Faculty Bibliography

Program cell death ligand-1 (PD-L1) membranous expression on >5% tumor cells (PD-L1 positive tumors) is an unfavorable prognostic marker in many solid tumors. We previously showed that approximately 40% of neurofibromatosis type 2 (NF2) meningiomas are PD-L1 positive tumors. However, due to the invasive nature of biopsies, collection of tumor tissue is not always feasible. Thus, a non-invasive alternative is needed to evaluate the status of tumor growth and confirm PD-L1 positive tumors before the consideration of immunotherapy. It has recently been revealed that expression of PD-L1 on tumor associated macrophages is also a strong prognostic indicator. We retrieved formalin-fixed paraffin-embedded (FFPE) tissue from 10 NF2 meningioma cases to identify PD-L1 expression on macrophages and/or monocytes. We found that 3 out of 4 PD-L1 positive tumors were associated with expression of PDL-1 on CD68 (-) monocyte-like cells located in the peri-and intravascular lumens. These cells were only observed in 1 out of 6 PD-L1 negative tumors. Compared to others, tumors with PD-L1 expression on monocyte-like cells presented a higher Ki-67 proliferative index that was above 10%. Our results suggest that PD-L1 positive circulating CD68 (-) monocyte-like cells are correlated with tumor cell PD-L1 expression and progression in NF2 meningiomas

We previously reported an increased prevalence of pre-treatment lymphopenia in pediatric patients with medulloblastoma, the most common childhood malignant brain tumor, suggesting tumor-induced systemic immune suppression present at the time of diagnosis. Tumor-induced systemic immune suppression, including lymphopenia, has been recognized in adult high-grade gliomas for several decades. Pediatric midline gliomas express distinct epigenetic and genetic alterations, such as the histone H3 K27M mutation. Patients with this mutation often experience a more aggressive clinical course and poor outcome. We confirmed the K27M mutation from tumor biopsies in 10 pediatric midline glioma patients by whole genome DNA methylation array analysis, and analyzed complete blood counts (CBC) at the time of diagnosis prior to surgery and chemotherapy. Compared to a control group of pilocytic astrocytoma, patients with K27M-mutated gliomas have higher monocyte-to-lymphocyte ratios (MLR) and absolute monocyte counts (AMC), reaching statistical significance (p<0.05). Furthermore, the prevalence of monocytosis, characterized by AMC above normal age-adjusted means, was statistically significantly higher (p<0.05) in the study group (80%) than the control group (33%). High pre-operative MLR has previously been reported in various solid tumors in association with worse prognosis and poor overall survival. This is the first study to our knowledge that identifies MLR as a potential valuable biomarker in pediatric gliomas. Our findings suggest monocytosis and overall tumor-induced systemic immune suppression present at the time of diagnosis in K27M mutant tumors. Aberrant circulating immune cell ratios may affect the development of immune therapies for malignant pediatric midline gliomas with the histone H3 K27M mutation

In contrast to adulthood, meningiomas are rare among children and adolescents. Although recent papers have characterized the genomics of adult meningiomas, the molecular profiles of childhood meningiomas have not been elucidated in detail. We analyzed 41 tumor samples from 37 pediatric meningioma patients (female: 17, male: 20; age range: 1-21 years). Atypical meningioma WHO grade II was the most frequent histological subtype (N=14, 38%). Most tumors were located at the convexity (N=18) or the skull base (N=15). Lack of SMO, AKT, KLF4/TRAF7 mutations by Sanger sequencing (n=22) prompted whole genome sequencing of a subset (n=7). All seven cases exhibited bi-allelic inactivation of NF2 (combined large deletion and germline (5/7) or somatic (2/7) base exchanges/frameshifts). Subsequently, tumor samples from all 37 patients were subjected to 450K DNA methylation profiling and targeted DNA sequencing using brain tumor specific gene panel. Loss of chromosome 22 was frequent (N=28, 76%), followed by loss of chromosome 1 (N=12, 32%) and chromosome 18 (N=7, 19%). Moreover, separation into three groups was evident: One encompassing all clear-cell meningiomas with enrichment for SMARCE1 mutations, a second dominated by atypical meningiomas, and a third group composed of benign meningiomas, as well as rare subtypes such as rhabdoid meningiomas. When analyzed with 105 adult tumors, most of pediatric meningiomas (28/37) clustered into a separate methylation group both by unsupervised hierarchical clustering and t-stochastic nearest neighbor embedding (t-SNE). Four recurrences were similar to the primary tumor. These data suggest that pediatric meningiomas are genetically distinct from adult counterparts

INTRODUCTION: Liquid biopsy has the potential to revolutionize diagnosis and management of cancer. In brain tumors, detection of cell free tumor DNA (ctDNA) and circulating tumor cells (CTC) is challenging due to low level of the circulating material. We present a novel workflow for detection and capture of CTCs. METHODS: Peripheral blood was obtained from five pediatric patients with astrocytoma (n=2), ependymoma (n=1), dysembryoplastic neuroepithelial tumor (n=1), and medulloblastoma (n=1) at initial resection (n=3) and relapse (n=2). Samples were enriched using the Clear-Bridge ClearCell FX1 system and the suspension stained with antibodies against CD56 and CD45. Samples were analyzed using Silicon Biosystems DEPArray to capture single and pooled CTCs. CTCs were identified by CD56 positivity, while leukocytes were positive for CD45 and NK cells double-positive for CD56 and CD45. RESULTS: CTCs were identified in all 5 patient samples. The number of CD56-positive cells isolated from each sample ranged from 1 to 25 (mean 9). The CD56-positive cells were on average 11.8 mum in diameter (range 9.0-15.7 mum), and CD45-positive cells were on average 10.8 mum in diameter (range 8.9-15.9 mum). Single CTCs and CTC pools are amenable to molecular analysis after whole genome amplification. CONCLUSION: We report a novel integrated workflow for capturing CTCs in pediatric patients with brain tumors. While rare, CTCs circulate in peripheral blood of patients with brain tumors regardless of their grade and are amenable for molecular analysis. This method has the potential to serve as a non-invasive diagnostic and monitoring method for pediatric brain tumors

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.

As oxygen is essential for many metabolic pathways, tumour hypoxia may impair cancer cell proliferation^1-4. However, the limiting metabolites for proliferation under hypoxia and in tumours are unknown. Here, we assessed proliferation of a collection of cancer cells following inhibition of the mitochondrial electron transport chain (ETC), a major metabolic pathway requiring molecular oxygen ⁵ . Sensitivity to ETC inhibition varied across cell lines, and subsequent metabolomic analysis uncovered aspartate availability as a major determinant of sensitivity. Cell lines least sensitive to ETC inhibition maintain aspartate levels by importing it through an aspartate/glutamate transporter, SLC1A3. Genetic or pharmacologic modulation of SLC1A3 activity markedly altered cancer cell sensitivity to ETC inhibitors. Interestingly, aspartate levels also decrease under low oxygen, and increasing aspartate import by SLC1A3 provides a competitive advantage to cancer cells at low oxygen levels and in tumour xenografts. Finally, aspartate levels in primary human tumours negatively correlate with the expression of hypoxia markers, suggesting that tumour hypoxia is sufficient to inhibit ETC and, consequently, aspartate synthesis in vivo. Therefore, aspartate may be a limiting metabolite for tumour growth, and aspartate availability could be targeted for cancer therapy.

Patients with DICER1 predisposition syndrome have an increased risk to develop pleuropulmonary blastoma, cystic nephroma, embryonal rhabdomyosarcoma, and several other rare tumor entities. In this study, we identified 22 primary intracranial sarcomas, including 18 in pediatric patients, with a distinct methylation signature detected by array-based DNA-methylation profiling. In addition, two uterine rhabdomyosarcomas sharing identical features were identified. Gene panel sequencing of the 22 intracranial sarcomas revealed the almost unifying feature of DICER1 hotspot mutations (21/22; 95%) and a high frequency of co-occurring TP53 mutations (12/22; 55%). In addition, 17/22 (77%) sarcomas exhibited alterations in the mitogen-activated protein kinase pathway, most frequently affecting the mutational hotspots of KRAS (8/22; 36%) and mutations or deletions of NF1 (7/22; 32%), followed by mutations of FGFR4 (2/22; 9%), NRAS (2/22; 9%), and amplification of EGFR (1/22; 5%). A germline DICER1 mutation was detected in two of five cases with constitutional DNA available. Notably, none of the patients showed evidence of a cancer-related syndrome at the time of diagnosis. In contrast to the genetic findings, the morphological features of these tumors were less distinctive, although rhabdomyoblasts or rhabdomyoblast-like cells could retrospectively be detected in all cases. The identified combination of genetic events indicates a relationship between the intracranial tumors analyzed and DICER1 predisposition syndrome-associated sarcomas such as embryonal rhabdomyosarcoma or the recently described group of anaplastic sarcomas of the kidney. However, the intracranial tumors in our series were initially interpreted to represent various tumor types, but rhabdomyosarcoma was not among the typical differential diagnoses considered. Given the rarity of intracranial sarcomas, this molecularly clearly defined group comprises a considerable fraction thereof. We therefore propose the designation "spindle cell sarcoma with rhabdomyosarcoma-like features, DICER1 mutant" for this intriguing group.

IDH-mutant astrocytomas are significantly less aggressive than their IDH-wildtype counterparts. We analyzed The Cancer Genome Atlas dataset (TCGA) and identified a small group of IDH-mutant, WHO grade II-III astrocytomas (n = 14) with an unexpectedly poor prognosis characterized by a rapid progression to glioblastoma and death within 3 years of the initial diagnosis. Compared with IDH-mutant tumors with the typical, extended progression-free survival in a control group of age-similar patients, the tumors in the rapidly progressing group were characterized by a markedly increased level of overall copy number alterations ([CNA]; p = 0.006). In contrast, the mutation load was similar, as was the methylation pattern, being consistent with IDH-mutant astrocytoma. Two of the gliomas (14%) in the rapidly progressing, IDH-mutant group but none of the other grade II-III gliomas in the TCGA (n = 283) had pathogenic mutations in genes (FANCB and APC) associated with maintaining chromosomal stability. These results suggest that chromosomal instability can negate the beneficial effect of IDH mutations in WHO II-III astrocytomas. The mechanism of the increased CNA is unknown but in some cases appears to be due to mutations in genes with a role in chromosomal stability. Increased CNA could serve as a biomarker for tumors at risk for rapid progression.

Background: Mucinous adenocarcinoma is a special type of colorectal cancer (CRC) that has poor response to the treatment, more aggressive behavior and poorer outcome than non-mucinous CRC. Its biological and clinical behavior is largely determined by its molecular genetics. However, the genetics of this group of CRC is yet to be defined. Design: 152 cases of resected CRC with sequencing data generated by Ion AmpliSeq Cancer Hotspot Panel (or 50 genes) in the past two years were retrospectively retrieved from the departmental databases. No neoadjuvant therapy was performed before surgery. CRCs were divided into two groups: 1). Mucinous CRC (MCRC) group (13 mucinous CRC and 19 CRC with at least 20% of mucinous component (n=32) and 2). Non-mucinous CRC (NMCRC) group without mucinous component (n=120). The type and frequency of gene mutations and microsatellite instability (MSI) status defined by immunohistochemistry for mismatch repair proteins were analyzed. Fisher exact test was employed for statistical analysis. Results: In MCRC group, 31 of 32 (97%) were positive for the mutation of either KRAS (15/32), BRAF (15/32) or double mutations (1/32). Only one case showed no mutation for KRAS or BRAF, but TP53. The highest frequent mutations in MCRC group were KRAS (16/32) and BRAF (16/32), PIK3CA (10/32), followed by APC (9/32) and TP53 (8/32). In NMCRC group, 64 of 120 (53%) cases had either KRAS (50/120, 42%) or BRAF (14/120, 12%) mutation, and no double mutations. TP53 mutation (65/120, 54%) is most frequent mutation in this group, followed by KRAS (50/120, 42%), APC (39/120, 33%), PIK3CA (26/120, 22%) and BRAF (14/120, 12%). MSI-high status is more frequently seen in BRAF mutated CRCs in MCRC group (12/15, 80%) than in BRAF mutated NMCRC group (50%) (p<0.05), suggesting that MSI-high status is more commonly related with epigenetic effect in MCRCs. Conclusions: The vast majority of mucinous CRC or CRC with mucinous component have the mutations either in KRAS or BRAF in EGFR signaling pathway, suggesting that dysregulation of EGFR pathway plays a critical role in the development of mucinous CRC or CRC with mucinous component. Poor response to the treatment of mucinous adenocarcinoma may be partially attributable to the unique genetics of this group of CRCs

Background: CRC is a heterogeneous and complex disease, harboring numerous genetic and epigenetic alterations acquired during cancer development. The genetics and epigenetics of CRCs may dictate their histology, biology, and clinical outcome. We reviewed the sequencing data generated from the next generation sequencing technology to analyze the relationship between the quantity of gene mutations and the biology of CRCs Design: 152 cases of resected CRCs without neoadjuvant therapy that have sequencing data generated by Ion AmpliSeq Cancer Hotspot Panel (or 50 genes panel) were retrospectively identified from the department database. These 152 cases also had immunostaining results for mismatch repair proteins (surrogate markers for Microsatellite instability status, or MSI). The CRCs were divided into two groups: hypermutated (3 or more gene mutations) and hypomutated (2 or less gene mutations). Tumor grade, T stage, lymph node metastasis, and MSI status in the two groups were compared and the data analyzed using Fisher's exact test. Results: Of the 152 cases, 93 (61.2%) were classified into the hypomutation group and 59 (38.8%) into the hypermutation groups. In the hypomutation group, 80 (86.0%) were low (well and moderately differentiated) and 13 (14.0%) were high grade (poorly differentiated) CRCs. In the hypermutation group, 37 (62.7%) were diagnosed as low and 22 (37.3%) as high grade CRCs. The hypermutation status is strongly associated with high tumor grade (P=0.0014). High T stages (stage 4) does not correlate with mutation status (25/93 in hypomutation group and 19/59 in hypermutation group, p>0.05). In addition, no correlation between hypermutation and positive lymph nodes or MSI-High was found (54/93 or 20/90 in hypomutation group and 29/59 or 18/57 in hypermutation group, p>0.05). Conclusions: CRCs with hypermutation detected by Ion AmpliSeq Cancer Hotspot Panel are more likely to have high grade of histology, suggesting accumulation of gene mutations leading to worse biological behavior