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Genetic manipulations with chemotherapy in pancreatic cancer
CHAPTER 7
Genetic manipulations with chemotherapy in pancreatic cancer Himanshu Tillu1, Pallaval Veera2 Bramhachari2 1
Sysmex India Private Limited, Mumbai, India; Department of Biotechnology, Krishna University, Machilipatnam, Andhra Pradesh, India
Abstract Pancreatic cancers are some of the most life-threatening malignancies plaguing the human race. The aggressiveness of the tumors, inherent genetic resistance to tumors, and late-stage diagnosis make it challenging to contain pancreatic tumors. The established chemotherapeutic regimen has been unable to bring down the high mortality rate associated with pancreatic ductal adenocarcinoma, necessitating newer therapeutic options. Altered genetic landscapes of tumors enabling tumor cells resist chemotherapy, which has been thought to be an insurmountable hurdle. Advances in gene transfer technologies have made alterations in the tumor genetic landscape technically and commercially feasible. Most strategies have focused on sensitizing tumor cells for chemotherapy by adenoviral transfer of relevant genes. Some of these strategies have reached to the clinical trial stage, where they are being evaluated with regard to their safety and efficacy. This review summarizes certain approaches of genetic manipulation aimed at improving the effectiveness of chemotherapy and thus the disease management of pancreatic cancers.
Keywords: Chemotherapy; Genetic manipulation; Pancreatic cancer; Tumor.
List of abbreviations BRCA1 Breast cancer type 1 CP450 Cytochrome P450 CPA Cyclophosphamide DCK:UMK Deoxycytidine kinaseeuridyl monophosphate kinase HSV-TK Herpes simplex virus type 1 thymidine kinase IPA Iphosphamide PDAC Pancreatic ductal adenocarcinoma PM Phosphoramide mustard RGD Arginine-glycine-aspartate SSTR2 Somatostatin receptor subtype 2 gene TNF-a Tumor necrosis factor-a TRAIL Tumor necrosis factorerelated apoptosis-inducing ligand G.P. Nagaraju, S. Ahmad (eds.) Theranostic Approach for Pancreatic Cancer ISBN 978-0-12-819457-7 https://doi.org/10.1016/B978-0-12-819457-7.00007-4
Copyright © 2019 Elsevier Inc. All rights reserved.
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Introduction Most malignant pancreatic neoplasms are adenocarcinoma, specifically pancreatic ductal adenocarcinoma (PDAC). Neuroendocrine tumors, acinar and colloid carcinomas, pancreatoblastomas, and solid-pseudopapillary neoplasms [1] are rare cancers. The incidence of PDAC is predicted to skyrocket to 4,20,000 by 2020, culminating in increasing death cases [2]. PDAC is currently the fourth leading cause of cancer-related mortality and has the potential to become the second most important cause in the next decade [3]. Tobacco smoking [4], obesity, a sedentary lifestyle [5,6]; a high intake of saturated fats, processed and red meats and a low intake of vegetables [7,8]; heavy, immoderate alcohol consumption [9]; type 2 diabetes mellitus [1]; and chronic pancreatitis [10] are predisposing factors for pancreatic cancers and account for 25%e33% of cases [1]. Certain genetic landscapes also predispose an individual to pancreatic cancer and include mutations in genes such as breast cancer type 1 (BRCA1) and BRCA2, cyclindependent kinase inhibitor 2A, ataxia-telangiectasia mutated, serine/threonine kinase 11, serine protease 1, MutL homolog 1, partner and localizer of BRCA2 [1], and the ABO blood group locus [11]. A familial history of pancreatic cancer has been observed in 10% of cases [12]. Around 7% of cases harbor mutations in DNA repair genes, and poly(adenosine phosphate-ribose) polymerase inhibitors are thought to be beneficial for these cases [13].
Pancreatic cancer: unique challenges The prognosis in pancreatic cancer is extremely poor (<7% 5-year survival rate) [14] for several reasons. First, it is diagnosed only in late stages owing to nonspecificity or, in some cases, the lack of symptoms, an unavailability of specific biomarkers, and difficulties in imaging early-stage tumors. Second, PDAC is aggressive, with perineural and vascular and early distant metastases that preclude surgical intervention. Third, PDAC tumors have dense and complex microenvironments with genetically and epigenetically altered cells displaying remarkable resistance to nonsurgical treatment options such as chemotherapy, radiotherapy, and molecular targeted therapies [15]. Genetic analyses revealed that PDAC is characterized by numerous exonic alterations in proteins involved in several signaling pathways; alterations vary significantly between patients. Moreover,
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the heterogeneity of primary tumors impedes the development of new chemotherapeutic agents [13,16,17]. Despite the different treatment alternatives, survival rates for PDAC have not improved for several decades[18].
Therapy Disease management in cases of pancreatic cancer is a formidable challenge because most patients present with locally advanced disease with vascular involvement and widespread metastases generally to the liver and peritoneum. More than 80% of cases have unresectable disease. In the few patients for which surgical intervention is a viable option, 80% of such cases relapse and finally succumb to the disease [1]. In 1997, gemcitabine, a nucleoside analogue, became a frontline drug for treating PDAC when it was approved by the Food and Drug Administration (FDA). Although gemcitabine did not make a significant impact on the survival of PDAC patients, the drug proved to be clinically beneficial from the point of view of symptom management [19]. In 2005, the combination of erlotinib and gemcitabine was approved by the FDA owing to a randomized trial that demonstrated improvement over gemcitabine monotherapy [20]. Furthermore, a combination of 5-fluorouracil, folinic acid (leucovorin), oxaliplatin, and irinotecan (FOLFIRINOX) demonstrated robust activity compared with gemcitabine monotherapy [21]. However, this regimen had significant toxicities, with patients presenting with nausea, diarrhea, fatigue, neuropathy, and myelosuppression. Symptoms were only partially controlled by antidiarrheal and antiemetic drugs. Hence, FOLFIRINOX is administered to patients only age 76 years of age or younger who have excellent performance. The regimen of gemcitabine and albumin-bound paclitaxel was introduced in 2012 after FDA approval, when the combination showed improved performance over gemcitabine alone. However, toxicities with this therapy are also significant: nausea, myelosuppression, fatigue, alopecia, and neuropathy. Nevertheless, the side effects are readily reversible with discontinuation of NAB-paclitaxel and a subsequent reduction in dose [22]. Several other regimens have been investigated, such as gemcitabine and cisplatin for patients with DNA repair mutations in their germline [23], gemcitabine, and capecitabine, based on a study showing significant improvement in progression-free survival [24], and fixed-dose rate gemcitabine, taxotere, and capecitabine [25]. Pharmacokinetic optimization of gemcitabine dose has also been attempted [26].
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The choice of second-line therapy is dictated by the choice of first-line drugs. A gemcitabine-based regimen is chosen if FOLFIRINOX was initially used. A fluorinated pyrimidine-based regimen is selected if gemcitabine and albumin-bound paclitaxel were initially used. The FDA has approved the combination of liposomal irinotecan and 5-fluorouracil and leucovorin after first-line gemcitabine treatment [27].
Newer therapeutic directions Extensive pharmaceutical research has not yet been able to circumvent these issues and confer a significant survival advantage to patients. Certain out of the box strategies have aimed at scaling these seemingly unsurmountable barriers by synergizing gene therapy with chemotherapy. Advanced genetic research has taken two main directions: assisted killing of tumor cells and sensitizing PDAC tumors to chemotherapy. The strategies are summarized in Fig 7.1.
Figure 7.1 Gene therapy and chemotherapy synergistic strategies. The genetically modified viral vectors cooperate with gemcitabine to mediate killing of pancreatic ductal adenocarcinoma tumor cells. The gene product cytochrome P450 (CP450) and the fusion gene product CYL-02 convert chemotherapeutic drugs into toxic forms. CPA, cyclophosphamide; GM, gemcitabine; IPA, iphosphamide; PDAC, pancreatic ductal adenocarcinoma; RGD, arginine-glycine-aspartate; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
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Assisted killing of tumor cells Oncolytic adenovirus mutants devoid of antiapoptotic E1B19K gene in combination with gemcitabine demonstrated increased pancreatic cancer cell killing by enhancing drug-induced apoptosis in xenografted mice. Killing of normal cells was not observed, which suggested that this combinatorial therapy could be well-tolerated [28]. The combinatorial therapy of hyaluronidase-expressing, replication-proficient adenovirus (VCN-01) with gemcitabine and Abraxane (Celgene, Uxbridge, UK) is being evaluated as a part of a clinical trial [NCT02045602]. VCN-01 presents enhanced infectivity through a modified fiber containing the RGD motif and an improved distribution through the expression of a soluble hyaluronidase (PH20) [29]. Hyaluronidase allows for greater penetration of several drugs including gemcitabine [30]. As in any other disorder, cytokines occupy a central niche in immunity to pancreatic cancers. True to its name, tumor necrosis factor-a (TNF-a) exerts antitumoral activity through TNF-a receptor 1 in the context of pancreatic cancer. Considering the systemic cytotoxicity of TNF-a, the strategy of producing a higher concentration of TNF-a within the tumor was thought to be more beneficial [31]. Intratumoral delivery of human TNF-a gene combined with gemcitabine delayed the growth of human pancreatic xenograft tumors relative to either agent alone in vivo [31]. A promising product was developed employing such a strategy: TNFerade [32]. TNFerade involved an AdEgr.TNF.11D construct in which human TNF-a cDNA completely replaces the E1 and E4 genes and partially replaced the E3 gene and is placed under the control of early growth response promoter. Gemcitabine was able to induce the expression of TNF-a in human pancreatic cancer cell lines MiaPaca2 and Panc1 infected with the AdEgr.TNF.11D construct. Gemcitabine in conjunction with AdEgr.TNF.11D markedly delayed tumor growth in nude mice grafted with human pancreatic cancer cell lines MiaPaca2 and AsPC1. The effect was significantly stronger than AdEgr.TNF.11D or gemcitabine alone, although it was not statistically significant. The dose used in the treatment was smaller than that used in actual treatment. Hence, at higher doses, the difference could be statistically significant. The combination therapy was tolerated well in the mice tested [31]. The combination therapy of gemcitabine plus TNFerade had raised the hopes of the pharmaceutical world. However, these hopes were dashed when it was concluded from the
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randomized phase III multiinstitutional study conducted that year that the regimen of standard of care plus TNFerade was safe but inefficacious. A gene therapy construct prepared on an adenoviral backbone by replacing the E1 gene of adenovirus by the TNF-related apoptosis-inducing ligand (TRAIL) placed under the control of human telomerase reverse transcriptase promoter has been shown to be a promising target for treating pancreatic cancer [33,34]. The vector is further modified by inserting the arginine-glycine-aspartate (RGD) motif in the H1 loop of the adenoviral fiber protein, which increases the transduction efficiency of the vector [35,36]. Gemcitabine and Ad/TRAIL-F/RGD displayed a synergistic effect in vitro on cell death AsPC-1 and Capan-1 pancreatic cancer cell lines. Cell death was caused by the induction of apoptosis. Gemcitabine and Ad/ TRAIL-F/RGD treatment of nude mice suppressed the growth of tumors induced by Capan-1 cells in the liver. The reduction in tumor site in the gemcitabine plus Ad/TRAIL-F/RGDetreated animals was significantly more pronounced than either of the therapeutic agents alone. Gemcitabine and Ad/TRAIL-F/RGD therapy was well-tolerated by all animals tested, as inferred by the normal levels of liver enzymes in these animals. Moreover, no treatment-related death was observed in these animals [33]. A phase I trial in metastatic cancer cases composed of intratumoral injection of two suicide fusion genes, yeast cytosine deaminase and a mutant form of herpes simplex virus type 1 thymidine kinase (HSV-1 TKSR39) and interleukin-12 on an adenoviral backbone with 5-fluorocytosine treatment is in progress. The primary end point is considered to be cytotoxicity at day 21. The intensity, persistence, and biodistribution of HSV-1 TK gene expression and the association of immunological parameters (cytokine levels and natural killer cytolytic activity) will be evaluated and correlated with cytotoxicity and clinical outcome (NCT03281382). The combination therapy of gemcitabine and Reolysin was found to induce reovirus replication, endoplasmic reticulum stress, and apoptosis in tumor cells in a single PDAC case. Although the tumor did not regress, the patient had stable disease for 2 years and reported symptomatic improvement with regard to cancer-related pain [37].
Sensitizing pancreatic ductal adenocarcinoma tumors to chemotherapy Cytochrome P450 (CP450) gene product was demonstrated to have the ability to render pancreatic cancer cells responsive to chemotherapeutic
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prodrugs cyclophosphamide (CPA) or iphosphamide (IPA). CP450 can convert CPA or IPA to phosphoramide mustard (PM), a toxic metabolite that is an alkylating agent responsible for generating DNA cross-links in a cell cycleeindependent manner. Phosphoramide is particularly lethal to dividing cells, a feature thought to confer tumor specific cytotoxicity. However, because CP450 is predominantly produced in the liver, toxicity of PM is systemic and not tumor-specific. Therefore, it was thought that intratumoral introduction of the CYP gene could be used to induce tumor regression. An in vitro study by Hlavaty et al. (2012) showed that retroviral transduction of CP450 2B1 enzymes led to an increase in the susceptibility of pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Hs-766T, PaCa-44, and PANC-1) to IPA for up to 13-fold [38]. The results of the in vitro studies were recapitulated in in vivo studies in a mouse model and human clinical trials as well. Retroviral CP450 2B1 transfer combined with CPA treatment of mice harboring pancreatic cancer cells NP-9, NP-18, and NP-31 led to a significant reduction in tumor volume compared with CPA treatment alone [39]. Furthermore, in a tumor-bearing mouse model, encapsulated cells genetically programmed to express CP450 could lead to significant tumor reduction when introduced intratumorally [40,41]. In addition, phase I/II trials in inoperable pancreatic carcinoma patients using these encapsulated cells were remarkably successful, with a doubled median survival and threefold increase in 1-year survival [42]. Some of the experimental therapies have reached clinical trial stage. Noteworthy are the Thergap trials, which deal with chemosensitization of PDAC tumors to gemcitabine. The strategy was intratumoral transfer of complementary somatostatin receptor subtype 2 gene (SSTR2) and deoxycytidine kinaseeuridyl monophosphate kinase (DCK:UMK) genes to inhibit PDAC tumor growth. The choice of genes was based on previous studies by several authors citing antitumor effects of SSTR2 and DCK:UMK upon in situ gene transfer [43e45]. The SSTR2 gene product had also demonstrated antiangiogenic properties and mediated chemosensitization of PDAC cells to gemcitabine. The DCK:UMK fusion gene product was able to phosphorylate gemcitabine, which is toxic to tumor cells. A clinical-grade gene therapy product, CYL-02, was developed by the authors; it is essentially a plasmid DNA encoding SSTR2 and DCK:UMK complexed to a synthetic polycationic carrier, polyethylenimine, with antineoplastic adjuvant. CYL-02 was found to be well-tolerated in advanced PDAC patients and was able to reduce tumor volume and cancer biomarker levels and contain metastatic spread. The median overall and
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progression-free survival rates were also significantly improved [46]. A Phase II trial is now under way (NCT02806687).
Conclusions and future perspectives Treatment of pancreatic cancer is fraught with several complications, such as disease too far progressed for surgical intervention, chemotherapeutic drugs unable to prolong survival or afford symptomatic improvements, and significant toxicities associated with conventional therapeutic drugs. With conventional therapies failing to improve disease outcome for most patients, the search for alternate or supplementary strategies has commenced with great urgency. One avenue explored is that of gene therapy. Despite the promise it has shown since its inception, gene therapy has been unable to gain momentum owing to several technical issues. Improper targeting and inadequate penetrance of the vector leading to loss in efficiency, lack of proper regulation of the inserted gene, and the high cost of procedures are some obstacles. Nevertheless, with advances in genetic modification techniques and technologies, the advent of gene therapy in mainstream medicine is imminent. In the context of pancreatic cancer, the objective of gene therapy is to increase the efficacy of conventional chemotherapy. It attempts to do so by sensitizing PDAC cells to chemotherapy and inducing apoptosis. Some strategies also aim to harness the antitumor effects of cytokines. The synergy of drugs and gene therapy has demonstrated an improved outcome in most cases. However, most strategies have been unable to reduce the toxicity of the chemotherapeutic regimen. Hence, it is likely that future research will aim to make chemotherapy safer for administration. Lentiviral transduction of the P140K mutant form of O6-methylguanine-DNA-methyl transferase and multi-drug resistance 1 in primary human hematopoietic stem cells afforded significant protection against the combination therapies of O(6)-BG/temozolomide and paclitaxel [47] and carmustine (bis-chloroethylnitrosourea) and doxorubicin [48]. Thus, it may be possible to use higher doses for the treatment, as was demonstrated in mice harboring transplanted allogenic stem cells [49]. Therefore, in the context of inoperable PDAC, gene therapy can partner chemotherapy to orchestrate the killing of tumor cells and mitigate the toxicity of the conventional therapeutic regime.
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Further reading Chopra M, Lang I, Salzmann S, Pachel C, Kraus S, Bäuerlein CA, Brede C, Garrote ALJ, Mattenheimer K, Ritz M, Schwinn S. Tumor necrosis factor induces tumor promoting and anti-tumoral effects on pancreatic cancer via TNFR1. PLoS One 2013;8(9):e75737. Jacob ST, Motiwala T. Epigenetic regulation of protein tyrosine phosphatases: potential molecular targets for cancer therapy. Cancer Gene Therapy 2005;12(8):665. Kleeff J, Korc M, Apte M, La Vecchia C, Johnson CD, Biankin AV, Neale RE, Tempero M, Tuveson DA, Hruban RH, Neoptolemos JP. Pancreatic cancer. Nat Rev Dis Prim 2016;2:16022. Thompson CB, Shepard HM, O’Connor PM, Kadhim S, Jiang P, Osgood RJ, Bookbinder LH, Li X, Sugarman BJ, Connor RJ, Nadjsombati S. Enzymatic depletion of tumor hyaluronan induces antitumor responses in preclinical animal models. Mol Cancer Ther 2010:1535e7163.
Genetic manipulations with chemotherapy in pancreatic cancer Himanshu Tillu1, Pallaval Veera2 Bramhachari2 1
Sysmex India Private Limited, Mumbai, India; Department of Biotechnology, Krishna University, Machilipatnam, Andhra Pradesh, India
Abstract Pancreatic cancers are some of the most life-threatening malignancies plaguing the human race. The aggressiveness of the tumors, inherent genetic resistance to tumors, and late-stage diagnosis make it challenging to contain pancreatic tumors. The established chemotherapeutic regimen has been unable to bring down the high mortality rate associated with pancreatic ductal adenocarcinoma, necessitating newer therapeutic options. Altered genetic landscapes of tumors enabling tumor cells resist chemotherapy, which has been thought to be an insurmountable hurdle. Advances in gene transfer technologies have made alterations in the tumor genetic landscape technically and commercially feasible. Most strategies have focused on sensitizing tumor cells for chemotherapy by adenoviral transfer of relevant genes. Some of these strategies have reached to the clinical trial stage, where they are being evaluated with regard to their safety and efficacy. This review summarizes certain approaches of genetic manipulation aimed at improving the effectiveness of chemotherapy and thus the disease management of pancreatic cancers.
Keywords: Chemotherapy; Genetic manipulation; Pancreatic cancer; Tumor.
List of abbreviations BRCA1 Breast cancer type 1 CP450 Cytochrome P450 CPA Cyclophosphamide DCK:UMK Deoxycytidine kinaseeuridyl monophosphate kinase HSV-TK Herpes simplex virus type 1 thymidine kinase IPA Iphosphamide PDAC Pancreatic ductal adenocarcinoma PM Phosphoramide mustard RGD Arginine-glycine-aspartate SSTR2 Somatostatin receptor subtype 2 gene TNF-a Tumor necrosis factor-a TRAIL Tumor necrosis factorerelated apoptosis-inducing ligand G.P. Nagaraju, S. Ahmad (eds.) Theranostic Approach for Pancreatic Cancer ISBN 978-0-12-819457-7 https://doi.org/10.1016/B978-0-12-819457-7.00007-4
Copyright © 2019 Elsevier Inc. All rights reserved.
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Introduction Most malignant pancreatic neoplasms are adenocarcinoma, specifically pancreatic ductal adenocarcinoma (PDAC). Neuroendocrine tumors, acinar and colloid carcinomas, pancreatoblastomas, and solid-pseudopapillary neoplasms [1] are rare cancers. The incidence of PDAC is predicted to skyrocket to 4,20,000 by 2020, culminating in increasing death cases [2]. PDAC is currently the fourth leading cause of cancer-related mortality and has the potential to become the second most important cause in the next decade [3]. Tobacco smoking [4], obesity, a sedentary lifestyle [5,6]; a high intake of saturated fats, processed and red meats and a low intake of vegetables [7,8]; heavy, immoderate alcohol consumption [9]; type 2 diabetes mellitus [1]; and chronic pancreatitis [10] are predisposing factors for pancreatic cancers and account for 25%e33% of cases [1]. Certain genetic landscapes also predispose an individual to pancreatic cancer and include mutations in genes such as breast cancer type 1 (BRCA1) and BRCA2, cyclindependent kinase inhibitor 2A, ataxia-telangiectasia mutated, serine/threonine kinase 11, serine protease 1, MutL homolog 1, partner and localizer of BRCA2 [1], and the ABO blood group locus [11]. A familial history of pancreatic cancer has been observed in 10% of cases [12]. Around 7% of cases harbor mutations in DNA repair genes, and poly(adenosine phosphate-ribose) polymerase inhibitors are thought to be beneficial for these cases [13].
Pancreatic cancer: unique challenges The prognosis in pancreatic cancer is extremely poor (<7% 5-year survival rate) [14] for several reasons. First, it is diagnosed only in late stages owing to nonspecificity or, in some cases, the lack of symptoms, an unavailability of specific biomarkers, and difficulties in imaging early-stage tumors. Second, PDAC is aggressive, with perineural and vascular and early distant metastases that preclude surgical intervention. Third, PDAC tumors have dense and complex microenvironments with genetically and epigenetically altered cells displaying remarkable resistance to nonsurgical treatment options such as chemotherapy, radiotherapy, and molecular targeted therapies [15]. Genetic analyses revealed that PDAC is characterized by numerous exonic alterations in proteins involved in several signaling pathways; alterations vary significantly between patients. Moreover,
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the heterogeneity of primary tumors impedes the development of new chemotherapeutic agents [13,16,17]. Despite the different treatment alternatives, survival rates for PDAC have not improved for several decades[18].
Therapy Disease management in cases of pancreatic cancer is a formidable challenge because most patients present with locally advanced disease with vascular involvement and widespread metastases generally to the liver and peritoneum. More than 80% of cases have unresectable disease. In the few patients for which surgical intervention is a viable option, 80% of such cases relapse and finally succumb to the disease [1]. In 1997, gemcitabine, a nucleoside analogue, became a frontline drug for treating PDAC when it was approved by the Food and Drug Administration (FDA). Although gemcitabine did not make a significant impact on the survival of PDAC patients, the drug proved to be clinically beneficial from the point of view of symptom management [19]. In 2005, the combination of erlotinib and gemcitabine was approved by the FDA owing to a randomized trial that demonstrated improvement over gemcitabine monotherapy [20]. Furthermore, a combination of 5-fluorouracil, folinic acid (leucovorin), oxaliplatin, and irinotecan (FOLFIRINOX) demonstrated robust activity compared with gemcitabine monotherapy [21]. However, this regimen had significant toxicities, with patients presenting with nausea, diarrhea, fatigue, neuropathy, and myelosuppression. Symptoms were only partially controlled by antidiarrheal and antiemetic drugs. Hence, FOLFIRINOX is administered to patients only age 76 years of age or younger who have excellent performance. The regimen of gemcitabine and albumin-bound paclitaxel was introduced in 2012 after FDA approval, when the combination showed improved performance over gemcitabine alone. However, toxicities with this therapy are also significant: nausea, myelosuppression, fatigue, alopecia, and neuropathy. Nevertheless, the side effects are readily reversible with discontinuation of NAB-paclitaxel and a subsequent reduction in dose [22]. Several other regimens have been investigated, such as gemcitabine and cisplatin for patients with DNA repair mutations in their germline [23], gemcitabine, and capecitabine, based on a study showing significant improvement in progression-free survival [24], and fixed-dose rate gemcitabine, taxotere, and capecitabine [25]. Pharmacokinetic optimization of gemcitabine dose has also been attempted [26].
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The choice of second-line therapy is dictated by the choice of first-line drugs. A gemcitabine-based regimen is chosen if FOLFIRINOX was initially used. A fluorinated pyrimidine-based regimen is selected if gemcitabine and albumin-bound paclitaxel were initially used. The FDA has approved the combination of liposomal irinotecan and 5-fluorouracil and leucovorin after first-line gemcitabine treatment [27].
Newer therapeutic directions Extensive pharmaceutical research has not yet been able to circumvent these issues and confer a significant survival advantage to patients. Certain out of the box strategies have aimed at scaling these seemingly unsurmountable barriers by synergizing gene therapy with chemotherapy. Advanced genetic research has taken two main directions: assisted killing of tumor cells and sensitizing PDAC tumors to chemotherapy. The strategies are summarized in Fig 7.1.
Figure 7.1 Gene therapy and chemotherapy synergistic strategies. The genetically modified viral vectors cooperate with gemcitabine to mediate killing of pancreatic ductal adenocarcinoma tumor cells. The gene product cytochrome P450 (CP450) and the fusion gene product CYL-02 convert chemotherapeutic drugs into toxic forms. CPA, cyclophosphamide; GM, gemcitabine; IPA, iphosphamide; PDAC, pancreatic ductal adenocarcinoma; RGD, arginine-glycine-aspartate; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
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Assisted killing of tumor cells Oncolytic adenovirus mutants devoid of antiapoptotic E1B19K gene in combination with gemcitabine demonstrated increased pancreatic cancer cell killing by enhancing drug-induced apoptosis in xenografted mice. Killing of normal cells was not observed, which suggested that this combinatorial therapy could be well-tolerated [28]. The combinatorial therapy of hyaluronidase-expressing, replication-proficient adenovirus (VCN-01) with gemcitabine and Abraxane (Celgene, Uxbridge, UK) is being evaluated as a part of a clinical trial [NCT02045602]. VCN-01 presents enhanced infectivity through a modified fiber containing the RGD motif and an improved distribution through the expression of a soluble hyaluronidase (PH20) [29]. Hyaluronidase allows for greater penetration of several drugs including gemcitabine [30]. As in any other disorder, cytokines occupy a central niche in immunity to pancreatic cancers. True to its name, tumor necrosis factor-a (TNF-a) exerts antitumoral activity through TNF-a receptor 1 in the context of pancreatic cancer. Considering the systemic cytotoxicity of TNF-a, the strategy of producing a higher concentration of TNF-a within the tumor was thought to be more beneficial [31]. Intratumoral delivery of human TNF-a gene combined with gemcitabine delayed the growth of human pancreatic xenograft tumors relative to either agent alone in vivo [31]. A promising product was developed employing such a strategy: TNFerade [32]. TNFerade involved an AdEgr.TNF.11D construct in which human TNF-a cDNA completely replaces the E1 and E4 genes and partially replaced the E3 gene and is placed under the control of early growth response promoter. Gemcitabine was able to induce the expression of TNF-a in human pancreatic cancer cell lines MiaPaca2 and Panc1 infected with the AdEgr.TNF.11D construct. Gemcitabine in conjunction with AdEgr.TNF.11D markedly delayed tumor growth in nude mice grafted with human pancreatic cancer cell lines MiaPaca2 and AsPC1. The effect was significantly stronger than AdEgr.TNF.11D or gemcitabine alone, although it was not statistically significant. The dose used in the treatment was smaller than that used in actual treatment. Hence, at higher doses, the difference could be statistically significant. The combination therapy was tolerated well in the mice tested [31]. The combination therapy of gemcitabine plus TNFerade had raised the hopes of the pharmaceutical world. However, these hopes were dashed when it was concluded from the
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randomized phase III multiinstitutional study conducted that year that the regimen of standard of care plus TNFerade was safe but inefficacious. A gene therapy construct prepared on an adenoviral backbone by replacing the E1 gene of adenovirus by the TNF-related apoptosis-inducing ligand (TRAIL) placed under the control of human telomerase reverse transcriptase promoter has been shown to be a promising target for treating pancreatic cancer [33,34]. The vector is further modified by inserting the arginine-glycine-aspartate (RGD) motif in the H1 loop of the adenoviral fiber protein, which increases the transduction efficiency of the vector [35,36]. Gemcitabine and Ad/TRAIL-F/RGD displayed a synergistic effect in vitro on cell death AsPC-1 and Capan-1 pancreatic cancer cell lines. Cell death was caused by the induction of apoptosis. Gemcitabine and Ad/ TRAIL-F/RGD treatment of nude mice suppressed the growth of tumors induced by Capan-1 cells in the liver. The reduction in tumor site in the gemcitabine plus Ad/TRAIL-F/RGDetreated animals was significantly more pronounced than either of the therapeutic agents alone. Gemcitabine and Ad/TRAIL-F/RGD therapy was well-tolerated by all animals tested, as inferred by the normal levels of liver enzymes in these animals. Moreover, no treatment-related death was observed in these animals [33]. A phase I trial in metastatic cancer cases composed of intratumoral injection of two suicide fusion genes, yeast cytosine deaminase and a mutant form of herpes simplex virus type 1 thymidine kinase (HSV-1 TKSR39) and interleukin-12 on an adenoviral backbone with 5-fluorocytosine treatment is in progress. The primary end point is considered to be cytotoxicity at day 21. The intensity, persistence, and biodistribution of HSV-1 TK gene expression and the association of immunological parameters (cytokine levels and natural killer cytolytic activity) will be evaluated and correlated with cytotoxicity and clinical outcome (NCT03281382). The combination therapy of gemcitabine and Reolysin was found to induce reovirus replication, endoplasmic reticulum stress, and apoptosis in tumor cells in a single PDAC case. Although the tumor did not regress, the patient had stable disease for 2 years and reported symptomatic improvement with regard to cancer-related pain [37].
Sensitizing pancreatic ductal adenocarcinoma tumors to chemotherapy Cytochrome P450 (CP450) gene product was demonstrated to have the ability to render pancreatic cancer cells responsive to chemotherapeutic
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prodrugs cyclophosphamide (CPA) or iphosphamide (IPA). CP450 can convert CPA or IPA to phosphoramide mustard (PM), a toxic metabolite that is an alkylating agent responsible for generating DNA cross-links in a cell cycleeindependent manner. Phosphoramide is particularly lethal to dividing cells, a feature thought to confer tumor specific cytotoxicity. However, because CP450 is predominantly produced in the liver, toxicity of PM is systemic and not tumor-specific. Therefore, it was thought that intratumoral introduction of the CYP gene could be used to induce tumor regression. An in vitro study by Hlavaty et al. (2012) showed that retroviral transduction of CP450 2B1 enzymes led to an increase in the susceptibility of pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Hs-766T, PaCa-44, and PANC-1) to IPA for up to 13-fold [38]. The results of the in vitro studies were recapitulated in in vivo studies in a mouse model and human clinical trials as well. Retroviral CP450 2B1 transfer combined with CPA treatment of mice harboring pancreatic cancer cells NP-9, NP-18, and NP-31 led to a significant reduction in tumor volume compared with CPA treatment alone [39]. Furthermore, in a tumor-bearing mouse model, encapsulated cells genetically programmed to express CP450 could lead to significant tumor reduction when introduced intratumorally [40,41]. In addition, phase I/II trials in inoperable pancreatic carcinoma patients using these encapsulated cells were remarkably successful, with a doubled median survival and threefold increase in 1-year survival [42]. Some of the experimental therapies have reached clinical trial stage. Noteworthy are the Thergap trials, which deal with chemosensitization of PDAC tumors to gemcitabine. The strategy was intratumoral transfer of complementary somatostatin receptor subtype 2 gene (SSTR2) and deoxycytidine kinaseeuridyl monophosphate kinase (DCK:UMK) genes to inhibit PDAC tumor growth. The choice of genes was based on previous studies by several authors citing antitumor effects of SSTR2 and DCK:UMK upon in situ gene transfer [43e45]. The SSTR2 gene product had also demonstrated antiangiogenic properties and mediated chemosensitization of PDAC cells to gemcitabine. The DCK:UMK fusion gene product was able to phosphorylate gemcitabine, which is toxic to tumor cells. A clinical-grade gene therapy product, CYL-02, was developed by the authors; it is essentially a plasmid DNA encoding SSTR2 and DCK:UMK complexed to a synthetic polycationic carrier, polyethylenimine, with antineoplastic adjuvant. CYL-02 was found to be well-tolerated in advanced PDAC patients and was able to reduce tumor volume and cancer biomarker levels and contain metastatic spread. The median overall and
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progression-free survival rates were also significantly improved [46]. A Phase II trial is now under way (NCT02806687).
Conclusions and future perspectives Treatment of pancreatic cancer is fraught with several complications, such as disease too far progressed for surgical intervention, chemotherapeutic drugs unable to prolong survival or afford symptomatic improvements, and significant toxicities associated with conventional therapeutic drugs. With conventional therapies failing to improve disease outcome for most patients, the search for alternate or supplementary strategies has commenced with great urgency. One avenue explored is that of gene therapy. Despite the promise it has shown since its inception, gene therapy has been unable to gain momentum owing to several technical issues. Improper targeting and inadequate penetrance of the vector leading to loss in efficiency, lack of proper regulation of the inserted gene, and the high cost of procedures are some obstacles. Nevertheless, with advances in genetic modification techniques and technologies, the advent of gene therapy in mainstream medicine is imminent. In the context of pancreatic cancer, the objective of gene therapy is to increase the efficacy of conventional chemotherapy. It attempts to do so by sensitizing PDAC cells to chemotherapy and inducing apoptosis. Some strategies also aim to harness the antitumor effects of cytokines. The synergy of drugs and gene therapy has demonstrated an improved outcome in most cases. However, most strategies have been unable to reduce the toxicity of the chemotherapeutic regimen. Hence, it is likely that future research will aim to make chemotherapy safer for administration. Lentiviral transduction of the P140K mutant form of O6-methylguanine-DNA-methyl transferase and multi-drug resistance 1 in primary human hematopoietic stem cells afforded significant protection against the combination therapies of O(6)-BG/temozolomide and paclitaxel [47] and carmustine (bis-chloroethylnitrosourea) and doxorubicin [48]. Thus, it may be possible to use higher doses for the treatment, as was demonstrated in mice harboring transplanted allogenic stem cells [49]. Therefore, in the context of inoperable PDAC, gene therapy can partner chemotherapy to orchestrate the killing of tumor cells and mitigate the toxicity of the conventional therapeutic regime.
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Further reading Chopra M, Lang I, Salzmann S, Pachel C, Kraus S, Bäuerlein CA, Brede C, Garrote ALJ, Mattenheimer K, Ritz M, Schwinn S. Tumor necrosis factor induces tumor promoting and anti-tumoral effects on pancreatic cancer via TNFR1. PLoS One 2013;8(9):e75737. Jacob ST, Motiwala T. Epigenetic regulation of protein tyrosine phosphatases: potential molecular targets for cancer therapy. Cancer Gene Therapy 2005;12(8):665. Kleeff J, Korc M, Apte M, La Vecchia C, Johnson CD, Biankin AV, Neale RE, Tempero M, Tuveson DA, Hruban RH, Neoptolemos JP. Pancreatic cancer. Nat Rev Dis Prim 2016;2:16022. Thompson CB, Shepard HM, O’Connor PM, Kadhim S, Jiang P, Osgood RJ, Bookbinder LH, Li X, Sugarman BJ, Connor RJ, Nadjsombati S. Enzymatic depletion of tumor hyaluronan induces antitumor responses in preclinical animal models. Mol Cancer Ther 2010:1535e7163.