Low-grade endometrial stromal sarcoma-like tumors in male with JAZF1 gene fusions.

Case 1 was a 58-year-old male with a history of rectal “gastrointestinal stromal tumor” (GIST), diagnosed incidentally 2 years prior to presentation while he underwent prostatectomy (without radiation therapy) for prostate cancer. His peri-rectal tumor progressed on tyrosine kinase inhibitor (imatinib and then sunitinib) treatment. Clinically, the mass was thought to arise between the rectum and the prostate (prostatectomy bed). Although slides from the original excision were not available for review, the tumor reportedly had 5 per 5 mm 2 and was ‘weakly KIT positive’. The current MRI studies revealed a large, 12 cm recurrent soft tissue mass in the pelvis, with extrinsic compression of the bowel ( Figure 1A ). In addition, there were multiple peritoneal and omental nodules resembling peritoneal sarcomatosis, with few of them being sampled for diagnosis. Histologic examination of the omental nodules revealed multiple cellular nodules ranging from 0.3 to 1.6 cm in greatest dimensions that were well demarcated from omental fat ( Figure 1B ). The tumor was composed of a cellular proliferation of uniformly round to ovoid spindled cells. The cells were monotonous and arranged in solid sheets and reticular growth patterns against a collagenous stroma. The background showed a delicate arteriolar vascular network ( Figure 1C ). The cells exhibited ovoid to fusiform nuclei with fine chromatin, inconspicuous nuclei, scant cytoplasm, indistinct cell borders, and lack nuclear pleomorphism ( Figure 1D ). Mitotic activity was 9 per 5 mm 2 . On immunohistochemistry, the neoplastic cells were diffusely positive for PR and AR, and focally positive for ER ( Figure 1E - G ). There was weak, nonspecific staining for CD117 (c-kit), and negative staining for DOG1, pancytokeratin, desmin, myogenin, BCOR, SALL4, CD34 and STAT6. MIB-1 index was approximately 40-50% ( Figure 1H ). The patient was started with bicalutamide (nonsteroidal anti-androgen) and megestrol acetate (progestin) followed by leuprolide acetate. Case 2 was a 57-year-old male presenting with a right paratesticular mass. Grossly, the tumor was a white-gray rubbery mass measuring 1.5 cm in greatest dimension, located at the inferior pole of the testis and was well-demarcated from the testicular parenchyma. Histologic examination demonstrates a paratesticular nodule that is relatively circumscribed and separated from adjacent unremarkable testicular parenchyma by a fibrous septa ( Figure 2A ). The tumor is composed of solid sheets and haphazardly arranged ovoid to plump spindled cells with scattered arteriolar vasculature ( Figure 2B ). Cytologically, the tumor cells were monotonous and demonstrate mild nuclear atypia, including distinct nucleoli. Mitotic activity was 1-2 per 10 high power fields ( Figure 2C ). The cells were cytologically monotonous, with scant amphophilic cytoplasm, indistinct cell borders, round to ovoid nuclei, vesicular chromatin, and prominent nucleoli ( Figure 2C ). Significant nuclear pleomorphism or tumor necrosis was absent. By immunohistochemistry, the lesional cells were diffusely positive for ER, PR, CD10 ( Figure 2D - F ), and WT1 (nuclear), and negative for pancytokeratin, CAM 5.2, SF1, inhibin, STAT6, melan A, synaptophysin, and calretinin CD31.

As the JAZF1-GLI3 fusion was novel, we questioned if this fusion may occur in the more common low grade ESS of the female GYN tract. And indeed, one of the 35 low grade ESS in our database showed an identical transcript, of JAZF1 exon 3 and GLI3 exon 7 by Archer. This case occurred in a 51-year-old female with a uterine low grade ESS. The tumor cells were arranged in sheets and focally in cords and trabeculae. Focally, the cells were arranged in cords and trabeculae. Areas with arteriolar vasculature and perivascular whorling of tumor cells were present ( Figure 4A , B ). Cytologically, the cells exhibited an ovoid to round morphology, mild nuclear atypia and minimal pleomorphism ( Figure 4C ). By immunohistochemistry, the cells were positive for ER and CD10 ( Figure 4D ). The findings were in keeping with a low-grade ESS with focal sex cord stromal differentiation.

Clinical data, including age, gender, and anatomic site were retrieved from pathology reports and clinical chart. Hematoxylin and eosin–stained slides from resection specimens were rereviewed. The study was approved by the Institutional Review Board. FISH on interphase nuclei from paraffin-embedded 4-μm sections was performed using custom probes of bacterial artificial chromosomes (BACs) flanking genes of interest, 16 including PHF1 and JAZF1 (see Supplementary Table 1 ). The BAC clones were obtained from BACPAC sources of Children’s Hospital of Oakland Research Institute (Oakland, CA; http://bacpac.chori.org ). DNA from individual BACs was isolated according to the manufacturer’s instructions, labeled with different fluorochromes in a nick translation reaction, denatured, and hybridized to pretreated slides. Slides were then incubated, washed, and mounted with DAPI (4′,6-diamidino-2-phenylindole) in an antifade solution. The genomic location of each BAC set was verified by hybridizing them to normal metaphase chromosomes. Two hundred successive nuclei were examined using a Zeiss fluorescence microscope (Zeiss Axioplan, Oberkochen, Germany), controlled by Isis 5 software (Metasystems, Waltham, MA). A positive FISH score was interpreted when at least 20% of the nuclei showed a break-apart signal. Nuclei with incomplete set of signals were omitted from the score. Detailed descriptions of MSK-Fusion, an amplicon-based targeted RNA NGS assay using the Archer™ FusionPlex™ standard protocol, were described previously. 17 Briefly, RNA is extracted from tumor formalin-fixed paraffin-embedded material followed by cDNA synthesis. cDNA libraries were made using the Archer™ FusionPlex™ standard protocol and supplied reagents including Archer® Universal RNA Reagent Kit for Illumina® (Catalog #AK-0040–8), Archer MBC adapters (Catalog #SA0040–45) and our custom designed Gene Specific Primer (GSP) Pool kit. Fusion unidirectional GSPs have been designed to target specific exons in 62 genes known to be involved in chromosomal rearrangements based on current literature. the final targeted amplicons are ready for 2×150bp sequencing on an Illumina MiSeq sequencer. FASTQ files are automatically generated using the MiSeq reporter software (Version 2.6.2.3) and analyzed using the Archer™ analysis software (Version 5.0.4). Each fusion call should be supported with a minimum of 5 unique reads and a minimum of 3 reads with unique start sites.

For case 1, an NGS Archer fusion panel confirmed an in-frame fusion between JAZF1 ( NM_175061 ) exon 3 and GLI3 ( NM_000168 ) exon 7 [chr7:27934839(-)::chr7:42079838(-)] ( Figure 3A ) (> 80% fusion-supporting reads spanning fusion junction). MSK-IMPACT demonstrated a KMT2D p.K1641Rfs*81 truncating mutation. For case 2, FISH analysis using custom BAC probes showed the presence of a JAZF1 and PHF1 gene rearrangements ( Figure 3B ) in keeping with a JAZF1-PHF1 gene fusion.

We report two cases of low-grade ESS-like sarcoma in male patients. Similar to the uterine or extrauterine low-grade ESS counterparts, histopathologically, both cases demonstrate monotonous proliferation of ovoid to fusiform cells with a background of arteriolar vascular network. Immunohistochemically, the neoplastic cells express ER, PR, and CD10, while lacking smooth muscle markers, e.g., desmin, consistent with the immunohistochemical profile of a low-grade ESS. 18 - 20 Importantly, both cases harbor JAZF1 gene fusions, which are detected in the majority of low grade ESS and not known as drivers in other tumor. 2 - 6 The paratesticular case harbored a JAZF1-PHF1 fusion, the second most common gene fusion in low-grade ESS, which has been associated with sex cord differentiation. 6 The periprostatic case revealed a JAZF1-GLI3 novel fusion which hitherto has not been reported in ESS. The fusion involves the first three exons of JAZF1 , with the same exon 3 breakpoint reported in other JAZF1 -related fusions in ESS. In keeping with this diagnostic consideration, we also report a separate case of a low-grade ESS with focal sex cord stromal elements arising in the uterine corpus of a 51-year-old female, harboring an identical JAZF1-GLI3 gene fusion transcript, supporting this as a bona fide gene fusion in low-grade ESS. In terms of differential diagnosis, the most common malignancies in the paratesticular soft tissue are dedifferentiated liposarcoma, rhabdomyosarcoma, and leiomyosarcoma. 21 In the abdominopelvic cavity, the differential diagnosis is rather broad and includes dedifferentiated liposarcoma, GIST, solitary fibrous tumor, 22 desmoplastic round cell tumor (DSRCT), synovial sarcoma, and other cellular soft tissue tumors with monotonous ovoid/spindle cells. The lack of an adjacent well-differentiated lipomatous component and MDM2 and CDK4 gene amplification excludes dedifferentiated liposarcoma, 23 , 24 while the absence of smooth muscle and skeletal muscle markers (desmin, myogenin, MyoD1) rules out leiomyosarcoma and rhabdomyosarcoma, 25 , 26 respectively. DOG1 is a highly useful marker and expressed in > 99% of GIST (Miettinen 2009), while the presence of KIT alone is nonspecific. The morphology of a cellular and monotonous round to ovoid soft tissue tumor also raises the differential diagnosis of DSRCT, which is excluded by the absence of cytokeratin and desmin expression and characteristic EWSR-WT1 fusion. 28 The “patternless” arrangement of monotonous fusiform spindle cells could also raise the consideration of a hemangiopericytoma-like solitary fibrous tumor, 29 which is ruled out by the lack of STAT6 immunostaining and NAB2-STAT6 gene fusion. Tumors of Müllerian origin in the male genitourinary tract are rare and largely recognized in the testis/paratestis. 30 , 31 They include paratesticular tumors of ovarian epithelial types: including serous cystadenomas with ovarian stromas, 30 - 35 mucinous tumors, 30 , 36 Brenner tumors; 30 sex cord stromal tumors: adult granulosa cell tumor; 37 pediatric DICER1 -associated sarcomas. 38 It is possible that they arise from Müllerian duct remnants in males, 39 which can give rise to developmental disorders such as persistent Müllerian duct syndrome. 40 To our knowledge, only one case of paratesticular low-grade ESS-like sarcoma has been reported in a 59-year-old male, which harbored JAZF1-SUZ12 gene fusion, 14 the most common fusion in low-grade ESS. Beyond the testis, one case of confirmed low-grade ESS with JAZF1-SUZ12 was reported in a tumor of purported prostatic stromal origin. 15 This group of prostatic stromal tumors are believed to be heterogeneous and may represent multiple histotypes, a subset of which could fall under the low-grade ESS category or be of Müllerian origin. Interestingly, an early report of sarcomas/proliferative lesions of the specialized prostatic stroma demonstrated ER and PR positivity in 1 of 7 and 6 of 7 cases, respectively. 41 Similarly, both of our cases express diffuse and strong PR by immunohistochemistry. To our knowledge, we report the second case of low-grade ESS-like sarcoma in the paratestis, confirmed by the presence of JAZF1-PHF1 fusion. Overall, the four cases have low mitotic count (< 10 per 10 HPF), no necrosis, and are ER and PR positive (not specified for the case for the tumor of prostatic stromal origin). Follow-up information is only available for one case (case 1 from the current study), where the tumor recurred and the patient is still alive on last follow-up. Table 1 summarizes the four reported cases of low-grade ESS-like tumors in males to date. Extrauterine ESS, or endometrioid stromal sarcoma, is thought to arise in association with endometriosis. 10 , 12 , 13 The pathogenesis of endometriosis is not well understood: the transplantation theory suggests retrograde implantation of shed, viable endometrial cells, whereas the celomic metaplasia hypothesis proposes that the peritoneal mesothelium undergoes metaplasia or transdifferentiation into endometrial tissue. 42 Endometriosis in males, which lends support to the celomic metaplasia theory, has been reported in isolated case reports in the pelvis, urinary bladder, paratestis, inguinal region, etc., and was attributed to prolonged estrogen exposure in the setting of prostate cancer hormonal therapy or cirrhosis. 43 - 46 For our unusual case of periprostatic ESS, the patient had a history of prostatic carcinoma status post prostatectomy. There is a distinct possibility that estrogen exposure in relation to the treatment of his prostate cancer led to endometriosis in the peritoneum, which in turn gave rise to ESS. On the other hand, the diffuse and strong AR nuclear expression and the fact that this mass was thought to arise between the prostate and rectum clinically suggests that this tumor is related to ESS-like tumors of prostatic stromal origin. In summary, we report two rare cases of low-grade ESS-like sarcoma in male patients, one as a paratesticular mass with JAZF1-PHF1 fusion, and the other as an extragenital pelvic mass with a novel JAZF1-GLI3 fusion. Larger studies of low-grade ESS-like sarcoma in male patients are needed to better understand this enigmatic entity.

Low-grade endometrial stromal sarcoma (ESS) is a low-grade malignant tumor resembling proliferative-phase endometrial stroma that most commonly occurs in the uterine corpus in women over a wide age range. 1 Low grade ESS are characterized by recurrent gene fusions, most commonly JAZF1-SUZ12 , followed by JAZF1-PHF1 , EPC1-PHF1 , and MEAF6-PHF1 . 2 - 6 The protein products of these genes are involved in the Polycomb chromatin remodeling complexes, while their fusion oncoproteins are predicted to inhibit apoptosis and promote cell proliferation and survival. 7 - 9 Endometrioid stromal sarcoma, also known as extrauterine ESS, is uncommon and arises at diverse anatomic sites, including ovaries, bowel wall, abdominal cavity/peritoneum, pelvis, and vagina, usually in association with endometriosis. 10 - 13 Endometrioid stromal sarcoma, or “low-grade ESS-like” sarcoma, is exceptionally rare in males, with only two cases been reported to date, one in the paratestis and one of prostatic stromal origin, 14 , 15 both cases confirmed by the presence of JAZF1-SUZ12 fusion. Herein, we report two new cases of low-grade ESS-like sarcoma in male patients, one as a paratesticular mass with JAZF1-PHF1 fusion and one as a recurrent periprostatic pelvic mass with a novel JAZF1-GLI3 fusion, which has not been previously reported in low-grade ESS.

Supplementary Table 1 : BAC clones.