Soma Sengupta

MEDULLOBLASTOMA EXOME SEQUENCING UNCOVERS SUBTYPE-SPECIFIC SOMATIC MUTATIONS

Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, β-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic β-catenin signalling in medulloblastoma.

Pleiotropic effects of miR-183~96~182 converge to regulate cell survival, proliferation and migration in medulloblastoma

Medulloblastomas are the most common malignant brain tumors in children. Several large-scale genomic studies have detailed their heterogeneity, defining multiple subtypes with unique molecular profiles and clinical behavior. Increased expression of the miR-183~96~182 cluster of microRNAs has been noted in several subgroups, including the most clinically aggressive subgroup associated with genetic amplification of MYC. To understand the contribution of miR-183~96~182 to the pathogenesis of this aggressive subtype of medulloblastoma, we analyzed global gene expression and proteomic changes that occur upon modulation of miRNAs in this cluster individually and as a group in MYC-amplified medulloblastoma cells. Knockdown of the full miR-183~96~182 cluster results in enrichment of genes associated with apoptosis and dysregulation of the PI3K/AKT/mTOR signaling axis. Conversely, there is a relative enrichment of pathways associated with migration, metastasis and epithelial to mesenchymal transition, as well as pathways associated with dysfunction of DNA repair in cells with preserved miR-183 cluster expression. Immunocytochemistry and FACS analysis confirm induction of apoptosis upon knockdown of the miR-183 cluster. Importantly, cell-based migration and invasion assays verify the positive regulation of cell motility/migration by the miR-183 cluster, which is largely mediated by miR-182. We show that the effects on cell migration induced by the miR-183 cluster are coupled to the PI3K/AKT/mTOR pathway through differential regulation of AKT1 and AKT2 isoforms. Furthermore, we show that rapamycin inhibits cell motility/migration in medulloblastoma cells and phenocopies miR-183 cluster knockdown. Thus, the miR-183 cluster regulates multiple biological programs that converge to support the maintenance and metastatic potential of medulloblastoma.

α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth

Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABRA5, which encodes the α5-subunit of the GABAA receptor complex, in aggressive MYC-driven, “Group 3” medulloblastomas. We hypothesized that modulation of α5-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABRA5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABRA5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOXA5 target gene expression. siRNA-mediated knockdown of HOXA5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOXA5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor.

Stereotactic Radiosurgery for Renal Cancer Brain Metastasis: Prognostic Factors and the Role of Whole-Brain Radiation and Surgical Resection.

Background. Renal cell carcinoma is a frequent source of brain metastasis. We present our consecutive series of patients treated with Stereotactic Radiosurgery (SRS) and analyse prognostic factors and the interplay of WBRT and surgical resection. Methods. This is a retrospective study of 66 patients with 207 lesions treated with the Cyberknife radiosurgery system in our institution. The patients were followed up with imaging and clinical examination 1 month and 2-3 months thereafter for the brain metastasis. Patient, treatment, and outcomes characteristics were analysed. Results. 51 male (77.3%) and 15 female (22.7%) patients, with a mean age of 58.9 years (range of 31–85 years) and a median Karnofsky Performance Status (KPS) of 90 (range of 60–100), were included in the study. The overall survival was 13.9 months, 21.9 months, and 5.9 months for the patients treated with SRS only, additional surgery, and WBRT, respectively. The actuarial 1-year Local Control rates were 84%, 94%, and 88% for SRS only, for surgery and SRS, and for WBRT and additional SRS, respectively. Conclusions. Stereotactic radiosurgery is a safe and effective treatment option in patients with brain metastases from RCC. In case of a limited number of brain metastases, surgery and SRS might be appropriate.

The evolution of medulloblastoma therapy to personalized medicine.

Recent advances in cancer genomics have revolutionized the characterization and classification of medulloblastomas. According to the current WHO guidelines, medulloblastomas are now classified into the following molecularly defined groups: Wnt signaling pathway (WNT)-activated, sonic hedgehog signaling pathway (SHH)-activated and tumor suppressor protein p53 (TP53)-mutant, SHH-activated and TP53-wildtype, and non-WNT/non-SHH (i.e. group 3 and group 4). Importantly, genomic, epigenomic, and proteomic advances have created a potential paradigm shift in therapeutic options. The challenge now is to (i) translate these observations into new therapeutic approaches and (ii) employ these observations in clinical practice, utilizing the classification following a molecular analysis for diagnosis and application of new subgroup-specific targeted therapeutics.

Dosimetric analysis of the alopecia preventing effect of hippocampus sparing whole brain radiation therapy

BackgroundWhole brain radiation therapy (WBRT) is widely used for the treatment of brain metastases. Cognitive decline and alopecia are recognized adverse effects of WBRT. Recently hippocampus sparing whole brain radiation therapy (HS-WBRT) has been shown to reduce the incidence of memory loss. In this study, we found that multi-field intensity modulated radiation therapy (IMRT), with strict constraints to the brain parenchyma and to the hippocampus, reduces follicular scalp dose and prevents alopecia.MethodsSuitable patients befitting the inclusion criteria of the RTOG 0933 trial received Hippocampus sparing whole brain radiation. On follow up, they were noticed to have full scalp hair preservation. 5 mm thickness of follicle bearing scalp in the radiation field was outlined in the planning CT scans. Conventional opposed lateral WBRT radiation fields were applied to these patient-specific image sets and planned with the same nominal dose of 30 Gy in 10 fractions. The mean and maximum dose to follicle bearing skin and Dose Volume Histogram (DVH) data were analyzed for conventional and HS-WBRT. Paired t-test was used to compare the means.ResultsAll six patients had fully preserved scalp hair and remained clinically cognitively intact 1–3 months after HS-WBRT. Compared to conventional WBRT, in addition to the intended sparing of the Hippocampus, HS-WBRT delivered significantly lower mean dose (22.42 cGy vs. 16.33 cGy, p < 0.0001), V24 (9 cc vs. 44 cc, p < 0.0000) and V30 (9 cc vs. 0.096 cc, p = 0.0106) to follicle hair bearing scalp and prevented alopecia. There were no recurrences in the Hippocampus area.ConclusionsHS-WBRT, with an 11-field set up as described, while attempting to conserve hippocampus radiation and maintain radiation dose to brain inadvertently spares follicle-bearing scalp and prevents alopecia.

CPT-11/bevacizumab for the treatment of refractory brain metastases in patients with HER2-neu-positive breast cancer.

Nervous system relapse of patients with advanced HER2–neu-positive breast cancer is an increasing problem, with one-third of women developing brain metastases. Standard therapies using steroids, surgery and radiotherapy do not provide a lasting response. We evaluated CPT-11 and bevacizumab, which can both cross the blood–brain barrier, as combination therapy to treat HER2–neu-positive breast cancer with brain metastases.

Posterior Reversible Encephalopathy Syndrome (PRES) Complicating Newly-Diagnosed Diffuse Large B-Cell Lymphoma

Patients with posterior reversible encephalopathy syndrome (PRES) might present with altered mental status, headache, nausea, vomiting, seizures, and visual disturbances. Posterior reversible encephalopathy syndrome might result from multiple etiologies including uncontrolled hypertension, toxic/metabolic abnormalities, medications, and malignancies. Management of PRES is directed at treating the underlying etiology. Biopsy should be considered for new lymphadenopathy or masses in a patient with history of cancer to determine whether the patient has recurrence or a new primary disease process.

Could α5-GABA-A receptor activation be used as a target for managing medulloblastomas?

‘Medulloblastoma cerebelli’ was initially described by Cushing and Bailey in 1925 [1]. Medulloblastomas are the most common malignant brain tumors in children and are a significant cause of cancer-related deaths in children [2]. Since their initial description, enormous headway has been made in the molecular characterization of this highly malignant predominantly pediatric brain tumor [3–6]. Recent genomic studies have delineated the molecular heterogeneity of these tumors. They are now classified into subtypes, each with a unique molecular signature and clinical outcome: SHH, WNT, group 3 and group 4 [3–6]. Interestingly, patients with group 3 medulloblastomas have a worse prognosis compared with the other medulloblastoma subtypes [7]. Indeed, 5-year progression-free survival is less than 20% for group 3 medulloblastomas after maximal therapy with externalbeam radiation and multidrug chemotherapy, as compared with 70% 5-year progression-free survival for all other medulloblastoma subtypes. Ten years after disease onset, almost all group 3 medulloblastoma patients will have succumbed to the disease. Therefore, it is of paramount importance to develop effective targeted therapy to manage this subtype of patients. Group 3 medulloblastomas are characterized by oncogenic MYC signaling, often through a high level of MYC amplification. In terms of the current arsenal of chemotherapeutic regimens and radiotherapy, this makes this subtype of medulloblastoma particularly resistant to standard chemotherapy. Unfortunately, attempts at developing drugs that target the MYC protein in group 3 medulloblastomas have so far been unsuccessful. Gene-expression profiling revealed an enrichment of genes associated with GABA pathway signaling in MYC-driven/group 3 medulloblastomas, largely from increased GABRA5 expression [3]. GABRA5 encodes the α5 subunit of the GABA-A receptor complex. This appears to be unique to the group 3 medulloblastomas. This receptor

Novel Use of Dual Immunomodulation for Treating Stiff-Person Syndrome, Cerebellar Variant

Stiff-person syndrome is an autoimmune syndrome defined by muscle spasms and rigidity of proximal and axial muscles. Glutamic acid decarboxylase is the autoantigen found in 60%‐80% of stiff person syndrome patients. 1,2 Stiff person syndrome, cerebellar variant was characterized in a subpopulation of patients (5 of 38) with stiff person syndrome and concomitant cerebellar dysfunction 3 and confirmed in another case series (3 of 61). 4 In both case series, increased intrathecal synthesis of anti‐glutamic acid decarboxylase antibodies was found in stiff person syndrome, cerebellar variant patients. Gamma-aminobutyric acid agonists such as diazepam and immunotherapies improved their stiffness but not cerebellar dysfunction. 3 In this case report we evaluate a therapy using rituximab and cyclophosphamide; this dual immunomodulation improved both stiffness and cerebellar function in our patient.