Alexander R. Judkins

The genetic landscape of the childhood cancer medulloblastoma

Genomic analysis of a childhood cancer reveals markedly fewer mutations than what is typically seen in adult cancers. Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Immunohistochemical analysis of hSNF5/INI1 in pediatric CNS neoplasms.

Atypical teratoid/rhabdoid tumor (AT/RT) may be misdiagnosed as primitive neuroectodermal tumor/medulloblastoma (PNET) and occasionally as other tumors. Molecular genetic analysis of AT/RT demonstrates deletion and mutation of the hSNF5/INI1 gene in most cases, with decreased or absent expression at the RNA or protein level. Immunohistochemistry with an antibody to INI1 was performed to determine whether this would be a sensitive and specific means of assessing INI1 loss in pediatric brain tumors. Fifty-three tumors consisting of 20 AT/RTs, 10 PNETs, and 23 other central nervous system tumors were examined. No nuclear staining was found in all 20 AT/RTs. Most other central nervous system tumors demonstrated nuclear staining. Eight cases in which classification as AT/RT or PNET was difficult were also examined. Seven cases had no chromosome 22 deletion or INI1 mutation; INI1 antibody showed nuclear staining in these cases. One case was a recurrent tumor with features consistent with an AT/RT. INI1 immunostaining was negative in this case, and a mutation in INI1 was subsequently identified. Immunohistochemical staining with an INI1 antibody correlates with molecular findings in AT/RT and may be useful in confirming the histologic diagnosis. INI1 immunostaining may have particular utility in the analysis of tumors with indeterminate histologic features or atypical immunophenotypic profiles.

Immunohistochemical analysis of hSNF5/INI1 distinguishes renal and extra-renal malignant rhabdoid tumors from other pediatric soft tissue tumors.

Malignant rhabdoid tumor (MRT) is a highly aggressive neoplasm that occasionally demonstrates phenotypic overlap with other soft tissue malignancies. Molecular genetic analysis of MRT frequently demonstrates deletion or mutation of the hSNF5/INI1 gene, with corresponding reduced expression at the protein level. INI1 immunohistochemistry was performed to determine the utility of this method in assessing loss of INI1 expression in rhabdoid tumors. Twenty-seven MRTs with molecular analysis (19 renal, 8 extra-renal) were evaluated. Seventeen additional MRT (10 renal, 7 extra-renal) without INI1 molecular analysis were also analyzed. Loss of INI1 expression was observed in the tumor cells in all 44 cases. To determine the specificity of this assay, a variety of 45 pediatric soft tissue tumors, some of which occasionally display rhabdoid differentiation, were investigated. These included Ewing’s sarcoma, Wilms’ tumor, desmoplastic small round cell tumor, clear cell sarcoma, congenital mesoblastic nephroma, synovial sarcoma, undifferentiated sarcoma, rhabdomyosarcoma, and epithelioid sarcoma. Positive nuclear staining was found in all nonrhabdoid tumors examined. Of interest, synovial and epithelioid sarcomas exhibited variable and/or focal staining. INI1 antibody immunohistochemistry is useful in confirming the histologic diagnosis of renal or extra-renal rhabdoid tumor, especially for cases with indeterminate histologic features, equivocal immunophenotypic profiles, or uninformative molecular studies.

Spectrum of SMARCB1/INI1 Mutations in Familial and Sporadic Rhabdoid Tumors

Germline mutations and deletions of SMARCB1/INI1 in chromosome band 22q11.2 predispose patients to rhabdoid tumor and schwannomatosis. Previous estimates suggested that 15–20% of rhabdoid tumors were caused by an underlying germline abnormality of SMARCB1. However, these studies were limited by case selection and an inability to detect intragenic deletions and duplications.

Duplication of 7q34 in Pediatric Low-Grade Astrocytomas Detected by High-Density Single-Nucleotide Polymorphism-Based Genotype Arrays Results in a Novel BRAF Fusion Gene

In the present study, DNA from 28 pediatric low‐grade astrocytomas was analyzed using Illumina HumanHap550K single‐nucleotide polymorphism oligonucleotide arrays. A novel duplication in chromosome band 7q34 was identified in 17 of 22 juvenile pilocytic astrocytomas and three of six fibrillary astrocytomas. The 7q34 duplication spans 2.6 Mb of genomic sequence and contains approximately 20 genes, including two candidate tumor genes, HIPK2 and BRAF. There were no abnormalities in HIPK2, and analysis of two mutation hot‐spots in BRAF revealed a V600E mutation in only one tumor without the duplication. Fluorescence in situ hybridization confirmed the 7q34 copy number change and was suggestive of a tandem duplication. Reverse transcription polymerase chain reaction‐based sequencing revealed a fusion product between KIAA1549 and BRAF. The predicted fusion product includes the BRAF kinase domain and lacks the auto‐inhibitory N‐terminus. Western blot analysis revealed phosphorylated mitogen‐activated protein kinase (MAPK) protein in tumors with the duplication, consistent with BRAF‐induced activation of the pathway. Further studies are required to determine the role of this fusion gene in downstream MAPK signaling and its role in development of pediatric low‐grade astrocytomas.

Comparative Measurements of Hypoxia in Human Brain Tumors Using Needle Electrodes and EF5 Binding

Hypoxia is known to be an important prognostic marker in many human cancers. We report the use of two oxygen measurement techniques in human brain tumors and compare these data with semiquantitative histological end points. Oxygenation was measured using the Eppendorf needle electrode and/or EF5 binding in 28 brain tumors. These data were compared with necrosis, mitosis, and endothelial proliferation. In some tumors, absolute EF5 binding was converted to tissue pO2 based on in vitro calibrations. Eppendorf electrode readings could not be used to identify WHO grade 1/2 versus WHO grade 3/4 tumors, they could not differentiate grade 3 versus grade 4 glial-derived neoplasms, nor did they correlate with necrosis or endothelial proliferation scores. EF5 binding increased as the tumor grade increased and was significantly associated with necrosis and endothelial proliferation. There was no statistically significant correlation between the two hypoxia detection techniques, although both methods indicated similar absolute ranges of tissue pO2. There was substantial inter- and intratumoral heterogeneity of EF5 binding in WHO grade 4 glial neoplasms. The majority of cells in glial-derived tumor had levels of hypoxia that were mild to moderate (defined herein as 10% to 0.5% pO2) rather than severe (defined as approximately 0.1% pO2). Immunohistochemical detection of EF5 binding tracks histological parameters in adult brain tumors, with increased binding associated with increasing necrosis and endothelial proliferation. The proportion of moderately to severely hypoxic cells is relatively low, even in the high-grade tumors. Human brain tumors are dominated by oxic to moderately hypoxic cells.

Genomic Analysis Using High-Density Single Nucleotide Polymorphism-Based Oligonucleotide Arrays and Multiplex Ligation-Dependent Probe Amplification Provides a Comprehensive Analysis of INI1/SMARCB1 in Malignant Rhabdoid Tumors

PURPOSE A high-resolution genomic profiling and comprehensive targeted analysis of INI1/SMARCB1 of a large series of pediatric rhabdoid tumors was done. The aim was to identify regions of copy number change and loss of heterozygosity (LOH) that might pinpoint additional loci involved in the development or progression of rhabdoid tumors and define the spectrum of genomic alterations of INI1 in this malignancy. EXPERIMENTAL DESIGN A multiplatform approach using Illumina single nucleotide polymorphism-based oligonucleotide arrays, multiplex ligation-dependent probe amplification, fluorescence in situ hybridization, and coding sequence analysis was used to characterize genome-wide copy number changes, LOH, and genomic alterations of INI1/SMARCB1 in a series of pediatric rhabdoid tumors. RESULTS The biallelic alterations of INI1 that led to inactivation were elucidated in 50 of 51 tumors. INI1 inactivation was shown by a variety of mechanisms, including deletions, mutations, and LOH. The results from the array studies highlighted the complexity of rearrangements of chromosome 22 compared with the low frequency of alterations involving the other chromosomes. CONCLUSIONS The results from the genome-wide single nucleotide polymorphism array analysis suggest that INI1 is the primary tumor suppressor gene involved in the development of rhabdoid tumors with no second locus identified. In addition, we did not identify hotspots for the breakpoints in sporadic tumors with deletions of chromosome 22q11.2. By employing a multimodality approach, the wide spectrum of alterations of INI1 can be identified in the majority of patients, which increases the clinical utility of molecular diagnostic testing.

Nonsense Mutation and Inactivation of SMARCA4 (BRG1) in an Atypical Teratoid/Rhabdoid Tumor Showing Retained SMARCB1 (INI1) Expression

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive brain tumors of early childhood poorly responding to therapy. The majority of cases show inactivation of SMARCB1 (INI1, hSNF5, BAF47), a core member of the adenosine triphosphate (ATP)-dependent SWI/SNF chromatin-remodeling complex. We here report the case of a supratentorial AT/RT in a 9-month-old boy, which showed retained SMARCB1 staining on immunohistochemistry and lacked genetic alterations of SMARCB1. Instead, the tumor showed loss of protein expression of another SWI/SNF chromatin-remodeling complex member, the ATPase subunit SMARCA4 (BRG1) due to a homozygous SMARCA4 mutation [c.2032C>T (p.Q678X)]. Our findings highlight the role of SMARCA4 in the pathogenesis of SMARCB1-positive AT/RT and the usefulness of antibodies directed against SMARCA4 in this diagnostic setting.

INI1 expression is retained in composite rhabdoid tumors, including rhabdoid meningiomas

Rhabdoid cells are encountered in specific entities, such as malignant rhabdoid tumor and atypical teratoid/rhabdoid tumor, as well as in composite rhabdoid tumors derived secondarily from other tumor types. Although rhabdoid tumors are uniformly aggressive, distinction of the entity from the phenotype remains important for its therapeutic implications. The majority of malignant rhabdoid tumors and atypical teratoid/rhabdoid tumors affect infants and young children, harbor chromosome 22q deletions, and inactivate the INI1/hSNF5/BAF47 tumor suppressor gene on 22q11.2. In contrast, most composite rhabdoid tumors are diagnosed in adults, with FISH detectable 22q losses the exception rather than the rule. However, this assay remains limited since 22q dosages are maintained in 20–30% of malignant rhabdoid tumors and atypical teratoid/rhabdoid tumors. Furthermore, chromosome 22 losses are common in some parent tumor types, particularly meningiomas. The recently developed INI1 antibody shows loss of nuclear expression in malignant rhabdoid tumors and atypical teratoid/rhabdoid tumors, though its status in composite rhabdoid tumors is largely unknown. Therefore, we utilized immunohistochemistry and FISH to study INI1 expression and 22q dosages, respectively, in 40 composite rhabdoid tumors, including 16 meningiomas, 15 carcinomas, three melanomas, two sarcomas, two glioblastomas, and 1 neuroblastoma. Approximately 70% of rhabdoid meningiomas had a 22q deletion, but this was rare in other tumor types. Except for one retroperitoneal leiomyosarcoma, nuclear INI1 expression was retained in all composite rhabdoid tumors, including meningiomas with 22q deletion. Therefore, we conclude that INI1 immunohistochemistry is a relatively simple, sensitive, and specific technique for distinguishing malignant rhabdoid tumor and atypical teratoid/rhabdoid tumor from composite rhabdoid tumor.

Asparagine Plays a Critical Role in Regulating Cellular Adaptation to Glutamine Depletion

Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.