Foreword

Uterine sarcoma is a malignant mesenchymal tissue-derived tumor, which accounts for about 1% of all female reproductive tract malignant tumors and 3%–7% of uterine body malignant tumors ^[1]. Uterine sarcomas often lack specific clinical manifestations, and the etiology remains unclear. The overall prognosis is poor. The five-year survival rate in stage I-II is 45–50%, while it plummets to 0–15% in the late stage ^[2]. Uterine sarcomas have complex tissue types. The most common type is Leiomyosarcoma of uterus (uLMS) (40%–50%), followed by low-grade endometrial stromal sarcoma (LGESS)^[1], and there are more than ten other rare subtypes. With the advancement of molecular pathology, gene detection has also been applied to the evaluation of uterine sarcoma ^[1], which is of great significance in diagnosis, prognosis and treatment.

Diagnosis and classification of uterine sarcoma

Uterine sarcomas often lack specific clinical manifestations. Patients mainly present with abnormal vaginal bleeding, pelvic masses, pelvic pain, and the etiology is still unclear. Ultrasonography, CT, MRI or PET/CT make it difficult to differentiate benign and malignant tumors preoperatively. Many patients are often diagnosed with benign uterine diseases at doctor's visit and uterine sarcoma is confirmed only through postoperative pathological examination. Diagnostic curettage or endometrial biopsy is helpful for the diagnosis of partial endometrial stromal sarcoma (ESS), but it is less sensitive and needs the help of pathological diagnosis (morphology, immunohistochemistry and molecular diagnosis) ^[1].The 5th edition (2020) of WHO Tumor Classification also classifies the benign and malignant female reproductive tumors and their molecular features ^[3].

WHO classification of uterine sarcoma ^[3]

Molecular mechanisms of uterine sarcoma

uLMS is the most common subtype of uterine sarcoma, accounting for 40%–50% of uterine sarcomas and 1%–2% of all malignant tumors of the uterine body. The symptoms and signs of patients are often similar to those of uterine leiomyomas, and it is difficult to distinguish them preoperatively ^[1]. The ratio of uterine leiomyoma to uLMS is about 800:1^[1], and histological diagnosis alone lacks a reliable prognosis. Molecular detection technology should be advocated for atypical cases ^[4]. The most common type of uLMS is the common type (spindle cell type) LMS, followed by epithelioid LMS, and the mucus type LMS is the rarest. The three are mainly distinguished by morphological characteristics and the number of mitotic figures ^[1].

A prospective study (NCT01775072) included 107 cases of uterine sarcoma, including 80 uLMS, 22 cases of high-grade non-leiomyosarcoma (HG non-LMS), 4 cases of LGESS, and 2 cases of smooth muscle tumor with undetermined malignant potential (SMTUMP). Tumor samples and matched blood samples were tested for MSK-IMPACT and MSK-Fusion. Function-deficient mutations in TP53(56%), RB1(51%), and ATRX(35%) were commonly found in uLMS. Homozygous deletions of BRCA2 occur in 5% of patients, more frequently than other cancers, and are limited to uLMS. The variation in PTEN is more common in metastatic samples than in primary samples (p=0.046)^[5].

Mutation map of uterine sarcoma ^[5]

A Yale University-led study involving 83 uLMS samples revealed hot spot mutations in TP53, MED12, and PTEN genes, with the most common somatic mutant genes including TP53, ATRX, PTEN, and MEN1. TP53 and ATRX mutations were associated with reduced survival. A variety of fusion forms have been detected, and ACTG2-ALK fusion has the potential of targeting. Copy number variation (CNV) analysis determined that 18.2%(12/66) of the samples had MYC amplification, with an additional 7 copies amplified and 29 copies lost. Twenty-five percent (12/48) of fresh frozen uLMS have homologous recombination repair defect (HRD) features ^[6]. In another study, which analyzed 525 samples from 509 patients by high-throughput sequencing (NGS), homologous recombination DNA damage repair (HR-DDR) gene was mutated in 28.2% of the samples, and the most common mutated genes were ATRX(18.2%), BRCA2(4%) and RAD51B(2.6%), with the highest mutation frequency in uLMS (35.4%) and the lowest in LGESS (2.9%)^[7].

TP53 (left) and ATRX (right) mutations have been associated with reduced overall survival (OS) ^[6]

ESS are rare and include the following two types, LGESS and HGESS. LGESS with an incidence of less than 1% of all uterine malignancies, is the second most common mesenchymal malignancy after uLMS. High-grade endometrial stromal sarcoma (HGESS) is an extremely rare and highly malignant tumor ^[1]. About two-thirds of LGESS have multiple gene fusion, with JAZF1-SUZ12 fusion most commonly occurring (> 50%) followed by JAZF1-PHF1, EPC1-PHF1, and MEAF6-PHF1 fusion, as well as MBTD1-CXorf67, BRD8-PHF1, EPC2-PHF1, and EPC1-SUZ12. Fluorescence in situ hybridization (FISH) or RNA targeting sequencing of LGESS-associated fusion can be used to confirm the diagnosis. The most common type of HGESS is YWHAENUTM2 A/B gene fusion, and the less common types are ZC3H7B-BCOR fusion or BCOR internal tandem repeat (ITD)^[8].

Common variations of subtypes of uterine sarcoma ^[8]

Targeted therapy for uterine sarcoma

In addition to adjuvant typing and prognosis, NCCN guidelines recommend that comprehensive genomic testing be used in metastatic patients to look for opportunities for pan-carcinomatous targeted therapy. For patients with recurrence, metastasis and inoperable diseases, TRK inhibitors can be used for NTRK gene fusion positive tumors, ALK inhibitors can be used for IMT of ALK translocation, and PARP inhibitors can be used for LMS of BRCA (mainly BRCA2) mutation ^[8].

In a prospective study (NCT01775072), potential operable mutations were identified in 48 patients (45%), of which 8 (17%) received matched therapy and 2 achieved clinical response. LBH-ALK fusion was detected in one patient who was treated with the ALK inhibitor crizotinib and was imagewise stable for more than 30 months. Sustained partial response to treatment with PARP inhibitors has been observed in uLMS patients with somatic BRCA2 variation ^[5].

Potential therapeutic markers of uterine sarcoma ^[5]

The research conducted by Yale University has revealed that Copanlisib(PI3K inhibitor), olapari (PARP inhibitor) and GS-626510(C-MYC/BET inhibitor) can inhibit cell proliferation in the human tumor tissue xenotransplantation model (PDX) of uLMS in vivo ^[6]. Currently, a Phase II clinical study (NCT03880019) of olapari combined with temozolomide in the treatment of advanced uLMS is ongoing, and preliminary data indicate an objective response rate (ORR) of 22.7% within six months after initiation of treatment ^[9,10]. There are also some cases reported that patients with metastatic uLMS with BRCA2 gene system mutation stable after treatment with olapari ^[9,11].

Detection method of uterine sarcoma

As the tissue types of uterine sarcomas are complex, and there is cross-expression between immunohistochemical markers of different types of uterine sarcomas, the specification for pathological diagnosis of uterine sarcomas indicates that molecular detection is recommended for conditional units, with FISH and NGS as the main detection methods. For some uterine sarcomas that failed to be classified in the past or new subtype classification, the molecular test results are required to support the diagnosis ^[12].

A total of 137 oncogenic fused transcripts of genes were detected in 158 samples based on link-dependent reverse transcriptase-polymerase chain reaction and targeted NGS (LD-RT-PCR-NGS), including 1 case of HGESS, 8 cases of LGESS, and 2 cases of PEComa. Compared with FISH and RT-PCR, the RNA-seq consensus rate reached 98.1%. Although simple and robust, FISH detection can only detect one fusion gene at a time. RT-PCR can screen multiple fusion genes simultaneously in one test, but its multiple detection ability is still limited. Sarcomas have diverse fusion variants, so multigenic combination therapy is needed to assist clinical prognosis evaluation of patients and guide clinical treatment plan ^[13].

Another retrospective study included 42 patients with morphologically diagnosed ESS who were negative for JAZF1 rearrangement (n=22), YWHAE rearrangement (n=11), or both (n=9) by FISH. Chromosome fusion was found in 31 (74%)FISH-negative samples using RNA-seq. RNA-seq reclassified 12 cases (29%), with 7 cases of LGESS reclassified as HGESS(n=2), malignant epithelioid tumor with GLI1 rearrangement (n=2), NCOA fusion-positive uterine tumor (n=2) or uterine myoma (n=1), 2 cases of HGESS reclassified as undifferentiated uterine sarcoma (UUS), and 3 cases of UUS reclassified as HGESS, NTRK rearranged uterine sarcoma and SDUS. RNA-seq can reclassify uterine sarcoma and guide therapeutic strategies ^[14].

Expression spectrum clustering analysis and survival analysis ^[14]

Summary

The tissue types and molecular mechanisms of uterine sarcoma are complex, and NGS is one of the main detection methods. Because of the complex combination of gene mutation, CNV and gene fusion in uterine sarcomas, co-detection at both DNA and RNA levels is desirable. The difficulty in the detection of fusion genes for uterine sarcoma is the detection of unknown fusions. The known fusion genes may be located at the 5' end or the 3' end, and there are many fusion partners, and there may be multiple breakpoints for each fusion. Using one-sided primer reverse transcription method to detect gene fusion at RNA level can detect unknown fusion without designing multiple pairs of primers.

Xiamen SpaceGen Uterine Sarcoma Gene Detection Project detected 37 genes (CNV with 6 genes) at DNA level and 28 genes (129 known and unknown fusion types of common breakpoints) at RNA level. SpaceGen is committed to providing the most innovative products and services for the individualized and precise medical detection of tumors, and continuously updates the existing products.

References

[1] Uterine sarcoma diagnosis and treatment guidelines (2021 edition)

[2] Pathology. 2007 Feb; 39(1):55-71.

[3] Fifth edition WHO gynecologic reproductive system tumor classification

[4] Gynecol Oncol Rep. 2021 Nov 17:38:100890.

[5] Clin Cancer Res. 2020 Jul 15; 26(14):3881-3888.

[6] Proc Natl Acad Sci U S A. 2021 Apr 13; 118(15):e2025182118.

[7] Gynecol Oncol. 2023 Oct:177:14-19.

[8] NCCN Guidelines for Diagnosis and Treatment of Uterine Tumor 2024 v3

[9] Gynecol Oncol Rep. 2022 Jul 14:42:101044.

[10] ClinicalTrials database

[11] Oncologist. 2019 Jul; 24(7):973-979.

[12] Specification for pathological diagnosis of uterine sarcoma

[13] Mod Pathol. 2022 May; 35(5):649-663.

[14] Cancers (Basel). 2020 Sep 11; 12(9):2604.

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