Faculty Bibliography

BACKGROUND: While most hemangioblastomas (~70%) are sporadic and occur predominantly in the cerebellum, they may present as well as familial form associated with von Hippel-Lindau (VHL) syndrome, an autosomal dominant disorder caused by germline mutations of the VHL gene that trigger nuclear translocation of hypoxia-inducible factor (HIF)- 1alpha and angiogenesis. Although inactivation of VHL, a tumor suppressor gene, has been observed in hemangioblastomas, the underlying pathogenic mechanisms responsible for familial and sporadic hemangioblastomas remain incompletely understood.
METHOD(S): Whole exome sequencing of cerebellar hemangioblastoma tumors and matched blood leukocytes from 24 patients, age 24-63, was performed. After preparation and amplification of barcoded libraries, exomes were captured using Kapa Biosystems methodology and paired-end sequenced on Illumina HiSeq 2500 to an average 100-fold coverage. Following read alignment to hg19 genome, tumor and germline (leukocyte) sequences were compared, and pathogenic single nucleotide variants (SNVs) identified and validated by re-sequencing followed by pathway analysis. Additionally, tumor RNA isolated using Maxwell Promega was sequenced on Illumina instrument and the expression counts determined and normalized.
RESULT(S): We found 314 pathogenic and/or highly deleterious mutations (both germline and somatic) with a median of 13 mutations per patient. Five patients had VHL syndrome (germline VHL mutation) and 4 carried somatic VHL mutations. Among the VHL tumors, 82 mutations were identified, including HNF1B, NOTCH1 and TCF7L1, suggesting a potential contribution of altered RNA metabolism based upon pathway analysis. Among all hemangioblastomas, germline growth factor receptor variants (FGFR4 p.G388R (14/23 (61%) patients), IGF1R, PDGFRA and TYK2) known to activate STAT3 signaling and induce HIF-1alpha and angiogenesis, were identified. Non-hierarchical clustering of RNA sequencing data revealed two transcriptionally-distinct subtypes of hemangioblastomas.
CONCLUSION(S): Our findings indicate that hemangioblastomas can also occur by germline mutations known to activate STAT3 signaling, which may have significant implication in genetic testing and counseling of patients with hemangioblastomas

BACKGROUND: There is no effective therapy for patients (pts) with IDH-mutant gliomas that progress after RT and chemotherapy. At time of progression, these tumors have often transformed to glioblastoma (GBM) and have increased numbers of somatic mutations, i.e. have a ?hypermutator phenotype?. We hypothesized that there is synergistic efficacy of Avelumab (anti-PD-L1) combined with HFRT in pts with secondarily trans- formed IDH-mutant GBMs. Safety-lead-in results will be presented.
METHOD(S): This is a phase II, open-label, single-arm, multicenter study of Avelumab with HFRT in adults with transformed IDH-mutant GBM who previously received RT and TMZ and/or PCV. All pts received Avelumab 10 mg/kg IV followed at Day 8 by HFRT (25 Gy in 5 daily 5-Gy fractions) and then Avelumab 10 mg/kg IV every 2 weeks. A 3 + 3 design was used for a 6-patient safety-lead-in cohort. Adverse events were recorded according to CTCAE.
RESULT(S): Six pts (F=4, M=2) with a median age= 45.5 yrs (range 31.5-54.4 yrs) were enrolled in the safety-lead-in cohort. No DLT was observed. Grade >= 3 AEs included increased cerebral edema (3 pts), hyponatremia (1 pt) and worsening hemiparesis (3 pts). Grade <= 2 AEs included nausea, hypothyroidism, lymphopenia, thrombocytopenia, transaminase elevation, and fever/chills. Median follow-up time was 8.9 mo. Best treatment response was SD in 1 patient. At time of last follow-up all pts have discontinued treatment for PD. Median PFS was 4.2 mo (range 1.4-5.7). Median OS was 10.1 (range 6.8-21+) mo. 4 pts (67%) died, 2 pts remain alive in follow-up at 6.9 and 21.6 months after treatment initiation. The study was closed after the safety lead-in completed enrollment due to slow accrual.
CONCLUSION(S): Avelumab combined with HFRT was tolerable without dose-limiting toxicity in this safety-lead-in cohort of adult patients with transformed IDH-mutant GBM. Further studies are necessary to determine efficacy of this treatment regimen

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis¹. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC^2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

IDH1/2 mutations are early drivers present in diverse human cancer types arising in various tissue sites. IDH1/2 mutation is known to induce a global hypermethylator phenotype. However, the effects on DNA methylation across IDH mutant cancers and functionally different genome regions, remain unknown. We analyzed DNA methylation data from IDH1/2 mutant acute myeloid leukemia, oligodendroglioma, astrocytoma, solid papillary breast carcinoma with reverse polarity, sinonasal undifferentiated carcinoma and cholangiocarcinoma, which clustered by their embryonal origin. Hypermethylated common probes affect predominantly gene bodies while promoters in IDH1/2 mutant cancers remain unmethylated. Enhancers showed global hypermethylation, however commonly hypomethylated enhancers were associated with tissue differentiation and cell fate determination. We demonstrate that some chromosomes, chromosomal arms and chromosomal regions are more affected by IDH1/2 mutations while others remain resistant to IDH1/2 mutation induced methylation changes. Therefore IDH1/2 mutations have different methylation effect on different parts of the genome, which may be regulated by different mechanisms.

Multifocal breast cancer (MFBC), ductal type, has been hypothesized to arise by one of two mechanisms: either through intramammary/intralymphatic spread from a single index tumor (MBC-1), or as multiple independent tumors with each focus carrying its corresponding ductal carcinoma in-situ (MBC-2). In order to improve our understanding of MFBC pathogenesis, we employed laser capture microdissection coupled with whole-exome sequencing to study clonal origin in MFBC. We selected three cases of MBC-1 (C1 to C3) and MBC-2 (C4 to C6) and analyzed three foci from each case. MBC-1 cases were histologically similar and showed a strong predilection for satellite foci, vascular invasion and nodal metastasis when compared to MBC-2. Our bioinformatics approach provided strong evidence for clonal relationships in MBC-1, as demonstrated by distinct clusters of genes conserved across all tumor foci. Conversely, no gene clusters were shared across all the foci in MBC-2, suggesting multiple independent tumors. These findings provide further support for the two distinct pathogenetic mechanisms in MFBC.