Faculty Bibliography

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

Background: Over 250,000 breast cancer cases are diagnosed annually in the US, of which 15% (n=37,500) are triple negative breast cancers (TNBC). TNBC has an aggressive clinical course with limited therapeutic options. Some studies estimate that a small proportion of TNBCs defined by immunohistochemical (IHC) stains alone, i.e. estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2)-negative, can, in fact, show HER2 amplification by fluorescent in situ hybridization (FISH) and, therefore, be reclassified as HER2-enriched. If properly classified, these patients can benefit from anti-HER2 therapy. To capture this subset of patients, we performed a now four-year-long prospective study, in which TNBCs were reflexed to HER2 by FISH. Herein, we present our findings with detailed clinicopathologic analysis of the reclassified cases. Design: TNBC cases and ER/PR-positive, HER2-negative breast cancers from 2014 to 2017 with concurrent IHC and FISH results were analyzed. The pathology slides were reviewed by two pathologists. Stromal tumor infiltrating lymphocytes (sTILs) were defined as the percentage of all mononuclear cells within tumor stroma not in direct contact with tumor cells. Results: Of the 253 TNBCs in the past 4 years, 13 tumors (5%) showed HER2 amplification by FISH (6 surgical excisions and 7 core biopsies). Two patients have previously identified BRCA1 mutation. Twelve of 13 tumors (92%) were grade 3. All tumors were invasive ductal carcinoma of no special type. No apocrine morphology was noted. Notably, five tumors had >=50% sTILs, which can be classified as lymphocyte-predominant breast cancer based on some studies. In the same time period, 41 ER/PR-positive/HER2-negative breast cancers were reflexed to HER2 by FISH, none of which showed amplification. All tumors were fixed in formalin for comparable amount of time (6-72 hours). After reclassification, approximately 40% of patients received anti-HER2 therapy. (Table Presented) Conclusions: Pathologists may consider reflexing HER2 evaluation to FISH in TNBCs with grade 3, high ki67 and high sTILs. If 5% of TNBCs can be reclassified as HER2-enriched tumors, based on the national statistics, annually approximately 1875 patients with TNBCs by IHC may actually benefit from anti-HER2 therapy

Epigenetic patterns on the level of DNA methylation have already been shown to separate clinically relevant subgroups of meningiomas. We here set out to identify potential prognostic implications of epigenetic modification on the level of histones with focus on H3K27 trimethylation (H3K27me3). H3K27me3 was assessed by immunohistochemistry on 232 meningiomas from 232 patients. In 194 cases, trimethylation was detected in tumor cells. In 25 cases, staining was limited to vessels while all tumor cells were negative. Finally, 13 cases yielded equivocal staining patterns. Reduced abundance of H3K27me3 in cases with staining limited to vessels was confirmed by mass spectrometry on a subset of cases. Lack of staining for H3K27me3 in all tumor cells was significantly associated with more rapid progression (p = 0.009). In line, H3K27me3-negative cases were associated with a DNA methylation pattern of the more aggressive types among the recently introduced DNA methylation groups. Also, NF2 and SUFU mutations were enriched among cases with complete lack of H3K27me3 staining in tumor cells (p < 0.0001 and p = 0.029, respectively). H3K27me3 staining pattern added significant prognostic insight into WHO grade II cases and in the compound subset of WHO grade I and II cases (p = 0.04 and p = 0.007, respectively). However, it did not further stratify within WHO grade III cases. Collectively, these data indicate that epigenetic modifications beyond DNA methylation are involved in the aggressiveness of meningioma. It also suggests that H3K27me3 immunohistochemistry might be a useful adjunct in meningioma diagnostics, particularly for cases with WHO grade II histology or at the borderline between WHO grade I and II.

Purpose: Prior studies have shown correlation between relative cerebral blood volume (rCBV) and patient survival and tumor genomics. The purpose of this study was to determine whether rCBV values correlate with isocitrate dehydrogenase (IDH) mutation status, genome-wide CNV (copy number variation) and patient overall survival in diffuse gliomas. Materials & Methods: 107 treatment naive gliomas (62 patients from TCGA/TCIA dataset and 45 patients from our institute) (44 glioblastoma and 63 lower grade gliomas) with DSC T2* perfusion data were included. IDH mutation and survival data were assayed by the TCGA, and pre-surgical imaging collected by The Cancer Imaging Archive. CNVabundance plots obtained with Illumina 850k EPIC DNA methylation arrays were reviewed in 19 patients. The association of rCBV with tumor genomics, CNV and overall survival were analyzed. Results: IDH-wildtype gliomas (44.8%) demonstrated higher rCBV values (rCBV = 6.87 +/- 3.09) than IDH-mutated gliomas (55.2%, rCBV =2.21 +/- 1.71 for 1p/19q codeleted gliomas and 2.09 +/- 2.00 for non-codeleted gliomas, ANOVA, p<0.0001). rCBV is a significant predictor of overall survival (HR 1.23, p<0.0001). Gliomas with rCBV < 3.80 showed better survival (n = 54, median survival time unobserved) than gliomas with rCBV > 3.8 (n = 53, median 18 months; log-rank p<0.0001). IDHwt gliomas with high rCBV had the worst survival (10.6% surviving at 3 years, 95% CI (4%, 30%)). CNV-S IDHmut 1p19q noncodeleted gliomas demonstrated significantly lower mean rCBV (1.4 +/- 0.4) than CNV-U gliomas (4.0 +/- 1.1, p = 0.009). Conclusion: IDHwt gliomas show higher rCBV than IDHmut gliomas irrespective of the glioma grade. Higher rCBV measurements are associated with poorer survival in the entire cohort and also within IDHmut and IDHwt gliomas. IDHmut 1p19q noncodeleted gliomas with higher CNV abundance (CNV-U) also show higher CBV when compared with those with lesser degree of CNVabundance (CNV-S)