Current therapies and their limitations in changing the natural course of disease
There are currently few FDA-approved treatments for metastatic melanoma 2, 3 which include biological agents and conventional chemotherapeutic agents. However, none of the currently available FDA-approved therapies clearly alter the natural history of the disease for the population as a whole. Interferon-a2b (IFN-a2b) is the most widely used adjuvant immunotherapy for stage III melanoma in the USA, although its efficacy is limited. In large-scale, randomized, observation-controlled trials, interferon-a2b has demonstrated a 10–20% improvement in relapse-free survival, but with no clear effect on melanoma-related mortality 2, 4, 5. Interleukin-2 (IL-2) is approved for stage IV melanoma, based on its ability to effect durable responses in 10–20% of patients, and is associated with severe, although short-lived, toxicities 6–8. Among conventional cytotoxic chemotherapies, the alkylating agent dacarbazine is the only FDA-approved agent. Responses are seen in approximately 5–10% of patients and are generally short-lived. Other related chemotherapeutic agents, including carmustine and temozolomide, and agents from other classes, such as taxanes and platinum-analogues, have similar efficacy profiles in the metastatic setting 2, 9, 10. The identification of signalling pathways critical to melanoma initiation and progression has opened up exciting new areas for the investigation of novel melanoma treatments. On the basis of these principles, several new targeted agents are currently being developed and tested alone or in combination with conventional chemotherapies.
Key oncogenic pathways and targeted therapies
Insights into melanoma at the molecular level have suggested a stepwise progression of mutations that transforms a normal melanocyte or naevus into a primary and then metastatic melanoma 11, 12. Multiple tumour-promoting events, including activation of oncogenes and inactivation of tumour suppressor genes, lead melanocytes through this transition. What follows will be an overview of selected pathways and oncogenes of demonstrated importance in the pathogenesis of melanoma, as well as targeted agents and other therapeutic approaches based on our current understanding of this disease.
RAS/mitogen-activated protein (MAP) kinase pathway
One growth factor pathway that has garnered considerable attention in the last few years is the RAS–RAF–MAPK–ERK signalling cascade. Oncogenic lesions introduce changes in the primary sequence of RAS so that the protein is constitutively active. Much of the attention surrounding this pathway in human melanoma focuses on the fact that, in virtually all cases, there is an alteration at some level in the RAS signalling cascade 13. Specifically, NRAS and BRAF mutations occur in about 80% of the most common types of melanoma 14. Their validation as therapeutic targets in melanoma in preclinical models has led to great enthusiasm to pursue drugs that selectively target this pathway 15.
The first agents to target this pathway were the RAS farnesyl transferase inhibitors (eg tipifarnib or R115777). It is believed that these farnesyl transferase inhibitors (FTIs) block post-translational modification of the RAS proteins, preventing their membrane localization and hence their activity 16. This agent was evaluated in a single-agent, single-arm phase II trial among patients with metastatic melanoma 17. The lack of responses among the first 14 patients led to early closure of the trial. However, these patients were unselected with respect to the presence or absence of NRAS mutations in their tumours. Given the approximate 15% prevalence of NRAS mutations across all types of melanoma, it is probable that only one or two patients in the trial harboured a NRAS mutation. Thus, a case could be made for further investigation of FTIs in patients who are determined prospectively to harbour NRAS mutations, although overall enthusiasm for FTIs as successful agents for RAS-family targeting remains reserved, at best. There is some evidence that RAS antagonism might enhance the effect of chemotherapies, although this approach has not been attempted clinically in melanoma 18, in large part because of the lack of effective RAS small molecule antagonists.
BRAF and other RAF family members
Given the relative frequency of BRAF mutations in the most common types of melanoma, much attention has been focused on the BRAF gene and its role in this disease. The BRAF gene encodes a protein belonging to the raf family of serine/threonine protein kinases, and lies downstream of RAS. BRAF is highly expressed in neuronal tissue and melanocytes, and is not likely an inherited cancer predisposition gene 19. Individuals with germline BRAF mutations develop cardio-facio-cutaneous syndrome, which is not associated with an increased risk of cancer or melanoma 20, 21.
Activating mutations in BRAF are found in approximately half of all melanomas, with the significant majority arising in skin with intermittent sun exposure, as compared with melanomas from chronically sun-exposed areas, acral, mucosal and uveal melanomas, suggesting an inverse association with high levels of cumulative sun exposure 22. The most common BRAF mutation (approximately 90% in clinical pathology samples) is the T1799A point mutation, in which a T → A transversion converts glutamic acid for valine at the 600 position of the amino acid sequence, BRAF, which results in the protein taking on a constitutive active configuration 23.
The finding that BRAF mutations are common in both benign and dysplastic naevi argues that such mutations are not sufficient for malignant transformation of melanocytes 24, 25. However, it does suggest a role in the earliest stages of neoplasia. This was first demonstrated in transgenic zebrafish, in which introduction of melanocyte-targeted BRAF resulted in fish-naevi which were stably growth arrested 26. In the presence of germline p53 mutation, the same BRAF transgene produced invasive melanomas, following a period of latency, and an incomplete penetrance. Similar phenotypes have been observed in BRAF knock-in mice in which BRAF activation alone triggers naevi, whereas combinatorial activation of BRAF plus deletion of either PTEN or p16/Ink4a results in melanoma 27, 28. The same mouse system has also begun to be used successfully for examining targeted therapies in genetically defined melanomas.
As demonstrated by Bevona et al, naevi are fundamentally growth-arrested and only rarely progress to melanoma 29. The introduction of a BRAF mutation into melanocytes has been shown to induce senescence and cell-cycle arrest. Animal studies have demonstrated that the coincidental mutation of p16 with BRAF permits transformation and the coincidental deletion of PTEN or p53 with BRAF results in the formation of invasive and metastatic melanoma in animal models 26, 30. The concomitant activation of BRAF and deletion of INK4A leads to invasive melanoma in mice. These findings support the human naevus observation that BRAF lesion, on its own, is not sufficient for malignant transformation of melanocytes.
More recent studies in mice have demonstrated that inducible knock-in of BRAF produces benign melanocytic neoplasms resembling naevi. In one case 27 the lesions were biologically stable, whereas in another 28 invasive melanomas formed after an extended latency period of many months. The differences between these models that are responsible for the distinct phenotypes are unknown, but the body of data still strongly suggests that BRAF mutation alone is primarily associated with naevus formation.
There remains uncertainty surrounding the factors driving BRAF mutation, specifically the role of ultraviolet (UV) exposure. However, one might theorize that melanocytes of persons who develop melanomas on intermittently sun-exposed skin have an inherent increased susceptibility to sun/UV exposure, leading to higher probability of acquiring BRAF mutation or development of proliferation in the setting of such a mutation. Epidemiological and animal studies suggest that, in this population, there may be a window of vulnerability to exposure to UV light early in life 31, 32. Arguing against this is that the T → A transformation is not classically associated with UV damage 33, and that genes such as BRAF and N-RAS that are commonly mutated in melanoma do not show typical UV ‘fingerprint’ mutations 14, 34, 35, particularly those associated with UVB wavelengths. The gene–environment interaction between UV exposure and BRAF mutation is further complicated by evidence of gene–gene interaction—that MC1R phenotype may modify the association between BRAF mutation, naevus burden and melanoma risk 36–38.
Given the prevalence of BRAF mutations in melanoma, there has been intense interest in selective BRAF inhibitors (SBIs). One of the earlier BRAF inhibitors investigated was the multi-kinase inhibitor sorafenib, which targets BRAF, CRAF and the VEGF and PDGF receptor tyrosine kinases (RTKs) 39. Sorafenib is a relatively non-selective BRAF inhibitor and it showed minimal activity against melanoma alone but showed greater activity when combined with chemotherapeutic agents, including carboplatin and paclitaxel or temozolomide 40, 41. In contrast, sorafenib exhibited significant clinical activity against renal cell carcinoma, where it is presumed to act against the VEGF receptor. Sorafenib's failure as monotherapy suggested the need for a more potent and targeted approach.
More selective BRAF inhibitors were subsequently developed, based upon a different selectivity model. The pharmacological strategy focused upon exploiting the fact that a large majority of BRAF-mutated melanomas exhibit the identical mutant allele (V600E). PLX4032 was developed with the goal of producing mutant-selective BRAF inhibition and it is thought to suppress BRAF with approximately 30-fold selectivity relative to wild-type BRAF kinase. This drug has completed phase I study, where it was reported to produce approximately 70% objective response rates in patients with metastatic melanoma containing BRAF42. The drug has proceeded to phase II/III trials. A close analogue of this compound was evaluated in preclinical models and demonstrated selective efficacy in BRAF models 43, 44.
There is clear evidence of single-agent activity with PLX4032 in melanoma patients with BRAF mutations, with an objective response rate of > 50% among those treated at the higher doses in the phase I trial 42. These remarkable preliminary results alone suggest that potent inhibition of BRAF is sufficient to induce cell death in these tumours. Furthermore, this result indicates that matching the relevant oncogene with a potent and selective antagonist can alter the viability and natural history of metastatic melanoma. Of note, approximately 20% of patients developed keratoacanthomas early in the course of therapy with PLX4032. The growth of these lesions in the setting of therapy could relate to the effect of selective BRAF inhibitors on the activity of the MAP kinase pathway in cells that lack BRAF mutations, although this hypothesis has not been investigated 45. There are several additional BRAF inhibitors in clinical development, including some that are similarly selective to PLX4032 46.
Besides BRAF, CRAF may be another target for therapy development for melanoma. CRAF is downstream to BRAF in mammalian cells and is required for melanoma proliferation 47, 48. Furthermore, CRAF, not BRAF, is noted to mediate MEK activation in RAS-mutated melanomas 49, and the ‘low-activity’ BRAF mutants (not V600 position) appear to activate CRAF as their primary mechanism of oncogenic signalling. There are ongoing trials with CRAF inhibitors focused on patients with these relatively uncommon BRAF mutation variants 49, 50.
MEK kinases lie immediately downstream of BRAF and they are a major mediator of oncogenic BRAF-induced melanoma formation. They have been considered a potential point of intervention in the MAP kinase pathway in BRAF- and NRAS-mutant melanoma. Melanoma cells with BRAF mutations appear to be more sensitive to MEK inhibitors than cells in which RAS is mutated 51, 52. Several MEK inhibitors have been tested in clinical trials in patients with advanced melanoma. PD0325901 was evaluated in a phase I trial which included extensive investigation of target inhibition in serial tumour biopsies. Significant reduction in ERK phosphorylation was noted in patients with metastatic melanoma. Of the 27 melanoma patients in the trial, two experienced an objective response (one with a BRAF mutation and the other with a NRAS mutation) while an additional five patients showed disease stabilization 53. These results demonstrated proof of concept that MEK inhibition could be an efficacious point of intervention. Phase II trials of this drug in non-small cell lung cancer, however, were suspended because of visual disturbance and limited activity 54.
In a phase I trial, AZD6244 showed only moderate effect among a very small group of patients with metastatic melanoma harbouring BRAF mutations 55. In a subsequent phase II trial, 12% of patients whose tumour harboured BRAF experienced significant, but incomplete, regression of tumour with AZD6244 53. This limited efficacy could be due to suboptimal target inhibition at clinically tolerable doses with the schedule of administration used, or that MEK inhibition alone does not fully antagonize the signalling effects of mutant BRAF. Of note, the anthrax lethal toxin, which selectively degrades and inactivates MEK1 and MEK2, is also being tested in melanoma clinical trials 15, 56.
c-KIT and imatinib
Melanoma cells variably express a number of growth factor receptors, including EGFR, PDGFR and KIT. KIT is an RTK that plays an important role in proliferation, development and survival of melanocytes, haematopoietic cells and germ cells and is ubiquitously expressed in mature melanocytes 57. Pathogenesis-activating KIT mutations have been observed in a variety of tumours, including gastrointestinal stromal tumours (GISTs), seminomas, a subset of thymic carcinomas and certain acute myeloid leukaemias 58.
The c-KIT RTK has been shown to be amplified or mutated in a subset of melanomas, specifically those that develop on body sites with little UV exposure, such as acral and mucosal melanomas 57, making this subgroup vulnerable to c-KIT inhibition. Mutations in c-KIT were also observed in melanomas arising within the context of chronically sun-damaged skin. Vulnerability of Kit-mutant melanomas to Kit-targeted small molecule kinase inhibitors has been supported by several case reports 59, 60 and subsequently confirmed in other melanoma cell lines harbouring an activating c-KIT mutation 61. The most common melanoma KIT mutation is exon 11 L576P, found in approximately one-third of cases 62. Activation of this tyrosine kinase results in the stimulation of the MAPK and PI3K–AKT pathways, producing both proliferative and survival advantages 63.
A significant proportion of mutations found in melanoma occur in the juxtamembrane region of c-KIT58. This is a well-studied KIT mutation that responds to therapy with imatinib, a therapeutic TKI with known efficacy in GIST. In a recent trial, four patients with acral and mucosal melanomas and documented KIT mutations were treated with either imatinib or sorafenib and all showed tumour regression. However, all cases also showed increased rates of CNS progression, thought to be due to limited penetration of the drug into the brain 64.
The beneficial effects of c-KIT inhibition by single-agent imatinib may be attributed to its inhibition of multiple signalling pathways, such as MAPK, PI3K–AKT and JAK–STAT 65. Several multicentre phase II trials are currently under way to evaluate KIT-targeted agents, including imatinib, sunitinib, nilotinib and dasatanib, in melanoma patients whose tumours harbour KIT aberrations. The current clinical trials will begin to assess whether differential sensitivities amongst the various KIT targeting agents exist or correlate with particular KIT mutations. Also to be determined are the relative clinical response implications for tumours with activating KIT mutations, as compared to tumours without mutations but with KIT amplification, as compared to tumours that possess both mutations and amplification. Improved understanding of the genomic mechanism for KIT amplification may improve our ability to target this subset of tumours.
Finally, as with BRAF-targeted therapies, understanding the potential mechanisms of resistance to KIT-targeted therapy is an active area of investigation. In GISTs, the acquisition of additional KIT mutations by tumours is a common mechanism of KIT-targeted drug resistance. Whether this is true for KIT-mutant melanomas, or whether additional mechanisms such as amplification or alternative signalling pathways are involved, remains to be determined. There remains great enthusiasm for targeting KIT in mucosal, acral and chronically sun-damaged melanoma subtypes, as KIT is a proven oncogene with validated inhibitors.
The PTEN gene, located on chromosome 10, encodes a tumour suppressor protein and has also gained considerable attention as our understanding of melanoma pathogenesis has increased 66. Mutations in PTEN are found in 10–20% of primary melanomas 67 and have also been associated with thyroid, breast and prostate cancer. The PTEN protein has lipid phosphatase activity, which prevents formation of intracellular signalling molecules, required for conformational change activating the AKT protein kinase family 68. The loss of PTEN in a significant subset of melanomas, particularly some of those with BRAF mutations, eliminates a mechanism of negative regulation on Akt and downstream components of the PI3 kinase pathway 69. Loss of PTEN may contribute a vital hit in the formation of invasive melanomas 70.
The independent therapeutic value of inhibiting the PI3 kinase pathway in melanoma has not been well-established, but a body of preclinical evidence supports this pathway as an important adjunct to MAP kinase pathway-targeted therapy. Recent studies have demonstrated that activation of the AKT pathway suppresses apoptosis 71, 72 through phosphorylation and inactivation of pro-apoptotic proteins 73–75. DNA copy gain of the AKT3 locus is found in 40–60% of melanomas and results in activation of AKT. Interestingly, AKT3 expression correlates with melanoma progression 76. Loss of PTEN function allows proliferation of the AKT pathway, which contributes to aberrant cell growth and escape from apoptosis, and evidence suggests that there is cooperation between loss of PTEN and BRAF mutations 77.
mTOR is a kinase that lies downstream of AKT signalling and can regulate the activity of AKT through a direct feedback mechanism. It can either positively or negatively impact AKT activity, depending on the composition of the mTOR signalling complex. mTOR forms at least two signalling complexes: (a) an active mTORC1 complex that suppresses AKT signalling; and (b) an mTORC2 complex that stimulates AKT signalling through phosphorylation. A number of small molecule inhibitors are available that target mTORC1 but not mTORC2 signalling, all of which are derivatives of rapamycin, which is FDA-approved for use as an immunosuppressive agent after solid organ transplantation. There is evidence that mTOR signalling is active in melanoma and that rapamycin has growth-inhibitory effects across a panel of human melanoma cell lines 78. However, this did not translate into clinical evidence of anti-tumour activity in a phase II trial in melanoma patients 79. There does appear to be synergy between sorafenib/MEK inhibitors and rapamycin in preclinical models of melanoma 78, 80, 81.
Overlap of somatic genetic changes in melanoma
None of the oncogenes or tumour suppressor genes identified in melanoma are thought to stand alone in the pathogenesis of the disease. In fact, there are relationships between somatic genetic changes in melanoma. For instance, NRAS, as mentioned above, activates Raf kinases in response to growth factor receptor activation and harbours activating mutations in 15–20% of melanomas 82, 83. As BRAF mutations confer RAS-independent activation of the MAP kinase pathway, it is not surprising that NRAS and BRAF mutations tend to be mutually exclusive 77, 84.
The loss of the p16 tumour suppressor is a relatively frequent event in melanoma 85, and there is significant overlap with BRAF mutation 86. PTEN mutations and deletion have been described in a minority of melanomas and, as noted above, these events appear to coincide with BRAF mutation 22, 77, 86. It has been suggested that the coincidence of BRAF mutation and PTEN loss speaks to the importance of the two Ras-effector pathways in melanoma, both of which would presumably reside downstream of Ras mutations. Less common genetic alterations, such as cyclin D amplification and CDK4 mutation, have also been identified in association with BRAF mutation 22, 87, 88. Other mechanisms of diminished tumour suppressor activity (such as gene silencing of p16/Ink4a) are also thought to operate in a fraction of melanomas.
It is clear that single-agent approaches in melanoma are infrequently capable of achieving cure. One explanation is that melanoma cells harbour compensatory or redundant signalling responses through which melanoma reconstructs itself after other growth pathways are disrupted. For melanomas that initially respond to a targeted agent but subsequently progress, it is likely that selection for resistant clones has permitted escape from the targeted drug. Therefore, one strategy is to use agents that target several pathways together.
Most preclinical attention has focused on the combined inhibition of signal transduction pathways, although another strategy is to combine targeted therapy agents with chemotherapy. It has been noted that sorafenib may enhance the activity of carboplatin/paclitaxel and dacarbazine 89. Treatment of cancer cells with paclitaxel activates the MAPK pathway, and inhibition of MEK in combination with paclitaxel leads to additive effects on cell growth inhibition and apoptosis induction 90.
In addition, the MEK inhibitor AZD6244 appears to enhance the pro-apoptotic effects of docetaxel in human melanoma cells 91. These combinations are now undergoing evaluation for a number of solid tumours in phase I and II clinical trials. Combination of sorafenib with carboplatin and paclitaxel is the focus of a phase III clinical trial for first-line treatment of patients with advanced melanoma 92. A combination trial which tests Glaxo-Smith-Kline's mutant-selective BRAF inhibitor plus its MEK inhibitor has recently opened, and is based upon the same strategy of blocking clones which may resist targeted BRAF suppression alone.
Other therapeutic approaches
Targeting downstream mediators of pathogenic pathways
While the concept of targeted inhibition of mutated kinase oncogenes is already validated in multiple settings of oncology, there are clearly limits to the efficacy of this approach. One limitation is that not every cancer may harbour an activated ‘druggable’ oncogene. Another is the resistance mechanisms may almost inaevitably arise, preventing the acquisition of stable cures of advanced disease. An approach to broaden the targeted-therapy portfolio is to identify and validate downstream mediators of mutated oncogenes. Examples of this strategy have already been shown to offer clinical benefit and will be briefly mentioned.
One contributing factor to tumour cell transformation is dysregulated apoptosis pathways 93. A number of anti-apoptotic proteins, including Bcl-2, Bcl-xL and X-linked inhibitor of apoptosis (XIAP), are over-expressed in melanoma and may confer resistance to chemotherapy 94, 95. Clinically, oblimersen is an antisense agent targeted to mitochondrial Bcl-2, and a phase I/II study suggested that it can sensitize melanoma cells to dacarbazine-based chemotherapy 96. In a randomized phase III clinical trial, oblimersen failed to improve overall survival in the entire cohort of metastatic melanoma patients accrued. A retrospective subset analysis suggested that those with low serum lactate dehydrogenase levels may have achieved some benefit 2, 97.
Angiogenesis is known to be necessary for the growth of primary tumours and metastases, and melanoma does not appear to be an exception 98, 99. Oncogenic BRAF mutations induce angiogenesis 100 and melanoma metastases tend to be very vascular 101. Angiogenic factors such as VEGF, IL-8, basic FGF and PDGF are released by melanoma and host cells into the tumour microenvironment 102.
VEGF has been implicated as the primary promoter of angiogenesis in renal cell, colorectal, non-small cell lung and breast cancers. However, in melanoma VEGF is only expressed in a minority of primary or metastatic tumours 103. bFGF expression is not associated with increasing stage of disease and the presence of bFGF in tumour blood vessels is paradoxically associated with good prognosis 104, 105. On the other hand, PDGF and IL-8 are over-expressed in the majority of melanomas, are associated with invasiveness and correlate with poor prognosis 102, 106–109.
The VEGF ligand-targeted monoclonal antibody bevacizumab and VEGF receptor inhibitors sorafenib and axitinib have anti-tumour activity in mice bearing human melanoma xenografts. However, clinical activity with single-agent becacizumab or sorafenib has been limited, and only axitinib has demonstrated appreciable single-agent activity. While anti-angiogenic agents do not appear to have a role as single-agent therapy, they may be useful in combination therapy with conventional chemotherapies, as seen in some cancer types 110, 111, although sorafenib did not significantly augment the efficacy of cytotoxic chemotherapy in randomized trials 89, 92.
The investigation of cancer immune therapies has been a major focus in melanoma, as it is a cancer that demonstrates evidence of immunogenicity, which includes evidence of spontaneous regression of primary tumours, pathological association of tumour-infiltrating lymphocytes and detection of antigen-specific cytotoxic T cells and antibodies in melanoma patients. Approaches that have shown some activity in patients with advanced melanoma include the use of recombinant biologics (interleukin-2, interferon), adaptive transfer of in vitro expanded tumour-infiltrating lymphocytes or lymphocytes with enforced expression of T cell receptors against melanocytic antigens, various vaccines, cytokines or immune checkpoint antagonists, which include blocking antibody against CTLA4 or PD1. The anti-CTLA4 antibody (ipilimumab) was recently shown to produce significant clinical benefit in patients 112 and represents a breakthrough in successful ‘targeted approaches’ to immune modulation for cancer. An exhaustive discussion of melanoma immunotherapy is beyond the scope of this review, but has been discussed in detail in a number of recent reviews on the topic 113–116.
A new era in melanoma care
While targeted therapies, specifically those directed against BRAF and mutant KIT, have produced major clinical responses, the conversion of transient remissions to stable cures remains a vital challenge. A next step into this new era in melanoma care is a push to identify mechanisms of resistance and strategies to overcome them. One such line of focus is the controversial role of melanoma stem cells, or melanoma-initiating cells. Several recent studies have suggested important roles for subpopulations of melanoma cells which may express specific identifiable markers and exhibit particularly enhanced capacity to produce distinct melanoma lesions (usually in mouse xenograft studies), in which this ‘stem cell’ compartment again represents only a small subset of the total melanoma population. The melanoma stem cell field has engendered considerable controversy because a seminal study by Sean Morrison and colleagues demonstrated altered xenografting propensity of melanomas, which was based upon the degree of immunodeficiency in the recipient mouse (rather than distinctive features of the melanoma subpopulations). Nonetheless this field continues to provide fascinating glimpses into the extraordinary propensity of melanoma cells to invade, survive and recapitulate new tumours with remarkable heterogeneity in terms of marker expression patterns 117–119.
The genetic heterogeneity of melanomas suggests that it is likely that ‘personalized’ combination therapies will need to be developed. Along these lines, future targeted therapy trials may need to be conducted in patients whose tumours have been scrutinized for the presence of the relevant combination of genetic aberrations. Making correlations between functionally associated molecular signatures and clinical response to therapy may permit for rational development of targeted therapy combinations to address coexistent alterations in alternative pathways. A better understanding about the complexity and redundancy that is responsible for melanoma progression will provide new direction for anti-cancer drug development. Agents or combination therapies exhibiting significant activity should be rapidly deployed in earlier stage patients in order to prevent, rather than treat, metastatic disease. Our understanding of the molecular basis of melanoma and the successes of targeted therapies has pushed melanoma care to the precipice of a new era. Indeed, the promise of genetic analysis of malignant tissue to inform choice of combination therapy is within the clinical realm and ‘personalized melanoma care’ will continue to gain momentum as we continue into the molecular age.