Mullerian adenosarcoma: clinicopathologic and molecular characterization highlighting recurrent BAP1 loss and distinctive features of high-grade tumors

Mullerian adenosarcoma is an uncommon mesenchymal tumor of the gynecologic tract. Most cases are low-grade, while high-grade adenosarcomas are rare and not well studied. Herein, we characterize the clinicopathologic and molecular features of 27 adenosarcomas of gynecologic origin, enriched for high-grade tumors subjected to targeted panel sequencing. Sarcomatous overgrowth was more frequently seen in high-grade compared to low-grade tumors (12/17, 71%, vs 1/10, 10%, p = 0.004) and heterologous elements were exclusive to high-grade cases (n = 7, p = 0.03). All deaths were from high-grade disease (advanced primary, n = 2, or recurrence, n = 5). Genetic alterations specific to high-grade adenosarcomas have known associations with chromosome instability, including TP53 mutations (n = 4) and amplifications of MDM2 (n = 2) and CCNE1 (n = 2). Somatic ATRX frameshift mutations were found in 2 patients with high-grade recurrences following a primary low-grade adenosarcoma and ATRX deletion in 1 high-grade adenosarcoma with an adjacent low-grade component. The fraction of genome altered by copy number alterations was significantly higher in high-grade compared to low-grade adenosarcomas (P = 0.001). Other recurrent genetic alterations across the entire cohort included BAP1 homozygous deletions (n = 4), DICER1 mutations (n = 4), ARID1A mutations (n = 3), TERT promoter mutations (n = 2) and amplification (n = 1), as well as alterations involving members of the PI3K and MAPK signaling pathways. One tumor harbored an ESR1-NCOA3 fusion and another had an MLH1 homozygous deletion. Immunohistochemical analysis for BAP1 revealed loss of nuclear expression in 6/24 (25%) cases, including all four tumors with BAP1 deletions. Notably, out of 196 mesenchymal neoplasms of gynecologic origin, BAP1 homozygous deletion was only found in adenosarcomas (P = 0.0003). This study demonstrates that high-grade adenosarcomas are heterogeneous at the molecular level and are characterized by genomic instability and TP53 mutations; ATRX loss may be involved in high-grade transformation of low-grade adenosarcoma; and BAP1 inactivation appears to be a specific pathogenic driver in a subset of adenosarcomas.


INTRODUCTION
Mullerian adenosarcoma is an uncommon gynecologic neoplasm, often found in the lower uterine corpus and cervix, and accounts for 5-7% of uterine sarcomas. It can also arise in ovaries or peritoneum, in some cases, associated with endometriosis 1-3 . As its name implies, adenosarcoma is a biphasic tumor composed of epithelial and mesenchymal components, with somatic genetic alterations confined to the latter 4,5 . The histomorphologic appearance is characterized by peri-glandular condensation and stromal expansion, imparting a leaf-like architecture, closely resembling Phyllodes tumor of the breast 6 .
Most adenosarcomas are low-grade mesenchymal neoplasms, comprising non-specific fibroblastoid spindled stroma or resembling endometrial stroma. These tumors tend to have indolent behavior and are typically curable with surgery. In some adenosarcomas, there is predominance of the mesenchymal component, termed "sarcomatous overgrowth" when pure sarcoma comprises over 25% of the tumor [7][8][9] . Areas of sarcomatous overgrowth are often composed of markedly atypical tumor cells with high mitotic activity, and high-grade sarcomatous overgrowth is associated advanced stage disease and poor prognosis 2,7,8 . However, sarcomatous overgrowth can rarely be encountered in low-grade adenosarcomas 4,5 , which in this context, is of unknown prognostic significance. Conversely, high-grade tumor cells, particularly when present only focally, can be observed in adenosarcomas lacking sarcomatous overgrowth 9 . A recent study suggests that even a minor component of high-grade histology may be associated with increased risk of recurrence, though the data on such rare cases are limited 9 . Approximately a quarter of adenosarcomas contain heterologous elements, most commonly in the form of rhabdomyosarcomatous differentiation 1 . Heterologous elements are more commonly seen in high-grade adenosarcomas with sarcomatous overgrowth 10 .
Molecular genetic profiling of adenosarcomas has been performed in several studies, which revealed these adenosarcomas to be genetically heterogeneous, but with recurrent pathogenic driver alterations identified, including rare cases with ESR1-NCOA2/3 fusions 5,9,[11][12][13] . DICER1 mutations are among the most common and of particular interest, as they have also been implicated in uterine and cervical embryonal rhabdomyosarcomas, which may show morphologic overlap with adenosarcoma 11,14 . In addition, a subset of high-grade adenosarcomas harbor TP53 genetic alterations with associated aberrant p53 immunohistochemical expression 9 . Mutations of genes within the PI3K/AKT/PTEN pathway, ATRX, FGFR2, and KMT2C have been reported, as well as amplifications of the MDM2/CDK4 locus and BAP1 deletions 5,9,[11][12][13] .
As adenosarcomas are relatively uncommon, our understanding of this entity is based on small series, mostly of uterine tumors. Herein, we describe the clinicopathologic and molecular features of a cohort of 27 adenosarcomas, including uterine and extrauterine primary sites, and enriched for high-grade tumors. The main goals of this study were to uncover oncogenic drivers of high-grade adenosarcoma and to identify recurrent alterations which may lead to development of clinically useful diagnostic or prognostic markers.

MATERIALS AND METHODS Case selection and review
Following institutional review board approval, 27 uterine or extrauterine Mullerian adenosarcomas were identified from our institutional database of tumors subjected to clinical targeted massively parallel sequencing of up to 505 cancer genes using the Memorial Sloan Kettering Cancer Center -Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platform 15 (from 2014 to 2020). Demographic and clinicopathologic data were extracted from electronic medical records. Of note, none of these cases were previously reported in the prior study of Mullerian adenosarcomas from our institution 5 .
Only cases with slides of the initial primary tumor available were included in this study and diagnoses were confirmed by a gynecologic pathologist (MHC). Histomorphologic review was performed on all available primary and recurrent tumors from each patient. In addition, the following features were evaluated: mitotic rate, presence/absence of: sarcomatous overgrowth, highgrade component, and heterologous elements (and type, if present). As there is no established system for assigning tumor grade in adenosarcoma, we adapted criteria previously described by Hodgson et al. 9 . Low-grade adenosarcomas displayed monotonous, small ovoid nuclei, resembling those seen in low-grade endometrial stromal sarcoma. High-grade atypia was defined as enlarged nuclei with coarse chromatin, exhibiting marked pleomorphism and prominent nucleoli, identifiable at low power magnification, as previously described 9 . We further subclassified the extent of highgrade histology as "focal" when it comprised <10% of a tumor that is predominantly low-grade.
The number of H&E-stained slides of tumor available for review for each case ranged from 2 to 25 (median 12, mean 12). Of note, for the focally high-grade tumors (n = 3), all tumor slides were reviewed (14-25 slides). For the 2 low-grade adenosarcomas that recurred as high-grade sarcoma, only a limited number of tumor slides of primary tumor were available (2 and 5, respectively), as these were received in consultation from other institutions.

Targeted next generation sequencing
Targeted panel sequencing of matched tumor and blood-derived normal DNA was performed using MSK-IMPACT, a hybridization capture-based next-generation sequencing assay targeting all exons and selected intronic regions of 410-505 cancer-related genes 15 . Sequencing data were analyzed as previously described 15,16 . Variants were annotated by OncoKB 17 . Fraction of genome altered by copy number alterations and tumor mutational burden were derived from MSK-IMPACT data. Total and allele-specific copy number was estimated using FACETS 18 .

Targeted RNA-sequencing
For the tumor with ESR1-NCOA3 gene rearrangement identified by MSK-IMPACT, targeted RNA-sequencing was performed using the MSK Solid Fusion assay (v3) 19 , for orthogonal validation. The assay incorporates the Archer™ FusionPlex™ and a custom designed Gene Specific Primer Pool kit, designed to target specific exons in 62 genes known to be involved in chromosomal rearrangements.

Microsatellite instability and MLH1 promoter methylation
For MA04, which harbored an MLH1 deletion, microsatellite instability (MSI) was assessed using the Idylla MSI TM Test. MLH1 promoter hypermethylation status was determined using the Illumina MethylationEPIC 850 K bead array platform.

Statistical analysis
For categorical data, comparisons between groups were analyzed by the Fisher's exact test. Group comparisons of continuous data were performed using the Mann-Whitney test. All statistical tests were two-tailed, with the threshold for statistical significance set at p < 0.05.

Clinical and histopathologic features of adenosarcomas
Primary sites of the 27 adenosarcomas included in this study were uterine corpus (n = 19), cervix (n = 3), ovary (n = 4), and pelvic peritoneum (n = 1; Table 1). The mean age at diagnosis was 56 years (range: 23-76 years). Apart from 1 patient with a biopsy diagnosis only, all patients underwent primary surgical resection.
All tumors showed peri-glandular stromal cuffing, stromal hypercellularity and atypia, and at least focal Phyllodes-like architecture, manifested by polypoid growth of stromal cells protruding into glands ( Fig. 1A-I). Mitotic activity was variable, ranging from 1 to 58 (median: 5) per 10 high-powered fields. The glandular component showed variable degrees of proliferation, with atypical endometrial hyperplasia present in five cases. Tumors arising in the cervix (n = 3) were lined by benign endocervical mucinous epithelium.
The median length of clinical follow-up, from the date of primary surgical resection, was 29 months (range: 1-69 months; Table 1). Two patients had extensive disease which could not be completely resected, and seven patients developed subsequent recurrence (all of whom achieved complete gross resection at primary surgery) and 18 patients remained disease-free at last follow-up. Sites of disease recurrence included abdomen, colonic serosa, vagina, pelvis, and chest wall. Median time to recurrence was 15 months (range: 1-52 months). For the recurrent cases, the original primary tumor was classified as low-grade (n = 3), focally high-grade (n = 1), or high-grade (n = 3); however, six of seven (86%) developed high-grade sarcomas at recurrence. Seven (26%) patients died of disease: five from recurrent high-grade sarcoma and two from extensive primary disease (both high-grade) that could not be completely resected.

Associations between histomorphologic and molecular features of Mullerian adenosarcomas
There was clear separation of low-grade and high-grade tumors with respect to the fraction of genome altered by copy number alterations (FGA; low-grade, median: 0.8% vs. high-grade, median: 17%; p = 0.002, Fig. 3). MA02 was an outlier amongst low-grade adenosarcomas, which demonstrated low-grade morphology, but displayed a high FGA. This patient subsequently developed a high-grade sarcoma recurrence. Tumors with only a focal highgrade component had low FGA values, similar to the low-grade group, likely attributable to only low-grade tumor or predominantly low-grade tumor present in the sample extracted for molecular analysis. Total mutation counts did not differ significantly across tumor grade. MA27, a predominantly low-grade adenosarcoma which focally showed nuclear atypia of indeterminate grade had a low FGA, but harbored the highest number of mutations across the cohort. There were no statistically significant associations between any specific genetic alteration and tumor grade, sarcomatous overgrowth or heterologous elements, though statistical analysis may not be meaningful, as each individual gene was altered in only up to a maximum of four cases. Nevertheless, there were some notable observations. All TP53-mutated tumors were high-grade (n = 4), two of which also displayed sarcomatous overgrowth, and were exemplified by high chromosomal instability (median FGA, TP53-mutated: 38% vs TP53-wildtype: 3%, p = 0.01). Immunohistochemistry confirmed the aberrant p53 expression in tumors harboring TP53 mutations, and a wildtype expression pattern in those lacking TP53 genetic alterations, including the two highgrade tumors with MDM2 amplification. Notably, in MA24, p53 immunohistochemical analysis demonstrated aberrant diffuse overexpression restricted to the focal high-grade area present only in the biopsy specimen ( Fig. 4A-C). Molecular analysis performed on available tumor tissue from the hysterectomy specimen, which consisted only of low-grade tumor, did not detect a TP53 mutation. Adenosarcomas with CCNE1 amplification (n = 2) were also high-grade. and total mutation counts are presented, stratified by histologic grade. Note that the low-grade group includes the tumor with focal indeterminate grade (MA27). MA08 (molecular analysis performed on recurrence) is excluded from these graphs. n.s. not significant, *p < 0.05, **p < 0.01, Mann-Whitney test. Fig. 2 Summary of somatic genetic alterations in Mullerian adenosarcomas. Recurrently altered genes in this cohort or reported in prior studies of adenosarcoma are presented, as well as the unusual finding of MLH1 deletion (n = 1). With exception of MA08, for which only the high-grade recurrence (MA08) was available, molecular profiling was performed on the primary tumors.
Two patients with somatic ATRX frameshift mutations (MA02, MA08) initially presented with low-grade adenosarcoma, but subsequently recurred with high-grade sarcoma and died of disease ( Fig. 4D-F). Another case of high-grade adenosarcoma (MA23, Fig. 4G-I) harbored an ATRX homozygous deletion and the tumor also displayed foci of low-grade adenosarcoma, compatible with a low-grade origin. Immunohistochemical analysis confirmed loss of ATRX expression in evaluable tumors from all 3 patients (primary low-grade tumor for MA02, recurrent high-grade tumor for MA08, both low-grade and high-grade components for MA23). In MA01 and MA24, which harbored an ATRX VUS, immunohistochemical staining revealed intact ATRX expression.
Of the four adenosarcomas with DICER1 mutations, three showed rhabdomyosarcomatous differentiation: one (MA16) showed extensive sarcomatous overgrowth by undifferentiated sarcoma, focally admixed with pleomorphic rhabdomyoblasts, while 2 (MA17 and MA25) displayed features of embryonal rhabdomysarcoma. For the latter 2 cases, the presence of focal areas with Phyllodes architecture, peri-glandular cuffing by stromal cells with low-grade fibroblastoid morphology were consistent with origin of the rhabdomysarcomatous component from a pre-existing adenosarcoma (Fig. 1G-I). Of note, rhabdomyosarcomatous elements were also observed in three adenosarcomas without DICER1 mutations and consisted of large, pleomorphic rhabdomyoblasts in areas of sarcomatous overgrowth (Fig. 1C, D). MA07, with the ESR1-NCOA3 fusion, was a 0.5 cm endometrialbased tumor with superficial (1 mm) myometrial invasion, which exhibited typical morphologic features of low-grade adenosarcoma (Fig. 1B), with mitotic activity reaching up to 3 per 10 highpowered fields. With exception of the fusion, no other somatic mutations, copy number alterations or structural variants were found in this tumor. Of note, ESR1-NCOA2/3 fusions have previously been reported in uterine tumors resembling ovarian sex cord tumor (UTROSCTs) 20 , however, we did not observe evidence of sex-cord differentiation in this case.
Given the frequent occurrence of endometrial glandular hyperplasia in adenosarcoma, we performed immunohistochemical analysis on cases with PTEN, ARID1A, and MLH1 genetic alterations to determine whether loss of expression was seen in the neoplastic mesenchymal component or the benign/hyperplastic glandular component (Fig. 5A-F). For cases harboring PTEN mutations, loss of PTEN expression was restricted to benign proliferative glands only in MA01 but was observed in the mesenchymal component in MA12 and MA22. For cases with ARID1A mutations detected by sequencing, loss of expression was detected in atypical hyperplasia only in MA27, with retained expression in the mesenchymal component. MA12 also showed retained expression in the mesenchymal component, which comprised the entirety of the sample submitted for sequencing. MA10 harbored a homozygous MLH1 deletion, and showed loss of MLH1 and PMS2 expression, confined to the stromal component, and to our knowledge, is the first reported case of a mismatch repair protein-deficient adenosarcoma. MLH1 promoter hypermethylation was negative. MSI analysis, however, demonstrated the tumor to be microsatellite stable, suggesting that MLH1 deletion may have occurred late in tumor progression and probably not a significant pathogenic driver. This tumor also harbored a concomitant BAP1 deletion.
BAP1 loss in Mullerian adenosarcomas and other mesenchymal neoplasms of gynecologic tract BAP1 homozygous deletion was identified in four adenosarcomas (high-grade, n = 3, and low-grade, n = 1; Fig. 6A, B). Immunohistochemical analysis of BAP1 in 24 adenosarcomas with available tissue confirmed loss of protein expression in 6 cases (25%), including all 4 tumors with BAP1 deletions, and 2 tumors lacking BAP1 genetic alterations (MA25 and MA14, with focal weak retained expression in the latter). MA08, which harbored an I675F VUS, showed retained expression, and hence this likely represents a non-pathogenic passenger mutation.

DISCUSSION
Mullerian adenosarcomas have a heterogeneous genomic landscape. Despite the lack of a pathognomonic molecular feature 5,9,13 , recurrent genetic alterations have been identified. Many of these are commonly mutated cancer genes that are not specific to adenosarcoma, including PI3K and MAPK pathway gene alterations, TP53 mutations, and MDM2/CDK4 amplification. As adenosarcomas are uncommon, study cohorts are generally small (less than 30 cases). Therefore, multiple studies of independent cohorts are needed to comprehensively characterize the spectrum and frequencies of genetic alterations in this disease. Our present study confirms prior findings, provides new insights on the molecular features distinguishing low-grade and high-grade adenosarcomas, and evidence supporting BAP1 deletion as a distinctive feature of a subset of adenosarcomas.
While sarcomatous overgrowth is well recognized as a poor prognostic feature in adenosarcoma, the clinical significance of histologic grading has not been addressed until relatively recently. Hodgson et al. demonstrated that adenosarcomas could be subdivided based on nuclear grade, independent of sarcomatous overgrowth, and that high-grade tumors have distinct clinical, morphologic and molecular characteristics 9 . In that study, highgrade morphology was associated with large tumor size, high mitotic index, sarcomatous overgrowth, and presence of TP53 mutations (observed in 6/9 cases). These tumors had aggressive clinical behavior, characterized by widespread metastasis and early recurrence, which was observed even in cases with a minor (<25%) high-grade component. In our cohort, almost all tumors with sarcomatous overgrowth were high-grade and heterologous elements were exclusive to high-grade adenosarcomas. All seven deaths were from high-grade disease: 2 of these were associated with exclusively low-grade adenosarcoma at initial presentation, and 1 had only a focal high-grade component.
We observed a few characteristic molecular features of highgrade adenosarcomas. Aside from TP53 mutations (n = 4), MDM2 amplification (n = 2) may serve as an alternative mechanism to cause p53 inactivation. CCNE1 amplification (n = 2) is known to induce chromosome instability, through centrosome amplification and chromosome missegregation 21 .
The significantly higher FGA in high-grade compared to lowgrade adenosarcomas, is consistent with chromosomal instability being a characteristic feature of high-grade tumors. Indeed, this is in keeping with the marked nuclear pleomorphism, and is analogous to other high-grade TP53-mutated tumors, such as high-grade serous carcinomas or uterine leiomyosarcomas. Interestingly, the only low-grade adenosarcoma with high FGA subsequently recurred as an overtly high-grade sarcoma. While total mutation counts did not vary significantly between lowgrade and high-grade tumors, it is notable that a case displaying nuclear irregularities and increased mitotic activity, but lacking pleomorphism, had a particularly high number of mutations.   6 BAP1 homozygous deletion is a distinctive feature of a subset of Mullerian adenosarcomas. A, B Representative adenosarcomas with BAP1 homozygous deletions: A MA10, which also harbors an MLH1 deletion (see Fig. 5 for MLH1/PMS2 immunohistochemistry) and B MA11. For each case, log2 copy number ratio plots across the whole genome (top) and chromosome 3, containing the BAP1 locus at 3p21 (middle, right), are presented. Estimated copy number plots for chromosome 3 (middle, left), with total (black line) and minor allele copy number (red line), demonstrate focal homozygous deletion of BAP1. Immunohistochemical staining for BAP1 (bottom) shows loss of expression in the neoplastic mesenchymal component. C BAP1 copy number alterations across the spectrum of gynecologic mesenchymal neoplasms.