YWHAE-FAM22 endometrial stromal sarcoma: diagnosis by reverse transcription–polymerase chain reaction in formalin-fixed, paraffin-embedded tumor

doi:10.1016/j.humpath.2012.08.007

Human Pathology

Volume 44, Issue 5, May 2013, Pages 837-843

https://doi.org/10.1016/j.humpath.2012.08.007 Get rights and content

Summary

A subset of endometrial stromal sarcoma harbors t(10;17)(q23;p13), which results in the genetic fusion between YWHAE and 1 of 2 highly homologous FAM22 family members—FAM22A or FAM22B. In contrast to classic low-grade endometrial stromal sarcoma with JAZF1-SUZ12 fusions, YWHAE-FAM22 endometrial stromal sarcoma displays high-grade histologic features and is associated with more aggressive disease course. Ancillary fluorescence in situ hybridization assay demonstrating the presence of YWHAE rearrangement can be used to support the diagnosis, but the detection of fusion transcript would be the most definitive test. We describe here an optimized reverse transcription–polymerase chain reaction assay for detection of YWHAE-FAM22 fusion transcript in formalin-fixed and paraffin-embedded tumor samples. We studied a series of 6 YWHAE-FAM22 endometrial stromal sarcomas, 7 JAZF-SUZ12 endometrial stromal sarcomas, 3 JAZF1-PHF1/EPC1-PHF1 endometrial stromal sarcomas, 6 undifferentiated endometrial sarcomas, 4 uterine leiomyosarcomas, and 4 uterine adenosarcomas. All 6 YWHAE-FAM22 endometrial stromal sarcomas were confirmed by fluorescence in situ hybridization assay, whereas all non–YWHAE-FAM22 tumors were confirmed to lack YWHAE rearrangement by fluorescence in situ hybridization assay. The reverse transcription–polymerase chain reaction assay optimized for formalin-fixed and paraffin-embedded samples detected YWHAE-FAM22 fusion transcripts in all 6 YWHAE-FAM22 endometrial stromal sarcomas and none of the 24 non–YWHAE-FAM22 uterine sarcomas. These findings show that this reverse transcription–polymerase chain reaction assay is sensitive and specific for detection of YWHAE-FAM22 fusion transcript and can serve as a useful adjunct diagnostic assay to confirm the diagnosis of YWHAE-FAM22 endometrial stromal sarcoma in formalin-fixed and paraffin-embedded tumor samples.

Introduction

Endometrial stromal sarcoma (ESS) is genetically, biologically, and histologically heterogeneous. Tumors showing classic low-grade histology frequently harbor translocation associated genetic fusions between JAZF1 and members of polycomb complex genes (SUZ12, PHF1, and EPC1) [1], [2], [3], [4], [5], [6], [7]. In contrast, tumors with high-grade (or mixed low- and high-grade) appearances frequently harbor chromosomal translocation t(10;17)(q22;p13), which results in YWHAE-FAM22A or YWHAE-FAM22B genetic fusions (collectively referred to as YWHAE-FAM22 ESS) [8], [9]. Clinically, YWHAE-FAM22 ESS is more aggressive than JAZF1 ESS (encompassing JAZF1/SUZ12/PHF1/EPC–rearranged cases), characterized by frequent and early recurrence (local and/or systemic). Furthermore, in contrast to JAZF1 ESS, the high-grade component of YWHAE-FAM22 ESS consistently lacks estrogen receptor and progesterone receptor expression, suggesting that hormonal therapy typically used in the management of JAZF1 ESS will likely be ineffective against YWHAE-FAM22 ESS [10], [11], [12]. The distinction between YWHAE-FAM22 ESS and JAZF1 ESS is therefore clinically relevant as the management would be different.

Although nearly all primary YWHAE-FAM22 ESS possess high-grade areas characterized by larger nuclei, more irregular nuclear contours and greater mitotic activity compared with JAZF1 ESS [8], [9], it is sometimes difficult to histologically distinguish between these 2 tumor types as approximately half of YWHAE-FAM22 ESS contain a histologically low-grade component, which can mimic JAZF1 ESS or other classic low-grade ESS morphologically. Immunohistochemically, in contrast to the high-grade component of YWHAE-FAM22 that shows diffuse strong nuclear cyclin D1 positivity in the absence of significant estrogen receptor (ER), progesterone receptor (PR), and CD10 immunostaining, the low-grade component typically displays diffuse strong ER, PR, and CD10 immunopositivity, with focal weak cyclin D1 nuclear staining, an immunoprofile that is similar to most JAZF1 ESS [13]. Because of such morphologic and immunophenotypic overlap, molecular characterization is necessary to determine the tumor genotypes in some cases. We have previously described fluorescence in situ hybridization (FISH) assay on formalin-fixed, paraffin-embedded (FFPE) tumor sample for diagnostic purposes, and we describe here a reverse transcription–polymerase chain reaction (RT-PCR) assay that is optimized for the detection of YWHAE-FAM22 in FFPE tumor samples as a definitive ancillary diagnostic test.

Section snippets

Study samples

FFPE tumor tissues were obtained from the pathology archives at Brigham and Women's Hospital and Vancouver General Hospital, and these include ESS cases in which FISH studies had demonstrated either YWHAE-FAM22 rearrangement (n = 6) or JAZF1/SUZ12/PHF1/EPC1 rearrangement (n = 10). The histologic features of all tumors were reviewed by 2 authors (C. H. Lee and M. R. Nucci) and previously described [8], [9]. For comparison, FFPE tumor tissues from 6 undifferentiated endometrial sarcomas, 4

Study sample

FFPE tumor blocks were obtained from 6 YWHAE-FAM22 ESSs. Five of the 6 were previously reported (Table) [8]. The 1 additional case (case no. 13) was a myopermeative uterine tumor that contained a mix of low- and high-grade areas as illustrated in Fig. 1. The low-grade component showed varying degree of cellularity and the stroma ranged from fibrous to myxoid (Fig. 1B-D). The more cellular area demonstrated histologic features that were well accepted for classic low-grade ESS (Fig. 1D), with

Discussion

We have validated in the current study an RT-PCR assay optimized for FFPE tumor samples that is sensitive and specific for detecting YWHAE-FAM22 fusion transcript in uterine sarcomas. Primer set 1 produces a 168-base product that can be subjected to sequence confirmation by a conventional sequence analyzer. This provides a definitive ancillary diagnostic method for identifying YWHAE-FAM22 ESS in clinical cases where only FFPE tissue is available. In particular, the distinction of YWHAE-FAM22

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The histological grading of endometrial stromal sarcoma (ESS) has been surrounded by confusion with definitions of low and high grade tumors changing over the years with the recognition that mitotic count was not an effective predictor of behavior [29,30]. With the advent of molecular pathology, it has been recognized that almost half are associated with JAZF1-SUZ12 fusion [29,30]. A further subgroup of endometrial stromal sarcoma is identified by fusion between YWHAE and FAM22A/B harboring t(10;17) (q22;p13) [29,30].
One of the most common challenges in everyday clinical practice of gynecological oncology is to identify the type and the primary origin of a tumor. This is a crucial step in the management, treatment, prognosis, and survival of patients suffering from a gynecological malignancy. Immunohistochemistry has been widely adopted over the last three decades in pathology laboratories all over the world. Recent advances in our understanding of the differentiation of gynecological tumors based on immunohistochemical expression have resulted in use of immunohistochemistry as a major diagnostic tool in gynecology, for precise tumor classification. More recently, advances in molecular pathology, have taken this disease sub-classification further resulting in more effective personalised treatment regimens. The aim of this review is to provide clinicians with up to date information on the various immunohistochemical and molecular tests used in the diagnosis of gynecological malignancies of the female genital tract and an understanding of how to interpret them.
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Development and Evaluation of a Pan-Sarcoma Fusion Gene Detection Assay Using the NanoString nCounter Platform
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An in-depth literature search was performed for gene translocations involved in sarcomas. The sequence across the fusion break was collected from relevant case reports when reported.12–94 When the exact sequence was not reported, the junction sequence was deduced from reported exon/exon break points.
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In some cases, HGESS is associated with more typical-appearing areas of LGESS.216,217 YWHAE rearrangements have not been found in other gynecologic tumors, and FISH and/or RT-PCR studies may serve as a useful adjunct to the histologic diagnosis.218,219 CyclinD1 IHC may be used as a marker for YWHAE rearrangement,202,217 although this is not entirely sensitive or specific, particularly when considering undifferentiated endometrial carcinoma and tumors outside the gynecologic tract (clear cell sarcoma of kidney),220–223 so confirmatory FISH studies are advised.
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Primer sets that cover the exonuclease domain regions of POLE in which mutations were previously identified in endometrial carcinomas were used to amplify exonuclease domain genomic regions — exon 9 (forward: 5′-TGTTCAGGGAGGCCTAATGG-3′; reverse: 5′-AACAAATACTAACAGTGGGG-3′), exon 10 (forward: 5′-GCTGCAATTCTGATCTGACG-3′; reverse: 5′-CAGCCTCTGACTTGTGCTGA-3′), exon 11 (forward: 5′-CTTCTGAACTTTGGGAGAGG-3′; reverse: 5′-CACCTCCTAAGTCGACATGG-3′), exon 12 (forward: 5′-GCATTAGAGCCTGACCTGC-3′; reverse: 5′-ACAGCACAGTCTGCAAGAGG-3′), exon 13 (forward: 5′-CGGGATGTGGCTTACGTGC-3′; reverse: 5′-TTGCATCTGTCTGTGTGGTG-3′), exon 14 (forward: 5′-TCTGTGCTTCACACTTGACC-3′; reverse: 5′-GACATCCACCTCCATTCAGC-3′). PCR amplifications were performed as previously described using 50 ng genomic DNA and the primer sets using High-Fidelity Tag DNA polymerase (Invitrogen, Carlsbad, CA, USA) [18]. Prior to sequencing, PCR amplicons were electrophoresed and visualized to confirm the presence of desired amplicons and the absence of off-target amplification products.
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^☆: Anna Isphording was supported by funding from the Interdisciplinary Oncology Program at the University of British Columbia, Vancouver, British Columbia, Canada.

^☆☆: Disclosure: The authors have no conflicts of interest to disclose.

¹: Both authors contributed equally to the study.

²: Co-senior authors.

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Original contributionYWHAE-FAM22 endometrial stromal sarcoma: diagnosis by reverse transcription–polymerase chain reaction in formalin-fixed, paraffin-embedded tumor☆,☆☆

Summary

Introduction

Section snippets

Study samples

Study sample

Discussion

J Mol Diagn

Ann Oncol

Frequency of known gene rearrangements in endometrial stromal tumors

Am J Surg Pathol

Molecular detection of JAZF1-JJAZ1 gene fusion in endometrial stromal neoplasms with classic and variant histology: evidence for genetic heterogeneity

Am J Surg Pathol

Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors

Proc Natl Acad Sci USA

Consistent rearrangement of chromosomal band 6p21 with generation of fusion genes JAZF1/PHF1 and EPC1/PHF1 in endometrial stromal sarcoma

Cancer Res

Molecular analysis of the JAZF1-JJAZ1 gene fusion by RT-PCR and fluorescence in situ hybridization in endometrial stromal neoplasms

Am J Surg Pathol

Original contribution
YWHAE-FAM22 endometrial stromal sarcoma: diagnosis by reverse transcription–polymerase chain reaction in formalin-fixed, paraffin-embedded tumor☆,☆☆