Youssef Yakkioui

Chordoma: the entity

Chordomas are malignant tumors of the axial skeleton, characterized by their locally invasive and slow but aggressive growth. These neoplasms are presumed to be derived from notochordal remnants with a molecular alteration preceding their malignant transformation. As these tumors are most frequently observed on the skull base and sacrum, patients suffering from a chordoma present with debilitating neurological disease, and have an overall 5-year survival rate of 65%. Surgical resection with adjuvant radiotherapy is the first-choice treatment modality in these patients, since chordomas are resistant to conventional chemotherapy. Even so, management of chordomas can be challenging, as chordoma patients often present with recurrent disease. Recent advances in the understanding of the molecular events that contribute to the development of chordomas are promising; the most novel finding being the identification of brachyury in the disease process. Here we present an overview of the current paradigms and summarize relevant research findings.

Changes in brainstem serotonergic and dopaminergic cell populations in experimental and clinical Huntington’s disease

The predominant motor symptom in Huntingtons disease (HD) is chorea. The patho-anatomical basis for the chorea is not well known, but a link with the dopaminergic system has been suggested by post-mortem and clinical studies. Our previous work revealed an increased number of dopamine-containing cells in the substantia nigra and ventral tegmental area in a transgenic rat model of HD (tgHD). Since there were no changes in the total number of cells in those regions, we hypothesized that changes in cell phenotype were taking place. Here, we tested this hypothesis by studying the dorsal raphe nucleus (DRN), which houses dopaminergic and non-dopaminergic (mainly serotonergic) neurons in tgHD rat tissue and postmortem HD human tissue. We found an increased number of dopamine and reduced number of serotonin-containing cells in the DRN of tgHD rats. Similar findings in postmortem HD brain tissue indicate that these changes also occur in patients. Further investigations in the tgHD animal tissue revealed the presence of dopaminergic cell bodies in the B6 raphe region, while in control animals exclusively serotonin-containing cells were found. These data suggest the existence of phenotype changes in monoaminergic neurons in the DRN in HD and shed new light on the neurobiology of clinical neurological symptoms such as chorea and mood changes.

A comparison of cell-cycle markers in skull base and sacral chordomas

OBJECTIVE Despite refinement of surgical techniques and adjuvant radiotherapy, the prognosis for patients with a chordoma remains poor. Identification of prognostic factors related to tumor biology might improve this assessment and result in molecular markers for targeted therapy. Limited studies have been performed to unravel the impact of cell-cycle markers in chordoma, and those performed have shown inconclusive results. In the current study, we aimed to discover the impact of cyclin-dependent kinase 4 (CDK4) expression and its relation to prognosis and other cell-cycle markers in chordoma. METHODS Twenty-five human formalin-fixed, paraffin-embedded chordoma specimens were examined by immunohistochemistry for the expression of CDK4, protein 53 (p53), and murine double minute 2 (MDM2). The MIB-1 labeling index and mitotic index were used for the examination of proliferation. We collected detailed demographic and clinical data. RESULTS Overexpression of CDK4, p53, and MDM2 was found in five (20%), seven (28%), and 14 (56%) of the cases, respectively. All three cell-cycle markers showed a significant correlation with MIB1 labeling index. Expression of CDK4 (P = 0.02) and p53 (P < 0.01) were both significantly correlated with poor overall survival. Also, histologically observed necrosis (P < 0.05) and a dedifferentiated tumor subtype (P < 0.01) were related to adverse patient outcome. CONCLUSION Our results show that the expression of CDK4 and p53 are related to cell proliferation capacity and worse outcome in patients with chordoma.

A neuroanatomical analysis of the effects of a memory impairing dose of scopolamine in the rat brain using cytochrome c oxidase as principle marker.

Acetylcholine plays a role in mnemonic and attentional processes, but also in locomotor and anxiety-related behavior. Receptor blockage by scopolamine can therefore induce cognitive as well as motor deficits and increase anxiety levels. Here we show that scopolamine, at a dose that has previously been found to affect learning and memory performance (0.1 mg/kg i.p.), has a widespread effect on cytochrome c oxidase histochemistry in various regions of the rat brain. We found a down-regulation of cytochrome c oxidase in the nucleus basalis, in movement-related structures such as the primary motor cortex and the globus pallidus, memory-related structures such as the CA1 subfield of the hippocampus and perirhinal cortex and in anxiety-related structures like the amygdala, which also plays a role in memory. However choline acetyltransferase levels were only affected in the CA1 subfield of the hippocampus and both, choline acetyltransferase and c-Fos expression levels were decreased in the amygdala. These findings corroborate strong cognitive behavioral effects of this drug, but also suggest possible anxiety- and locomotor-related changes in subjects. Moreover, they present histochemical evidence that the effects of scopolamine are not ultimately restricted to cognitive parameters.

Integrated and Quantitative Proteomics of Human Tumors

Quantitative proteomics represents a powerful approach for the comprehensive analysis of proteins expressed under defined conditions. These properties have been used to investigate the proteome of disease states, including cancer. It has become a major subject of studies to apply proteomics for biomarker and therapeutic target identification. In the last decades, technical advances in mass spectrometry have increased the capacity of protein identification and quantification. Moreover, the analysis of posttranslational modification (PTM), especially phosphorylation, has allowed large-scale identification of biological mechanisms. Even so, increasing evidence indicates that global protein quantification is often insufficient for the explanation of biology and has shown to pose challenges in identifying new and robust biomarkers. As a consequence, to improve the accuracy of the discoveries made using proteomics in human tumors, it is necessary to combine (i) robust and reproducible methods for sample preparation allowing statistical comparison, (ii) PTM analyses in addition to global proteomics for additional levels of knowledge, and (iii) use of bioinformatics for decrypting protein list. Herein, we present technical specificities for samples preparation involving isobaric tag labeling, TiO2-based phosphopeptides enrichment and hydrazyde-based glycopeptides purification as well as the key points for the quantitative analysis and interpretation of the protein lists. The method is based on our experience with tumors analysis derived from hepatocellular carcinoma, chondrosarcoma, human embryonic intervertebral disk, and chordoma experiments.

Kinome and phosphoproteome of high-grade meningiomas reveal AKAP12 as a central regulator of aggressiveness and its possible role in progression

There is a need to better understand meningioma oncogenesis for biomarker discovery and development of targeted therapies. Histological or genetic criteria do not accurately predict aggressiveness. Post-translational studies in meningioma progression are lacking. In the present work, we introduce a combination of mass spectrometry-based phosphoproteomics and peptide array kinomics to profile atypical and anaplastic (high-grade) meningiomas. In the discovery set of fresh-frozen tissue specimens (14), the A-kinase anchor protein 12 (AKAP12) protein was found downregulated across the grades. AKAP12 knockdown in benign meningioma cells SF4433 increases proliferation, cell cycle, migration, invasion, and confers an anaplastic profile. Differentially regulated pathways were characteristic of high-grade meningiomas. Low AKAP12 expression in a larger cohort of patients (75) characterized tumor invasiveness, recurrence, and progression, indicating its potential as a prognostic biomarker. These results demonstrate AKAP12 as a central regulator of meningioma aggressiveness with a possible role in progression.

Kinase Activity in Recurring Primary Skull Base Chordomas and Chondrosarcomas: Identification of Novel Pathways of Oncogenesis and Potential Drug Targets

BACKGROUND Chordomas and chondrosarcomas can occur in the skull base. Currently, 45% of chordomas and 56% of chondrosarcomas recur within 5 years of surgery. The role of adjuvant therapy is highly debated. No pharmacotherapies have been approved by the U.S. Food and Drug Administration for chordomas or chondrosarcomas. High propensity for recurrence and lack of definitive adjuvant therapy necessitate additional basic science research to identify molecular anomalies associated with recurrent disease. METHODS We pooled tumor lysates from patients based on clinical criteria into 4 groups: primary chordomas, primary chordomas that recurred, primary chondrosarcomas, and primary chondrosarcomas that recurred. We used a peptide labeling method, isobaric tags for relative and absolute quantitation, to uniquely identify each tumor group. Phosphorylated peptides were identified and quantified via mass spectroscopy to determine and predict active kinases. RESULTS Six groups of phosphorylated peptides were associated with primary tumors that later recurred. Specific kinases associated with primary chordomas that recurred were FES and FER. Specific kinases associated with primary chondrosarcomas that recurred were FES, FER, SRC family kinases, PKC, ROCK, and mitogen-activated protein kinase signaling (JNK, ERK1, p38). CONCLUSIONS These data provide clinicians with a means to screen skull base chordomas and chondrosarcomas to help identify tumors with a propensity to recur. Many of these kinases can be efficaciously inhibited by Food and Drug Administration-approved drugs that have not yet been used in clinical trials for treatment of skull base chordomas or chondrosarcomas. Validation of kinases identified in this study may advance treatment options for patients with these tumors.

Validation of reference genes in human chordoma

Background: Chordoma are rare slow-growing tumors of the axial skeleton, which are thought to arise from remnants of the notochord. Little is known about the underlying mechanisms that drive this tumor. However, the assessment of gene expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) is hampered due to a lack of validated reference genes. Using an unstable reference gene in qRT-PCR may lead to irreproducible results. Methods: The expression of 12 candidate reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ) was analyzed by qRT-PCR in flash frozen chordoma samples from 18 patients. GeNorm and NormFinder algorithms were used to rank the stability of the genes. Results: From most to least stably expressed, the top six genes found by geNorm were PGK1, YWHAZ, ACTB, HPRT, EF1A, and TBP. When analyzed by NormFinder, the top six genes were ACTB, YWHAZ, PGK1, B2M, TBP, and HPRT. GAPDH alone, which is often used as a reference gene in chordoma gene expression studies, is not stable enough for reliable results. Conclusion: In gene expression studies of human chordomas, PGK1, ACTB, and YWHAZ are more stably expressed, and therefore, are preferred reference genes over the most often used reference gene so far, GAPDH.

A novel drainage approach in patients with cholesterol granuloma : From petrous apex to mastoid air cell

Background: Cholesterol granulomas (CG) of the petrous apex (CGPA) are benign lesions that have high recurrence rates after surgical intervention. We describe the use of a robust silicon drain between the petrous apex and mastoid air cells to allow constant aeration of the lesion for preventing recurrence. Case Description: A retrospective analysis was performed using the data of four patients treated at the Maastricht University Medical Centre between 2014 and 2016. Using the middle fossa approach, the petrous apex was reached, the cyst was opened, and the content aspirated. Subsequently, a robust silicon drain was placed between the cyst and mastoid air cell system. The outcome measures were clinical improvement of the symptoms and radiological parameters. The patients were female (n = 2) and male (n = 2) with an age range between 33 and 53 years at the time of the operation. Computed tomography and magnetic resonance imaging scans were used to confirm CG diagnosis. The most common presenting symptoms in our population were diplopia and headaches. The symptoms improved after surgery and there were no complications. Thus far, no recurrence has been observed and imaging shows aeration in the lesion area. Conclusion: The use of a robust drain seems to be an effective, safe, and feasible option to prevent recurrences in patients with CG.

Notochord isolation using laser capture microdissection

BACKGROUND Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. METHODS Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. RESULTS This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. CONCLUSION LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.