Methylation subgroup and molecular heterogeneity is a hallmark of glioblastoma: implications for biopsy targeting, classification and therapy.

BACKGROUND: Intratumoral heterogeneity at the cellular and molecular level is a hallmark of glioblastoma (GB) that contributes to treatment resistance and poor clinical outcome. Little is known regarding epigenetic heterogeneity and intratumoral phylogeny and their implication for molecular classification and targeted therapies. PATIENTS AND METHODS: Multiple tissue biopsies (238 in total) were sampled from 56 newly-diagnosed, treatment-naive GB patients from a prospective in-house cohort and publicly available data and profiled for DNA methylation using the Illumina MethylationEPIC array. Methylation-based classification using the glioma classifier developed by Ceccarelli et al. and estimation of the MGMT promoter methylation status via the MGMT-STP27 model were carried out. In addition, copy number variations (CNVs) and phylogeny were analyzed. RESULTS: Almost half of the patients (22/56, 39%) harbored tumors composed of heterogeneous methylation subtypes. We found two predominant subtype combinations: classic-/mesenchymal-like, and mesenchymal-/pilocytic astrocytoma-like. Nine patients (16%) had tumors composed of subvolumes with and without MGMT promoter methylation, whereas 20 patients (36%) were homogeneously methylated, and 27 patients (48%) were homogeneously unmethylated. CNV analysis revealed high variations in many genes, including CDKN2A/B, EGFR, and PTEN. Phylogenetic analysis correspondingly showed a general pattern of CDKN2A/B loss and gain of EGFR, PDGFRA, and CDK4 during early stages of tumor development. CONCLUSIONS: (Epi)genetic intratumoral heterogeneity is a hallmark of GB, both at DNA methylation and CNV level. This intratumoral heterogeneity is of utmost importance for molecular classification as well as for defining therapeutic targets in this disease, as single biopsies might underestimate the true molecular diversity in a tumor.

Pan-cancer analysis of genomic scar patterns caused by homologous repair deficiency (HRD).

Homologous repair deficiency (HRD) is present in many cancer types at variable prevalence and can indicate response to platinum-based chemotherapy and PARP inhibition. We developed a tumor classification system based on the loss of function of genes in the homologous recombination repair (HRR) pathway. To this end, somatic and germline alterations in BRCA1/2 and 140 other HRR genes were included and assessed for the impact on gene function. Additionally, information on the allelic hit type and on BRCA1 promoter hypermethylation was included. The HRDsum score including LOH, LST, and TAI was calculated for 8847 tumors of the TCGA cohort starting from genotyping data and for the subcohort of ovarian cancer also starting from WES data. Pan-cancer, deleterious BRCA1/2 alterations were detected in 4% of the tumors, while 18% of the tumors were HRD-positive (HRDsum ≥ 42). Across 33 cancer types, both BRCA1/2 alterations and HRD-positivity were most prevalent in ovarian cancer (20% and 69%). Pan-cancer, tumors with biallelic deleterious alterations in BRCA1/2 were separated strongly from tumors without relevant alterations (AUC = 0.89), while separation for tumors with monoallelic deleterious BRCA1/2 alterations was weak (AUC = 0.53). Tumors with biallelic deleterious alterations in other HHR genes were separated moderately from tumors without relevant alterations (AUC = 0.63), while separation for tumors with such monoallelic alterations was weaker (AUC = 0.57). In ovarian cancer, HRDsum scores calculated from WES data correlated strongly with HRDsum scores calculated from genotyping data (R = 0.87) and were slightly (4%) higher. We comprehensively analyzed HRD scores and their association with mutations in HRR genes in common cancer types. Our study identifies important parameters influencing HRD measurement and argues for an integration of HRDsum score with specific mutational profiles.

[Homologous recombination repair deficiency as a predictive biomarker : Basic mechanisms and detection methods]. / Defizienz der homologen Rekombinationsreparatur als prädiktiver Marker : Grundlagen und Nachweis.

BACKGROUND: DNA double-strand breaks may evoke cell death or cancer. Cells have developed two fundamentally different mechanisms of DNA double-strand repair: the accurate mechanism of homologous recombination repair and the error-prone nonhomologous end joining. The deficiency of homologous recombination repair (HRD) is a frequent feature of several solid tumor entities and is associated with sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitor therapy. OBJECTIVES: Among other biomarkers, HRD testing provides an opportunity to guide PARP inhibitor therapy in solid tumors, but the basic principles are complex and the use and benefits of the different methodologies remain controversial. Knowledge of the underlying mechanisms at the molecular level will advance our understanding and will pave the way to introduce the testing of new biomarkers. METHODS: An overview of the fundamental mechanisms of DNA repair is provided. Important terms like HRR, HRD, and BRCAness are defined, and analysis methods are described, especially with regard to their integration in molecular pathology routine diagnostics. RESULTS: Currently, at least testing of the BRCA mutation status and genomic instability using a composite HRD score should be implemented in laboratories to identify subgroups of patients who might benefit from PARP inhibitor therapies. A broad range of testing methods is available with pros and cons for introduction in the clinical setting. They have to be validated carefully to reliably inform treatment selection. CONCLUSIONS: Biomarkers to identify current homologous recombination deficiency status are needed to predict the benefit from PARP inhibitors and stratify their use in clinical management. Besides commercial assays, different tests might be used for the analysis of HRD. The application depends, among other things, on the local situation and has to be extensively validated.

[Status of the availability and use of next generation sequencing (NGS) in bladder cancer-a questionnaire within the uropathology working group]. / Status der Verfügbarkeit und Anwendung von "next generation sequencing" (NGS) beim Harnblasenkarzinom ­ eine Umfrage in der Arbeitsgemeinschaft Uropathologie.

BACKGROUND: Technical advancement and availability of high-throughput analysis has advanced molecular subtyping of most cancers. Thus, new possibilities for precision oncology have emerged. AIM: Therefore, we aimed to collect data regarding availability and use of next generation sequencing (NGS) for urothelial cancer within the uropathology working group of the German Society of Pathology. METHODS: We collected data by questionnaires and additionally asked for sequencing results of bladder cancers in the participating institutions. RESULTS: A total of 13 university-affiliated institutes of pathology took part in the survey. All university institutes offer NGS-based molecular panel diagnostics and provide panels covering between 15 and 170 genes. Altogether, only 20 bladder cancers were sequenced in routine diagnostics and for 10 cancers potential targeted treatment options were available. DISCUSSION: So far, despite availability of NGS diagnostics at university institutes of pathology, only few bladder cancer samples have been sequenced. Based on current data from the molecular subtyping of bladder cancers, we recommend a step-by-step protocol with basic immunohistochemistry analysis and subsequent subtype-dependent analyses, e.g., alterations of the fibroblast growth factor receptors (FGFR) or comprehensive gene panel analyses.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Molecular signaling in multiple myeloma: association of RAS/RAF mutations and MEK/ERK pathway activation.

Multiple myeloma (MM) is a plasma cell malignancy that is still considered to be incurable in most cases. A dominant mutation cluster has been identified in RAS/RAF genes, emphasizing the potential significance of RAS/RAF/MEK/ERK signaling as a therapeutic target. As yet, however, the clinical relevance of this finding is unclear as clinical responses to MEK inhibition in RAS-mutant MM have been mixed. We therefore assessed RAS/RAF mutation status and MEK/ERK pathway activation by both targeted sequencing and phospho-ERK immunohistochemistry in 180 tissue biopsies from 103 patients with newly diagnosed MM (NDMM) and 77 patients with relapsed/refractory MM (rrMM). We found a significant enrichment of RAS/BRAF mutations in rrMM compared to NDMM (P=0.011), which was mainly due to an increase of NRAS mutations (P=0.010). As expected, BRAF mutations were significantly associated with activated downstream signaling. However, only KRAS and not NRAS mutations were associated with pathway activation compared to RAS/BRAFwt (P=0.030). More specifically, only KRASG12D and BRAFV600E were consistently associated with ERK activation (P<0.001 and P=0.006, respectively). Taken together, these results suggest the need for a more specific stratification strategy consisting of both confirmation of protein-level pathway activation as well as detailed RAS/RAF mutation status to allow for a more precise and more effective application of targeted therapies, for example, with BRAF/MEK inhibitors in MM.

[Molecular diagnostics of non-small cell lung cancer: New markers and technologies]. / Molekulardiagnostik des nichtkleinzelligen Lungenkarzinoms: Neue Marker und Technologien.

Lung cancer is the prototypical tumor entity for the development of new diagnostic and individualized therapeutic strategies based on molecular patient stratification. Developments in this field specifically concentrate on predictive biomarkers for the response to conventional therapeutic agents, novel drugs targeting specific mutations and also new immunomodulatory drugs. The multitude of upcoming new predictive biomarkers requires the development and implementation of efficient test strategies and comprehensive technical methods, specifically when tissue restrictions inherent to lung cancer diagnostics are also taken into account. Novel procedures and technical aspects of these issues are discussed in this review.

Congenital hypothyroidism caused by a novel homozygous mutation in the thyroid peroxidase gene.

UNLABELLED: Congenital primary hypothyroidism occurs in one out of 4,000 births. About 20% of cases are due to defects in thyroid hormonogenesis. We report on a German girl with congenital hypothyroidism due to a mutation in the thyroid peroxidase (TPO) gene who had elevated serum levels of thyroglobulin during periods of hyperthyrotropinemia. METHODS: The TPO gene was sequenced directly from genomic DNA. RESULTS: The patient had a novel homozygous mutation (R314W) in the TPO gene. The unaffected parents were non-consanguineous and both heterozygous carriers of the mutation. Fifty normal individuals did not harbor the mutation ruling out a common polymorphism. CONCLUSION: The identified TPO gene mutation (R314W) is very likely the genetic cause for hypothyroidism in the reported child. R314W has not been described before and codes for a presumably inactive TPO molecule.

Mild congenital primary hypothyroidism in a Turkish family caused by a homozygous missense thyrotropin receptor (TSHR) gene mutation (A593 V).

Congenital primary hypothyroidism (CH) occurs in 1 of 4000 births. The majority of the cases are due to agenesis or dysgenesis of the thyroid gland, which can be caused by mutations in genes encoding for transcriptional factors that are responsible for the development of the thyroid gland. It is also known that the thyrotropin receptor (TSHR), a G-protein coupled receptor, is involved in late stages of thyroid organogenesis. Thus, mutations in the TSHR gene can cause congenital hypothyroidism. However, the clinical spectrum of thyroid abnormalities due to mutant TSHRs is wide and ranges from severe hypoplasia to an almost normal sized and structured thyroid gland. So far, 23 distinct loss-of-function mutations in the TSHR gene have been reported, occurring in families of different ethnic backgrounds and geographical areas. Here we report on a Turkish kindred in which two children were diagnosed to have very mild congenital primary hypothyroidism and one child had subclinical hypothyroidism. A novel homozygous missense mutation in codon 593 (A593 V) of the TSHR gene was identified in the affected individuals as the underlying molecular defect. This mutation substitutes a non-polar amino acid (alanine) with a non-polar amino acid (valine), so that only a minimal impairment of the TSHR function is expected. Indeed, the molecular finding is in agreement with the observed mild phenotype of the affected individuals. Our conclusion is that in mild primary hypothyroidism or subclinical hypothyroidism, mutations in the TSHR gene have to be considered as the molecular cause, especially in patients who have no detectable thyroid autoantibodies and have thyroid glands of normal size and in normal location.