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Chemical patterns on the surface of DNA, including a process known as methylation, can regulate the translation of genetic information into functional proteins. This regulation helps ensure that an organism’s cells can specialise in specific tasks, even though all the cells in a human being contain the same DNA information. Genetic information that is not required in a liver cell can thus be ‘switched off’, whilst the same sections may be activated in a lung cell, providing the blueprint for cellular components that support lung function.

Numerous studies have shown that tumours also differ in their DNA methylation. These differences can be used to diagnose tumours more accurately and classify them into subgroups than is possible through histological examination under a microscope.

To this end, the DNA methylation of a tumour sample under investigation is compared with a reference library of tumours. This reference library and the associated algorithm were developed in Heidelberg.

As this method analyses hundreds of thousands of positions on the DNA, it is also possible to determine whether certain DNA segments are present in unusually high or low numbers – in other words, whether copy-number variations are present. 

This is particularly significant for investigating a 1p/19q co-deletion in cases of suspected oligodendroglioma, the presence of EGFR amplification in glioblastomas, or amplifications of MYC, MYCN, GLI, or deletions of TP53 or PTCH1 in medulloblastomas. 

In some cases, this analysis can also provide evidence of structural changes in the DNA, such as KIAA1549::BRAF, c11orf95::RELA, NTRK1/2/3 or FGFR1/2/3 fusions. Mutations cannot be detected using this method. The MGMT promoter status is also determined.

The duration and interpretability of the test depend largely on the quality (condition, tumour cell content) and quantity of the sample. A low tumour cell content or a small sample size may complicate or prevent interpretation, or necessitate repeat analyses.  

Information on requesting the analyses can be found under ‘Information for Submitters’

Mutations – that is, changes in the sequence of DNA components – can lead to the increased activation of certain genes that may result in tumour growth (oncogenes), or to the deactivation of others that might otherwise prevent tumour growth (tumour suppressor genes). This can be caused by the replacement of a single DNA building block, which lies below the detection threshold of DNA methylation analysis.

These ‘point mutations’ and other minor alterations can be identified using gene panel sequencing. This involves reading the sequence of the DNA building blocks, the bases. The ‘Heidelberg Brain Tumour Panel’ is specifically targeted at genes whose mutations are relevant to brain tumours. The selection of target genes is configured in such a way that all diagnostically relevant and all potentially therapeutically relevant genes are examined. This broad coverage is made possible by our use of a hybrid capture method. The Heidelberg Brain Tumour Panel thus overcomes the technical limitation of previous methods to smaller DNA segments (Sanger sequencing, amplicon panel sequencing), whilst at the same time avoiding the detection of numerous variants of unclear relevance, as occurs with exome sequencing.

For specific investigative purposes, we also carry out whole-exome or whole-genome sequencing.

The simultaneous analysis of numerous genes allows for a more efficient and comprehensive investigation than testing for mutations using traditional methods such as Sanger sequencing. Furthermore, next-generation sequencing technology also allows for the quantification of mutations, enabling even more precise assessments of the proportion of mutated cells.

The advantages of panel sequencing lie in its sensitivity and efficiency: in the case of diffusely growing brain tumours, the proportion of tumour cells in the sample may be low. Panel sequencing can detect even the smallest amounts of mutated DNA in the material.

The detection of a specific mutation can be a decisive factor for diagnosis (e.g. IDH1/2, H3F3A mutations) or for treatment (e.g. mutations in BRAF, PTCH1, SMO or AKT1). 

DNA methylation analysis and sequencing complement one another: whilst methylation analysis can determine the tumour type and detect copy-number alterations, sequencing reveals the exact mutations within the genes. For example, DNA methylation analysis may indicate a medulloblastoma of the SHH group; however, various mutations with differing responses to treatment may occur within this group (PTCH1, SMO, SUFU). These can only be distinguished from one another by means of panel sequencing. On the other hand, the detection of a BRAF mutation in panel sequencing can occur in various tumours (ganglioglioma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma). Methylation analysis is therefore required as a supplementary test to make this distinction.

Information on requesting the analyses can be found under ‘Submitter Information’

Whilst DNA sequencing is primarily used to detect point mutations and minor changes, many brain tumours arise from the joining of two previously separate gene segments – known as gene fusions. In this process, parts of two genes are joined together in such a way that a new, pathologically active gene product is created, which can drive tumour growth. Such fusions can be detected particularly reliably at the RNA level – that is, the transcript of the DNA that serves as a template for protein synthesis.

In RNA sequencing, the RNA present in the cell is converted into a stable form and then sequenced. As the non-coding regions of a gene are spliced out during RNA synthesis, the breakpoints of two fused genes can be visualised directly here, without the interfering intervening regions. This makes RNA sequencing the method of choice for detecting gene fusions.

The detection of certain fusions can be crucial for both diagnosis and treatment. These include, for example, KIAA1549::BRAF fusions in pilocytic astrocytoma, c11orf95::RELA fusions in ependymoma, or fusions of the NTRK1/2/3 and FGFR1/2/3 genes, for which targeted therapies are available. Although DNA methylation analysis may already provide indications of structural alterations, it is only through RNA sequencing that these can be precisely characterised down to the exact breakpoint.

DNA and RNA sequencing are therefore complementary: whilst DNA sequencing detects point mutations and small alterations, RNA sequencing fills the diagnostic gap for gene fusions and structural rearrangements. As with the other methods, the duration and reliability of the analysis depend largely on the quality and quantity of the material submitted.

Information on commissioning the analyses can be found under ‘Submitter Information’

Comprehensive sequencing is not always necessary for specific, clearly defined questions. In such cases, we use digital PCR (dPCR), a method that allows individual genetic changes to be detected particularly quickly, sensitively and precisely. Unlike conventional PCR, the sample is divided into many thousands of tiny sub-reactions, each of which is analysed individually. This not only allows us to determine whether a specific alteration is present, but also to determine very precisely what proportion of the material it constitutes.

This high sensitivity is particularly advantageous when only a small amount of material is available or the proportion of tumour cells in the sample is low. dPCR can reliably detect even the smallest quantities of a mutation and is therefore also suitable for the targeted confirmation of individual findings.

An important area of application is the determination of MGMT promoter methylation status. Methylation of the MGMT promoter leads to reduced production of the associated repair enzyme. Tumours with a methylated MGMT promoter therefore generally respond better to treatment with alkylating chemotherapeutic agents such as temozolomide, meaning that this parameter plays an important role in treatment planning for glioblastomas. dPCR enables the MGMT status to be determined quantitatively and with a high degree of accuracy.

Furthermore, dPCR is suitable for the targeted detection of individual changes relevant to diagnosis or treatment, as well as for confirming findings from other methods. It thus complements DNA methylation analysis and sequencing methods by providing a rapid and particularly sensitive tool for specific, targeted investigations.

Information on commissioning the analyses can be found under ‘Submitter Information’

In addition to established methods, we are trialling the use of nanopore sequencing for rapid intraoperative molecular diagnostics. We would like to expressly point out that this is an experimental procedure which is currently under development and validation and is not part of routine diagnostic practice.

In nanopore sequencing, the DNA strand is passed through a tiny pore; the resulting characteristic changes in the signal are directly translated into the sequence of the DNA building blocks. Unlike other sequencing methods, this technique delivers results very rapidly and also allows chemical patterns in the DNA, such as methylation, to be detected immediately.

These properties open up the possibility of obtaining diagnostically relevant information whilst surgery is still in progress. Within a short time, it is thus possible to determine a methylation-based classification indicator, as well as selected copy-number variations and MGMT promoter status. Such information can provide the surgical team with clues as to the nature of the tumour whilst the procedure is still underway, thereby supporting the surgical approach. The analysis concept used for this purpose (Rapid-CNS) was developed in Heidelberg.

As the procedure is still in the trial phase, the results obtained intraoperatively do not in any way replace the final, comprehensive diagnosis. They are always confirmed by established methods – DNA methylation analysis, sequencing and histological examination – and incorporated into the final report.

Information on commissioning the analyses can be found under ‘Information for Submitters’

Tumours of the central nervous system release small amounts of their DNA into the surrounding bodily fluids. In the case of brain tumours, and particularly those associated with the internal and external cerebrospinal fluid spaces, this so-called cell-free tumour DNA can be detected in the cerebrospinal fluid. The analysis of this freely circulating DNA is known as a liquid biopsy and enables molecular diagnosis without the need for surgical removal of tumour tissue.

This approach is particularly important when tissue sampling is not possible, or only possible to a limited extent – for example, in the case of deep-seated or diffusely growing tumours – or when the course of the disease needs to be closely monitored. As cerebrospinal fluid can be collected repeatedly and relatively non-invasively, liquid biopsy also makes it possible to monitor the response to treatment or the recurrence of tumour growth over time.

As the amount of cell-free tumour DNA in the cerebrospinal fluid can be very low, highly sensitive methods such as sequencing and digital PCR are used, which can detect even the smallest amounts of tumour-specific alterations. The diagnostic value depends largely on the extent to which the tumour releases DNA into the cerebrospinal fluid; consequently, a normal result does not definitively rule out the presence of a tumour.

The processing of cerebrospinal fluid samples is carried out in close collaboration with the neurology department’s cerebrospinal fluid laboratory, which possesses the necessary expertise in cerebrospinal fluid diagnostics and the pre-analytical processing of these sensitive samples. This collaboration ensures that the material is collected under optimal conditions and prepared for subsequent molecular analysis.

Information on requesting the analyses can be found under ‘Information for Submitters’

Microscopic examination of the tissue remains the cornerstone of neuropathological diagnosis. Wafer-thin sections are prepared from the submitted material and treated with various stains that reveal different tissue components. The classic haematoxylin-eosin stain provides an initial overview of the tissue structure, cell density and any abnormalities, whilst specialised stains specifically highlight individual structures such as connective tissue, myelin sheaths or deposits.

Conventional histology is supplemented by immunohistochemistry. This involves the use of antibodies that bind specifically to certain proteins in the tissue, thereby revealing their presence and distribution. In this way, cell types can be identified more precisely, the origin of a tumour can be narrowed down, and it is now also possible to detect numerous molecular alterations directly on the tissue section – for example, using mutation-specific antibodies that recognise the altered gene product. Immunohistochemistry thus combines the spatial information from the tissue section with findings at the protein level, providing important clues for interpreting the subsequent molecular analyses.

Furthermore, specialised processing and staining techniques are used for the diagnosis of disorders of the muscles and peripheral nerves. Muscle and nerve tissue is generally processed unfixed and deep-frozen for this purpose, as many of the necessary examinations must be carried out on fresh tissue. In nerve tissue, the ‘teasing’ technique enables the assessment of individual nerve fibres and their myelin sheaths, thereby allowing conclusions to be drawn about the nature of nerve damage. In the case of muscle tissue, in addition to histological staining, enzyme histochemical techniques are used in particular, in which the activity of specific metabolic enzymes is visualised directly in the tissue section. These stains provide information on fibre type, fibre distribution and changes in energy metabolism, such as those occurring in inflammatory, neurogenic or metabolic muscle disorders.

Information on commissioning these analyses can be found under ‘Information for Submitters’