INSM1 Expression in Mesenchymal Tumors and Its Clinicopathological Significance

Background Insulinoma-associated protein 1 (INSM1) has been identified as a nuclear marker of neuroendocrine tumors. Although INSM1 appears to be a subtle and specific biomarker for neuroendocrine tumor, its expression and clinicopathological significance in mesenchymal tumors remain unclear. Methods We analyzed INSM1 mRNA level in GEO database and conducted immunohistological staining to detect the expression of INSM1 on 576 mesenchymal tumors from pathology department of Tongji Hospital. Results At transcription level, INSM1 expression in AITL (angioimmunoblastic T-cell lymphoma) was higher than their adjacent normal tissues as well as Hodgkin's lymphoma. Moreover, INSM1 expression in well-differentiated liposarcoma (WDLPS) was significantly higher than normal fat (P = 0.014) and dedifferentiated liposarcoma (DDLPS) (P = 0.0248). At protein level, the positive rate of INSM1 in AITL was 18/48 (47.4%), while in DDLPS was 9/20 (45%). INSM1 expression in AITL was significantly higher than Hodgkin's lymphoma (P = 0.008). And INSM1 expression in WDLPS was significantly lower than DDLPS (P = 0.015). Conclusion The combination of GEO data and immunohistochemistry data indicated that the expression level of INSM1 was higher in AITL compared with normal control, suggesting that INSM1 may be involved in pathogenesis of AITL. The abnormal expression of INSM1 was found in WDLPS, and the positive rate of INSM1 was higher in DDLPS than in WDLPS. INSM1 may be involved in the regulation of liposarcoma development. There were significant differences in the expression of INSM1 between AITL and Hodgkin's lymphoma and WDLPS and DDLPS. These findings may assist in the differential diagnosis of these tumors when common markers are difficult to identify, enriching the diagnostic index system of mesenchymal tumors.

Some sporadic case studies have shown that neuroendocrine indicators can also be expressed in soft tissue tumors, such as synapsin (Syn) and chromogranin A (CgA) expressed in extrasolar myxochondrosarcoma, alveolar rhabdomyosarcoma, and angiosarcoma [20][21][22][23][24]. As an emerging neuroendocrine marker, whether INSM1 is beneficial to diagnose soft tissue tumors with neuroendocrine differentiation aroused the interest of some researchers. It has been found that INSM1 is positively expressed in 90% of extracorporeal myxochondrosarcoma [25]. INSM1 is expected to become a potential marker for the diagnosis of this tumor. Although the proportion was only 26% in angiosarcomas, we cannot ignore the presence of angiosarcomas with abnormal INSM1 expression, especially when evaluating poorly differentiated and inadequately sampled samples [25,26]. In addition, a case study reported nuclear translocation t(12; 14) (q23.2; Q32.3) may make ASCL bind to INSM1 enhancer, upregulating INSM1 expression in chronic lymphocytic leukemia. This also leads us to explore the possibility of "reexpression" of INSM1 in blood diseases [27].
Although INSM1 appears to be a subtle and specific biomarker for neuroendocrine tumors, its expression in mesenchymal tumors and its clinicopathological significance are still unclear [28]. Therefore, whether INSM1 can continue to maintain excellent diagnostic performance in mesenchymal tumors is indeed a question worth exploring. Although some researchers have added mesenchymal tumors in the study of neuroendocrine tumors, more tissue types have not been involved. The study on the expression of INSM1 in mesenchymal tumors is still limited. Here, we use immunohistochemical method to detect the expression of INSM1 in 576 mesenchymal tumors. In addition, we also used R language to analyze GEO databases due to limited samples in our hospital to evaluate its potential value in clinical diagnosis and treatment.

INSM1 mRNA Level of Mesenchymal Tumors in GEO
Database. Data analysis showed that the expression of AITL and ALCL was different from that of normal lymph node/ peripheral blood. The INSM1 mRNA of AITL was higher than that of normal control, and the INSM1 mRNA of ALCL was lower than that of normal control. The INSM1 mRNA of AITL was significantly higher than that of most lymphomas (there was no statistically significant difference between AITL and NK/T-cell lymphoma) (Figures 1-3). The abbreviations of hematologic neoplasms are as shown in Table 1. Other lymphoma pairwise comparison is shown in Table S2.
INSM1 mRNA level between multiple subtypes of soft tissue tumor showed significant differences (Table S3). Many tumor types were lack of corresponding normal control. Thus, we focus on liposarcoma, rhabdomyosarcoma, and bone tumor. We found that the INSM1 mRNA content in WDLPS was significantly higher than that in normal fat, while there was no significant difference in the INSM1 mRNA level between bone tumor/rhabdomyosarcoma compared with their normal control ( Figure 4 and Figure S1, 2).    Table 2). The HE and IHC staining of each lymphoma is shown in Figures 5 and 6 and Figure S3. Immunohistochemical staining showed that the level of INSM1 protein in AITL was significantly higher than that in many other lymphomas, consistent with in GEO database analysis. As mentioned above, we found significant differences in INSM1 mRNA expression levels between AITL and other lymphomas except for NK/T-cell lymphoma. We validated AITL at the protein level and found significant differences between AITL and Hodgkin's lymphoma, T/B lymphoblastic lymphoma, mantle cell lymphoma, small lymphocyte lymphoma, NK/T-cell lymphoma, and anaplastic large cell lymphoma (Figure 7). It is partly consistent with the results of bioinformatics analysis.  Table 3. The representative HE and IHC staining of each soft tissue tumor is shown in Figure 8 and Figure S4-7. According to GEO analysis mentioned above, we further explored INSM1 expression rates between dedifferentiated liposarcomas (DDLPS) and other subtypes of liposarcomas. Positive rates of INSM1 in DDLPS were significantly higher than well-differentiated liposarcoma (WDLPS) (P = 0:015) (Figure 9), while GEO analysis showed that DDLPS was significantly lower than WDLPS (P = 0:0239) in terms of INSM1 transcription level. INSM1 mRNA was not significant between DDLPS and myxoid liposarcoma (MLS), which was consistent with our immunochemical results ( Figure S1, 2).

Discussion
Insulinoma-associated protein 1 (INSM1) has been considered as a novel immunostaining marker for neuroendocrine tumors (NETs) and is hypothesized to be more reliable than first-generation NET biomarkers, such as  [26].
In this study, we found that INSM1 might help in the differential diagnosis between AITL and Hodgkin's lymphoma. AITL originates from TFH cells (germinal center follicular helper T-cells) [29]. The most significant histological feature that distinguishes AITL from other peripheral Tcell lymphomas is the abundance of high endothelial microveins and clear cells in the tumor tissue [28]. The diagnosis of AITL requires the expression of at least two TFH markers (CD10, BCL6, PD1, ICOS, CXCL13, and CXCR5). Sometimes, the diagnosis of AITL may be confused with  Table 1. Hodgkin's lymphoma [29,30]. We found that the mRNA and protein levels of INSM1 were significantly different between AITL and Hodgkin's lymphoma. INSM1 may contribute to diagnosis between AITL and Hodgkin's lymphoma, but more clinical practice is needed.
AITL is characterized by high frequency of epigenetic modification-related mutations such as TET2, IDH2, and DNMT3A [30]. Epigenetic therapy has proven to be a desirable option in AITL. Clinical studies have shown that the overall response rate (ORR) of the histone deacetylase inhibitors romidepsin or belinostat alone in AITL is 30% and 45%, respectively, higher than that of other T-cell lymphomas. The ORR was 75%, and the complete response (CR) rate was 42%, also higher than other subtypes, and all patients who benefit from epigenetic therapy had TET2 mutations. The combination of romidepsin and 5azacytidine increased the ORR and CR of AITL to 83% and 50%, respectively [28][29][30][31].
INSM1 has a positive rate of nearly 50% in our samples. It is worth pondering whether INSM1 can become a unique marker of a certain subtype of AITL. Regrettably, we did not step further due to incomplete genetic molecular data in our patients. Several preclinical studies of INSM1-targeted therapies have shown promising results; then perhaps, patients 9 BioMed Research International who do not benefit from epigenetic therapy will benefit from INSM1-targeted therapies in the future.
Moreover, INSM1 may also help in the differential diagnosis between WDLPS and DDLPS. Genetic studies have shown that MDM2/CDK4 is the oncogene frequently amplified [32,33]. MDM2 and CDK4 amplification can be seen in more than 90% of liposarcoma patients, but the karyotype and gene profile of DDLPS are far more complex than that of WDLPS [34].
Overexpression of INSM1 inhibits the binding of cyclin D1 and CDK4, induces cell cycle arrest, and inhibits the growth of panc-1 pancreatic cancer cell line [6]. INSM1 is an important transcription factor regulating the development of the nervous system and the differentiation of neural stem cells. The mechanism of interaction between INSM1 and CDK4 may be involved in the development of liposarcoma, which needs to be further explored.
Limitations of this study are as follows: firstly, the sample size of our study is relatively small. The findings remain further investigation to be more confirmed before clinic practice. Secondly, in diagnosis, it seems that other markers perform superiorly than INSM1. The differential diagnostical value of INSM1 seemed limited in particular cases. Finally, the function of INSM1 in non-NETs was not clear, and this study lacks deeper insights on how INSM1 expression influences the mesenchymal tumors.

Conclusion
Mesenchymal neoplasms, especially lymphomas and sarcomas, often progress rapidly, respond poorly to treatment, and have a poor prognosis. INSM1 is a reliable diagnosis marker in neuroendocrine tumors that has attracted much attention in recent years. Our study confirmed that there were statistically significant differences in the positive rate of INSM1 between AITL/Hodgkin's lymphomas and DDLPS/WDLPS, which may assist in the differential diagnosis of these tumors. What is more significant, targeted signaling pathways related to INSM1 contribute to the exploration of new treatment strategies. However, whether INSM1 can become a therapeutic target in mesenchymal tumors lacks theoretical and experimental bases. Here, our study systematically explored the expression of INSM1 in different mesenchymal tumors, laying a foundation for the diagnosis and targeted therapy of INSM1 in mesenchymal tumors.

Data Availability
The figure and table data used to support the findings of this study are included within the article and the supplementary information files. eosinophilic epithelioid and spindle cells with vacuolar nuclei. Collagen deposition was observed between cells (400x). (E, F) Alveolar soft tissue sarcoma: the tumor cells were arranged "organ-like" with fibrous interspersed with different widths (400x); (G, H) clear cell sarcoma of soft tissue: the tumor cells are polygonal and oval, with faint cytoplasm and scattered hyaline areas, with large myxoid areas at the right. INSM1 staining in the figure was negative (400x). Figure