Precision Oncology

“Precision oncology” refers to the use of molecular tumor characterization followed by personalized disease management. Molecular testing and molecularly stratified therapies are already standard practice for various tumor entities (e.g., non-small cell lung cancer [NSCLC], colorectal cancer [CRC], hematologic neoplasms, and many others). Reference is made to the respective entity-specific guidelines.

However, in addition to guideline-based personalized oncology, effective treatment options can also be identified through molecular tumor analysis in patients with other tumor types. This is underscored by the increasing number of predictive biomarkers with cross-entity relevance. Predictive biomarkers can be characterized by alterations in specific molecules (e.g., BRAF mutations, NTRK fusions, RET fusions, HER2 overexpression) or, as complex biomarkers, be based on the identification of numerous alterations (e.g., microsatellite instability or tumor mutational burden). In addition to predictive biomarkers, diagnostic, prognostic, predisposing, or pharmacogenomic biomarkers can also be identified and used for personalized disease management. Biomarkers can be detected in various analytes (DNA, RNA, protein) using a growing number of (high-throughput) methods. Therefore, broad molecular analyses are often performed to identify diverse potential biomarkers. Furthermore, even within the framework of guideline-based molecular diagnostics, alterations may be identified that are potentially therapeutically relevant but are not addressed by current guidelines. Proper indication, test selection, and clinical interpretation of molecular alterations - with the goal of evidence-based, personalized treatment guidance despite often limited clinical data - present a challenge and require multidisciplinary expertise within the framework of specialized molecular tumor boards.

Conducting and interpreting comprehensive molecular testing requires multidisciplinary expertise. This includes proper indication, the molecular analysis itself, the evaluation and interpretation of the results, and their contextualization within the individual clinical setting aiming at making a treatment recommendation [15]. A schematic overview of the workflow in precision oncology is shown in Figure 1.

In cases of clear, i.e., guideline-based indications and corresponding approvals, referral of the patient to an organ-specific tumor board is adequate (e.g., typical EGFR mutation in NSCLC, BRAF p.V600E mutation in melanoma, RET mutations in medullary thyroid carcinoma). In cases of complex findings not covered by guidelines - most commonly in the context of re-biopsies in cases of recurrence or in advanced stages of disease - the case should be presented to a molecular tumor board (MTB).

Outside of clinical trials, the decision to order molecular diagnostics should always be based on the potential clinical relevance of the results. Factors to be considered include the patient’s eligibility and preference for treatment, current guidelines, the approval status of any therapy that may result from molecular testing, the available clinical and scientific findings, and, if applicable, the decision of a (molecular) tumor board.

Molecular diagnostics are increasingly being incorporated into guideline-based standard therapy for first-line or later-line treatment, and are part of the standard diagnostic workup for the respective tumor type. Examples include many driver mutations in NSCLC, BRAF mutations in melanoma and CRC, HER2 amplifications in breast cancer, RET mutations in medullary thyroid carcinoma, and many others. For these patients, once molecular diagnostics have been performed, referral to an organ-specific tumor board is generally sufficient for making a treatment decision.

Additional indications for molecular diagnostics include meeting the inclusion criteria for molecularly stratified clinical trials. Here, too, molecular diagnostics is increasingly being incorporated into previous lines of treatment. For example, the centers of the German National Network for Genomic Medicine in Lung Cancer (nNGM) integrate study-relevant molecular markers into their Next Generation Sequencing (NGS)-based primary diagnostics in addition to approved molecular therapy options.

For patients whose standard treatment options - including approved molecular-targeted therapies - have failed, the indication for (possibly expanded) molecular diagnostics can be determined by an organ-specific tumor board. The resulting findings should then be discussed in an MTB. MTBs are established at most Centers of Excellence (CCCs) for oncology and, increasingly, at other cancer centers as well.

In accordance with recommendations of the German Network for Personalized Medicine (DNPM), also included in the German Cancer Society’s certification criteria for Centers for Personalized Medicine, the following inclusion criteria for access to MTBs (with corresponding comprehensive molecular characterization) are established (Table 1):

The decision to pursue expanded molecular diagnostics after standard therapies have been exhausted typically requires individualized decision-making in a multidisciplinary tumor board. Particularly in patients with rapidly progressive tumors, the choice of an appropriate method, the expected duration of the diagnostic process, and the subsequent clinical implementation should be considered, and corresponding analyses should therefore be initiated at an early stage.

Increasingly, the molecular characterization of tumors is being used as part of routine diagnostics and, based on guidelines, even early in the course of treatment - for example, before the decision on first-line therapy is made. In the majority of cases, an informed patient consent form is not required here. However, with the increasing use of methods for parallel DNA and RNA sequencing of numerous gene regions, genetic aberrations are also being identified whose biological and clinical relevance is not yet established (e.g., atypical EGFR mutations) or complex molecular changes such as the simultaneous presence of multiple potentially predictive biomarkers. The clinical interpretation of such findings, as well as of variants of uncertain significance (VUS), may require the additional involvement of a medical geneticist.

Figure 2 illustrates a possible framework for integrating comprehensive molecular diagnostics and specialized discussion of findings into the clinical treatment/decision-making algorithm.

In addition to patient selection (see Table 1), the selection of adequate samples to be examined also plays a central role. In this context, both the representativeness of the available material and the fixation technique and age of the sample must be taken into account. Table 2 lists criteria that can be used when deciding whether to obtain a repeat sample.

In addition to repeat sampling, liquid biopsy techniques can also be used for certain diagnostic questions. It should be noted that for many markers - particularly fusion genes and amplifications - these techniques have a high specificity but (as yet) lower sensitivity compared to tissue biopsies. Therefore, tissue-based diagnostic procedures currently remain the standard, particularly in primary diagnosis, while liquid biopsy is increasingly used in cases of recurrence where biopsy sampling is difficult (note that reimbursement options may be limited in such cases) and, increasingly, in clinical trials for treatment monitoring. For certain clinical settings (e.g., detection of ESR1 mutations in breast cancer, relapse of lung cancer in specific cases within the context of nNGM), liquid biopsy currently can be performed as part of routine diagnostics.

Therapeutically relevant molecular alterations can be identified at numerous levels of cellular regulation. Table 3 summarizes possible levels of cellular dysregulation.

New technologies enable the rapid and simultaneous analysis of numerous potential biomarkers using high-throughput methods. In the field of precision oncology, DNA and RNA sequencing currently play the most important role alongside immunohistochemistry. Available diagnostic methods are summarized in Table 4 [1, 8].

The selection of an appropriate molecular diagnostic test should also take into account previous analyses as well as the pre-test probability of relevant biomarkers (Tables 8 and 9). Currently, in Germany, for defined clinical situations and at participating centers, reimbursement is available for whole-genome sequencing as part of the Genome Sequencing Pilot Project (German SGB V, §64e).

The performance of molecular testing and the identification of molecular alterations is a largely standardized process and falls within the domains of human genetics, (molecular) pathology, and bioinformatics, among others, and should be conducted in a quality-assured environment, preferably at an institute or laboratory accredited for molecular diagnostics. Ideally, molecular diagnostics should be embedded in a clinical setting that ensures a multidisciplinary approach with regard to the interpretation and clinical annotation of the results. The report should include information on the (tumor) material used, material quality, tumor cell content, the type of analysis performed, a list of identified alterations using standardized coding, variant allele frequency, and a functional assessment of the variants. Human genetics should be consulted for the evaluation of possible germline variants.

The functional evaluation of molecular alterations should be based on current guidelines and Standard Operating Procedures (SOPs) (e.g., [6]).

Specific molecular alterations may contain relevant information for the clinical management of malignancies. The identification of these biomarkers is therefore the goal of molecular testing. The clinical annotation of a variant serves to evaluate it as a biomarker. In this context, the following potential biomarkers must be distinguished (Table 5) [16].

Annotating a predictive biomarker often requires a comprehensive literature review. Numerous databases facilitate the search for information, although the content of these databases often does not overlap (e.g., civicdb.org, oncokb.org, ckb.genomenon.com/), and analysis of the primary literature is essential for interpreting the findings [14].

To evaluate the relevant biomarker in the context of the respective tumor entity, levels of evidence have been defined that should be used for assessment [7, 10, 13]. In German-speaking countries, the NCT/ZPM levels of evidence are the most widely used (Table 6). In addition to these, other levels of evidence are available, such as AMP/ASCO/ACP [11] or ESMO-ESCAT [12].

However, in addition to interpreting potential predictive biomarkers, other relevant biomarkers should also be evaluated (e.g., DPYD testing, if included). If germline testing has been performed, this requires the expertise and co-evaluation of a human geneticist. Due to the significant time required and the necessary research, the clinical classification and interpretation of the findings often take place before the final decision regarding clinical application is made.

The evaluation and clinical interpretation of germline findings fall within the scope of human genetics. It must be noted that variants identified in a purely somatic tumor analysis may be attributable to underlying germline alterations. The possibility of a germline alteration should therefore also be evaluated when performing purely somatic tumor analyses. In this context, both the probability of a germline alteration and the clinical implications should be considered if such an alteration is present in the germline.

According to ESMO recommendations, the following applies to variants with the following characteristics:

1. Minor allele frequency <0.01

2. Resulting in protein truncation and/or classified as pathogenic/likely pathogenic

3. Variant allele frequency >30% (SNVs) or >20% (small insertions/deletions).

in the genes listed below (Table 7), with a probability of ≥ 5% for a germline origin (in a cross-tumor analysis) of the variants detected in the somatic tumor analysis, as well as a possible clinical implication [18].

Subsequent germline analysis of the identified variants should therefore be offered based on the probability and potential clinical implications.

*See Appendix: Approval Status (in German language, for Germany, Swiss and Austria only)

There is a growing number of predictive biomarkers with tumor-agnostic approvals for targeted drugs (corresponding to at least ESCAT Level I-C, NCT m1A, JCR Tier 1 A.1). Corresponding predictive biomarkers with EMA approval are presented in Table 8. In Europe, there are currently only five tumor-agnostic approvals for NTRK inhibitors (larotrectinib, entrectinib, and repotrectinib) and RET inhibitors (selpercatinib) in the presence of NTRK1-3 or RET fusions, as well as for trastuzumab deruxtecan in cases of HER2 overexpression (immunohistochemical 3+, according to ASCO-CAP [19] for gastric cancer) and pembrolizumab in cases of MSI-H or dMMR for CRC, endometrial, gastric, small intestine, or biliary tract cancers.

Accordingly, the following treatment algorithm applies in a cross-entity setting, taking into account FDA approvals (Figure 3). However, it should be noted that there are data on the efficacy of additional targeted therapies based on other predictive biomarkers that are currently not approved by either the FDA or the EMA.

Table 9 summarizes the prevalence of these biomarkers (see Table 8) in various tumor types.

The final application of the identified and evaluated biomarkers in specific clinical situations requires broad multidisciplinary expertise and should take place at designated centers (e.g., German Cancer Society-certified centers for personalized medicine). According to the criteria of the German Network for Personalized Medicine (DNPM; [9]) and the German Cancer Society, the participating disciplines of an MTB team include, at a minimum, hematology and medical oncology, pathology, molecular pathology, molecular biology, bioinformatics, human genetics, as well as case-specific disciplines and radiology as needed. In addition, this team should possess sufficient and comprehensive expertise in the interpretation, evaluation, and integration of molecular findings into the clinical course of therapy, which is ensured by sufficient experience in molecular analysis and a sufficient number of MTB cases.

The high degree of individual variation, as well as the varying levels of evidence supporting resulting recommendations, requires close monitoring of the clinical course and careful consideration when integrating potentially experimental treatment options into further therapy.

Ideally, off-label treatment options should be administered within the framework of clinical trials; the availability of such trials should therefore be evaluated as part of the treatment recommendation and assessed at least at the national level. However, implementing the treatment recommendation often requires off-label use of medications (usually following a prior application to the health insurance provider for coverage in Germany). In such cases, particular attention should be paid to the level of evidence supporting the recommended therapy in light of the expected patient benefit. Off-label therapies should be monitored as part of registry studies (see below).

In addition to predictive biomarkers, diagnostic biomarkers play an important role in treatment recommendations made by molecular tumor boards. Reclassification of tumor diseases based on molecular findings can open up further treatment options beyond classic targeted therapies.

The high frequency of unapproved treatment recommendations and the lack of clinical data suggest the need to integrate research-based care into precision oncology. In particular, implementation rates of targeted therapies and treatment responses to off-label therapies should be comprehensively tracked and systematically documented (see core data sets of the Medical Informatics Initiative, https://www.medizininformatik-initiative.de). Ideally, this should be conducted as part of a prospective registry study and data should be consolidated within a network; several scientific networks are currently making important contributions in this area. Consistent recording of molecular alterations can thus lead, in the long term, to an improved understanding of molecular alterations and more effective treatment options, even for rarer tumors.

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according to the rules of the responsible Medical Societies.