Jacquelyn J. Roth

Diagnostic application of high resolution single nucleotide polymorphism array analysis for children with brain tumors

Single nucleotide polymorphism (SNP) array analysis is currently used as a first tier test for pediatric brain tumors at The Childrens Hospital of Philadelphia. The results from 100 consecutive patients are summarized in the present report. Eighty-seven percent of the tumors had at least one pathogenic copy number alteration. Nineteen of 56 low grade gliomas (LGGs) demonstrated a duplication in 7q34, which resulted in a KIAA1549-BRAF fusion. Chromosome band 7q34 deletions, which resulted in a FAM131B-BRAF fusion, were identified in one pilocytic astrocytoma (PA) and one dysembryoplastic neuroepithelial tumor (DNT). One ganglioglioma (GG) demonstrated a 6q23.3q26 deletion that was predicted to result in a MYB-QKI fusion. Gains of chromosomes 5, 6, 7, 11, and 20 were seen in a subset of LGGs. Monosomy 6, deletion of 9q and 10q, and an i(17)(q10) were each detected in the medulloblastomas (MBs). Deletions and regions of loss of heterozygosity that encompassed TP53, RB1, CDKN2A/B, CHEK2, NF1, and NF2 were identified in a variety of tumors, which led to a recommendation for germline testing. A BRAF p.Thr599dup or p.V600E mutation was identified by Sanger sequencing in one and five gliomas, respectively, and a somatic TP53 mutation was identified in a fibrillary astrocytoma. No TP53 hot-spot mutations were detected in the MBs. SNP array analysis of pediatric brain tumors can be combined with pathologic examination and molecular analyses to further refine diagnoses, offer more accurate prognostic assessments, and identify patients who should be referred for cancer risk assessment.

Chromosome band 7q34 deletions resulting in KIAA1549-BRAF and FAM131B-BRAF fusions in pediatric low grade gliomas

The majority of pediatric low‐grade gliomas (LGGs) are characterized by constitutive activation of the mitogen‐activated protein kinase (MAPK) pathway through various mechanisms including BRAF mutations, inactivation of NF1, and KIAA1549‐BRAF and FAM131B‐BRAF fusions. The KIAA1549‐BRAF fusion typically results from a 2.0 Mb tandem duplication in chromosome band 7q34. In the present study, single nucleotide polymorphism (SNP)‐based array analysis of three LGGs demonstrated deletions in 7q34 that resulted in a BRAF fusion. Case 1 was likely a pilocytic astrocytoma (PA) with three deletions in 7q33q34 and an exon 15‐9 KIAA1549‐BRAF fusion. SNP array analysis of case 2, a possible dysembryoplastic neuroepithelial tumor (DNT), revealed a 2.6 Mb deletion, which included the 5′ end of BRAF and extended to the 3′ end of FAM131B. In case 3, deletions involving BRAF and FAM131B were observed in both a primary and a recurrent PA. RNA‐based sequence analysis of cases 2 and 3 confirmed a fusion between FAM131B exon 2 and BRAF exon 9. The presence of fusion transcripts in these three LGGs highlights the utility of SNP array analysis to identify deletions that are suggestive of fusion proteins. BRAF fusions can result from multiple non‐overlapping deletions, suggesting various complex mechanisms of formation.

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution.

Single nucleotide polymorphism array analysis of bone marrow failure patients reveals characteristic patterns of genetic changes.

The bone marrow failure syndromes (BMFS) are a heterogeneous group of rare blood disorders characterized by inadequate haematopoiesis, clonal evolution, and increased risk of leukaemia. Single nucleotide polymorphism arrays (SNP‐A) have been proposed as a tool for surveillance of clonal evolution in BMFS. To better understand the natural history of BMFS and to assess the clinical utility of SNP‐A in these disorders, we analysed 124 SNP‐A from a comprehensively characterized cohort of 91 patients at our BMFS centre. SNP‐A were correlated with medical histories, haematopathology, cytogenetic and molecular data. To assess clonal evolution, longitudinal analysis of SNP‐A was performed in 25 patients. We found that acquired copy number‐neutral loss of heterozygosity (CN‐LOH) was significantly more frequent in acquired aplastic anaemia (aAA) than in other BMFS (odds ratio 12·2, P < 0·01). Homozygosity by descent was most common in congenital BMFS, frequently unmasking autosomal recessive mutations. Copy number variants (CNVs) were frequently polymorphic, and we identified CNVs enriched in neutropenia and aAA. Our results suggest that acquired CN‐LOH is a general phenomenon in aAA that is probably mechanistically and prognostically distinct from typical CN‐LOH of myeloid malignancies. Our analysis of clinical utility of SNP‐A shows the highest yield of detecting new clonal haematopoiesis at diagnosis and at relapse.

Whole Chromosome 7 Gain Predicts Higher Risk of Recurrence in Pediatric Pilocytic Astrocytomas Independently From KIAA1549-BRAF Fusion Status.

The most frequent genetic alteration identified in pediatric pilocytic astrocytomas and pilomyxoid variant is the KIAA1549-BRAF fusion, which typically results from a 2.0 Mb tandem duplication in chromosome band 7q34. Less frequent abnormalities include fusion genes, BRAF, FGFR, KRAS, and NF1 point mutations, and whole chromosome gains. To correlate genetic alterations with clinical course data, we retrospectively analyzed the tumors with pilocytic and pilomyxoid histology of a cohort of 116 pediatric patients, aged 5 months to 23 years. Gross total resection was associated with a decreased risk of recurrence (p = 0.001), supporting previous findings that complete tumor excision correlates with long-term and disease-free survival. We found no significant association between recurrence rate and the presence of the KIAA1549-BRAF fusion or BRAF mutation (p = 0.167). Interestingly, gain of whole chromosome 7 (WC7) was associated with a 4.7-fold increased risk of tumor recurrence, even after adjusting for surgical status (p = 0.025), and other genetic alterations. Using fluorescence in situ hybridization, we demonstrated that when WC7 gain accompanies the KIAA1549-BRAF fusion, the fusion likely arises first. This study highlights the utility of genetic studies for risk assessment of pilocytic and pilomyxoid astrocytomas, which may impact treatment selections.

Common polymorphic deletion of glutathione S-transferase theta predisposes to acquired aplastic anemia: Independent cohort and meta-analysis of 609 patients

Acquired aplastic anemia (AA) is a rare life‐threatening bone marrow failure syndrome, caused by autoimmune destruction of hematopoietic stem and progenitor cells. Epidemiologic studies suggest that environmental exposures and metabolic gene polymorphisms contribute to disease pathogenesis. Several case–control studies linked homozygous deletion of the glutathione S‐transferase theta (GSTT1) gene to AA; however, the role of GSTT1 deletion remains controversial as other studies failed to confirm the association. We asked whether a more precise relationship between the GSTT1 null polymorphism and aplastic anemia could be defined using a meta‐analysis of 609 aplastic anemia patients, including an independent cohort of 67 patients from our institution. We searched PubMed, Embase, and the Cochrane Database for studies evaluating the association between GSTT1 null genotype and development of AA. Seven studies, involving a total of 609 patients and 3,914 controls, fulfilled the eligibility criteria. Meta‐analysis revealed a significant association of GSTT1 null genotype and AA, with an OR = 1.74 (95% CI 1.31–2.31, P < 0.0001). The effect was not driven by any one individual result, nor was there evidence of significant publication bias. The association between AA and GSTT1 deletion suggests a role of glutathione‐conjugation in AA, possibly through protecting the hematopoietic compartment from endogenous metabolites or environmental exposures. We propose a model whereby protein adducts generated by reactive metabolites serve as neo‐epitopes to trigger autoimmunity in aplastic anemia. Am. J. Hematol. 88:862–867, 2013.

Copy number alterations determined by single nucleotide polymorphism array testing in the clinical laboratory are indicative of gene fusions in pediatric cancer patients

Gene fusions resulting from structural rearrangements are an established mechanism of tumorigenesis in pediatric cancer. In this clinical cohort, 1,350 single nucleotide polymorphism (SNP)‐based chromosomal microarrays from 1,211 pediatric cancer patients were evaluated for copy number alterations (CNAs) associated with gene fusions. Karyotype or fluorescence in situ hybridization studies were performed in 42% of the patients. Ten percent of the bone marrow or solid tumor specimens had SNP array‐associated CNAs suggestive of a gene fusion. Alterations involving ETV6, ABL1‐NUP214, EBF1‐PDGFRB, KMT2A(MLL), LMO2‐RAG, MYH11‐CBFB, NSD1‐NUP98, PBX1, STIL‐TAL1, ZNF384‐TCF3, P2RY8‐CRLF2, and RUNX1T1‐RUNX1 fusions were detected in the bone marrow samples. The most common alteration among the low‐grade gliomas was a 7q34 tandem duplication resulting in a KIAA1549‐BRAF fusion. Additional fusions identified in the pediatric brain tumors included FAM131B‐BRAF and RAF1‐QKI. COL1A1‐PDGFB, CRTC1‐MAML2, EWSR1, HEY1, PAX3‐ and PAX7‐FOXO1, and PLAG1 fusions were determined in a variety of solid tumors and a novel potential gene fusion, FGFR1‐USP6, was detected in an aneurysmal bone cyst. The identification of these gene fusions was instrumental in tumor diagnosis. In contrast to hematologic and solid tumors in adults that are predominantly driven by mutations, the majority of hematologic and solid tumors in children are characterized by CNAs and gene fusions. Chromosomal microarray analysis is therefore a robust platform to identify diagnostic and prognostic markers in the clinical setting.

Abstract 2977: Most patients with acquired aplastic anemia develop clonal hematopoiesis early in disease

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Clonal hematopoiesis is an expansion of hematopoietic stem cells, caused by somatic mutations or epigenetic changes that confer a growth advantage to the host cell. Although recently recognized as a phenomenon of aging, clonal hematopoiesis has been traditionally associated with pre-cancerous states and malignant transformation. Acquired aplastic anemia (AA), a non-neoplastic autoimmune blood disorder occurring in children and adults, has been associated with clonal hematopoietic disorders; transformation to myelodysplastic syndrome (MDS) or acute leukemia is a late complication in 10-15% of AA patients. Based on the association of AA with clonal disorders, we hypothesized that clonal hematopoiesis is a general phenomenon in AA, and can be seen in the majority of AA patients, including children. To evaluate somatic genetic changes in AA, we used a combination of single nucleotide polymorphism array (SNP-A) genotyping and comparative whole exome sequencing of paired bone marrow aspirates and skin in twenty nine patients with AA. All somatic mutations were validated by bi-directional Sanger sequencing. The median age of diagnosis was 14 years (range 1.5-65). Patients were analyzed at a median of 1.1 years from diagnosis. None of the patients had histopathological evidence of MDS at the time of analysis. Somatic mutations were identified in the majority of patients, including patients with pediatric-onset AA. Three patients (10%) had somatic loss-of-function mutations in HLA class I alleles. Although MDS-associated mutations were identified in 2 of 29 patients, the majority of mutations were not in genes associated with MDS and hematologic malignancies. Pathway analysis of mutated genes revealed an enrichment of genes in pathways of immunity and transcriptional regulation. Comparison of somatic mutations in AA to a patient with a 30-year history of AA who progressed to MDS revealed that, unlike in AA, which was characterized by diverse and frequently oligoclonal hematopoiesis, progression to MDS was associated with an expansion of a dominant clone carrying multiple classical mutations linked to malignancy: pathogenic mutations in SUZ12 (homozygous for the mutated region due to copy number-neutral loss of heterozygosity (CN-LOH) at the chromosomal region 17q11.2qter), ASXL1, RUNX1, and PHF6. In conclusion, our data show that clonal hematopoiesis emerges in the majority of patients with AA, including children and young adults, can be detected early in disease, and has a mutational spectrum largely distinct from MDS. Our results highlight that in the absence of morphologic features of myelodysplasia, the presence of clonal hematopoiesis with somatic mutations cannot be used to distinguish MDS from AA. Future longitudinal studies of clonal hematopoiesis in AA will help to explain differences in patients’ disease course, and will enable personalized treatment approaches in AA. Citation Format: Daria V. Babushok, Nieves Perdigones, Juan C. Perin, Timothy S. Olson, Wenda Ye, Jacquelyn J. Roth, Curt Lind, Carine Cattier, Yimei Li, Helge Hartung, Michele E. Paessler, Dale M. Frank, Hongbo M. Xie, Tracy M. Busse, Shanna Cross, Gregory M. Podsakoff, Dimitrios Monos, Jaclyn A. Biegel, Philip J. Mason, Monica Bessler. Most patients with acquired aplastic anemia develop clonal hematopoiesis early in disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2977. doi:10.1158/1538-7445.AM2015-2977