Synthesis and Antitumour Evaluation of Tricyclic Indole-2-Carboxamides against Paediatric Brain Cancer Cells.

Antitumour properties of some cannabinoids (CB) have been reported in the literature as early as 1970s, however there is no clear consensus to date on the exact mechanisms leading to cancer cell death. The indole-based WIN 55,212-2 and SDB-001 are both known as potent agonists at both CB1 and CB2 receptors, yet we demonstrate herein that only the former can exert in vitro antitumour effects when tested against a paediatric brain cancer cell line KNS42. In this report, we describe the synthesis of novel 3,4-fused tricyclic indoles and evaluate their functional potencies at both cannabinoid receptors, as well as their abilities to inhibit the growth or proliferation of KNS42 cells. Compared to our previously reported indole-2-carboxamides, these 3,4-fused tricyclic indoles had either completely lost activities, or, showed moderate-to-weak antagonism at both CB1 and CB2 receptors. Compound 23 displayed the most potent antitumour properties among the series. Our results further support the involvement of non-CB pathways for the observed antitumour activities of amidoalkylindole-based cannabinoids, in line with our previous findings. Transcriptomic analysis comparing cells treated or non-treated with compound 23 suggested the observed antitumour effects of 23 are likely to result mainly from disruption of the FOXM1-regulated cell cycle pathways.

Synthesis and antitumour evaluation of indole-2-carboxamides against paediatric brain cancer cells.

Paediatric glioblastomas are rapidly growing, devastating brain neoplasms with an invasive phenotype. Radiotherapy and chemotherapy, which are the current therapeutic adjuvant to surgical resection, are still associated with various toxicity profiles and only marginally improve the course of the disease and life expectancy. A considerable body of evidence supports the antitumour and apoptotic effects of certain cannabinoids, such as WIN55,212-2, against a wide spectrum of cancer cells, including gliomas. In fact, we previously highlighted the potent cytotoxic activity of the cannabinoid ligand 5 against glioblastoma KNS42 cells. Taken together, in this study, we designed, synthesised, and evaluated several indoles and indole bioisosteres for their antitumour activities. Compounds 8a, 8c, 8f, 12c, and 24d demonstrated significant inhibitory activities against the viability (IC50 = 2.34-9.06 µM) and proliferation (IC50 = 2.88-9.85 µM) of paediatric glioblastoma KNS42 cells. All five compounds further retained their antitumour activities against two atypical teratoid/rhabdoid tumour (AT/RT) cell lines. When tested against a medulloblastoma DAOY cell line, only 8c, 8f, 12c, and 24d maintained their viability inhibitory activities. The viability assay against non-neoplastic human fibroblast HFF1 cells suggested that compounds 8a, 8c, 8f, and 12c act selectively towards the panel of paediatric brain tumour cells. In contrast, compound 24d and WIN55,212-2 were highly toxic toward HFF1 cells. Due to their structural resemblance to known cannabimimetics, the most potent compounds were tested in cannabinoid 1 and 2 receptor (CB1R and CB2R) functional assays. Compounds 8a, 8c, and 12c failed to activate or antagonise both CB1R and CB2R, whereas compounds 8f and 24d antagonised CB1R and CB2R, respectively. We also performed a transcriptional analysis on KNS42 cells treated with our prototype compound 8a and highlighted a set of seven genes that were significantly downregulated. The expression levels of these genes were previously shown to be positively correlated with tumour growth and progression, indicating their implication in the antitumour activity of 8a. Overall, the drug-like and selective antitumour profiles of indole-2-carboxamides 8a, 8c, 8f, and 12c substantiate the versatility of the indole scaffold in cancer drug discovery.

Design, synthesis and evaluation of novel indole-2-carboxamides for growth inhibition of Mycobacterium tuberculosis and paediatric brain tumour cells.

The omnipresent threat of tuberculosis (TB) and the scant treatment options thereof necessitate the development of new antitubercular agents, preferably working via a novel mechanism of action distinct from the current drugs. Various studies identified the mycobacterial membrane protein large 3 transporter (MmpL3) as the target of several classes of compounds, including the indole-2-caboxamides. Herein, several indoleamide analogues were rationally designed, synthesised, and evaluated for their antitubercular and antitumour activities. Compound 8g displayed the highest activity (MIC = 0.32 µM) against the drug-sensitive (DS) Mycobacterium tuberculosis (M. tb) H37Rv strain. This compound also exhibited high selective activity towards M. tb over mammalian cells [IC50 (Vero cells) = 40.9 µM, SI = 128], suggesting its minimal cytotoxicity. In addition, when docked into the MmpL3 active site, 8g adopted a binding profile similar to the indoleamide ligand ICA38. A related compound 8f showed dual antitubercular (MIC = 0.62 µM) and cytotoxic activities against paediatric glioblastoma multiforme (GBM) cell line KNS42 [IC50 (viability) = 0.84 µM]. Compound 8f also showed poor cytotoxic activity against healthy Vero cells (IC50 = 39.9 µM). Compounds 9a and 15, which were inactive against M. tb, showed potent cytotoxic (IC50 = 8.25 and 5.04 µM, respectively) and antiproliferative activities (IC50 = 9.85 and 6.62 µM, respectively) against KNS42 cells. Transcriptional analysis of KNS42 cells treated with compound 15 revealed a significant downregulation in the expression of the carbonic anhydrase 9 (CA9) and the spleen tyrosine kinase (SYK) genes. The expression levels of these genes in GBM tumours were previously shown to contribute to tumour progression, suggesting their involvement in our observed antitumour activities. Compounds 9a and 15 were selected for further evaluations against three different paediatric brain tumour cell lines (BT12, BT16 and DAOY) and non-neoplastic human fibroblast cells HFF1. Compound 9a showed remarkable cytotoxic (IC50 = 0.89 and 1.81 µM, respectively) and antiproliferative activities (IC50 = 7.44 and 6.06 µM, respectively) against the two tested atypical teratoid/rhabdoid tumour (AT/RT) cells BT12 and BT16. Interestingly, compound 9a was not cytotoxic when tested against non-neoplastic HFF1 cells [IC50 (viability) = 119 µM]. This suggests that an indoleamide scaffold can be fine-tuned to confer a set of derivatives with selective antitubercular and/or antitumour activities.

Polo-Like Kinase 4 (PLK4) Is Overexpressed in Central Nervous System Neuroblastoma (CNS-NB).

Neuroblastoma (NB) is the most common extracranial solid tumor in pediatrics, with rare occurrences of primary and metastatic tumors in the central nervous system (CNS). We previously reported the overexpression of the polo-like kinase 4 (PLK4) in embryonal brain tumors. PLK4 has also been found to be overexpressed in a variety of peripheral adult tumors and recently in peripheral NB. Here, we investigated PLK4 expression in NBs of the CNS (CNS-NB) and validated our findings by performing a multi-platform transcriptomic meta-analysis using publicly available data. We evaluated the PLK4 expression by quantitative real-time PCR (qRT-PCR) on the CNS-NB samples and compared the relative expression levels among other embryonal and non-embryonal brain tumors. The relative PLK4 expression levels of the NB samples were found to be significantly higher than the non-embryonal brain tumors (p-value < 0.0001 in both our samples and in public databases). Here, we expand upon our previous work that detected PLK4 overexpression in pediatric embryonal tumors to include CNS-NB. As we previously reported, inhibiting PLK4 in embryonal tumors led to decreased tumor cell proliferation, survival, invasion and migration in vitro and tumor growth in vivo, and therefore PLK4 may be a potential new therapeutic approach to CNS-NB.

A functional screening of the kinome identifies the Polo-like kinase 4 as a potential therapeutic target for malignant rhabdoid tumors, and possibly, other embryonal tumors of the brain.

PURPOSE: Malignant rhabdoid tumors (MRTs) are deadly embryonal tumors of the infancy. With poor survival and modest response to available therapies, more effective and less toxic treatments are needed. We hypothesized that a systematic screening of the kinome will reveal kinases that drive rhabdoid tumors and can be targeted by specific inhibitors. METHODS: We individually mutated 160 kinases in a well-characterized rhabdoid tumor cell line (MON) using lentiviral clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The kinase that most significantly impaired cell growth was further validated. Its expression was evaluated by microarray gene expression (GE) within 111 pediatric tumors, and functional assays were performed. A small molecule inhibitor was tested in multiple rhabdoid tumor cell lines and its toxicity evaluated in zebrafish larvae. RESULTS: The Polo-like kinase 4 (PLK4) was identified as the kinase that resulted in higher impairment of cell proliferation when mutated by CRISPR/Cas9. PLK4 CRISPR-mutated rhabdoid cells demonstrated significant decrease in proliferation, viability, and survival. GE showed upregulation of PLK4 in rhabdoid tumors and other embryonal tumors of the brain. The PLK4 inhibitor CFI-400945 showed cytotoxic effects on rhabdoid tumor cell lines while sparing non-neoplastic human fibroblasts and developing zebrafish larvae. CONCLUSIONS: Our findings indicate that rhabdoid tumor cell proliferation is highly dependent on PLK4 and suggest that targeting PLK4 with small-molecule inhibitors may hold a novel strategy for the treatment of MRT and possibly other embryonal tumors of the brain. This is the first time that PLK4 has been described as a potential target for both brain and pediatric tumors.

Stabilization of HIF-1α and HIF-2α, up-regulation of MYCC and accumulation of stabilized p53 constitute hallmarks of CNS-PNET animal model.

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice's brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients' PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

Inhibition of polo-like kinase 4 (PLK4): a new therapeutic option for rhabdoid tumors and pediatric medulloblastoma.

Rhabdoid tumors (RT) are highly aggressive and vastly unresponsive embryonal tumors. They are the most common malignant CNS tumors in infants below 6 months of age. Medulloblastomas (MB) are embryonal tumors that arise in the cerebellum and are the most frequent pediatric malignant brain tumors. Despite the advances in recent years, especially for the most favorable molecular subtypes of MB, the prognosis of patients with embryonal tumors remains modest with treatment related toxicity dreadfully high. Therefore, new targeted therapies are needed. The polo-like kinase 4 (PLK4) is a critical regulator of centriole duplication and consequently, mitotic progression. We previously established that PLK4 is overexpressed in RT and MB. We also demonstrated that inhibiting PLK4 with a small molecule inhibitor resulted in impairment of proliferation, survival, migration and invasion of RT cells. Here, we showed in MB the same effects that we previously described for RT. We also demonstrated that PLK4 inhibition induced apoptosis, senescence and polyploidy in RT and MB cells, thereby increasing the susceptibility of cancer cells to DNA-damaging agents. In order to test the hypothesis that PLK4 is a CNS druggable target, we demonstrated efficacy with oral administration to an orthotropic xenograft model. Based on these results, we postulate that targeting PLK4 with small-molecule inhibitors could be a novel strategy for the treatment of RT and MB and that PLK4 inhibitors (PLK4i) might be promising agents to be used solo or in combination with cytotoxic agents.

Overexpression of TEAD4 in atypical teratoid/rhabdoid tumor: New insight to the pathophysiology of an aggressive brain tumor.

BACKGROUND: Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant embryonal brain tumor that occurs mainly in early childhood. Although most of the tumors are characterized by inactivating mutations of the tumor suppressor gene, SMARCB1, the biological basis of its tumorigenesis and aggressiveness is still unknown. PROCEDURE: We performed high-throughput copy number variation analysis of primary cell lines generated from primary and relapsed tumors from one of our patients to identify new genes involved in AT/RT biology. The expression of the identified gene was validated in 29 AT/RT samples by gene expression profiling, quantitative real-time polymerase chain reaction, and immunohistochemistry (IHC). Furthermore, we investigated the function of this gene by mutating it in rhabdoid tumor cells. RESULTS: TEAD4 amplification was detected in the primary cell lines and its overexpression was confirmed at mRNA and protein levels in an independent cohort of AT/RT samples. TEAD4's co-activator, YAP1, and the downstream targets, MYC and CCND1, were also found to be upregulated in AT/RT when compared to medulloblastoma. IHC showed TEAD4 and YAP1 overexpression in all samples. Cell proliferation and migration were significantly reduced in TEAD4-mutated cells. CONCLUSIONS: We report the overexpression of TEAD4 in AT/RT, which is a key component of Hippo pathway. Recent reports revealed that dysregulation of the Hippo pathway is implicated in tumorigenesis and poor prognosis of several human cancers. Our results suggest that TEAD4 plays a role in the pathophysiology of AT/RT, which represents a new insight into the biology of this aggressive tumor.

Loss of expression of the Neural Cell Adhesion Molecule 1 (NCAM1) in atypical teratoid/rhabdoid tumors: a new diagnostic marker?

Background: Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive embryonal tumors of the central nervous system. They are largely characterized by inactivating mutations of the SMARCB1 tumor suppressor gene. AT/RT patients have a very poor prognosis and no standard therapeutic protocol has been defined yet. Recently, multimodal therapy with multiple drug combinations has slightly improved the overall survival, however drug toxicity remains high. In this scenario, a better understanding of the pathophysiology of the disease is needed. Methods: We evaluated the gene expression profile of AT/RT samples to find new genetic factors contributing to the pathophysiology of the disease. We found target genes significantly differentially expressed between AT/RT and medulloblastoma (MB), the most common embryonal brain tumor. The mRNA expression was validated by quantitative real-time PCR and, at the protein level, expression was validated by immunohistochemistry in an independent set of tumors. Results: The Neural cell adhesion molecule 1 (NCAM1) gene was found to be consistently downregulated in AT/RT samples when compared to MB and normal brain tissue. Immunohistochemistry showed that the expression of NCAM1 in AT/RT was significantly lower than that of MB. Conclusion: NCAM1 is an important molecule involved in neuron-to-neuron and neuron-to-muscle adhesion during development. Downregulation of NCAM1 has been implicated in several human cancers suggesting that it might have a tumor repressor role. In this study we found a significantly reduced expression of NCAM1 in AT/RT when compared to MB and we suggest that this feature can be used as a diagnostic marker, along with demonstration of SMARCB1 (INI1) or SMARCA4 (BRG1) inactivation. The roles of NCAM1 in the pathophysiology of AT/RT are still to be determined (AU)

Spontaneous involution of pediatric low-grade gliomas: high expression of cannabinoid receptor 1 (CNR1) at the time of diagnosis may indicate involvement of the endocannabinoid system.

BACKGROUND: Pediatric low-grade gliomas (P-LGG) consist of a mixed group of brain tumors that correspond to the majority of CNS tumors in children. Notably, they may exhibit spontaneous involution after subtotal surgical removal (STR). In this study, we investigated molecular indicators of spontaneous involution in P-LGG. METHODS: We performed an integrated molecular analysis including high throughput gene expression (GE), microRNA (miRNA) expression data of primary, untreated tumors from patients with P-LGG who underwent STR at our institution, with at least 10 years follow-up. RESULTS: We identified a set of protein-coding genes and miRNAs significantly differentially expressed in P-LGG that presented spontaneous involution (involution-I) or without progression (stable-S) after STR alone. The cannabinoid receptor 1 (CNR1 or CB1) gene (FC = 2.374; p value = 0.007) was at the top of the list and predicted to be regulated by hsa-miR-29b-3p (FC = -2.353, p value = 0.0001). CNR1 also showed a trend to be higher expressed in S/I by immunohistochemistry. CONCLUSIONS: The P-LGG, which remained stable or that presented spontaneous involution after STR, showed significantly higher CNR1 expression at the time of diagnosis. We hypothesize that high expression levels of CNR1 provide tumor susceptibility to the antitumor effects of circulating endocannabinoids like anandamide, resulting in tumor involution. This corroborates with reports suggesting that CNR1 agonists and activators of the endocannabinoid system may represent therapeutic opportunities for children with LGG. We also suggest that CNR1 may be a prognostic marker for P-LGG. This is the first time spontaneous involution of P-LGG has been suggested to be induced by endocannabinoids.

Extensive miRNA expression analysis in craniopharyngiomas.

PURPOSE: Craniopharyngiomas are benign tumors of the sellar or parasellar regions. They arise from the remnants of Rathke's pouch and are considered a "developmental disease." microRNAs are short non-coding RNAs that play a key regulatory role in the control of expression of entire gene networks. We performed an extensive analysis of miRNAs in craniopharyngiomas aiming to identify a miRNA expression signature that might aid in the prognosis of disease progression and outcome. METHODS: Thirty-seven craniopharyngioma samples from twenty-three patients, ten age-matched controls from autopsy, and ten infant controls from the developing pituitary from autopsy were evaluated for the expression of 754 miRNAs using TaqMan® Low Density Arrays (TLDAs) v2.0 (Applied Biosystems, Foster City, CA). RESULTS: Among the most differentially expressed miRNAs, downregulation of miR-132 appears to be a marker of aggressiveness and also plays a role in epithelial-mesenchymal transition. CONCLUSIONS: This is the first time that an extensive study of miRNA expression has been performed in craniopharyngiomas. Further research needs to be performed to investigate the potential role of miR-132 in the development and progression of craniopharyngiomas, and its value as a prognostic marker of aggressiveness.

Atypical teratoid rhabdoid tumors of the posterior fossa in children.

PURPOSE: Atypical teratoid rhabdoid tumors (AT/RT) are rare, aggressive, central nervous system neoplasms that typically affect children under 3 years of age and have a very poor prognosis. Early case series consistently demonstrated rapid recurrence with progression to death, but more recent experience has shown significant improvements in progression free and overall survival. METHODS: A retrospective analysis of the clinical data of children diagnosed with AT/RT at the Ann & Robert H. Lurie Children's Hospital of Chicago (formerly Children's Memorial Hospital) between 2000 and 2014 was performed. Overall survival (OS) was used to describe outcome. Our small sample size and the utilization of different adjuvant regimens over the study period precluded a detailed statistical analysis. RESULTS: Eight children with AT/RT of the posterior fossa were included in our report. Gross total resection (GTR) was achieved in five children (63 %), two children underwent subtotal resection (25 %), and there was one who underwent biopsy. Patients were treated with various combinations of chemotherapy with or without conformal radiation therapy (RT). Median overall survival was 5 months (range 1 to 107 months) with two patients achieving sustained responses to 45 and 107 months. CONCLUSIONS: Our experience is in line with prior reports that show that children diagnosed with AT/RT of the posterior fossa have a poor prognosis, but that long-term survival is possible. These tumors provide many challenges, but contemporary series are beginning to show improvements in survival.

A mouse model of human primitive neuroectodermal tumors resulting from microenvironmentally-driven malignant transformation of orthotopically transplanted radial glial cells.

There is growing evidence and a consensus in the field that most pediatric brain tumors originate from stem cells, of which radial glial cells constitute a subtype. Here we show that orthotopic transplantation of human radial glial (RG) cells to the subventricular zone of the 3rd ventricle--but not to other transplantation sites--of the brain in immunocompromised NOD-SCID mice, gives rise to tumors that have the hallmarks of CNS primitive neuroectodermal tumors (PNETs). The resulting mouse model strikingly recapitulates the phenotype of PNETs. Importantly, the observed tumorigenic transformation was accompanied by aspects of an epithelial to mesenchymal transition (EMT)-like process. It is also noteworthy that the tumors are highly invasive, and that they effectively recruit mouse endothelial cells for angiogenesis. These results are significant for several reasons. First, they show that malignant transformation of radial glial cells can occur in the absence of specific mutations or inherited genomic alterations. Second, they demonstrate that the same radial glial cells may either give rise to brain tumors or differentiate normally depending upon the microenvironment of the specific region of the brain to which the cells are transplanted. In addition to providing a prospect for drug screening and development of new therapeutic strategies, the resulting mouse model of PNETs offers an unprecedented opportunity to identify the cancer driving molecular alterations and the microenvironmental factors that are responsible for committing otherwise normal radial glial cells to a malignant phenotype.

Histone deacetylases expression in atypical teratoid rhabdoid tumors.

PURPOSE: Atypical teratoid rhabdoid tumors (ATRTs) are rare, highly malignant central nervous system tumors that occur during infancy and early childhood. Their poor outcome and resistance to conventional chemotherapies and radiotherapy, urges the development of new therapies. Recent studies have evaluated the effects of histone deacetylase inhibitors (HDACi) as a new potential treatment for ATRTs. However, most HDACi act unselectively against all, or at least several, histone deacetylase (HDAC) family members. We hypothesized that specific HDAC family members are deregulated in ATRT and therefore a more selective class of HDACi would be beneficial to patients with ATRT. METHODS: To test our hypothesis, we evaluated the expression level of different HDAC family members in ATRTs. Eight ATRTs were compared to six medulloblastoma samples in regards to the level of expression of the 18 HDAC family members as determined by microarray gene expression profiling. RESULTS: HDAC1 was the only member of the HDAC family to be significantly differentially expressed in ATRTs (FC = 4.728; p value = 0.00003). CONCLUSIONS: A class of HDACi specifically targeting HDAC1 may allow for the desired therapeutic benefits with fewer side effects for children with ATRT.

Rhabdoid tumor: gene expression clues to pathogenesis and potential therapeutic targets.

Rhabdoid tumors (RT) are aggressive tumors characterized by genetic loss of SMARCB1 (SNF5, INI-1), a component of the SWI/SNF chromatin remodeling complex. No effective treatment is currently available. This study seeks to shed light on the SMARCB1-mediated pathogenesis of RT and to discover potential therapeutic targets. Global gene expression of 10 RT was compared with 12 cellular mesoblastic nephromas, 16 clear cell sarcomas of the kidney, and 15 Wilms tumors. In all, 114 top genes were differentially expressed in RT (P<0.001, fold change >2 or <0.5). Among these were downregulation of SMARCB1 and genes previously associated with SMARCB1 (ATP1B1, PTN, DOCK4, NQO1, PLOD1, PTP4A2, PTPRK); 28/114 top differentially expressed genes were involved with neural or neural crest development and were all sharply downregulated. This was confirmed by Gene Set Enrichment Analysis (GSEA). Neural and neural crest stem cell marker proteins SOX10, ID3, CD133, and Musashi were negative by immunohistochemistry, whereas Nestin was positive. Decreased expression of CDKN1A, CDKN1B, CDKN1C, CDKN2A, and CCND1 was identified, while MYC-C was upregulated. GSEA of independent gene sets associated with bivalent histone modification and polycomb group targets in embryonic stem cells showed significant negative enrichment in RT. Several differentially expressed genes were associated with tumor suppression, invasion, and metastasis, including SPP1 (osteopontin), COL18A1 (endostatin), PTPRK, and DOCK4. We conclude that RTs arise within early progenitor cells during a critical developmental window in which loss of SMARCB1 directly results in repression of neural development, loss of cyclin-dependent kinase inhibition, and trithorax/polycomb dysregulation.

Upregulation of mir-221 and mir-222 in atypical teratoid/rhabdoid tumors: potential therapeutic targets.

PURPOSE: The aim of this study is to search for new therapeutic targets for atypical teratoid-rhabdoid tumors (ATRT). METHODS: To achieve this, we compared the expression of 365 microRNAs among ATRT, medulloblastomas, and normal brain. RESULTS: MiR-221 and miR-222 were within the top differentially expressed microRNAs. The deregulated expression of miR221/222 was demonstrated to inhibit the expression of the tumor suppressor and inhibitor of cell cycle p27(Kip1). Here, we demonstrated the negative regulation of p27(Kip1) by miR-221/222 in ATRT using microarray, real-time reverse transcriptase polymerase chain reaction, and immunohistochemistry. CONCLUSION: As anti-miR therapy was recently proposed as an alternative treatment for cancer, these findings suggest that anti-miR-221/222 therapy might have therapeutic potential in ATRT.

Hepatoblastomas and liver development: a study of cytokeratin immunoexpression in twenty-nine hepatoblastomas.

Hepatoblastomas (HBs) recapitulate liver development. It is possible that HBs result from malignant transformation of hepatic precursor cells, and they may reflect a blockage in normal development. Here we study the expression of cytokeratins (CKs) in order to delineate the immunoprofile and relationship with liver development, as well as vimentin and alphafetoprotein (AFP), of HBs. Immunohistochemistry was performed in a tissue microarray (TMA) containing representative areas of 18 HBs (fetal and/or embryonal and/or mesenchymal); we also reviewed 11 cases not included in the TMA. No cases stained for CKs 1, 5/6, 7, 10, 13, 15, 16, 20, and 34betaE12. CK8 stained 73.07% of fetal, 50% of embryonal, and 18% of mesenchymal areas. CK18 stained 100% of epithelial areas. CK19 staining was intense and diffuse in 100% of embryonal samples, but it was weaker in fetal areas (66.66%). AE1 stained epithelial areas in all cases, and it stained 29.41% of mesenchymal areas. AE3 stained 84.61% of embryonal and 60% of fetal components. AE1/AE3 showed stronger staining in embryonal (100%) than in fetal areas (76.92%). Vimentin staining was strong in embryonal (66.66%) and mesenchymal (84.61%) components but weak in fetal areas (8%). Alphafetoprotein was positive in only 20% of fetal and 70% of embryonal areas. Our results support the hypothesis that immunoexpression of HBs follows the stages of normal liver development. Embryonal areas look less differentiated, expressing vimentin and biliary epithelium CKs, whereas fetal areas display a more developed phenotype, similar to that of mature hepatocytes. These data aid in understanding the ontogenesis of HBs and may be used in histopathological diagnosis.