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| Year : 2021 | Volume
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| Issue : 1 | Page : 96-101 |
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| FISH for EWSR1 in Ewing's sarcoma family of tumors: Experience from a tertiary care cancer center |
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Sudha S Murthy1, Sandhya Devi Gundimeda1, Sundaram Challa1, V Manjula1, Daphne Fonseca1, Vishal B Rao1, Senthil J Rajappa2, K V. V N Raju3, T Subramanyeshwar Rao3
1 Department of Pathology and Laboratory Medicine, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
2 Department of Medical, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
3 Department of Surgical Oncology, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
Click here for correspondence address and email
| Date of Submission | 30-Mar-2020 |
| Date of Decision | 14-May-2020 |
| Date of Acceptance | 08-Sep-2020 |
| Date of Web Publication | 8-Jan-2021 |
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 Abstract | | |
Background: Molecular confirmation of histologic diagnosis has become mandatory for the diagnosis of Ewing sarcoma family of tumors (ESFT). Aim: To validate the diagnosis made by morphology and immunohistochemistry (IHC) by fluorescence in-situ hybridization (FISH) for EWSR1 rearrangement on formalin fixed paraffin embedded (FFPE) tissues. Settings and design: A retrospective and prospective observational study. Material and methods: All patients who had FISH studies for EWSR1 rearrangement for small round cell tumors during 10 years period were included. Demographic, clinical and radiological details were obtained from medical records. Morphology was reviewed with IHC by CD99, FLI1 and others. FISH studies were performed using the break apart probe. Additional molecular studies and IHC were done to resolve the diagnosis in EWSR1 rearranged tumors. Final diagnosis was made by integrating clinical, morphology, IHC and molecular features. Results: There were 81 patients (M: F 45:36, median age 21 years) with 32 skeletal and 49 extra skeletal tumors. CD 99 was positive in 94.52%. FISH for EWSR1 were positive in 59, negative in 13 and failed in 9. The final diagnosis was made as ESFT in 67, angiomatoid fibrous histiocytoma in 3, desmoplastic small round cell tumor in 3, myxoid chondrosarcoma in 2, unclassified in one, synovial sarcoma in 3, and one each of lymphoma and small cell neuroendocrine carcinoma. FISH was positive for ESFT in 89.83% of EWSR1 rearranged tumors. FISH validated the diagnosis made on IHC in 79.10%. FISH resolved the diagnosis in 1.49% CD99 negative tumors. Conclusion: FISH is a reliable ancillary technique for the diagnosis of ESFT on FFPE tissues.
Keywords: Ewing's sarcoma family of tumors, EWSR1 rearranged tumors, fluorescence in-situ hybridization, immunohistochemistry
How to cite this article:
Murthy SS, Gundimeda SD, Challa S, Manjula V, Fonseca D, Rao VB, Rajappa SJ, N Raju K V, Rao T S. FISH for EWSR1 in Ewing's sarcoma family of tumors: Experience from a tertiary care cancer center. Indian J Pathol Microbiol 2021;64:96-101 |
How to cite this URL:
Murthy SS, Gundimeda SD, Challa S, Manjula V, Fonseca D, Rao VB, Rajappa SJ, N Raju K V, Rao T S. FISH for EWSR1 in Ewing's sarcoma family of tumors: Experience from a tertiary care cancer center. Indian J Pathol Microbiol [serial online] 2021 [cited 2023 Mar 30];64:96-101. Available from: https://www.ijpmonline.org/text.asp?2021/64/1/96/306502 |
| Introduction | |  |
Ewing's sarcoma family of tumors (ESFT) are aggressive bone and soft tissue tumors, characterized by small round cell morphology and specific chromosomal translocations resulting in a fusion of the EWSR1 (Ewing sarcoma breakpoint region 1) gene with one of the members of the ETS family of transcription factors, or rarely FUS substitutes EWSR1.[1],[2] Establishing the correct diagnosis of ESFT and differentiating from other small round cell tumors is important for therapy and prognosis.[3] The gold standard for the diagnosis of ESFT is confirmation of histologic diagnosis by cytogenetics or molecular studies.[2],[3],[4] Molecular studies may involve demonstration of chimeric fusion transcript by reverse transcription polymerase chain reaction (RT-PCR) or gene rearrangement by fluorescence-in-situ hybridization (FISH). Both techniques can be applied to formalin-fixed paraffin-embedded (FFPE) tissues and require a small sample of tissue. However, FISH is a more sensitive and reliable ancillary technique than RT-PCR. RT-PCR has a better yield on fresh tissue and has the advantage of identifying the fusion partner.[5],[6],[7] A combination of techniques was recommended to enhance the accuracy of the diagnosis.[2],[5],[7]
With the availability of FISH in diagnostic surgical pathology and its applicability to FFPE, more small round cell tumors with the possible diagnosis of ESFT are subjected to FISH studies with EWSR1 to confirm the diagnosis. The present study aims to study the role of FISH in establishing the diagnosis of ESFT.
| Materials and Methods | | |
All the patients evaluated for EWSR1 rearrangement by FISH during the period of September 2009 to August 2019 were included in the study. Institutional review board approval was taken. Demographic, clinical, and radiologic findings were noted from the medical records. The samples included biopsies and resected specimens of patients diagnosed at our institute as well as those referred to our center for review. The samples were fixed in 10% neutral buffered formalin and processed for paraffin sections. The morphology was reviewed on hematoxylin and eosin (H&E) stained sections, along with immunohistochemistry (IHC) for the possibility of ESFT and other EWSR1 rearranged tumors. Depending on the morphological differential diagnosis considered, IHC was performed with a panel of markers.
Immunohistochemistry: Briefly, 4 μm sections were mounted on poly-L-lysine coated slides, deparaffinized with xylene and hydrated through graded alcohols. IHC was performed on BOND-III fully automated IHC stainer as per the manufacturer's instructions. The following antibodies were used: cluster of differentiation 99 (CD99; Cell Marque, EPR3097Y, 1:100); friend leukemia integration 1 transcription factor (FLI1; MRQ-1, Cell Marque, 1:50); leukocyte common antigen (LCA; Dako, 2B11 + PD7/26, Ready to use (RTU); vimentin (Thermo; V9, RTU); desmin (Biocare, D33), B-cell lymphoma 2 (Bcl-2; Dako, 124, RTU); pan cytokeratin (panCK; DAKO, AE1/AE3, 1:50); synaptophysin (Cell Marque, MRQ-40, 1:100); chromogranin (Thermo; SP12; 1:00); terminal deoxynucleotidyl transferase (TdT; Cell Marque polyclonal, 1:100); epithelial membrane antigen (EMA; Biocare E29, 1:100); HMB45 (DAKO, RTU); Melan A (DAKO; A103, RTU); S-100 (DAKO, polyclonal RTU); glial fibrillary acidic protein (GFAP; Cell Marque, EP672Y, 1:25), transducer-like enhancer of split 1 (TLE1;Cell Marque, 1F5; 1:100); Wilms tumor protein (WT1; DAKO, 6F-H2, RTU), CD56 (Cell Marque; MRQ-42; 1:100) Ki67 (Dako, MIB1, RTU).
Diffuse membranous staining was considered positive for CD99; weak/moderate/strong staining was considered as positive staining and equivocal staining as negative staining. Nuclear positivity was considered positive for FLI1; focal staining in the tumor cells was reported as focal positivity, no staining as negative; endothelial cells and lymphocytes were used as internal controls.
FISH procedure: EWSR1 gene rearrangement was assessed by FISH on 81 FFPE tissues. The tumor area was marked on the blocks, avoiding areas of necrosis and crush artifacts. 3–4 μm sections were cut and mounted on poly-L-lysine slides, deparaffinized in xylene, and dehydrated in absolute ethyl alcohol. The sections were pretreated with sodium thiocyanate, protease solution, and dehydrated in absolute ethyl alcohol. Locus specific, Vysis dual-color break-apart probe for EWSR1 gene, located at 22q12 was used. Four microliters of undiluted probe were applied to the target area in the dark and the slides were coverslipped and sealed with rubber cement. The 5' probe was labeled in spectrum orange (O) and 3' probe in spectrum green (G). The target DNA in section and probe were co-denatured in Thermobrite hybridizer at 85°C for 5 mi?n and incubated overnight at 37°C. The following day, the coverslips were removed and sections were dipped in preheated (72°C) post hybridization buffer consisting of 2 x SSC and NP40 for few seconds to remove the excess unbound probe. The slides were dried completely in the dark and the nuclei were counterstained with di-amino-phenyl indole. The hybridization signals in 5?0 nonoverlapping nuclei were visualized under the Olympus BX41 fluorescence microscope in oil immersion using appropriate filters.
Interpretation of normal and abnormal signal patterns: Normal cells without the EWSR1 gene rearrangement displayed 2 fusion (F) or closely opposed orange and green signals, representing two intact copies of EWSR1 genes at 22q12. Nuclei with EWSR1 rearrangement displayed typical and atypical hybridization signal patterns. In nuclei with a typical signal pattern, the probe proximal to EWSR1 breakpoint—the centromeric sequences (Spectrum Orange)(O) remain on the native chromosome (22q12) and the probe distal to EWSR1 breakpoint—telomeric (Spectrum Green)(G) segregates to the partner chromosome (unknown) thus generating separate orange and green signal. These correspond to the two rearranged chromosomes, containing a part of the sequences of EWSR1 gene. The remaining yellow (F) signal represents the normal chromosome 22. Atypical signal patterns demonstrate 1 fusion and 1 orange (1FIO) signal pattern (which connotes EWSR1 break with loss of 3' telomeric sequences) and 1 fusion and 1 green (1F1G) (which connotes EWSR1 rearrangement with loss of 5' sequence, centromeric of EWSR1 breakpoint). A mix of typical and atypical patterns was also noted. The cutoff was taken as 15%.
RT-PCR procedure: EWS-CREB1 was assessed by RT-PCR in 4 patients. Two samples were outsourced (SRL Centre of Excellence, Mumbai) for EWS-CREB1 fusion transcript by RT-PCR, which were used as index samples for in-house validation. Two other samples were processed in-house. The primer sequences for EWS-CREB translocation used were
EWS Forward primer 5' TCC TAC AGC CAA GCT CCA AGT C 3'.
CREB1 Reverse primer 5'-ACAATGGTACCGATGGGGTAC-3'.
EWS-CREB1 fusion transcript amplification was performed using 10 pm each of both primers, with 2.5 mM of dNTPs, 1U of Amplicon Taq gold, and 1 μg of RNA template following 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. An amplicon of 200 bp on agarose gel was considered as EWS-CREB1 fusion transcript positive.
A final diagnosis was rendered correlating the clinical, radiological, morphological, immunohistochemical, and molecular features.
| Results | | |
In the study period, 81 patients were assessed for the status of EWSR1 rearrangement by FIS?H. The age ranged from 7 months to 65 years (median 21 years) and comprised 45 males and 36 females. Tumors were located in bone in 32 patients and were extraskeletal in 49 patients. Among the extraskeletal cases, 29 patients had tumors in soft tissues and 20 patients had tumors in the viscera. The samples included 22 core biopsies, 52 excision, and 7 resection specimens.
All the tumors were cellular, most of which were round cells with a high mitotic rate and foci of necrosis. Spindle cells, pleomorphic cells, and prominent vascular channels were seen in few cases. There was no glandular differentiation?. CD99 expression was seen as diffuse membranous positivity in 67 [Figure 1] and as focal positivity in 2 of the 73 samples tested (95%) and it was not done in 8 (referral cases submitted for FISH studies only). CD99 was negative in one each of ESFT, desmoplastic small round cell tumor (DSRCT), unclassified tumor, and lymphoma. | Figure 1: Photomicrograph shows (a) diffuse sheets of round cells (hematoxylin and eosin ×100); (b) rosettes with vesicular nuclei with marginated nuclei (hematoxylin and eosin ×400); (c) Immunohistochemistry (IHC) for CD 99 showing membrane positivity (CD 99 ×400); (d) IHC for FLI1 showing nuclear positivity (FLI1 ×400)
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Assessment for EWSR1 rearrangement by FISH was positive in 59 (73%), negative in 13 (16%), and failed/inconclusive in 9 patients (11%) [Table 1]. Tumors with EWSR1 rearrangement displayed either typical break-apart signal pattern in 48/59 (81%) patients or atypical signal patterns in 11/59 (19%) with positivity rates ranging from 19-92% and 26-92%, respectively [Figure 2]. Atypical 1F1O signal pattern was noted in 4 patients. Seven patients displayed admixture of cells with submicroscopic deletion of 5' and 3' sequences of EWSR1 gene at the breakpoint. One patient with no EWSR1 gene rearrangement had an additional copy of EWSR1 gene. The diagnoses were resolved using an additional panel of IHC markers and further molecular tests?. | Figure 2: Fluorescence.in.situ hybridization (FISH) image of two specimens showing positivity for EWSR1 gene rearrangement; (a) shows both typical (1Fusion, 1 green &1 orange signal) and atypical signal pattern (1 fusion &1 orange, or 1 fusion & 1green). (b) shows a typical break.apart pattern and separated orange and green signals (shown with arrows)
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The EWSR1 rearranged tumors were diagnosed as ESFT in 53 (90%), angiomatoid fibrous histiocytoma (AFH) in 2, DSRCT in 2, extraskeletal myxoid chondrosarcoma (EMC) in one, and unclassified (negative for CD99) in one patient. The EWSR1 negative cases were resolved as ESFT in 6 and one each of AFH (morphology and IHC), DSRCT, 3 synovial sarcomas, and one lymphoma. All synovial sarcomas were positive for TLE1 and two for Bcl2 and one was confirmed for rearrangement involving the SS18 (SYT) gene by FISH. Samples where FISH studies were inconclusive were resolved by IHC as ESFT in 8 and one small cell neuroendocrine carcinoma [Table 2]. | Table 2: Demographic morphologic and molecular details of cases diagnosed as Ewing sarcoma family of tumors (n=67) and non ESFT
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In four patients, AFH was suspected on morphology, though clinical and radiological features favored ESFT and hence were subjected to RT-PCR for EWS-CREB1. RT-PCR resolved the diagnosis as AFH in 2. One had small round cell morphology and another had classic morphology with spindle cells and dilated ectatic vessels. EWSRI rearrangement by FISH was positive in one and negative in the other. One other patient with a mass on the right leg demonstrated EWSR1 rearrangement by FISH; RT-PCR for EWS-CREB1 revealed a faint band on the gel electrophoresis. A repeat could not be done because of the lack of tissue and was considered as AFH after correlating with morphology, IHC, and FISH. The fourth case was a referral (tissue processed elsewhere) and morphology was that of small round cells; though RT- PCR was attempted in this case, there was RNA fragmentation and, therefore, results were considered inconclusive.
Correlating with clinical, radiology, morphology, and IHC, the final diagnosis was made as ESFT in 67, AFH in 3, DSRCT in 3, EMC in 2, unclassified in one, synovial sarcoma in 3, and one each of lymphoma and small cell neuroendocrine carcinoma.
Ewing sarcoma family of tumors (n = 67): Th?e demographic, pathologic, and molecular details of patients in this group are summarized in [Table 2]. The patients were predominantly males (Male:Female ratio; 1.16:1.0) with a median age of 21 years. Molecular characterization of tumors by FISH in extraskeletal location was 55.2% which comprised soft tissue locations (59.4%) and visceral organs (40.5%). CD99 was positive in 98.3% tested samples in ESFT and 97.8% FISH positive ESFT. FLI1 was positive in 98.3% tested samples of ESFT and was positive in 36/39 (92.3%) FISH positive tumors. Of the 59 EWSR1 rearranged tumors, 53/59 (90%) cases were resolved as ESFT. FISH validated the diagnosis made on IHC in 53/67 (79.1%). FISH resolved the diagnosis in one (1.4%) CD99 negative tumor.
| Discussion | | |
ESFT is a translocation-related sarcoma involving EWSR1 gene. Initially, EWSR1 gene rearrangement was thought to be specific for ESFT; however, this translocation was documented in a number of tumors of both mesenchymal and nonmesenchymal lineage, including DSRCT, AFH, EMC, clear cell sarcoma, and others.[7],[8],[9] EWSR1 rearrangements can be detected by FISH with break-apart probes on FFPE tissues. FISH is one of the most widely used platforms for the molecular diagnosis of ESFT as it can be performed on a small amount of tissue and its applicability to FFPE. It is being more frequently used in many centers for molecular confirmation of ESFT. In the present study, FISH studies were performed on 81 samples that were suspected on clinical features, radiology, and morphology, and the diagnosis of ESFT could be confirmed in 53/59 (90%) EWSR1 rearranged tumors. FISH hybridization signals patterns may be typical break apart as seen in 42/53 (79%) or atypical in 11 (21%) patients with positivity rates ranging from 19%–92% and 26%–92%, respectively. Atypical hybridization signal patterns 1F1O and 1 F1G patterns are also interpreted as a break in the EWSR1 gene, i.e., there is an EWSR1 gene rearrangement with submicroscopic loss of only telomeric sequences or centromeric or both. Various publications have described EWSR1-rearranged tumors diagnosed with ESFT in 144 of 156 (92%),[2] 89 of 109 (82%),[7] 91% in a series of 22 with no false positives,[6] and 15/18 (83%) cases.[5] Indian studies described EWSR1 positive results by FISH in a very small sample size, in 10/11 (91%)[10] and 12/13 (92%)[11] cases. Our sample size is larger and results were comparable to these previously reported series. Due importance needs to be given to the preanalytics of the specimen, validation of the probe, establishing the cut-off, adherence to the protocol, and awareness of signal patterns for correct interpretation of the status of EWSR1 rearrangement.
FISH can be performed using either a fusion or break-apart strategy and both the techniques give concordant results.[6] FISH break-apart strategy is used when more translocation partners are involved, is cost-effective and signals are relatively easier to interpret with brighter and larger signals; however, this method has the disadvantage due to its inability to differentiate ESFT from other EWSR1 rearranged tumors.[6] In the present study, break-apart method was used; EWSR1 rearrangement was seen in 10% of tumors which were not ESFT. Additional molecular methods and IHC were required to resolve the diagnosis.
The tumors with EWSR1 rearrangement other than ESFT constituted 6/59 (10%) in the present study and included AFH [Figure 3] DSRCT and EMC. Translocations involving EWSR1 and its partner genes are not specific for a tumor type?. Noujaim et al. reported FISH positivity for DSRCT, myxoid liposarcoma, EMC, AFH, and clear cell sarcoma in 86.3%, 4.3%, 58.5%, 60.0%, and 87.9%, respectively, in a series of 210 EWSR1 rearranged tumors.[7] In the present study, additional molecular tests and IHC resolved the diagnosis as AFH in 2 and DSRCT in 2. One tumor was unclassified as morphology and IHC (CD99 was negative, S100, CD56, and vimentin were positive; and panCK, HMB45, and MelanA were negative) were not conclusive for any particular diagnosis. Noujaim et al.[7] described 13/210 (6%) FISH-positive cases that remained unclassifiable. These tumors, perhaps represent yet, the uncharacterized types, harboring the EWSR1 gene rearrangements with unknown partner genes.[7] False-positive results of EWSR1 rearrangement can occur in INI-1 deficient neoplasms due to the proximity of SMARCB1 and EWSR1 genes on chromosome 22.[12] | Figure 3: Photomicrograph of Angiomatoid Fibrous Histiocytoma demonstrating diffuse sheets of oval cells with interspersed histiocytes (a: H and E ×100); Cells are oval to spindle.shaped (myofibroblast like) with large vesicular nuclei with a sprinkling of RBC (b: H and E ×400); IHC for CD99 demonstrates focal cytoplasmic reactivity (c: IHC; CD99 ×100); MIB1 proliferation index 35% (d: IHC; Ki67)
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Thirteen tumors were negative by FISH and were resolved as ESFT in 6, one each of AFH, DSRCT and EMC, lymphoma and as Synovial Sarcoma in 3. Correlating with morphology and IHC, the remaining seven cases could have been the undifferentiated sarcomas with round cell phenotype (non-ESFT). Rare variants involving the FUS gene have been reported in the literature.[13],[14],[15] Some publications recommend that tumors negative for EWSR1 rearrangement should be tested for translocations involving FUS gene before labeling it as undifferentiated sarcomas with round cell phenotype.[2],[16] Recent studies suggested assessment of gene fusion transcripts like CIC-DUX4, BCOR-CCNB3 by RT-PCR in rare/infrequent EWSR1 negative undifferentiated round cell sarcomas due to the possibility of adverse prognosis.[3],[8],[17],[18] Published studies indicated that this subset of tumors can be picked up by IHC as well.[18],[19] However, these studies could not be performed due to the lack of availability/need in the present study. Final diagnosis should always be done by correlating the clinical, radiological, morphological, immunohistochemical, and molecular features.
FISH for EWSR1 was inconclusive in one case and based on IHC, it was resolved as small cell carcinoma. The failure rate of FISH varied from 2.5% to 23.4% in various reported series.[2],[5],[6],[7] and was attributed to the low percentage of tumor cells, more stromal or immune component, and the fixation/processing of samples.[7] The failure rate was 9/81 (11%) in the present study. All the nine samples were referral samples submitted for review and hence the preanalytical factors like fixation may be the reason for the inconclusive results.[2],[5],[6],[7] However, increased awareness for molecular testing improved the fixation and processing techniques as well as the optimal utilization of samples, resulting in the reduction of the technical failure rate?s.[20]
The absence of molecular confirmation should not rule out the diagnosis of ESFT but should prompt a review of clinical, histological, and IHC features.[4],[21],[22] Immunohistochemistry was very helpful for the diagnosis, though less specific. Both CD99 and FLI1 were very sensitive for the diagnosis of ESFT with FISH positive tumors in 98% and 92%, respectively. One CD99 negative tumor had EWSR1 rearrangement. Similar observations were made earlier.[2],[23]
FISH studies detected EWSR1 rearrangement in 53 (90%) ESFT. FISH validated the diagnosis made on IHC in 53/67 (79%). FISH resolved the diagnosis in one (1%) CD99 negative tumor. These results were in agreement with earlier studies.[2],[7] The limitation of this study is that CIC-DUX4 and BCOR studies have not been performed which would have completed the spectrum of round cell sarcoma?s.[24]
To conclude, FISH is a robust and reliable complementary technique for the diagnosis of ESFT, especially in resource-limited settings where extensive molecular assays may not be available to pinpoint the translocation partner. This study illustrates the importance of FISH as an ancillary diagnostic tool in the diagnosis of EWSR1 rearranged neoplasms. However, with the increasing numbers of tumors associated with EWSR1 gene, highly sensitive and robust technologies like next-generation sequencing (RNA sequencing) might address the challenges associated with FISH.
Acknowledgements
We would like to thank the technical team of Histopathology, IHC, FISH including U Ravinder, Khaja Ather Hussain, Ms. M. Padma, for rendering technical support in all areas of services.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Sudha S Murthy
Department of Pathology and Laboratory Medicine, Basavatarakam Indo.American Cancer Hospital and Research Institute (BIACH and RI), Road No 10, Banjara Hills, Hyderabad, Telangana
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/IJPM.IJPM_267_20
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2] |
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