DÄ internationalArchive15/2021Routine Molecular Pathology Diagnostics in Precision Oncology

Review article

Routine Molecular Pathology Diagnostics in Precision Oncology

Dtsch Arztebl Int 2021; 118: 255-61. DOI: 10.3238/arztebl.m2021.0025

Wenzel, C; Herold, S; Wermke, M; Aust, D E; Baretton, G B

Background: Technical advances in the field of molecular genetics permit precise genomic characterization of malignant tumors. This has not only improved our understanding of tumor biology but also paved the way for molecularly stratified treatment strategies in routine clinical practice.

Methods: A selective search of PubMed to identify literature on molecular pathology methods, their indications, the challenges associated with molecular findings, and future developments.

Results: Tumors can be characterized with the aid of immunohistochemistry, in-situ hybridization, and sequencing of DNA or RNA. The benefits of molecularly stratified tumor treatment have been demonstrated by randomized clinical trials on numerous tumor entities, e.g., non–small-cell lung cancer, colorectal cancer, and breast cancer. Therefore, initiation of specific treatment for these entities should be preceded by molecular pathology biomarker analyses, generally carried out on tumor tissue. Randomized controlled trials and non-controlled studies show that enhanced progression-free survival ensues if the pharmacological treatment is oriented on the findings of molecular pathology diagnostics. In next-generation sequencing, numerous relevant gene sequences or even whole genes can be sequenced in parallel, dispensing with complex staged diagnostics and reducing the use of biomaterials. These new methods also complement the currently relevant predictive biomarkers by permitting the investigation of genetic alterations presently of interest in the context of clinical studies. Prior to widespread routine clinical application, however, sequencing of large gene panels or whole genomes or exomes need to be even more stringently validated.

Conclusion: Quality-assured molecular pathology assays are universally available for the determination of currently relevant predictive biomarkers. However, the integration of extensive genomic analyses into routine molecular pathology diagnostics represents a future challenge in precision oncology.

LNSLNS

Molecular pathology analyses extend beyond the genomic characterization of malignant tumors by delivering insights into the behavior of a tumor and its response to various oncological treatment strategies. On the basis of randomized clinical trials (RCTs), numerous specific treatments have been approved over the last decades; as a perquisite for their use, valid molecular testing for biomarkers has to be performed on tumor material. Cross-institutional comparative studies have been conducted in an effort to harmonize the methods used (e1). This is complemented by regular interlaboratory comparisons (ILCs) organized by independent institutions as an instrument of nationwide quality assurance (e2). First studies have shown that quality-assured molecular pathology diagnostics has the potential to not only guide physicians in their treatment decisions, but also greatly influence the disease courses of patients (1). For this reason, routine molecular pathology testing of various biomarkers has become firmly established in tumor diagnostics. However, especially with regard to sequencing of large gene panels or whole genomes or exomes (WGS/WES), it should be noted that the clinical relevance of many genetic aberrations is often poorly understood. In addition, the cells of a tumor of a single patient display genomic heterogeneity which has an effect on the clonal evolution of the primary tumor and its metastases. Consequently, the role of molecular pathologists and biologists has now evolved beyond providing appropriate tests to interpreting test results in the context of clinical findings.

Indications for molecular pathology-based tumor analysis

Molecular pathology tumor analyses provide evidence of abnormal nucleic acid sequences or proteins which induce malignancies or define specific properties of tumors. Most of these aberrations can be measured by appropriate tests and serve as oncological biomarkers. Biomarkers can be detected both in tumor tissue and in bodily fluids. In routine clinical practice, molecular pathology assays are used to identify diagnostic, prognostic and predictive biomarkers. While diagnostic biomarkers can, in combination with tumor morphology, contribute to establishing the definite diagnosis (e3), prognostic biomarkers provide information about the behavior of a tumor (e4). On a routine basis, however, predictive biomarkers are the biomarkers most frequently investigated. They provide information about how tumors will respond to a molecularly stratified treatment strategy and have as a requirement for the use of numerous drugs become an integral part of tumor diagnostics (Table 1).

Selected predictive biomarkers and their rates of occurrence in solid tumors in routine clinical pathology
Table 1
Selected predictive biomarkers and their rates of occurrence in solid tumors in routine clinical pathology

Overview of molecular pathology methods

Various methods are available for molecular pathology diagnostics (Figure, Table 2). In pathology, formalin-fixed and paraffin-embedded (FFPE) tissue samples are almost always used for genomic analyses of malignant tumors. In the process of tissue fixation, however, there is a potential for artefact formation which should be taken into account when interpreting analysis results (e5).

From tissue block to biomarker. Schematic overview of analysis duration and complexity of various molecular pathology methods
Figure
From tissue block to biomarker. Schematic overview of analysis duration and complexity of various molecular pathology methods
Selected examples of molecular pathology biomarker testing und their expressions
Table 2
Selected examples of molecular pathology biomarker testing und their expressions

Immunohistochemistry

Immunohistochemistry is one of the most widely used methods in tumor diagnostics. Numerous antibodies are available to detect genetic alterations. Especially, translocations involving ALK, ROS1 and NTRK can be detected, based on protein overexpression. Furthermore, immunohistochemistry provides information about amplifications of individual genes (e6). Immunohistochemistry is primarily a screening method to detect rare genetic alterations (e7). Positive and inconclusive results should be validated using a second molecular pathology method (e8). In addition, immunohistochemistry is a valid screening method to detect potential microsatellite instability. It can identify solid tumors with microsatellite instability (MSI) based on a lack of expression of proteins involved in base excision repair (mismatch repair, MMR). The cause of this microsatellite instability, again, can only be conclusively established by additional molecular analyses (e9). Furthermore, immunohistochemistry is the method of choice to determine the expression of programmed cell death ligand 1 (PD-L1) on invasive tumor cells and tumor-associated immune cells (e10).

In-situ hybridization

In-situ hybridization (ISH) is a specific method for the detection of chromosomal alterations. Using dye-labelled probes, specific DNA segments can be detected. Depending on the probe system used, it is possible to identify translocations and amplifications in tissue section. Even though interpretation algorithms are now available for numerous biomarkers (e11), ISH diagnostics requires experienced technical staff and properly equipped laboratories. Aberrant signals can complicate the interpretation of the analyses. Given the increasing use of high-throughput methods, currently ISH diagnostics for the detection of translocations is primarily used for validation purposes in routine molecular pathology (e12). However, ISH continues to be the gold standard for the detection of amplifications (e13).

DNA and RNA sequencing

Methods for sequencing specific genes and/or gene segments are gaining in importance. Since some entities require simultaneous testing of several biomarkers, the molecular analysis spectrum has increasingly shifted from single-gene analysis to gene panel sequencing which allows for the analysis of several pre-defined genetic hotspots or whole genes. Thus, second-generation high-throughput sequencing (next-generation sequencing, NGS) is increasingly used in routine laboratory testing. By means of parallel sequencing, it is possible to detect not only single-nucleotide variants (SNV), insertions and deletions, but also gene fusions and—to some extent—gene amplifications. In contrast to immunohistochemistry and most probe-based systems of ISH diagnostics, RNA-based NGS methods used to analyze translocations also provide information about the translocation partner—and patient outcome may be influenced by the respective translocation partner (e14). Regardless of the sequencing method used, adequate tumor cell content in the FFPE sample to be analyzed and tissue-sparing fixation during the pre-analytic workflow are essential requirements for the detection of genuine genetic variants. In contrast to the methods mentioned above, panel sequencing is significantly more time-consuming and expensive (e15). On the other hand, parallel sequencing of multiple biomarkers reduces the use of precious biomaterial and dispenses with complex staged diagnostics. A cross-institutional comparative study showed that NGS panel diagnostics is a sensitive and robust method to detect genomic variants in malignant tumors (e16). A systematic review of studies with a variety of designs (case series, observational studies, one RCT) showed, using the CUP (cancer of unknown primary) syndrome as an example, that molecular targets, which fundamentally enable selective pharmacological treatment, can be detected by panel sequencing. However, no associated clinical benefit has yet been demonstrated (2).

Liquid biopsy

Liquid biopsy is the term used for a molecular pathology analysis of bodily fluids, in particular blood. Malignant tumors can release individual tumor cells or nucleic acids which accumulate in the patient’s blood plasma. Next generation sequencing-based whole blood analysis can detect genetic aberrations in the genetic material released by the tumor. Thus, liquid biopsy enables the detection of resistance mutations, e.g. in non-small cell lung cancer (NSCLC) with known driver mutation, in tyrosine kinase inhibitor (TKI)-treated patients with clinical progression (e17), especially if not enough tumor tissue is available (3). Yet, there are also other diagnostic areas where a liquid biopsy-based approach could be useful, including tumor monitoring and screening for malignancies (e18). Besides a higher degree of representativeness of the whole tumor genome compared to localized tissue samples (e19), liquid biopsy offers the great advantage of minimally invasive sampling. However, the concentration of tumor DNA circulating in the blood is often very low, rendering a large proportion of samples unsuitable for diagnostic decision-making, even though the analytic approach as such is reliable.

Molecularly stratified tumor treatment in routine clinical practice

The methods described above enable, alone and in combination, a valid and quality-assured diagnostic assessment of oncology biomarkers which are required for targeted tumor treatment of selected entities (Table 1). Below and in Table 2, the most important entities of current routine molecular pathology diagnostics are listed as examples.

Entity-specific indications

Non-small cell lung cancer plays a leading role in the precision oncology of solid tumors. Numerous targeted treatments are available that are used based on the specific molecular aberrations displayed by the tumor. Consequently, in all patients with advanced cancer and metastatic disease at the time of first diagnosis, molecular pathology diagnostics for mutations in EGFR and BRAF as well as for translocations in ALK and ROS1 should be performed (3). RCTs showed a longer progression-free survival in patients who received—based on molecular pathology diagnostics—targeted treatments with matching inhibitors (18.9 months versus 10.2 months and 10.9 months versus 7.7 months, respectively) (4, 5). These results were confirmed by uncontrolled studies, showing an overall response rate of 64% (6) and a progression-free survival of 19.2 months (7). In addition, an immunohistochemical analysis of PD-L1 expression should be performed (3, 8). At the same time, resistance diagnostics in NSCLC with driver mutations in patients treated with TKI therapy is becoming increasingly important. Preclinical (9) and clinical studies (10) showed divergent response rates for different targeted treatments, depending on possible ALK resistance mutations. Numerous additional biomarkers of future relevance are currently undergoing clinical testing. Given the favorable results of studies on RET-translocated and MET exon 14 skipping-mutated NSCLC, an approval is expected to be granted soon (11, 12).

In the field of gastrointestinal tumors, extensive research efforts have been made in recent years, focusing on molecular characterization (13, 14). However, none of these insights has so far been incorporated into the clinical routine. While in stomach cancer only HER2 is being tested as a predictive biomarker (15), molecular testing for KRAS, NRAS and BRAF mutations is key in colorectal cancer (16, 17). With the approval of encorafenib in combination with cetuximab, a targeted treatment for BRAF-V600E-mutated colorectal cancer has become available for the first time (18). Promising approaches from a phase 2 trial are also at hand for colorectal cancer with HER2 overexpression/amplification (19); approval is pending. Olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, is approved for use in patients with pancreatic cancer with BRCA1/BRCA2 germline mutations (20).

BRCA1/BRCA2 mutations also play a decisive role in about 5% of breast cancer and ovarian cancer cases. However, in 37% of the BRCA1 variants and 45% of the BRCA2 variants, their clinical significance is undetermined as yet (21). While several PARP inhibitors are currently approved for all types of epithelial ovarian cancer with pathogenic BRCA mutations (22), their use in breast cancer is limited to patients with advanced breast cancer with germline mutations in BRCA1/BRCA2 (23). Additionally, testing of the HER2 status is required in breast cancer patients, since potential HER2 overexpression/amplification may entail treatment with HER2-targeted inhibitors (24). Furthermore, last year a first PIK3CA-targeted inhibitor became available for the treatment of hormone receptor-positive breast cancer which increased progression-free survival in an RCT from 5.7 to 11 months (25). A requirement for its use is the detection of PIK3CA mutations which occur in about 28% of breast cancer cases (25).

Besides lung cancer and colorectal cancer, malignant melanomas in particular have BRAF V600 mutations in about 50% of cases. This subgroup was one of the first entities for which a targeted treatment with BRAF inhibitors and MEK inhibitors became available. In an RCT, the combination therapy with dabrafenib and trametinib achieved a better overall survival rate at 12 months compared to vemurafenib monotherapy (72%, 95% confidence interval: [67%; 77%] versus 65% [59%; 70%]) (26).

Cross-entity indications

With the approval of numerous PD-1 and PD-L1 antibodies, immuno-oncology therapy is now available for many solid tumors. The biomarkers to be tested as a requirement for the use of immuno-oncology therapies include the expression of PD-L1 on tumor cells as well as on peri- and intratumoral immune cells. For the use of anti-PD1 and anti-PD-L1 antibodies to treat non-small cell lung cancer, it is necessary to determine the PD-L1-expressing tumor cells in relation to total tumor cells (tumor proportion score, TPS). In an RCT, pembrolizumab for NSCLC with TPS of 50% or greater significantly increased overall survival to 30 months compared to 14.4 months in the control group (27). An explorative analysis of an RCT with triple-negative breast cancer patients found prolonged overall survival with atezolizumab compared to placebo (25 months versus 18 months) in tumors with a PD-L1 expression of at least 1% of the peri- and intratumoral immune cells (28). Here, the PD-L1-expressing tumor-associated immune cells in relation to the total tumor area were calculated (so-called IC score). In the second-line therapy for advanced urothelial carcinoma, it is relevant for the use of pembrolizumab to calculate the Combined Positivity Score (CPS) which is the number of PD-L1-positive tumor and immune cells divided by the total number of tumor cells. In an RCT, the overall survival in the pembrolizumab arm was eight months for tumors with CPS ≥ 10 compared to 5.2 months in the control arm (29). Recently, pembrolizumab has been approved by the US Food and Drug Administration (FDA) for the treatment of microsatellite instability-high solid tumors. Microsatellite instability (MSI) is the result of mutations or epigenetic changes in genes of the base excision repair pathway (deficient mismatch repair; dMMR). The loss of the corresponding proteins is causing an increased number of mutations in the DNA which, in turn, promotes the formation of neoantigens, potentially increasing the immunogenicity of these tumors (30). First successes were also noted for nivolumab in the treatment of various entities with dMMR in a sample of selected previously treated patients—42 of 4902 screened patients were included—where it achieved an objective response rate of 36% (31). In addition, with the approval of larotrectinib for solid tumors with identified NTRK translocation, the cross-entity use of a tyrosine kinase inhibitor is now possible (32). However, a multicenter RCT found that the molecular marker-based treatment beyond the defined indication areas was ineffective in pretreated patients with various types of cancer (33). Two non-controlled prospective trials indicate that molecular pathology analysis may—by enabling targeted treatment—improve progression-free survival in heavily pretreated patients (34, 35).

Future developments and challenges in molecular pathology

With the expanding scope of molecular analyses, a growing number of potential predictive biomarkers for the management of malignant neoplasms have been discovered in recent years. In the field of immuno-oncology, tumor mutational burden (TMB) has been discussed as a potential predictive biomarker, in addition to PD-L1 and microsatellite instability. However, since first clinical trials have not shown the successes that were hoped for, the significance of tumor mutational burden as a predictive biomarker has yet to be conclusively determined (36). Research programs rely on whole-exome or whole-genome analyses. Supplementing the analysis of typical hot-spot regions of numerous genes as well as the calculation of tumor mutational burden, these tests also provide information about non-coded regions and thus cover splice mutations and genetic aberrations in the promoter region of genes (37). In addition, WES/WGS analyses provide the means to study microsatellite instability and mutation signatures. Mutation signatures are the sum of multiple co-occurring genetic aberrations (38). Since, due to the lack of RCTs, no universal treatment recommendations have yet been established, levels of evidence for their assessment have been developed which are now used by local tumor boards where specialists in the various disciplines formulate personalized treatment recommendations based on molecular data (Table 3) (39). Studies have shown that on the basis of genomic analyses alone an optimized targeted therapy cannot be achieved. Rather, a comprehensive morphomolecular assessment as well as the incorporation of functional analyses are critical for the advancement of precision oncology in the future (40).

Clinical levels of evidence for the creation of personalized treatment recommendations in molecular tumor boards
Table 3
Clinical levels of evidence for the creation of personalized treatment recommendations in molecular tumor boards

Conflict of interest statement

Dr. Wermke received consultancy and lecture fees from Novartis, Roche, Bristol Myers Squibb, MSD, Amgen, AstraZeneca, Pfizer, and Takeda. He received reimbursement of congress fees and travel expenses from Roche, AstraZeneca, Pfizer, Novartis, and Bristol Myers Squibb. He received study support (third party funding) from Novartis, BMS, MSD, AstraZeneca, and Pfizer.

Prof. Aust received consultancy fees as well as reimbursement of travel expenses from Pfizer, AstraZeneca, Roche, and MSD. She received lecture fees from Roche, Pfizer and AstraZeneca.

Prof. Baretton received consultancy fees, reimbursement of travel expenses and lecture fees from Pfizer, Roche, MSD, and AstraZeneca.

The remaining authors declare not to have any conflicts of interest.

Manuscript received on 26 May 2020, revised version accepted on 1 December 2020

Translated from the original German by Ralf Thoene, MD.

Corresponding author
Prof. Dr. med. Gustavo B. Baretton
Institut für Pathologie
Universitätsklinikum Carl Gustav Carus an der TU Dresden
Fetscherstraße 74, 01307 Dresden, Germany
gustavo.baretton@ukdd.de

Cite this as:
Wenzel C, Herold S, Wermke M, Aust DE, Baretton GB: Routine molecular pathology diagnostics in precision oncology. Dtsch Arztebl Int 2021; 118: 255–61.
DOI: 10.3238/arztebl.m2021.0025

Supplementary material

eReferences:
www.aerzteblatt-international.de/m2021.0025

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Institute of Pathology, University Hospital Carl Gustav Carus Dresden, TU Dresden, Dresden: Dr. med. Carina Wenzel, Dr. rer. nat. Sylvia Herold, Prof. Dr. med. Daniela E. Aust, Prof. Dr. med. Gustavo B. Baretton
Medical Department I, University Hospital Carl Gustav Carus Dresden, TU Dresden, Dresden: Dr. med. Martin Wermke
From tissue block to biomarker. Schematic overview of analysis duration and complexity of various molecular pathology methods
Figure
From tissue block to biomarker. Schematic overview of analysis duration and complexity of various molecular pathology methods
Selected predictive biomarkers and their rates of occurrence in solid tumors in routine clinical pathology
Table 1
Selected predictive biomarkers and their rates of occurrence in solid tumors in routine clinical pathology
Selected examples of molecular pathology biomarker testing und their expressions
Table 2
Selected examples of molecular pathology biomarker testing und their expressions
Clinical levels of evidence for the creation of personalized treatment recommendations in molecular tumor boards
Table 3
Clinical levels of evidence for the creation of personalized treatment recommendations in molecular tumor boards
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