When evaluating the role of novel therapies, it is important to understand potential interactions with the disease in question, specifically focusing on disease factors that could impact on drug delivery, tolerability or dosing for example. PDAC is associated with multiple complexities which have precluded the development of novel approaches and therefore require due consideration.
PDAC is a disease of the elderly, presenting on average at 71 years of age. Up to 9% of patients present with localised disease however, the majority are diagnosed with either locally advanced or metastatic disease at their first consultation. Due its anatomical location the symptoms associated with PDAC tend to occur insidiously therefore contributing to the often delayed time to investigation and subsequent diagnosis. Such patient factors have important implications on treatment decisions which may in part explain the limited use of FOLFIRINOX in many patients despite its improved median OS, PFS and objective response.
Given the advanced age and stage at diagnosis the traditional focus on clinical outcomes and survival for interventional studies of PDAC management have shifted with increasingly more importance being placed on patient reported outcomes such as pain management and appetite. This is of particular importance for advanced PDAC given its poor prognosis. Without acknowledging the impact the treatment may have on patient reported outcomes, therapeutic advancements may be futile, thus developing drugs that are minimally toxic would be beneficial, yet to date, therapeutic advancements have continued to cause very similar side effect profiles to one another.
Genetic basis of PDAC
Inherited predispositions to PDAC account for 5%-10% of cases. In ongoing studies through the Australasian Pancreatic Cancer Genome Initiative, Humphris et al described the manifestations of inherited PDAC as occurring in 3 distinct settings, hereditary tumour predisposition syndromes, hereditary pancreatitis and familial pancreatic cancers which were further defined as occurring in a kindred in whom at least 2 first degree relatives have PDAC without diagnostic criteria for an inherited cancer syndrome[19,20]. Interestingly some of the genes identified in hereditary forms of PDAC affect pathways involved in DNA repair such as BRCA1/2 and PALB2 which have been noted in more recent detailed sequencing/mutational studies presented by Waddell et al and Bailey et al in two separate comprehensive analyses. While inherited forms of cancer are of interest the vast majority of genetic/metabolic pathway abnormalities are not inherited and form the basis of most PDAC cases.
Four major driver genes, KRAS, TP53, CDKN2A and SMAD4 have been identified in the development of PDAC, each sharing common oncogenic signalling pathways. KRAS mutations occur in > 90% of tumours and may represent the underlying insult to numerous subsequent events contributing to disease development. This mutation is widely accepted as a requirement for “reprogramming” pancreatic cell metabolism to facilitate the acidic environment needed for extracellular matrix breakdown and tumour invasion common to PDAC[23,24].
Of interest, the complex genetic and metabolic pathways associated with PDAC have identified various interactions and sites which can be utilised for therapeutic means. Among these include certain growth factor receptors such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor which have important roles in the RAS/RAF pathway as outlined in Figure 1. Furthermore it is now clear that drugs such as nab-paclitaxel have therapeutic actions against some of these pathways either directly or downstream accounting for their efficacy.
Figure 1 Schematic of major pathways associated with pancreatic ductal adenocarcinoma and site of action of current treatments.
Multiple pathways and receptors are associated with the development of pancreatic ductal adenocarcinoma (PDAC) including epidermal growth factor receptors (EGFR), human epidermal growth factor receptor 2 (Her2), and vascular endothelial growth factor receptor (VEGFR). All of these have important roles in the RAS/RAF/MEK/ERK and AKT/PI3K/mTOR pathways involved in cell growth. EGFR also has a role in the JAK/STAT pathway necessary for activation of signalling cascades and gene transcription. Transforming growth factor (TGF-β) is a multifunctional cytokine involved in various processes some of which are mediated by SMAD 4, a known mutation associated with development of PDAC. Current therapeutics target these processes at various sites.
Recent data published in Nature by Waddell et al reported the complex mutational landscape of PDAC, identifying multiple point mutations and structural variations in key genes. This data confirmed that KRAS abnormalities were almost ubiquitous while TP53 lesions were noted in 74% of samples, closely followed by CDKN2A lesions (35%) and SMAD4 abnormalities (31%). Through their analysis, PDAC was sub classified into 4 subtypes based on the distribution of events such that samples fell into either stable, scattered, unstable or locally arranged groupings. Of interest, the same study established a relationship between mutational load and abnormalities affecting DNA maintenance genes, specifically BRCA and PALB2. It is not surprising that PDAC associated with either PALB2 or BRCA2 mutations should behave in a similar fashion given their roles in DNA damage and repair. PALB2 binds and co-localizes with BRCA2 in order to facilitate double stranded DNA damage. In cases where PDAC harbours BRCA2 mutations there is good evidence for improved response to platinum containing therapies unlike other forms of PDAC. For PALB2 mutant disease there have been a number of reports noting improved outcomes in response to platinum based therapies or mitomycin C when compared with gemcitabine[27,28] and there is potential for targeted treatment with poly ADP ribose polymerase (PARP) inhibitors especially in the setting of BRCA2 as seen in breast and ovarian cancers.
Further to the comprehensive studies of Waddell et al, Bailey et al identified aggregates of point mutations in core molecular pathways affecting cellular functions including DNA damage and repair pathways, cell cycle regulation, transforming growth factor beta (TGFβ) signalling, chromatin regulation and axonal guidance. Based on the expression of 32 recurrently mutated genes found to aggregate into ten distinct pathways. From this analysis four subtypes were identified, comprising of, squamous, pancreatic progenitor, immunogenic and aberrantly differentiated endocrine and exocrine categories. Similar to Waddell et al the implications of these findings may potentially identify opportunities for therapeutic development. While these data have provided valuable insights into the complex molecular basis of PDAC many studies have previously attempted to manipulate some of these genes and/or pathways at various levels already and will be briefly discussed here.
Given the near ubiquitous nature of KRAS mutations in PDAC, targeting this gene and its associated pathways has been an area of interest however thus far this has not translated to clinically significant outcomes. In an effort to target as many known mutations/pathways as possible various studies have focused on novel therapies in combination with known chemotherapeutics in the hopes that treatment outcomes improve.
Selumetinib, an orally bioavailable selective MEK1/2 inhibitor showed promise in preclinical studies but failed to demonstrate a survival advantage in the second line setting when compared with the oral equivalent to fluorouracil, capecitabine. Similarly, attempts at targeting other known PDAC genes/pathways including P13K/Akt/mTOR have been clinically disappointing[30,31].
Her2/neu amplification is well characterised in a number of malignancies which have shown response when used as druggable targets in breast and gastric malignancies for example[32-34]. In PDAC, Her2/neu is amplified in up to 45% of cases, particularly in the advanced setting. Unfortunately, while initial pre-clinical studies indicated a potential role for Her-2 directed therapy specifically trastuzumab as a monotherapy or in combination with gemcitabine it did not prove clinically advantageous. Similarly, investigations combining trastuzumab with capecitabine were disappointing. In a related fashion EGFR which is known to co-express with Her2 has also been investigated with statistical gains noted for the EGFR inhibitor erlotinib in conjunction with gemcitabine, however this was only in the order of two weeks survival benefit and little mention of the related quality of life impact secondary to treatment was reported.
The JAK/STAT pathway has also been implicated as a regulator in the development of PDAC via its role in activating signalling cascades and gene transcription. Stimulated by oxidative stress, this pathway ultimately induces the production of inflammatory cytokines as well as cell proliferation, malignant transformation and inhibition of apoptosis in the pancreas[37,38]. This association with inflammation has prompted trials of the JAK-1/2 inhibitor ruxolotinib in combination with capecitabine for patients with metastatic PDAC after failure of first line therapy if patients expressed elevated inflammatory markers as assessed by C-reactive protein. In this phase II randomised trial, 127 patients were treated with either ruxolotinib and capecitabine or capecitabine and placebo. However, interim analyses failed to demonstrate sufficient efficacy with ruxolotinib combination therapy and further investigations of this drug has been suspended.
Together this information provides us with a raft of data for potential therapeutic targets, whether via drivers, alterations in signalling pathways or susceptibility genes. For example in the case of PALB2 mutation-associated disease there are multiple reports suggesting improved outcomes in response to platinum based therapy or mitomycin C when compared with gemcitabine[27,28] suggesting that a more comprehensive understanding of the genetic complexity of PDAC will assist in treatment decisions. Thus understanding the similarities and differences between the “poor” and “exceptional responders” may provide biomarkers to identify patients who might benefit from these treatments and improve outcomes.
Stromal microenvironment and drug delivery
PDAC is characterised by the surrounding cells, specifically activated fibroblasts, myofibroblasts and pancreatic stellate cells which contribute to the composition of the surrounding matrix, elements such as hyaluronan, growth factors (e.g., TGF-β) and secreted protein acidic rich in cysteine (SPARC)[42,43]. These result in a unique stromal microenvironment which may not only promote tumour initiation and progression but also create a barrier to drug delivery thus rendering PDAC relatively chemoresistant. Consequently, much effort has focused on ways to deplete or manipulate the stromal microenvironment and improve therapeutic outcomes.
SPARC is a glycoprotein believed to be involved in cancer development via its modulation of cell proliferation, progression, angiogenesis, migration, metastasis and apoptosis. It’s normal role in cellular functions is thought to be multifactorial with effects on cell dispersion and chemosensitization as well as induction of apoptosis but also has antiangiogenic properties[45-48]. While the intricacies of SPARC and cancer are yet to be fully elucidated its potential to increase the invasive capacity of malignant cells and possible association with poor prognosis is recognised. Of interest, SPARC methylation leading to pathogenesis correlates with both tobacco smoking and alcohol consumption, which are known associated modifiable risk factors for PDAC.
Interestingly gemcitabine has been reported to alter SPARC expression in a dose dependent manner in cell lines however, there is also evidence to suggest that SPARC overexpression enhances PDAC cell chemosensitivity to gemcitabine. In fact, SPARC may actually assist in the delivery of nab-paclitaxel to the tumour due to its affinity for albumin. Nab-paclitaxel is formulated with human albumin at concentrations that closely resemble physiological albumin levels. This feature seems to enable nab-paclitaxel to penetrate the stromal environment and reach the tumour more efficiently. Despite these studies however the role for SPARC within the stromal micro-environment and its implications on therapy remain controversial. For example data presented by Hidalgo et al reported no clear association between SPARC levels and treatment efficacy with combination therapy using gemcitabine and nab-paclitaxel or gemcitabine alone in metastatic PDAC.
Growth factors such as TGF-β are produced by cells within the stromal microenvironment. Interestingly TGF-β levels correlate with tumour metastases and progression as well as poorer patient outcomes. The specific role for TGF-β in this case is not clear but may involve regulation of cell cycle arrest, apoptosis, immune response and/or wound healing. Activated TGF-β signalling is mediated via SMADs, a known driver of PDAC, which is also reported to correlate with worse prognosis or disseminated disease[56,57].
Intriguingly TGF-β has been reported as a tumour suppressor in early stages of malignancy but a promoter in established disease further emphasising the complex nature of PDAC.
Despite the steady accumulation of knowledge from studies into genetic and molecular pathways, complex stromal characteristics and better drug delivery remains an ongoing issue further prompting investigations of novel approaches such as nucleolar stress pathways and ribosome biogenesis.