Transcript dual color, break apart rearrangement probe

A novel unknown translocation partner on
chromosome 2 or chromosome 5 for the
EWSR1 gene in Ewing Sarcoma
S.Shetty1, N. Uddin1,2,K.B. Geiersbach1, S.T. South1,2.
1) University of Utah, School of Medicine, Department of Pathology, Salt lake City, UT and Cytogenetics/Molecular Cytogenetics, ARUP Laboratories, Salt Lake City, UT
2) University of Utah, School of Medicine, Department of Pediatrics, Salt lake City, UT and Cytogenetics/Molecular Cytogenetics, ARUP Laboratories, Salt Lake City, UT
Abstract
Ewing sarcoma is a common malignant round cell tumor of childhood and adolescence. Cytogenetic analysis to identify a common t(11;22)(q23;q12) or less frequently a t(21;22)(q22;q12) or t(7;22)(p22;q12) plays an important
role in confirmation of the clinical diagnosis. The genetic mechanism, in majority of the cases, involves fusion of EWSR1 gene on 22q12 with a member of the ETS family of transcription factors. We report a case of a 20 year old
male who had a soft tissue tumor on the left scapula. Conventional cytogenetic analysis on unstimulated suspension culture revealed a complex karyotype with multiple numerical and structural abnormalities; all cells from the
monolayer culture were karyotypically normal. No chromosomal rearrangement involving the EWSR1 region was recognized. However, the biopsy results were consistent with the diagnosis of Ewing sarcoma.
Immunohistochemistry was positive for CD99 and vimentin and negative for AE1/3, CAM 5.2, CD45 and Fli1. To identify a cryptic rearrangement, fluorescence in situ hybridization (FISH) on interphase cells was performed
utilizing the LSI EWSR1, dual-color break-apart rearrangement probe. FISH findings revealed two normal fusion signals and an extra signal for the 5’ EWSR1 region suggestive of a rearrangement and deletion of the 3’
EWSR1. FISH on G banded metaphases from unstimulated suspension culture identified that the extra 5’ EWSR1 signal was on chromosome 2. Based on G-banding and FISH findings, the derivative chromosome 2 was shown to
consist of a translocation between 2q and 5q with an insertion of the long arm of the 22q12 (approximately between bands 2q12~13 and 5q13. These findings suggest that an insertion event involving the EWSR1 locus on
chromosome 22 is another mechanism that could lead to EWSR1-unknown partner gene fusion. To our knowledge, this is the first case report of an insertion of a segment of the EWSR1 region with a possible novel translocation
partner on either chromosome 2q12~13 or 5q13 in Ewing sarcoma. This case also highlights the importance of chromosome studies in addition to FISH to understand the genetic mechanism and aid in identifying new partners for
the EWSR1 gene on unstimulated suspension cultures in round cell tumors. Based on FISH findings alone, rearrangement of EWSR1 in this case would have been just a postulate and the rearrangements would have been missed
if only monolayer culture was analyzed.
Introduction
Fig 1A
Fig 1B
Results
Fig 2
Fig 3
Fig 3
Ewing sarcoma/peripheral primitive neuroectodermal tumor (PNET) is a round-cell sarcoma that may show
varying degrees of neuroectodermal differentiation. These tumors are identified by a characteristic round-cell
morphology and immunohistochemical profile, as well as by specific translocations involving the EWS gene on
chromosome 22 and the 3' portion of the E26 transformation-specific (ETS) family of transcription factors. These
translocations result in fusion proteins that act as aberrant transcription factors. The majority of Ewing sarcoma
cases are characterized by a balanced t(11;22)(q24;q12) translocation resulting in a EWSR1-FLI1 fusion.
Specific chromosomal abnormalities often correlate with distinct morphologic or phenotypic subtypes of tumors
and play an important role in prognosis 1,2. Here we describe the molecular cytogenetic investigation of a case of
Ewing sarcoma in the left scapula of a 22-year-old male using fluorescent in situ hybridization and G-banding.
This case shows the importance of combining cytogenetics and molecular cytogenetics to identify uncommon
rearrangements in sarcomas and emphasizes the developing understanding of the biological nature of different
tumor types.
Figure 1A: Photomicrograph of the tumor (Hematoxylin-Eosin, X40): It is showing a
confluent sheet of small, uniform looking cells with round to oval nuclei, fine chromatin, moderate
amount of lightly eosinophilic cytoplasm & indistinct cytoplasmic membranes.
Figure 1B: Photomicrograph of the CD99/O13 Immunostain (immunoperoxidase,
X40): Majority of the tumor cells are showing a characteristic membranous staining by O-13
antibody against CD99 antigen.
Fig 4A
Objectives of Study
•
•
Fig 4B
Figure 2: Representative abnormal Gbanded Karyotype: One of the abnormal
karyotyped cells from the suspension culture is
captured, showing the der(2) chromosome with its
long arm composed of a translocation between the
long arms of chromosomes 2 & 5. In addition, also
shown are the aberrations of -5, +?del(12)(q24.1),
all marked by arrows. The -11 & -21 present in this
cell were not found to be a clonal aberration after
complete analysis of 20 cells.
Figure 3: Interphase FISH
with Vysis® LSI EWSR1
(22q12) dual color, break
apart rearrangement
probe: Showing two yellow
(fusion) signals for two intact
EWSR1 loci & one red signal
(for 5’ EWSR1) suggestive of
EWSR rearrangement with
deletion of the 3’ EWSR1 (FISH
probe fluorophore designation:
5’ EWSR1 – Red, 3’ EWSR1 –
Green)
Cytogenetics in diagnosis of blue round cell tumors
Molecular based approach to identify unknown translocation partners
Methods
Abnormality detected by Immunohistochemistry, Chromosomes, and FISH
Fig 2
1) Immunohistochemistry: CD99, Vimentin, AE1/3, CAM5.2,CD45 and FLI1
2) Culture type: Monolayer and Suspension
3) Molecular cytogenetics: Commercially available EWSR1 FISH Probe
Figure 4 A & B: Metaphase FISH with Vysis® LSI EWSR1 (22q12) dual color, break apart rearrangement probe: The metaphase FISH on abnormal
1B (for 5’ EWSR1) on the der(2) consistent with EWSR
metaphase (4A) shows two yellow (fusion) signals for intact EWSR1 at two normal chromosome 22 & one redFig
signal
rearrangement with deletion of the 3’ EWSR1. On the right (4B) the same abnormal metaphase is shown by inverted dapi staining method to identify the specific chromosomes
showing hyberdization signals. (FISH probe fluorophore designation: 5’ EWSR1 – Red, 3’ EWSR1 – Green)
4) Molecular approach to identify unknown partner (Study in progress)
Discussion and Conclusion
Cytogenetic studies have been instrumental in mapping cancer-related genes located at genomic sites that are visibly involved in neoplasia-associated chromosomal rearrangements. The importance of cytogenetic characterization
of solid tumors is twofold. First, recurrent aberrations provide insight into the pathogenetic mechanisms. They point to the areas in the human genome that might carry genes or regulatory sequences whose function is disrupted
in neoplastic cells. Secondly, even before a fundamental understanding of the mechanism is achieved, the cytogenetic aberrations have direct clinical importance. The finding of an acquired clonal chromosomal abnormality
identifies the presence of a neoplastic process, and the specific type of aberration may reveal the true nature of the tumor and thus improve the diagnostic precision.
Our experience with culturing round cell tumors have shown that the suspension cultures (tube culture) work better than monolayer cultures (flask culture) and invariably clonal abnormalities are detected in the suspension
culture. Our hypothesis is that the round cells tumors may be derived from the primitive and pluripotent cells that do not necessarily grow as a monolayer on a support (e.g., flask or coverslip), unlike other neoplastic cells
(fibroblasts, endothelial, adipocytes etc.) that grow well in monolayer cultures.
In our patient, all cells from the monolayer culture showed a normal chromosome complement; these normal cells likely represent non-neoplastic stromal cells. All cells from the suspension culture showed complex clonal
abnormalities. This patient had diagnosis of Ewing sarcoma based on morphology and immunohistochemistry. Despite the complex chromosomal abnormalities, there was no cytogenetic evidence of a EWSR1 rearrangement.
FISH analysis using the EWSR1 break-apart probe showed FFR signal pattern consistent with two normal chromosomes 22 (two fusion EWSR1 signals) and an additional red signal for the 5’ end of EWSR1 probe. FISH was
also performed on a G-banded slide in order to locate the abnormal metaphases and understand the mechanism of the abnormal signal pattern. Metaphase FISH revealed insertion of the 5’ EWSR1 probe into the derivative
chromosome 2 due to an unbalanced translocation between chromosomes 2 and 5. This has prompted us to look for a novel translocation partner for EWSR1 on chromosomes 2 and 5 using molecular based approaches (study
ongoing).
In conclusion, this case emphasizes on the role cytogenetics plays in mapping genes and identifying new translocation partners that may be prognostically significant. In addition, this case highlights the importance of an
understanding of the biological nature of the tumor cells in culture in order to successfully grow them in culture.
References
1) Pfeifer JD: Molecular Genetic Testing in Surgical Pathology Edited by Pfeifer JD Philadelphia, Lippincott Williams and Wilkins, 2006, pp.186-231
2) Delattre et al. The Ewing family of tumors--a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 1994: 331:294-299.
Acknowledgement: We would like to thank Joyce Stratton for her technical expertise with round cell tumor culturing and analysis.