Introduction

Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm composed of myofibroblastic and fibroblastic spindle cells with a background of mixed inflammatory cells [1]. It is extremely rare in the female genital tract [1,2,3,4]. Similar to IMTs in other anatomic sites, most IMTs in the uterus demonstrate indolent behavior, while a subset of uterine IMT with aggressive biological behavior has been described [2, 5, 6]. Although smooth muscle and endometrial stromal tumors are most common uterine mesenchymal neoplasms, morphologic and immunophenotypic overlaps between them and IMTs lead to diagnostic challenge. Approximately half of all IMTs harbor fusions of the anaplastic lymphoma kinase (ALK) gene at 2p23 [7] which may be susceptible to targeted therapy. Recently in IMTs, translocations of genes encoding other tyrosine kinases including ROS1, NTRK3, PDGFRb, and RET may also be therapeutically targetable [3, 7,8,9,10]. Therefore, identification of uterine IMTs demonstrates clinical importance as patients with IMTs may benefit from targeted therapy.

Here we present a series of 4 uterine mesenchymal tumor cases with aggressive behavior, initially diagnosed as leiomyosarcoma, demonstrating ALK or ROS1 rearrangements. Besides TNS1-ALK gene fusion, novel ROS1-NUDCD3 and NRP2-ALK fusions were identified by next-generation sequencing (NGS). Finding of specific gene rearrangement suggested uterine IMTs may be more common than expected.

Materials and methods

Four consulting cases of primary uterine IMTs were acquired from the achieves of Department of Pathology, Zhongshan Hospital, Fudan University. All four patients received surgery resection as the primary treatment in other hospitals and came to our hospital for further treatment recommendations. Hematoxylin and eosin (H&E)-stained slides and immunohistochemical (IHC) stains of the primary tumors were available for all cases, and tissue from the recurrent tumors was available in 3 cases. H&E-stained sections and immunohistochemistry of all cases were reviewed by two pathologists (Lili Zhang and Lijuan Luan) to confirm the diagnosis. Details of treatment and follow-up were obtained from the institutional medical records or from contributing pathologists.

Immunochemistry

IHC staining was performed on a Dako or VENTANA Autostainer following the manufacturer’s instructions. Primary antibodies are listed as follows: smooth muscle actin (clone 1A4, 1:100 dilution, Thermo), desmin (D33, ready-to-use, Dako), CD10 (56C6, 1:300 dilution, Leica Biosystems), ALK (D5F3, ready-to-use, Roche), ER (6F11, 1:80 dilution, Leica Biosystems), PR (16, 1:100, Leica Biosystems), cyclin D1 (EPR2241, 1:100 dilution, Abcam), ROS1 (D4D6, 1:250 dilution, Cell Signaling Technology).

Fluorescence in situ hybridization (FISH)

FISH on interphase nuclei from paraffin-embedded 4-micron sections was performed applying commercial available break-apart probes (ALK (2p23) Probe, lot: F.01330–01; ROS1 (6q22) Probe, lot: F.01086; Ret (10q11) Probe, lot: F.01104–01; YWHAE (17p13) Probe, lot: F.01183–01; JAZF1 (7p15) Probe, lot: F.01352–01; BCOR (Xp11) Probe, lot: F.01220–01, LBP Medicine Science and Technology Company, LTD, Guangzhou, China) according to manufacturer’s instructions. In short, slides of tumor sections were deparaffinized, pretreated, and hybridized with denatured probes overnight, followed by post-hybridization washes and interphase nucleus counter staining with DAPI. Microscopic analysis was performed using a Leica DM6000B microscope (Leica Microsystems, Wetzlar, Germany) by Leica DFC310 FX imaging system. FISH images were processed by the LAS V4.5 (Leica Microsystems) software. For examination, at least 100 nonoverlapping tumor nuclei were analyzed. Gene rearrangement was considered if ≥ 15% of tumor nuclei demonstrated break-apart signals.

Next-generation sequencing

Primary tumor samples of two patients (cases 1 and 3) and recurrence tumor samples of 3 patients (Case 1, 2 and 4) were targeted-sequenced by the GeneseeqOne™ pancancer gene panel (425-cancer-relevant genes, Geneseeq Technology Inc.). Briefly, for the formalin-fixed paraffin-embedded samples, five 10-μm tumor slices were used for DNA extraction using the QIAamp DNA FFPE Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. DNA quality was assessed by spectrophotometry with absorbance at 230, 260, and 280 nm, and quantified by Qubit 2.0. Libraries were prepared by 1 μg of fragmented genomic DNA underwent end-repairing, A-tailing and ligation with indexed adapters sequentially, followed by size selection using Agencourt AMPure XP beads (Beckman Coulter). Hybridization-based target enrichment was carried out with GeneseeqOne™ pancancer gene panel (425-cancer-relevant genes, Geneseeq Technology Inc.), and xGen Lockdown Hybridization and Wash Reagents Kit (Integrated DNA Technologies). Captured libraries by Dynabeads M-270 (Life Technologies) were amplified in KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and quantified by qPCR using the KAPA Library Quantification kit (KAPA Biosystems). Target enriched libraries were sequenced on the HiSeq4000 platform (Illumina) with 2 × 150 bp pair-end reads. Sequencing data were demultiplexed by bcl2fastq (v2.19), analyzed by Trimmomatic to remove low-quality (quality < 15) or N bases, and mapped to the reference hg19 genome (Human Genome version 19) using the Burrows-Wheeler Aligner. PCR duplicates were removed by Picard (available at: https://broadinstitute.github.io/picard/). The Genome Analysis Toolkit (GATK) was used to perform local realignments around indels and base quality reassurance. SNPs and indels were called by VarScan2 and HaplotypeCaller/UnifiedGenotyper in GATK, with the mutant allele frequency (MAF) cutoff as 0.5% for tissue samples and a minimum of three unique mutant reads. Gene fusions were identified by FACTERA and copy number variations (CNVs) were analyzed with ADTEx.

Results

Main clinicopathological parameters were summarized in Table 1. Patient age was 53, 51, 64, and 44 years respectively. Two patients (cases 1 and 3) presented with abnormal painless vaginal bleeding. In three cases, the primary tumors located in the uterus body. Cases 1 and 2 demonstrated multiple masses with the largest lesion 54 mm and 88 mm in diameter, respectively. Histologically, in case 1, case 2, and case 4, tumor cells were spindled containing eosinophilic cytoplasm and elongated nuclei in fascicular arrangement with mild to moderate nuclear atypia. In case 3, nuclear atypia was moderate to severe with multinucleated and bizarre cells. Necrosis was identified in cases 1 and 3. Prominent inflammatory cell infiltration was present in one tumor (case 2), while scant inflammatory cells were found in the other three tumors (cases 1, 3, and 4). Immunochemically, smooth muscle markers (a-SMA, desmin) were positive in all four tumors; CD10 was diffusely positive in case 1, partially positive in cases 3 and 4 and negative in case 2; ALK was positive in three tumors (cases 2, 3, and 4), while negative in one tumor (case 1). FISH analysis for ALK rearrangements was performed in all four cases and was positive in three tumors (cases 2, 3, and 4). ROS1 rearrangement was identified in case 1. NGS results demonstrated two novel fusions including NUDCD3-ROS1 (NUDCD3: exon2-ROS1: exon36) fusion (case 1) and NRP2-ALK (NRP2: exon8-ALK: exon19) fusion (case 2); and previously reported TNS1-ALK fusion (TNS1: exon16-ALK:exon18) fusion (case 3) and TNS1: exon19-ALK: exon12 (case 4). With regard to treatment, four patients received surgery as primary treatment and following adjuvant treatments: chemotherapy (3 patients), chemotherapy and targeted therapy (case 1), targeted therapy (case 4), and reoperation (cases 1, 2, and 4). Recurrences occurred in all 4 patients, and time to recurrence ranged from 1 month to 7 years. Follow-up ranged from 5 months to 15 years. Two patients were alive with disease (cases 1 and 3), and the other 2 patients died of disease progression (cases 2 and 4).

Table 1 Clinicopathologic features of uterine IMTs
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Clinicopathologic details of each case are presented as follows.

Case presentations

Case 1

A 53-year-old female patient went to hospital for abnormal menstruation in February 2020. Ultrasound showed multiple heterogeneous hypoechoic masses in the wall of uterus, among which the largest was 54 × 41 mm in size. She was admitted to hospital with abnormal vaginal bleeding for 17 days and anemia in July 2020. Gynecological examination found uterus enlargement as 3 months pregnancy. She underwent laparoscopic total hysterectomy combined with bilateral adnexectomy, pelvic lymphadenectomy, and pelvic adhesiolysis.

Gross examination revealed multiple masses in the uterus wall. The largest mass was 8 cm in diameter, poorly circumscribed, gray-white with partial necrosis, and fish-flush cut surface. The initial pathological diagnose was uterine leiomyosarcoma.

In August 2020, the patient accepted 4 cycles of chemotherapy (gemcitabine + docetaxel). Five months after surgery in December 2020, FDG-PET/CT showed metastatic nodules in liver surface, pelvic omentum, and mesentery. The patient was referred to our hospital and treated by palliative chemotherapy (dacarbazine + domesol + arotinib) since Jan 2021. After 2 months’ treatment, the patient achieved partial regression (PR).

During pathological consultation, histology of the primary tumor was under re-evaluation. Generally, the tumor showed infiltrative margins, composed of fascicular spindle cells with hypercellular area alternating with hypocellular region (Fig. 1A). Tumor cells were spindle with eosinophilic cytoplasm and elongated nuclei. Nuclear atypia was mild to moderate, while in some areas multinucleated cells and necrosis was observed. Mitoses count was approximately 10/10 HPF (Fig. 1B). Infiltration of inflammatory cells was not prominent. Immunochemically, tumor cells were positive for smooth muscle markers, including a-SMA and desmin (Fig. 2A), as well as CD10, ER, and PR. They were negative for ALK, ROS1, and CyclinD1. For differential diagnosis, FISH analysis was used to evaluate ALK, ROS1, JAZF1, YWHAE, and BCOR. FISH results demonstrated the tumor was positive for ROS1 rearrangement in 40% tumor cell, and negative for the other 4 genes (Fig. 3A). Moreover, subsequent NGS analysis revealed a NUDCD3-ROS1 (NUDCD3: exon2-ROS1: exon36, AF 21.40%) fusion (Fig. 4A). The diagnosis of IMT was established.

Fig. 1
figure 1

Tumor cells are spindled containing eosinophilic cytoplasm and elongated nuclei, which is composed of fascicular spindled cells with hypercellular area alternating with hypocellular region (× 4) (case 1; A). Hypercellular region with mitoses and multinucleated cells at × 400 (case 1; B). Spindled tumor cells in fascicular arrangement, while inflammatory cell infiltration is present (× 200) (case 2; C). Spindled and epithelioid tumor cells (× 100) (case 3; D). Bizarre and multinucleated cells with mitoses (× 400) (case 3; E). Infiltrative tumors showed percolation of tumor cells into the myometrium (× 100) (case 4; F)

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Fig. 2
figure 2

Tumor cells positive for desmin (case 1; A). Tumors showing a granular cytoplasmic and perinuclear ALK staining pattern (case 2; B, case 3; C, and case 4; D)

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Fig. 3
figure 3

Positive results detected by break-apart FISH showing ROS1 (case 1; A) and ALK rearrangement (case 2; B and case 3; C). MDM2 amplification of case 4 (D)

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Fig. 4
figure 4

NGS showing NUDCD3-ROS1 fusion (NUDCD3: exon2-ROS1:exon36, AF 21.4%) (case 1; A), NRP2-ALK fusion (NRP2:exon8-ALK:exon19, AF 64.1%) (case 2; B), TNS1-ALK fusion (TNS1:exon16-ALK:exon18, AF 30.36%) (case 3; C), and TNS1-ALK fusion (TNS1: exon19-ALK: exon 12, AF 86.29) (case 4; D)

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The patient continued chemotherapy (dacarbazine + domesol + arotinib) until May 2021. In June 2021, the patient achieved continuous partial remission (PR). Considering ROS1 gene fusion as a target, the patient was given crizotinib for maintenance treatment. After 25 days on crizotinib, CT scan indicated progressive disease. She switched back to chemotherapy (dacarbazine + domesol + arotinib). On July 27th, she developed further progressive disease and a post-line regimen of arotinib + eribulin + Keytruda was introduced. Chemotherapy was paused due to increase of creatinine. In September 2021, pelvic lesion resection combined with right hemicolectomy, partial rectal resection, descending colostomy, vaginal stump formation, and complex intestinal adhesion lysis were performed. The patient was alive with disease at last follow-up (17 months after the first operation).

Case 2

A 51-year-old female was admitted to hospital in June 2021 due to failure of stool and gas pass for 1 week, accompanied by lower abdominal pain, abdominal distension without chills, and high fever. PET-CT examination showed multiple low-density foci in pelvic and peritoneal cavity with elevated FDG, considering metastasis as well as multiple nodules in both lungs with partial increase of FDG. She had a history of “uterine smooth muscle sarcoma” and underwent total hysterectomy combined with double adnexectomy in September 2020.

To relief intestinal obstruction, excision of pelvic and abdominal lesions, partial resection of small intestine, and enterostomy were performed on June 24th 2021. Gross examination revealed multiple masses with fish-flesh cut surface, which was prone to bleed. Histologically, tumor cells were spindled with eosinophilic cytoplasm and elongated nuclei in fascicular arrangement (Fig. 1C). Nuclear atypia was mild to moderate. Mitoses were observed (9/10 HPF). Inflammatory cell infiltration was seen in some areas of stroma (Fig. 1C). IHC showed positive expression of SMA and desmin while negative for CD10. Tumor cells were strongly and diffusely positive for ALK protein (Fig. 2B). FISH analysis revealed 60% of tumor cells with isolated red signal (Fig. 3B). NGS analysis identified a NRP2-ALK fusion (NRP2: exon8-ALK: exon19, AF 64.11%) (Fig. 4B). After consultation, the initial diagnosis of smooth muscle sarcoma was corrected to IMT. The patient died of disease progression in September 2021 after adjuvant chemotherapy (specific drug unknown).

Case 3

A 64-year-old female presented with painless vaginal bleeding for a month. She went to a local hospital in July 2021, and ultrasound analysis showed multiple uterine intramural myoma with the largest 88 × 74 × 88 mm in size. The patient has a 20 years history of uterine fibroids. She underwent laparoscopic total hysterectomy with bilateral adnexectomy on September 22nd 2021 in the local hospital. Grossly, the uterus was broken with necrosis, which was difficult to measure. Initial diagnosis favored leiomyosarcoma. On Oct 27th 2021, PET-CT scan revealed right upper lung metastasis. The patient was referred to our hospital and treated by palliative chemotherapy (dacarbazine + epirubicin).

Sections of the primary tumor were sent to our hospital for pathological consultation. Histologically, tumor cells were densely composed of spindled and epithelioid cells with eosinophilic cytoplasm. Nuclear atypia was moderate to severe, while multinucleated and bizarre cells could easily be seen (Fig. 1D). Mitoses were 5–6/10 HPF (Fig. 1E). Inflammatory cells were scant. Immunochemically, tumor cells expressed smooth muscle markers including SMA and desmin. ALK staining was diffusely positive (Fig. 2C). ALK FISH analysis confirmed ALK translocation in 70% of the tumor cells (Fig. 3C). NGS analysis showed TNS1-ALK fusion (TNS1: exon16-ALK: exon18, AF 30.36%) (Fig. 4C). Therefore, the initial diagnosis was corrected to uterine IMT.

The patient achieved partial response (PR) after 4 cycles of chemotherapy. She was alive with disease at last follow-up (5 months after the first operation).

Case 4

A 59-year-old female was admitted to hospital in March 2018 because of retroperitoneal tumor for 1 year with pain of both lower limbs for a month. The lesion was diagnosed as smooth muscle tumor by biopsy in another hospital. She had a history of “uterine myoma” in November 2003, and “smooth muscle tumor of undetermined significance” of the right ovary in October 2010. The patient received apatinib treatment from May 2017 and had disease progression after 8 months.

In pathological consultation, the slides of uterine and ovarian tumor were reviewed. Histologically, tumor cells in the uterus were spindled in fascicular arrangement with mild nuclear atypia, while mitosis was scarcely observed (Fig. 1F). In the meanwhile, ovarian tumor was more cellular with mild to moderate nuclear atypia and brisker mitosis (12/50 HPF). Both uterine and ovarian tumor cells expressed SMA, desmin, and CD10, as well as ALK (Fig. 2D). ALK translocation was confirmed by FISH analysis. The diagnosis of the uterine and ovarian tumors was corrected to IMT.

In order to relieve compression of the retroperitoneal tumor, the patient underwent retroperitoneal lesion resection in February 2018. Gross examination revealed one enveloped gray and brown tumor with fish-flesh cut surface, size 15 × 14 × 4 cm. Grayish yellow necrosis was seen in some areas. Histologically, tumor cells showed similar features to primary uterine tumor but with more severe nuclear atypia and brisk mitosis (38/50 HPF). IHC results revealed SMA, desmin, CD10, and ALK expression. ALK translocation in 80% of the tumor cells and MDM2 amplification were confirmed by FISH (Fig. 3D). NGS analysis identified TNS1-ALK fusion (TNS1: exon19-ALK: exon12, AF 86.29%) (Fig. 4D). After 5 months after surgery, CT scan follow-up showed multiple recurrence lesions in pelvic and peritoneal cavity, along with left lung metastasis. The patient received crizotinib targeted therapy since then. After 2 months of crizotinib treatment, she developed further progressive disease and was treated with alectinib instead. The patient stayed SD (stable disease) for 4 months with alectinib treatment. She died of disease progression in September 2019.

Mutation profiles

We investigated the genetic profiles of all four cases, including post-crizotinib (RC) samples of two cases (case 1 and case 4). Top 30 gene mutations and somatic copy number variants of four patients were shown in Fig. 5A. Cell cycle and p53 signaling pathway related gene aberrations (TP53, MYC, MDM2, CDK4, CDKN2A) were identified in all the 4 cases. In case 1, mutations including hotspot TP53(p.E258K), TP53(p. L252del), ATRX(p.M1556Nfs*22), and NUDCD3:exon2-ROS1:exon36 were identified both tumors before and post crizotinib treatment. However, MYC copy number gain was found only in tumor before treatment, Moreover, THADA gene fusion and other mutations were only found in the recurrent tumors after crizotinib treatment (Fig. 5A case 1, RC1-4). In case 4, CDK4(AMP), MDM2(AMP), CDKN2A(DEL) together with TNS1:exon19 ~ ALK:exon12 were identified in tumors before and post crizotinib treatment. Hotspot TP53 (p.C238Y) was identified in case 3, and IGR (upstream DMRTA1)-CDKN2A:exon2 was found in case 4. Generally, besides ALK translocation, the 4 cases shared limited gene aberrations. The multiple gene alterations in the 4 cases indicated that the IMT was a heterogenous entity. To understand the biological importance of the mutations, we performed the KEGG pathway analyses of mutated genes of 4 patients. We found that despite of diversity in the altered genes, these aberrations mainly involve several enriched pathways include the PI3K-Akt signaling pathway, MAPK signaling pathway, and RAS signaling pathway as shown in Fig. 5B.

Fig. 5
figure 5

Genetic alteration and somatic copy number variants of uterine IMTs from primary tumor (PT), recurrences (R), and recurrences after crizotinib (RC) (A). KEGG pathway analyses of mutated genes of 4 patients with inflammatory myofibroblastic tumor (B)

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Discussion

In this article, we presented 4 uterine IMTs with novel gene fusions. IMT is a mesenchymal neoplasm of intermediate biologic potential, which occurs mostly in the lung and abdomen cavity [1]. Histologically, it is characterized as a neoplasm composed of myofibroblastic and fibroblastic spindle cells with a background of mixed inflammatory cells [1].

IMTs rarely arise from the uterus or cervix. Less than 150 uterine IMTs were reported in the English literatures [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39]. Clinicopathologic features of these cases were summarized in Table 2. The age at presentation of the reported cases ranged from 3.5 to 78 years, with median age of 39 years old. About 70% (96/126) of the cases are arising from the uterus body; 15% (19/126) cases affect cervix. There were 33 IMT cases associated with the placenta during pregnancy [5, 8, 9, 12, 14, 20, 26]. Uterine IMTs can be single or multiple mass. Tumor size ranged from 5 to 200 mm (median, 45 mm). The margins of tumors are described in 77 cases, from which about half of the tumors are well circumscribed (37/77), the others are infiltrated (28/77), irregular (9/77), and pushing (3/77).

Table 2 Clinical, histologic, and molecular features of uterine IMTs reported in the literature
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Histologically, IMTs are typically consisted of fibroblastic/myofibroblastic spindle cells in a myxoid background which is accompanied by inflammatory infiltrates, including lymphocytes, plasma cells, foamy histiocytes, neutrophils, and eosinophils. There are three histologic patterns described in the literature: (1) a hypocellular/loose myxoid pattern; (2) a hypercellular/compact pattern, characterized by cellular areas arranged in fascicular or storiform architecture; and (3) a hyalinized pattern, composed of loosely arranged tumor cells in an abundant collagenous matrix. Nuclear atypia is at most mild, and the mitotic index is usually low (mostly less than 5/10 HPF). Three of our cases have features consistent with the pattern 2, while the fourth case was densely composed of spindled and epithelioid cells with multinucleated and bizarre cells. Nuclear atypia in our cases was moderate to severe. Only one case showed prominent inflammatory cells, making it difficult to make a differential diagnosis with smooth muscle tumors.

Immunochemically, IMTs variably express SMA, actin, desmin, and CD10. Usually the extent and density of smooth muscle markers is not as strong as smooth muscle tumors. In this study, all the 4 cases showed strong and diffuse IHC staining of SMA and desmin. These IHC findings and histological features were the main reasons leading to the misdiagnosis of leiomyosarcoma. Studies showed that the expression of smooth muscle makers in uterine IMT was inversely correlated to the degree of myxoid stroma [14]. Tumors with more myxoid stroma are less likely to show smooth muscle expression compared with more cellular areas with less myxoid stroma.

In the literatures, ALK protein expression is observed in about 87% (103/119) of uterine IMTs, However, one ALK IHC negative case was identified ALK rearrangement by FISH [2]. Several gene fusion partners for ALK have been reported in uterine IMTs (Table 3) including TIPM3(16), THBS1(12), IGFBP5(10), FN1(3), TNS1(5), DES (3), DCTN1(2), and RANBP2(1) [3,4,5, 9, 11, 12, 14,15,16, 18, 23]. Recently, although rare, alternative translocations including ROS1 (2 cases, TIMP3-ROS1, FN1-ROS1), RET (2 cases, TIMP3-RET, SORBS1-RET), IGFBP5-PDGFRB (1 case), and NTRK3 (1 case, NTRK3-ETV6) have been identified in a subset of ALK-negative uterine IMT tumors [8,9,10, 39]. In the present study, novel NUDCD3-ROS1 (case 1) and NRP2-ALK (case 2) fusions were identified for the first time. Beside ALK and ROS1 gene status, we further investigated the genetic profiles of the 4 IMTs by NGS. We also compared the changes of mutations and copy number variation before and after crizotinib treatment. Notably, TP53 mutations including a hotspot mutation (p.E258K) and MYC copy number gain were identified in case 1. However, patients harboring ROS1 fusion with concomitant TP53 mutations or MYC overexpression may have influence on tyrosine kinase inhibitor (TKI) resistance in lung cancer [40, 41]. Moreover, it is reported in lung cancer that MDM2 amplification plays an important role in TKI resistance [42], which may explain the failure of crizotinib treatment in case 4.

Table 3 Summary of reported gene rearrangements in uterine IMTs
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IMTs are considered to have intermediate biologic potential with a local recurrence and distant metastasis rate of about 25% [1]. In the reported uterine IMTs, most cases showed indolent behaviors without follow-up evidence of recurrence or metastasis. However, cases with aggressive behaviors in the uterus IMTs have been described [2, 5, 6, 15, 16, 19,20,21,22,23, 37]; the recurrence and metastasis rate is about 29% (22/77). Histologically, approximately 73% (16/22) aggressive tumors had infiltrative margins. Necrosis was more frequently seen in aggressive IMTs (10/22) than in indolent tumors [5, 6]. Aggressive biological behaviors may be related to tumor size, higher grade of atypia, and mitoses index [2, 5, 6]. In our study, all the four cases show aggressive behavior. Necrosis are seen in two cases, while mitoses index are relatively high in all four cases (> 5/10 HPF).

To better understand the underlying mechanism, we performed NGS analysis to the primary tumors of all the 4 cases and recurrent tumors from 2 cases. We identified different alterations in multiple genes. These findings supported IMT as a heterogenous entity. Despite of the diversity of gene mutations, several enriched pathways which involved in the IMTs include the PI3K-Akt signaling pathway, MAPK signaling pathway, and RAS signaling pathway were confirmed [43]. Furthermore, we discovered TP53 mutations, CDKN2A structure variation, and MDM2 amplification in the 4 cases. These alterations may lead to activation of P53 signaling pathway which may play important roles in the adverse outcome of these uterine IMTs. Theses finding provided new insights to the understanding of this rare entity and the underlying mechanisms are to be further elucidated.

The main differential diagnosis of uterine IMTs is smooth muscle tumors due to morphologic and immunophenotypic overlap between the two entities. Parra-Herran et al. [22] reported 4 cases out of 30 cases previously diagnosed as uterine smooth muscle tumor to be IMTs with positive ALK IHC or FISH results. In the current cases, histologically, all the cases were indistinguishable from smooth muscle tumors due to lack of inflammatory cell infiltration, and were positive for smooth muscle markers such including SMA and desmin; therefore, all of them were initially diagnosed as smooth muscle tumors. The final diagnosis was established after the confirmation of ROS1 and ALK gene rearrangement. Thus, distinguishing uterus IMTs from smooth muscle tumors can be quite challenging especially for ALK negative IMTs. Ancillary modalities including IHC, FISH, and NGS may help to make the correct diagnosis and find gene rearrangement potentially for targeted therapy.

In conclusion, we presented 4 rare uterus IMTs with malignant biological behaviors and identified novel NUDCD3-ROS1 and NRP2-ALK fusions. All these cases were misdiagnosed as smooth muscle tumors at beginning and correct diagnosis was established after ALK and ROS analysis in consultation, indicating great challenges in the pathology diagnosis of this rare entity. It might be reasonable to include ALK in the IHC panel for differential diagnosis of uterus mesenchymal tumors in routine practice. NGS analysis revealed certain gene alterations involved in the P53 pathway which may contributed to the malignant behaviors. These findings shed new lights on the understating of this rare entity, and further mechanism studies are expected.