Kipp, Benjamin R.

AbstractBackgroundOligodendroglioma is genetically defined by concomitant IDH (IDH1/IDH2) mutation and whole-arm 1p/19q codeletion. Codeletion of 1p/19q traditionally evaluated by fluorescence in situ hybridization (FISH) cannot distinguish partial from whole-arm 1p/19q codeletion. Partial 1p/19q codeletion called positive by FISH is diagnostically a “false-positive” result. Chromosomal microarray (CMA) discriminates partial from whole-arm 1p/19q codeletion. Herein, we aimed to estimate the frequency of partial 1p/19q codeletion that would lead to a false-positive FISH result.MethodsFISH 1p/19q codeletion test probe coordinates were mapped onto Oncoscan CMA data to determine the rate of partial 1p/19q codeletion predicted to be positive by FISH. Diffuse astrocytic gliomas with available CMA data (2015–2018) were evaluated and classified based on IDH1-R132H/ATRX/p53 immunohistochemistry, IDH/TERT promoter targeted sequencing, and/or CMA according to classification updates. Predicted false-positive cases were verified by FISH whenever possible.ResultsThe overall estimated false-positive FISH 1p/19q codeletion rate was 3.6% (8/223). Predicted false positives were verified by FISH in 6 (of 8) cases. False-positive rates did not differ significantly (P = .49) between IDH-mutant (4.6%; 4/86) and IDH-wildtype (2.9%; 4/137) tumors. IDH-wildtype false positives were all WHO grade IV, whereas IDH-mutant false positives spanned WHO grades II-IV. Testing for 1p/19q codeletion would not have been indicated for most false positives based on current classification recommendations.ConclusionSelective 1p/19q codeletion testing and cautious interpretation for conflicting FISH and histopathological findings are recommended to avoid potential misdiagnosis.

Abstract Objectives Sclerosing pneumocytomas are rare pulmonary neoplasms that are typically benign. However, rare patients experience progressive disease, and therapy targeting specific genetic underpinnings could be an attractive therapeutic option. Recent studies have found recurrent AKT 1 mutations in sclerosing pneumocytoma, but little is known about whether oncogenic fusion genes may also be present. Methods To better understand the genetic background, 10 sclerosing pneumocytomas were subjected to next-generation sequencing cancer mutation panel testing (n = 9) and/or RNA sequencing (n = 3). The patients were all women (average age, 47 years; range, 17-74 years). Results Eight patients had solitary sclerosing pneumocytomas, while one had two tumors, and one had many bilateral tumors. Recurrent mutations were noted in genes involved in the mTOR pathway, including AKT1, PIK3R1, and PTEN. AKT1 alterations were particularly common, present in 78%. No recurrent genetic fusions were identified. The patient in our study with multiple bilateral lesions was treated with the mammalian target of rapamycin (mTOR) inhibitor everolimus, with no objective radiographic evidence of treatment response after 4 months. Conclusions Our data further support that abnormal activation of the mTOR pathway is a consistent genetic event in sclerosing pneumocytoma. This warrants further exploration to determine if mTOR pathway inhibitors may be effective in patients with metastatic or recurrent disease.

Abstract Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytoma predominantly affecting children and young adults. We performed comprehensive genomic characterization on a cohort of 67 patients with histologically defined PXA (n = 53, 79%) or anaplastic PXA (A‐PXA, n = 14, 21%), including copy number analysis (ThermoFisher Oncoscan, n = 67), methylation profiling (Illumina EPIC array, n = 43) and targeted next generation sequencing (n = 32). The most frequent alterations were CDKN2A/B deletion (n = 63; 94%) and BRAF p.V600E (n = 51, 76.1%). In 7 BRAF p.V600 wild‐type cases, alternative driver alterations were identified involving BRAF , RAF1 and NF1 . Downstream phosphorylation of ERK kinase was uniformly present. Additional pathogenic alterations were rare, with TERT , ATRX and TP53 mutations identified in a small number of tumors, predominantly A‐PXA. Methylation‐based classification of 46 cases utilizing a comprehensive reference tumor allowed assignment to the PXA methylation class in 40 cases. A minority grouped with the methylation classes of ganglioglioma or pilocytic astrocytoma (n = 2), anaplastic pilocytic astrocytoma (n = 2) or control tissues (n = 2). In 9 cases, tissue was available from matched primary and recurrent tumors, including 8 with anaplastic transformation. At recurrence, two tumors acquired TERT promoter mutations and the majority demonstrated additional non‐recurrent copy number alterations. Methylation class was preserved at recurrence. For 62 patients (92.5%), clinical follow‐up data were available (median follow‐up, 5.4 years). Overall survival was significantly different between PXA and A‐PXA (5‐year OS 80.8% vs. 47.6%; P = 0.0009) but not progression‐free survival (5‐year PFS 59.9% vs. 39.8%; P = 0.05). WHO grade remained a strong predictor of overall survival when limited to 38 cases defined as PXA by methylation‐based classification. Our data confirm the importance of WHO grading in histologically and epigenetically defined PXA. Methylation‐based classification may be helpful in cases with ambiguous morphology, but is largely confirmatory in PXA with well‐defined morphology.

Introduction: Multikinase inhibitors have clinical activity in radioactive iodine refractory (RAIR) differentiated thyroid cancers (DTCs) but are not curative; optimal management and salvage therapies remain unclear. This study assessed clinical effects of pazopanib therapy in RAIR-DTC patients with progressive disease, examining in parallel biomarker that might forecast/precede therapeutic response. Methods: Assessment of responses and toxicities and of any association between thyroglobulin (Tg) changes cycle 1 and RECIST (response evaluation criteria in solid tumors) response to pazopanib therapy were prospectively undertaken in Tg antibody negative RAIR-DTC patients. RECIST progressive metastatic disease <6 months preceding enrollment was required. With a sample size of 68 (assuming 23 attaining partial response [PR]), there would be 90% chance of detecting a difference of >30% when the proportion of patients attaining PR whose Tg values decrease by >50% is >50% cycle 1 (one-sided α = 0.10, two sample test of proportions). Mean corpuscular volume (MCV) change or mutational status or pretreatment were also explored as early correlates of eventual RECIST response. Results: From 2009 to 2011, 60 individuals were treated and evaluated; (one additional patient withdrew; another was found ineligible before therapy initiation); 91.7% had previous systemic therapy beyond RAI. Adverse events included one death (thromboembolic) deemed possibly pazopanib associated. Twenty-two confirmed RECIST PRs resulted (36.7%, confidence interval; CI [24.6–50.1]); mean administered 4-week cycles was 10. Among 44 fully accessible patients, the Tg nadir was greater among the 20 attaining PR (median: −86.8%; interquartile range [IQR]: −90.7% to −70.9%) compared with the 28 who did not (median: −69.0%; IQR: −78.1% to −27.7%, Wilcoxon rank-sum test: p = 0.002). However, the difference in the proportion of PRs among those whose Tg fell ≥50% after cycle 1 versus those that did not were not significantly correlated (−23.5% [CI: −55.3 to 8.3]; Fisher's exact test p -value = 0.27). RECIST response was also not correlated with/predicted by early MCV change, receipt of prior therapy, or tumor mutational status. Conclusions: This trial prospectively confirmed pazopanib to have clinical activity and manageable toxicities in patients with progressive RAIR-DTC. Response to pazopanib, however, was not robustly forecast by early associated changes in Tg or MCV, by prior therapy, or by tumor mutational status. ClinicalTrials.gov NCT00625846.

11119 Background: Evaluation of prognostic factors in carcinoid tumors of the lung is limited due to the rarity of disease. This study assessed Ki-67 expression and other clinical variables as prognostic factors in 262 patients. Methods: A systematic search of Mayo Clinic lung cancer epidemiology and tumor registry databases from 1997 to 2009 identified 449 consecutive patients, with 262 having available tissue blocks [40 atypical carcinoids (AC) and 222 typical carcinoids (TC)]. Clinical data were collected by chart review. Tissue blocks were reviewed by 1/3 pathologists using WHO criteria. Tumors were tested for the Ki-67 index using digital image analysis (tumor tracing) by two operators. The association and predictive value of the factors with recurrence-free and overall survival (RFS and OS) were explored using univariable Cox proportional Hazards model and concordance (c) index. Results: Age, stage, smoking history, lymph node (LN) involvement and Ki-67 index were significant prognostic factors for RFS and OS. Median follow-up on alive-patients is 5 years (range: 0.006-5). Median percentage of Ki-67 index of AC and TC were 1.61% and 0.56% (P<0.0001), respectively. Patients with Ki-67 ≥ 1% had significantly worse RFS (HR=3.69, P<0.0001) and OS (HR=3.69, P=0.0007) compared with Ki-67 < 1% group. The c-index of Ki-67 (0.65) was comparable to the pathologic distinction between AC and TC (0.62 for original diagnosis and 0.63 for central-reviewed diagnosis). Conclusions: Ki-67 index cutoff value of 1% is a valuable prognostic biomarker for pulmonary carcinoids based on this large cohort. Our data also provide strong evidence for clinical variables such as age, stage, smoking history, and LN involvement as clinical prognostic factors in pulmonary carcinoids. A prognostic calculator incorporating Ki-67 and clinical variables is under development. [Table: see text]

Papillary fibroelastoma (PFE) is an increasingly recognized cardiac tumor. Despite its prevalence, controversy exists as to whether it represents a reactive or neoplastic process due to histopathologic similarities with Lambl excrescences (LEs), an accepted reactive phenomenon. Recently, KRAS mutations were reported in a small collection of PFEs, but the incidence of mutations and conditions in which they arise in are unknown. Furthermore, the relationship between PFE and LE has yet to be investigated. Institutional archives were queried for cases of PFE (2001-2017). Paraffin-embedded tissue was microdissected for tumor isolation. Prospectively identified LEs (2018) were collected and wholly isolated. Extracted DNA underwent droplet digital polymerase chain reaction analysis of the most common KRAS mutations (codons 12/13 and 61). Relevant clinical information was abstracted from the medical record. Fifty-two PFEs were tested from 50 patients (32 women). The median patient age was 67 years. Seventeen (33%) PFEs harbored pathogenic variants in tested KRAS codons (12 in codons 12/13; 5 in codon 61). Mutations were mutually exclusive. No clinical or pathologic correlates differed significantly from cases without detectable pathogenic variants. No pathogenic mutation were detected in LEs (n=20; P=0.002). Herein, we report on the largest series of PFE tested for KRAS mutations and present the largest cohort of KRAS-mutant PFEs to date, providing evidence in support of the notion that at least a subset of PFEs represents neoplasia. Moreover, the lack of KRAS mutations in LEs provides evidence as to the separate etiology of this accepted reactive lesion.

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations. Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed. Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon). Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis. Disclosures Jen: Celgene: Employment.

Abstract Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and genetic MAPK pathway activation through alterations such as BRAF V600E mutation, and FGFR2-KIAA1598/CTNNA3 and FGFR3-TACC3 fusions. We report the molecular profiling of PLNTY in 9 patients, 7 female: 2 male, median age at diagnosis of 16 years (range, 5–34). All tumors were supratentorial with diagnostic morphological features of PLNTY including oligodendroglioma-like areas and strong diffuse CD34 immunostaining. Four (of 9; 44%) tumors were positive for BRAF V600E immunostain, indicating the presence of a BRAF V600E mutation. The 6 cases evaluated for IDH status were negative by IDH1-R132H immunostain (n=5; 3 BRAF V600E-negative and 2 BRAF V600E-positive) or by NGS (n=1; BRAF V600E-negative). Oncoscan chromosomal microarray performed in 5 BRAF V600E-negative tumors showed recurrent copy number changes including gain of whole chromosomes 5, 7, 8, 9, 12, 18, 19, 20, 21 and X, and loss of chromosomes 1, 2, 10q, 13, 22 and Xq. The 10q losses (n=2) were highly suggestive of an FGFR2-KIAA1598 and of a potentially novel FGFR2 underlying fusion event in one case each. Custom targeted neuro-oncology DNA and RNA NGS panel performed in 2 cases revealed a fusion similar to fusions previously reported in pilocytic astrocytoma: one with a KIAA1549-BRAF (exon 15-exon 9) fusion associated with a 7q34 ~785 kilobase deletion rather than the characteristic ~2 megabase duplication seen in pilocytic astrocytoma, and another with a QKI-NTRK2 (exon 6-exon 15) fusion associated with a 9q21 ~191 kilobase duplication disrupting NTRK2. The KIAA1549-BRAF fusion-positive case also had a TP53 mutation with loss of whole chromosome 17, suggesting TP53 complete inactivation. This study confirms that PLNTY is a low-grade neuroepithelial tumor with frequent MAPK pathway activation and expands the spectrum of MAPK activating alterations observed in PLNTY.

AbstractMAPK pathway activation has been recurrently observed in desmoplastic infantile ganglioglioma/astrocytoma (DIG/DIA) with reported disproportionally low mutation allele frequencies relative to the apparent high tumor content, suggesting that MAPK pathway alterations may be subclonal. We sought to expand the number of molecularly profiled cases and investigate if tumor cell composition could account for the observed low mutation allele frequencies. Molecular (targeted neuro-oncology next-generation sequencing/RNA sequencing and OncoScan microarray) and immunohistochemical (CD68-PGM1/CD163/CD14/CD11c/lysozyme/CD3/CD20/CD34/PD-L1) studies were performed in 7 DIG. Activating MAPK pathway alterations were identified in 4 (57%) cases: 3 had a BRAF mutation (V600E/V600D/V600_W604delinsDQTDG, at 8%–27% variant allele frequency) and 1 showed a TPM3-NTRK1 fusion. Copy number changes were infrequent and nonrecurrent. All tumors had at least 30% of cells morphologically and immunophenotypically consistent with microglial/macrophage lineage. Two subtotally resected tumors regrew; 1 was re-excised and received adjuvant treatment (chemotherapy/targeted therapy), with clinical response to targeted therapy only. Even with residual tumor, all patients are alive (median follow-up, 83 months; 19–139). This study further supports DIG as another MAPK pathway-driven neuroepithelial tumor, thus expanding potential treatment options for tumors not amenable to surgical cure, and suggests that DIG is a microglia/macrophage-rich neuroepithelial tumor with frequent low driver mutation allele frequencies.