How will the identification and therapeutic intervention of genetic targets in oncology evolve for future therapy?

ABSTRACT

Introduction

Mapping of the human genome, together with the broad understanding of new biomolecular pathways involved in cancer development, represents a huge dividing line for advances in cancer treatment. This special article aims to express the next evolution of cancer therapy, while also considering the challenges and uncertainties facing future directions.

Area covered

The recent achievements of medical science in the oncology field concern both new diagnostic techniques, such as liquid biopsy, and therapeutic strategies with innovative anticancer drugs. Although several molecular characteristics of tumors are linked to the tissue of origin, some mutations are shared by multiple tumor histologies, thus paving the way for what is called ‘precision oncology.’ The article highlights the importance of identifying new mutations and biomolecular pathways that can be pursued with new anticancer drugs.

Expert opinion

Oncology and medical science have made great progress in understanding new molecular targets; being able to early identify tumor markers that are not confined to a single organ through minimally invasive diagnostic techniques allows us to design new effective therapeutic strategies. Multidisciplinary teams now aim to evaluate the most appropriate and personalized diagnostic/therapeutic approach for the individual patient.

KEYWORDS:

• Biomolecular targets
• druggable targets
• mutation
• precision oncology
• safety

1. Introduction

Medical science, and the pharmaceutical world (and its business) live this era trying to give less fragile hope to all cancer patients, but also to the whole of humanity. Fortunately, scientific research is accelerating, and the mapping of the human genome, together with a broad understanding of new biomolecular pathways involved in the development of cancer, represent a huge dividing line for progress in cancer treatment. However, it is still impressive to consider the global cancer statistics. In 2018, one in every six deaths worldwide could be globally attributed to cancer, with a predicted 1,261,990 deaths for all cancer in 2023 in the EU-27 [1,2]. However, cancer mortality rates have been declined since 1991, with an overall decrease of 33% and about 3.8 million cancer deaths prevented in the U.S.A [3]. The overall burden can change from year to year, and since 2019 some health systems have reduced access to routine care due to the COVID-19 pandemic. These measures have disrupted cancer screening, diagnosis and treatment in many cancer clinics. At the same time, survival rate has increased considerably. For example, according to Italian regional data, at 5-years relative survival at diagnosis of any cancer (excluding non-melanoma skin cancer) for diagnoses made between 1995 and 2017, increased from 46% to 59% for men and from 54% to 66% for women, after adjustment for age [4]. However, significant differences are observed by neoplastic sites. These advances can be attributed to a number of factors, such as new diagnostic techniques, e.g. liquid biopsy, and a deep understanding of new biomolecular pathways and ‘actionable’ targets expressed by different forms of cancer, no longer limited only to organ-specific forms. This has opened the door to innovations that can now offer ‘precision oncology’ and ‘agnostic’ treatments. Great progress has also been made in what we call the comprehensive pharmaceutical care for cancer patients. While, the typical chemotherapy-induced toxicity profile, which has an extremely heavy impact on patients, has certainly not been completely eliminated, but today’s therapies are much more specific in ‘locking in’ the aberrant molecular target and more selective to discriminate between healthy cells/tissues and cancer cells, thus helping us to predict the nature of adverse reactions (e.g. from immunotherapy) and to manage them more effectively. Patients’ quality of life is also greatly improved by the availability of a large number of orally administered drugs, which allow patients to be treated at home, in as private and comfortable environment as possible.

Herein we discuss some of the most relevant precision oncology challenges.

2. New discoveries in biomolecular cancer targets: the importance of mutation research

Since 2002, at least 22 druggable targeted mutations have been identified and approved for clinical use by the U.S. Food and Drug Administration (FDA). In addition, many other mutations that have not been fully approved by the FDA continue to be studied [5].

The first approach to targeting was to counteract a single mutation in an oncogenic gene with monoclonal antibodies. This required the expression of a specific antigen on the cell surface that is altered in its amino acid structure or has higher cellular expression than the corresponding non-cancerous cell. This approach, due to the limited accessibility and availability of the targets on cell surface and their widespread expression even in healthy cells has led to the identification of very few genes as potential drug targets. In addition, the approval of a drug for the treatment of cancer depends also on its pharmacokinetics, its ability to reach and effectively target tumor cells, and its overall toxicity profile. Today’s these complex property requirements are aided by integration of artificial intelligence with multi-omics data and in silico approaches for identifying active scaffolds of molecules, and plausible binding pocket evaluation [6]. Currently, there are no drugs produced by using artificial intelligence on the market, and its use must be subject to strict legal regulations; nevertheless, this approach could reduce the time to market of a drug, and thus the costs of production and marketing, but leading to a reduction in human jobs. Today, these approaches have mainly focused to the discovery of technologies that enable the intracellular transport and delivery of drugs, such as nanoparticles [7]. When stimulated or changed by appropriate factors, such as thermal temperature, these smart particles efficiently aggregate at the target site and release their drugs. New classes of nanoparticles being studied include extracellular vesicles and thermal nanomaterials. Toxicology is the main obstacle to the use of these nanoparticles because of their composite structure makes their toxicity difficult to detect, and there is no validated model suitable for testing fine biological interactions, particularly nano-immuno interactions, so very few nanoparticles have received FDA approval to date [8]. In 2011, a class of targeted therapies known as immune check-point inhibitors was introduced in the field of cancer treatment. These drugs by blocking certain proteins that function as negative regulators of the immune system, unlike traditional drugs that aim to boost the immune response, revolutionized therapeutic strategies, and became more effective in killing cancer cells. Ipilimumab, was the first drug in this class to be approved [9]. Subsequently, a significant advance in the field of immunotherapy was made with Chimeric Antigens Receptor Cells-T (CAR-T) [10]. This innovative approach involves genetically modifying the patient’s immune cells so that they express a synthetic receptor for a target antigen. After the infusion of these modified CAR T cells into the patient, they are able to effectively localize and destroy cancer cells expressing the target antigen. Two of the best-known CAR T-cell therapies, Kymriah and Yescarta, received European Union approval in 2018 for the treatment of certain types of hematological malignancies. Interestingly, some CAR molecules are able to cross the blood-brain barrier and enter neurons to be effective [e.g. Neurotrophic tyrosine receptor kinase (NTRK) family drugs] [11]. However, their use in clinical applications is hampered by high cost, complex manufacturing processes and cytotoxicity. Moreover, their efficacy and durability are limited and influenced by many factors, including the design of the CAR molecule, the cell type constituting the tumor microenvironment and the mechanism of action. Several strategies are currently being explored mainly to improve their durability. These include arming CAR-T to improve tumor penetration and avoid immune cell exhaustion. With these goals in mind, the current studies seek to understand whether combing CAR T therapy with immune checkpoint inhibitors can improve T-cell persistence to increase relapse-free survival. Genomic editing of CAR T can now be facilitated by the use of the Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein (CRISPR-cas) tool, which allows a precise DNA region to be edited quickly and inexpensively, but opens up many new bioethical questions [12].

An example of a recent successful targeted therapy is the direct targeting of the Kirsten rat sarcoma virus (KRAS) G12C mutant protein, which results in constitutive activation of the gene, and has been unsuccessfully targeted by traditional pharmacological methods [13]. Functional proteomics technologies, such as Activity-Based Protein Profiling (ABPP) using small chemical probes to understand interaction mechanisms between compounds and targets [14]. [Wang, S.; Tian, Y.; Wang, M.; Wang, M.; Sun, G.; Sun, X. Advanced Activity-Based Protein Profiling Application Strategies for Drug Development. Front. Pharmacol. 2018, 9.] helped to identify effective inhibitors that covalently bind to a specific pocket in the protein’s inactive state, stabilizing it and preventing its activation, even before the molecular structure of the protein is obtained and allowing the time required to achieved the same result to be considerably reduced. Interestingly, the same KRAS G12C was found in different tumor histotypes (i.e. non-small-cell lung cancer, colorectal cancer and pancreatic ductal adenocarcinoma) and can now be used in all these tumor subtypes. Advances in covalent drug discovery have subsequently led to other successful drugs against e.g. multiple KRAS mutations, epidermal growth factor receptor (EGFR) and Bruton Tyrosine Kinase (BTK). Numerous computational datasets are now available and will be explored as part of an effort to introduce the concept of a translational chain involving known druggable targets. However, it is essential to note that the potential pool of identified targets is drastically reduced by the lack of efficacy observed in preclinical studies. Moreover, only a limited number of these targets can be applied in vivo due to the lack of knowledge on drug safety and patient health conditions [15].

In certain cases, diagnosing genetic alterations have been made more complex due to the fusion of one gene with many other genes (e.g. NTRK1-2-3 fusion gene). This problem is now being solved by the introduction of advanced technologies such as next-generation sequencing (NGS) or Nanopore sequencing, which enable the use of specific primers for the NTRK1-2-3 gene and the identification of the fused gene by extensive DNA or improved RNA sequencing. The example of the NTRK 1-2-3 fusion gene highlights the importance of not only characterizing a single mutation, but also understanding the pathway it affects, and its consequences. Efficacy and resistance in many cases result from a complex and heterogeneous tumor microenvironment. For instance, NTRK1-2-3 mutations have been shown to interact with the RAS pathway, and tumors with mutated RAS genes have been found to be refractory to treatments against genes from NTRK family.

Until now, one of the main challenges in cancer therapy has been the lack of effective targets for tumor suppressor genes, such as the anti-apoptotic tumor protein p53 (TP53), which are particularly difficult to target because restoring the function of a mutant protein product is more difficult than inhibiting it. There are currently, no approved drugs for this type of gene alteration, but some studies have reached advanced and promising preclinical trials [16]. One strategy is that of mouse double minute 2 (MDM2) proteolysis-targeting chimeras (PROTACs) strategy that target and degrade MDM2, a protein that ubiquitin TP53, and induces its degradation [17]. A second strategy uses small molecules capable of covalently binding to the mRNA of the mutated tumor suppressor gene alone and directing its degradation, leaving the normal P53 protein intact [18]. Another approach involves interfering with the expression of tumor suppressor gene by modulating regulatory molecules at the post-transcriptional level, such as microRNAs (miRNAs) [19]. Another, but more complicated approach, is to directly modify the tumor suppressor gene. This could involve using gene editing techniques such as CRISPR method to correct or change the mutation in the gene [20]. These approaches are a range of strategies being explored and hold promise for development of new treatments, but further research and clinical trials are needed to determine their safety and efficacy.

Taken together, the above examples highlight the significance of using omics-based approaches to explore not only the pinpoint druggable mutations, but also to understand the intricate oncogenic pathways and tumor microenvironment (TME) in which these variants occurred. Finally, it is important to note that patient samples are heterogenous, being composed of both somatic DNA from healthy tissue and heterogenous tumor cells. This heterogeneity can lead to a low variant allele frequency (VAF), which can result from the presence of normal cells alongside the heterogeneous tumor cells, occasionally leading to errors in mutations identification [21]. Consequently, it is often required to confirm the presence of mutations using more than one method to ensure the accuracy and reliability of the result obtained. Immune checkpoint inhibitors (ICIs) just introduced, have demonstrated notable effectiveness in treating patients whose malignancies express a lot of tumor-related antigens. Mutations in the function of DNA repair genes, including the Breast Cancer gene (BRCA)1/2 mutation are often responsible for this enhanced immunogenicity [22]. Additionally, ICIs have been proven to be beneficial in cases where a gene alteration, besides its primary oncogenic function, also induces an immunosuppressive tumor microenvironment, as seen in the context of mutated Kirsten rat sarcoma virus (KRAS) [23]. These factors contribute to the enhanced efficacy of ICIs in certain patient populations and highlight the significance of understanding the molecular characteristics of both the tumor and its microenvironment in the context of immunotherapy. Given that tumors are complex and heterogeneous diseases, it is becoming clear that the most effective way to treat a patient is frequently to combine a number of druggable therapies [24]. To identify the co-occurrence of multiple or rare mutations and detect the emergence of metastatic clones next-generation sequencing (NGS) is a useful technique. It is unimportant to note that many mutations have unclear clinical consequences, which has led to their classification as Variants of Uncertain Significance (VUS). Despite increasing knowledge of the biology of these mutations, clear guidelines for diagnosis and optimal therapy may still be lacking.

According to Tumor boarding (TMB) analysis, at least 1 megabase (MB) of genomic DNA should be sequenced using a panel of at least 300 genes, some of which are druggable. In order to classified the impact of germline mutations it may occasionally be required to test a non-tumor sample. For the successful usage of ICIs, a cutoff of more than 10 mutations per megabase MB has been suggested, while the threshold for TMB is still somewhat debatable. In order to give a standard molecular categorization of mutations and probable clinical classifications as pathogenic, likely pathogenic, or benign, several public databases now collect mutation and clinical information from patients with the variants.

NGS can also be applied to liquid biopsies, often referred to as circulating free DNA (cfDNA), which allow for noninvasive monitoring of genetic changes in the bloodstream. Its sensitivity and specificity are significantly lower than tissue NGS, especially when it is used in detecting very low VAF of 0.5%. Furthermore, cfDNA often requires a specific genetic panel tailored for liquid biopsies. Nonetheless, cfDNA offers a few notable advantages. It can identify tumors in a wide range of situations, regardless of their histology, heterogeneity or primary site of origin, making it a powerful tool for cancer identification. Furthermore, by identifying new mutations, cfDNA can detect early recurrences, potentially providing benefits for early cancer detection and surveillance. However, because the overall information is so complex, a diverse team of experts with backgrounds in oncology, genetics, molecular biology, and bioinformatics, is required to properly use cfDNA in cancer diagnosis and treatment. This collaborative effort is essential to ensuring that patients receive the most appropriate and successful treatments, especially in cases where precision medicine and targeted therapies are involved.

Another important consideration in cancer treatment concerns the emergence of a treatment-resistant cell clones. These clones might be no detectable by the analysis of the small number of cells in the tumor with a specific alteration or by the addition of new mutation induced by the treatment itself. This underlines the critical need to identify specific and sensitive biomarkers for early detection and appropriate treatment. Currently, the number of patients who can benefit from targeted therapy is often limited, primarily due to a limited number of targetable mutations, the sometimes-insufficient quality or quantity of samples analyzed, and the advanced stage of the patient’s disease. To improve this situation, particular attention is currently being paid on the impact of additional demographic and molecular information. This includes exploring epigenetic modifications, exosome analysis, metabolic and proteomic profiling, which can lead to changes in the phenotype of tumor cells. These changes may not always be identifiable by genomic analysis alone. Additionally, novel strategies to drug design are being evolving to increase the pool of patients who could eventually benefit from the same targeted therapy.

3. Precision oncology and the evolution of therapy in cancer

Precision oncology, defined as the molecular profiling of cancers to find druggable targets, is increasingly recognized as a promising therapeutic approach. However, the concept of targeting an altered protein or pathway is not new. For example, the anti-breakpoint cluster region protein- tyrosine-protein kinase ABL1 (BCR-ABL) fusion protein Imatinib and the anti-receptor tyrosine-protein kinase erbB-2 (HER2) antibody Trastuzumab have dramatically improved the prognosis of chronic myeloid leukemia and breast cancer, respectively. As the cost of gene sequencing has dropped, a substantial amount of cancer mutation data has accumulated in recent years, highlighting the distinct molecular landscape of various tumor histologies. Nevertheless, genome sequencing has also revealed that alterations in certain genes or pathways occur consistently regardless of the cancer’s tissue of origin. Therefore, in parallel with the increasing availability of pharmacological compounds, the idea of matching a specific genomic aberration with a corresponding drug seemed obvious. Although this approach seems plausible from a biological point of view, many challenges are emerging from the precision oncology trials (e.g. NCI-MATCH, MOSCATO 01, IMPACT/COMPACT, ProfiLER) [25] and three of them deserve a mention.

Firstly, the number of molecular aberrations tested and the number of available drugs to target them. Precision oncology trials are based on the assumption that many cancers have at least one alteration that could be targeted pharmacologically. However, sequencing a small number of genes or having a small number of drugs could lead to a disproportionated effort between recruitment and clinical actionability. This discrepancy may explain, at least in part, the low proportion of patients matched in some trials (e.g. IMPACT/COMPACT) [26]. Secondly, not all mutations are equal, nor are all mutations predictive of response. Indeed, several arms of precision oncology trials have shown futility. This could be caused by targeting passenger or subclonal mutations or by compensatory resistance mechanisms. One possible solution could be to match drugs to genes that have already been validated as predictive of response (e.g. ESMO ESCAT scale) [27]. This strategy has already shown efficacy, for example targeting BRCA1/2 mutations through PARP inhibitors (e.g. SAFIR02-BREAST trial) [28]. Another different strategy could be the combination of drugs in order to avoid or limit primary resistance (e.g. ComboMATCH trial, NCT05564377) [29]. It is also important to note that even the same gene mutation might be predictive only in a specific histological context. For example, BRAF V600E predicts a high probability of response to BRAF inhibitor monotherapy in melanoma, but not in colorectal cancer [30]. It is therefore pivotal to collect data on the biological context (e.g. tissue of origin of the cancer).

Thirdly, one in four patients is affected by a rare cancer, characterized by a prevalence of less than 1.5 cases per 10,000 individuals. Rare cancers typically offer only a few treatment choices. Nevertheless, the occurrence of practically targetable genetic mutations and the advantages of tailored therapies are on par with those seen in common cancers [31]. This highlights the importance and ethical value of the precision oncology approach in such diseases. However, it will be crucial that these patients are referred to experienced centers involved in precision oncology trials. Collectively, precision oncology could dramatically change the way cancer is treated but more knowledge needs to be gained in order to turn hope into fact.

4. Expert opinion

Tumor characterization based on the identification of molecular targets is set to become the standard approach for future oncology. New insights into the biomolecular mechanisms expressed by cancer cells, together with new pharmaceutical technologies to produce increasingly specific anti-cancer drugs, have made it possible to increase life expectancy after a cancer diagnosis, giving humanity more hope for the near future. This will certainly impact real-world outcomes on both the diagnostic and cancer care sides. Innovative and sophisticated tools such as NGS, liquid biopsy, and modern imaging technologies aim to increasingly reduce invasiveness for patients, and in parallel to increase the sensitivity and specificity of diagnosis and treatment. Additionally, the rapid development of artificial intelligence may help aggregate the outputs of these innovative methods by making the delivery of health care more efficient, improving the quality of life during illness [32]. In parallel, clinical practice in oncology is also changing. Where individual oncology departments used to work individually, advanced multidisciplinary teams and Molecular Tumor Boards are now being created to assess what is the best therapeutic-diagnostic approach for each individual clinical case.

However, some challenges need to be addressed before hopes can be turned into reality. First, it is crucial to test a relevant number of molecular aberrations and to have the corresponding drugs available; second, mutations need to be classified according to their predictive value (e.g. ESMO ESCAT scale); third, patients with rare cancers could benefit greatly from precision oncology and should be referred to centers involved in precision oncology trials. Genetic mutations are still subject to different interpretations and fit into multiple or coincident pathways: this may generate different views and different assumptions about therapeutic decisions; moreover, knowledge about the oncogenicity of some variants is not exhaustive to date. Another major problem of molecular oncology are assumptions about the development of resistance. The innovativeness of new treatments and therapeutic potential often leads oncologists and family members of the cancer patient to ‘see’ only the prospective benefits and not consider safety issues. Great progress has also been made in the management of side effects and drug safety, thanks to increased awareness of pharmacovigilance among healthcare providers, but the phenomenon of under-reporting of ADR, mainly in oncology, still remains a very important challenge. Predominantly in the field of oncology, in fact, the clinician is inclined to see adverse effects as something known, therefore not to be reported, or a consequence related to the intrinsic efficacy of anticancer therapy. This – as reported in some reviews – is in any case in contradiction with what the regulations of health care systems in advanced countries provide, and with respect to the organization of international Pharmacovigilance networks, which allow monitoring of the risk-benefit relationship of drugs throughout the post-marketing life cycle [33,34].

In conclusion, we can therefore say that, like all branches of modern Medicine, oncology must increasingly move toward an approach that is not only ‘molecular’ but above all ‘patient-oriented.’ Despite all the modern diagnostic and interventional technologies, despite new insights in molecular mechanisms, and despite all the opportunities offered by information systems and data analysis, it will only be by considering the patient as a ‘unique’ individual with a ‘unique’ disease, and only by listening to his or her values and preferences at the time of treatment, will we have clinical care that is not only more effective, but also more humane and acceptable.

Article highlights

• New knowledge about the biomolecular mechanisms expressed by cancer cells, together with new pharmaceutical technologies, enable the production of innovative cancer drugs

• Some forms of cancer express mutations and molecular targets that are not relegated only to single organ disease, but are common to multiple cancer histologies

• Also, new diagnostic techniques, such as liquid biopsy, allow better cancer prevention and more precise identification of tumor markers

• Advanced Multidisciplinary Teams and the Molecular Tumor Committees are now being established to evaluate and choose what is the best diagnostic-therapeutic approach for each individual clinical case.

• This opens the door wide to precision oncology and personalized cancer therapy

• These great advances in oncology give humanity greater hope for the near future

Abbreviation /Acronim list

FDA	=	Food and Drug Administration
BCR-ABL	=	breakpoint cluster region / Abelson gene
HER2	=	Human Epidermal Growth Factor Receptor 2
CAR-T	=	Chimeric Antigens Receptor Cells-T
CRISPR	=	clustered regularly interspaced short palindromic repeats-associated protein
NTRK	=	neurotrophic tyrosine receptor kinase drugs
KRAS	=	Kirsten Rat Sarcoma viral oncogene homolog
ABPP	=	activity-based protein profiling
NSCLC	=	non-small cell lung cancer
CRC	=	colorectal cancer
EGFR	=	epidermal growth factor receptor
BTK	=	Bruton’s tyrosine kinase
NGS	=	next-generation sequencing
TME	=	tumor microenvironment
VAF	=	variant allele frequency
ICI	=	immune checkpoint inhibitor
PD-1	=	programmed death 1
PD-L1	=	programmed death ligand 1
TMB	=	tumor boarding / tumor molecular board
VUS	=	variant or uncertain significance
ESMO	=	European Society of Medical Oncology
ESCAT	=	Esmo Scale for Clinical Actionability of Molecular Targets

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This paper was funded by the Italian Ministry of Health, Ricerca Corrente.