How Molecular Testing is Transforming Brain Tumor Diagnosis and Treatment

Table of Contents

• Introduction: Why Molecular Testing Matters for Brain Tumors
• Key Molecular Tests for Brain Tumor Diagnosis and Treatment
• DNA Methylation Profiling: A Revolutionary Diagnostic Tool
• DNA and RNA Sequencing: Detecting Critical Mutations
• Additional Diagnostic Tools and Techniques
• Applying Molecular Testing to Brain Tumor Diagnosis
• Adult Diffuse Gliomas: How Molecular Testing Guides Diagnosis
• What This Means for Patients: Clinical Implications
• Understanding the Limitations of Molecular Testing
• Recommendations for Patients Facing Brain Tumor Diagnosis
• Source Information

Introduction: Why Molecular Testing Matters for Brain Tumors

Molecular profiling has completely transformed how doctors diagnose and classify central nervous system (CNS) tumors in recent years. The latest World Health Organization (WHO) classification of CNS tumors, published in 2021, now requires combining traditional microscopic examination with molecular testing to achieve accurate, reproducible diagnoses that directly impact patient care.

This integrated approach means pathologists must now work with multiple molecular tests, primarily including DNA methylation profiling and DNA/RNA next-generation sequencing. These advanced techniques help classify tumors more precisely, identify specific genetic mutations, and even reveal potential treatment targets that weren't previously detectable through conventional methods alone.

The significance of molecular profiling has expanded dramatically, with all major CNS tumor types now requiring evaluation of specific molecular markers for proper diagnosis. Many new tumor types have actually been discovered through these technologies, particularly DNA methylation analysis, highlighting how essential they've become in modern neuropathology.

Key Molecular Tests for Brain Tumor Diagnosis and Treatment

Molecular neuropathology now includes a wide range of tests with different purposes, complexity levels, availability, and costs. Understanding these differences is crucial because it helps doctors choose the most appropriate test for each specific diagnostic challenge and correctly interpret the results.

The current molecular toolbox includes several essential technologies that provide complementary information about brain tumors. Each test has specific strengths and applications that make it particularly valuable for certain diagnostic situations or tumor types.

DNA Methylation Profiling: A Revolutionary Diagnostic Tool

DNA methylation profiling has arguably been the most impactful molecular tool in recent years for diagnosing brain tumors. This approach uses the unique epigenetic profile of tumor cells, which reflects both the characteristics of their original tissue and the changes acquired during cancer development, creating a specific signature for each tumor type.

Practically, DNA methylation profile is currently assessed using the MethylationEPIC array beadchip (850K), which investigates the methylation status of several hundred thousand CpG islands across the entire genome. The raw data is then uploaded to a dedicated platform and matched against a comprehensive repository of CNS tumors and other selected entities.

A matching score of ≥ 0.9 strongly supports the diagnostic classification, though experts always review these results alongside clinical, imaging, and histopathological findings. It's important to note that DNA methylation profiling using this platform isn't a certified assay, so different countries may have varying regulations about its diagnostic use.

This technology has proven extremely valuable for both research and daily diagnostic purposes. For research, many new tumor types and subtypes have been discovered through unsupervised analysis of large brain tumor datasets. Many newly identified tumors show significant overlaps with other entities in terms of morphological features and/or have very low incidence, explaining why they weren't previously recognized as distinct neoplasms.

In clinical practice, multiple groups have published their experience with this technology in both pediatric and adult settings. Overall, a matching score (≥ 0.9) is achieved in about 50-65% of samples, and a significant impact on diagnosis occurs in about 10-20% of cases with potential clinical consequences. This remarkable finding justifies the quick adoption of this tool in just a few years.

Higher median classification scores are usually observed in cases analyzed to confirm a diagnosis or assess specific tumor subtypes. A wider range of scores appears among challenging samples or smaller specimens. Lower scores in complex cases can result from multiple factors, including small, poor quality, or unrepresentative biopsies that might be unclassifiable through both traditional pathology and methylation profiling.

Ideally, 200 nanograms of DNA with ≥ 60% tumor cell concentration is desirable, though diagnostic classification can be achieved with significantly lower amounts. Formalin fixed paraffin embedded (FFPE) tissue blocks typically provide similar results to fresh frozen samples, and analysis of older specimens can also yield correct classification.

DNA methylation profiling has shown particular usefulness for reclassifying rare tumor types with unspecific histopathological characteristics. While now a key tool for diagnosing CNS tumors, this approach poses significant challenges in terms of required technological facilities, costs, turnaround time (multiple days), and expertise needed for execution and interpretation.

DNA and RNA Sequencing: Detecting Critical Mutations

Many CNS tumors feature specific point mutations detectable by DNA sequencing or gene fusions mainly identified through RNA sequencing. For example, mutations in IDH1/IDH2 and H3-coding genes characterize specific subsets of adult and pediatric gliomas, respectively. Other mutations, like BRAF V600E, can appear in multiple tumor types with varying frequencies but still contribute to diagnostic assessment and enable targeted treatment.

Multiple assay types can be used for DNA sequencing, including single gene direct sequencing (Sanger sequencing) and next-generation sequencing (NGS)-based approaches like targeted panel sequencing and whole exome/genome sequencing (WES/WGS). These analyses detect single nucleotide variations (SNV), small insertions/deletions (InDel), and copy number variations (CNV).

Within NGS assays, targeted gene panel sequencing is currently the most relevant tool for daily molecular diagnostic workup of CNS tumors. It allows analysis of relatively large sets of relevant genes with acceptable costs, turnaround times, and interpretation feasibility. Many genes most relevant for brain tumor diagnosis are relatively specific to these neoplasms, warranting customized or larger panels.

The diagnostic efficacy of medium-sized gene panels has been demonstrated, detecting mutations and CNV even with limited input material. More recent studies using larger gene panels (including IDH1/IDH2, TERT, TP53, ATRX, BRAF, H3F3A, H3F3B) confirmed these results. These assays detect diagnostically relevant alterations in more than half of analyzed CNS tumors, with informative CNV detected in 57% of cases even when NGS results were noncontributory.

Laboratory protocols are critical for sequencing success. DNA quality and tumor cell rate should be maximized, with adequate coverage/read depth achieved according to assay type and sample characteristics. The data analysis pipeline and expertise of the reporting molecular pathologist are equally important for correct variant calling and interpretation.

Analysis of circulating tumor DNA (ctDNA) from blood and/or cerebrospinal fluid represents another approach to tumor molecular profiling through minimally invasive liquid biopsy. Technical challenges have so far limited implementation in daily practice, but recent data shows comprehensive NGS panels can detect CNV and address intratumoral heterogeneity even in ctDNA.

For RNA sequencing, the main diagnostic purpose is detecting gene fusions, which characterize many CNS tumors. For instance, pilocytic astrocytomas frequently harbor the KIAA1549::BRAF fusion, and specific molecular supratentorial ependymoma subtypes are defined by ZFTA or YAP1 fusions. Gene fusions can represent exploitable therapeutic targets, as seen in infant-type hemispheric gliomas frequently characterized by NTRK1/NTRK2/NTRK3, ROS1, ALK, or MET gene fusions with available effective inhibitors.

Studies focused on RNA NGS significance in CNS tumors show this tool is especially valuable for pediatric neoplasms, which more frequently feature these events. In adult brain neoplasms, gene fusions are relatively rare and usually don't represent therapeutic targets.

Additional Diagnostic Tools and Techniques

Microarray-based assessment of whole-genome copy number variations (CNV) is another relevant diagnostic tool frequently used to molecularly characterize CNS tumors, especially before DNA methylation profiling became available. These assays detect many chromosomal alterations (deletions, amplifications, loss of heterozygosity, copy-neutral loss of heterozygosity, chromothripsis) that serve as diagnostic and/or prognostic hallmarks of specific tumor types.

Molecular profiling doesn't necessarily mean simultaneous analysis of multiple alterations. Fluorescence in situ hybridization (FISH) can evaluate specific DNA loci directly on tissue slides, useful for validation purposes or when a specific alteration is strongly suspected based on tumor characteristics or when material is insufficient for other analyses.

Instead of nucleic acids, proteins can be evaluated using widely available, fast, and inexpensive immunohistochemical stainings. Immunohistochemistry can establish presence of mutant proteins (IDH1 R132H, p53, H3 K27M, H3 G34R/V, BRAF V600E), loss of normal/functioning proteins (ATRX, H3 K27me3, INI1, BRG1), or hyperactivation of aberrant pathways (EZHIP).

Assessment of MGMT promoter methylation remains crucial for IDH-wildtype glioblastoma molecular characterization due to its prognostic and predictive relevance. Multiple assays can investigate this marker, with no clear superiority in clinical correlations. Since no equivalence criteria exist between different assays, understanding the characteristics of the locally available assay is important. MGMT immunohistochemistry is not a reliable surrogate for these assays.

Applying Molecular Testing to Brain Tumor Diagnosis

Molecular analyses contribute to or are required for diagnosing many tumor types recognized by the 2021 WHO classification. The most relevant molecular tools vary by specific neoplasm, with selected examples demonstrating the significance of these tools in current neuropathology practice.

Adult Diffuse Gliomas: How Molecular Testing Guides Diagnosis

According to the 2021 WHO classification, adult diffuse gliomas are mainly stratified by IDH1/IDH2 status. This division is well justified based on different tumor biology, oncogenic mechanisms, and clinical implications associated with this molecular marker.

Glioblastoma, IDH-wildtype is the most frequent diffuse glioma, usually occurring in older adults with dismal prognosis. It's a morphologically and molecularly heterogeneous neoplasm typically showing poorly differentiated astrocytic features with infiltrative growth, high proliferation, microvascular proliferation, and necrosis. With consistent morphology, doctors must exclude IDH1/IDH2 mutation to rule out IDH-mutant astrocytoma or IDH-mutant, 1p/19q-codeleted oligodendroglioma.

Multiple options exist for this task: immunohistochemistry for IDH1 R132H can exclude the most frequent mutation (about 90% of supratentorial IDH-mutant gliomas). This strategy works adequately for patients aged 55 and older, where the probability of finding an alternative mutation is less than 1%. However, if history suggests a previous lower grade glioma, sequencing is necessary.

If morphological features of glioblastoma are lacking but suspected, the WHO 2021 classification allows molecular diagnosis of glioblastoma. This requires either a consistent DNA methylation profile or at least one of three markers: chromosome 7 gain plus chromosome 10 loss, TERT promoter mutation, or EGFR amplification. These markers show relative specificity in the right context, with similar patient outcomes to those diagnosed by morphological features.

For IDH-mutant diffuse gliomas, diagnosis requires histopathological findings consistent with an infiltrating diffuse glioma with IDH1/IDH2 mutation and ATRX loss/mutation or exclusion of 1p/19q codeletion. IDH-mutant and 1p/19q codeleted oligodendroglioma requires whole-arm combined 1p/19 codeletion. Alternatively, diagnosis can be based on detecting the corresponding methylation class.

ATRX status can be assessed by immunohistochemistry (watching for artifacts from necrosis or intermixed positive non-neoplastic reactive astrocytes) or sequencing to detect loss of function mutation. TP53/p53 evaluation also helps since it's frequently present in IDH-mutant astrocytomas.

For diagnosing IDH-mutant, 1p/19q-codeleted tumors, the critical hallmark is whole-arm 1p/19q codeletion, investigable by multiple assays including FISH. However, FISH's limited targeting of single loci can produce false positive results, especially in tumors with complex karyotypes. False positive FISH assessments frequently occur in cases where 1p/19q status evaluation wouldn't have been warranted, emphasizing the importance of appropriate test use.

For grading IDH-mutant diffuse gliomas, morphological features remain critical, but the 2021 WHO classification added CDKN2A/B status evaluation as a grading criterion for IDH-mutant astrocytomas. With homozygous CDKN2A/B deletion, grade 4 is assigned due to association with unfavorable outcome. CDKN2A/B status can be evaluated through DNA methylation profiling CNV plots, DNA NGS, or FISH, though no conclusive cut-off value has been established.

What This Means for Patients: Clinical Implications

The integration of molecular testing into brain tumor diagnosis represents a fundamental shift in patient care. These advanced techniques provide more accurate diagnoses, better prognostic information, and identify potential treatment targets that weren't previously detectable.

For patients, this means more personalized treatment approaches based on the specific genetic characteristics of their tumor. Molecular profiling can identify targeted therapies that may be more effective than conventional treatments, particularly for tumors with specific mutations like BRAF V600E or gene fusions involving NTRK, ROS1, ALK, or MET genes.

The ability to classify tumors more precisely also improves prognostic accuracy, helping patients and doctors make more informed decisions about treatment intensity and follow-up care. For example, the distinction between IDH-mutant and IDH-wildtype gliomas significantly impacts expected outcomes and treatment approaches.

Molecular testing also enables identification of patients who might benefit from clinical trials of novel targeted therapies, expanding treatment options beyond standard approaches. This is particularly important for aggressive or rare tumor types where conventional treatments offer limited benefits.

Understanding the Limitations of Molecular Testing

While molecular testing has revolutionized brain tumor diagnosis, it's important to understand its limitations. These technologies require significant expertise to interpret correctly, and results must always be considered alongside clinical, imaging, and traditional pathological findings.

Technical challenges include the need for sufficient tumor material of adequate quality. Small biopsies, poor preservation, or low tumor cell content can limit the effectiveness of molecular testing. Some assays also have relatively long turnaround times (multiple days) compared to traditional pathological examination.

Cost and availability remain significant barriers to universal implementation of comprehensive molecular profiling. Not all medical centers have access to these advanced technologies, particularly in resource-limited settings.

While molecular classification provides valuable information, it doesn't always translate directly to treatment decisions. Some molecular alterations don't yet have corresponding targeted therapies, and the clinical significance of newly discovered genetic changes may not be fully understood.

False positive and false negative results can occur with any test, emphasizing the importance of expert interpretation and correlation with other diagnostic information. This is particularly relevant for tests like FISH, where technical limitations can sometimes produce misleading results.

Recommendations for Patients Facing Brain Tumor Diagnosis

For patients diagnosed with or suspected of having a brain tumor, several recommendations can help navigate the diagnostic process:

1. Seek comprehensive molecular testing: Ask your medical team about available molecular profiling options, including DNA methylation profiling and targeted sequencing panels, as these can provide critical diagnostic and prognostic information.
2. Understand your specific tumor characteristics: Request clear explanations of any molecular findings and how they impact your diagnosis, prognosis, and treatment options.
3. Consider second opinions: Molecular testing interpretation requires significant expertise. Seeking review of your case at a specialized neuro-oncology center can ensure the most accurate diagnosis.
4. Discuss clinical trial options: If your tumor has specific molecular characteristics, ask about targeted therapy clinical trials that might be appropriate for your situation.
5. Preserve tumor tissue: If undergoing surgery, discuss with your surgical team the importance of preserving adequate tumor material for potential molecular testing, both initially and for future retesting if needed.
6. Genetic counseling consideration: Some molecular findings may suggest inherited cancer predisposition. Discuss with your doctor whether genetic counseling might be appropriate.

Remember that molecular testing is most valuable when integrated with other clinical information. Work with your medical team to understand how these advanced diagnostics fit into your overall treatment plan and what they mean for your specific situation.

Source Information

Original Article Title: Molecular neuropathology: an essential and evolving toolbox for the diagnosis and clinical management of central nervous system tumors

Authors: Luca Bertero, Luca Mangherini, Alessia Andrea Ricci, Paola Cassoni, Felix Sahm

Publication: Virchows Archiv (2024) 484:181–194

DOI: https://doi.org/10.1007/s00428-023-03632-4

This patient-friendly article is based on peer-reviewed research and aims to make complex scientific information accessible to patients and caregivers while preserving all essential medical information from the original study.