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Targeted molecular therapy: the cancer paradigm
C H A P T E R
29 Targeted molecular therapy: the cancer paradigm Dyfud Mark Davies Department of Oncology, Singleton Hospital, Swansea, Wales, United Kingdom; Division of Cancer & Genetics, Institute of Medical Genetics, Cardiff University, Cardiff, United Kingdom
29.1 Introduction Significant strides have been made in our understanding of the genetics of cancer, beginning with the initial identification of oncogenes and tumor suppressor genes and culminating in the genomic profiling of tumors being routinely available in clinical practice. This progress has also led to some remarkable successes in molecularly targeted therapy, with the development of agents that target genetically driven tumor dependencies and vulnerabilities. This chapter will discuss the broad principles underlying targeted therapy and the difficulties that have been encountered, using illustrative examples from a range of cancer types.
29.2 Oncogene addiction The concept of “oncogene addiction,” where the survival of cancer cells is highly dependent on the activity of a single-gene product, underpins many molecularly targeted therapies [1]. The treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs) is a paradigm for this approach. Here, a single dysregulated protein is a fundamental driver of the disease; the protein has a “druggable” gain of function, and this protein is expressed in [2] leukemic cells but not in normal cells [3]. In CML the “druggable” target arises from a gene fusion. In 1960 a small derivative chromosome was noted to be consistently seen in the bone marrow cells of CML patients [4]. This chromosome was named the “Philadelphia” (Ph) chromosome, after the city in which it was first reported. The Ph chromosome was shown to arise from a reciprocal translocation between chromosomes 9 and 22: t(9;22)(q34;q11) [5]. This translocation results in the juxtaposition of the Abelson 1 (ABL1) gene, located on the long arm of chromosome 9, with a gene called BCR for breakpoint cluster region, on the long arm of chromosome 22 [2]. The normal ABL1 protein is a tyrosine kinase involved in a wide range of cellular processes, including regulation of cell growth, survival, and migration, interacting with several intracellular signaling pathways including the RAS/RAF/MEK pathway, the JAK2/STAT pathway, and the PI3K/mTOR pathway. The protein arising from the chimeric BCR ABL1 gene had tyrosine kinase activity, derived from ABL1 but deregulated as a consequence of the translocation. The first TKI to be used in the treatment of CML was imatinib [6]. This compound was initially identified from a chemical library screen of inhibitors of protein kinase A but was then subsequently shown to be an inhibitor of multiple kinases including ABL1. Early phase trials of imatinib in CML showed dramatic responses, and a subsequent phase III trial demonstrated the superiority of imatinib to interferon, the standard treatment for CML at that time [7]. The introduction of imatinib revolutionized the treatment of CML, leading to a dramatically better outcome for patients and providing the one of the first examples of a molecularly targeted approach to cancer treatment.
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Oncogene addiction has also been successfully exploited in the treatment of melanoma and nonsmall cell lung cancer (NSCLC). Over 80% of melanomas harbor a mutation in v-raf murine sarcoma viral oncogene homolog b1(BRAF) [8], a serine/threonine protein kinase that plays a crucial role in the mitogen-activated protein kinase (MAPK) signaling pathway, involved in cell growth, proliferation, survival, and differentiation. The most frequent BRAF mutation is a glutamic acid base substitution for valine at codon 600 (BRAF V600E). This mutation causes constitutive activation of the MAPK pathway, which, in turn, drives tumor progression. The first drug developed to target BRAF V600E specifically was vemurafenib, a small molecule reversible inhibitor with specific affinity for the adenosine tri-phosphate (ATP)-binding pocket of BRAF V600E [9]. Early phase I trials of vemurafenib in melanoma showed unprecedented clinical response rates (over 50%). BRIM-3, a phase III trial of vemurafenib in patients with BRAF V600 mutation-positive metastatic melanoma, reported an improved progression-free survival (PFS) and overall survival (OS) for vemurafenib compared with dacarbazine [10]. A second mutant BRAF inhibitor, dabrafenib has comparable efficacy, and both drugs have been approved for use in advanced malignant melanoma harboring a BRAF V600 mutation [11]. In many cases, NSCLC exhibits addiction to mutation within the epidermal growth factor receptor (EGFR) gene. These mutations are found in approximately 15% of cases in Western populations. In the early 2000s EGFR TKIs were initially trialed in an unselected NSCLC patient population based on the high proportion of NSCLCs known to express EGFR [12]. The results from these studies were disappointing, but the analysis of tumor tissue from responders demonstrated a link between certain EGFR kinase domain mutations and response [13]. In phase II studies, which enrolled patients with NSCLC harboring these mutations, treatment with EGFR TKIs resulted in an objective response rate of 65% 78% and PFS of 8.9 9.7 months [14]. The importance of molecular stratification was highlighted in the landmark IPASS trial that showed patients who had tumors with EGFR sensitizing mutations had significantly better PFS when treated with the TKI, gefitinib, compared with chemotherapy, whereas those with wild-type EGFR had significantly worse PFS with gefitinib [15]. The success of EGFR TKIs led to efforts to identify other actionable targets in NSCLC. Anaplastic lymphoma kinase (ALK) rearrangements occur in approximately 5% of NSCLCs [16]. In 95% of cases, ALK is fused to EML4 through a translocation, leading to activation of ALK. Crizotinib, a first-generation multitargeted TKI that inhibits ALK, improved PFS compared to chemotherapy in the first-line setting. More potent and selective ALK TKIs (ceritinib, alectinib, and brigatinib) have been subsequently developed and approved for treatment of ALK-rearranged NSCLC. ROS1 rearrangements occur in 1% 2% of NSCLC patients [17]. The most frequent fusion partner is CD74 (40% 45%), but a larger number of fusion partners have been identified in ROS1-rearranged NSCLC than ALKrearranged NSCLC. ROS1 is a tyrosine kinase that promotes survival and proliferation through downstream signaling via SHP-1/SHP-2, JAK/STAT, PI3K/AKT/mTOR, and MAPK/ERK pathways. ROS1 translocations lead to fusions of an intact ROS1 tyrosine kinase domain with partner genes. Crizotinib, which potently targets ROS1, has been approved for treatment of ROS1-rearranged NSCLC. Despite deep and sometimes durable responses of cancer to targeted drugs resistance invariably occurs, often within a year of starting treatment. Resistance to targeted agents can be classified as intrinsic, adaptive, or acquired [18,19]. Some tumors exhibit intrinsic resistance and do not respond to initial treatment. In other patients, there is an initial response, but adaptive resistance occurs when the tumor cells undergo changes in cell functioning, allowing survival during therapy. Acquired resistance can arise from selection for preexisting genetic alterations within a heterogeneous cancer cell population and the acquisition of new alterations during treatment. Resistance can develop by mechanisms that are either (1) “on target,” which is direct target reactivation mutations, or (2) “off target,” including activation of upstream effectors or downstream bypass signaling pathways and engagement of adaptive survival mechanisms. Restoring the biologic function of targeted oncoproteins is a critical mechanism by which cancer cells can overcome targeted therapy. Acquired resistance to imatinib in BCR ABL1 fusion-positive CML can be driven by secondary mutations in the drug-binding site, the ATP-binding pocket in the catalytic domain [3]. In NSCLC, EGFR mutations most commonly involve a deletion in exon 19 (19 Del) or substitution of leucine with arginine at codon 858 in exon 21 (L858R) [20]. EGFR exon 20 insertion mutations are the third most common type of EGFR mutation encountered in NSCLC. These mutations are present in 4% 9% of EGFR-mutant NSCLCs and confer intrinsic resistance to first-generation EGFR TKIs. The most common mechanism of acquired resistance is a secondary single base substitution in EGFR exon 20, resulting in a T790M mutation [21]. The third-generation EGFR inhibitor osimertinib was designed to target the T790M clone with maintained activity against the original exon 19del and L858R mutations. Osimertinib initially gained US Food and Drug Administration (FDA) approval for patients with metastatic EGFR T790M-mutant NSCLC after progressing on first- or second-generation EGFR TKI, but it subsequently gained approval in the first-line treatment of EGFR-mutant lung cancer. A phase III clinical
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trial comparing osimertinib to first-generation EGFR TKIs reported a significant improvement in PFS with osimertinib (18.9 vs 10.2 months) [22]. This demonstrates the benefit of the anticipation and targeting of common resistance mechanisms. The initial use of BRAF inhibitors in BRAF V600E-positive melanoma demonstrated that responses to the single agent were often dramatic but of short duration, typically lasting 6 8 months [23]. Multiple resistance mechanisms have been described, which often lead to the restoration of MAPK signaling, amplification of the mutated BRAF allele, and activating mutations in NRAS or NF1 [24 27]. Moreover, a considerable number of patients treated with single-agent BRAF inhibitors developed secondary skin cancers, including squamous cell carcinoma and keratoacanthoma, due to paradoxical activation of the MAPK pathway in BRAF wild-type cells [28]. In light of these findings, clinical trials were conducted treating patients with combined BRAF and MEK inhibition. A phase III study in BRAF V600-mutant melanoma patients showed the superiority of the BRAF inhibitor, dabrafenib, also the MEK inhibitor, trametinib, compared with dabrafenib alone. Patients in the combination arm had a median PFS of 11 months and OS of 25.1 months as compared with PFS of 8.8 months and OS of 18.7 months in those who only received dabrafenib [29]. Furthermore, the number of skin cancers was much lower in the combination arm compared with the dabrafenib only arm. Adaptive responses by cells are a means of developing resistance to a drug, which does not require additional genetic events [30]. For example, an adaptive upregulation of NF-κB pathway appears to be a mediator of resistance to EGFR TKI treatment in NSCLC [31]. Identification of such adaptive responses may allow the rational selection of drug combinations designed to inhibit such early prosurvival adaptive responses.
29.3 Synthetic lethality Synthetic lethality refers to a situation where the loss of either one of two genes has little or no effect, but the combination of the loss of both genes is lethal [32]. Within a cancer cell, a genetic alteration, such as a defect in a tumor suppressor gene, can cause a second gene to become essential for cell survival. Targeting of the product of this second gene might be lethal to cancer cells but relatively nontoxic to normal cells. The most clinically advanced use of synthetic lethality involves targeting tumors harboring BRCA1/2 loss-offunction with poly(ADP-ribose) polymerase (PARP) inhibitors [33]. PARP1 and PARP2 sense single-stranded DNA breaks and other types of DNA damage and upregulate the DNA-damage response (DDR) [34]. The tumor suppressor genes, BRCA1 and BRCA2, encode proteins involved in the repair of double-stranded DNA breaks by homologous recombination (HR) [35]. Heterozygous mutation of BRCA1 and BRCA2 in the germ line leads to an increased risk of breast, ovarian, prostate, and other cancers [36]. Tumors arising in individuals with a germline BRCA1 or BRCA2 mutation often have an acquired “second” hit, a somatic loss-of-function mutation, in the corresponding wild-type BRCA allele and therefore have defective HR. The synthetic lethality between PARP inhibition and loss of BRCA function is thought to be related to an increased number of double-strand DNA breaks or collapsed replication forks induced by PARP inhibition or PARP trapping on DNA [37]. Early phase trials demonstrated deep and durable responses in BRCA1- or BRCA2-deficient cancers to the PARP inhibitor olaparib. These findings have been confirmed in phase III trials, and PARP inhibitors are now in clinical use in BRAC1- or BRCA2-deficient breast or ovarian cancer [37]. Acquired resistance to PARP inhibitors has been observed by mechanisms, which restore HR functioning of such reversion mutation in BRCA1 or BRCA2 leading to or alterations in other components of the DDR [38]. A synthetic lethal relationship between PARP inhibitors and another component of the HR system might exist. HR deficiency results in a characteristic mutation signature, and identification of the presence of this signature might predict response to PARP inhibitors when a mutation in a specific DDR gene has not been identified [39].
29.4 Histology agnostic treatment Traditionally, cancer drug development has followed a well-established pathway with drugs being developed to treat tumor types defined by their presumed anatomical origin, for example, lung cancer or colorectal cancer. However, the recent approval of programed cell death-1 (PD-1) inhibitory antibodies, pembrolizumab and the tropomyosin receptor kinase (TRK) inhibitor, larotrectinib for use in a histological agnostic manner has established a new paradigm. These agents can be used in any tumor type, provided a biomarker predicting response is present.
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Microsatellite instability (MSI) is the biomarker that predicts response to pembrolizumab [40]. Microsatellites are repetitive sequences found throughout the human genome. These sequences are prone to accumulation of mutations, mainly due to slippage of polymerases during DNA synthesis. The mismatch repair (MMR) system is responsible for excising mismatched nucleotide that can result from polymerase misincorporation errors, following recombination and from chemical or physical damage to nucleotides. Mutations in the genes that encode MMR components can lead to defective MMR (dMMR) and an accumulation of mutations, which can be detected as MSI. Tumors with significant MSI are termed MSI high (MSI-H) [41]. MSI-H has been observed in multiple tumor types, including colorectal, gastric, endometrial, and ovarian. Approximately 15% of colorectal cancers exhibit MSI-H owing to either epigenetic silencing of MLH1 or a germline mutation in one of the MMR genes MLH1, MSH2, MSH6, or PMS2 [42]. When anti-PD-1 antibodies were first tested in patients with colorectal cancer, a response was only seen in MSI-H tumors [43]. It was hypothesized that dMMR led to an increase in tumor mutational burden, which could be recognized by the patient’s immune system following immune checkpoint blockade, leading to dramatic responses to treatment. This hypothesis was tested in some trials, which showed a very much higher response rate to PD-1 antibodies in patients with MSI-H tumors. In 2017 the FDA approved pembrolizumab for the treatment of adult and pediatric patients with unresectable or metastatic solid tumors that have been identified as having MSI-H or dMMR. This was the first FDA approval for cancer treatment based on a biomarker rather than the anatomical site of the tumor. Later in 2017 the FDA granted approval for nivolumab, another PD-1 inhibitory antibody, as a treatment for patients with MSI-H or dMMR metastatic colorectal cancer after progression on standard chemotherapy. The histology agnostic indication in MSI-H and dMMR tumors was approved after pembrolizumab had already obtained approvals for multiple, histologically defined indications, such melanoma and NSCLC and the safety profiles of the drug had been well established. The trials that lead to the approval of larotrectinib were explicitly done in a tissue agnostic fashion in patients with tumors with neurotrophic receptor tyrosine kinase (NTRK) fusions. The NTRK genes NTRK1, NTRK2, and NTRK3 encode the proteins TRKA, TRKB, and TRKC, respectively [44]. Recurrent chromosomal fusion events involving the carboxy-terminal kinase domain of TRK have been identified around 1% of all solid tumors that occur in children and adults. These fusions lead to overexpression of the chimeric protein and constitutively active downstream signaling. The efficacy of targeting TRK fusions in the broad range of cancer types has been tested using a basket trial approach. Here patients are selected by the molecular pathology of their tumors rather than by histological classification, based on the organ of origin. Patients whose tumors contain the qualifying genomic alterations can be entered, irrespective of cancer type. A basket trial of larotrectinib, a highly selective TRK inhibitor, included patients of any age and with any tumor type if a TRK fusion was present and reported a response rate of 75% [45]. In November 2018 the FDA granted accelerated approval to larotrectinib for adult and pediatric patients with solid tumors that have a NTRK gene fusion. This is a significant change of perspective in oncology drug development moving from seeking activity in a histology defined tumor group to taking a histology agnostic approach from the outset. This is likely to be a recurrent theme in the development of the next generation of cancer drugs, but the context in which a biomarker occurs will often remain of importance. For example, BRAF V600 mutations occur in a range of tumor types in addition to melanoma, but the sensitivity that these mutations confer to vemurafenib varies significantly between tumor type. BRAF V600 mutations are found in NSCLC, colorectal cancer, papillary thyroid cancer, cholangiocarcinoma, Langerhans cell histiocytosis, and Erdheim Chester disease. In some of these tumor types the incidence of mutations BRAF V600 mutations is around 50%, but in more than half the incidence of mutations is less than 5%. In NSCLC, BRAF V600 is found in approximately 3% of cases. A basket trial of vemurafenib in patients in the broad range of cancer types, which contained with V600 mutations, showed a high response rate in patients with NSCLC, Erdheim Chester disease, and Langerhans cell histiocytosis; but for some tumor types, such as colon cancer, the V600 mutations were associated with a low response rate [46].
29.5 Limitations of molecularly targeted therapy in cancer There has been undoubted success with molecular targeted therapy in cancer, but there are also ongoing constraints. A recent randomized trial (the SHIVA study) found equivalent outcomes between patients with a range of tumor types who were randomized to receive therapy matched to genomic abnormalities and for those who received conventional treatment [47]. The criticisms of this trial reflect the main challenges associated with targeted therapy. A key question is how to identify and classify the variants in genes that encode potential targets.
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Advances in sequencing technology have revealed the genomic landscape of multiple cancer types, but the understanding of the functional consequences of these genetic changes has lagged. Being able to discriminate between which variants do, or do not, confer susceptibility to a drug is the key to maximizing response rates. As the number of potential targets increases, then annotating the functional consequences of variants becomes a huge bioinformatic challenge. Robust evidence that matches a drug with a given predictive biomarker is also essential. The SHIVA trial was criticized for not optimizing the match between variant and drug, in part, due to the limited number of targeted drugs currently available. There is also an increasing appreciation of the influence of tumor heterogeneity on initial response and acquired resistance [48]. Tumor heterogeneity refers to the existence of cell subpopulations with distinct phenotypic characteristics, within (intratumor heterogeneity) and between tumors (intertumor heterogeneity). Cancers evolve from an ancestral cell, this evolution can be fueled by genomic instability and shaped by the selection pressures exerted by treatment. Tumor evolution can be depicted as a branching tree with early truncal mutations being shared by all sites of disease, even in patients with advanced cancers. Mutations arising over time are restricted to sets of subclones, giving rise to a branch-like pattern. For example, BRAF V600 mutations occur in dysplastic nevi before they progress to malignant melanoma and remain as an oncogenic driver during progression to malignant melanoma. Targeting of these truncal mutations might be expected to lead to an initial response. However, the selection pressure exerted may allow resistant subclonal populations to become the dominant cell population, manifesting as treatment failure and disease progression. But even where complex polyclonal resistance emerges, the resistance mechanisms may converge on specific pathways, suggesting this might be managed with drugs that target this pathway [49]. This highlights the importance of increasing the understanding of the evolution of resistance. Genetic profiling to date has been performed on a tissue sample obtained by a biopsy or resection. However, a tumor’s genetic makeup may vary from one part of the tumor to another, from the primary tumor compared to metastatic sites and may change over time. It is not feasible to obtain multiple biopsies from the tumor and all sites of spread nor to repeat biopsies multiple times throughout treatment. Cell-free circulating tumor DNA (ctDNA) consists of DNA fragments released from tumors into the circulation [50]. Multiple cells from within the primary tumor and from metastatic sites contribute to the ctDNA pool, which consequently reflects tumor genetic heterogeneity. As the ctDNA is obtained from blood samples, it is relatively easy to obtain multiple samples, so changes in the amount of ctDNA and the mutational profile can be captured over time. However, ctDNA is present in small amounts in the blood, and most circulating DNA is derived from normal cells. A fundamental challenge in the analysis of ctDNA is that mutated DNA fragments in the blood are present at low concentrations and mutations of interest may be present at very low allele frequency. Detection of mutations at such very low allele frequency requires deep sequencing coverage and error suppression techniques to distinguish true lowlevel variants from amplification and sequencing errors. Technical advances are allowing the development of strategies that will allow ctDNA analysis to become part of routine clinical practice and changes in a cancers’ genetic profile to be tracked during treatment.
29.6 Making common cancer rare By taking common diseases such as breast or colorectal cancer and stratifying them according to, often individually rare genetic variants, we have converted a small number of common conditions into a large number of individually rare conditions. This has a broad range of implications on areas such as trial design, on the level of the evidence required for regulatory approval and on reimbursement of treatment costs. Large randomized phase III trials may be of decreasing practicality if entry is based on molecularly stratified subtypes rather than broad diagnostic groups. Larger number of potential participants will need to be screened to identify those that are eligible, and treatment centers may need to offer a bigger portfolio of trials to allow the matching of patients with the appropriate experimental targeted drug. Lessons may need to be learned from the rare disease community, including the need for regulators to accept lower quality levels of evidence than that obtained from large phase III trials, which in some cases may be too difficult to conduct.
29.7 Conclusion Advances in genomics are driving new approaches in many areas of medicine, including the treatment of cancer. Genomics is leading to fundamental insights into the biology of cancer and a new taxonomy of cancer, which
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may complement or replace traditional histological classifications systems. This genomic taxonomy may be more informative in terms of predicting prognosis and response and will identify new potential targets. But achieving the full potential of this genetic-based approach requires an increased understanding of the functional consequences of genetic changes, so that therapies can be rationally designed and selected. In the foreseeable future, resistance will remain the key reason why most treatments ultimately fail. Developing strategies to overcome resistance may have as much impact on outcome as further increasing the number and range of targeted drugs. There have been some spectacular successes for molecularly targeted therapies in oncology. The pace of development is likely to increase further, and drugs for today’s intractable conditions will become tomorrow’s successes.
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29 Targeted molecular therapy: the cancer paradigm Dyfud Mark Davies Department of Oncology, Singleton Hospital, Swansea, Wales, United Kingdom; Division of Cancer & Genetics, Institute of Medical Genetics, Cardiff University, Cardiff, United Kingdom
29.1 Introduction Significant strides have been made in our understanding of the genetics of cancer, beginning with the initial identification of oncogenes and tumor suppressor genes and culminating in the genomic profiling of tumors being routinely available in clinical practice. This progress has also led to some remarkable successes in molecularly targeted therapy, with the development of agents that target genetically driven tumor dependencies and vulnerabilities. This chapter will discuss the broad principles underlying targeted therapy and the difficulties that have been encountered, using illustrative examples from a range of cancer types.
29.2 Oncogene addiction The concept of “oncogene addiction,” where the survival of cancer cells is highly dependent on the activity of a single-gene product, underpins many molecularly targeted therapies [1]. The treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs) is a paradigm for this approach. Here, a single dysregulated protein is a fundamental driver of the disease; the protein has a “druggable” gain of function, and this protein is expressed in [2] leukemic cells but not in normal cells [3]. In CML the “druggable” target arises from a gene fusion. In 1960 a small derivative chromosome was noted to be consistently seen in the bone marrow cells of CML patients [4]. This chromosome was named the “Philadelphia” (Ph) chromosome, after the city in which it was first reported. The Ph chromosome was shown to arise from a reciprocal translocation between chromosomes 9 and 22: t(9;22)(q34;q11) [5]. This translocation results in the juxtaposition of the Abelson 1 (ABL1) gene, located on the long arm of chromosome 9, with a gene called BCR for breakpoint cluster region, on the long arm of chromosome 22 [2]. The normal ABL1 protein is a tyrosine kinase involved in a wide range of cellular processes, including regulation of cell growth, survival, and migration, interacting with several intracellular signaling pathways including the RAS/RAF/MEK pathway, the JAK2/STAT pathway, and the PI3K/mTOR pathway. The protein arising from the chimeric BCR ABL1 gene had tyrosine kinase activity, derived from ABL1 but deregulated as a consequence of the translocation. The first TKI to be used in the treatment of CML was imatinib [6]. This compound was initially identified from a chemical library screen of inhibitors of protein kinase A but was then subsequently shown to be an inhibitor of multiple kinases including ABL1. Early phase trials of imatinib in CML showed dramatic responses, and a subsequent phase III trial demonstrated the superiority of imatinib to interferon, the standard treatment for CML at that time [7]. The introduction of imatinib revolutionized the treatment of CML, leading to a dramatically better outcome for patients and providing the one of the first examples of a molecularly targeted approach to cancer treatment.
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Oncogene addiction has also been successfully exploited in the treatment of melanoma and nonsmall cell lung cancer (NSCLC). Over 80% of melanomas harbor a mutation in v-raf murine sarcoma viral oncogene homolog b1(BRAF) [8], a serine/threonine protein kinase that plays a crucial role in the mitogen-activated protein kinase (MAPK) signaling pathway, involved in cell growth, proliferation, survival, and differentiation. The most frequent BRAF mutation is a glutamic acid base substitution for valine at codon 600 (BRAF V600E). This mutation causes constitutive activation of the MAPK pathway, which, in turn, drives tumor progression. The first drug developed to target BRAF V600E specifically was vemurafenib, a small molecule reversible inhibitor with specific affinity for the adenosine tri-phosphate (ATP)-binding pocket of BRAF V600E [9]. Early phase I trials of vemurafenib in melanoma showed unprecedented clinical response rates (over 50%). BRIM-3, a phase III trial of vemurafenib in patients with BRAF V600 mutation-positive metastatic melanoma, reported an improved progression-free survival (PFS) and overall survival (OS) for vemurafenib compared with dacarbazine [10]. A second mutant BRAF inhibitor, dabrafenib has comparable efficacy, and both drugs have been approved for use in advanced malignant melanoma harboring a BRAF V600 mutation [11]. In many cases, NSCLC exhibits addiction to mutation within the epidermal growth factor receptor (EGFR) gene. These mutations are found in approximately 15% of cases in Western populations. In the early 2000s EGFR TKIs were initially trialed in an unselected NSCLC patient population based on the high proportion of NSCLCs known to express EGFR [12]. The results from these studies were disappointing, but the analysis of tumor tissue from responders demonstrated a link between certain EGFR kinase domain mutations and response [13]. In phase II studies, which enrolled patients with NSCLC harboring these mutations, treatment with EGFR TKIs resulted in an objective response rate of 65% 78% and PFS of 8.9 9.7 months [14]. The importance of molecular stratification was highlighted in the landmark IPASS trial that showed patients who had tumors with EGFR sensitizing mutations had significantly better PFS when treated with the TKI, gefitinib, compared with chemotherapy, whereas those with wild-type EGFR had significantly worse PFS with gefitinib [15]. The success of EGFR TKIs led to efforts to identify other actionable targets in NSCLC. Anaplastic lymphoma kinase (ALK) rearrangements occur in approximately 5% of NSCLCs [16]. In 95% of cases, ALK is fused to EML4 through a translocation, leading to activation of ALK. Crizotinib, a first-generation multitargeted TKI that inhibits ALK, improved PFS compared to chemotherapy in the first-line setting. More potent and selective ALK TKIs (ceritinib, alectinib, and brigatinib) have been subsequently developed and approved for treatment of ALK-rearranged NSCLC. ROS1 rearrangements occur in 1% 2% of NSCLC patients [17]. The most frequent fusion partner is CD74 (40% 45%), but a larger number of fusion partners have been identified in ROS1-rearranged NSCLC than ALKrearranged NSCLC. ROS1 is a tyrosine kinase that promotes survival and proliferation through downstream signaling via SHP-1/SHP-2, JAK/STAT, PI3K/AKT/mTOR, and MAPK/ERK pathways. ROS1 translocations lead to fusions of an intact ROS1 tyrosine kinase domain with partner genes. Crizotinib, which potently targets ROS1, has been approved for treatment of ROS1-rearranged NSCLC. Despite deep and sometimes durable responses of cancer to targeted drugs resistance invariably occurs, often within a year of starting treatment. Resistance to targeted agents can be classified as intrinsic, adaptive, or acquired [18,19]. Some tumors exhibit intrinsic resistance and do not respond to initial treatment. In other patients, there is an initial response, but adaptive resistance occurs when the tumor cells undergo changes in cell functioning, allowing survival during therapy. Acquired resistance can arise from selection for preexisting genetic alterations within a heterogeneous cancer cell population and the acquisition of new alterations during treatment. Resistance can develop by mechanisms that are either (1) “on target,” which is direct target reactivation mutations, or (2) “off target,” including activation of upstream effectors or downstream bypass signaling pathways and engagement of adaptive survival mechanisms. Restoring the biologic function of targeted oncoproteins is a critical mechanism by which cancer cells can overcome targeted therapy. Acquired resistance to imatinib in BCR ABL1 fusion-positive CML can be driven by secondary mutations in the drug-binding site, the ATP-binding pocket in the catalytic domain [3]. In NSCLC, EGFR mutations most commonly involve a deletion in exon 19 (19 Del) or substitution of leucine with arginine at codon 858 in exon 21 (L858R) [20]. EGFR exon 20 insertion mutations are the third most common type of EGFR mutation encountered in NSCLC. These mutations are present in 4% 9% of EGFR-mutant NSCLCs and confer intrinsic resistance to first-generation EGFR TKIs. The most common mechanism of acquired resistance is a secondary single base substitution in EGFR exon 20, resulting in a T790M mutation [21]. The third-generation EGFR inhibitor osimertinib was designed to target the T790M clone with maintained activity against the original exon 19del and L858R mutations. Osimertinib initially gained US Food and Drug Administration (FDA) approval for patients with metastatic EGFR T790M-mutant NSCLC after progressing on first- or second-generation EGFR TKI, but it subsequently gained approval in the first-line treatment of EGFR-mutant lung cancer. A phase III clinical
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trial comparing osimertinib to first-generation EGFR TKIs reported a significant improvement in PFS with osimertinib (18.9 vs 10.2 months) [22]. This demonstrates the benefit of the anticipation and targeting of common resistance mechanisms. The initial use of BRAF inhibitors in BRAF V600E-positive melanoma demonstrated that responses to the single agent were often dramatic but of short duration, typically lasting 6 8 months [23]. Multiple resistance mechanisms have been described, which often lead to the restoration of MAPK signaling, amplification of the mutated BRAF allele, and activating mutations in NRAS or NF1 [24 27]. Moreover, a considerable number of patients treated with single-agent BRAF inhibitors developed secondary skin cancers, including squamous cell carcinoma and keratoacanthoma, due to paradoxical activation of the MAPK pathway in BRAF wild-type cells [28]. In light of these findings, clinical trials were conducted treating patients with combined BRAF and MEK inhibition. A phase III study in BRAF V600-mutant melanoma patients showed the superiority of the BRAF inhibitor, dabrafenib, also the MEK inhibitor, trametinib, compared with dabrafenib alone. Patients in the combination arm had a median PFS of 11 months and OS of 25.1 months as compared with PFS of 8.8 months and OS of 18.7 months in those who only received dabrafenib [29]. Furthermore, the number of skin cancers was much lower in the combination arm compared with the dabrafenib only arm. Adaptive responses by cells are a means of developing resistance to a drug, which does not require additional genetic events [30]. For example, an adaptive upregulation of NF-κB pathway appears to be a mediator of resistance to EGFR TKI treatment in NSCLC [31]. Identification of such adaptive responses may allow the rational selection of drug combinations designed to inhibit such early prosurvival adaptive responses.
29.3 Synthetic lethality Synthetic lethality refers to a situation where the loss of either one of two genes has little or no effect, but the combination of the loss of both genes is lethal [32]. Within a cancer cell, a genetic alteration, such as a defect in a tumor suppressor gene, can cause a second gene to become essential for cell survival. Targeting of the product of this second gene might be lethal to cancer cells but relatively nontoxic to normal cells. The most clinically advanced use of synthetic lethality involves targeting tumors harboring BRCA1/2 loss-offunction with poly(ADP-ribose) polymerase (PARP) inhibitors [33]. PARP1 and PARP2 sense single-stranded DNA breaks and other types of DNA damage and upregulate the DNA-damage response (DDR) [34]. The tumor suppressor genes, BRCA1 and BRCA2, encode proteins involved in the repair of double-stranded DNA breaks by homologous recombination (HR) [35]. Heterozygous mutation of BRCA1 and BRCA2 in the germ line leads to an increased risk of breast, ovarian, prostate, and other cancers [36]. Tumors arising in individuals with a germline BRCA1 or BRCA2 mutation often have an acquired “second” hit, a somatic loss-of-function mutation, in the corresponding wild-type BRCA allele and therefore have defective HR. The synthetic lethality between PARP inhibition and loss of BRCA function is thought to be related to an increased number of double-strand DNA breaks or collapsed replication forks induced by PARP inhibition or PARP trapping on DNA [37]. Early phase trials demonstrated deep and durable responses in BRCA1- or BRCA2-deficient cancers to the PARP inhibitor olaparib. These findings have been confirmed in phase III trials, and PARP inhibitors are now in clinical use in BRAC1- or BRCA2-deficient breast or ovarian cancer [37]. Acquired resistance to PARP inhibitors has been observed by mechanisms, which restore HR functioning of such reversion mutation in BRCA1 or BRCA2 leading to or alterations in other components of the DDR [38]. A synthetic lethal relationship between PARP inhibitors and another component of the HR system might exist. HR deficiency results in a characteristic mutation signature, and identification of the presence of this signature might predict response to PARP inhibitors when a mutation in a specific DDR gene has not been identified [39].
29.4 Histology agnostic treatment Traditionally, cancer drug development has followed a well-established pathway with drugs being developed to treat tumor types defined by their presumed anatomical origin, for example, lung cancer or colorectal cancer. However, the recent approval of programed cell death-1 (PD-1) inhibitory antibodies, pembrolizumab and the tropomyosin receptor kinase (TRK) inhibitor, larotrectinib for use in a histological agnostic manner has established a new paradigm. These agents can be used in any tumor type, provided a biomarker predicting response is present.
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Microsatellite instability (MSI) is the biomarker that predicts response to pembrolizumab [40]. Microsatellites are repetitive sequences found throughout the human genome. These sequences are prone to accumulation of mutations, mainly due to slippage of polymerases during DNA synthesis. The mismatch repair (MMR) system is responsible for excising mismatched nucleotide that can result from polymerase misincorporation errors, following recombination and from chemical or physical damage to nucleotides. Mutations in the genes that encode MMR components can lead to defective MMR (dMMR) and an accumulation of mutations, which can be detected as MSI. Tumors with significant MSI are termed MSI high (MSI-H) [41]. MSI-H has been observed in multiple tumor types, including colorectal, gastric, endometrial, and ovarian. Approximately 15% of colorectal cancers exhibit MSI-H owing to either epigenetic silencing of MLH1 or a germline mutation in one of the MMR genes MLH1, MSH2, MSH6, or PMS2 [42]. When anti-PD-1 antibodies were first tested in patients with colorectal cancer, a response was only seen in MSI-H tumors [43]. It was hypothesized that dMMR led to an increase in tumor mutational burden, which could be recognized by the patient’s immune system following immune checkpoint blockade, leading to dramatic responses to treatment. This hypothesis was tested in some trials, which showed a very much higher response rate to PD-1 antibodies in patients with MSI-H tumors. In 2017 the FDA approved pembrolizumab for the treatment of adult and pediatric patients with unresectable or metastatic solid tumors that have been identified as having MSI-H or dMMR. This was the first FDA approval for cancer treatment based on a biomarker rather than the anatomical site of the tumor. Later in 2017 the FDA granted approval for nivolumab, another PD-1 inhibitory antibody, as a treatment for patients with MSI-H or dMMR metastatic colorectal cancer after progression on standard chemotherapy. The histology agnostic indication in MSI-H and dMMR tumors was approved after pembrolizumab had already obtained approvals for multiple, histologically defined indications, such melanoma and NSCLC and the safety profiles of the drug had been well established. The trials that lead to the approval of larotrectinib were explicitly done in a tissue agnostic fashion in patients with tumors with neurotrophic receptor tyrosine kinase (NTRK) fusions. The NTRK genes NTRK1, NTRK2, and NTRK3 encode the proteins TRKA, TRKB, and TRKC, respectively [44]. Recurrent chromosomal fusion events involving the carboxy-terminal kinase domain of TRK have been identified around 1% of all solid tumors that occur in children and adults. These fusions lead to overexpression of the chimeric protein and constitutively active downstream signaling. The efficacy of targeting TRK fusions in the broad range of cancer types has been tested using a basket trial approach. Here patients are selected by the molecular pathology of their tumors rather than by histological classification, based on the organ of origin. Patients whose tumors contain the qualifying genomic alterations can be entered, irrespective of cancer type. A basket trial of larotrectinib, a highly selective TRK inhibitor, included patients of any age and with any tumor type if a TRK fusion was present and reported a response rate of 75% [45]. In November 2018 the FDA granted accelerated approval to larotrectinib for adult and pediatric patients with solid tumors that have a NTRK gene fusion. This is a significant change of perspective in oncology drug development moving from seeking activity in a histology defined tumor group to taking a histology agnostic approach from the outset. This is likely to be a recurrent theme in the development of the next generation of cancer drugs, but the context in which a biomarker occurs will often remain of importance. For example, BRAF V600 mutations occur in a range of tumor types in addition to melanoma, but the sensitivity that these mutations confer to vemurafenib varies significantly between tumor type. BRAF V600 mutations are found in NSCLC, colorectal cancer, papillary thyroid cancer, cholangiocarcinoma, Langerhans cell histiocytosis, and Erdheim Chester disease. In some of these tumor types the incidence of mutations BRAF V600 mutations is around 50%, but in more than half the incidence of mutations is less than 5%. In NSCLC, BRAF V600 is found in approximately 3% of cases. A basket trial of vemurafenib in patients in the broad range of cancer types, which contained with V600 mutations, showed a high response rate in patients with NSCLC, Erdheim Chester disease, and Langerhans cell histiocytosis; but for some tumor types, such as colon cancer, the V600 mutations were associated with a low response rate [46].
29.5 Limitations of molecularly targeted therapy in cancer There has been undoubted success with molecular targeted therapy in cancer, but there are also ongoing constraints. A recent randomized trial (the SHIVA study) found equivalent outcomes between patients with a range of tumor types who were randomized to receive therapy matched to genomic abnormalities and for those who received conventional treatment [47]. The criticisms of this trial reflect the main challenges associated with targeted therapy. A key question is how to identify and classify the variants in genes that encode potential targets.
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Advances in sequencing technology have revealed the genomic landscape of multiple cancer types, but the understanding of the functional consequences of these genetic changes has lagged. Being able to discriminate between which variants do, or do not, confer susceptibility to a drug is the key to maximizing response rates. As the number of potential targets increases, then annotating the functional consequences of variants becomes a huge bioinformatic challenge. Robust evidence that matches a drug with a given predictive biomarker is also essential. The SHIVA trial was criticized for not optimizing the match between variant and drug, in part, due to the limited number of targeted drugs currently available. There is also an increasing appreciation of the influence of tumor heterogeneity on initial response and acquired resistance [48]. Tumor heterogeneity refers to the existence of cell subpopulations with distinct phenotypic characteristics, within (intratumor heterogeneity) and between tumors (intertumor heterogeneity). Cancers evolve from an ancestral cell, this evolution can be fueled by genomic instability and shaped by the selection pressures exerted by treatment. Tumor evolution can be depicted as a branching tree with early truncal mutations being shared by all sites of disease, even in patients with advanced cancers. Mutations arising over time are restricted to sets of subclones, giving rise to a branch-like pattern. For example, BRAF V600 mutations occur in dysplastic nevi before they progress to malignant melanoma and remain as an oncogenic driver during progression to malignant melanoma. Targeting of these truncal mutations might be expected to lead to an initial response. However, the selection pressure exerted may allow resistant subclonal populations to become the dominant cell population, manifesting as treatment failure and disease progression. But even where complex polyclonal resistance emerges, the resistance mechanisms may converge on specific pathways, suggesting this might be managed with drugs that target this pathway [49]. This highlights the importance of increasing the understanding of the evolution of resistance. Genetic profiling to date has been performed on a tissue sample obtained by a biopsy or resection. However, a tumor’s genetic makeup may vary from one part of the tumor to another, from the primary tumor compared to metastatic sites and may change over time. It is not feasible to obtain multiple biopsies from the tumor and all sites of spread nor to repeat biopsies multiple times throughout treatment. Cell-free circulating tumor DNA (ctDNA) consists of DNA fragments released from tumors into the circulation [50]. Multiple cells from within the primary tumor and from metastatic sites contribute to the ctDNA pool, which consequently reflects tumor genetic heterogeneity. As the ctDNA is obtained from blood samples, it is relatively easy to obtain multiple samples, so changes in the amount of ctDNA and the mutational profile can be captured over time. However, ctDNA is present in small amounts in the blood, and most circulating DNA is derived from normal cells. A fundamental challenge in the analysis of ctDNA is that mutated DNA fragments in the blood are present at low concentrations and mutations of interest may be present at very low allele frequency. Detection of mutations at such very low allele frequency requires deep sequencing coverage and error suppression techniques to distinguish true lowlevel variants from amplification and sequencing errors. Technical advances are allowing the development of strategies that will allow ctDNA analysis to become part of routine clinical practice and changes in a cancers’ genetic profile to be tracked during treatment.
29.6 Making common cancer rare By taking common diseases such as breast or colorectal cancer and stratifying them according to, often individually rare genetic variants, we have converted a small number of common conditions into a large number of individually rare conditions. This has a broad range of implications on areas such as trial design, on the level of the evidence required for regulatory approval and on reimbursement of treatment costs. Large randomized phase III trials may be of decreasing practicality if entry is based on molecularly stratified subtypes rather than broad diagnostic groups. Larger number of potential participants will need to be screened to identify those that are eligible, and treatment centers may need to offer a bigger portfolio of trials to allow the matching of patients with the appropriate experimental targeted drug. Lessons may need to be learned from the rare disease community, including the need for regulators to accept lower quality levels of evidence than that obtained from large phase III trials, which in some cases may be too difficult to conduct.
29.7 Conclusion Advances in genomics are driving new approaches in many areas of medicine, including the treatment of cancer. Genomics is leading to fundamental insights into the biology of cancer and a new taxonomy of cancer, which
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may complement or replace traditional histological classifications systems. This genomic taxonomy may be more informative in terms of predicting prognosis and response and will identify new potential targets. But achieving the full potential of this genetic-based approach requires an increased understanding of the functional consequences of genetic changes, so that therapies can be rationally designed and selected. In the foreseeable future, resistance will remain the key reason why most treatments ultimately fail. Developing strategies to overcome resistance may have as much impact on outcome as further increasing the number and range of targeted drugs. There have been some spectacular successes for molecularly targeted therapies in oncology. The pace of development is likely to increase further, and drugs for today’s intractable conditions will become tomorrow’s successes.
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