IFITM1, CD10, SMA, and h-caldesmon as a helpful combination in differential diagnosis between endometrial stromal sarcoma and cellular leiomyoma

Background : The differential diagnosis of endometrial stromal sarcoma (ESS) and uterine cellular leiomyoma (CL) remains a challenge in clinical practice. Cluster of differentiation 10 (CD10) and smooth muscle actin (SMA) are commonly used in the differential diagnosis of ESS and CL . However, the current combination of immunohistochemical antibodies has been shown to be inaccurate, suggesting the need for novel immunomarkers panels for differentiating between ESS and CL. Interferon-induced transmembrane protein 1 (IFITM1) is a novel immunomarker for endometrial stromal cells, h-caldesmon is an immunomarker for smooth muscle cells and has a higher specificity than SMA. So this study aimed to investigate IFITM1, CD10, SMA, and h-caldesmon as a useful combination of biomarkers for diagnosing between ESS and CL. Methods : Tissue microarrays were used to detect IFITM1, CD10, SMA, and h-caldesmon immunohistochemical staining in 30 ESS and 33 CL cases. Results : The expressions of IFITM1 and CD10 were high in ESS (86.7% and 63.3%, respectively) but low in CL (18.2% and 21.2%), whereas those of h-caldesmon and SMA were high in both CL (87.9% and 100%) and low in ESS (6.9% and 40%). In diagnosing ESS, IFITM1 had better sensitivity and specificity (86.7% and 81.8%, respectively) than CD10 (63.3% and 78.8%). The specificity of h-caldesmon in diagnosing CL was significantly higher (93.1%) than that of SMA (60%). When all four antibodies were combined for the differential diagnosis, the area-under-the-curve predictive value was 0.995. The most sensitive and specific combinations for diagnosing ESS were IFITM1(+) or CD10(+) and h-caldesmon(-) ( sensitivity 86.7%, specificity 93.9%), IFITM1(+) and h-caldesmon(-) (sensitivity 80%, specificity 100%). The most sensitive and specific combinations for diagnosing CL were h-caldesmon(+) and SMA(+)(sensitivity 87.9%, specificity 100%), h-caldesmon(+) or SMA(+) and IFITM1(-)(sensitivity 81.8% , specificity 93.1%). Conclusions : Therefore, IFITM1, CD10, SMA, and h-caldesmon are a good combination of biomarkers for the differential diagnosis of ESS and CL.


Background
Endometrial stromal sarcoma (ESS) is a rare malignant mesenchymal tumor of the uterine. In 2014, the World Health Organization classified ESS as low-grade ESS, highgrade ESS, and undifferentiated endometrial sarcoma [1]. However, there is a overlap in morphology and immunohistochemistry between ESS and leiomyoma, especially for the low grade ESS from cellular leiomyoma (CL) . Currently, cluster of differentiation 10 (CD10) has been considered as the best immunomarker for endometrial stromal cells [2][3][4][5][6], but it not expressed in all mesenchymal tumors [7][8][9]. Rather, CD10 is sometimes expressed in leiomyoma [10,11]. Smooth muscle actin (SMA) is a common biomarker for smooth muscle, however, SMA is sometimes expressed in ESS [12][13][14][15], suggesting the need for novel immunomarkers and immunohistochemical panels for differentiating between ESS and CL.
Interferon-induced transmembrane protein 1 (IFITM1), also called CD225, is a novel immunomarker for endometrial stromal cells and tumors [16,17] and outperforms CD10 in distinguishing LG ESS from CL [18,19]. Meanwhile, h-caldesmon is an immunomarker for smooth muscle cells and has a higher specificity than smooth muscle actin (SMA). However, there has been no study on the combined use of IFITM1, CD10, SMA, and h-caldesmon in distinguishing between ESS and CL. This study aimed to investigate IFITM1, CD10, SMA, and h-caldesmon as a useful combination of biomarkers for the differential diagnosis of ESS and CL. years and mainly showed clinical manifestations of dysmenorrhea, prolonged menstrual period, and increased menstrual volume. All pertinent clinical information was obtained from the hospital electronic medical records. All patients had complete medical history and clinicopathologic data, and all cases were confirmed by surgery and pathology.

Tissue microarray building
Paraffin blocks and corresponding hematoxylin and eosin (HE)-stained sections were collected, and the HE-stained sections were evaluated by two senior pathologists.
Morphologically representative regions were carefully selected on each individual paraffin-embedded block, and a hollow needle (1.0 mm diameter) was used to puncture the selected area to a new small wax block. Considering the specificity of the tumor and the tendency of the paraffin tissue to flake off, two punctures were performed in different areas of each tumor wax block.

Immunohistochemistry
For immunohistochemical analysis, biopsy specimens were fixed in 10% neutralbuffered formalin and routinely processed. The paraffin-embedded blocks were sectioned (4 μm thickness), stained with HE, and observed by microscopy. The twostep immunohistochemical EnVision method was applied. The primary antibodies used are listed in Table 1. The extent of staining was evaluated as 0%, 0-25%, 26-50%, 51-75%, and 76-100%, and the intensity of staining as absent (0), weak (1+), moderate (2+), and strong (3+). The second staining was conducted after 1 month, and the results were interpreted as described above. When a different staining evaluation was used, the higher intensity score was used as the final score.

Statistical analysis
Differences between the two groups were compared using the chi-squared test. The sensitivity, specificity, and positive predictive values (PPVs) were calculated from the screening and diagnostic tests. The staining score was obtained by multiplying the extent with the intensity, and the resulting score was used for the receiver operating characteristic curve. All statistical analyses were performed using SPSS version 17.0. A p-value of <0.05 was considered as statistically significant.

Results
The immunohistochemical results are summarized in Table 2 and illustrated in Figure   1. The sensitivity, specificity, PPVs, and negative predictive values (NPVs) are summarized in Tables 3-6

SMA and h-caldesmon
SMA was positive in 12 (40%) of the 30 ESS cases ( Fig. 1-G). The staining in these cases was expressed in the cytoplasm and was moderate to strong (2+ to 3+) in 8 cases and weak (1+) in 4 cases. All 33 (100%) CL cases expressed SMA ( Fig. 1-H), and among them, the staining was moderate to strong (2+ to 3+) in 11 cases and weak (1+) in the remaining cases. The average intensity score was 1.7. Meanwhile, hcaldesmon was expressed in the cell cytoplasm of only 2 (6.7%) of the 30 ESS cases of 93.5%, and an NPV of 87.5%. SMA had the highest sensitivity (100%), but its specificity was 60%, significantly lower than that of h-caldesmon. SMA had a PPV and an NPV of 73.3% and 100%, respectively (Tables 3 and 4). IFITM1, CD10, h-caldesmon, and SMA as a useful combination for differential diagnosis Based on the expressions of the four antibodies and their receiver operating characteristic curve(the area-under-the-curve predictive value was 0.995 (Fig. 2), we speculate that their combinations could be helpful in the differential diagnosis of ESS and CL.

Discussion
The standard routine immunomarker panel used by most pathologists to distinguish ESS from CL consists of CD10, h-caldesmon, and SMA [10,[20][21][22], and an immunoprofile of CD10(+), h-caldesmon(-), and SMA(-) supports the diagnosis of ESS [15]. However, the current combination of immunohistochemical antibodies has been shown to be inaccurate, especially when diagnosing endometrial stromal tumors using CD10 alone [3,10] . CD10 is not merely expressed in endometrial stromal tumors but is also positively expressed in 20-30% of smooth muscle tumors [13,15]. SMA is a common muscle marker for endometrial stromal tumors and therefore has a very low specificity. Although h-caldesmon has a higher specificity that of SMA, its sensitivity is worse [10,13,15,23]. Thus, the need for a novel biomarker or a new immunohistochemical combination is imperative. IFITM1 is a novel biomarker for endometrium stromal cells and is reported to be more valuable than CD10 [19,24]. According to Busca et al [19] , IFITM1 and CD10 were expressed in 14 ESS cases, and although their sensitivities were 83% and 91%, respectively, IFITM1 showed a higher specificity than CD10, that is, 70% vs. 45%.
Based on the expressions of the four antibodies and their receiver operating characteristic curve, we speculate that their combination could be useful in the clinical and differential diagnosis of ESS and CL. When such combination was used for diagnosis, the area-under-the-curve predictive value was 0.995. However, there are certain limitations in that the receiver operating characteristic curve cannot completely show the positive and negative expressions of the antibodies. We therefore compared the sensitivities and specificities of the four combined antibodies using a screening test and selected the best biomarker panel. For the diagnosis of ESS, the combination IFITM1(+) or CD10(+) had the highest sensitivity (93.3%), followed by IFITM1(+) or CD10(+) and h-caldesmon(-) (86.7%). In terms of specificity, the combinations IFITM1(+) and h-caldesmon(-), IFITM1(+) and SMA(-), and CD10(+) and SMA(-) were the best panels, having a specificity of up to 100%, while IFITM1(+) or CD10(+) was the worst panel, with a specificity of only 66.7%. Considering both sensitivity and specificity, the best panel for diagnosing ESS was IFITM1(+) or CD10(+) and h-caldesmon(-), with a sensitivity of 86.7% and a specificity of 93.9%. The other good predictive panel for distinguishing ESS from CL was IFITM1(+) and hcaldesmon(-), with a sensitivity of 80% and a specificity of 100%.
In general, no one immunomarker is sensitive and specific enough to make an accurate diagnosis, and so we usually use an immunohistochemical panel for differential diagnosis. This study revealed that the combination of IFITM1, CD10, SMA, and h-caldesmon comprised the best immunohistochemical panel for differentiating between ESS and CL. Considering the costs, we also recommend the combinations IFITM1 and h-caldesmon for the same purpose.

Ethics approval and consent to participate
Ethical approval was obtained from the Institutional Ethics Review Board (IERB) of the First Affiliated Hospital of School of Medicine, Shihezi University.Research was conducted according to all ethical standards, and written informed consent was obtained from all patients.

Consent for publication
Consent to publish has been obtained from all authors.

Availability of data and materials
All data in our study are available upon request.