS NM_004960 4,5,6,7,8,9,
10,11,14
3' Fusion
GLI1 NM_005269 4,5,6,7 5' Fusion
GLI1 NM_005269 4,5,6,7 3' Fusion
HMGA2 NM_003483 1,2,3,4,5 3' Fusion
JAZF1 NM_175061 2,3,4 3' Fusion
MEAF6 NM_001270875 4,5 3' Fusion
MKL2 NM_014048 11,12,13 5' Fusion
NCOA2 NM_006540 11,12,13,14 5' Fusion
NTRK3 NM_002530 13,14,15,16 5' Fusion
NTRK3 NM_002530 13,14,15 3' Fusion
PDGFB NM_002608 2,3 5' Fusion
PLAG1 NM_002655 1,2,3,4 5' Fusion
ROS1 NM_002944 31,32,33,34,
35,36,37
5' Fusion
SS18 NM_001007559 10,11 5' Fusion
SS18 NM_001007559 4,5,6,8,9,10 3' Fusion
STAT6 NM_001178078 1,2,3,4,5,6,7,
16,17,18,19
5' Fusion
TAF15 NM_139215 6,7 5' Fusion
TAF15 NM_139215 5,6,7 3' Fusion
TCF12 NM_207036 4,5,6 3' Fusion
TFE3 NM_006521 3,4,5,6 5' Fusion
TFG NM_006070 4,5,6,7 3' Fusion
TFG NM_006070 6 5' Fusion
USP6 NM_004505 1,2,3 5' Fusion
YWHAE NM_006761 5 3' Fusion

What is sarcoma?

Sarcoma is broadly defined as a cancer arising from cells of mesenchymal origin. Mesenchymal cells develop into and comprise connective tissues such as bone and cartilage as well as tissues of the lymphatic and circulatory systems. Sarcomas are relatively rare—only about 15,000 new cases are reported each year in the United States (1). Nevertheless, sarcomas account for more than 20% of all childhood malignancies. The majority of sarcoma cases are soft tissue sarcomas originating in fat, muscle and tissue of the trunk, arms or legs; however, malignant bone tumors also comprise over 10% of sarcoma instances. Because early-stage sarcomas frequently elude identification until substantial metastasis has occurred, sarcomas bear a particularly high mortality rate and represent an important challenge for cancer research (1).

Section of rhabdomyosarcoma (right) with normal muscle tissue (left)

Section of rhabdomyosarcoma (right) with normal muscle tissue (left).


Translocations in sarcoma

Chromosomal translocations are genomic rearrangements that result in gene fusions that drive the pathogenesis of many types of cancer. Current estimates attribute 20-30% of sarcomas to these translocations (3,4). Several translocations were found to be recurrently associated with specific sarcoma subtypes. For example, translocations between the EWSR1 and FLI1 genes are thought to underlie 85% of Ewing sarcomas, a rare bone cancer that occurs most frequently in adolescents (4). Similarly, in the case of synovial sarcoma, which can be difficult to identify histologically, the prevalence of SS18-SSX1 translocations is so dramatic that molecular detection of this fusion might be necessary to make a conclusive determination (5).

Tumor Type Translocation Gene(s)
Aneurysmal bone cyst t(16;17)(q22;p13)
t(1;17)(p34.3;p13)
t(3;17)(q21;p13)
t(9;17)(q22;p13)
t(17;17)(q21;p13)
CDH11-USP6
THRAP3-USP6
CNBP-USP6
OMD-USP6
COL1A1-USP6
Angiofibroma t(5;8)(p15;q13) AHRR-NCOA2
Angiomatoid fibrous histiocytoma t(12;22)(q13;q12)
t(2;22)(q33;q12)
t(12;16)(q13;p11)
EWSR1-ATF1
EWSR1-CREB1
FUS-ATF1
Alveolar rhabdomyosarcoma t(2;13)(q35;q14)
t(1;13)(p36;q14)
PAX3-FKHR
PAX7-FKHR
Alveolar soft-part sarcoma der(17)t(X;17)(p11;q25) ASPSCR1-TFE3
Chondroid lipoma t(11;16)(q13;p13) C11orf95-MKL2
Clear-cell sarcoma t(12;22)(q13;q12)
t(2;22)(q33;q12)
EWSR1-ATF1
EWSR1-CREB1
Congenital/infantile fibrosarcoma t(12;15)(p13;q25) ETV6-NTRK3
Dermatofibrosarcoma protuberans/
giant cell fibroblastoma
t(17;22)(q21;q13) COLIA1-PDGFB
Desmoplastic small round-cell tumor t(11;22)(p13;q12) EWSR1-WT1
Endometrial stromal sarcoma t(7;17)(p15;q11)
t(6;7)(p21;p15)
t(6;10)(p21;p11)
t(t(10;17)(q22;p13)
t(1;6)(p32~34;p21)
JAZF1-JJAZ1(SUZ12)
JAZF1-PHF1
EPC-PHF1
YWHAE-FAM22A/B
MEAF6-PHF1
Epithelioid hemangioendothelioma t(1;3)(p36;q25)
t(X;11)(p11.2;q22.1)
WWTR1-CAMTA1
TFE3-YAP1
Ewing sarcoma;
Primitive neuroectodemal tumor (PNET)
t(11;12)(q24;q12)
t(21;22)(q22;q12)
t(7;22)(p22;q12)
t(2;22)(q33;q12)
t(17;22)(q12;q12)
inv(22)(q21;12)
t(16;21)(p11;q22)
EWSR1-FLI1
EWSR1-ERG
EWSR1-ETV1
EWSR1-FEV
EWSR1-E1AF
EWSR1-ZSG
FUS-ERG
Ewing-like bone sarcoma inv(X)(p11.4p11.22)
t(20;22)(q13;q12)
BCOR-CCNB3
EWSR1-NFATC2
Extraskeletal myxoid
chondrosarcoma
t(9;22)(q22;q12)
t(9;17)(q22;q11)
t(9;15)(q22;q21)
t(3;9)(q11;q22)
EWSR1-NR4A3
TAF15-NR4A3
TCF12-NR4A3
TFG-NR4A3
Giant cell fibroblastoma t(17;22)(q21;q13) COLIA1-PDGFB
Inflammatory myofibroblastic tumor t(1;2)(q22;p23)
t(2;19)(p23;p13)
t(2;17)(p23;q23)
t(2;2)(p23;q13)
t(2;11)(p23;p15)
inv(2)(p23;q35)
t(2;4)(p23;q21)
TPM3-ALK
TPM4-ALK
CLTC-ALK
RANBP2-ALK
CARS-ALK
ATIC-ALK
SEC31A-ALK
Lipoblastoma t(7;8)(p22;q13) COL1A2-PLAG1
Lipoma t(3;12)(q27-28;q14-q15) HMGA2-LPP
LPP-HMGA2
Low-grade fibromyxoid sarcoma t(7;16)(q33;p11)
t(11;16)(p11;p11)
FUS-CREB3L2
FUS-CREB3L1
Meningeal hemagiopericytoma inv(12)(q13q13) NAB2-STAT6
Mesenchymal chondrosarcoma t(8;8)(q13;q21) HEY1-NCOA2
Myoepithelial tumor of
soft tissue and bone
t(6;22)(p21;q12)
t(19;22)(q13;q12)
t(1;22)(q23;q12)
t with 16p11
EWSR1-POU5F1
EWSR1-ZNF444
EWSR1-PBX1
FUS
Myxoid/round-cell liposarcoma t(12;16)(q13;p11)
t(12;22)(q13;q12)
FUS-DD1T3
EWSR1-DD1T3
Nodular fasciitis t(17;22)(p13;q13) MYH9-USP6
Pericytoma t(7;12)(p22;q13) ACTB-GLI1
Pulmonary myxoid sarcoma t(2;22)(q34;q12) EWSR1-CREB1
Sclerosing epithelioid
fibrosarcoma
t(7;16)(p22;q24)
t(11;16)(p13;p11.2)
FUS-CREB3L2
FUS-CREB3L1
Solitary fibrous tumor inv(12)(q13q13) NAB2-STAT6
Spindle cell rhabdomyosarcoma t(6;8)(p21;q13)
t(8;11)(q13;p15)
SRF-NCOA2
TEAD-NCOA2
Synovial sarcoma t(X;18)(p11;q11) SS18-SSX1
SS18-SSX2
SS18-SSX4
Undifferentiated small round
blue cell tumor
t(4;19)(q35;q13)
t(10;19)(q26;q13)
CIC-DUX4
CIC-DUX4

Limitations to current translocation detection techniques

Chromosomal translocations have been traditionally detected using methods that vary in sensitivity, scalability and the ability to multiplex. These methods include:

  • Fluorescence in situ hybridization (FISH)
  • Immunohistochemistry (IHC)
  • Reverse transcription polymerase chain reaction (RT-PCR)

IHC is a relatively straightforward method to detect proteins in fixed tissue sections using antibodies specific to the target protein. IHC can indirectly detect translocations if the gene fusion leads to overexpression of a fusion protein above background levels, with the intensity of the staining indicative of fusion protein expression level. Although not technically challenging, a key limitation to IHC as an effective translocation detection strategy is the need for antibodies that target one of the fusion partners. Also, IHC only provides a qualitative analysis because of the nonlinear chromogenic signal, and the limited number of chromogenic signals available prevents the use of IHC for multiplexing.

FISH relies on a fluorescent DNA probe that hybridizes to the target gene in chromosomal DNA, and translocations are often detected by visually determining the colocalization of two fluorescent probes that hybridize to flanking sequences in the target fusion event. FISH is more objective than IHC, because colocalization of the two fluorescent probes is positive for the fusion event. But there is some subjectivity because of the level of colocalization, especially when the two genes are normally in close proximity. FISH is also technically challenging, laborious by both the preparation and preview, poorly scalable and limited in multiplexing.

RT-PCR is an inexpensive and robust method to detect gene fusions using very low input amounts by reverse transcribing messenger RNA into complementary DNA (cDNA) and then amplifying and detecting the target genes. The method yields a relatively straightforward yes-or-no readout, but the sequences of both fusion partners must be known to craft forward and reverse primers to amplify the fusion sequence. RT-PCR also is limited in scalability and clinical sensitivity.

Gene fusions can be detected by histological and molecular methods, including IHC, FISH and RT-PCR (from left to right).

Gene fusions can be detected by histological and molecular methods, including IHC, FISH and RT-PCR (from left to right)

Because of limitations associated with these traditional methods, cutting-edge technologies are increasingly defining novel sarcoma subtypes through the identification of new chromosomal translocations (6-8).

Detecting fusions using the Archer FusionPlex Sarcoma Kit

ArcherDX has developed a kit for the rapid detection of sarcoma-associated translocations from total nucleic acid isolated from tumor samples—including FFPE preserved specimens. Anchored Multiplex PCR (AMP) enables rapid preparation of highly multiplexed next generation sequencing (NGS) libraries for targeted capture of mRNAs produced from fusion genes. The Archer technology permits the simultaneous detection of both known recurrent fusions as well as previously unidentified fusions at key breakpoints in target genes. Archer’s sarcoma kit offers a complete fusion detection solution, from library preparation through data analysis, for both the Illumina® and Ion Torrent™ platforms.

Learn more about:

Overview of Anchored Multiplex PCR (AMP)


References

  1. Burningham Z, Hashibe M, Spector L, Schiffman JD. The epidemiology of sarcoma. Clin. Sarcoma Res. Oct 4;2(1), 14 (2012).
  2. Seattle Cancer Care Alliance, Sarcoma Website http://www.seattlecca.org/diseases/types-subtypes-sarcoma.cfm
  3. Demicco EG, Maki RG, Lev DC, Lazar AJ. New therapeutic targets in soft tissue sarcoma. Adv. Anat. Pathol. May 19(3), 170-80 (2012).
  4. Ducimetiere F, Lurkin A, Ranchere-Vince D, et al. Incidence of sarcoma histotypes and molecular subtypes in a prospective epidemiological study with central pathology review and molecular testing. PLoS One (2011).
  5. Coindre JM, Pelmus M, Hostein I, Lussan C, Bui BN, Guillou L. Should molecular testing be required for diagnosing synovial sarcoma? A prospective study of 204 cases. Cancer Dec 15 98(12) (2003).
  6. Errani C, Zhang L, Sung YS, Hajdu M, Singer S, Maki RG, Healey JH, Antonescu CR. A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioid hemangioendothelioma of different anatomic sites. Genes Chromosomes Cancer Aug 50(8), 644-53 (2011).
  7. Tanas MR, Sboner A, Oliveira AM, Erickson-Johnson MR, Hespelt J, Hanwright PJ, Flanagan J, Luo Y, Fenwick K, Natrajan R, Mitsopoulos C, Zvelebil M, Hoch BL, Weiss SW, Debiec-Rychter M, Sciot R, West RB, Lazar AJ, Ashworth A, Reis-Filho JS, Lord CJ, Gerstein MB, Rubin MA, Rubin BP. Identification of a disease-defining gene fusion in epithelioid hemangioendothelioma. Sci. Transl. Med. Aug 31 3(98), 98ra82 (2011).
  8. Pierron G, Tirode F, Lucchesi C, Reynaud S, Ballet S, Cohen-Gogo S, Perrin V, Coindre JM, Delattre O. A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion. Nat. Genet. Mar 4 44(4), 461-6 (2012).
  9. Taylor BS, Barretina J, Maki RG, Antonescu CR, Singer S, Ladanyi M. Advances in sarcoma genomics and new therapeutic targets. Nat. Rev. Cancer. Jul 14 11(8), 541-57 (2011).

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For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.