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

This study enrolled 30 patients with EST (5 with ESNs, 16 with low-grade ESSs, 5 with high-grade ESSs, and 4 with undifferentiated endometrial sarcoma) and 33 patients with CL. Data were collected from 2012 to 2017 from the Department of Pathology of the First Affiliated Hospital of Shihezi University School of Medicine and the Department of Pathology of Xinjiang Uygur Autonomous Region People’s Hospital. 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.

For tumor microarray construction, paraffin-embedded tissues of 63 cases were included as mentioned above [20]. 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. One section was stained with H&E to evaluate the presence of the tumour by light microscopy.

For immunohistochemical analysis, biopsy specimens were fixed in 10% neutral-buffered formalin and routinely processed. The paraffin-embedded blocks were sectioned (4 μm thickness), stained with HE, and observed by microscopy. The two-step immunohistochemical EnVision method was applied. The primary antibodies used were ITIFM1 (Sigma, 1:400), CD10 (ZSGB-BIO, 1:50), SMA (ZSGB-BIO, 1:100) and h-caldesmon (ZSGB-BIO, 1:100). CD10 uses EDTA for antigen retrieval, and all other antibodies use citrate. The staining of IFITM1 and CD10 is located in the cytoplasm and membrane, h-caldesmon and SMA are positive in the cytoplasm. The evaluation of the four biomarkers was assessed twice by two gynecological pathologists with intermediate professional title or above, separated by one-month period. 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+). When a different staining evaluation was used, the higher intensity score was used as the final score. The staining score was obtained by multiplying percentage with intensity and this score was used for our statistics analysis. The results were interpreted as described above.

The major purpose of statistical comparison was to seek helpful antibodies to differential diagnosis between EST and CL. First, composition scores for the 4 antibodies tested were determined based on immunohistochemical grades (range 0–12) as intensity (range 0–3) multiplied by percent expression (range 0–4). Then, the expression patterns of the four antibodies were checked, the chi-square test was used to compare the differences between the two groups, and Fisher’s exact test was performed on each marker. The sensitivity, specificity, positive predictive values (PPVs) and negative predictive values (NPVs) were calculated from the screening and diagnostic EST. Among the statistically significant biomarkers, we perform receiver operating characteristic (ROC) curve analysis in descending order, add each biomarker one by one, and use the area under the curve (AUC) to indicate statistical significance [21]. All statistical analyses were performed using SPSS version 17.0. A p-value of < 0.05 (all, two-tailed test) was considered as statistically significant.

The median age of 30 EST patients was 49.5 (27–73) years, and the main clinical symptoms were irregular vaginal bleeding, abdominal pain, postmenopausal vaginal bleeding, and uterine fibroids. The 33 CL patients had an median age of 41 (26–60) years and mainly showed clinical manifestations of dysmenorrhea, prolonged menstrual period, and increased menstrual volume.

The immunohistochemical results are summarized in Table 1 and illustrated in Fig. 1. The ROC values, sensitivity, specificity, PPVs, and NPVs are summarized in Tables 2, 3, 4 and 5 and shown in Fig. 2.

Both EST (Fig. 1a) and CL cases (Fig. 1b) showed a dense spindle-cell braid-like arrangement. Among the 30 EST cases, 26 (86.7%) demonstrated IFITM1 cytoplasmic positivity (Fig. 1c). The staining intensity was strong (3+) in 8 cases, moderate (2+) in 8 cases, and weak (1+) in 10 cases. Of the 33 CL cases, only 6 (18.2%) demonstrated IFITM1 nuclear positivity (Fig. 1d), all of which scored weak (1+) in intensity. CD10 was expressed in 19 (63.3%) of the 30 EST cases (Fig. 1e). The staining in these cases occurred in the cell cytoplasm and was strong (3+) in 6 cases, moderate (2+) in 7 cases, and weak (1+) in 6 cases. Only 7 (21.2%) of the 33 CL cases were CD10(+), and all positive cases had a weak (1+) intensity (Fig. 1f).

SMA was positive in 12 (40%) of the 30 EST cases (Fig. 1g). 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. 1h), and among them, the staining was moderate to strong (2+ to 3+) in 11 cases and weak (1+) in the remaining cases. Meanwhile, h-caldesmon was expressed in the cell cytoplasm of only 2 (6.7%) of the 30 EST cases (Fig. 1i), and the staining in these positive cases were weak (1+). However, 29 (87.9%) of the 33 CL cases exhibited h-caldesmon positivity (Fig. 1j). In these 33 CL cases, the staining was strong (3+) in 4 cases, moderate (2+) in 10 cases, and weak (1+) in 15 cases.

In order to avoid the influence of hormones on tumor expression, combined with clinical information, we divided the patients into two groups of pre- and post-menopausal women, and compared the expression of IFITM1, CD10, h-caldesmon, and SMA in EST and CL between pre- and post-menopausal women (Table 6). The results showed that in the same tumor, the expressions of IFITM1, CD10, h-caldesmon, and SMA were not statistically different between pre- and post-menopausal groups. In the two groups of pre- and post-menopausal, the expressions of IFITM1, SMA, and h-caldesmon were significantly different in EST and leiomyomas and showed the same trend. CD10 was slightly different, and its expression was significant difference in premenopausal EST and CL, but there was no statistical difference in post-menopausal group. From the above results, we believe that hormones have no significant effect on the expression of tumor antibodies.

In the diagnosis of EST, IFITM1 showed a sensitivity of 86.7%, a specificity of 81.8%, a PPV of 81.3%, and an NPV of 87.1%. For CD10, the sensitivity, specificity, PPV, and NPV were 63.3, 78.8, 73.1, and 70.3%, respectively. h-caldesmon positivity may support a diagnosis of CL, showing a sensitivity of 87.9%, a specificity of 93.3%, a PPV 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 (Table 2).

Based on the expressions of the four antibodies and their ROC curve, the combination of IFITM1, CD10, SMA, and h-caldesmon four antibodies showed the highest predictive value of AUC, and the ROC values of other combinations are lower than this type of combination (Table 3, Fig. 2), we speculate that their combinations could be helpful in the differential diagnosis of EST and CL.

When all four antibodies were combined for the EST diagnosis (Table 4), The three most sensitive combinations in descending order were IFITM1 (+) or CD10 (+), IFITM1 (+) or CD10 (+) and h-caldesmon (−), IFITM1 (+) and h-caldesmon (−), with their sensitivity of 93.3, 86.7, 80%, respectively. The combination of antibodies greatly increased the specificity of EST diagnosis, the specificity of combinations of IFITM1 (+) and h-caldesmon (−), IFITM1 (+) and CD10 (+) and h-caldesmon (−) and SMA (−), and IFITM1 (+) or CD10 (+) and h-caldesmon (−) and SMA (−) were 100%. Considering both sensitivity and specificity, the combination with the best diagnostic value for EST was IFITM1 (+) or CD10 (+) and h-caldesmon(−), with a sensitivity and a specificity of 86.7 and 93.9%, respectively.

In diagnosing CL (Table 5), the three most sensitive combinations in descending order were h-caldesmon (+) or SMA (+), h-caldesmon (+) and SMA (+), and h-caldesmon (+) or SMA (+) and IFITM1 (−), with sensitivity values of 100, 87.9, and 81.8%, respectively. On the other hand, h-caldesmon (+) and IFITM1 (−), h-caldesmon (+) and SMA (+), h-caldesmon (+) and SMA(+) and IFITM1 (−) showed better specificity for predicting CL from EST with all specificity were 100%. Taking into account sensitivity and specificity, h-caldesmon (+) and SMA (+) was the best combination for distinguishing CL from EST, with a sensitivity of 87.9% and a specificity of 100%. The second-best combination for distinguishing CL from EST was h-caldesmon (+) or SMA (+) and IFITM1 (−), with a sensitivity of 81.8% and a specificity of 93.1%.