Peggy N. Atmodimedjo

The impact of surgery in molecularly defined low-grade glioma: an integrated clinical, radiological, and molecular analysis

Background Extensive resections in low-grade glioma (LGG) are associated with improved overall survival (OS). However, World Health Organization (WHO) classification of gliomas has been completely revised and is now predominantly based on molecular criteria. This requires reevaluation of the impact of surgery in molecularly defined LGG subtypes. Methods We included 228 adults who underwent surgery since 2003 for a supratentorial LGG. Pre- and postoperative tumor volumes were assessed with semiautomatic software on T2-weighted images. Targeted next-generation sequencing was used to classify samples according to current WHO classification. Impact of postoperative volume on OS, corrected for molecular profile, was assessed using a Cox proportional hazards model. Results Median follow-up was 5.79 years. In 39 (17.1%) histopathologically classified gliomas, the subtype was revised after molecular analysis. Complete resection was achieved in 35 patients (15.4%), and in 54 patients (23.7%) only small residue (0.1-5.0 cm3) remained. In multivariable analysis, postoperative volume was associated with OS, with a hazard ratio of 1.01 (95% CI: 1.002-1.02; P = 0.016) per cm3 increase in volume. The impact of postoperative volume was particularly strong in isocitrate dehydrogenase (IDH) mutated astrocytoma patients, where even very small postoperative volumes (0.1-5.0 cm) already negatively affected OS. Conclusion Our data provide the necessary reevaluation of the impact of surgery in molecularly defined LGG and support maximal resection as first-line treatment for molecularly defined LGG. Importantly, in IDH mutated astrocytoma, even small postoperative volumes have negative impact on OS, which argues for a second-look operation in this subtype to remove minor residues if safely possible.

Diagnostic Detection of Allelic Losses and Imbalances by Next-Generation Sequencing: 1p/19q Co-Deletion Analysis of Gliomas

Cancer cells are genomically unstable and accumulate tumor type-specific molecular aberrations, which may represent hallmarks for predicting prognosis and targets for therapy. Co-deletion of chromosomes 1p and 19q marks gliomas with an oligodendroglioma component and predicts a better prognosis and response to chemotherapy. In the current study, we present a novel method to detect chromosome 1p/19q co-deletion or loss of heterozygosity (LOH) in a diagnostic setting, based on single-nucleotide polymorphism (SNP) analysis and next-generation sequencing (NGS). We selected highly polymorphic SNPs distributed evenly over both chromosome arms. To experimentally determine the sensitivity and specificity of targeted SNP analysis, we used DNAs extracted from 49 routine formalin-fixed, paraffin-embedded glioma tissues and compared the outcome with diagnostic microsatellite-based LOH analysis and calculated estimates. We show that targeted SNP analysis by NGS allows reliable detection of 1p and/or 19q deletion in a background of 70% of normal cells according to calculated outcomes, is more sensitive than microsatellite-based LOH analysis, and requires much less DNA. This specific and sensitive SNP assay is broadly applicable for simultaneous allelic imbalance analysis of multiple genomic regions and can be incorporated easily into NGS mutation analyses. The combined mutation and chromosomal imbalance analysis in a single NGS assay is suited perfectly for routine glioma diagnostics and other diagnostic molecular pathology applications.

PTEN in colorectal cancer: a report on two Cowden syndrome patients.

Kersseboom R, Dubbink HJ, Corver WE, van Tilburg AJP, Poley JW, van Leerdam ME, Atmodimedjo PN, van de Laar IMBH, Collée JM, Dinjens WNM, Morreau H, Wagner A. PTEN in colorectal cancer; a report on two Cowden syndrome patients.

Identification of mutations in Cell-free Circulating Tumor DNA in Adrenocortical Carcinoma: a Case Series.

Context The disease course of adrenocortical carcinoma (ACC) patients is heterogeneous. A marker for prognosis and treatment response would facilitate choices for diagnosis and therapy. In other cancer types, circulating cell-free tumor DNA predicted tumor dynamics. Case Descriptions The present pilot study included six patients. Next-generation sequencing (NGS) showed mutations in three ACC cases. From these patients, blood was drawn before (1 to 2 weeks) and after surgery and cell-free circulating DNA (cfDNA) was isolated. Tumor-specific mutations were found in the cfDNA of one of the three patients, with metastasized ACC at diagnosis. NGS of the tumor showed an NRAS mutation (c.182A>G:p.Q61R) in 78%, a TP53 mutation (c.856G>A:p.E286K) in 60%, and a TERT gene mutation (1295250C>T) in 28% of the reads. The preoperative cfDNA showed the same mutations at a frequency of 64%, 32%, and 2%, respectively. The postoperative cfDNA showed the same mutations but at lower frequencies (52%, 16%, and 3%, respectively). The patient was postoperatively treated with mitotane and chemotherapy. No mutations were detected in the corresponding leukocyte DNA or in the cfDNA from the two other patients. Conclusions To the best of our knowledge, we report for the first time mutations occurring at high levels in cfDNA collected before and after surgery from one of three patients, after previous identification in the tumor. However, in the cfDNA from two patients with known mutations, we were unable to reliably detect mutations in the cfDNA. Our results indicate that mutation detection in cfDNA can vary among ACC patients, and other approaches might be required to detect the tumor response and monitor progressive disease.

Molecular and clinical heterogeneity of adult diffuse low-grade IDH wild-type gliomas: assessment of TERT promoter mutation and chromosome 7 and 10 copy number status allows superior prognostic stratification

have been suggested. However, as this is not a well-defined separate entity, the question remains whether IDHwt DLGG classified according to current WHO classification qualifies as a single entity, with sufficient information to estimate prognosis adequately, and therefore, guide treatment, or if the assessment of additional markers is necessary and if so which. A recent study by Aibaidula et al. showed that IDHwt DLGG are prognostically heterogeneous and that markers like TERTp, EGFR amplification and H3F3A mutation could be of additional value [1]. The prognostic role of + 7/− 10q and its relationship with TERTp mutations were not reported, however. In this study, we report on the impact of additional molecular markers, including + 7/− 10q and TERTp, on overall survival in adult IDHwt DLGG. In our institute targeted next-generation sequencing is part of routine diagnostics for DLGG. We assess copy number changes of chromosome 1, 7 (including EGFR amplification), 9p, 10, 12, 19, and mutational status of genes IDH1/2, TP53, ATRX, CIC, FUBP1, EGFR, PIK3CA, CDKN2A, PTEN, H3F3A, BRAF, NOTCH1, TERTp. In our routine diagnostics, we use gain of whole chromosome 7 and loss of whole chromosomal arm 10q as criterion for + 7/− 10q status. Our sequencing protocol has been described previously [3, 4]. Between January 2003 and January 2017, we sequenced a total of 639 tumors as part of daily diagnostic routine (samples since 2013) and as part of a project on extent of resection in DLGG (samples since 2003) [8]. Of these, 510 tumors were histologically classified as DLGG and on sequencing 74 were IDHwt. We collected Karnofsky Performance Status (KPS) at diagnosis, age, gender and overall survival which were defined as time between date of diagnostic imaging and date of death. Patients that were alive at the time of analysis were censored. Further stratification of these IDHwt DLGG showed a molecularly heterogeneous group of tumors. Only 52.7% of With the 2016 revision of the World Health Organization classification of tumors of the central nervous system (WHO 2016) testing for the presence of mutations in isocitrate dehydrogenase 1 and 2 (IDH) and chromosome 1p/19q status is the cornerstone of glioma classification [5]. Approximately 80% of diffuse low-grade (grades II and III) gliomas (DLGG) are IDH mutated and have a relatively favorable prognosis compared to their IDH wild-type (IDHwt) counterparts [2]. The prognosis of IDHwt DLGG is almost similar to primary glioblastoma and genetic aberrations that are seen in primary glioblastoma are also reported in IDHwt DLGG: the combination of trisomy of whole chromosome 7 and loss of chromosomal arm 10q (+ 7/− 10q), and telomerase reverse transcriptase gene promoter (TERTp) mutations [2, 6, 7]. However, + 7/− 10q or TERTp mutations are not part of the WHO 2016 criteria and not all IDHwt DLGG have these specific molecular aberrations [2, 5]. Although clinical trials have not been performed, in view of their poor prognosis, aggressive treatment regimens for IDHwt DLGG

SNPitty: An Intuitive Web Application for Interactive B-Allele Frequency and Copy Number Visualization of Next-Generation Sequencing Data

Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic aberrations is, however, lacking. Therefore, the authors developed the web application SNPitty that allows interactive visualization and interrogation of variant call format files by using B-allele frequencies of single-nucleotide polymorphisms and single-nucleotide variants, coverage metrics, and copy numbers analysis results. SNPitty displays variant alleles and allelic imbalances with a focus on loss of heterozygosity and copy number variation using genome-wide heterozygous markers and somatic mutations. In addition, SNPitty is capable of generating predefined reports that summarize and highlight disease-specific targets of interest. SNPitty was validated for diagnostic interpretation of somatic events by showcasing a serial dilution series of glioma tissue. Additionally, SNPitty is demonstrated in four cancer-related scenarios encountered in daily clinical practice and on whole-exome sequencing data of peripheral blood from a Down syndrome patient. SNPitty allows detection of loss of heterozygosity, chromosomal and gene amplifications, homozygous or heterozygous deletions, somatic mutations, or any combination thereof in regions or genes of interest. Furthermore, SNPitty can be used to distinguish molecular relationships between multiple tumors from a single patient. On the basis of these data, the authors demonstrate that SNPitty is robust and user friendly in a wide range of diagnostic scenarios.

Double somatic mutations in mismatch repair genes are frequent in colorectal cancer after Hodgkin's lymphoma treatment

Objective Hodgkins lymphoma survivors who were treated with infradiaphragmatic radiotherapy or procarbazine-containing chemotherapy have a fivefold increased risk of developing colorectal cancer (CRC). This study aims to provide insight into the development of therapy-related CRC (t-CRC) by evaluating histopathological and molecular characteristics. Design 54 t-CRCs diagnosed in a Hodgkins lymphoma survivor cohort were analysed for mismatch repair (MMR) proteins by immunohistochemistry, microsatellite instability (MSI) and KRAS/BRAF mutations. MSI t-CRCs were evaluated for promoter methylation and mutations in MMR genes. Pathogenicity of MMR gene mutations was evaluated by in silico predictions and functional analyses. Frequencies were compared with a general population cohort of CRC (n=1111). Results KRAS and BRAF mutations were present in 41% and 15% t-CRCs, respectively. Compared with CRCs in the general population, t-CRCs had a higher MSI frequency (24% vs 11%, p=0.003) and more frequent loss of MSH2/MSH6 staining (13% vs 1%, p<0.001). Loss of MLH1/PMS2 staining and MLH1 promoter methylation were equally common in t-CRCs and the general population. In MSI CRCs without MLH1 promoter methylation, double somatic MMR gene mutations (or loss of heterozygosity as second hit) were detected in 7/10 (70%) t-CRCs and 8/36 (22%) CRCs in the general population (p=0.008). These MMR gene mutations in t-CRCs were classified as pathogenic. MSI t-CRC cases could not be ascribed to Lynch syndrome. Conclusions We have demonstrated a higher frequency of MSI among t-CRCs, which results from somatic MMR gene mutations. This suggests a novel association of somatic MMR gene mutations with prior anticancer treatment.

157 Sensitive and specific detection of 1p/19q codeletion in gliomas by next generation sequencing

utility. Recently, inhibitors of spindle-regulatory proteins have attracted considerable attention, and transforming acidic coiled-coil-3 (TACC3) is a spindle-regulatory protein overexpressed in many human cancers. We have developed a protein knockdown system to induce degradation of target proteins via the ubiquitin–proteasome system in cells with hybrid molecules named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser). In this study, we designed and synthesized novel SNIPER(TACC3)s that target TACC3 for degradation, and evaluated their activity in vitro. SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3, and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C mediates the SNIPER(TACC3)-induced degradation of TACC3. Cancer cells express larger amount of TACC3 than do normal fibroblasts, and SNIPER(TACC3) selectively induced cell death in cancer cells. These results suggest protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy to treat cancers overexpressing the TACC3 protein.