Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

BACKGROUND: As a dedifferentiated tumor, small cell endometrial neuroendocrine tumors (NETs) are rare and frequently diagnosed at an advanced stage with a poor prognosis. Current treatment recommendations are often extrapolated from histologically similar tumors in other sites or based on retrospective studies. The exploration for diagnostic and therapeutic markers in small cell NETs is of great significance. METHODS: In this study, we conducted single-cell RNA sequencing on a specimen obtained from a patient diagnosed with small cell endometrial neuroendocrine carcinoma (SCNEC) based on pathology. We revealed the cell map and intratumoral heterogeneity of the cancer cells through data analysis. Further, we validated the function of ISL LIM Homeobox 1 (ISL1) in vitro in an established neuroendocrine cell line. Finally, we examined the association between ISL1 and tumor staging in small cell lung cancer (SCLC) patient samples. RESULTS: We observed the significant upregulation of ISL1 expression in tumor cells that showed high expression of the neuroepithelial markers. Additionally, in vitro cell function experiments demonstrated that the high ISL1 expression group exhibited markedly higher cell proliferation and migration abilities compared to the low expression group. Finally, we showed that the expression level of ISL1 was correlated with SCLC stages. CONCLUSIONS: ISL1 protein in NETs shows promise as a potential biomarker for diagnosis and treatment.

Clinical Routine Application of the Second-generation Neuroendocrine Markers ISL1, INSM1, and Secretagogin in Neuroendocrine Neoplasia: Staining Outcomes and Potential Clues for Determining Tumor Origin.

Neuroendocrine neoplasms (NENs) have traditionally been identified via expression of proteins associated to the regulation of secretory vesicles and granules. We report the clinical usage of the "second-generation" proteins ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSM1), and secretagogin (SECG) as immunohistochemical markers of neuroendocrine differentiation since their introduction in clinical routine and compare the results with the established proteins chromogranin A (CGA) and synaptophysin (SYP). In total, 161 tumors, including 139 NENs and 22 "non-NENs" (unrelated tumors with an initial suspicion of NEN), were informatively stained for ISL1, and subsets were also interrogated for INSM1 and/or SECG. Diffuse or focal positive immunoreactivity was noted for ISL1 in 91/139 NENs (65%) and in 6/22 (27%) non-NENs, for INSM1 in 76/85 NENs (89%) and in 2/5 (40%) non-NENs, and for SECG in 49 out of 64 NENs (77%) and in 0/5 non-NENs (0%). Generally, ISL1, INSM1, and SECG exhibited sensitivities in line with or slightly below that of CGA and SYP-largely attributable to tissue-specific patterns regarding tumoral origin. Moreover, for pancreatic and small intestinal NENs, the two largest subgroups, ISL1 staining results were consistent irrespectively of tumor source and WHO grade. We verify previously suggested immunohistochemical schemes of neuroendocrine markers of first- and second-generations to facilitate the diagnostic process for NENs and confirm that the second-generation neuroendocrine markers display tissue-specific patterns. We therefore recommend their implementation in tertiary endocrine pathology centers, not least to aid in the identification of primary tumors when analyzing metastases.

Expression of SOX11, PAX5, TTF-1 and ISL-1 in medulloblastoma.

The aim of our study was to evaluate the immunohistochemical expression of SOX11, PAX5, TTF-1 and ISL-1 in medulloblastoma (MB) to investigate their diagnostic usefulness. METHODS: Immunohistochemical expression of PAX5 (two antibodies: Dako, DAK-Pax5; and BD, clone 24), TTF-1 (Dako, 8G7G3/1), SOX11 (CL0142; Abcam) and ISL-1 (1 H9, Abcam) was analyzed using the h-score and Remmele score in 25 cases of MB. RESULTS: There were 18 MBs of classic and 7 of desmoplastic type. SOX11 was strongly expressed in all tumors. The expression of PAX5 was higher and more frequent in a case of DAK-Pax5 clone (25/25) than clone 24 (6/25). ISL-1 was positive in 11 (44%) and TTF-1 in 3 (12%) cases. ISL-1 expression correlated positively (p<0.001), while TTF-1 correlated negatively with the age of patients (p=0.039). PAX5 expression correlated with ISL-1 (p=0.039) and showed a trend toward higher expression in the desmoplastic subtype (p=0.069). CONCLUSIONS: SOX11 is strongly and robustly expressed in MBs. PAX5 expression pattern differs substantially among two antibody clones. TTF-1 and ISL-1 is associated with the age of patients.

Expression pattern of ISL-1, TTF-1 and PAX5 in olfactory neuroblastoma.

Olfactory neuroblastoma (ONB) is a rare neoplasm of the sinonasal area with neuroendocrine differentiation. ISL-1, TTF-1 and PAX5 are transcription factors that are frequently upregulated in tumors showing neuroendocrine differentiation. The aim of our study was to evaluate these markers in a group of ONBs. We included 11 ONBs from 4 large university hospitals. Immunohistochemical expression of TTF-1, PAX5 and ISL-1 was evaluated. TTF-1, ISL-1 and PAX5 were expressed in 3/11 cases (27.27%, h-score: 3-45), 7/11 cases (63.64%, h-score: 23-200), and in 3/11 cases (27.77%, h-score 3-85), respectively. The patient with the strongest PAX5 reactivity exhibited an aggressive clinical course with rapid dissemination to the spine and death shortly after the diagnosis. No significant correlation in the expression of PAX5 and TTF-1 ( = 0.43; p = 0.18) was observed. ISL-1 is widely expressed in tumors with neuroendocrine differentiation and therefore of limited value in their differential diagnosis. TTF-1 positivity does not exclude the diagnosis of primary ONB, although usually only a small percentage of cells are positive. PAX5 expression is infrequent (27.27%) in ONB; however, if present it can be associated with a very aggressive clinical course.

Identification of common and differential mechanisms of glomerulus and tubule senescence in 24-month-old rats by quantitative LC-MS/MS.

Kidney aging together with related renal disease had become a major clinical problem. Understanding the mechanisms of aging was important for suspending senescence and decreasing the incidence of aging-related diseases. In the present work, 24-month-old F344 rats were used as aging rats and 3-month-old rats were used as young controls. Senescence-associated-ß-galactosidase staining results showed that the degree of senescence in renal tubules was more severe than that in glomeruli. We performed quantitative LC-MS to assess the differential protein expression profiles of senescent glomeruli and tubules. Bioinformatics analysis showed that aging, response to oxidative stress, nucleotide metabolism, amine acid metabolism, and inflammatory response were common mechanisms of glomerulus and tubule senescence. Differentially expressed proteins network mediated Golgi vesicle transport, actin filament based process, and regulation of cell death were associated with tubule senescence. More importantly, we found that the changes of four and a half LIM protein 2 (FHL2) were opposite in senescent glomeruli and tubules, and FHL2 could regulate p16 by suppressing T-box 3, which was involved in regulation of senescence in glomeruli and tubules. In conclusion, we assessed the mechanisms of senescence in aging glomeruli and tubules, and the results yielded new insight into kidney senescence.

Enhanced FHL2 and TGF-ß1 Expression Is Associated With Invasive Growth and Poor Survival in Malignant Melanomas.

OBJECTIVES: This study examines the expression and the role of four-and-a-half LIM domains protein 2 (FHL2) and transforming growth factor ß1 (TGF-ß1) in human malignant melanoma. It is determined whether both proteins influence melanoma survival time. METHODS: We analyzed the immunohistochemical staining intensities of FHL2 and TGF-ß1 in normal skin and in 50 malignant melanomas with different mutation status (BRAF-V600E, NRAS codon 61 mutation, and wild type). Survival data were available for 45 cases. RESULTS: In melanocytes of nonneoplastic human skin, FHL2 expression was absent. In contrast, 38 (76%) of 50 melanomas showed strong cytoplasmic and partly nuclear FHL2 expression. At the invasion front, cytoplasmic TGF-ß1 staining was observed in 32 (64%) of 50 melanomas, and a correlation of FHL2 and TGF-ß1 staining intensities was detectable. In follow-up analyses, enhanced FHL2 and TGF-ß1 staining intensities in the tumor invasion front were associated with poor survival. CONCLUSIONS: Enhanced FHL2 and TGF-ß1 expression is correlated with poor survival in human malignant melanoma. Protumorigenic effects of autocrine TGF-ß1 secretion might be exerted by induction of FHL2 expression in melanoma cells. Since melanomas treated with targeted therapies often do not show sufficient response rates, inhibition of FHL2 and/or TGF-ß1 might be a promising therapeutic approach.

Expression of the LIM homeobox domain transcription factor ISL1 (Islet-1) is frequent in rhabdomyosarcoma but very limited in other soft tissue sarcoma types.

The transcription factor ISL1 (islet-1) has emerged as a useful marker for metastatic pancreatic well differentiated neuroendocrine neoplasms, but recent studies showed wider expression in poorly differentiated neuroendocrine carcinomas from different sites as well as poorly differentiated neuroblastoma. Expression of ISL1 in soft tissue sarcomas has not been studied before.We evaluated ISL1 expression in 249 soft tissue tumour specimens from 249 patients and 17 precursor cell lymphoblastic lymphomas (ALL). ISL1 was not detected in any of 63 liposarcomas of different subtypes, 55 leiomyosarcomas, 22 solitary fibrous tumours, 20 undifferentiated pleomorphic/spindle cell sarcomas, 13 small cell synovial sarcomas and 17 ALL cases. Variable nuclear expression was detected in rhabdomyosarcoma (15/25, 60%), rhabdomyoblastic areas of malignant müllerian mixed tumours (5/5), Ewing sarcoma (2/12, very weak) and monophasic fibrous synovial sarcoma (2/29). More extensive staining (moderate to strong) was restricted to rhabdomyosarcoma. Taken by histological subtype, ISL1 was expressed more frequently in alveolar (9/11, 82%) versus non-alveolar (6/14, 43%) rhabdomyosarcoma. ISL1 is commonly expressed in rhabdomyosarcoma, particularly the alveolar subtype and should be distinguished from poorly differentiated neuroendocrine and neuroblastic neoplasms. Awareness of this finding helps to avoid misinterpretation as neuroendocrine neoplasms that would result in inappropriate therapeutic and prognostic consequences.

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

ISL1 expression is not restricted to pancreatic well-differentiated neuroendocrine neoplasms, but is also commonly found in well and poorly differentiated neuroendocrine neoplasms of extrapancreatic origin.

The human insulin gene enhancer-binding protein islet-1 (ISL1) is a transcription factor involved in the differentiation of the neuroendocrine pancreatic cells. Recent studies identified ISL1 as a marker for pancreatic well-differentiated neuroendocrine neoplasms. However, little is known about ISL1 expression in pancreatic poorly differentiated and in extrapancreatic well and poorly differentiated neuroendocrine neoplasms. We studied the immunohistochemical expression of ISL1 in 124 neuroendocrine neoplasms. Among pancreatic neuroendocrine neoplasms, 12/13 with poor differentiation were negative, whereas 5/7 with good differentiation but a Ki67 >20% were positive. In extrapancreatic neuroendocrine neoplasms, strong positivity was found in Merkel cell carcinomas (25/25), pulmonary small cell neuroendocrine carcinomas (21/23), medullary thyroid carcinomas (9/9), paragangliomas/pheochromocytomas (6/6), adrenal neuroblastomas (8/8) and head and neck neuroendocrine carcinomas (4/5), whereas no or only weak staining was recorded in pulmonary carcinoids (3/15), olfactory neuroblastomas (1/4) and basaloid head and neck squamous cell carcinomas (0/15). ISL1 stained the neuroendocrine carcinoma component of 5/8 composite carcinomas and also normal neuroendocrine cells in the thyroid, adrenal medulla, stomach and colorectum. Poorly differentiated neuroendocrine neoplasms, regardless of their ISL1 expression, were usually TP53 positive. Our results show the almost ubiquitous expression of ISL1 in extrapancreatic poorly differentiated neuroendocrine neoplasms and neuroblastic malignancies and its common loss in pancreatic poorly differentiated neuroendocrine neoplasms. These findings modify the role of ISL1 as a marker for pancreatic neuroendocrine neoplasms and suggest that ISL1 has a broader involvement in differentiation and growth of neuroendocrine neoplasms than has so far been assumed.

Islet-1 is a sensitive but not entirely specific marker for pancreatic neuroendocrine neoplasms and their metastases.

Islet-1 (Isl1) is a transcription factor involved in the embryogenesis of islets of Langerhans. Immunohistochemically, Isl1 is a sensitive lineage-specific marker for pancreatic neuroendocrine neoplasms (NENs) and their metastases. Its specificity has not been documented, nor have large numbers of NENs from other parts of the gut or other organs been studied. We examined Isl1 expression in 203 primary NENs (gastroenteropancreatic, lung, breast, and ovarian neoplasms) and 40 hepatic NEN metastases (enteropancreatic and lung neoplasms) from known primaries. The correlation between Isl1 and CDX2 expression was studied using a tissue microarray containing 46 pancreatic NENs. Immunostaining for Isl1 and CDX2 was also performed in selected NENs from other sites. Isl1 was positive in 90% of pancreatic, 89% of duodenal, 100% of rectal, 38% of colonic, 14% of appendiceal, and 6% of ileal primaries. Isl1 was negative in all other NENs. Among metastatic neoplasms, 76% of pancreatic and 2 of 2 rectal NEN metastases were Isl1 positive, whereas all other tested metastases were negative. The overall sensitivity and specificity of Isl1 in identifying primary pancreatic NENs was 88% and 80%, respectively. Thirty-six of 46 pancreatic NENs in the tissue microarray were Isl1 positive; 4 were Isl1 negative but CDX2 positive. Our findings confirm that Isl1 is a sensitive marker of pancreatic origin in cases of metastatic NEN. However, Isl1 does not distinguish pancreatic NEN from duodenal and colorectal NEN, even when used in association with CDX2.

Value of Islet 1 and PAX8 in identifying metastatic neuroendocrine tumors of pancreatic origin.

Neuroendocrine tumors can present as liver metastases before discovery of the primary tumor. Islet 1 and PAX8 have recently been proposed as markers for neuroendocrine tumors of pancreatic origin. In this study, we compared the utility of Islet 1 and PAX8 in distinguishing pancreatic neuroendocrine tumors from neuroendocrine tumors of other sites and determined the usefulness of an immunohistochemical panel, including TTF1, CDX2, Islet 1 and/or PAX8, in identifying metastatic pancreatic neuroendocrine tumors. A total of 110 primary neuroendocrine tumors (33 pancreatic, 31 pulmonary, 23 ileal, 14 rectal, and 9 gastric) and 73 metastatic neuroendocrine tumors (28 pancreatic, 5 pulmonary, 37 ileal, 1 rectal, 1 colonic, and 1 duodenal) were studied. Islet 1 and PAX8 were positive in 27/33 (82%) and 29/33 (88%), respectively, of primary pancreatic neuroendocrine tumors, and in 19/28 (68%) and 15/28 (54%), respectively, of metastatic pancreatic neuroendocrine tumors. No cases of primary (0/23) or metastatic (0/37) ileal neuroendocrine tumors were positive with either Islet 1 or PAX8. There was Islet 1 positivity in 2/31 (6%) primary pulmonary, 12/14 (86%) primary rectal, and 1/1 metastatic rectal neuroendocrine tumors, and PAX8 positivity in 7/31 (23%) primary pulmonary, 11/14 (79%) primary rectal, and 2/9 (22%) primary gastric neuroendocrine tumors. ROC curve analysis incorporating sensitivity and specificity data of immunohistochemical panels for metastatic pancreatic neuroendocrine tumors showed that a four-stain panel, including Islet 1, PAX8, TTF1, and CDX2 significantly outperformed a three-stain panel composed of PAX8, TTF1, and CDX2 (P=0.019), and also showed a trend for better performance compared with a three-stain panel composed of Islet 1, TTF1, and CDX2 (P=0.072). Both Islet 1 and PAX8 are reliable immunohistochemical markers for pancreatic neuroendocrine tumors and would be useful adjuncts to other markers (TTF1, CDX2) currently used to work up a metastatic neuroendocrine tumor of unknown primary.