Next-Generation Sequencing Analysis of 3 Uterine Adenosarcomas with Heterogeneously Differentiated Genomic Mutations

Uterine adenosarcoma (UA) is an uncommon mixed tumor containing a benign to at most mildly atypical epithelial component and a sarcoma-like stroma, usually a low-grade, stromal component, with rare heterogeneous elements. Currently, tumor etiology is largely unknown. To better understand the gene mutations in UA, next-generation sequencing (NGS) technology analysis was performed. This study showed that two low-grade UAs with heterologous components had ATRX gene frameshift mutation, and one patient had a MED12 missense mutation. Copy number amplification genes were mainly observed on chromosome 12q13–15. In this study, PIK3/AKT/PTEN pathway mutations were found to be common in adenosarcoma. In addition, a rare BCORL1-PRR14L fusion mutation was also identified. These findings provide a basis for future research into these molecular changes in tumorigenesis and targeted therapy.


Introduction
Uterine adenosarcoma (UA) is an uncommon mixed tumor containing a benign to at most mildly atypical epithelial component and a sarcoma-like stroma and accounts for approximately 5% of uterine sarcomas [1].Te epithelial component is Müllerian-derived, mainly endometrioid epithelium and a few visible tubal epithelium and squamous epithelium; the glandular epithelium may be accompanied by diferent atypical degrees.Te stroma is generally a low-grade uterine sarcoma, mostly endometrial stromal sarcoma [2], which may contain heterologous components, such as the skeletal muscle, cartilage, and smooth muscle, among others.However, adenosarcoma with heterologous components is rare [3], and the molecular mechanism is unclear.Two of the three UAs reported in this study had heterologous components, and the molecular changes were detected by next-generation sequencing (NGS) to understand the pathogenesis and identify diagnostic or prognostic biomarkers.

Case Selection.
Te specimen-related and existing clinical data of three patients with pathologically diagnosed UA treated in our hospital from 2017 to 2022 were obtained.

Next-Generation Sequencing (NGS)
. DNA was extracted from tissue samples based on the instructions in the QIA-GEN DNA FFPE extraction kit (QIAGEN, Valencia, CA, USA).Te quality control samples were arranged for the construction of the library.Te 481 gene probe was used for targeted enrichment of the DNA samples, and the target enrichment library was sequenced on the NovaSeq 6000 platform (Illumina).Finally, comprehensive information regarding genetic mutations, such as point mutation, insertion and deletion mutations, and gene copy number change, was obtained from the sequencing data using the genome analysis toolkit.

Clinicopathological Features.
Te clinicopathological characteristics of the patients are summarised in Table 1.Te age of the three patients ranged from 57 to 76 years (average, 67 years).Te sites of occurrence of the tumors were the uterine fundus, corpus, and cervix.Tree patients were admitted because of abnormal vaginal bleeding.All three patients underwent abdominal hysterectomy and bilateral adnexectomy.Te postoperative pathological macroscopic image revealed that the uterus was enlarged and single or multiple polypoid masses protruded into the uterine cavity or cervical canal.Te average diameter of the tumors was 6.9 cm (range, 2.8-14 cm).Te surface of the tumors was smooth, and the cut surface was fsh fesh-like, with grey red color and tender texture.Te tumors had a saccular shape with local bleeding and necrosis.Microscopically, the tumor tissue was composed of endometrioid glands and proliferative spindle cells.A large number of tumor cells were difused and distributed in a woven and cord-like arrangement, and local tumor cells were concentrated around the gland, forming a cuf structure around the gland (Figure 1).Fusiform nuclei with mild to moderate atypia and local mitotic images are easy to see (Figure 2).One case had heterogeneous diferentiation of tumor cells into chondrocytes (Figure 3), and another case had smooth muscle diferentiation.No defnite tumor thrombus was found in the vasculature.
Immunohistochemical examination showed positive expression of CK7 and EMA in glandular epithelium.CD10 and vimentin were positive in the interstitium; desmin, SMA, and PR were partially positive in one patient, and ER was partially positive in two patients.P53, inhibin, caldesmon, myogenin, and MyoD1 were negative in all three patients, and the positivity rate of Ki67 was 5%-15%.Combined with the histopathological and immunohistochemical fndings of the patients, adenosarcoma of the uterus was diagnosed.None of the patients received radiotherapy or chemotherapy after surgery.Te follow-up time ranged from 2 months to 36 months.One patient had vaginal recurrence after 17 months.

Discussion
UA, also known as Müllerian adenosarcoma [3], was defned by the World Health Organization as a mixed epithelial and mesenchymal tumor [4].UA occurs most frequently in the uterine body, followed by the cervix, and about 1/4 showed other heterologous interleafet components, with striated muscle diferentiation being the most common [5,6].In this study, it is rare that one patient had tumors in the uterine cavity and cervix simultaneously.Te age of onset of UA ranges from 10 to 94 years (median age, above 50 years) [7] and is more common in postmenopausal women.Overall, UA has a better prognosis than other uterine sarcomas and carcinosarcomas [8,9].However, Heveder et al. [10][11][12] suggested a 50% reduction in survival in UA patients with adverse prognostic factors.Although surgery is the major treatment [13], the therapeutic efect of chemotherapy in UA has been reported in the literature [14].Some commonalities were identifed between copy number variations (CNVs) and mutations.A study has shown that UA and its variants are genetically heterogeneous with frequent CNVs in SO [15].Meanwhile, MDM2, CDK4, HMGA2, and GLI1 gene amplifcations are common molecular events in Müllerian adenosarcoma, often seen in patients with SO [16].Consistently, in this study, gene amplifcation was seen in recurrent UAs and UAs with heterologous diferentiation without SO.
Furthermore, the MED12 gene is closely related to uterine leiomyoma [17].Te 70% of uterine leiomyomas have point mutations in the MED12 gene, and all relevant point mutations are concentrated in exon 2 [17].MED12 mutations alter the functioning of the MED12 protein, thus disrupting normal cell signalling and repair regulation of cell growth and other functions, resulting in uncontrolled cell growth and tumorigenesis [17].In contrast, this study has detected a missense mutation in exon 43 of the MED12 gene, p.Q2097L, resulting in the change of the 6290th base from A to T and the change of the 2097 th amino acid from glutamine to leucine.However, the clinical signifcance of this mutation is unclear.Nevertheless, if the protein functions abnormally, it may afect downstream signalling pathways and be involved in tumorigenesis and progression of cancer.
Moreover, ATRX (located on chromosome Xq21.1)encodes a chromatin remodelling protein which is thought to be important in regulating DNA methylation and telomere stability [18].Howitt et al. [16] reported that ATRX mutations were present in 50% of UAs with SO (including one case with distant metastasis), while ATRX mutations were not present in UAs without SO, suggesting that ATRX may be a poor prognostic feature of MA [19,20].Tis is inconsistent with fndings of this study, in which ATRX mutations were occurred in low-grade UAs with heterologous components but not with SO.Terefore, there requires further studies to confrm these fndings.
In addition, structural variation (SV) is relatively low in UAs.Tis study reported a fusion mutation in the BCORL1-PRR14L gene.Meanwhile, the diagnosis of BCOR overexpressing uterine sarcomas and high-grade endometrial mesenchymal sarcomas carrying these mutations is suggested by identifying ZC3H7B-BCOR fusion mutations or BCOR-ITD [21].However, the clinical signifcance of this mutation in UAs is currently unclear.If the protein functions abnormally, it may afect downstream signalling pathways involved in tumor development and progression.Analysis of these gene fusions may provide clues to further understanding of the genetic mechanisms involved in UA tumorigenesis.
PIK3/AKT/PTEN pathway mutations are most common in adenosarcoma [16].Te fndings of this study consistently indicated that targeting this pathway may be a potential therapeutic target in UA treatment.Moreover, surgical resection is the main treatment for UA, and hysterectomy with bilateral adnexectomy is the basic surgical method [22].Previous studies have reported that the recurrence rate of UA is 14.3-46%, and the local recurrence rate is higher than the distant recurrence rate [8,23].Te high-risk factors might afect the prognosis of adenosarcoma [24] and therefore individualized treatment should be considered.In terms of adjuvant therapy, it has been shown that adenosarcoma recurrence with or without sarcoma overgrowth responds to the treatment regimen of ifosfamide or doxorubicin [25].However, radiotherapy does not beneft the overall survival of patients, and there is insufcient evidence regarding the benefts of chemotherapy and hormone therapy [25].Further studies are needed to determine the most efective adjuvant therapy.

Conclusion
In conclusion, UA is a rare uterine sarcoma.Tis study showed that the ATRX gene was mutated in low-grade UA with a heterozygous component, possibly having important prognostic implications.Meanwhile, molecular evaluation of mutations in the BCOR and BCORL1 genes in the diagnosis of uterine sarcomas overexpressing BCOR is recommended to diferentiate high-grade endometrial mesenchymal sarcomas with BCOR fusions from rare adenosarcomas with BCORL1 gene rearrangements and BCORL1-PRR14L fusions, potentially broadening the genetic spectrum of adenosarcomas.Future studies should be conducted on a larger sample, along with a detailed study of the mutated genes.

Figure 1 :
Figure 1: Local tumor cells were concentrated around the gland, forming a cuf structure around the gland (H&E ×200).

Figure 3 :
Figure 3: Fusiform nuclei with mild to moderate atypia, and local mitotic images are easy to see (HE ×200).

Figure 4 :
Figure 4: Target validation of the gene mutation types and distribution of 27 genes in 3 UAs by next-generation sequencing (NGS).Rows represent individual genes and columns represent individual tumors.Mutated genes are sorted according to frequency in this cohort.Colors indicate the mutation type detected in each tumor.

Figure 5 :
Figure 5: Copy number variation data for chromosome 12 in CNV analysis, with the plot of copy number variation by chromosome (colorcoded).Te vertical axis is the ratio of the number of reads for this specimen versus a panel of normals in log base 2 scale.A value of 0 denotes no diference from normal (diploid).Te horizontal axis shows the chromosomes.

Figure 6 :
Figure 6: KEGG pathway analysis of oncogenes/tumor suppressor genes altered in UAS; the redder the circle color, the more signifcant the enrichment, and the larger the circle, the more enriched the genes.