The Use of Personalized Medicine in Pancreatic Ductal Adenocarcinoma (PDAC): New Therapeutic Opportunities

Abstract

Pancreatic cancer constitutes a significant cause of cancer-related fatalities, with a five-year survival rate of only 12%. The most prevalent form of this disease is pancreatic ductal adenocarcinoma (PDAC). Given that a single therapeutic intervention has proven inadequate for the treatment of PDAC, it is essential to identify distinct molecular signatures that could improve treatment efficacy and alleviate the economic burden on patients. Surgery is recognized as the most effective treatment option for PDAC; however, only a small percentage of patients are candidates for this procedure due to the advanced stage of the disease at the time of diagnosis. In this context, we propose to explore the biology of PDAC with a focus on microbiome, epigenetics, and genetics. Our objective is to examine the existing knowledge in these areas and to identify potential pathways for personalized medicine. This approach holds promise for advancing our understanding of PDAC development, progression, and resistance to standard therapy.

1. Introduction

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the lowest five-year survival rate among all cancers in both the United States and Europe [1,2,3]. It ranks as the third leading cause of cancer-related mortality among both men and women in the United States, and it is the seventh leading cause of cancer death globally [4]. A significant factor contributing to the poor prognosis associated with pancreatic cancer is that approximately 90% of tumors are identified at an advanced stage, after they have already metastasized beyond the pancreas, with systemic spread occurring in more than 50% of cases [5,6]. This situation emphasizes the urgent need for the development of diagnostic tools that can facilitate the early detection of PDAC prior to its dissemination beyond the pancreas. Additionally, there is a critical requirement for therapeutic interventions that more effectively target and eliminate PDAC cells following metastasis [7].

Personalized medicine endeavors to tailor medical treatments to individual patients by considering their unique characteristics, which include genetic factors, physiological conditions, and environmental influences [8]. In the context of cancer therapy, personalized medicine emphasizes the development of strategies specifically customized to meet the needs of each patient, thereby enhancing patient outcomes [9]. In fact, recent advancements in our understanding of tumor biology, coupled with the integration of more personalized treatment methodologies, have resulted in significant improvements in the prognosis of cancer patients [10,11]. The development of personalized medicine strategies for PDAC is still in its early stages compared to other types of cancer. However, there has been a significant global increase in efforts in recent years, demonstrating positive outcomes that emphasize the feasibility and clinical advantages of precision medicine-based approaches for PDAC. Nevertheless, the use of molecularly matched treatments is currently limited to a small group of patients. The molecular biology of PDAC is complex, highlighting the significant challenges in translating scientific advancements and the urgent need to realize the potential of precision therapeutics in PDAC treatment [12].

This review explores the intricate biology of PDAC, with a particular focus on its genetic, epigenetic, and microbiome characteristics (Figure 1). The primary aim is to elucidate the current understanding of PDAC while also presenting new insights that could guide future research endeavors in the field of personalized medicine. This information may be instrumental in formulating strategies to address drug resistance in a more personalized and effective manner.

A literature search was conducted in the PubMed and B-ON databases using the keywords “PDAC Genome” and “Microbiome”, individually or in combination with other relevant terms such as “Genomic Instability” or “Epigenetic Modifications”. The search focused on full-text articles in English published from 2015 onwards, while some older articles were also consulted to provide historical context and ensure a broader understanding of the topic. Articles were selected based on their relevance to the themes of PDAC Genetics, Microbiome, and Resistance to standard chemotherapies, without strictly applying predefined inclusion or exclusion criteria. This review aims to offer insights into current knowledge in these areas as part of the broader discussion on personalized medicine.

2. Pancreatic Ductal Adenocarcinoma (PDAC): A Global Health Concern

Pancreatic cancer is a significant health concern, accounting for 8.2% of cancer-related deaths and ranking as the third leading cause of cancer-related mortality. The five-year survival rate for pancreatic cancer is a mere 12% [1]. PDAC represents the primary form of pancreatic cancer, where tissue containing neoplastic cells coexists with interstitial tissue comprising immune cells, fibroblasts, and vascular epithelial cells [13]. Due to the intricate nature of PDAC, relying on a single therapeutic intervention or drug to achieve a universal treatment outcome is not only ineffective but also exacerbates the financial burden placed on patients [14].

PDAC is a highly aggressive malignancy, and surgery remains the most effective treatment option. However, only a small proportion of patients are eligible for surgical intervention due to the advanced stage of the disease [15,16]. It is noteworthy that the radiologic stage and histopathological features of unresectable PDACs can be similar, yet the response to chemotherapeutic agents can vary significantly, leading to either complete unresponsiveness or partial response. The latter may provide an opportunity for subsequent surgical intervention [17].

2.1. Dilemma in Diagnosis of PDAC

The majority of patients with pancreatic cancer do not present symptoms until the disease has reached an advanced stage. When symptoms do occur, they are often vague and nonspecific, leading to delays in diagnosis that can span several months [18]. The prognosis for pancreatic cancer is notably poor, with median survival rates of approximately 10 to 12 months for those receiving treatment and 5 to 6 months for those who do not [7].

Ongoing research into the molecular alterations and tumor microenvironmental changes that influence pancreatic cancer development, progression, and metastasis is identifying distinct, targetable pathways among specific patient subpopulations. Early identification of these populations during the diagnostic phase may facilitate the personalization of therapeutic strategies, potentially leading to improved treatment outcomes (Figure 1). As our knowledge expands regarding the driving pathways associated with PDAC, there will be opportunities to create innovative drug therapies and combinations that could further enhance therapeutic efficacy for a broader range of patients [7].

2.2. Dilemma in Chemotherapy of PDAC

As previously mentioned, surgical intervention is a viable option for certain cases; however, the recurrence and metastasis rates following surgery remain high. Therefore, it is essential to consider comprehensive treatment modalities, including chemotherapy and targeted therapy. Unfortunately, PDAC exhibits low sensitivity to most chemotherapy drugs, resulting in poor chemotherapy efficacy [19]. Pancreatic cancer is a challenging disease to manage, particularly when it is advanced or unresectable. In such cases, systemic chemotherapy has emerged as the preferred treatment option. Combination regimens like FOLFIRINOX, which incorporates 5-fluorouracil (5-FU)/leucovorin, irinotecan, and oxaliplatin, or gemcitabine (GEM) and nab-paclitaxel, have become first-line standards for patients with preserved performance status [20].

GEM (Scheme 1) remains a standard cytotoxic agent for treating metastatic pancreatic cancer in patients with poor performance status [20]. However, its effectiveness is limited, and the administration rates of second-line and third-line chemotherapy have been found to be relatively low in such cases [21]. Optimizing first-line chemotherapy is critical, given the aggressive nature of advanced pancreatic cancer, which is characterized by progressive therapeutic resistance [22].

Fluoropyrimidines, a class of chemotherapeutic agents including 5-FU, capecitabine, and TAS-1, are frequently co-administered with GEM (Scheme 1) for solid digestive tumors, including PDAC chemotherapy [23]. However, the effectiveness of such treatment is often limited by drug-induced resistance or toxicity [24]. Consequently, the treatment may have to be administered at lower doses or terminated prematurely, resulting in inadequate treatment outcomes [25].

3. Standard Chemotherapy of PDAC

3.1. Antimetabolite Drug 5-FU in PDAC

Antimetabolite drugs are a class of chemotherapeutic agents that inhibit essential biosynthetic processes or are incorporated into macromolecules, such as DNA and RNA, thereby impeding their normal function. One such drug, 5-fluorouracil (5-FU, Scheme 1), is a fluoropyrimidine that accomplishes both of these mechanisms. The development of fluoropyrimidines occurred in the 1950s following the observation that rat hepatomas metabolized uracil, one of the four bases found in RNA, at a faster rate than normal tissues. This suggested that uracil metabolism could be a potential target for antimetabolite chemotherapy [26]. The use of the antimetabolite drug 5-FU in PDAC is still a subject of debate due to the heterogeneity of the disease and the variability of patient response. Further studies are required to optimize the use of this drug in PDAC treatment and to identify predictive biomarkers that could aid in patient selection and treatment monitoring.

3.1.1. 5-FU Mechanism of Action

5-FU is transported into cells through facilitated transport, utilizing the same mechanism as uracil. Following transportation, 5-FU undergoes metabolic conversion via two competing pathways (Figure 2) [27]. Upon intravenous administration, 5-FU undergoes a series of anabolic reactions, leading to the production of several active metabolites. These metabolites have been observed to inhibit the activity of thymidylate synthase (TS), disrupting DNA/RNA synthesis. Consequently, this interference ultimately leads to DNA/RNA damage and subsequent cell death (Figure 2) [28].

Once inside the cells, 5-FU is converted to 5-fluorouridine monophosphate (FUMP) through a direct pathway utilizing orotate phosphorylase (OPRT) as a cofactor, or an indirect pathway involving uridine phosphorylase (UP) and uridine kinase (UK) through Fluorouridine (FUR). FUMP is further phosphorylated to form fluorouridine diphosphate (FUDP). Subsequently, FUDP can be enzymatically converted to the active metabolite fluorodeoxyuridine triphosphate (FUTP) or to fluorodeoxyuridine diphosphate (FdUDP) by the enzyme ribonucleotide reductase (RNR) [28]. The active metabolites generated after the administration of fluoropyrimidine-based chemotherapeutic agents are known for their potential to be incorrectly incorporated into RNA and DNA, replacing uridine-5′-triphosphate/2-deoxythymidine-5′-triphosphate (UTP/dTTP). For instance, 5-FU is transformed into fluorodeoxyuridine monophosphate (FdUMP) through the actions of thymidine phosphorylase (TP) and thymidine kinase (TK), or via the reduction of FUDP by ribonucleotide reductase (RNR), representing the primary process [27].

The enzyme TS plays a crucial role in the biosynthesis of thymidylate by catalyzing the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) in the presence of 5,10-methylenetetrahydrofolate (CH₂THF). The binding of FdUMP to TS forms a stable ternary complex that inhibits the synthesis of dTMP by blocking the access of dUMP. This inhibition causes the cytotoxic effects of 5-FU, making it an effective chemotherapeutic agent. A reduction in the levels of dTMP can result in the depletion of deoxythymidine triphosphate (dTTP) levels. The pivotal role of dTMP in dTTP biosynthesis impacts the levels of other deoxynucleotides, significantly disrupting DNA synthesis and repair. The inhibition of TS leads to the accumulation of dUMP, which increases deoxyuridine triphosphate (dUTP) and fluorodeoxyuridine triphosphate (5-FdUTP)/dTTP levels. This can result in the misincorporation of dUTP and FdUTP in DNA, causing irreversible damage. To mitigate the accumulation of dUTP, dUTPase (DUT) can be employed to generate dUMP, thereby preventing the misincorporation of dUTP into DNA. This may improve the efficacy of 5-FU-based cancer therapy (Figure 2) [29,30].

The catabolic pathway of 5-FU is characterized by the rate-limiting conversion of 5-FU into dihydrofluorouracil (DHFU) by the enzyme dihydropyrimidine dehydrogenase (DPD). This metabolic process leads to the formation of α-2-fluoro-β-alanine (FBAL) and α-2-fluoro-β-ureido propionic acid (FUPA), which are excreted through the kidneys, with more than 80% being degraded in the liver [28]. Kinetic studies have shown that intravenous bolus administration of 5-FU leads to the excretion of 60–90% of 5-FU as FBAL or CO₂ via urine or exhalation within 24 h. However, oral administration of 5-FU pro-drugs, due to variability in DPD activity, exhibits poor bioavailability (which can be attributed to circadian/genetic variation), resulting in unpredictable 5-FU levels in the plasma. This unpredictability is further compounded by intra- and inter-patient variability in the adsorption/elimination of 5-FU [30], leading to variations in the plasma levels of the drug (15). To address the severe toxicities associated with 5-FU administration in DPD-deficient cancer patients, several 5-FU pro-drugs have been developed and are currently being used to modulate the efficacy of 5-FU therapy [27].

3.1.2. Chemoresistance to and Cytotoxicity of 5-FU

The FOLFIRINOX regimen, a combination of 5-FU, irinotecan, leucovorin, and oxaliplatin, has been established as a standard treatment for metastatic PDAC following the success of the phase III clinical trial PRODIGE-4/ACCORD-11. More recently, the results of the phase III multicenter, randomized clinical trial PRODIGE 24/CCTG PA.6 have supported the use of the FOLFIRINOX regimen in potentially curable PDAC as an alternative to GEM monotherapy [31]. However, although the benefit of this three-drug regimen is promising, its associated increased toxicity profile has raised concerns. As such, several studies are underway to evaluate potential predictive or monitoring tools to optimize this therapy [31]. The initial rapid degradation of 5-FU by DPD, the first and rate-limiting enzyme of the pyrimidine catabolic pathway, reduces the efficacy of the drug and necessitates the application of high doses. Furthermore, the breakdown of 5-FU produces fluorinated products that lead to multiple side effects in the nervous system [32]. Therefore, DPD is considered a critical target for improving the efficacy of 5-FU. Mammalian DPD polymorphisms have been extensively studied for their potential to predict fluoropyrimidine-based toxicity and aid in determining the appropriate 5-FU dose [33,34].

3.2. Gemcitabine in PDAC

Currently, GEM-based chemotherapy remains a primary therapeutic approach for PDAC [35,36]. Upon entry into the tumor cells, GEM derivatives disrupt the synthesis of cellular DNA, resulting in the cell cycle arrest [37]. The potential for GEM to enhance the median survival time of PDAC patients is recognized; however, its overall impact on survival rates and prognosis is still limited [38]. According to statistics, more than 74% of patients experience disease recurrence or progression despite GEM chemotherapy [39]. Notably, the resistance of PDAC to GEM presents a significant difficulty and formidable obstacle in the treatment of this disease [40].

In our previous review study [41], we provided an explanation of the mechanism of action of gemcitabine, as well as potential pathways to address microbiome-acquired resistance in PDAC. The purpose of the current review is to explore how a more thorough understanding of PDAC biology can advance personalized treatment approaches. This insight has the potential to enhance therapeutic outcomes and effectively combat drug resistance.

4. Pathogenesis of PDAC

PDAC emerges from noninvasive precancerous lesions that possess the potential for curability if detected and managed at an early stage. These precancerous lesions are categorized according to their size and their extent of involvement with the pancreatic ductal system. The preponderance of PDAC cases is attributed to microscopic pancreatic intraepithelial neoplasm (PanIN), a neoplasm that affects the pancreatic ducts and is defined as being less than 5 mm in size [42]. Conversely, a lesser fraction of PDAC cases, constituting less than 10%, arises from intraductal papillary mucinous neoplasms (IPMNs), which are macrocystic lesions that impact the pancreatic ductal system [42,43,44]. Mucinous cystic neoplasms, although the least common precancerous neoplasm, exhibit distinct clinical and pathological features. Unlike other neoplasms, mucinous cystic neoplasms do not involve the ductal system and are characterized by the presence of ovarian-type stroma. These neoplasms predominantly affect women [45].

The unique pathological characteristics of each lesion present specific clinical challenges. For instance, IPMNs are often detected incidentally during abdominal imaging due to their larger size, necessitating data-driven approaches for effective surveillance and intervention [46]. Conversely, PanINs are infrequently identified incidentally. While the pathological features of these precancerous lesions are well-established, the debate regarding the cell of origin for pancreatic cancer persists. The anatomical positioning of these precancers within the ductal system may indicate a ductal cell origin; however, numerous studies involving murine models suggest that acinar cells can also lead to the development of PanINs following pancreatic injury and metaplasia. Confirming these findings in human samples remains a considerable challenge. This ongoing controversy has been the subject of recent comprehensive reviews [47].

4.1. Genetics and PDAC

PDAC is primarily driven by somatic mutations in oncogenes and tumor suppressor genes. Key genes commonly mutated in PDAC include the oncogene KRAS and the tumor suppressor genes CDKN2A, TP53, and SMAD4. These mutations were initially identified in the 1980s and 1990s through targeted molecular biology techniques and sequencing methods [48,49,50,51]. In 2008, the first comprehensive analysis of the PDAC exome was published, highlighting the genomic landscape of PDAC, which consists of four principal “mountains” (KRAS, CDKN2A, TP53, and SMAD4) alongside a significant number of less frequently mutated “hills” [52]. Subsequent large-scale sequencing studies, including efforts by The Cancer Genome Atlas and The International Cancer Genome Consortium [53,54,55,56], have refined this landscape with remarkable detail. These studies have characterized several important groups of “hills”, which include genes implicated in DNA repair, chromatin remodeling, and axon guidance. Some of these groups correlate with clinically significant subcategories that respond to specific therapeutic interventions [7]. Recent research has also identified the phenomenon of kataegis, which involves clustered nucleotide substitutions in PDAC, likely associated with the activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes [57]. In addition to identifying small somatic mutations within coding regions, the broadening of genomic analysis in PDAC to include whole genome sequencing has resulted in the comprehensive documentation of significant chromosomal alterations. These alterations encompass copy number variations, chromosomal rearrangements, and the phenomenon referred to as chromothripsis [57]. The term “chromothripsis”, which derives from the Greek word for “shattering”, pertains to a condition wherein one or a limited number of chromosomes exhibit an extensive array of clustered genomic rearrangements [57]. Certain researchers posit that these alterations may be the consequence of catastrophic DNA damage events, which facilitate a pattern of punctuated evolution as opposed to a gradual evolutionary process [58]. A continuous challenge in the interpretation of these alterations resides in the identification of potential driver genes within large altered regions and the differentiation of driver mutations from passenger mutations that emerge as a result of the genomic instability inherent to PDAC [7].

4.2. Epigenetics and PDAC

While alterations in DNA sequences represent the most extensively examined characteristics of PDAC pathogenesis, it is important to recognize that other molecular modifications also significantly contribute to the etiology of this condition. Epigenetic modifications—including DNA methylation and histone modifications, such as acetylation and methylation—have the capacity to heritably influence chromatin architecture and gene expression. Significantly, the inactivation of the tumor suppressor gene CDKN2A through DNA methylation has been frequently observed; however, this mechanism is less common for the inactivation of other critical PDAC drivers, including TP53 and SMAD4 [59,60].

A meticulous methylation profiling study conducted by The Cancer Genome Atlas identified two distinct clusters of PDACs, classified by the extent of DNA hypermethylation. Furthermore, an integrated analysis that encompassed both DNA methylation and messenger RNA expression data revealed nearly 100 genes that were recurrently silenced as a result of DNA methylation, among them ZPF82, PAPR6, and DNAJC15 [56]. Moreover, comprehensive epigenomic reprogramming has been demonstrated to influence the PDAC genome. Investigations utilizing human autopsy samples have indicated substantial alterations in histone states within metastatic tissues in comparison to primary tumors, thereby suggesting that epigenetic modifications may play an integral role in the progression of metastasis [61]. Such epigenetic alterations have also been documented in precancerous lesions, including PanIN and IPMN [62,63,64,65].

4.3. Microbiome and PDAC

Recent studies indicate that variations in the gut, oral, and intratumoral microbiomes may significantly influence the development and progression of PDAC [66,67,68].

Establishing a clear causal relationship between gut microbiome dysbiosis—characterized by alterations in the composition, diversity, and metabolic activity of the gut microbiome with potential pathogenic implications—and pancreatic diseases presents a significant challenge in observational studies. This complexity arises from confounding factors and the possibility of reverse causality. One hypothesized mechanism by which the gut microbiome may influence carcinogenesis is through the modulation of circulating metabolite concentrations. Notably, elevated circulating and tumor levels of gut-derived lipopolysaccharide, a pro-inflammatory component of bacterial membranes, have been observed in murine models of PDAC. These findings are correlated with a decline in gut barrier function [69]. Furthermore, the concentration of various bacteria-related circulating metabolites has been linked to both increased and decreased risks of gastrointestinal cancers. This includes metabolites such as secondary bile acids [70,71,72,73,74], amino acid derivatives [75], tryptophan derivatives [76], and short-chain fatty acids [77]. Mendelian randomization (MR) is a robust genetic epidemiology technique that employs genetic variants as instruments to evaluate the impact of an exposure on a specific outcome. Since alleles are randomly segregated during meiosis, this method effectively eliminates concerns related to environmental confounding variables. Additionally, because these alleles exist prior to the onset of PDAC, the potential for reverse causation is also removed [78]. Therefore, MR serves as the most appropriate strategy for investigating the causal relationship between the gut microbiome, associated circulating metabolites, and PDAC risk. Daniel et al. [79] conducted a comprehensive investigation into the associations between genetically predicted abundances of individual gut microbiota and the genetically predicted circulating concentrations of microbiome-associated metabolites, specifically in the context of PDAC, employing the methodology of MR. The results of this study, although predicated exclusively on genetically predicted characteristics of the gut microbiome and their corresponding metabolite concentrations, furnish compelling evidence for causal associations with the etiology of pancreatic carcinogenesis [79].

5. Personalized Approaches That Influence Standard Therapies Response in PDAC

5.1. Genetic Polymorphism of DPYD

Fluoropyrimidine treatment has the potential to induce severe toxicity in as many as 30% of patients. This adverse effect is frequently associated with diminished activity of the pivotal metabolic enzyme DPD, which is predominantly influenced by genetic variants within the gene that encodes for DPD (DPYD) [80]. The four DPYD variants that are regarded as most clinically significant, demonstrating a statistically significant association with severe toxicity, include DPYD*2A (rs3918290, c.1905 + 1G > A, IVS14 + 1G > A), c.2846A > T (rs67376798, D949V), c.1679T > G (rs55886062, DPYD*13, I560S), and c.1236G > A (rs56038477, E412E, in haplotype B3) [81,82,83]. Current evidence indicates that heterozygous carriers of these variants may experience an average reduction in DPD enzyme activity ranging from approximately 25% (for c.2846A > T and c.1236G > A) to as much as 50% (for DPYD*2A and c.1679T > G) [83].

Implementing prospective DPYD genotyping, coupled with dose reductions for heterozygous carriers of these variant alleles, presents a promising strategy to mitigate the risk of severe and potentially life-threatening fluoropyrimidine-related toxicities while maintaining the efficacy of treatment. A comprehensive study conducted by Henricks et al. [84] elucidates the feasibility of employing genotype-guided dosing within clinical practice. The individualization of therapeutic doses resulted in a significant reduction of the risk associated with severe toxicity (grade 3 or higher) among carriers of the DPYD*2A variant. Furthermore, the protocol demonstrated safety for an isolated carrier of the c.1679T > G variant while concurrently diminishing the toxicity risk for carriers of the c.2846A > T variant; it is crucial to underscore that the associated risk for these individuals remains elevated in comparison to wild-type patients. In contrast, a 25% dose reduction for carriers of the c.1236G > A variant proved inadequate in alleviating severe treatment-related toxicity. The conclusions drawn from their study suggest that DPYD genotype-guided dose individualization possesses considerable potential to enhance patient safety, evidenced by the reduced toxicity risk in three of the four variants under investigation [84]. Considering that fluoropyrimidines are among the most commonly employed agents in oncological therapy, the findings support the implementation of DPYD genotype-guided individualized dosing as an imperative new standard of care [84].

5.2. Chronomodulated Treatment with 5-FU

Cellular circadian clocks play a significant role in modulating the metabolic pathways associated with 5-FU. These clocks affect both catabolic pathways, such as the metabolism of DPD, and anabolic pathways, including those mediated by uridine monophosphate synthase. Notably, diurnal variation has a considerable impact on the expression of DPYD mRNA, consequently influencing DPD biosynthesis. This variation results in distinct diurnal patterns in plasma levels of the endogenous substrate uracil, as well as variations in the dihydrouracil-to-uracil (DHU/U) ratio [85,86,87,88]. Recent studies have highlighted diurnal variations in the plasma concentrations during continuous constant-rate infusions, as documented in the literature [89,90,91,92]. Notably, these investigations have identified both inter- and intra-individual variability regarding the timing and magnitude of peak DPD activity [87,93,94].

Mathematical analyses employing semi-mechanistic models have revealed significant interpatient variability in the pharmacokinetics associated with chronomodulated therapy utilizing 5-FU [95]. Notably, despite all patients receiving the same peak delivery rate and relative dose per body surface area, marked differences in maximum plasma concentrations and areas under the plasma concentration–time curve have been observed. Given that 5-FU clearance is closely linked to DPD activity, the inter-individual variability in DPD-mediated 5-FU metabolism may significantly contribute to the variations seen in 5-FU plasma exposure [95,96]. Therefore, examining diurnal variations in DPD activity may facilitate the exploration of personalized chronomodulated 5-FU therapy by allowing for the assessment of alternative individualized dosing strategies.

5.3. cNEK6 Induces GEM Resistance

Currently, there is a notable absence of clear markers that can accurately determine whether pancreatic cancer exhibits resistance to GEM. Therefore, it is essential to identify reliable biomarkers that can effectively indicate the sensitivity of PDAC to GEM. This identification will facilitate the development of appropriate strategies for chemotherapy sensitization [97].

There is an increasing body of evidence suggesting that tumor metabolism, including glucose, lipid, and amino acid metabolism, substantially influences the efficacy of chemotherapy in PDAC [98]. Tumors predominantly depend on the energy and metabolites generated through glycolysis [99]. Oxidative stress induced by reactive oxygen species generated during the administration of GEM results in the inactivation of pivotal glycolytic enzymes, specifically pyruvate kinase M2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This process significantly enhances tumor eradication’s efficacy [100,101]. The promotion of glycolysis in pancreatic cancer cells facilitates the utilization of glycolytic metabolites, thereby undermining the therapeutic effectiveness of GEM [99]. A profound and comprehensive elucidation of the molecular mechanisms underlying the glycolytic processes associated with PDAC is essential for the formulation of strategies aimed at augmenting the sensitivity of these cells to GEM-based chemotherapy [97].

A study conducted by Li et al. [97] presents a significant finding regarding cNEK6, a circular RNA (circRNA), which has been shown to induce GEM resistance through glycolysis for the first time. This research highlights cNEK6 as a potential biomarker and therapeutic strategy for enhancing the sensitivity of patients with PDAC to GEM chemotherapy. The elevated levels of cNEK6 observed in both tumor tissue and peripheral blood of PDAC patients strongly indicate a correlation with GEM resistance.

Future research initiatives should prioritize the identification of robust biomarkers that can accurately indicate the intrinsic resistance of PDAC to GEM. Such advancements would be instrumental in guiding chemotherapy sensitization strategies, enabling the prediction of resistance ahead of time and facilitating the direction of patients toward more tailored therapeutic approaches.

6. Precision Medicine: Engineering the Microbiome to Improve Drug Outcomes

Notwithstanding the recognition afforded nearly a century ago regarding the clinically pertinent role of the human microbiome in drug disposition, the elucidation of mechanistic insights and the exploration of translational applications continues to be markedly constrained [102]. The field of pharmacomicrobiomics is still in its early stages of development. While there is a substantial and historical body of research focused on antibiotic resistance mechanisms in pathogenic bacteria [103], our understanding of how pharmaceutical agents interact with resident microbial communities remains limited. Furthermore, the mechanisms by which the microbiome influences drug pharmacokinetics and pharmacodynamics are not yet fully elucidated [102]. Several key questions require further investigation: To what extent are various drugs influenced by the microbiome? Which microbial species, genes, and enzymes are responsible for the direct biotransformation of these drugs? Additionally, do certain medications exert their effects, at least in part, by modulating the microbiome? How does microbial colonization impact host metabolic pathways related to drug metabolism, absorption, or mechanisms of action? Given the inherent complexity of the microbiome, is it feasible to create targeted approaches for managing microbial metabolic activity and their interactions with host tissues [102]?

The extensive evolutionary and metabolic diversity present within host-associated microbial communities can be quite complex. A well-documented example is found in the genes associated with carbohydrate metabolism. While the human genome encodes approximately two dozen distinct enzymes, the variety of carbohydrate-active enzyme families present within the microbiome encompasses hundreds of different types [104]. One indication of the complexity of the enzymatic machinery involved is the expanding catalog of pharmaceuticals that can be metabolized by the human gut microbiome [105]. These compounds exhibit diverse characteristics, including variations in structure, molecular weight, polarity, and solubility. This diversity strongly suggests the involvement of a broad array of enzymes in their biotransformation processes [102].

The impact of host-associated bacteria on drug metabolism is applicable across various bodily environments [106] and even in diseased tissues [107]. Studies comparing the vaginal microbiotas of patients undergoing treatment with the antiretroviral medication tenofovir demonstrated that drug efficacy was significantly enhanced in microbiotas dominated by Lactobacillus, in contrast to those with a high prevalence of Gardnerella vaginalis [106]. Measurements of drug concentrations, both in patients and through in vitro incubation with G. vaginalis, indicated that bacterial inactivation of the drug contributed to the observed reduction in efficacy. Interestingly, a recent investigation into PDAC reached a similar conclusion, albeit through a different lens [107]. Bacterial contamination of cancer cell lines was found to confer resistance to the chemotherapeutic agent GEM. This resistance was attributed to the long isoform of the bacterial enzyme cytidine deaminase. Furthermore, intratumoral injections of cytidine-deaminase-expressing isolates from gut bacteria hindered disease treatment in mouse models, with 76% of the analyzed human cancer samples exhibiting detectable bacterial presence, predominantly from Gammaproteobacteria [102,107].

Personalized Alteration of the Gut Microbiota: Future Therapy Opportunities

Developing a comprehensive encyclopedia of drug–microbiome interactions will enhance our understanding of the diverse ways in which host-associated microorganisms interact with physiological molecules, ultimately influencing host health. A pertinent inquiry that follows is how we can effectively translate this knowledge to enhance disease treatment. While the concept of manipulating the gut microbiota has been discussed for some time, existing clinical strategies often lack the necessary precision; they tend to induce extensive changes in the microbiome when more targeted modifications are intended. For instance, antibiotics are frequently prescribed for infections caused by specific pathogens, yet their use can lead to significant and lasting alterations in the microbiota [108].

Recent reviews have highlighted the advancements in microbiome-targeted therapeutics, as well as the ongoing challenges within this field [109]. Notably, several intriguing examples have emerged that detail innovative strategies for the precise manipulation of gut microbiota. These strategies can be categorized into four overarching categories:

(1)

Inhibition of metabolic activities performed by gut bacterial enzymes: Microbial enzymes that catalyze undesirable reactions within the gastrointestinal tract present potential targets for therapeutic intervention. The development of selective and non-lethal enzyme inhibitors aimed at these enzymes could offer a promising approach to modulating gut health [110].

(2)

Removal of specific bacterial species or strains: An additional method for microbiome modification entails the targeted depletion of specific strains exhibiting detrimental activities, particularly those that metabolize pharmacological agents into toxic metabolites. This strategy seeks to improve the safety and efficacy of therapeutic applications [102,111].

(3)

Introduction or engrafting of engineered strains into the gut: In addition to efforts aimed at eliminating specific strains, there is a growing focus on the introduction of engineered strains into the host as live bacterial therapeutics. This approach bears similarities to the application of probiotics, which are designed to provide beneficial functions [102,112].

(4)

Direct genetic modification of bacterial cells present within the gastrointestinal tract: Recent advancements in the modification of gut microbiota indicate a paradigm shift from the introduction of engineered strains to the direct genetic modification of bacterial populations that naturally colonize the gastrointestinal tract. This progressive approach, known as in vivo or in situ engineering, involves the execution of genetic modification techniques within the host organism, as opposed to traditional methodologies conducted within a laboratory environment [102,113].

Notwithstanding the substantial body of evidence, the translation of clinical interventions that target microbial agents into fully developed applications for patients with PDAC remains inadequate. This phenomenon can be attributed to a multifaceted array of factors, including the pronounced individual variability in sensitivity to the same microbial agents. The inquiry into whether microbiome-targeted interventions can be seamlessly incorporated into existing PDAC management frameworks to yield more comprehensive and advantageous antitumor effects endures as an open question. As the matter remains unresolved, there exists a pressing necessity for additional preclinical investigations and prospective clinical trials to elucidate the challenges involved. Ultimately, although numerous obstacles persist, the critical importance and extensive potential of gut microbiota in the advancement of novel anticancer strategies cannot be overstated. Thus, it is imperative to pursue a holistic approach that integrates microbial modulation therapy within the prevailing PDAC management paradigms.

7. New Therapeutic Strategies: Future Directions

Recent advancements in the field of omics, particularly concerning DNA sequencing and genetic engineering, have provided promising methodologies for the enhancement of therapeutic options for PDAC. Although our comprehension of the microbiome and its interactions with PDAC remains in its nascent stages, we recognize the significant potential for utilizing the microbiome as a strategic instrument in developing innovative therapeutic approaches for the treatment of this malignancy.

In this chapter, we provide a summary of recent research studies focused on improving PDAC outcomes. Our objective is to explore both enhancements to standard chemotherapeutic approaches and the development of novel strategies. We aim to present current avenues of research that could potentially be pursued as part of a personalized treatment plan.

7.1. Probiotic-Based Regimens Combined with Chemotherapy Drugs

The study conducted by Chen et al. [114] aimed to investigate the potential of probiotics as an adjuvant during chemotherapy for pancreatic cancer. The combined treatment of GEM and probiotics showed a reduction in PanIN formation grade. Mice treated with both GEM and probiotics demonstrated lower levels of aspartate aminotransferase and alanine aminotransferase. Notably, high-dose probiotics alone, without GEM, also had an inhibitory effect on PanIN changes and serum liver enzyme elevation. The results indicate that probiotics could potentially augment the efficacy of conventional chemotherapy and enhance a patient’s ability to tolerate the treatment. Nevertheless, forthcoming research endeavors should prioritize conducting randomized controlled trials involving human subjects to ascertain the precise role of probiotics in the context of PDAC treatment [115].

7.2. Gene Therapy and Oncolytic Virotherapy for PDAC

The presence of microorganisms has been associated with the development of PDAC. However, it is important to note that these microorganisms also have potential applications in the biotechnological field, particularly in gene therapy.

In recent years, clinical trials focusing on gene therapy and oncolytic virotherapy for cancer have demonstrated viability and safety [116,117,118,119]. These trials encompass early- and advanced-phase clinical trials targeted at treating PDAC [120,121,122,123,124]. Although the primary objective of phase I/II trials is not to directly assess therapeutic efficacy, early-phase trials for advanced-stage PDAC have not met the anticipated outcomes from preclinical studies [120,123,124]. Furthermore, the initial phase III trial of TNF gene transfer therapy using a replication-defective adenoviral vector in patients with locally advanced PDAC exhibited a favorable safety profile but did not result in improved survival [122]. The clinical complexities associated with treating PDAC, characterized by high metastatic potential and rapid progression, present significant challenges for gene therapy. An especially promising strategy currently undergoing clinical evaluation involves optimizing gene delivery efficiency through the utilization of tumor-selective retroviral replicating vectors (RRVs) for PDAC. RRVs are capable of efficient replication and selective transduction specifically within cancer cells, as they can only infect actively dividing cells and are constrained in normal tissues by innate and adaptive immunity, which are compromised or suppressed in the tumor microenvironment [125,126].

The progression of preclinical development of “Toca 511” (vocimagene amiretrorepvec), an optimized RRV-encoding yeast cytosine deaminase (yCD), which transforms the antifungal prodrug 5-fluorocytosine (5-FC) into the anticancer drug 5-fluorouracil (5-FU), has laid the groundwork for the clinical application of RRV as a potential innovative cancer therapy [126,127].

A study by Inoko et al. [128] represents the initial preclinical investigation indicating that RRV-mediated prodrug activator gene therapy holds promise as a new approach against PDAC. They hypothesized that Toca 511/5-FC gene therapy (Figure 3), which generates tumor-localized highly concentrated 5-FU, may have more therapeutic effect in PDAC [128]. The study reveals that RRV effectively propagates and integrates into PDAC cells both in vitro and in vivo; RRV Toca 511 proficiently delivers the yCD prodrug activator gene to PDAC cells, resulting in substantial cancer cell-selective cytotoxicity in vitro and anti-tumor effectiveness in vivo upon 5-FC prodrug treatment; and RRV copy numbers in normal tissues were low, especially in immunocompetent hosts [128]. The findings demonstrate the potential of Toca 511 and 5-FC therapy for clinical translation, indicating its promise as a therapeutic strategy in PDAC (Figure 3) [128].

7.3. Epigenetics Engineering: Modifying miRNAs with GEM and 5-FU for Developing miRNA-Based Therapeutics

MicroRNAs (miRNAs) are non-coding RNA molecules that play a significant role in epigenetic mechanisms, particularly in regulating gene expression. A recent study [129] has demonstrated the effectiveness of miRNA mimics in inhibiting colon, lung, and pancreatic cancer cells. The miRNA mimics were engineered by substituting the uracils on the guide strand of the miRNA with 5-FU, an antimetabolite chemotherapeutic. These mimics exhibit specific binding to their mRNA targets and possess the capability to penetrate cancer cells unassisted, addressing a critical challenge in nucleic acid-based drug development. To enhance this approach, a distinct miRNA modification technique involving GEM was formulated to produce more potent miRNA mimics tailored for the treatment of PDAC. GEM-modified miRNA mimics of miR-15a and miR-194 were developed. The research [129] revealed that the GEM-modified miRNA mimics effectively functioned as miRNAs and demonstrated significant efficacy in restraining cell proliferation, inducing cell-cycle arrest, and promoting apoptosis in vitro, with or without the use of a delivery vehicle. Notably, the findings underscore the potential of GEM-miR-15a as a robust therapeutic agent for PDAC in vivo. This study has laid the groundwork for a versatile approach to modifying miRNAs as multi-targeted cancer therapeutics, offering the promise of enhancing current anti-cancer treatments [129].

7.4. Bacteria and Aptamer-Drug Conjugates (ApDCs)

Aptamer-drug conjugates (ApDCs) represent a significant advancement in the domain of nucleic acid-based therapeutic approaches for cancer treatment. Nonetheless, their clinical application has been considerably constrained by several challenges, including the intrinsic instability of nucleic acid drugs in vivo and the avascular characteristics of pancreatic cancer, which is marked by a dense stromal composition. Notably, VNP20009, a genetically modified strain of Salmonella typhimurium, demonstrates a pronounced preference for anaerobic environments. Despite its cytotoxic properties and lack of specificity, this strain possesses the potential to serve as an effective delivery vehicle for ApDCs, thereby augmenting their therapeutic efficacy in the context of complex cancer pathophysiology.

An investigation conducted by Xiao et al. [130] has elucidated a synergistic therapeutic strategy through the covalent attachment of ApDCs to the surface of VNP20009, resulting in the creation of drug-loaded bacteria. This innovative methodology leverages the inherent advantages of both the bacterial delivery vehicles and ApDCs, facilitating targeted delivery to the tumor microenvironment (TME). The intrinsic affinity of bacteria for hypoxic conditions enhances the capacity of ApDCs to penetrate deeply into tumor tissue, thereby augmenting both drug delivery and therapeutic efficacy. Furthermore, the proliferation of bacteria within the tumor microenvironment engenders an immune response, thereby providing a comprehensive therapeutic approach to the treatment of pancreatic cancer. This strategy not only optimizes the delivery of therapeutic agents to the tumor site but also addresses the limitations associated with conventional drug delivery systems in the context of pancreatic cancer treatment, particularly with respect to overcoming the obstacles intrinsic to the TME [130].

8. Conclusions

PDAC constitutes a formidable global health challenge necessitating immediate and effective interventions aimed at improving survival rates. The pathobiology of this malignancy is characterized by a high degree of complexity; nonetheless, the present article delineates fundamental pillars that significantly contribute to the etiology and progression of PDAC. While genetic determinants have historically been regarded as the principal factors, emerging evidence suggests that epigenetic mechanisms and the influence of the microbiome considerably impact the disease’s pathology, thereby presenting opportunities for personalized therapeutic approaches (Figure 4).

The discipline of personalized medicine underscores the importance of exploring innovative strategies that facilitate the identification of individual patient characteristics. Such insights are critical for informing optimal treatment regimens tailored to the distinctive needs of each patient. Recent advancements in genetic methodologies have rendered it increasingly practicable to integrate these innovations into clinical practice.

Furthermore, the microbiome holds a substantial promise for revolutionizing the future of PDAC treatment modalities. Future research endeavors should prioritize the examination of the topics herein discussed, with the intent of engineering the microbiome to establish personalized treatment approaches that are aligned with specific therapeutic objectives.