INSM1 Expression Research Articles

The Common Expression of INSM1 in HPV-related Oropharyngeal Squamous Cell Carcinomas Is Not Associated With True Neuroendocrine Transformation or Aggressive Behavior.

INSM1 has rapidly emerged as a robust marker for neuroendocrine (NE) differentiation, prompting the use of INSM1 as a standalone marker for NE differentiation in various sites. INSM1 staining could be an especially practical tool for evaluating HPV-associated oropharyngeal squamous cell carcinoma (HPV-OPSCCs) because high-grade NE transformation may be exceedingly difficult to recognize on morphologic grounds alone, even though it portends highly aggressive clinical behavior. The purpose of this study was to determine the incidence of INSM1 expression in HPV-OPSCC and study its role in determining NE transformation and clinical aggressiveness. Forty-six consecutive nonselected HPV-OPSCCs were stained with INSM1 and synaptophysin. In addition, 2 clinically matched cohorts of aggressive (n=28) versus nonaggressive (n=26) HPV-OPSCCs were retrospectively stained with the INSM1 and synaptophysin to determine the prognostic significance of INSM1 staining. In all, 42% of HPV-OPSCC showed positive INSM1 staining, but synaptophysin was not co-expressed in even a single case. In selected cases based on clinical behavior, INSM1 staining was observed more frequently in nonaggressive than aggressive tumors (50.0% vs. 21.4%, P=0.03). INSM1 expression is a common finding in HPV-OPSCC and is usually not linked with true NE transformation or aggressive behavior. Indeed, INSM1 expression is more commonly observed in nonaggressive cancers. Reliance on INSM1 staining alone in recognizing NE differentiation in HPV-OPSCC could be calamitous.

Evaluation of immunohistochemical expression of novel neuroendocrine marker INSM1 and histological tumor growth pattern in well-differentiated neuroendocrine tumors of the appendix: 15-year single tertiary center experience.

Well-differentiated neuroendocrine tumors of the appendix (A-WDNET) are the most common neoplasms of the appendix but are rare and incidental. INSM1 is a novel transcription factor marker with high sensitivity and specificity for neuroendocrine differentiation. It may display architecturally diverse tumor growth patterns including solid, nest, insular trabecular, and acinar. The aim of this study was to determine the staining expression of INSM1 in A-WDNETs and detail the morphological tumor growth patterns. INSM1 immunohistochemistry was performed on 35 A-WDNET patients. Tumors were histologically classified according to their growth patterns. Thirty-five patients, 60% of whom were male, had a mean age of 30 years at diagnosis and a mean tumor size of 0.9 cm. Eight percent of the tumors invaded the mucosa/submucosa, 34.3% showed invasion into the muscularis propria, 57.1% showed invasion into the subserosa or mesoappendix, LN metastasis was observed in two patients, lymph-vascular invasion in two patients, and perineural invasion in five patients. Ninety-four percent of the tumors were grade 1, the mitotic rate was >2% in two cases, and Ki-67 PI was >3% in two cases. INSM1 was positive in all cases (100%), 1+ 8.6%, 2+ 5.7%, 3+ 17.1%, 4+ 68.6%, weak staining in 11.4%, moderate staining in 22.9%, and strong staining in 67.7%. Type A histological growth pattern was observed in 54.3%, type B in 31.4%, and type C in 14.3% architecturally. INSM1 was positive in all A-WDNET morphological patterns and was 100% sensitive. INSM1 IHC can be used as an alternative to traditional neuroendocrine markers or in combination with the diagnosis of neuroendocrine tumors.

INSM1 expression in primary and metastatic neuroendocrine neoplasms at distinct locations

BackgroundInsulinoma-associated protein 1 (INSM1) has been reported as a valuable marker for neuroendocrine neoplasms (NENs). The aims of this study were to evaluate any change in INSM1 expression between primary and metastatic NENs in distinct locations, as well as the expression of INSM1 at different differentiation levels. Furthermore, we would also investigate the significance of INSM1 expression in non-neuroendocrine neoplasms (non-NENs). MethodsWe collected 78 cases with primary NENs and 16 cases with metastatic NENs. An addition 7 cases of non-NENs with neuroendocrine (NE) differentiation and 84 cases of other non-NENs, respectively, were included as controls. ResultsIn our cohort, 82% of primary NENs and 88% of metastatic NENs expressed INSM1 with no difference between them. There was no difference in the expression of INSM1 in the lung and digestive system, and its staining pattern was independent of tumor differentiation or location. The proportion of INSM1 -positive in non-NENs with NE differentiation was significantly higher than that in other non-NENs. INSM1 sensitivity for primary NENs (82%) was comparable to Chromogranin A (82%), less than that of Synaptophysin (96%) and CD56 (94%); specificity was higher (96% vs 94%, 82%, and 89%, respectively). The sensitivity of INSM1 for well differentiated NENs was significantly higher than that of poorly differentiated NENs (100% vs 79%). ConclusionsINSM1 is a useful neuroendocrine marker in primary and metastatic NENs, helping to identify primary NENs with different degrees of differentiation. The expression of INSM1 was independent of tumor location. It should be with caution to interpret the expression of INSM1 in non-NENs that morphologically resemble NENs.

Comprehensive analysis of cancer of unknown primary and recommendation of a histological and immunohistochemical diagnostic strategy from China

BackgroundPrevious studies on cancer of unknown primary (CUP) mainly focus on treatment and prognosis in western populations and lacked clinical evaluation of different IHC markers, so this study aimed to evaluate characteristics of CUP and recommend a diagnostic strategy from a single center in China.Methods and resultsData of 625 patients with CUP were retrospectively collected and reviewed. The patients ranged in age from 20 to 91 years, with a female-to-male ratio of 1.3:1. The predominant histological type was poor or undifferentiated adenocarcinomas (308; 49.3%). The results of Canhelp-Origin molecular testing for the identification of the tissue of origin in 262 of 369 patients (71.0%) were considered predictable (similarity score > 45), with the most common predicted primary tumor site being the breast (57, 21.8%). Unpredictable molecular results correlated with more aggressive clinical parameters and poor survival. Thee positivity rates of several targeted antibodies (GATA3, GCDFP15, TTF1, Napsin A, and PAX8), based on the clinically predicted site, were lower than those reported for the corresponding primary tumors. Nonetheless, TRPS1 and INSM1 were reliable markers of predicted breast carcinoma (75.0%) and neuroendocrine tumors (83.3%), respectively. P16 expression, as well as HPV and EBER testing contributed significantly to the diagnosis of squamous cell carcinomas. Survival analysis revealed that older ages (> 57), ≥ 3 metastatic sites, non-squamous cell carcinomas, bone/liver/lung metastases, unpredictable molecular results, and palliative treatment correlated with poor overall survival.ConclusionsWe recommend a CUP diagnostic strategy involving the use of targeted antibody panels as per histological findings that is potentially applicable in clinical practice. The markers TRPS1, INSM1, and P16 expression, as well as HPV and EBER testing are particularly valuable in this aspect. Molecular testing is also predictive of survival rates.

Comparison of INSM1 immunostaining with established neuroendocrine markers Synaptophysin and Chromogranin A in over 14,000 neuroendocrine and non-neuroendocrine tumors

Abstract Introduction/Objective INSM1 is a transcription factor protein which is increasingly used as an immunohistochemical marker for neuroendocrine differentiation. Methods/Case Report To determine the prevalence of INSM1 expression in tumors and its expression pattern in normal tissues, tissue microarrays containing 14,908 samples from 117 different tumor types/subtypes as well as 76 different normal tissues were analyzed by immunohistochemistry. Results (if a Case Study enter NA) INSM1 was positive in 89.2% of 471 neuroendocrine neoplasms (NEN) and in 3.5% of 11,815 non-neuroendocrine neoplasms that were successfully analyzed. INSM1 positivity occurred in 59 non- neuroendocrine tumor entities, of which 15 entities contained at least one strongly positive case. Comparison with synaptophysin and chromogranin A revealed that in NEN, synaptophysin showed the highest sensitivity (93.3%), followed by INSM1 (89.2%) and chromogranin A (87.5%). In neuroendocrine carcinomas (NEC), sensitivity was highest for INSM1 (88.0%), followed by synaptophysin (86.5%) and chromogranin A (66.4%). The additional use of INSM1 increased the sensitivity for detecting neuroendocrine differentiation in NEN from 88.2% (synaptophysin and chromogranin A) to 91.2% (synaptophysin, chromogranin A and INSM1). Conclusion Our study shows that INSM1 is a useful additional marker for neuroendocrine differentiation that shows a particularly high sensitivity in NEC. The additional use of INSM1 results in a higher sensitivity for the identification of a neuroendocrine differentiation than what can be obtained by using only synaptophysin and chromogranin A.

Insm1 regulates mTEC development and immune tolerance.

The expression of self-antigens in medullary thymic epithelial cells (mTECs) is essential for the establishment of immune tolerance, but the regulatory network that controls the generation and maintenance of the multitude of cell populations expressing self-antigens is poorly understood. Here, we show that Insm1, a zinc finger protein with known functions in neuroendocrine and neuronal cells, is broadly coexpressed with an autoimmune regulator (Aire) in mTECs. Insm1 expression is undetectable in most mimetic cell populations derived from mTECs but persists in neuroendocrine mimetic cells. Mutation of Insm1 in mice downregulated Aire expression, dysregulated the gene expression program of mTECs, and altered mTEC subpopulations and the expression of tissue-restricted antigens. Consistent with these findings, loss of Insm1 resulted in autoimmune responses in multiple peripheral tissues. We found that Insm1 regulates gene expression in mTECs by binding to chromatin. Interestingly, the majority of the Insm1 binding sites are co-occupied by Aire and enriched in superenhancer regions. Together, our data demonstrate the important role of Insm1 in the regulation of the repertoire of self-antigens needed to establish immune tolerance.

Comparison of INSM1 immunostaining with established neuroendocrine markers synaptophysin and chromogranin A in over 14,000 neuroendocrine and non-neuroendocrine tumors

INSM1 is a transcription factor protein which is increasingly used as an immunohistochemical marker for neuroendocrine differentiation. To determine the prevalence of INSM1 expression in tumors and its expression pattern in normal tissues, tissue microarrays containing 14,908 samples from 117 different tumor types/subtypes as well as 76 different normal tissues were analyzed by immunohistochemistry. INSM1 was positive in 89.2% of 471 neuroendocrine neoplasms (NEN) and in 3.5% of 11,815 non-neuroendocrine neoplasms that were successfully analyzed. At least an occasional weak INSM1 positivity was observed in 59 different non-neuroendocrine tumor entities, of which 15 entities contained at least one case with strong INSM1 staining. A comparison with synaptophysin and chromogranin A staining revealed that in NEN, synaptophysin showed the highest sensitivity (93.3%), followed by INSM1 (89.2%) and chromogranin A (87.5%). In neuroendocrine carcinomas (NEC), sensitivity was highest for INSM1 (88.0%), followed by synaptophysin (86.5%) and chromogranin A (66.4%). If INSM1 was used as an additional marker, the sensitivity for detecting neuroendocrine differentiation in NEN increased from 96.6% (synaptophysin and chromogranin A) to 97.2% (synaptophysin, chromogranin A and INSM1). Our study shows that INSM1 is a useful additional marker for neuroendocrine differentiation with high sensitivity, particularly in NEC.

Small Molecules Targeting INSM1 for the Treatment of High-Risk Neuroblastoma.

Human neuroblastoma (NB) is the most common childhood extracranial tumor arising from the sympathetic nervous system. It is also a clinically heterogeneous disease that ranges from spontaneous regression to high-risk stage 4 disease. The cause of this disease remains elusive. However, the amplification of NMYC oncogene occurred in roughly 30% of NB patients, which strongly correlated with the advanced stage of disease subtype and the worse prognosis status. We discovered that N-Myc oncoprotein binds and activates INSM1, a zinc-finger transcription factor of neuroendocrine tumors. We also found that INSM1 modulates N-Myc stability mediated through PI3K/AKT/GSK3β signaling pathway. Therefore, INSM1 emerges as a critical co-player with N-Myc in facilitating NB tumor cell growth and sustaining the advanced stage of malignancy. Using an INSM1-promoter driven luciferase screening-platform, we have recently identified fifteen small molecules that negatively regulate INSM1 expression. Interestingly, the identified small molecules can be divided into four large groups of compounds such as cell signaling inhibitor, DNA/RNA inhibitor, HDAC inhibitor, and cardiac glycoside. These findings support the presence of a unique mechanism associated with INSM1 and N-Myc interplay, which is critical in regulating NB tumor cell growth. We discuss the feasibility of identifying novel or repurposing small molecules targeting INSM1 as a potential treatment option for high-risk NB.

SDH-B, INSM1, and GATA3 expression in cauda equina neuroendocrine tumors: report on 21 cases from a North indian tertiary care center

Primary neuroendocrine tumors (NETs) of the central nervous system are rare, primarily seen in the cauda equina region, known as cauda equina NETs. This study was carried out to evaluate the morphological and immunohistochemical characteristics of cauda equina NETs. All cases of histologically proven NETs that originated within the spinal cord from 2010 to 2021 were retrieved from the surgical pathology electronic database. For each case, the clinical presentation, site, radiological features, functional status, and preoperative diagnosis were recorded. Immunohistochemical stains for GFAP, synaptophysin, chromogranin A, cytokeratin 8/18, INSM1, Ki-67, GATA3, and SDH-B were performed for every case using an automated immunostainer. GATA3 immunohistochemistry was repeated manually. A retrospective probe of records revealed 21 cases of NETs having a mean age of 44 years and slight male dominance (M:F ratio: 1.2:1). Cauda equina was the most prevalent site of involvement (19, 90.5%). The most typical presentation was lower backache and weakness of bilateral lower limbs. The histopathological features were similar to NETs seen at other sites. Reactivity for at least one neuroendocrine marker was seen in all cases while GFAP was negative. Cytokeratin 8/18 was expressed in the majority (88.9%) of cases. INSM1 and GATA3 expression was seen in 20 (95.2%) and 3 (14.3%) cases, respectively. All cases retained SDH-B cytoplasmic staining. Higher Ki-67 index (≥3%) was associated with a higher risk of recurrence. Cauda equina NETs rarely express GATA3 and are unlikely to be associated with SDH mutations. Recurrent cases may be negative for synaptophysin, chromogranin, and cytokeratin; thus, INSM1 immunohistochemistry is helpful.

Heterogeneity of canonical prostate cancer markers across lesions in metastatic castration-resistant prostate cancer.

219 Background: Metastatic castration-resistant prostate cancers (mCRPC) show a diversity of clinical responses and phenotypes, suggesting molecular heterogeneity. Understanding molecular phenotypes is critical for patient stratification and selection of therapeutic targets. Treatment with androgen receptor (AR) pathway inhibitors are thought to drive AR-negative disease and the emergence of neuroendocrine lineages. There are several emerging biomarkers for mCRPC, including novel targets, such as PSMA and STEAP1, and markers of neuroendocrine and aggressive disease, such as INSM1, DLL3, and loss of p53, RB1 and PTEN. Our understanding of the co-expression of such biomarkers across multiple metastatic sites is currently limited. This study aimed to determine biomarker expression in multiple anatomical sites within individual mCRPC patients. Methods: Through the rapid-autopsy CASCADE program at the Peter MacCallum Cancer Centre, tissue was obtained from 5-14 tumor sites from 12 individual mCRPC patients yielding samples from a total of 110 tumor sites (8 primary and 102 metastatic). Ten patients were diagnosed with prostate adenocarcinoma (89 sites), and 2 patients with neuroendocrine histopathology in all sites (21 sites). Tissue was stained for AR, PSMA, STEAP1, INSM1, DLL3 p53, RB1 and PTEN by immunohistochemistry (IHC). Serial sections were reviewed and scored by a pathologist. IHC results were corroborated with genetic information derived from DNA sequencing. Results: In the adenocarcinoma cohort, on average 77% of sites of individual patients had AR expression, suggesting intra-patient heterogeneity. Co-expression of AR and PSMA was common (55/89 sites), but 6/10 adenocarcinoma patients had 1 to 6 PSMA+AR- lesions. Co-expression of PSMA and STEAP1 was also common in adenocarcinoma patients (60/89 sites), although 8/10 patients had 1 to 2 PSMA-STEAP1+ lesions, and 7 patients had 1 to 3 PSMA+STEAP1- lesions. Expression of INSM1 and DLL3 was mostly seen in the 2 patients (20/21 sites) with neuroendocrine prostate cancer. In contrast, only 2 INSM1+DLL3+ tumor sites in liver and 1 INSM1-DLL3+ prostate cancer sample were detected in the adenocarcinoma cohort. Of the 110 sites, p53 loss was observed in 9, RB1 loss in 30 and PTEN loss in 25 sites. Of these, 8/9, 14/30 and 8/25 occurred in INSM1+DLL3+ sites, suggesting that loss of these markers does not always correlate with INSM1 and DLL3 expression. Conclusions: While AR, PSMA and STEAP1 expression are frequently detected in a single site of disease, this co-expression is not consistent across all sites. In this dataset, INSM1 and DLL3 expression was almost exclusively confined to neuroendocrine prostate cancer. Loss of RB1 and PTEN often occurs without INSM1 and DLL3 expression. Understanding differential biomarker expression in mCRPC may guide future targeted treatments and warrants further research.

Overexpression of INSM1, NOTCH1, NEUROD1, and YAP1 genes is associated with adverse clinical outcome in pediatric neuroblastoma.

Pediatric neuroblastoma is responsible for approximately 8-10% of pediatric tumors, and it is one of the leading causes of tumor-related deaths in children. Although significant progress has been made in the characterization of neuroblastoma in recent years, the mechanisms influencing the prognosis of neuroblastoma patients remain largely unknown. Our aim was to investigate if the major neuroendocrine-associated transcriptional drivers, including ASCL1, NEUROD1, DLL3, NOTCH1, INSM1, MYCL1, POU2F3 and YAP1 are correlated with specific clinical and pathological characteristics. We selected a retrospective series of 46 primary pediatric neuroblastoma, composed of 30 treatment-naïve and 16 post-chemotherapy cases. Gene expression levels were explored by means of quantitative real-time PCR. An increased expression of NOTCH1 (p = 0.005), NEUROD1 (p = 0.0059), and YAP1 (p = 0.0008) was found in stage IV tumors, while the highest levels of MYCL1 and ASCL1 were seen in stages IVS and III, respectively (p = 0.0182 and p = 0.0134). A higher level of NOTCH1 (p = 0.0079) and YAP1 (p = 0.0026) was found in cases with differentiating morphology, while high mitosis-karyorrhexis index cases demonstrated significantly lower levels of POU2F3 (p = 0.0277). High expression of NOTCH1 (p = 0.008), NEUROD1 (p = 0.026), INSM1 (p = 0.010), and YAP1 (p = 0.005) together with stage IV (p = 0.043) was associated with shorter disease-free survival. In summary, our data indicate that the assessment of gene expression levels of neuroendocrine-lineage transcription factors might help to identify neuroblastoma patients with the risk of relapse.

Abstract 6028: Patient derived organoids demonstrate hallmark characteristics of small cell lung cancer

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers with an overall 5-year survival rate of 5-10%. Although transgenic mouse models and patient derived xenograft models have been shown to capture the heterogeneity and complexity of SCLC, the costs involved and timeframe of generating these models can be extensive. Furthermore, these models lack the ability to study the patients’ immune components and tumor microenvironment. Alternatively, in other cancer types in vitro methods to generate organoids have been successful in the hope to develop personalized patient derived organoids for screening and further downstream analyses. Here, we have generated and characterized small cell lung cancer patient-derived tumor organoids and confirmed that the organoids possess similar molecular characteristics, genetic profiles and morphological architectures to the corresponding patient tumor tissue. Organoids were shown to express hallmark features for SCLC, which included expression of neuroendocrine markers CD56, CHGA, INSM1 and SYP shown by immunohistochemistry. We suggest utilizing these organoids as SCLC patient models would be ideal for investigating the tumor microenvironment in SCLC, mechanisms between tumor immune cell interactions as well as assessing patients’ responses to therapy. Citation Format: Joshua D'Rozario, Julie George, Roman Thomas. Patient derived organoids demonstrate hallmark characteristics of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6028.

Expression of novel neuroendocrine markers in breast carcinomas: a study of INSM1, ASCL1, and POU2F3

INSM1, ASCL1, and POU2F3 are novel transcription factors involved in neuroendocrine (NE) differentiation of neoplasms in several organs, but data on their expression in breast carcinomas (BCs) are limited. We retrospectively evaluated the expression of these markers in a series of 97 BCs (58 with NE morphology and 39 with otherwise uncommon morphology) tested prospectively using immunohistochemistry (IHC). Nuclear staining in >50% of the cells was used as the positive cut-off. Thirty-two of the 97 BCs (33%) were INSM1-positive. INSM1-positivity correlated significantly with histologic type and presence of stromal mucin. INSM1 also correlated with synaptophysin and chromogranin, established markers of NE differentiation (P<.0001 and P=.0023, respectively). In BC with NE morphology, the expression of INSM1 supported NE differentiation, and INSM1 was more specific than synaptophysin and more sensitive and specific than chromogranin. INSM1 was the most expressed NE marker in 17BCs. INSM1-positive BCs included 56% of solid papillary BCs, 88% of BCs with solid papillary features, and 75% of high-grade NE carcinomas. Of 35 BCs tested for POU2F3 and ASCL1, only 1 and 4 cases were positive, respectively. Our results show that INSM1 is a sensitive marker of NE differentiation in BC and should be included with synaptophysin and chromogranin in the IHC panel used to evaluate NE differentiation in BC with NE morphology. ASCL1 and POU2F3 are uncommon in BC and their routine assessment is not warranted.

Molecular Subtypes of Extra-pulmonary Neuroendocrine Carcinomas Identified by the Expression of Neuroendocrine Lineage-Specific Transcription Factors

Extra-pulmonary neuroendocrine carcinomas (EPNEC) represent a group of rare and heterogenous neoplasms with adverse clinical outcome. Their molecular profile is largely unexplored. Our aim was to investigate if the major transcriptional drivers recently described in high-grade pulmonary neuroendocrine carcinomas characterize distinct molecular and clinical subgroups of EPNEC. Gene expression of ASCL1, NEUROD1, DLL3, NOTCH1, INSM1, MYCL1, POU2F3, and YAP1 was investigated in a series of 54 EPNEC (including 10 cases with mixed components analyzed separately) and in a group of 48 pulmonary large cell neuroendocrine carcinomas (P-LCNEC). Unsupervised hierarchical cluster analysis classified the whole series into four major clusters. P-LCNEC were classified into two major clusters, the first ASCL1/DLL3/INSM1-high and the second (including four EPNEC) ASCL1/DLL3-low but INSM1-high. The remaining EPNEC cases were sub-classified into two other clusters. The first showed INSM1-high and alternative ASCL1/DLL3 or NEUROD1 high expression. The second was characterized mainly by MYCL1 and YAP1 overexpression. In the ten cases with mixed histology, ASCL1, DLL3, INSM1, and NEUROD1 genes were significantly upregulated in the neuroendocrine component. Higher gene-expression levels of NOTCH1 and INSM1 were associated with lower pT stage and negative nodal status. Low INSM1 gene expression was associated with shorter overall survival in the entire case series (p = 0.0017) and with a trend towards significance in EPNEC, only (p = 0.06). In conclusion, our results show that EPNEC possess distinct neuroendocrine-lineage-specific transcriptional profiles; moreover, low INSM1 gene expression represents a novel potential unfavorable prognostic marker in high-grade NECs including those in extra-pulmonary location.

Clinical-Pathological Evaluation and Prognostic Analysis of 228 Merkel Cell Carcinomas Focusing on Tumor-Infiltrating Lymphocytes, MCPYV Infection and ALK Expression

Merkel cell carcinoma is a rare and aggressive primary neuroendocrine carcinoma of the skin, whose pathogenesis can be traced back to UV radiation damage or Merkel cell polyomavirus (MCPyV) infection. Despite some improvements on the characterization of the disease partly due to its increased incidence, crucial pathogenetic and prognostic factors still need to be refined. A consecutive series of 228 MCC from three hospitals in Turin was collected with the aim of both analyzing the apparent increase in MCC incidence in our area and investigating the distribution and prognostic role of clinical-pathological parameters, with a focus on MCPyV status, ALK tumor expression and tumor infiltrating lymphocytes (TILs). Review of morphology and conventional immunohistochemical staining was possible in 191 cases. In 50 cases, the expression of the novel neuroendocrine marker INSM1 was additionally assessed. Fourteen cases of MCC of unknown primary skin lesion were identified and separately analyzed. While confirming an exponential trend in MCC incidence in the last decades and providing a description of histological and cytological features of a large series of MCC, the present study concludes that 1) INSM1 is a highly sensitive marker in both skin and lymph node primary MCC; 2) positive MCPyV status, brisk TILs and lower tumor size and thickness are independent positive prognostic parameters, and the combination of the former two may provide a novel tool for prognostic stratification; 3) ALK is expressed 87% of MCC and associated with positive viral status, and could represent a prognostic biomarker, if validated in larger series.

Neuroendocrine Carcinoma of the Larynx and Pharynx: A Clinical and Histopathological Study.

Simple SummaryNeuroendocrine carcinomas (NECs) of the head and neck are rare. The presented series of 20 patients with laryngeal and pharyngeal NECs is population-based and one of the largest published to date. We analyzed the treatment results according to the type of therapy and the role of various standard (synaptophysin-chromogranin-CD56, Ki-67, p16, HPV, and EBV) and some novel (INSM1 and PD-L1) neuroendocrine markers or potential prognosticators. The results indicate the following: (1) laryngeal and pharyngeal NECs accounted for 0.43% and 0.17% of the cases in the corresponding tumor groups, respectively; (2) neuroendocrine differentiation can be reliably determined by INSM1 immunohistochemistry; (3) the prognosis was determined by the nodal stage and TNM stage but not by the histological grade (which refers to moderately and poorly differentiated NECs); (4) except in well-differentiated NECs and early-stage (T1-2N0-1) moderately/poorly differentiated NECs, aggressive multimodal therapy is needed; and (5) the p16, HPV, and EBV statuses failed to show any prognostic value.Neuroendocrine carcinomas (NECs) of the head and neck are rare and the experience scanty. The Cancer Registry of Slovenia database was used to identify cases of laryngeal and pharyngeal NECs diagnosed between 1995–2020. Biopsies were analyzed for the expression of standard neuroendocrine markers (synaptophysin, chromogranin, CD56), INSM1, Ki-67, p16, and PD-L1 (using the combined positive score, CPS). In situ hybridization for human papillomavirus (HPV) and Epstein–Barr virus (EBV) was performed. Twenty patients (larynx, 12; pharynx, 8) were identified. One tumor was well differentiated (WD), five were moderately differentiated (MD), and 14 were poorly differentiated (PD). Disease control was achieved solely by surgery in 4/4 MD/PD T1-2N0-1 tumors. Eight patients died of the disease, seven of which were due to distant metastases. All three traditional markers were positive in 11/17 NECs and the INSM1 marker in all 20 tumors. Two of fourteen p16-positive tumors were HPV-positive, but all three nasopharyngeal NECs were EBV-negative. Three tumors had CPSs ≥ 1. In conclusion, INSM1 was confirmed to be a reliable marker of neuroendocrine differentiation. Except in WD and early-stage MD/PD tumors, aggressive multimodal therapy is needed; the optimal systemic therapy remains to be determined. p16, HPV, and EBV seem to bear no prognostic information.

Insm1, Neurod1, and Pax6 promote murine pancreatic endocrine cell development through overlapping yet distinct RNA transcription and splicing programs.

Insm1, Neurod1, and Pax6 are essential for the formation and function of pancreatic endocrine cells. Here, we report comparative immunohistochemical, transcriptomic, functional enrichment, and RNA splicing analyses of these genes using gene knock-out mice. Quantitative immunohistochemical analysis confirmed that elimination of each of these three factors variably impairs the proliferation, survival, and differentiation of endocrine cells. Transcriptomic analysis revealed that each factor contributes uniquely to the transcriptome although their effects were overlapping. Functional enrichment analysis revealed that genes downregulated by the elimination of Insm1, Neurod1, and Pax6 are commonly involved in mRNA metabolism, chromatin organization, secretion, and cell cycle regulation, and upregulated genes are associated with protein degradation, autophagy, and apoptotic process. Elimination of Insm1, Neurod1, and Pax6 impaired expression of many RNA-binding proteins thereby altering RNA splicing events, including for Syt14 and Snap25, two genes required for insulin secretion. All three factors are necessary for normal splicing of Syt14, and both Insm1 and Pax6 are necessary for the processing of Snap25. Collectively, these data provide new insights into how Insm1, Neurod1, and Pax6 contribute to the formation of functional pancreatic endocrine cells.

Cross-talk among MEN1, p53 and Notch regulates the proliferation of pancreatic neuroendocrine tumor cells by modulating INSM1 expression and subcellular localization.

Genomic analysis of Pancreatic Neuroendocrine Tumors (PanNETs) has revealed that these tumors often lack mutations in typical cancer-related genes such as the tumor suppressor gene p53. Instead, PanNET tumorigenesis usually involves mutations in specific PanNET-related genes, such as tumor suppressor gene MEN1. Using a PanNET mouse model, human tissues and human cell lines, we studied the cross-talk among MEN1, p53 and Notch signaling pathways and their role in PanNETs. Here, we show that reactivation of the early developmental program of islet cells underlies PanNET tumorigenesis by restoring the proliferative capacity of PanNET cells. We investigated the role of INSM1, a transcriptional regulator of islet cells’ development, and revealed that its expression and subcellular localization is regulated by MEN1 and p53. Both human and mouse data show that loss of MEN1 in a p53 wild-type genetic background results in increased nuclear INSM1 expression and cell proliferation. Additionally, inhibition of Notch signaling in a p53 wild-type background reduces the proliferation of PanNET cells, due to repression of INSM1 transcription and nuclear localization. Our study elucidates the molecular mechanisms governing the interactions of INSM1 with MEN1, p53 and Notch and their roles in PanNET tumorigenesis, suggesting INSM1 as a key transcriptional regulator of PanNET cell proliferation.

INSM1 Expression in Breast Neoplasms with Neuroedocrine Features

According to the 2019 WHO classification of breast tumors, neuroendocrine neoplasms (NENs) are classified into well-differentiated NE tumors (NET) and poorly differentiated NE carcinomas (NEC), while other breast cancers (BCs) of special and no special type with neuroendocrine (NE) features are not incorporated in this scheme anymore. We aimed to assess whether INSM1, a novel NE marker, could have a role in breast NEN subtyping. We selected 63 BCs operated from 2003 to 2018, classified as BCs with NE features, with available clinico-pathological data. Following 2019 WHO criteria, this cohort was reclassified into 37 NETs/NECs, the remaining 26 tumors representing solid-papillary (7), mucinous (7), and mixed type (12) carcinomas with NE differentiation. Chromogranin A (CGA) and synaptophysin (SYN) immunostains were reviewed, and INSM1 was tested by immunohistochemistry. Thirty CGA- and SYN-negative no special type BCs served as negative control. INSM1 was expressed in 52/63 cases of the whole cohort (82.54%). INSM1 positive and negative cases had no significantly different clinico-pathological characteristics. INSM1 expression was not significantly different between the newly reclassified NET/NEC group and other BCs with NE features. No immunoexpression was observed in control BCs. The sensitivity and specificity of INSM1 for the NE phenotype was 82.5% and 100%, respectively, compared to 61.9% and 100% for CGA, and 95.2 and 100% for SYN. In conclusion, INSM1 is as accurate as traditional NE biomarkers to identify NE differentiation in BC. In analogy to standard NE markers, INSM1 could not distinguish NET and NEC from the other BC histotypes with NE differentiation.

Application of INSM1 in Diagnosis and Grading of Laryngeal Neuroendocrine Carcinoma.

Chromogranin (CHG), synaptophysin (Syn), and CD56 are generally used in a panel to support diagnoses of laryngeal neuroendocrine carcinomas (NECs). However, the absence of expression of these markers does not completely exclude the diagnosis. INSM1 is a novel marker that is considered sufficiently sensitive and specific for NE differentiation. The aim of this study is not only to detect its sensitivity and specificity, but also to evaluate its application in grading for laryngeal NECs. The clinicopathological characteristics of the 25 cases with laryngeal NECs were retrospectively analyzed. The expressions of INSM1, CHG, Syn, and CD56 were detected by immunohistochemistry. Of the 25 laryngeal NECs, INSM1 had higher sensitivity (92%) than Syn (84%), CHG (76%) and CD56 (76%). The average H scores of INSM1, CD56, Syn, and CHG were 160, 37.5, 300, 300 for well-differentiated neuroendocrine carcinoma (WD-NEC); 190, 149, 209, 215 for moderately differentiated neuroendocrine carcinoma (MD-NEC); 251, 208, 104, 25 for poorly differentiated neuroendocrine carcinoma with small cell (SCNEC); 109, 160, 98, 26 for large cell types (LCNEC), respectively. Of these 98 non-neuroendocrine tumors, INSM1 expression was seen in nine (9%) tumors, all were squamous cell carcinoma. And INSM1 staining was generally focal. INSM1 has high sensitivity and specificity in diagnosis of laryngeal NECs. For grading laryngeal NECs, Syn and CHG showed significant advantages in the diagnosis of WD-NEC and MD-NEC, whereas INSM1 and CD56 showed greater diagnostic value in the diagnosis of SCNEC and LCNEC, especially in SCNEC. 4 Laryngoscope, 131:E2662-E2668, 2021.