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Article Type : Review

Endometrial stromal sarcoma- the new genetic paradigm

Cheng-Han Lee, MD, PhD. 1, Marisa R Nucci, MD 2

1

Department of Laboratory Medicine and Pathology, Royal Alexandra Hospital, University

of Alberta, Edmonton, Alberta, Canada 2

Department of Pathology, Brigham and Women's Hospital, Boston, United States

Running Title: Updates on endometrial stromal sarcoma

Corresponding authors: Marisa R. Nucci, M.D. Associate Professor, Department of Pathology, Harvard Medical School, Associate Pathologist, Brigham and Women's Hospital, 75 Francis Street, Amory 3 Boston, MA, USA. 02115 Office: 617-732-5054 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/his.12594 This article is protected by copyright. All rights reserved.

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Fax: 617-277-9015 E-mail: [email protected]

Keywords: Endometrial stromal sarcoma, uterine sarcoma, YWHAE, NUTM2

Disclosure/Conflict of Interest: The authors declare no conflict of interest.

Abstract Endometrial stromal sarcoma (ESS) is a gynecologic sarcoma that is composed of cells that resemble proliferative phase endometrial stroma.

The 2014 World Health Organization

(WHO) tumor classification system separates ESS into low-grade and high-grade types, which are histologically, genetically and clinically distinct from undifferentiated uterine sarcoma (UUS).

Low-grade ESSs frequently contain chromosomal rearrangement that

results in JAZF1-SUZ12 or equivalent genetic fusions. While most low-grade ESSs display classic histologic features that closely resemble proliferative phase endometrial stroma, there are several histologic variants that are associated with the same genetic fusions as seen in the classic type. High-grade ESS is defined by the presence of YWHAE-NUTM2A/B (YWHAEFAM22A/B) genetic fusion. High-grade ESSs are clinically more aggressive than low-grade ESSs but are associated with lower mortality rate than UUSs. The histologic and immunophenotypic features of these different types of ESSs, and their diagnostic considerations are the subjects of this review.

Introduction Endometrial stromal sarcoma (ESS) is a mesenchymal malignancy that occurs predominantly in the uterine corpus, although origin from extrauterine sites including the ovary and peritoneum has also been well-documented. In the uterine corpus, ESS characteristically This article is protected by copyright. All rights reserved.

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differentiated endometrial sarcoma exhibited greater degree of mitotic activity on average and more frequent tumor necrosis, mitotic rate was not by itself an important prognosticator.

In 1990, Chang et al examined the clinicopathologic features of 109 ESSs, the largest series published to date.1

They defined endometrial stromal tumor as primary uterine tumor

(measuring >0.5 cm in size) composed almost exclusively of uniform cells with scant cytoplasm that resembles the cells of proliferative phase endometrium. The finding of serpentine infiltration through the myometrium (myoinvasion) and/or intravascular growth was needed for the designation as ESS.

The presence of focal sex cord differentiation,

smooth muscle differentiation and focal glandular elements was allowed. However, spindle cell neoplasms with a high degree of pleomorphism, tumor giant cells, malignant glandular elements or the presence of more than scanty cytoplasm in a significant portion of the tumor were excluded.

Although highly pleomorphic tumors were excluded, nuclear atypia was

graded from 1 to 3 based on nuclear hyperchromasia, nucleomegaly, nucleolar prominence and irregularity of nuclear membrane. Chang et al that found 14% of the tumors had mitotic index greater than 10 MF/10 HPF and 23% of the tumors showed grade 2 to 3 nuclear atypia. In patients with stage 1 disease (disease confined to the uterus), mitotic index was not predictive of disease recurrence. While the correlation between cytologic atypia and mitotic index was weak for stage 1 tumors, there was an apparent correlation between cytologic atypia and mitotic index in stage 3 and 4 tumors. These findings reinforced the notion that mitotic index alone should not be used to separate ESS into low-grade and high-grade groups. Even though it was not specifically emphasized by the authors (perhaps due to the small sample size), 4 of 8 patients with tumors showing concomitant grade 2-3 nuclear atypia and elevated mitotic index (> 10 MF/ 10 HPF) presented with unresectable disease and subsequently died from the disease (average 6 months after the initial diagnosis, ranged from This article is protected by copyright. All rights reserved.

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2 to 7 months), in contrast to 11 of 82 patients with tumors lacking these concurrent features who died from the disease (average 78 months after the initial diagnosis, ranged from 14 to 360 months). This finding suggests that there may be different disease subgroups within this series of ESS and that a combined assessment for the presence of cytologic/nuclear atypia and mitotic index may provide better prognostic information that that provided by either feature alone.

Based on these earlier findings, the 2003 WHO system adopted a classification scheme that recognized only low-grade ESS and UES, and the term high-grade ESS was excluded.3 The intent was to discourage the designation of otherwise classic low-grade ESS with increased mitotic rate as high-grade ESS and to reflect the recognition that most of what were formerly categorized as high-grade ESSs were actually undifferentiated pleomorphic uterine sarcomas that bear no histologic resemblance to endometrial stroma. However, as shown by Chang et al in their strictly defined series,1 there is a subset of ESSs that exhibits mitotic activity and nuclear atypia that are greater than that seen in usual low-grade ESSs, and with the ensemble of these more aggressive histologic features, it would be counterintuitive for most pathologists to designate these tumors as low-grade ESS. It is in this grey zone where more descriptive diagnosis such as “histologically high-grade ESS” or “ESS with high grade histologic features” emerges. In an attempt to resolve this issue, Kurihara et al proposed the term UES with nuclear uniformity (UES-U) in contrast to UES with nuclear pleomorphism (UES-P) that is used to describe high-grade undifferentiated pleomorphic uterine sarcomas.18 Their study sample size was small and the significance of such distinction was unclear as there were no apparent differences clinically with regards to patient outcome between UES-U and UES-P in their series.

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disease at presentation and disease recurrence, either locally or at distant sites such as the lungs occurs more frequently and more rapidly.52, 64

All YWHAE-NUTM2 ESSs reported to date all appear to have originated from the uterus and typical form a grossly apparent myopermeative uterine mass which can range from 1 to 12 cm in size, though the size of the extrauterine tumor extension/deposit may reach a size larger than the intrauterine component. Similar to low-grade ESSs, uterine YWHAE-NUTM2 ESS consistently displays a highly permeative growth pattern with extensive tongue-like myoinvasion and vascular invasion (Figure 3A).52,

61, 64

pleomorphism at low magnification (4-10 × objective).

There is no apparent nuclear A rich fine arborizing stromal

capillary network is consistently present with occasional small concentric arterioles (Figure 3B), and this is an important defining feature of this tumor type. In contrast to low-grade ESS, all primary YWHAE-NUTM2 tumors contain high-grade round cell area, either in part or exclusively. These tumor cells in the high-grade round cell area demonstrate a combination of nucleomegaly (4 to 6 times the size of stromal lymphocyte nuclei, with no tumor giant cells), slightly more irregular nuclear contour (discernible at 20x objective) and increased mitotic activity (with all cases showing > 10 MF/10 HPF) compared to classic low-grade ESS (Figure 3C-D). The nucleoli are non-prominent and atypical mitoses are not present. This appears similar to what Chang et al had previously described for a subset of ESS, which exhibits grade 2 to 3 nuclear atypia accompanied by increased mitotic activity.1 The amount of cytoplasm seen in the high-grade round cells can range from scanty to moderate in amount. In the area where cells possess scanty cytoplasm, the combination of high cellularity and high-grade nuclear features imparts a malignant small round blue cell appearance (Figure 3C),65 while area with a moderate amount of faintly eosinophilic cytoplasm can assume an epithelioid appearance, with a vaguely nested pattern imparted the rich fine stromal capillary This article is protected by copyright. All rights reserved.

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to be arising from the uterus, it is important to exclude a mixed epithelial and mesenchymal malignancy such as carcinosarcoma and adenosarcoma,85, 86 particularly in older patients as the sarcomatous component of carcinosarcoma and adenosarcoma can become the dominant component of the tumor. Adequate tumor sampling, with particular focus on the endometrial surface aspects including the adjacent background endometrium may provide evidence to support a diagnosis of carcinosarcoma or adenosarcoma.

For a resection specimen (i.e. total hysterectomy), when a mixed epithelial and mesenchymal tumor has been excluded after reasonable sampling effort (at least one section per cm of tumor with directed sampling of the surface of the intracavitary tumor and adjacent endometrium), it is then important to determine whether one is dealing with a pleomorphic uterine sarcoma or monomorphic uterine sarcoma based on low power examination. If significant nuclear pleomorphism is encountered, the differential would include UUS, leiomyosarcoma and distinctively rare heterologous sarcomas such as pleomorphic rhabdomyosarcoma, among others87. If there is no significant nuclear pleomorphism present, the differential then includes ESS, smooth muscle tumor (leiomyosarcoma or intravascular leiomyomatosis), dedifferentiated/undifferentiated endometrial carcinoma, and the rare uterine PEComa and Ewing sarcoma.87

For tumor showing exclusively classic low-grade ESS growth pattern and morphology, including tumors with focal smooth muscle differentiation, the differential is primarily between JAZF1 LGESS and ESS with no demonstrable genetic rearrangement (as YWHAENUTM2 ESS typically always contains admixed low and high-grade areas, and YWHAENUTM2 ESS has not been documented to display smooth muscle differentiation).

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Genetic

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subtyping (FISH and/or RT-PCR analysis for LGESS genetic fusions) is optional at the present time as the prognosis is similar between them and there is currently no genotypespecific targeted therapy.

This however applies only to hysterectomy specimen with

adequate tumor sampling (at least one section per cm of area involved by tumor). ER status should be determined in these cases as it presents a potential therapeutic target. In tumors with extensive smooth muscle differentiation and intravascular tumor extension, an important differential to include is uterine leiomyoma with intravascular leiomyomatosis and genetic analysis may help to resolve this issue.

For tumors consisting of a monomorphic ovoid/spindle cell proliferation with fibrous to myxoid stroma that lacks significant nuclear atypia and mitotic activity, the differential includes fibrous/fibromyxoid variant of low-grade ESS (with or without demonstrable genetic rearrangement), sarcomatous overgrowth of adenosarcoma, a smooth muscle tumor and the low-grade component of YWHAE-NUTM2 ESS. The presence of an intersecting fascicular growth pattern and positive immunostaining for smooth muscle actin, caldesmon and/or desmin would favor a smooth muscle tumor. Attempt should also be made to exclude the possibility of an adenosarcoma with sarcomatous overgrowth, given its higher recurrence risk compared to low-grade ESS. If a smooth muscle tumor has been excluded and there are no adenosarcoma or high-grade round cell areas (YWHAE-NUTM2 ESS) identified after adequate tissue sampling, a diagnosis of low-grade ESS (fibrous/fibromyxoid variant) would be favored, particularly if the tumor is immunoreactive for CD10 and ER/PR.

For tumor consisting purely of monomorphic spindle to round tumor cells with increased mitotic activity and/or moderate to high grade nuclear atypia (but non-pleomorphic), the

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Titles and legends to Tables Table 1: Comparison of the pathologic features between low-grade ESS (LGESS; with or without genetic rearrangements), YWHAE-NUTM2 ESS, and UUS. * Occasional tumors with diffuse cyclin D1 nuclear immunoreactivity are CD10 positive.

Titles and legends to Figures Figure 1: Representative images of classic low-grade ESSs with JAZF1-SUZ12 genetic fusion showing, A) myopermeative growth, B) rich arteriolar vascular network, C) bland monomorphic nuclear features with scanty cytoplasm with classic appearance, D) focal tumor necrosis, E-F) stromal hyalinization in the form of fibrous plaques and bands.

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Classic low-grade ESS without demonstrable genetic rearrangements Currently, there remains a small group of classic low-grade ESS with prototypic morphology and immunophenotype that lacks demonstrable genetic rearrangement of known ESS fusion genes. Given that some of the ESSs display isolated JAZF1 or PHF1 genetic rearrangements with no rearranged partner genes identified,42, 43, 60 it is likely that additional genetic fusion types such as the recently characterized MEAF6-PHF1, MBTD1-CXORF67 and ZC3H7BBCOR genetic fusions will be identified in the remaining low-grade ESS subset, particularly with the increasing application of next generation sequencing technology. In term of their clinical behavior, preliminary data from our group suggest that JAZF1 LGESS and classic low-grade ESS with no demonstrable genetic rearrangements both behave in an indolent manner clinically, with low-stage disease at presentation and low incidence of disease recurrence.

YWHAE-NUTM2 High-grade ESS About half of tumors previously designated as UES (2003 WHO) with uniform nuclear features harbours a recurrent chromosomal translocation - t(10;17)(q22;p13).61

This

translocation was found to result in YWHAE-NUTM2A or YWHAE-NUTM2B genetic fusions (collectively referred to as YWHAE-NUTM2 in this review).61 NUTM2A/B is formerly known as FAM22A/B, and the change of nomenclature reflects its sequence homology to NUT protein (encoded by NUTM1) which is important in NUT midline carcinoma.62 The finding of frequent YWHAE-NUTM2 in what was described as UES with nuclear uniformity has now also confirmed by other groups.51,

63, 64

YWHAE-NUTM2 genetic rearrangements and

JAZF1/SUZ12/EPC1/PHF1 genetic rearrangements are mutually exclusive. When compared to JAZF1 LGESS, YWHAE-NUTM2 ESS is more frequently associated with high stage

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disease at presentation and disease recurrence, either locally or at distant sites such as the lungs occurs more frequently and more rapidly.52, 64

All YWHAE-NUTM2 ESSs reported to date all appear to have originated from the uterus and typical form a grossly apparent myopermeative uterine mass which can range from 1 to 12 cm in size, though the size of the extrauterine tumor extension/deposit may reach a size larger than the intrauterine component. Similar to low-grade ESSs, uterine YWHAE-NUTM2 ESS consistently displays a highly permeative growth pattern with extensive tongue-like myoinvasion and vascular invasion (Figure 3A).52,

61, 64

pleomorphism at low magnification (4-10 × objective).

There is no apparent nuclear A rich fine arborizing stromal

capillary network is consistently present with occasional small concentric arterioles (Figure 3B), and this is an important defining feature of this tumor type. In contrast to low-grade ESS, all primary YWHAE-NUTM2 tumors contain high-grade round cell area, either in part or exclusively. These tumor cells in the high-grade round cell area demonstrate a combination of nucleomegaly (4 to 6 times the size of stromal lymphocyte nuclei, with no tumor giant cells), slightly more irregular nuclear contour (discernible at 20x objective) and increased mitotic activity (with all cases showing > 10 MF/10 HPF) compared to classic low-grade ESS (Figure 3C-D). The nucleoli are non-prominent and atypical mitoses are not present. This appears similar to what Chang et al had previously described for a subset of ESS, which exhibits grade 2 to 3 nuclear atypia accompanied by increased mitotic activity.1 The amount of cytoplasm seen in the high-grade round cells can range from scanty to moderate in amount. In the area where cells possess scanty cytoplasm, the combination of high cellularity and high-grade nuclear features imparts a malignant small round blue cell appearance (Figure 3C),65 while area with a moderate amount of faintly eosinophilic cytoplasm can assume an epithelioid appearance, with a vaguely nested pattern imparted the rich fine stromal capillary This article is protected by copyright. All rights reserved.

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network (Figure 3B and 3D). Furthermore, additional variant features including focal sexcord like differentiation, focal pseudoglandular and pseudopapillary appearance and focal rosette-like formation have been described in YWHAE-NUTM2 ESS.52,

65

Importantly,

smooth muscle differentiation that is present in a subset of JAZF1 LGESS has not been observed in YWHAE-NUTM2 ESS to date.

In about half of YWHAE-NUTM2 ESS (at both the primary uterine site and metastatic sites), there is an accompanying histologically low-grade component where the tumor cells appear ovoid to spindle in shape (Figure 4A). The high-grade and low-grade components are typically closely admixed, but it can appear well-demarcated in some cases. The low-grade component is less cellular than the high-grade component (low to moderate cellularity), and frequently contains fibrous to fibromyxoid background stroma (Figure 4). The juxtaposition between the highly cellular high-grade component and the moderately cellular low-grade component can impart an apparent “biphasic” appearance at low-magnification. In contrast to the tumor cells in the high-grade component, the ovoid to spindle shaped tumor cells in the low-grade component exhibits smooth nuclear contour and lack significant mitotic activity (up to 3 MF/10 HPF).

The low-grade component can assume a number of different

architectural patterns that include a classic low-grade ESS-like pattern (Figure 4B-C), a fibroblastic/fibromyxoid low-grade ESS pattern (Figure 4D-E) and a fascicular spindle cell growth pattern (Figure 4F). The classic low-grade ESS-like area of YWHAE-NUTM2 ESS can be histologically similar to classic JAZF1 LGESS, except for the presence of the highgrade component in YWHAE-NUTM2 ESS. The fibrous/fibromyxoid low-grade ESS area of YWHAE-NUTM2 ESS also resembles the fibroblastic/fibromyxoid variant of low-grade ESS,16 though none of these reported cases showed an accompanying high-grade component. The fascicular spindle cell low-grade area of YWHAE-NUTM2 can mimic a smooth muscle This article is protected by copyright. All rights reserved.

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tumor but it lacks the presence of well defined eosinophilic cytoplasm and immunohistochemical evidence of smooth muscle differentiation.

It can also mimic

sarcomatous overgrowth of müllerian adenosarcoma, though the fascicular spindle cell lowgrade area of YWHAE-NUTM2 is less mitotically active and lacks the other characteristic features of an adenosarcoma(e.g. phyllodes growth, periglandular cuffing) .

While YWHAE-NUTM2 genetic fusion is specific for ESS in the context of uterine tumors and adult mesenchymal tumors, the same chromosomal translocation – t(10;17)(q22;p13), which occurs in a subset of clear cell sarcoma of the kidney also results in the same YWHAENUTM2 genetic fusion.66

There however is no clinical or anatomic overlap between

YWHAE-NUTM2 ESS and clear cell sarcoma of the kidney, as the later disease entity occurs exclusively in the pediatric age group and arises from the kidney. It is worth noting that Suzuki et al recently reported on the findings of concurrent YWHAE, NUTM2A and NUTM2B genomic breakages as shown by FISH in an uterine angiosarcoma but no YWHAENUTM2A/B fusion transcript was identified.67

This is different from the genomic

rearrangements (either concurrent YWHAE and NUTM2A rearrangement or concurrent YWHAE and NUTM2B rearrangement) seen in YWHAE-NUTM2 high-grade ESS.

The

findings in this uterine angiosarcoma thus most likely reflect the presence of a highly complex pattern of genomic rearrangement akin to what is seen with chromothripsis.

Immunohistochemically, the high-grade and low-grade components of YWHAE-NUTM2 ESS shows different staining patterns.52

The low-grade area consistently displays diffuse

immunostaining for ER, PR and CD10, in contrast to the high-grade area which lacks significant ER, PR and CD10 immunoreactivity (70%) and strong nuclear cyclin D1 immunostaining (Figure 5A-B),51,

63, 64, 68

whereas the low-grade area shows typically weak and focal cyclin D1

immunostaining (Figure 5C-D). It is important to note that such diffuse strong cyclin D1 immunohistochemical expression is very uncommon among other types of uterine sarcomas and the combination of diffuse cyclin D1 immunostaining coupled with negative CD10 and ER immunostaining in a high-grade monomorphic endometrial/uterine sarcoma would support a diagnosis of YWHAE-NUTM2 ESS. The high-grade area also consistently displays moderate to strong cytoplasmic KIT immunostaining while the staining in the low-grade area is typically weak to absent (Figure E-F); DOG1 is consistently negative in both the highgrade and low-grade areas.64, 69 Increased expression of cytoplasmic beta-catenin is observed in the high-grade area of YWHAE-NUTM2 ESS but there does not appear to be nuclear accentuation in our experience. Immunohistochemical p53 expression is typically patchy in both high-grade and low-grade areas, with no evidence to suggest the presence of TP53 mutation/loss. CD99 has been reported to be positive in YWHAE-NUTM2 ESS,65 but its staining pattern has not been systemically examined. YWHAE-NUTM2 ESS is negative for pankeratin, EMA, smooth muscle actin, desmin, caldesmon, S100, HMB-45 and Melan-A.52, 65

In terms of nomenclature, both high-grade ESS and UUS/UES-U have been previously used to describe ESS with YWHAE-NUTM2 genetic rearrangement. We do not favor the use of UUS with nuclear uniformity (formerly UES-U) for YWHAE-NUTM2 uterine sarcoma because of the following. undifferentiated.

Firstly, YWHAE-NUTM2 uterine sarcoma is not truly

About half of the tumor possesses an admixed low-grade area that

resembles usual low-grade ESS and its histologic variants. Furthermore, the low-grade area of YWHAE-NUTM2 tumors displays diffuse CD10/ER/PR-positive immunoprofile, akin to This article is protected by copyright. All rights reserved.

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that seen in usual low-grade ESS. Secondly, there is at the present time no evidence for recurrent genetic fusions in de novo UUS (excluding dedifferentiated ESS). Thirdly, UUS represent a highly aggressive and lethal group of uterine malignancy.18, 70-76 It is generally non-responsive to conventional therapy and the prognosis is similar to that of carcinosarcoma and high-grade leiomyosarcoma.70-73, 75 While it is clear that YWHAE-NUTM2 ESS is more aggressive than the usually indolent JAZF1 LGESS,52, 61 our preliminary data based suggests that it does not behave as aggressively as UUS.77 More specifically, long term survival is possible even for patients with high-stage YWHAE-NUTM2 ESS disease when adjuvant chemotherapy and/or radiation therapy are given.

Because of the difference in histology, genetics and clinical behavior between UUS and YWHAE-NUTM2 ESS, it is important to separate them as different disease entities. Based on currently available evidence, we believe that YWHAE-NUTM2 tumors represent a type of high-grade ESS and this is the terminology adopted by the 2014 WHO gynecologic tumor classification system.27 This tumor is distinct from other types of histologically high-grade ESS such as dedifferentiated ESS (JAZF1-SUZ12).

The term dedifferentiated ESS is

inappropriate for YWHAE-NUTM2 ESS as the high-grade area (presumed dedifferentiated component) is always present in the primary tumor without evidence of low-grade component in half of the cases. Furthermore, we have observed admixed low-grade and high-grade components in metastatic foci of tumor deposits; it would be unusual for the well differentiated tumor to metastasize together with the dedifferentiated tumor to the same distant site.

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Undifferentiated uterine sarcoma (formerly known as undifferentiated endometrial sarcoma) The 2014 WHO gynecologic tumor classification system replaced the old terminology of UES with UUS.27 This acknowledges the understanding that not all UES arose from the endometrium and a descriptive terminology of UUS is therefore likely more accurate. UUS is a high-grade sarcoma and exhibits a combination of severe nuclear atypia and high mitotic rate.

Tumor necrosis is frequently present. The majority of UUS show prominent nuclear

pleomorphism, though a subset of cases displays more uniform albeit high-grade nuclear features. Rhabdoid morphology can be present in a focal to diffuse manner in some cases. As its name implies, UUS lack specific lines of mesenchymal differentiation and it is thus a diagnosis of exclusion. It is therefore important to rule out the possibility of other uterine sarcoma types (i.e. leiomyosarcoma, rhabdomyosarcoma, high-grade ESS), mixed epithelialmesenchymal uterine tumors (sarcoma-predominant carcinosarcoma or sarcomatous overgrowth of adenosarcoma), uterine carcinomas (undifferentiated or dedifferentiated endometrial carcinoma) and secondary involvement of the uterus by extrauterine soft tissue sarcomas. However, despite our best efforts, it is likely that a subset of UUS may represent highly poorly differentiated leiomyosarcoma or carcinosarcoma in which the sarcomatous component has either completely replaced or near completely outgrown the carcinomatous component. Genetically, there is currently little known about UUS. Cytogenetic analysis of UUS show a complex karyotype, with many structural and numerical chromosomal aberrations.78 Sequencing analysis showed that a subset of UUS harbors missense TP53 mutations.18 The 2014 WHO tumor classification includes (high-grade) dedifferentiated ESS into UUS category,27 but this is a very rare phenomenon and likely accounts for a very small subset of UUS. As discussed, these dedifferentiated ESS-type UUSs harbor the same genetic rearrangement

as

seen

in

the

accompanying

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low-grade

ESS

component.

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Immunohistochemically, UUS by definition is undifferentiated. It can however be positive for CD10 and hormone receptors, hence it is important to not regard CD10 as evidence of endometrial stromal differentiation.

It may show very focal positive staining for smooth

muscle actin, but the presence of positive staining for more than one smooth muscle markers should raise the suspicion for leiomyosarcoma or malignant PEComa. Focal keratin and EMA staining, when encountered in a suspected UUS that demonstrates nuclear uniformity, should prompt a careful investigation into the possibility of undifferentiated or dedifferentiated endometrial carcinoma. It is important to note that the distinction between undifferentiated/dedifferentiated endometrial carcinoma and UUS with nuclear uniformity can be very difficult, particular in limited biopsy sample.

2014 WHO classification of endometrial stromal sarcoma A comparison of the pathologic features between YWHAE-NUTM2 ESS, low-grade ESS (with or without genetic rearrangement) and UUS is shown in Table 1. JAZF1 LGESS and classic low-grade ESS with no demonstrable genetic rearrangements share the same histologic and immunophenotypic features, and are grouped under the same low-grade ESS category. YWHAE-NUTM2 ESS is categorized as high-grade ESS, which is distinct from UUS.

This classification scheme confers prognostically important and therapeutically

relevant information (Figure 6).

JAZF1 LGESS and classic low-grade ESS with no

demonstrable genetic rearrangements generally present with resectable stage 1 disease and have an excellent prognosis with a small risk of recurrence (10-20%).52, 61 When the tumor recurs, it is often occurs several years later. These tumors are almost always ER and PRpositive, and anti-estrogenic therapy including the better-tolerated aromatase inhibitors appears to have some value in controlling the disease.79-84 Adjuvant chemotherapy and/or

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radiation therapy are typically not warranted unless the disease is extensive. In comparison, YWHAE-NUTM2 ESS typically presents with advanced stage disease (stage 2-4) and frequently recurs, usually within a few years after initial surgery. Their lack of ER and PR immunopositivity in the high-grade component suggests that anti-estrogenic therapy will likely be ineffective in controlling tumor growth. Furthermore, while our experience is limited, some of the long term survivor of patients with stage 2 or higher YWHAE-NUTM2 ESS received adjuvant chemotherapy and radiation therapy, suggesting that adjuvant therapy may provide survival benefit in patients with YWHAE-NUTM2 ESS. In comparison, UUS as currently defined is a highly aggressive malignancy. Patients with high stage (stage 2 or greater) UUS have a dismal prognosis and these tumors are generally non-responsive to conventional chemotherapy and/or radiation therapy.70-75

Diagnostic considerations In the work-up of a malignant uterine mesenchymal tumor, the differential diagnosis can be broad. The uterus is situated in the pelvis where a number of primary soft tissue based tumors can occur, such as dedifferentiated liposarcoma, PEComa, gastrointestinal stromal tumors, peripheral nerve sheath tumors and low-grade fibromyxoid sarcoma. Conversely, uterine mesenchymal tumors can spread beyond the uterus and form an adjacent pelvic mass. Therefore, the most fundamental distinction is whether the malignant uterine mesenchymal tumor represents a primary uterine tumor or secondary involvement by an extra-uterine tumor. This distinction is usually more straightforward after a hysterectomy and tumor debulking surgery, where one can more definitively assess the histologic features and distribution of the disease. The challenge is with limited tissue biopsy sample (core biopsy or laparoscopic tissue biopsy) when radiologic findings are inconclusive. If the tumor appears

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to be arising from the uterus, it is important to exclude a mixed epithelial and mesenchymal malignancy such as carcinosarcoma and adenosarcoma,85, 86 particularly in older patients as the sarcomatous component of carcinosarcoma and adenosarcoma can become the dominant component of the tumor. Adequate tumor sampling, with particular focus on the endometrial surface aspects including the adjacent background endometrium may provide evidence to support a diagnosis of carcinosarcoma or adenosarcoma.

For a resection specimen (i.e. total hysterectomy), when a mixed epithelial and mesenchymal tumor has been excluded after reasonable sampling effort (at least one section per cm of tumor with directed sampling of the surface of the intracavitary tumor and adjacent endometrium), it is then important to determine whether one is dealing with a pleomorphic uterine sarcoma or monomorphic uterine sarcoma based on low power examination. If significant nuclear pleomorphism is encountered, the differential would include UUS, leiomyosarcoma and distinctively rare heterologous sarcomas such as pleomorphic rhabdomyosarcoma, among others87. If there is no significant nuclear pleomorphism present, the differential then includes ESS, smooth muscle tumor (leiomyosarcoma or intravascular leiomyomatosis), dedifferentiated/undifferentiated endometrial carcinoma, and the rare uterine PEComa and Ewing sarcoma.87

For tumor showing exclusively classic low-grade ESS growth pattern and morphology, including tumors with focal smooth muscle differentiation, the differential is primarily between JAZF1 LGESS and ESS with no demonstrable genetic rearrangement (as YWHAENUTM2 ESS typically always contains admixed low and high-grade areas, and YWHAENUTM2 ESS has not been documented to display smooth muscle differentiation).

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Genetic

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subtyping (FISH and/or RT-PCR analysis for LGESS genetic fusions) is optional at the present time as the prognosis is similar between them and there is currently no genotypespecific targeted therapy.

This however applies only to hysterectomy specimen with

adequate tumor sampling (at least one section per cm of area involved by tumor). ER status should be determined in these cases as it presents a potential therapeutic target. In tumors with extensive smooth muscle differentiation and intravascular tumor extension, an important differential to include is uterine leiomyoma with intravascular leiomyomatosis and genetic analysis may help to resolve this issue.

For tumors consisting of a monomorphic ovoid/spindle cell proliferation with fibrous to myxoid stroma that lacks significant nuclear atypia and mitotic activity, the differential includes fibrous/fibromyxoid variant of low-grade ESS (with or without demonstrable genetic rearrangement), sarcomatous overgrowth of adenosarcoma, a smooth muscle tumor and the low-grade component of YWHAE-NUTM2 ESS. The presence of an intersecting fascicular growth pattern and positive immunostaining for smooth muscle actin, caldesmon and/or desmin would favor a smooth muscle tumor. Attempt should also be made to exclude the possibility of an adenosarcoma with sarcomatous overgrowth, given its higher recurrence risk compared to low-grade ESS. If a smooth muscle tumor has been excluded and there are no adenosarcoma or high-grade round cell areas (YWHAE-NUTM2 ESS) identified after adequate tissue sampling, a diagnosis of low-grade ESS (fibrous/fibromyxoid variant) would be favored, particularly if the tumor is immunoreactive for CD10 and ER/PR.

For tumor consisting purely of monomorphic spindle to round tumor cells with increased mitotic activity and/or moderate to high grade nuclear atypia (but non-pleomorphic), the

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differential includes UUS with nuclear uniformity, YWHAE-NUTM2 high-grade ESS, epithelioid leiomyosarcoma, dedifferentiated endometrial adenocarcinoma, and more rarely malignant PEComa and uterine Ewing sarcoma. The expression of smooth muscle markers and melanocytic markers can be evaluated if an epithelioid leiomyosarcoma and malignant PEComa are suspected.87,

88

In cases where UUS with nuclear uniformity and YWHAE-

NUTM2 high-grade ESS are considered, CD10, cyclin D1, ER and PR immunostaining should be performed. Strong diffuse cyclin D1 nuclear staining, in the absence of CD10, ER and PR staining suggests a diagnosis of YWHAE-NUTM2 ESS. Even though a subset of UUS with uniform nuclei can show diffuse cyclin D1 immunpositivity, they typically also exhibits focal CD10, ER or PR staining, which is not seen in the high-grade area of YWHAE-NUTM2 ESS. It is also important to exclude the possibility of undifferentiated or dedifferentiated endometrial carcinomas as they may exhibit diffuse cyclin D1 nuclear staining based on our experience.

YWHAE-NUTM2 high-grade ESS does not express epithelial markers, hence

even focal positive staining with epithelial markers should dissuade against the possibility of high-grade ESS. Examining for the expression of mismatch repair (MMR) proteins may also be helpful in evaluating for the possibility of undifferentiated/dedifferentiated endometrial carcinoma as these tumors frequently exhibit loss of MMR protein(s).89 While extra-uterine Ewing sarcoma can show diffuse strong cyclin D1 staining,90 all molecularly confirmed primary uterine Ewing sarcomas reported to date formed an expansile mass,91-93 and none showed the myopermeative growth pattern seen in YWHAE-NUTM2 ESS. CD99 and FLI-1 may be useful in the diagnosis of uterine Ewing sarcomas, although YWHAE-NUTM2 ESS may show focal CD99 positivity as well and FLI1 has not been systemically examined in YWHAE-NUTM2 ESS.65

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For monomorphic tumor showing an apparent biphasic appearance with a combination or admixture of histologically low-grade ovoid/spindle cell component and high-grade round cell component, YWHAE-NUTM2 ESS should be strongly considered, with the differential that includes dedifferentiated ESS.

Our experience with dedifferentiated ESS (JAZF1-

SUZ12) is limited at the present; one of the two reported cases lacked significant cyclin D1 staining and contains admixed classic low-grade ESS areas, hence cyclin D1 may be useful in distinguishing YWHAE-NUTM2 ESS and dedifferentiated ESS in uterine sarcoma with admixed low-grade ESS and high-grade components.18 The findings of diffuse cyclin D1 positivity in the high-grade round cell component, together with CD10/ER/PR-negative immunoprofile in the high-grade round cell component and CD10/ER/PR-positive immunoprofile in the low-grade component are diagnostic for YWHAE-NUTM2 high-grade ESS.

The diagnostic approach described above pertains only to resection/hysterectomy specimen. In the scenario when only a limited tissue sample is obtained e.g. core biopsy, ancillary tests including immunohistochemical characterization and molecular study are important, as the nuclear features of high-grade YWHAE-NUTM2 high-grade ESS may be less apparent in core biopsy sample. This is illustrated by a case depicted in Figure 7, in which a 24 year old female presented with a large pelvic mass that was queried as a uterine mass radiologically. A core biopsy demonstrates a myopermeative, cellular neoplasm with round to fusiform nonpleomorphic tumor cells, with some degree of crush artefact (Figure 7A-B). Mitotic activity is low. This appearance would favor a diagnosis of low-grade endometrial stromal sarcoma morphologically but immunohistochemical characterization is necessary for diagnostic confirmation. The tumor cells are negative for CD10, ER, pankeratin, EMA, smooth muscle actin, caldesmon and desmin immunohistochemically, and showed diffuse strong nuclear This article is protected by copyright. All rights reserved.

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cyclin D1 immunopositivity.

The subsequent hysterectomy specimen showed typical

appearance of YWHAE-NUTM2 high-grade ESS (Figure 7C-D) and the presence of YWHAENUTM2 was molecularly confirmed.

Conclusion The classification of uterine sarcoma can be challenging at times, and this is illustrated by how the classification of ESS and UUS has evolved over the past few decades. Because of greater understanding of tumor genetics, we are now able to recognize YWHAE-NUTM2 high-grade ESS as a distinct entity apart from UUS and low-grade ESS.

Part of the

diagnostic challenge in approaching uterine sarcoma or sarcomatous neoplasm is that karyotypically simple translocation associated sarcoma (i.e. low-grade ESS and high-grade ESS) and karyotypically complex sarcoma (i.e. leiomyosarcoma and UUS) can both occur with regularity in the uterus, and this is further compounded by the presence of mixed epithelial-mesenchymal tumors, in which the epithelial elements can be overgrown by sarcomatous elements. With the application of next generation sequencing technology, it is anticipated that there will be more genetic insights gained in uterine sarcomas (and mixed epithelial/mesenchymal tumors). This will likely propel further evolution in uterine sarcoma classification. Ultimately, one hopes that this will provide us with more objective methods to reproducibly and meaningfully classify uterine sarcomas for optimal clinical management.

Acknowledgement Both authors (C.H. Lee and M.R. Nucci) contributed equally to the preparation and the writeup of this review article.

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Titles and legends to Tables Table 1: Comparison of the pathologic features between low-grade ESS (LGESS; with or without genetic rearrangements), YWHAE-NUTM2 ESS, and UUS. * Occasional tumors with diffuse cyclin D1 nuclear immunoreactivity are CD10 positive.

Titles and legends to Figures Figure 1: Representative images of classic low-grade ESSs with JAZF1-SUZ12 genetic fusion showing, A) myopermeative growth, B) rich arteriolar vascular network, C) bland monomorphic nuclear features with scanty cytoplasm with classic appearance, D) focal tumor necrosis, E-F) stromal hyalinization in the form of fibrous plaques and bands.

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Figure 2: Representative images on the histologic variants of low-grade ESSs with genetic fusions. A) low-grade ESS with smooth muscle differentiation (JAZF1-SUZ12), B) low-grade ESS with sex-cord differentiation (JAZF1-PHF1), C) fibrous variant of low-grade ESS (PHF1-rearranged), D) epithelioid variant of low-grade ESS (JAZF1-SUZ12), E-F) a dedifferentiated JAZF1-SUZ12 ESS showing classic low-grade area in (E) and high-grade dedifferentiated area in (F).

Figure 3: Representative images of high-grade area of YWHAE-NUTM2 ESS and JAZF1-SUZ12 low-grade ESS. A) A YWHAE-NUTM2 ESS showing myopermeative growth pattern with vascular invasion, B) rich fine capillary vascular network of a YWHAE-NUTM2 ESS, which imparts a nested appearance, C-F) YWHAE-NUTM2 ESS (C and D) demonstrates greater nuclear atypia (nucleomegaly and irregular nuclear contour) and mitotic activity compared to JAZF1-SUZ12 low-grade ESS (E and F) (images C-F are taken at the same magnification).

Figure 4: Representative images of admixed low-grade areas of YWHAE-NUTM2 ESS. A) a YWHAE-NUTM2 ESS with admixed high-grade area (upper right corner) and low-grade area (lower left corner). B-C) low-grade component of YWHAE-NUTM2 ESSs showing appearance that mimic classic low-grade ESS, D-E) low-grade component of YWHAENUTM2 ESSs showing appearance that mimic fibromyxoid and fibrous variant of low-grade ESS, F) low-grade component of YWHAE-NUTM2 ESS showing a moderately cellular bland fascicular spindle cell proliferation.

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Figure 5: Cyclin D1 and KIT immunostaining in YWHAE-NUTM2 ESS. A-B) YWHAENUTM2 ESS shows strong diffuse nuclear cyclin D1 immunoreactivity, in contrast to the virtually negative immunostaining of adjacent normal endometrium. C-D) YWHAE-NUTM2 ESS showing diffuse strong nuclear cyclin D1 immunoreactivity in its high-grade component (lower right corner) and focal weak cyclin D1 immunoreactivity in its low-grade component (upper left corner). E-F) YWHAE-NUTM2 ESS showing diffuse cytoplasmic KIT immunostaining in the high-grade area.

Figure 6: Schematic diagram depicting the current classification of ESS and UUS. * UUS includes the rare examples of dedifferentiated ESS.

Figure 7: A case of YWHAE-NUTM2 ESS in a 24 year old female. A-B) low and high magnification images of the core biopsy. C-D) low and high power images of the myopermeative tumor in the hysterectomy specimen.

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Table 1: Comparison of the pathologic features between low-grade ESS (LGESS; with LGESS Growth features Myopermeative growth Vascular invasion Biphasic (LG+HG) Stromal vasculature

YWHAE-NUTM2 ESS

UUS

Present

Present

Occasional

Frequent

Frequent

Occasional

Rare

~50% of cases

Rare

Prominent arterioles (periarteriolar whorling of cells)

Prominent thin-wall capillary network in the round cell component

Nonconspicuous

Architecture/Growth Classic LGESS Variant LGESS pattern

LG component Classic LGESSlike Fibroblastic LGESS-like Fascicular spindle cell

HG component Nested epithelioid/round cell or small round blue cell

Fascicular to sheet-like

Round

Round to spindle

Large (3~5 x of lymphocyte nuclei)

Variable ± tumor giant cells

Cytologic features Nuclear shape

Round to fusiform

Nuclear size

Small (1.5~3x of lymphocyte nuclei)

Nuclear membrane Nuclear pleomorphism

Cytoplasm

Mitotic activity Necrosis

Fusiform to spindle Similar to the size of normal smooth muscle nuclei

Very smooth contour nonpleomorphic (very uniform) Scanty to moderate eosinophilic cytoplasm Usually < 5 MF/10 HPF Sometimes

Slightly irregular contour non-pleomorphic non-pleomorphic (up to 2x (very uniform) variation in size) Scanty to Scanty/nonmoderate distinct eosinophilic cytoplasm

Negative to Focal ( 10 MF/10 HPF

Absent

Frequent

Variable amount ± rhabdoid features > 10 MF/10 HPF Frequent

Negative to Focal Diffuse Diffuse

Diffuse (>70%) Negative Negative

Negative (majority)* Variable Variable

Immunophenotype Cyclin D1 CD10 ER/PR

or without genetic rearrangements), YWHAE-NUTM2 ESS, and UUS. * Occasional tumors with diffuse cyclin D1 nuclear immunoreactivity are CD10 positive.

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