O’Neill, Brian Patrick

AbstractBackgroundGlioblastoma (GBM) exhibits profound intratumoral genetic heterogeneity. Each tumor comprises multiple genetically distinct clonal populations with different therapeutic sensitivities. This has implications for targeted therapy and genetically informed paradigms. Contrast-enhanced (CE)-MRI and conventional sampling techniques have failed to resolve this heterogeneity, particularly for nonenhancing tumor populations. This study explores the feasibility of using multiparametric MRI and texture analysis to characterize regional genetic heterogeneity throughout MRI-enhancing and nonenhancing tumor segments.MethodsWe collected multiple image-guided biopsies from primary GBM patients throughout regions of enhancement (ENH) and nonenhancing parenchyma (so called brain-around-tumor, [BAT]). For each biopsy, we analyzed DNA copy number variants for core GBM driver genes reported by The Cancer Genome Atlas. We co-registered biopsy locations with MRI and texture maps to correlate regional genetic status with spatially matched imaging measurements. We also built multivariate predictive decision-tree models for each GBM driver gene and validated accuracies using leave-one-out-cross-validation (LOOCV).ResultsWe collected 48 biopsies (13 tumors) and identified significant imaging correlations (univariate analysis) for 6 driver genes: EGFR, PDGFRA, PTEN, CDKN2A, RB1, and TP53. Predictive model accuracies (on LOOCV) varied by driver gene of interest. Highest accuracies were observed for PDGFRA (77.1%), EGFR (75%), CDKN2A (87.5%), and RB1 (87.5%), while lowest accuracy was observed in TP53 (37.5%). Models for 4 driver genes (EGFR, RB1, CDKN2A, and PTEN) showed higher accuracy in BAT samples (n = 16) compared with those from ENH segments (n = 32).ConclusionMRI and texture analysis can help characterize regional genetic heterogeneity, which offers potential diagnostic value under the paradigm of individualized oncology.

Abstract Background Pilocytic astrocytoma is a rare tumor in adults. This report is of a prospective clinical trial with long-term follow-up. Methods Between 1986 and 1994, 20 eligible adults with supratentorial pilocytic astrocytomas were enrolled in a prospective intergroup trial of radiotherapy (RT) after biopsy (3 patients) or observation after gross (11 patients) or subtotal (6 patients) resection. Results At the time of analysis (median follow-up, 20.8 years), 2 patients (10%) have died and 18 patients (90%) are alive. Neurologic and cognitive function were stable or improved over time for the majority of patients. No toxic effects of treatment or malignant transformations have been recorded at last follow-up. For the entire cohort the 20-year time to progression and overall survival rates are 95% and 90% respectively. The cause of death (2.2 and 16.1 years after enrollment) in both patients was unrelated to tumor although both were biopsy-only patients. One subtotally resected tumor progressed 1 month after enrollment requiring P32 injection into an enlarging cyst. Because of further progression this patient required RT 18 months later. This patient is alive without evidence of progression 18 years after RT. Conclusion The long-term follow-up results of this prospective trial confirm that adults with pilocytic astrocytomas have a favorable prognosis with regard to survival and neurologic function. Close observation is recommended for adults with pilocytic astrocytomas, reserving RT for salvage, as the majority remain stable after gross or subtotal resection and no adjuvant therapy.

Abstract Purpose: Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma confined to the central nervous system. Whether there is a PCNSL-specific genomic signature and, if so, how it differs from systemic diffuse large B-cell lymphoma (DLBCL) is uncertain. Experimental Design: We performed a comprehensive genomic study of tumor samples from 19 immunocompetent PCNSL patients. Testing comprised array-comparative genomic hybridization and whole exome sequencing. Results: Biallelic inactivation of TOX and PRKCD was recurrently found in PCNSL but not in systemic DLBCL, suggesting a specific role in PCNSL pathogenesis. In addition, we found a high prevalence of MYD88 mutations (79%) and CDKN2A biallelic loss (60%). Several genes recurrently affected in PCNSL were common with systemic DLBCL, including loss of TNFAIP3, PRDM1, GNA13, TMEM30A, TBL1XR1, B2M, CD58, activating mutations of CD79B, CARD11, and translocations IgH-BCL6. Overall, B-cell receptor/Toll-like receptor/NF-κB pathways were altered in >90% of PNCSL, highlighting its value for targeted therapeutic approaches. Furthermore, integrated analysis showed enrichment of pathways associated with immune response, proliferation, apoptosis, and lymphocyte differentiation. Conclusions: In summary, genome-wide analysis uncovered novel recurrent alterations, including TOX and PRKCD, helping to differentiate PCNSL from systemic DLBCL and related lymphomas. Clin Cancer Res; 21(17); 3986–94. ©2015 AACR.

Abstract Background: Specific germline alterations within the TERT, EGFR, CCDC26, CDKN2A/B, PHLDB1, TP53 and RTEL1 regions are associated with development of glioma. While some of these germline variants are associated with all gliomas (e.g. rs2736100 in TERT), others are associated with a specific morphologic and/or molecular subtype. For example, the RTEL1 region variants are associated with primary glioblastoma and rs55705857 in CCDC26 is associated with 1p/19q co-deleted oligodendrogliomas and with IDH mutant astrocytic gliomas. We hypothesized that germline variants in these regions will be associated with other molecular subtypes. The Cancer Genome Atlas (TCGA) and other groups have described molecular subtypes of glioma based on acquired somatic mutation patterns. Herein, we grouped glioma patients into mutation-based molecular subgroups based on three molecular alterations and determined if germline polymorphisms are associated with these mutation-based molecular subtypes. Methods: Five molecular subtypes were defined based on combinations of the following molecular alterations: TERT promoter mutation (TERTmut vs. TERTwt), IDH1/2 mutation (IDH mut vs. IDH wt) and 1p/19q co-deletion (1p19qcodel vs. 1p19q noncodel). Germline single nucleotide polymorphisms (SNPs) were evaluated by custom Illumina array analysis of blood-derived DNA and 1K genome imputation. Results: One hundred ninety-nine gliomas (24 oligodendrogliomas, 59 mixed oligoastrocytomas, 41 grade 2-3 astrocytomas and 75 glioblastomas) had both SNP data and mutation data available in order to assign them into the five molecular subgroups. The prevalence of each of the molecular subtypes was as follows: TERTmut/IDHmut/1p19qcodel (20%), TERTmut/IDHmut/1p19qnoncodel (5%), TERTmut/IDHwt/1p19qnoncodel (32%), TERTwt/IDHmut/1p19qnondel (35%) and TERTwt/IDHwt/1p19qnoncodel (8%). The TERT SNP (rs2736100) was protective for TERTmut/IDHmut/1p19qnoncodel and TERTwt/IDHmut/1p19qnoncodel gliomas. The CCDC26 SNP (rs55705857) was strongly associated with the risk of developing TERTmut/IDHmut/1p19qcodel and TERTwt/IDHmut/1p19qnoncodel gliomas. The TP53 SNP (rs7837822) was strongly associated with risk of developing TERTmut/IDHmut/1p19qnoncodel and TERTwt/IDHmut/1p19qnoncodel gliomas. Interestingly, while one RTEL1 region SNP (rs6062297) was strongly associated with glioma risk, RTEL1 SNPs rs6010620 and rs2297440 were protective for the development of TERTmut/IDHwt/1p19qnoncodel gliomas. We are currently in the process of validating these results using the TCGA glioblastoma and low grade glioma data. Conclusions: Glioma patients can be meaningfully classified according to their acquired mutation subtype. Importantly, our results suggest that there are significant associations between germline polymorphisms and mutation-based glioma molecular subtypes. Citation Format: Jeanette E. Eckel-Passow, Thomas M. Kollmeyer, Gobinda Sarkar, Anisha Chada, Paul A. Decker, Matthew L. Kosel, Alissa A. Caron, Hugues Sicotte, Kannabiran Nandakumar, Naresh Prodduturi, Brian P. O'Neill, Daniel H. Lachance, Robert B. Jenkins. The association of glioma germline risk SNPs with mutation-based molecular subgroups. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4714. doi:10.1158/1538-7445.AM2014-4714

Abstract Background: Gliomas are primary malignant brain tumors with significant morbidity and mortality. Single nucleotide polymorphisms (SNPs) mapped to 8q24 have been shown to be associated with glioma development. Recently, we identified seven SNPs (through tag SNP genotyping/imputation, pooled next-generation sequencing using long-range PCR, and validation SNP genotyping) that are highly associated with the risk of developing glioma. After stratifying by histologic and tumor subtype, one SNP, rs55705857, in the 8q24.21 region near CCDC26 was significantly associated with oligodendroglial tumors and IDH mutated astrocytic gliomas with high odds ratios (ORs ∼6.0) (Jenkins et al. Nature Genet. 44:1122;2012). By array comparative genomic hybridization (aCGH), a significant proportion of oligodendroglial tumors and IDH mutated astrocytomas have been shown to duplicate 8q24 (Kitange et al., Genes Chr Cancer 42:68;2005; Nousmehr et al. Cancer Cell 17:510;2010). We were interested in learning if the germline risk allele of rs55705857 is associated with gliomas that duplicate 8q24. We were also interested in learning if the risk allele is preferentially gained in gliomas that duplicate 8q24. Methods: We performed aCGH on 217 tumor samples. rs55705857 germline genotype was available on 120 of the patients; of which 30 carried the risk (G) allele. In addition, we sequenced the rs55805857 region in 48 gliomas, 15 of which exhibited gain or duplication of chromosome 8, 8q, or 8q24. The ratio of the Sanger-sequencing peak heights at and around rs55705857 was used to determine which allele (risk or wild-type) was gained. Results: All 15 duplications included rs55705857 and the immediately adjacent regions. Of the 30 patients who carried the germline risk (G) allele for rs55707857, 8 (26.7%) exhibited duplication of 8q24 upon aCGH analysis of their glioma. Of the 90 patients who only carried the wild-type (A) allele, 7 (7.5%) exhibited duplications of 8q24 upon aCGH analysis of their glioma. This difference in proportion is statistically significant (p=0.024, Fischer exact test). Of the 15 tumors with duplication of 8q24, 8 came from patients who were germline rs55705857 heterozygotes (AG). Of these 8 germline heterozygotes with duplication of 8q24, 4 gained the risk (G) allele, 2 gained the wild-type (A) allele and 2 exhibited gain of both alleles in their tumor specimens. Conclusions: While the results require replication, our data suggest that carrying the risk allele for rs55705857 predisposes to the development of gliomas that subsequently duplicate 8q24. However, gliomas with duplication of 8q24 do not preferentially gain the risk rs55705857 allele. This result implies that, while the duplication always includes the rs55705857 region, the risk allele or region is not the direct target of the duplication. Citation Format: Amanda L. Rynearson, Kirsten A. Schowalter, Stephanie R. Fink, Thomas M. Kollmeyer, Chandralekha Halder, Gobinda Sarkar, Alissa A. Caron, Rachel A. Pauley, Daniel H. Lachance, Brian Patrick O'Neill, Robert B. Jenkins. The germline rs55705857 risk allele is not preferentially gained in gliomas with 8q24 duplication. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4207. doi:10.1158/1538-7445.AM2013-4207

Abstract Introduction. Up to now PCNSL incidence in the US was presumed to be increasing in all age groups independent of changes in nosology, new diagnostic techniques, and HIV infection. The intent of our study was, firstly, to extend our prior study of PCNSL incidence in the US (Olson et al, Cancer 2002;95:1504-10) with an additional decade of SEER data; and, secondly, to determine time trends during the HAART era in the US. Methods. PCNSL age-adjusted incidence rates from 1973-2007 were obtained from the nine SEER registries used in the Olson et al. study. Similarly, the same exclusion criteria were used to remove PCNSL most likely associated with HIV (never married males and females; persons of unknown marital status; all cases reported in the San Francisco registry). To assess the accuracy of the criteria in removing HIV associated cases, incidence rates of Kaposi sarcoma (KS) were assessed with and without the use of the same exclusion criteria. Results. The incidence of PCNSL in the general population decreased from 1995 to 2000. Following this decrease, the incidence stabilized and is now beginning to increase in the last 5 years. Stratifying the incidence rate by age group and employing the exclusion criteria, PCNSL incidence has remained stable in the 20-64 year old population, but has steadily increased in the 65+ population. Removing the exclusion criteria did not alter the data for the 65+ population but demonstrated a sharp drop in incidence after 1996 for the 20-64 year old population. Applying the same HIV exclusion criteria the incidence of KS resulted in no appreciable change in incidence rates over the same time frame. Conclusion. The incidence rate of PCNSL is increasing. This increase is driven by a continued increase in incidence among the 65+ population while the incidence for those aged 20-64 has stabilized during the same time frame. An effect of HAART on PCNSL incidence in the US is suggested by the decline in the incidence among the 20-64 year old population without the HIV exclusion criteria. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1926. doi:10.1158/1538-7445.AM2011-1926

There has been an overall decline in the United States incidence of Primary CNS Lymphoma (PCNSL) from 1998 to 2008. This study's intent was to characterize the cohorts contributing to it. First, calculated the PCNSL incidence rates from nine Surveillance, Epidemiology, and End Results (SEER) registries for time period 1973 to 2008. Second, examined the time trends overall and by age and gender. Third, used 1992–2008 SEER data from the same registries to obtain overall trends for diffuse large B‐cell lymphoma (DLBCL). Last, rates were age‐adjusted to the 2000 US standard population and reported per 100,000 person‐years. Rates continued to increase in women at all ages and men aged 65 and older. In men aged 20–39 and 40–64 years incidence rates peaked in 1995 and then declined dramatically, stabilizing after 1998. The trends in the incidence of PCNSL over this time frame were significantly different from DLBCL for ages 20–39 (P < 0.001) and 40–64 (P < 0.001) years but were not different for the 65 years and older age group (P = 0.99). The overall PCNSL incidence rate declined since 1995 and was driven primarily by the changing incidence in young and middle‐aged men. The rate has continued to increase in men aged 65 years and older and in women. The trends in incidence in the younger age groups over this time period did not parallel those observed for DLBCL. Am. J. Hematol. 88:997–1000, 2013. © 2013 Wiley Periodicals, Inc.

Abstract Introduction: Only few genetic studies have been performed in PCNSL, partly due to the rarity of the tumors and the limited amount of available tissue. Unlike nodal DLBCL, PCNSL is poorly understood in terms of its precise histogenetic origin and molecular pathogenesis. It is still a matter of debate whether PCNSL differs from nodal DLBCL with respect to their molecular features and pathogenesis and also if there is a genomic signature PCNSL-specific. Materials and Methods: To determine the pattern of genetic alterations, 17 immunocompetent (EBV-, HIV-) PCNSL were analyzed by aCGH. Seven cases were analyzed using fresh tumor samples and 10 using formalin-fixed paraffin embedded (FFPE) tissues. B-cell differentiation status was characterized by immunostains for CD10, MUM-1, and BCL-6. The aCGH profile from PCNSL cases was compared with aCGH data obtained from 59 nodal DLBCL. Results: We found a complex karyotype with a median of 21 copy-number abnormalities (CNA) per case (range 10-49). Overall, 18 regions (10 losses and 8 gains) were affected in &gt;20% of cases. Loss of 9p21 (CDKN2A) was the commonest CNA (82% of cases). Other common CNAs were loss of 6q23.3 (TNFAIP3; 59%), 6p21 (HLA genes; 53%), 6q21 (PRDM1; 47%) and gain of 12q21-q24 (53%), 7q21-q31 (35%) and 19q13 (35%). Five novel recurrent focal CNAs were found in PCNSL; loss of 3p21.31, 3q26.32, 10p15.3-p14, 12q24.31 and 16q12.1-q21 (29% of cases each). All of them, excluding –16q12.1-q21, were found either in less than 5% or absent in nodal DLBCL, thus suggesting to be unique to PCNSL. Recurrent homozygous deletions were found in CDKN2A, TMEM30A, TOX and CD58, the latter two involved in T-cell development and activation. CD58 was also recurrently affected by small focal deletions in the Ig-like C2-type domain. Furthermore, DNA sequencing analysis showed missense mutations in the Toll/IL-1 receptor domain of MYD88 in 35% of PCNSLs. Another 34 genes, including B2M, ETV6, MHC class II genes, PRDM1, and TNFRSF10A were homozygously deleted. Bioinformatics analysis including the most commonly affected genes showed an enrichment of networks associated with immune response, NF-kB pathway, proliferation, regulation of the apoptosis and lymphocyte differentiation and activation. Conclusions: We showed evidence of a highly complex genome and identified a subset of genes with potential relevance in PCNSL pathogenesis. The genomic profile described here reinforces the existence of a specific molecular signature in PCNSL, thus helping to genetically differentiate this entity from the nodal DLBCL and related lymphomas. Additionally, the results obtained from FFPE samples are encouraging and larger archival tissue collections can now be analyzed in order to complement the still fragmented knowledge we have of the genetic basis of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5081. doi:1538-7445.AM2012-5081

Abstract Objective. Molecularly distinct PCNSL signatures with divergent clinical behavior are now recognized. Implied within this heterogeneity are mechanisms for treatment response and pattern of failure. Recent cytogenetic and genomic studies demonstrated influences of select chromosomal regions on outcome to treatment and survival. These included genetic and epigenetic alterations affecting CDKN2A (9p21.3) that distinguished PCNSL from its systemic counterpart. We now report that deletion of 9p21.3/p16 is associated with a negative effect on survival. Methods and Patients. Well-annotated and population-based formalin fixed paraffin embedded (FFPE) patient samples were sourced from the SEER TRP. Specimens were retrieved from the Hawaii, Iowa and Los Angeles County (LA Co) tissue repositories. “Home Brew” fluorescent in situ hybridization (FISH) probes for BCL6, and ptpr-α were prepared according to a standard protocol (Cady et al. J Clin Oncol 2008;26:4814-9). FISH probes for 9p.21 were prepared similarly and then compared to a commercially available probe. Survival was based on the SEER TRP clinical annotations. Results. Thirty five patients were sourced - twelve from Iowa, 13 from Hawaii, and 10 from LA County. The test cohort was 23 from Iowa and Hawaii; the FISH probe failed in 9 and three had insufficient remaining tumor for analysis. For the Iowa and Hawaii registries and for the registries combined the lack of FISH probe detection of 9p.21/p16 correlated with survival (p=0.0159) and was independent of BCL6 (p=0.8788) and ptpr-α (p=0.3724). Conclusion. Deletion of p16 confers a negative effect on survival of PCNSL patients and appears to be independent of treatment, age, BCL6 and ptpr-α status. These results need to be confirmed in a larger sample size. 9p21.3 detection by FISH in feasible in surgical specimens and may aid in the stratification of such patients into different and/or more intensive treatment regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4507. doi:1538-7445.AM2012-4507