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Performance of the new CE-IVD-marked HercepTest™ mAb pharmDx (Dako Omnis) assay (HercepTest (mAb)) was compared against the PATHWAY® anti-HER-2/neu (4B5) (PATHWAY 4B5) assay using 119 pre-selected breast cancer samples covering the entire range of HER2 immunohistochemistry (IHC) expression scores (0, 1 + , 2 + , 3 +). The sensitivity and specificity of both assays were assessed based on consensus IHC scores and amplification status, as determined by fluorescence in situ hybridization (FISH) according to 2018 ASCO/CAP testing guidelines. There was a high concordance between results from the HercepTest (mAb) and PATHWAY 4B5 assays for HER2-negative (IHC 0, 1 + , 2 + and FISH negative) and HER2-positive (IHC 3 + , 2 + and FISH positive) breast carcinomas (98.2%). Regarding individual IHC scores, complete agreement was achieved in 69.7% (83/119) of cases, and all but one of the discordant cases were due to higher HER2-status scoring using the HercepTest (mAb). Thus, more tumors were overscored as IHC 2 + by HercepTest (mAb) (27 versus 15) as evidenced by their lower FISH positivity rate (48.1% versus 80%). However, two amplified tumors identified as IHC 2 + by HercepTest (mAb) were missed by PATHWAY 4B5 (IHC 1 +). Four additional cases identified as IHC 2 + by HercepTest (mAb), with FISH ratio < 2 but elevated gene counts (≥ 4 to < 6), were recorded negative by PATHWAY 4B5. The HercepTest (mAb) detects HER2 expression with higher sensitivity in tumors with gene amplification (ISH group 1) and increased gene counts (ISH group 4) as well as in HER2-low tumors (HER2 IHC2 + /FISH negative or IHC 1 +). Future studies will demonstrate whether this translates into improved patient selection especially for new HER2-directed therapies., (© 2022. The Author(s).)

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 (IC 50 = 2.34-9.06 μM) and proliferation (IC 50 = 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 (CB 1 R and CB 2 R) functional assays. Compounds 8a , 8c , and 12c failed to activate or antagonise both CB 1 R and CB 2 R, whereas compounds 8f and 24d antagonised CB 1 R and CB 2 R, 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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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 [IC 50 (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 [IC 50 (viability) = 0.84 μM]. Compound 8f also showed poor cytotoxic activity against healthy Vero cells (IC 50 = 39.9 μM). Compounds 9a and 15, which were inactive against M. tb , showed potent cytotoxic (IC 50 = 8.25 and 5.04 μM, respectively) and antiproliferative activities (IC 50 = 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 (IC 50 = 0.89 and 1.81 μM, respectively) and antiproliferative activities (IC 50 = 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 [IC 50 (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., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Cancer cells have high demands for energy to maintain their exceedingly proliferative growth. However, the mechanism of energy expenditure in cancer is not well understood. We hypothesize that cancer cells might utilize energy-rich inorganic polyphosphate (polyP), as energetic reserve. PolyP is comprised of orthophosphates linked by phosphoanhydride bonds, as in ATP. Here, we show that polyP is highly abundant in several types of cancer cells, including brain tumor-initiating cells (BTICs), i.e., stem-like cells derived from a mouse brain tumor model that we have previously described. The polymer is avidly consumed during starvation of the BTICs. Depletion of ATP by inhibiting glycolysis and mitochondrial ATP-synthase (OXPHOS) further decreases the levels of polyP and alters morphology of the cells. Moreover, enzymatic hydrolysis of the polymer impairs the viability of cancer cells and significantly deprives ATP stores. These results suggest that polyP might be utilized as a source of phosphate energy in cancer. While the role of polyP as an energy source is established for bacteria, this finding is the first demonstration that polyP may play a similar role in the metabolism of cancer cells., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2020 Boyineni et al.)

Polo-like kinase 4 (PLK4) is a cell cycle-regulated protein kinase (PK) recruited at the centrosome in dividing cells. Its overexpression triggers centrosome amplification, which is associated with genetic instability and carcinogenesis. In previous work, we established that PLK4 is overexpressed in pediatric embryonal brain tumors (EBT). We also demonstrated that PLK4 inhibition exerted a cytostatic effect in EBT cells. Here, we examined an array of PK inhibitors (CFI-400945, CFI-400437, centrinone, centrinone-B, R-1530, axitinib, KW-2449, and alisertib) for their potential crossover to PLK4 by comparative structural docking and activity inhibition in multiple established embryonal tumor cell lines (MON, BT-12, BT-16, DAOY, D283). Our analyses demonstrated that: (1) CFI-400437 had the greatest impact overall, but similar to CFI-400945, it is not optimal for brain exposure. Also, their phenotypic anti-cancer impact may, in part, be a consequence of the inhibition of Aurora kinases (AURKs). (2) Centrinone and centrinone B are the most selective PLK4 inhibitors but they are the least likely to penetrate the brain. (3) KW-2449, R-1530 and axitinib are the ones predicted to have moderate-to-good brain penetration. In conclusion, a new selective PLK4 inhibitor with favorable physiochemical properties for optimal brain exposure can be beneficial for the treatment of EBT.

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.

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC's orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype., Competing Interests: CONFLICTS OF INTEREST The authors have declared that no competing interests exist.

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., Competing Interests: CONFLICTS OF INTEREST Although we have no conflicts of interest, we would like to declare that David R. Piper and Tufan Gokimak are employees of Thermo Fisher Scientific. They participated in the confluency assays, as well as in the review, editing and approval of the manuscript.

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., (© 2017 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals, Inc.)

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., (© 2016 Wiley Periodicals, Inc.)

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.

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.

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.

Objective: To identify differentially expressed microRNA (miRNA) in muscle biopsies (MBx) from 15 untreated children with juvenile dermatomyositis (JDM) compared with 5 controls., Methods: Following MBx miRNA profiling, differentially expressed miRNA and their protein targets were validated by quantitative real-time PCR (qRT-PCR) and immunological assay. The association of miRNA-10a and miRNA-10b with clinical data was evaluated, including Disease Activity Score (DAS), von Willebrand factor antigen (vWF:Ag), nailfold capillary end row loops, duration of untreated disease, and tumor necrosis factor (TNF)-α-308A allele., Results: In JDM, 16/362 miRNA were significantly differentially expressed [false discovery rate (FDR) < 0.05]. Among these, miRNA-10a was the most downregulated miRNA in both FDR and ranking of fold change: miRNA-10a = -2.27-fold, miRNA-10b = -1.80-fold. Decreased miRNA-10a and miRNA-10b expressions were confirmed using q, Rt-Pcr: -4.16 and -2.59 fold, respectively. The qRT-PCR documented that decreased miRNA-10a expression was related to increased vascular cell adhesion molecule 1 in 13 of these JDM cases (correlation -0.67, p = 0.012), unlike miRNA-10b data (not significant). Concurrent JDM plasma contained increased levels of interleukin (IL) 6 (p = 0.0363), IL-8 (p = 0.0005), TNF-α (p = 0.0011), and monocyte chemoattractant proteins 1 (p = 0.0139). Decreased miRNA-10a, but not miRNA-10b, was associated with the TNF-α-308A allele (p = 0.015). In the 15 JDM, a trend of association of miRNA-10a (but not miRNA-10b) with vWF:Ag and DAS was observed., Conclusion: MiRNA-10a downregulation is an important element in untreated JDM muscle pathophysiology. We speculate that muscle miRNA expression in adult dermatomyositis differs from muscle miRNA expression in untreated childhood JDM.

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.

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.

Rhabdoid tumors (RT), or malignant rhabdoid tumors, are among the most aggressive and lethal forms of human cancer. They can arise in any location in the body but are most commonly observed in the brain, where they are called atypical teratoid/rhabdoid tumors (AT/RT), and in the kidneys, where they are called rhabdoid tumors of the kidney. The vast majority of rhabdoid tumors present with a loss of function in the SMARCB1 gene, also known as INI1, BAF47, and hSNF5, a core member of the SWI/SNF chromatin-remodeling complex. Recently, mutations in a 2nd locus of the SWI/SNF complex, the SMARCA4 gene, also known as BRG1, were found in rhabdoid tumors with retention of SMARCB1 expression. Familial cases may occur in a condition known as rhabdoid tumor predisposition syndrome (RTPS). In RTPS, germline inactivation of 1 allele of a gene occurs. When the mutation occurs in the SMARCB1 gene, the syndrome is called RTPS1, and when the mutation occurs in the SMARCA4 gene it is called RTPS2. Children presenting with RTPS tend to develop tumors at a younger age, but the impact that germline mutation has on survival remains unclear. Adults who carry the mutation tend to develop multiple schwannomas. The diagnosis of RTPS should be considered in patients with RT, especially if they have multiple primary tumors, and/or in individuals with a family history of RT. Because germline mutations result in an increased risk of carriers developing RT, genetic counseling for families with this condition is recommended.

Atypical teratoid/rhabdoid tumors (AT/RTs) are aggressive brain tumors that are commonly associated with a dismal prognosis. However, there have been isolated reports of long-term survival that was not necessarily correlated with other prognostic factors such as age, clinical stage, or extent of surgical resection. Here, we report the case of a 6-year-old boy with AT/RT who remained disease-free for 8 years after undergoing subtotal surgical resection followed only by radiation therapy. On recurrence, the tumor rapidly progressed, leading to the patient's death a short time later. To further characterize this case and learn more about the tumor biology of long-term survivors, we compared the gene expression (GE) profiles from representative samples obtained from primary, recurrent, and progressive disease tumors of the above-mentioned patient along with a cohort of primary untreated AT/RT samples using cDNA microarrays. Global GE analysis and unsupervised hierarchical clustering showed the three events clustered together and distinctly apart from the rest of the samples. This indicates a GE background that is maintained throughout the course of the disease. This unique case suggests that there may be specific clinical characteristics associated with distinctive molecular subtypes of AT/RT. The identification and characterization of AT/RT subtypes could lead to advances in both prognosis and treatment of these tumors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: Malignant rhabdoid tumors (MRT) can occur in a variety of anatomical sites. The most frequent locations are the brain, where they are named atypical teratoid/rhabdoid tumors (AT/RT), and the kidney, where they are named rhabdoid tumors of the kidney (RTK). MRTs at all sites are recognized as the same entity due to their similar morphology, aggressive behavior, and a common genetic abnormality, an inactivating mutation of the SMARCB1/INI-1/hSNF5/BAF47 gene. We aim to investigate potential molecular differences between AT/RT and RTK., Methods: We analyzed the microRNA (miRNA) and gene expression (GE) profiles of 10 RTK, 13 AT/RT, and 2 human MRT cell lines (G401-RTK and MON-AT/RT). Illumina V2 MicroRNA Chips (Illumina, Inc., CA, USA) were used for miRNA analysis, and Illumina HT-12 whole genome expression arrays were used for GE analysis., Results: The distribution of p values from GE showed a significant difference between RTK and AT/RT, with 20 % of the genes having p values ≤0.05 and the principal component analysis of the GE data showed separation between RTK and AT/RT. However, the miRNA expression failed to identify the different tumor groups. Among the 122 genes significantly differentially expressed between AT/RT and RTK, we found both genes related to brain development (i.e., FABP7, 22-fold increase in AT/RT) and genes related to kidney development (i.e., TCF21, sixfold increase in RTK)., Conclusion: Based on our results, we hypothesized that although MRT are indeed the same tumor, independent of the site of origin, the GE differences reflect the influence of microenvironment over tumor development.

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.

Objective: To evaluate the effect of duration of untreated disease on vascular cell adhesion molecule 1 (VCAM-1) and microRNA (miRNA) expression in muscle biopsy samples from children with juvenile dermatomyositis (DM) as well as its effect on soluble VCAM-1 (sVCAM-1) and tumor necrosis factor α (TNFα) concentrations in sera from these children., Methods: We enrolled 28 untreated children with juvenile DM and 8 pediatric controls. Eleven children with juvenile DM had short duration of untreated disease (symptoms for ≤2 months before muscle biopsy), and 17 had long duration of untreated disease (symptoms for >2 months before muscle biopsy). Vascular structures, characterized by immunofluorescence using antibodies against von Willebrand factor, VCAM-1, and α-smooth muscle actin, were measured for total area and intensity. Circulating sVCAM-1 and TNFα levels were determined in patients with short duration of untreated disease, patients with long duration of untreated disease, and controls. Differential expression of microRNA-126 (miR-126) in muscle biopsy samples from the 2 patient groups and the control group was detected by miRNA expression profiling and confirmed by quantitative reverse transcription-polymerase chain reaction in muscle biopsy samples from the 3 groups., Results: Juvenile DM patients with short duration of untreated disease had significantly higher total positive area and intensity/high power field of VCAM-1 expression than did juvenile DM patients with long duration of untreated disease (P = 0.043 and P = 0.015, respectively) or controls (P = 0.004 and P = 0.001, respectively). Von Willebrand factor antigen-positive vasculature displayed greater VCAM-1 intensity in patients with short duration of untreated disease than in patients with long duration of untreated disease (P = 0.001). Circulating levels of sVCAM-1 and TNFα were significantly higher in patients with short duration of untreated disease than in controls (P = 0.013 and P = 0.048, respectively). The miRNA miR-126, a negative regulator of VCAM-1 expression, was significantly decreased (3.39-fold; P < 0.006) in patients with short duration of untreated disease compared to controls, while miR-126 expression in patients with long duration of untreated disease did not differ significantly compared to controls., Conclusion: In patients with short duration of untreated disease, miR-126 down-regulation is associated with increased VCAM-1 in both muscle and blood, suggesting that VCAM-1 plays a critical role early in juvenile DM disease pathophysiology, augmented by TNFα., (Copyright © 2012 by the American College of Rheumatology.)

Background: Aging is a complex process that involves the interplay of genetic, epigenetic, and environmental factors. Identifying aging-related biomarkers holds great potential for improving our understanding of complex physiological changes, thereby providing a means to investigate the mechanism by which aging influences various diseases., Method and Results: We performed a parallel study of microRNA and gene expression profiling of peripheral blood in a group of healthy young adult women, among which 13 were aged 22-25 and 9 were aged 36-39 years old. We identified a significantly distinct pattern of microRNA, but not gene expression profiling, between these two young adult women groups. We also performed correlation analysis of expression levels between all pairs of age-associated microRNAs and genes and identified a weak global correlation between these two types of expression levels. A significant involvement of estrogen regulation was observed by pathway analysis of the most differentially expressed microRNAs that included miR-155, -18a, -142, -340, -363, -195, and -24., Conclusion: Our results suggest that the change in global microRNA expression in the peripheral blood is associated with normal aging in young adult women. This change may precede global gene expression changes. Future studies are needed to investigate the regulatory mechanism of the estrogen-related microRNAs and associated diseases.

Introduction: We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers., Materials and Methods: We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features., Results: We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival., Conclusion: We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.

Retinoblastoma is the most frequent intra-ocular malignant tumor of the childhood, occurring in 1 of 18,000-30,000 live births. Little is known about the causes of sporadic retinoblastoma and only a few authors have investigated the etiologic role of human papillomavirus (HPV), with controversial results. Formalin-fixed, paraffin-embedded tissue blocks containing retinoblastoma were retrieved from the archives of the Department of Pathology at Hospital A C Camargo, São Paulo, Brazil. All patients were treated with enucleation (21 children had both eyes enucleated). Retinoblastoma and, when possible, normal retina of each specimen, were micro-dissected under direct light microscopic visualization by using a PixCell II Laser Capture Micro-dissection System. The DNA quality was evaluated by polymerase chain reaction (PCR) amplification of 110 base pairs fragment of the human β-globin gene using primers PCO3+/PCO4+. All globin positive specimens were analyzed by PCR for the presence of HPV DNA using consensus primers GP5+/GP6+. A total of 154 specimens were evaluated. Forty-four patients also had normal retinal specimens available for analysis of DNA HPV. The DNA HPV prevalence among all tumor specimens was 4.6% (95% CI 2.0; 8.8) (7 positive specimens/153 adequate specimens). Among normal retinal specimens, the DNA HPV prevalence was 9.1% (95% CI 2.9; 20.5) (4 positive specimens/44 specimens). There was no statistically significant difference between these rates (P = 0.318). Excluding any experimental failure, our results indicate a low prevalence of HPV DNA in retinoblastomas. We were therefore unable to conclude about the association between these oncogenic viruses and this rare pediatric neoplasm., (© 2010 Wiley-Liss, Inc.)

Background: Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established--namely, the Swarm Rat Chondrosarcoma (SRC)--and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy., Methods: To examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC., Results: The site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-β4, c-fos, and CTGF may play in chondrosarcoma development and progression., Conclusion: This report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-β4 may have a role in chondrosarcoma metastasis.

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.

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.

Abnormal patterns of DNA methylation are observed in several types of human cancer. While localized DNA methylation of CpG islands has been associated with gene silencing, the effect that genome-wide loss of methylation has on tumorigenesis is not completely known. To examine its effect on tumorigenesis, we induced DNA demethylation in a rat model of human chondrosarcoma using 5-aza-2-deoxycytidine. Rat specific pyrosequencing assays were utilized to assess the methylation levels in both LINEs and satellite DNA sequences following 5-aza-2-deoxycytidine treatment. Loss of DNA methylation was accompanied by an increase in invasiveness of the rat chondrosarcoma cells, in vitro, as well as by an increase in tumor growth in vivo. Subsequent microarray analysis provided insight into the gene expression changes that result from 5-aza-2-deoxycytidine induced DNA demethylation. In particular, two genes that may function in tumorigenesis, sox-2 and midkine, were expressed at low levels in control cells but upon 5-aza-2-deoxycytidine treatment these genes became overexpressed. Promoter region DNA analysis revealed that these genes were methylated in control cells but became demethylated following 5-aza-2-deoxycytidine treatment. Following withdrawal of 5-aza-2-deoxycytidine, the rat chondrosarcoma cells reestablished global DNA methylation levels that were comparable to that of control cells. Concurrently, invasiveness of the rat chondrosarcoma cells, in vitro, decreased to a level indistinguishable to that of control cells. Taken together these experiments demonstrate that global DNA hypomethylation induced by 5-aza-2-deoxycytidine may promote specific aspects of tumorigenesis in rat chondrosarcoma cells.

Object: Direct delivery of chemotherapeutic agents for the treatment of brain tumors is an area of focus in the development of therapeutic paradigms because this method of delivery circumvents the blood-brain barrier without causing adverse systemic side effects. Few studies have investigated longitudinal tumor response to this type of therapy. In this study, the authors examined the time course of tumor response to direct delivery of a chemotherapeutic agent in a rodent malignant glioma model., Methods: To visualize tumor response to chemotherapy, the authors used bioluminescence imaging in a rodent model. Rat 9L gliosarcoma cells expressing a luciferase gene were inoculated into adult male rat striata. Ten days following surgery the animals were randomly divided into 4 groups. Groups 1 and 2 received 20 and 40 microl carboplatin (1 mg/ml), respectively, via convection-enhanced delivery (CED); Group 3 received 60 mg/kg carboplatin intraperitoneally; and Group 4 received no treatment. Tumor growth was correlated with luminescence levels twice weekly., Results: Differential growth curves were observed for the 4 groups. Systemically treated rats showed decreasing photon flux emission at 15.0 + or - 4.7 days; rats treated with 20- or 40-microl CED showed decreased emissions at 4.0 + or - 2.0 and 3.2 + or - 1.3 days after treatment, respectively. Histopathologically, 6 of 12 CED-treated animals exhibited no residual tumor at the end point of the study., Conclusions: Direct and systemic delivery of carboplatin was examined to determine how the method of drug delivery affects tumor growth. The present report is one of the first in vivo studies to examine the time course of tumor response to direct drug delivery. The results indicate that direct drug delivery may be a promising option for treating gliomas.

Purpose: Recent studies suggest that children <24 months with stage I favorable histology Wilms tumors <550 g [very low risk Wilms tumors (VLRWT)] have an excellent prognosis when treated with nephrectomy only, without adjuvant chemotherapy. The identification of risk categories within VLRWT may enable refinement of their definition and optimization of their therapy., Experimental Design: To define biologically distinct subsets, global gene expression analysis was done on 39 VLRWT that passed all quality-control parameters and the clusters identified were validated in an independent set of 11 VLRWT. Validation of select differentially expressed genes was done with immunohistochemistry on a tissue microarray from 20 of 39 tumors. Loss of heterozygosity (LOH) for 11p15, 1p, and 16q was analyzed in 52 tumors using PCR., Results: Two distinctive clusters were identified. One cluster included 9 tumors with epithelial differentiated tubular histology, paucity of nephrogenic rests, lack of LOH for 1p, 16q, and 11p, absence of relapse, and a unique gene expression profile consistent with arrest following mesenchymal-to-epithelial transition. The second cluster included 13 tumors with mixed histology, intralobar nephrogenic rests, and decreased expression of WT1. Three of 6 relapses occurred in this cluster. Of 43 informative tumors, 11p LOH was present in 5 of 5 relapses and 11 of 38 nonrelapses., Conclusions: Two subsets comprising a total of 56% of VLRWT are identified that have pathogenetic and molecular differences and apparent differences in risk for relapse. If these predictors can be prospectively validated, this would enable the refinement of clinical stratification and less arbitrary definition of VLRWT.

Objective: To investigate the possible role of chromatin texture parameters, nuclear morphology, DNA ploidy and clinical functional status in discriminating benign from malignant adrenocortical tumors (ACT)., Patients and Methods: Forty-eight cases of clinically benign (n=40) and clinically malignant (n=8) ACT with a minimum of 5-years' follow-up were evaluated for chromatin texture parameters (run length, standard deviation, configurable run length, valley, slope, peak and other 21 Markovian features that describe the distribution of the chromatin in the nucleus), nuclear morphology (nuclear area, nuclear perimeter, nuclear maximum and minimum diameter, nuclear shape), and DNA ploidy. Nuclear parameters were evaluated in Feulgen-stained 5 mum paraffin-sections analyzed using a CAS 200 image analyzer., Results: Since ACTs present different biological features in children and adults, patients were divided into two groups: children (< or = 15 years) and adults (>15 years). In the group of children DNA ploidy presented a marginal significance (p=0.05) in discriminating ACTs. None of the parameters discriminated between malignant and benign ACT in the adult group., Conclusion: ACTs are uncommon and definitive predictive criteria for malignancy remain uncertain, particularly in children. Our data point to DNA content evaluated by image analysis as a new candidate tool for this challenging task. Texture image analysis did not help to differentiate malignant from benign adrenal cortical tumors in children and adults.

Purpose: Local direct delivery of chemotherapeutic agents for the treatment of brain tumors is an area of focus in the development of new therapeutic paradigms. These techniques need improvement, especially in terms of drug retention in brain tissue., Materials and Methods: In this study, we used a rat glioma model to examine carboplatin distribution, as measured by platinum penetration, after delivery via interstitial continuous (i.c.) infusion. We also examined rat survival times in response to carboplatin and oxaliplatin. I.C. infusion, a modified version of convection-enhanced delivery (CED) for local drug delivery, uses low volume (1 microl per hour) continuous infusion directly into the tumor., Results: I.C. infusion produced a nearly 360-fold higher concentration of platinum in tumor tissue and significantly prolonged rodent survival time compared to intraperitoneal (i.p.) infusion., Conclusions: We showed i.c. infusion allows for circumvention of the blood-brain barrier, focused drug distribution, and sustained drug delivery. This method could be a promising strategy for treating brain tumors.

Purpose: Currently, there is no conclusive treatment for brainstem tumor. To facilitate the development of new treatments, it is essential to establish predictive preclinical in vivo models in which therapeutic modalities can be evaluated. Although a few rodent models have been reported, there is no novel approach that can monitor tumor response qualitatively and quantitatively., Materials and Methods: Bioluminescence imaging was used to characterize a rat brainstem tumor model. In this model, 9L gliosarcoma cells, transduced with an onco-retroviral vector containing the luciferase coding sequence, were inoculated into Fisher 344 rats., Result: Histopathological assessment showed successful cell implantation into the brainstem. There was a strong correlation between pathological tumor volume and luminescence strength. Longitudinal quantitative responses of the tumor after application of a therapeutic agent were also demonstrated., Conclusion: This study demonstrates a robust rodent model with the ability to monitor brainstem tumor growth and response to chemotherapeutic agents.

Purpose: The past two decades has seen significant improvement in the overall survival of patients with favorable histology Wilms tumor (FHWT); however, this progress has reached a plateau. Further improvements may rely on the ability to better stratify patients by risk of relapse. This study determines the feasibility and potential clinical utility of classifiers of relapse based on global gene expression analysis., Experimental Design: Two hundred fifty FHWT of all stages enriched for relapses treated on National Wilms Tumor Study-5 passed quality variables and were suitable for analysis using oligonucleotide arrays. Relapse risk stratification used support vector machine; 2- and 10-fold cross-validations were applied., Results: The number of genes associated with relapse was less than that predicted by chance alone for 106 patients (32 relapses) with stages I and II FHWT treated with chemotherapy, and no further analyses were done. This number was greater than expected by chance for 76 local stage III patients. Cross-validation including an additional 68 local stage III patients (total 144 patients, 53 relapses) showed that classifiers for relapse composed of 50 genes were associated with a median sensitivity of 47% and specificity of 70%., Conclusions: This study shows the feasibility and modest accuracy of stratifying local stage III FHWT using a classifier of <50 genes. Validation using an independent patient population is needed. Analysis of genes differentially expressed in relapse patients revealed apoptosis, Wnt signaling, insulin-like growth factor pathway, and epigenetic modification to be mechanisms important in relapse. Potential therapeutic targets include FRAP/MTOR and CD40.

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common deregulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset., (Copyright 2008 S. Karger AG, Basel.)

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.

Context and Objective: Carcinoid tumors are very rare both in children and adults. About 85% of these tumors develop in the gastrointestinal tract. The objective of the present study was to describe our experience with children treated of carcinoid tumors, and investigate the frequency morphological findings and results., Design and Setting: Report on case series, at the Department of Pediatrics of Centro de Tratamento e Pesquisa Hospital do Câncer, São Paulo., Methods: This was a retrospective analysis of clinical pathological data and outcomes among children (< 18 years old) with carcinoid tumors admitted from January 1, 1990, to December 31, 2001., Results: Nine patients (mean age 12.2 years) were included: six girls and three boys (2:1), all of them Caucasian. In eight cases (89%), the primary tumor site was the appendix and in one (11%) it was the left bronchus. For those with primary tumor in the appendix, the main complaint was abdominal pain, which led to appendectomy. Only one patient underwent right hemicolectomy due to tumor extension into the serosa. The patient with bronchial tumor underwent left pneumonectomy. All patients had localized disease and are alive and free of disease. They have had follow-ups lasting from 1 to 11 years (mean of 3.5 years)., Conclusion: Although the majority of carcinoid tumors arise from the appendix, these tumors can also occur in other primary sites. Surgical resection at an early stage allows for good prognosis without the need for any adjuvant treatment.

Wilms' tumor is one of the most common solid tumors in children and is an interesting model for understanding the pathogenesis of embryonal tumors. Cytokeratins are intracellular fibrous proteins present in tissue of epithelial origin. The immunoexpression of the pan-cytokeratin AE1AE3 was studied in paraffin-embedded tissue sections from 24 Wilms' tumors (12 with nephrogenic rests) and also tissue samples from 15 corresponding normal kidneys, to evaluate epithelial differentiation in the genesis of Wilms' tumor. We observed that the intensity of the expression of AE1AE3 in the epithelial component of Wilms' tumors directly correlated with the degree of maturity of the epithelial structures correspondent to the collecting ducts.

We report a case of a 16-month-old girl with a primary endodermal sinus tumor (EST) of the parotid gland. The girl was admitted to the hospital with a left side cervical tumor with a quick growth (within 3 wk). The tumor was surgically resected. The gross examination showed a 7.0-cm hemorrhagic and fragmentized mass. Diagnosis of an EST was established on the hematoxylin and eosin-stained slides from the resected material. Elevated serum alpha-fetoprotein (AFP) levels were detected and follow-up examinations of the patient did not show ESTs in other locations or other manifestations due to the tumor. The patient received chemotherapy after the surgery and remained alive without evidence of disease at the time of this writing (2 years after the diagnosis). As far as we know, this is the second report in the literature of a parotid gland's EST.

Meningiomas are rare in children and a small number of cases has been published. They are tumors typically indolent, but sometimes they show an extreme aggressive behavior. The gold standard of treatment is surgery with total remove of tumor. If the surgery is not possible to do, the options of treatment are few. We describe a case of an atipical meningioma in a 3 years-old girl.

Aims: To demonstrate the correlation between morphological and proliferation parameters with the biological behaviour of adrenocortical tumours (ACTs) in different age groups., Methods: Seventy-six cases of ACT were evaluated comparing tumours of children (< or =15 years, n=24) and adults (>15 years, n=52) regarding morphological features and proliferation rates. These parameters were also compared in clinically benign (CB) and clinically malignant (CM) tumours. Tumours were analysed according to Weiss' histological parameters. Proliferation rates were assessed by MIB-1 labelling index and silver binding nucleolar organiser regions (AgNORs) were quantified by image analysis., Results: ACTs in children presented higher proliferative activity than in adults. In adult's ACTs, MIB-1 labelling index (cut-off, 10%) (P=0.037) and the AgNOR pattern (P<0.001) had influence over the disease-free survival (DFS). The AgNOR pattern type I was associated with CB-ACT and the AgNOR pattern type II with CM-ACT. The AgNOR pattern type III was characteristic of children's ACTs and type IV was characteristic of non-neoplasic adrenal glands., Conclusion: ACTs in children had a different morphological spectrum and higher proliferative activity than those tumours in adults, which should be always analysed as a separate group. In adults, our findings reinforce that the morphological criteria proposed by Weiss are an efficient way of differentiating CB-ACT from CM-ACT.

Mutations of the tumor suppressor gene p53 have been considered to be important determinants in several kinds of human cancer. Accumulation of p53 protein has been reported to correlate with more aggressive clinical behavior in some neoplasms. The role of p53 expression in adrenal cortical tumors (ACT) has not been elucidated but some studies have suggested its correlation with malignant behavior. Our objective was to determine if there is a correlation between the expression of immunoreactive p53 and the biological behavior of ACT. Fifty-seven ACT (21 from children and 36 from adults) were evaluated for p53 expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue and analyzed in terms of outcome. The p53 parameter was utilized semiquantitatively. Tumors were classified as p53 negative when no positivity was observed, or when only few cells showed weak positivity (0/1+) and scored as p53 positive when there was a diffuse and strong nuclear positivity (2+/3+). In children, p53 positivity was associated with clinically malignant ACT and p53 negativity was associated with clinically benign ACT (P = 0.026). In adults' ACT, p53 positivity had an effect on disease-free survival (P<0.001) and also correlated with Weiss score, with a cutoff = 4 (P = 0.04). p53 expression was related to the clinical behavior of ACT in both children and adults and these findings seem to support a role for p53 in ACT progression.

Background: Mutations of the tumor suppressor gene p53 are commonly found in several kinds of human cancer. In some types of neoplasms, accumulation of p53 protein has been reported to correlate with more aggressive clinical behavior. The role of p53 expression in Wilms tumors (WT) is not clear yet, but most studies have confirmed its correlation with anaplasia and advanced stage disease., Procedure: Ninety-seven WT were evaluated for p53 expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue and correlated with outcome. Tumors were classified as p53-Negative (p53-N) when no positivity was observed or only few cells showed weak positivity (0/1+) and p53-Positive (p53-P) when there was a diffuse and strong nuclear positivity (2+/3+)., Results: p53-P was detected in 13 out of 97 tumors and was associated with disease relapse (39 vs.17%; P = 0.06) but not with anaplasia. Among p53-N patients only 5% had metastatic disease compared with 31% of the p53-P sample. (P = 0.038). Overall survival was 94% for patients with p53-N vs. 85% for patients with p53-P at 1 year (P = 0.34)., Conclusions: p53 expression in Wilms tumor detected by immunohistochemistry seems to be associated with advanced disease and relapse., (Copyright 2001 Wiley-Liss, Inc.)

OBJECTIVE: To describe a case of a congenital hepatic hemangioma treated with surgery. METHODS: We report the case of a 6-day-old boy who presented a giant hepatic hemangioma, and describe its evolution. RESULTS: The child developed hemodynamic instability secondary to consumption coagulopathy and respiratory failure. The image studies were inconclusive. He was submitted to surgery with complete resection of the tumor. Pathology confirmed it was hemangioma. The child was discharged after 15 days and is well, without symptoms. CONCLUSIONS: Hepatic hemangiomas should be treated conservatively, with surgery reserved for intractable cardiac failure and/or refractory consumptive coagulopaty.