Via Research Recognition Day Program VCOM-Carolinas 2025

Clinical Educational Research

Can personalized medicine be applied to the combination therapies used to treat Pancreatic Ductal Adenocarcinoma? Jasmine A Williams, OMS-III 1 , Steven A. Enkemann, Ph.D 2 . VCOM-Carolinas, Spartanburg, SC. 2. Institution Name, Dept., City, State. Co-authorship should be given to collaborators such as a Statistician, who have made intellectual contribution in terms of writing, Mentor should be named last in list. Introduction Results Results

Human variation is known to lead to extreme toxicity with 5FU and many new enzymes may have a role in efficacy.

Pancreatic ductal adenocarcinoma (PDAC), which has dismal 5-year survival rating of roughly 10 percent, is quickly becoming a leading cause of cancer related deaths 1 . Often, the initial genetic defect in PDAC occurs in KRAS, which leads to a cascade of events that result in the inactivation of Rb and p53 tumor suppressors. Other genes mutated or activated in the development of PDAC include SMAD4 (mothers against decapentaplegic homolog 4), various hepatocyte growth factors, fibroblast growth factors, and insulin-like growth factor 2 . The combination of mutations that give rise to PDAC is one reason that tumors are difficult to treat. Diagnosis of PDAC often occurs late because early stages are typically asymptomatic. The disease classically manifests with jaundice, scleral icterus, elevated liver enzymes, early satiety, and abdominal pain that radiates to the back although imaging and tumor biopsies are usually required to make an official diagnosis of PDAC. The late stage at which the tumor is detected also contributes to the difficulty in effectively treating PDAC. Management of PDAC depends on the stage of disease at the time of diagnosis 3 . Stage one tumors are clearly resectable and the most likely to be cured 3,4 . Other forms of management include radiation and chemotherapy for more advanced tumors 3,4 . Because tumors are often diagnosed as advanced disease, combination therapy has become the norm for PDAC treatment 4 . This can mean a combination of treatment types, or a combination of drugs used in chemotherapy. The most commonly used chemotherapy combination is FOLFIRINOX (or modified FOLFIRINOX) which consists of 5-Fluorouracil, Folinic acid (leucovorin), irinotecan, and oxaplatin 6 . The decision of which therapy to use is based on the patient’s strength, health status, age, and ability to tolerate treatment. Healthier, stronger patients receive FOLFIRINOX⁶ while those with comorbidities and poorer health states receive a moderate regimen consisting of fewer agents. This difference is due to the toxicities of the multidrug therapy within FOLFIRINOX. The current research was performed to address the possibility of whether tolerance to toxicities could be predicted based on the genetics of the patient. This research sought to identify the role individual enzymes play in the efficacy and toxicity of the individual drugs used in the most common chemotherapy of PDAC.

The most commonly used combination therapy is FOLFIRINOX

5-Fluorouracil

FOLFIRINOX

Enzyme(s)

Effect/Influence

Irinotecan

5-fluorouracil

Leucovorin* Oxaliplatin

Cytidine deaminase (CDA) Thymidine phosphorylase

Inactivate drug to non-cytotoxic forms

Enhancer for 5FU, leads to increased pools of reduced folate cofactors

Mechanism of Action

Topoisomerase I inhibitor

Nucleotide analog that inhibits the enzyme thymidylate synthase. This blocks synthesis of the pyrimidine thymidylate (dTMP), required for DNA replication Nausea, Diarrhea, Anemia, Neutropenia, Bruising/bleeding, sore mouth/sores, increased blood clot risk Metabolism is hepatic. The catabolic metabolism of fluorouracil results in degradation products (e.g., CO2, urea and a-fluoro- β alanine) which are inactive.

Binds preferentially to the guanine and cytosine moieties of DNA, leading to cross-linking of DNA, thus inhibiting DNA synthesis and transcription. Nausea, constipation, diarrhea, anemia, neutropenia, bruising/bleeding, peripheral neuropathy, mouth sores, increased blood clot risk Undergoes nonenzymatic conversion in physiologic solutions to active derivatives via displacement of the labile oxalate ligand freeing several transient platinum derivatives, which covalently bind with DNA blocking repair enzymes. There is no evidence of cytochrome P450-mediated metabolism in vitro.

Increased TP expression = prognostic for malignancy due to enhanced angiogenesis Increased Uridine phosphorylase expression leads to enhanced neoplastic effect of 5-FU

Uridine phosphorylase

Carboxylase

X Exact mechanism unknown

Thymidylate synthase (TYMS)

Fluorouracil covalently binds TYMS to inhibit conversion of uracil leads to thymidylate, thus blocks DNA & RNA synthesis & leads to cell death; incorporation into RNA in place of uridine triphosphare (UTP) blocks RNA processing & protein synthesis

Side effects rare; may have high fever, but most likely side effects are from the chemotherapy drug, not the folinic acid It is metabolized by intestinal and hepatic mucosa with the main metabolite being active 5 methyl tetrahydrofolate

Side Effects

Peripheral neuropathy

Methylene Tetrahydrofolate Reductase (MTHFR)

Increased MTHFR leads to increased sensitivity to 5-FU based therapy

Orotate phosphoribosyltransferase (OPRT)

Overexpression of OPRT enhances susceptibility to 5-FU, but downregulation of OPRT may simulate synthesis of FdUMP leading to DNA dysfunction and increased susceptibility to 5-FU

CMPK1 CMPK2

CMPK1 participates in second phosphorylation step of drug CMPK2 participates in second phosphorylation step of drug

Metabolism Mostly hepatic metabolism

Glutathione S-transferase P

Alterations (A;A)/(A;G) leads to increased toxicity risk with fluorouracil NDPK phosphorylates drug into triphosphorylated (active) form

where irinotecan is converted to the active metabolite SN-38 by carboxylesterase enzymes and SN-38 is conjugated predominantly by the enzyme UDP-glucuronosyl transferase 1A1 (UGT1A1) to form an excreted glucuronide metabolite.

Nucleoside diphosphate kinase (NDPK) ATP-binding cassette subfamily C member 4 (ABCC4)

Decreased expression of ABCC4 on tumor cells leads to enhanced sensitivity to 5-FU chemo (due to ABCC4 not being able to pump chemo out of tumor cells) Tegafur is metabolized to 5-FU by CYP2A6; Variations to CYP2A6 leads to decreased or no enzyme activity leads to poor metabolism and production of 5-FU

CYP2A6

Dihydropyrimidine dehydrogenase (DPD; DPYD)

Catalyzes the first step in detoxifying 5FU; Mutations leads to decreased metabolism of 5-FU leads to increased toxicity

Table 3. Enzymes involved in 5-Fluorouracil metabolism and ultimately the therapeutic effects. Enzymes of importance include variants of MTHFR, TYMS, Glutathione S-transferase P, and DPYD, which result in increased risk for toxicity and possibly death. This list includes many newer enzymes involved in metabolism, such as the Carboxylase enzymes, that require further research as their exact mechanism of action within the metabolism of 5FU is unknown. Irinotecan was the first drug recognized to be influenced by human variation. Toxicity is now known to depend on variation in many different enzymes.

Table 1. Individual components of the FOLFIRINOX combination therapy and their metabolism and possible side effects. Although each drug has a different mechanism of action, the drugs collectively result in the inhibition of cell replication by affecting DNA replication. Irinotecan has the least associated side effects, with the greatest quantity of effects occurring from 5-FU and Oxaliplatin toxicity. 5-FU and Oxaliplatin have overlapping associated side effects, making these the most commonly reported with FOLFIRINOX use. *It is worth noting that although Leucovorin is utilized in FOLFIRINOX, Leucovorin is not a chemotherapy drug and only functions to enhance the effectiveness of 5-FU.

Human variation in many different enzymes could affect Oxaliplatin efficacy.

Methods

Irinotecan

Oxaliplatin

Enzyme(s)

Effect/Influence

Enzyme(s)

Effect/Influence

Literature searches were conducted using PubMed and Google Scholar databases for published articles that have examined the use of FOLFIRINOX and other treatment options for PDAC treatment. The individual agents that comprise FOLFIRINOX (5-Fluorouracil (5-FU), Folinic acid (leucovorin), irinotecan, and oxaliplatin) were reviewed for the enzymes involved in their metabolism to determine whether genetic variation may attribute to rapid or poor metabolism of FOLFIRINOX, which could result in suboptimal treatment.

Table 2. Enzymes involved in oxaliplatin metabolism. This chart shows important enzymes such as SLC31A1, OCT-1, OCT-2, and RAD50, where human variants result in increased risk for toxicity and adverse side effects, while others influence tumor efficacy, such as ERCC1,2, and 5. In addition, some enzymes, such as FAF1 and VSNL1, require further research as their exact mechanism of action within the metabolism of Oxaliplatin is unknown. Downregulation leads to decreased intracellular platinum levels; Rs10981694 leads to increased ototoxicity w/ cisplatin; Rs7851395 leads to renoprotective effects OCT-1/2, aka SLC22A1/SLC22A2 enhance uptake and cytotoxicity; overexpression leads to increased intracellular platinum levels; Competitive inhibition w/ atropine leads to decreased intracellular oxaliplatin; SLC22A2 is associated w/ nephrotoxicity and ototoxicity; Oxaliplatin peripheral neuropathy SLC31A2 [CTR2] Downregulation leads to higher intracellular concentration (less drug removed) hCTR1 (human copper transporter 1) CTR1 leads to oxaliplatin uptake into the cells ATP7A Overexpression leads to platinum resistance; Absence leads to increased efficacy of platinum agents ATP7 Absence leads to improved chemotherapeutic effect ERCC1, ERCC2, ERCC5 Overexpression leads to platinum resistance RAD50 Deficiency leased to increased cisplatin toxicity and efficacy BRCA1 Deficiency/mutation leads to decreased recognition of platinum-adducts, increased efficacy Sec61 Sec61 knockout leads to increased resistance to platinum therapy MMP7 tumor cells have overexpression of MMP7 lead to chemo resistance to oxaliplatin c-FLIP Downregulation of c-FLIP leads to enhanced chemo induced apoptosis (increased c-FLIP leads to chemoresistance) FOS Oxaliplatin activates FOS signaling FOSB Oxaliplatin activates FOSB FOSL1 / FOSL2 Oxaliplatin activates FOSL1 & FOSL2 NOTCH Oxaliplatin activates NOTCH signaling FAF1 X Exact mechanism unknown VSNL1 X Exact mechanism unknown SLC31A1 [CTR1]

CYP3A4 CYP3A5

poor metabolism leads to higher active metabolite poor metabolism leads to higher active metabolite rapid metabolism leads to higher active metabolite rapid metabolism leads to higher active metabolite poor metabolism leads to higher active metabolite poor metabolism leads to higher active metabolite

CES1 CES2

UGT1A1 UGT1A8

Results

Table 4. Enzymes involved in Irinotecan metabolism and how altered metabolism leads to toxicity. Enzymes resulting in “higher active metabolite” increases the risk for Irinotecan toxicity and its associated adverse effect of peripheral neuropathy.

Conclusions The metabolism of pharmaceutical drugs is becoming increasingly important in prescribing decisions. Despite many chemotherapy agents being influenced by the standard CYP enzymes, few prescribing decisions are made based on the genetic make-up of the patient. As chemotherapy continues to trend towards combination therapy, the importance of genetics in the prescription process holds great significance. Tolerance to FOLFIRINOX involves tolerance to 4 different drugs. Current research indicates that at minimum 13, and as many as 41 different enzymes, should be evaluated for poor or ultrarapid metabolism when FOLFIRINOX is prescribed. The enzymes to consider include CES1-2, UGT1A1, and UGT1A8 for Irinotecan metabolism; TYMS, DPYD, and glutathione s-transferase for 5-FU metabolism; and SLC31A1, ATP7B, and RAD50 in oxaliplatin metabolism, in addition to several CYP enzymes. In researching these enzymes, identification of candidates who may encounter the greatest challenges with FOLFIRINOX therapy can be identified and receive PDAC management with greater tolerability and success.

The main source of patient toxicity to chemotherapy results from an increased concentration of the active compound within the patient. This can occur by rapid activation of the infused compound to its active form, or by slow clearance of the active compound due to poor metabolism and/or excretion. Research has expanded beyond the traditional liver CYP enzymes as indicated in the following tables.

References

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