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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. Heterozygous germline PTEN mutations cause Cowden syndrome. The risk of colorectal cancer in Cowden patients, however, remains a matter of debate. We describe two patients presenting with colorectal cancer at a young age (28 and 39 years) and dysmorphisms fitting the Cowden spectrum. Heterozygous germline mutations in PTEN were found in both patients. Moreover, analysis of the resected colorectal cancer specimens revealed loss of heterozygosity at the PTEN locus with retention of the mutated alleles, and greatly reduced or absent PTEN expression. Histologically and molecularly, the tumours showed resemblance with sporadic colorectal cancers, although they had prominent fibrotic stroma. Our data indicate that PTEN loss was involved in carcinogenesis in the two patients, supporting that colorectal cancer is part of the Cowden syndrome-spectrum. This is in line with data on sporadic colorectal cancer, mice studies and emerging epidemiological data on Cowden syndrome. Although the exact role of germline PTEN mutations in the carcinogenesis of colorectal cancer remains unclear, we think that Cowden syndrome should be in the differential diagnosis of colorectal cancer certainly in view of the possible prognostic and therapeutic consequences. Prospective follow-up and surveillance of PTEN mutation carriers from the age of 25 to 30 years in a study setting should clarify this issue. [ABSTRACT FROM AUTHOR]

Introduction: For patients with KRAS G12C -mutated NSCLC who are treated with sotorasib, there is a lack of biomarkers to guide treatment decisions. We therefore investigated the clinical utility of pretreatment and on-treatment circulating tumor DNA (ctDNA) and treatment-emergent alterations on disease progression., Methods: Patients with KRAS G12C -mutated NSCLC treated with sotorasib were prospectively enrolled in our biomarker study (NCT05221372). Plasma samples were collected before sotorasib treatment, at first-response evaluation and at disease progression. The TruSight Oncology 500 panel was used for ctDNA and variant allele frequency analysis. Tumor response and progression-free survival were assessed per Response Evaluation Criteria in Solid Tumors version 1.1., Results: Pretreatment KRAS G12C ctDNA was detected in 50 of 66 patients (76%). Patients with detectable KRAS G12C had inferior progression-free survival (hazard ratio [HR] 2.13 [95% confidence interval [CI]: 1.06-4.30], p = 0.031) and overall survival (HR 2.61 [95% CI: 1.16-5.91], p = 0.017). At first-response evaluation (n = 40), 29 patients (73%) had a molecular response. Molecular nonresponders had inferior overall survival (HR 3.58 [95% CI: 1.65-7.74], p = 0.00059). The disease control rate was significantly higher in those with a molecular response (97% versus 64%, p = 0.015). KRAS amplifications were identified as recurrent treatment-emergent alterations., Conclusions: Our data suggest detectable pretreatment KRAS G12C ctDNA as a marker for poor prognosis and on-treatment ctDNA clearance as a marker for treatment response. We identified KRAS amplifications as a potential recurring resistance mechanism to sotorasib. Identifying patients with superior prognosis could aid in optimizing time of treatment initiation, and identifying patients at risk of early progression could allow for earlier treatment decisions., Competing Interests: Disclosure Dr. Mathijssen reports receiving institutional fees for investigator-initiated trials from Astellas, Bayer, Boehringer-Ingelheim, Cristal Therapeutics, Deuter Oncology, Nordic Pharma, Novartis, Pamgene, Pfizer, Roche, Sanofi, and Servier, outside the current work. Dr. Paats reports receiving institutional fees from AstraZeneca, Bayer, Eli Lilly, Janssen, Novartis, Pfizer, Roche, and Takeda, outside the current work. Dr. Koolen reports receiving speaker fees from Promise Proteomics, outside the current work. Dr. von der Thüsen reports receiving advisory board and speaker fees from Eli Lilly, Bristol-Myers Squibb, Merck Sharp & Dohme, AstraZeneca, Bayer, Janssen, and Pfizer, outside the current work. Dr. Aerts reports receiving advisory board and speaker fees from Eli Lilly, Bristol-Myers Squibb, Merck Sharp & Dohme, AstraZeneca, Bayer, and Amphera and is a stock owner of Amphera, outside the current work. Dr. Dubbink reports receiving translational research funding and support from AstraZeneca, Merck Sharp & Dohme, and Illumina; advisory board fees from AbbVie, AstraZeneca, Bayer, Janssen, Lilly, Merck Sharp & Dohme, and Pfizer; honorarium from AstraZeneca, Lilly, Novartis, and Pfizer; and consultant fees from Bayer. Dr. Dingemans reports receiving institutional fees from Roche, Eli Lilly, Boehringer Ingelheim, AstraZeneca, Janssen, Chiezi, Amgen, Pfizer, Bayer, Takeda, Pharmamar, Sanofi, and Daiichi, outside the current work. All other authors report no disclosures., (Copyright © 2024 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Active surveillance instead of standard surgery after neoadjuvant chemoradiotherapy (nCRT) has been proposed for patients with oesophageal cancer. Circulating tumour DNA (ctDNA) may be used to facilitate selection of patients for surgery. We show that detection of ctDNA after nCRT seems highly suggestive of major residual disease. Tumour biopsies and blood samples were taken before, and 6 and 12 weeks after, nCRT. Biopsies were analysed with regular targeted next-generation sequencing (NGS). Circulating cell-free DNA (cfDNA) was analysed using targeted NGS with unique molecular identifiers and digital polymerase chain reaction. cfDNA mutations matching pre-treatment biopsy mutations confirmed the presence of ctDNA. In total, 31 patients were included, of whom 24 had a biopsy mutation that was potentially detectable in cfDNA (77%). Pre-treatment ctDNA was detected in nine of 24 patients (38%), four of whom had incurable disease progression before surgery. Pre-treatment ctDNA detection had a sensitivity of 47% (95% CI 24-71) (8/17), specificity of 85% (95% CI 42-99) (6/7), positive predictive value (PPV) of 89% (95% CI 51-99) (8/9), and negative predictive value (NPV) of 40% (95% CI 17-67) (6/15) for detecting major residual disease (>10% residue in the resection specimen or progression before surgery). After nCRT, ctDNA was detected in three patients, two of whom had disease progression. Post-nCRT ctDNA detection had a sensitivity of 21% (95% CI 6-51) (3/14), specificity of 100% (95% CI 56-100) (7/7), PPV of 100% (95% CI 31-100) (3/3), and NPV of 39% (95% CI 18-64) (7/18) for detecting major residual disease. The addition of ctDNA to the current set of diagnostics did not lead to more patients being clinically identified with residual disease. These results indicate that pre-treatment and post-nCRT ctDNA detection may be useful in identifying patients at high risk of disease progression. The addition of ctDNA analysis to the current set of diagnostic modalities may not improve detection of residual disease after nCRT. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Purpose: In a post hoc analysis of the CATNON trial (NCT00626990), we explored whether adding temozolomide to radiotherapy improves outcome in patients with IDH1/2 wildtype (wt) anaplastic astrocytomas with molecular features of glioblastoma [redesignated as glioblastoma, isocitrate dehydrogenase-wildtype (IDH-wt) in the 2021 World Health Organization (WHO) classification of central nervous system tumors]., Patients and Methods: From the randomized phase III CATNON study examining the addition of adjuvant and concurrent temozolomide to radiotherapy in anaplastic astrocytomas, we selected a subgroup of IDH1/2wt and H3F3Awt tumors with presence of TERT promoter mutations and/or EGFR amplifications and/or combined gain of chromosome 7 and loss of chromosome 10. Molecular abnormalities including MGMT promoter methylation status were determined by next-generation sequencing, DNA methylation profiling, and SNaPshot analysis., Results: Of the 751 patients entered in the CATNON study, 670 had fully molecularly characterized tumors. A total of 159 of these tumors met the WHO 2021 molecular criteria for glioblastoma, IDH-wt. Of these patients, 47 received radiotherapy only and 112 received a combination of radiotherapy and temozolomide. There was no added effect of temozolomide on either overall survival [HR, 1.19; 95% confidence interval (CI), 0.82-1.71] or progression-free survival (HR, 0.87; 95% CI, 0.61-1.24). MGMT promoter methylation was prognostic for overall survival, but was not predictive for outcome to temozolomide treatment either with respect to overall survival or progression-free survival., Conclusions: In this cohort of patients with glioblastoma, IDH-wt temozolomide treatment did not add benefit beyond that observed from radiotherapy, regardless of MGMT promoter status. These findings require a new well-powered prospective clinical study to explore the efficacy of temozolomide treatment in this patient population., (©2022 American Association for Cancer Research.)

Up to 14% of large cell neuroendocrine carcinomas (LCNECs) are diagnosed in continuity with nonsmall cell lung carcinoma. In addition to these combined lesions, 1% to 7% of lung tumors present as co-primary tumors with multiple synchronous lesions. We evaluated molecular and clinicopathological characteristics of combined and co-primary LCNEC-adenocarcinoma (ADC) tumors. Ten patients with LCNEC-ADC (combined) and five patients with multiple synchronous ipsilateral LCNEC and ADC tumors (co-primary) were included. DNA was isolated from distinct tumor parts, and 65 cancer genes were analyzed by next generation sequencing. Immunohistochemistry was performed including neuroendocrine markers, pRb, Ascl1 and Rest. Pure ADC (N = 37) and LCNEC (N = 17) cases were used for reference. At least 1 shared mutation, indicating tumor clonality, was found in LCNEC- and ADC-parts of 10/10 combined tumors but only in 1/5 co-primary tumors. A range of identical mutations was observed in both parts of combined tumors: 8/10 contained ADC-related (EGFR/KRAS/STK11 and/or KEAP1), 4/10 RB1 and 9/10 TP53 mutations. Loss of pRb IHC was observed in 6/10 LCNEC- and 4/10 ADC-parts. The number and intensity of expression of Ascl1 and neuroendocrine markers increased from pure ADC (low) to combined ADC (intermediate) and combined and pure LCNEC (high). The opposite was true for Rest expression. In conclusion, all combined LCNEC-ADC tumors were clonally related indicating a common origin. A relatively high frequency of pRb inactivation was observed in both LCNEC- and ADC-parts, suggesting an underlying role in LCNEC-ADC development. Furthermore, neuroendocrine differentiation might be modulated by Ascl1(+) and Rest(-) expression., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC ( n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies ( n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ±0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

Quantification of tumor-specific variants (TSVs) in cell-free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next-generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell-free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell-free DNA to its full potential., (© 2020 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: The association between contrast enhanced (CE) and non-contrast enhanced (NCE) tumor resection and survival in patients with glioblastoma in relation to molecular subtypes is poorly understood. The aim of this study was to assess the association between CE and NCE tumor resection and survival in light of MGMT promoter methylation in newly diagnosed IDH-wildtype glioblastoma. Materials and methods: Patients with newly diagnosed IDH-wildtype glioblastoma who underwent surgery were eligible. CE and NCE tumor volumes were assessed on pre- and post-operative MRI scans and extent of resection was calculated. The association between CE and NCE tumor resection and survival was evaluated using multivariable Cox proportional hazards models and Kaplan Meier estimates. Results: Three hundred and twenty-six patients were included: 177 (54.3%) with and 149 (45.7%) without MGMT methylation. Multivariable Cox proportional hazards models stratified for MGMT methylation identified age ≤ 65y (HR 0.63; 95% CI, 0.49-0.81; p < 0.0001), chemoradiation (HR 0.13; 95% CI, 0.09-0.19; p < 0.0001), maximal CE tumor resection (HR 0.58; 95% CI, 0.39-0.87; p = 0.009), ≥ 30% NCE tumor resection (HR 0.71; 95% CI, 0.53-0.93; p = 0.014), and minimal residual CE tumor volume (HR 0.64; 95% CI, 0.46-0.88 p = 0.007) as being associated with longer overall survival. Kaplan Meier estimates showed that extensive surgery was more beneficial for patients with MGMT methylated glioblastoma. Conclusions: This study shows an association between maximal CE tumor resection, ≥30% NCE tumor resection, minimal residual CE tumor volume, and longer overall survival in patients with newly diagnosed IDH wildtype glioblastoma. Intraoperative imaging and stimulation mapping may be used to pursue safe and maximal resection. In future research, the safety aspect of maximizing tumor resection needs to be addressed., (Copyright © 2020 Incekara, Smits, van der Voort, Dubbink, Atmodimedjo, Kros, Vincent and van den Bent.)

Introduction: O 6 - methylguanine-methyltransferase (MGMT) promoter methylation and isocitrate dehydrogenase (IDH) mutation status are important prognostic factors for patients with glioblastoma. There are conflicting reports about a differential topographical distribution of glioblastoma with vs. without MGMT promoter methylation, possibly caused by molecular heterogeneity in glioblastoma populations. We initiated this study to re-evaluate the topographical distribution of glioblastoma with vs. without MGMT promoter methylation in light of the updated WHO 2016 classification. Methods: Preoperative T2-weighted/FLAIR and postcontrast T1-weighted MRI scans of patients aged 18 year or older with IDH wildtype glioblastoma were collected. Tumors were semi-automatically segmented, and the topographical distribution between glioblastoma with vs. without MGMT promoter methylation was visualized using frequency heatmaps. Then, voxel-wise differences were analyzed using permutation testing with Threshold Free Cluster Enhancement. Results: Four hundred thirty-six IDH wildtype glioblastoma patients were included; 211 with and 225 without MGMT promoter methylation. Visual examination suggested that when compared with MGMT unmethylated glioblastoma, MGMT methylated glioblastoma were more frequently located near bifrontal and left occipital periventricular area and less frequently near the right occipital periventricular area. Statistical analyses, however, showed no significant difference in topographical distribution between MGMT methylated vs. MGMT unmethylated glioblastoma. Conclusions: This study re-evaluated the topographical distribution of MGMT promoter methylation in 436 newly diagnosed IDH wildtype glioblastoma, which is the largest homogenous IDH wildtype glioblastoma population to date. There was no statistically significant difference in anatomical localization between MGMT methylated vs. unmethylated IDH wildtype glioblastoma., (Copyright © 2020 Incekara, van der Voort, Dubbink, Atmodimedjo, Nandoe Tewarie, Lycklama, Vincent, Kros, Klein, van den Bent and Smits.)

Background: The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) has recommended that isocitrate dehydrogenase 1 and 2 wildtype (IDH1/2wt) diffuse lower-grade gliomas (LGGs) World Health Organization (WHO) grade II or III that present with (i) a telomerase reverse transcriptase promoter mutation (pTERTmt), and/or (ii) gain of chromosome 7 combined with loss of chromosome 10, and/or (iii) epidermal growth factor receptor (EGFR) amplification should be reclassified as diffuse astrocytic glioma, IDH1/2 wildtype, with molecular features of glioblastoma, WHO grade IV (IDH1/2wt astrocytomas WHO IV). This paper describes the overall survival (OS) of IDH1/2wt astrocytoma WHO IV patients, and more in detail patients with tumors with pTERTmt only., Methods: In this retrospective multicenter study, we compared the OS of 71 IDH1/2wt astrocytomas WHO IV patients, with radiological characteristics of LGGs, with the OS of 197 IDH1/2wt glioblastoma patients. Moreover, we compared the OS of 22 pTERTmt only astrocytoma patients with the OS of the IDH1/2wt glioblastoma patients., Results: Median OS was similar for IDH1/2wt astrocytoma WHO IV patients (23.8 mo) and IDH1/2wt glioblastoma patients (19.2 mo) (Cox proportional hazards model: hazard ratio [HR] 1.27, 95% CI: 0.85-1.88, P = 0.242). OS was also similar in patients with IDH1/2wt astrocytomas WHO IV, pTERTmt only, and IDH1/2wt glioblastomas (HR 1.15, 95% CI: 0.64-2.10, P = 0.641)., Conclusions: The presented data confirm the cIMPACT-NOW recommendation and we propose that IDH1/2wt astrocytomas WHO IV in the absence of other qualifying mutations should be classified as IDH1/2wt glioblastomas., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Intratumour heterogeneity fuels carcinogenesis and allows circumventing specific targeted therapies. HER2 gene amplification is associated with poor outcome in invasive breast cancer. Heterogeneous HER2 amplification has been described in 5-41% of breast cancers. Here, we investigated the genetic differences between HER2-positive and HER2-negative admixed breast cancer components. We performed an in-depth analysis to explore the potential heterogeneity in the somatic mutational landscape of each individual tumour component. Formalin-fixed, paraffin-embedded breast cancer tissue of ten patients with at least one HER2-negative and at least one HER2-positive component was microdissected. Targeted next-generation sequencing was performed using a customized 53-gene panel. Somatic mutations and copy number variations were analysed. Overall, the tumours showed a heterogeneous distribution of 12 deletions, 9 insertions, 32 missense variants and 7 nonsense variants in 26 different genes, which are (likely) pathogenic. Three splice site alterations were identified. One patient had an EGFR copy number gain restricted to a HER2-negative in situ component, resulting in EGFR protein overexpression. Two patients had FGFR1 copy number gains in at least one tumour component. Two patients had an 8q24 gain in at least one tumour component, resulting in a copy number increase in MYC and PVT1. One patient had a CCND1 copy number gain restricted to a HER2-negative tumour component. No common alternative drivers were identified in the HER2-negative tumour components. This series of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. Many other molecular aberrations are likely to act as alternative or collaborative drivers. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile, of which some genetic aberrations will be crucial for cancer progression, and others will be mere 'passenger' molecular anomalies., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Familial atypical multiple mole melanoma (FAMMM) syndrome is a hereditary syndrome characterized by multiple dysplastic nevi and melanoma. Patients with FAMMM may have a heterozygous, inactivating, pathogenic germline variant in the CDKN2A gene, especially the NM&#95;000077.4: c.225&#95;243del19 (p.p75fs) variant, also known as p16-Leiden variant. Patients with this variant are at high risk for developing melanomas and pancreatic cancer due to somatic inactivation of the wild-type CDKN2A allele. The combination of an inactivating germline CDKN2A mutation and somatic inactivation of the wild-type CDKN2A allele in the same cell results in tumor formation. It has been suggested that carriers of a germline CDKN2A mutation are also at increased risk for several other cancer types, including esophageal cancer. Here, we describe two unrelated patients with the p16-Leiden variant who developed esophageal squamous cell cancer. Evidence of loss of the wild-type CDKN2A allele was obtained in the tumor tissue of both patients indicating biallelic inactivation of p16 in the tumor cells. These results suggest that these patients developed esophageal squamous cell cancer in the context of FAMMM syndrome., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Several studies reported a correlation between anatomic location and genetic background of low-grade gliomas (LGGs). As such, tumor location may contribute to presurgical clinical decision-making. Our purpose was to visualize and compare the spatial distribution of different WHO 2016 gliomas, frequently aberrated single genes and DNA copy number alterations within subgroups, and groups of postoperative tumor volume., Methods: Adult grade II glioma patients (WHO 2016 classified) diagnosed between 2003 and 2016 were included. Tumor volume and location were assessed with semi-automatic software. All volumes of interest were mapped to a standard reference brain. Location heatmaps were created for each WHO 2016 glioma subgroup, frequently aberrated single genes and copy numbers (CNVs), as well as heatmaps according to groups of postoperative tumor volume. Differences between subgroups were determined using voxelwise permutation testing., Results: A total of 110 IDH mutated astrocytoma patients, 92 IDH mutated and 1p19q co-deleted oligodendroglioma patients, and 22 IDH wild-type astrocytoma patients were included. We identified small regions in which specific molecular subtypes occurred more frequently. IDH -mutated LGGs were more frequently located in the frontal lobes and IDH wild-type tumors more frequently in the basal ganglia of the right hemisphere. We found no localizations of significant difference for single genes/CNVs in subgroups, except for loss of 9p in oligodendrogliomas with a predilection for the left parietal lobes. More extensive resections in LGG were associated with frontal locations., Conclusions: WHO low-grade glioma subgroups show differences in spatial distribution. Our data may contribute to presurgical clinical decision-making in LGG patients., (© The Author(s) 2019. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Since 2013 next-generation sequencing (NGS) targeting genes mutated in diffuse gliomas is part of routine diagnostics in our institute. In the present report, we evaluate the use of this custom tailored NGS platform on 434 samples. The NGS panel assesses mutations in ATRX, CIC, EGFR, FUBP1, NOTCH1, PTEN; H3F3A, IDH1/2, PIK3CA, and BRAF, amplifications in EGFR or MDM2 and copy number alterations (CNA) of chromosome 1p, 7, 10 and 19q. TERT promoter mutations were assessed separately when indicated. Of the 433 samples of individual tumors with NGS data available, 176 cases were diagnosed as grade 2 or 3 glioma (40.6) and in 201 patients a glioblastoma (46.4%). Of the remaining 56 patients, 22 had inconclusive histology. In 378 cases (87.1%) a diagnosis solely based on glioma-targeted NGS could be established and resulted in a different diagnosis in ~ 1/4 of the cases. In 17 out of 22 cases without a conclusive histological diagnosis NGS resulted in a molecular diagnosis.The current study on a large cohort of patients confirms the diagnostic strength of the platform we developed, with a clear separation of glioma subgroups with different outcomes. It demonstrates the diagnostic value and the efficiency of glioma-targeted NGS for routine glioma diagnostics allowing with a single assay a glioma diagnosis in the large majority of cases. It allows in one run the molecular assessments required for the WHO classification of diffuse gliomas, including the recent recommendations to assess copy number alterations of chromosome 7 and 10, and of the TERT promoter region in IDHwt lower grade glioma.

Background: At current prognostication of low grade glioma remains suboptimal and might be improved with additional markers. These may guide treatment decisions, in particular on early adjuvant therapy versus wait and see after surgery., Methods: We used a targeted Next-Generation Sequencing panel to assess mutational and copy number status of selected genes and chromosomes in a consecutive series of adult grade II supratentorial glioma, and assessed the impact of molecular markers of interest on overall survival., Results: 207 IDH mutated grade II glioma samples were analyzed with a median follow-up of 6.9 years. Loss of region 9p21.3 did not show a correlation with outcome in IDH mutated 1p/19q-codeleted oligodendroglioma or IDH mutated astrocytoma. We found a significant shorter overall survival with univariable analysis in IDH mutated astrocytoma patients with trisomy of chromosome 7 (Log rank P = 0.044) and in IDH mutated 1p/19q-codeleted oligodendroglioma patients with a PTEN mutation (Log rank P = 0.033). We could not validate these findings in multivariate analysis or in the TCGA dataset., Conclusions: Loss of 9p21.3 is not associated with outcome in a molecularly defined cohort of grade II glioma and therefore it remains unclear if loss of 9p21.3 can be used as additional marker of anaplasia or to guide treatment decisions. Trisomy of chromosome 7 in IDH mutated astrocytoma and PTEN mutations in IDH mutated oligodendroglioma are potential markers of poor prognosis, but require confirmation in larger series.

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., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: Hodgkin's 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 Hodgkin's 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., Competing Interests: Competing interests: MEvL obtained funding from the Dutch Society of Gastroenterology and Hepatology (Maag Lever Darm Stichting (MLDS) funding project FP14-04)., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

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., (Copyright © 2017 Endocrine Society)

Aims: Malignant melanoma is the most aggressive form of skin cancer, and metastatic dissemination to regional and visceral sites is responsible for the majority of melanoma-related mortalities. In a recent study by our group, we observed reduced expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in the majority of stage III melanoma samples studied. TIMP3 has been reported as a tumour suppressor in several human malignancies, with reduced expression correlating with poor clinical outcome. In this study, we investigated the changes in TIMP3 expression during melanoma progression., Patients and Methods: TIMP3 expression was analysed by immunohistochemistry in sequential archived tumour material from stage I/II, stage III and stage IV samples from melanoma patients (n = 33). Protein expression was investigated for associations with disease-free survival and overall survival. Methylation status of the gene promoter was determined using methylation-specific PCR. In vitro assays were used to investigate the functional consequences of TIMP3 expression on behavioural aspects of melanoma cells., Results: We show that TIMP3 expression decreases with melanoma progression although no significant clinical associations were obtained. Analysis of the status of promoter methylation using methylation-specific PCR revealed it to be a low-frequency event in melanoma. Additionally, through gene modulation experiments in melanoma cell lines, we show that TIMP3 negatively regulates cell migration, invasion and anoikis resistance., Conclusions: Collectively, our data suggests that TIMP3 functions as a tumour suppressor in melanoma and negatively regulates several aspects of the metastatic cascade., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Histopathological diagnosis of diffuse gliomas is subject to interobserver variation and correlates modestly with major prognostic and predictive molecular abnormalities. We investigated a series of patients with locally diagnosed anaplastic oligodendroglial tumors included in the EORTC phase III trial 26951 on procarbazine/lomustine/vincristine (PCV) chemotherapy to explore the diagnostic, prognostic, and predictive value of targeted next-generation sequencing (NGS) in diffuse glioma and to assess the prognostic impact of FUBP1 and CIC mutations., Methods: Mostly formalin-fixed paraffin-embedded samples were tested with targeted NGS for mutations in ATRX, TP53, IDH1, IDH2, CIC, FUBP1, PI3KC, TERT, EGFR, H3F3A, BRAF, PTEN, and NOTCH and for copy number alterations of chromosomes 1p, 19q, 10q, and 7. TERT mutations were also assessed, with PCR., Results: Material was available from 139 cases, in 6 of which results were uninformative. One hundred twenty-six tumors could be classified: 20 as type II (IDH mutation [mut], "astrocytoma"), 49 as type I (1p/19q codeletion, "oligodendroglioma"), 55 as type III (7+/10q- or TERTmut and 1p/19q intact, "glioblastoma"), and 2 as childhood glioblastoma (H3F3Amut), leaving 7 unclassified (total 91% classified). Molecular classification was of clear prognostic significance and correlated better with outcome than did classical histopathology. In 1p/19q codeleted tumors, outcome was not affected by CIC and FUBP1 mutations. MGMT promoter methylation remained the most predictive factor for survival benefit of PCV chemotherapy., Conclusion: Targeted NGS allows a clinically relevant classification of diffuse glioma into groups with very different outcomes. The diagnosis of diffuse glioma should be primarily based on a molecular classification, with the histopathological grade added to it. Future discussion should primarily aim at establishing the minimum requirements for molecular classification of diffuse glioma., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Several anti-tumour properties have been ascribed to the tissue inhibitor of matrix metalloproteinases-3 (TIMP3) gene, including inhibition of neovascularisation in tumour xenografts. Reduced protein expression has been linked to promoter hypermethylation and allelic loss of heterozygosity in various human malignancies. In melanoma-positive lymph nodes from patients, we evaluated the association between TIMP3 expression, vessel density, macrophage infiltration and potential correlations with disease-free survival (DFS) and overall survival (OS)., Patients and Methods: TIMP3 expression was analysed by immunohistochemistry (IHC) in melanoma lymph node biopsies of stage III melanoma patients (n = 43). Blood vessel density and macrophage infiltration were quantitatively assessed and correlation with TIMP3 expression was investigated. Methylation status of the gene promoter was determined using methylation-specific polymerase chain reaction (MSP). Protein expression and promoter methylation status were investigated for associations with DFS and OS., Results: Reduced expression of TIMP3, as determined by IHC, was observed in 74% of the cases (32 in 43). A significant inverse correlation was observed between TIMP3 expression and vessel density (p = 0.031). Correlation between TIMP3 expression and macrophage infiltration was not statistically significant (p = 0.369). MSP analysis revealed methylation of the gene promoter in 18% (7 in 38) of the analysed cases. No differences in OS and DFS were observed between cases with high and low TIMP3 expression. Gene promoter methylation was significantly associated with both poor 5-year DFS (p = 0.024) and OS (p = 0.034)., Conclusions: Our data indicate that TIMP3 is a dominant negative regulator of angiogenesis in cutaneous melanoma and gene silencing by promoter methylation is associated with poor outcome., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Patients with multiple tumors, either synchronous or metachronous, can have metastatic disease or suffer from multiple independent primary tumors. While proper diagnosis of these patients is important for prognosis and treatment, this can be challenging using only clinical and histological criteria. The aim of the present study was to evaluate the value of mitochondrial D310 mutation analysis in diagnostic questions regarding tumor clonality for a wide range of tumor types. Sanger sequencing of D310 was performed on a diagnostic cohort of 382 patients with 857 tumors that were previously analyzed using routine molecular analysis on genomic DNA. The D310 mononucleotide repeat was frequently somatically mutated (56/321, 17 %) in several tumor types, including breast, head and neck, gynecological, lung, colorectal, and skin tumors. For 84/327 (26 %) patients, a D310 mutation was detected in at least one of their tumors; for these patients, D310 can be used to determine the clonal relationship between their multiple tumors. Clonality assessments based on mitochondrial DNA (mtDNA) and routine genomic DNA analysis were concordant in 52/73 (71 %) patients. We conclude that D310 mutation status might aid in determining clonality of clinically challenging synchronous or metachronous tumors. To this end, next generation sequencing targeted genomic DNA assays should be complemented with mtDNA markers, such as the D310 repeat.