Preliminary Findings on the Use of Targeted Therapy in Combination with Sodium Phenylbutyrate in Recurrent Advanced Pancreatic Cancer—a Potential Strategy for Improved Survival

Metastatic pancreatic cancer carries an estimated five-year survival rate of only 2%. Gemcitabine-based chemotherapy remains a first-line standard-of-care treatment for elderly patients with advanced pancreatic cancer. Combination chemotherapy FOLFIRINOX offers better results, but it is not recommended for the older patient population due to substantial toxicity. Standard-of-care second-line treatment is not yet established and is used in approximately 30% of patients since performance status is too low to consider further therapy. Targeted therapies with a single agent and in combinations have been tested in numerous clinical trials, but except for the combination of gemcitabine and erlotinib, have not yet proven efficacy. Here, we present preliminary findings of improved overall survival (OS) using a combination of sodium phenylbutyrate with various chemotherapeutic and targeted agents in stage IV A and B pancreatic cancer patients who failed at least one line of chemotherapy. The results suggest a strategy of simultaneous interruption of signal transmission involving multiple pathways in the second-line treatment that are believed to interfere with cell cycle, cancer cell metabolism, autophagy and maintenance of cancer stem cells and promote apoptosis. In this group of patients, median OS was higher compared to other second-line therapies (10.5 months compared to between 2.9 and 6.5 months in other studies, and in the best supportive care group, 2.3 months). Given the understanding that our findings are preliminary, we propose the validation of our initial results using a well-designed Phase I/II trial in recurrent advanced pancreatic cancer.


Introduction
The American Cancer Society (ACS Cancer Statistics 2014) estimates that 39,590 patients will die from the pancreatic cancer and the number of new cases in the United States will increase to 46,420 [1].Projected deaths from pancreatic cancer will surpass breast, prostate, and colorectal cancers and will become the second leading cause of cancer-related death by 2030 [2].Furthermore, for patients with metastatic pancreatic cancer, the five-year survival rate is only 2% and is surpassed only by glioblastoma multiforme (GBM) [3]- [5].Gemcitabine was introduced as a first-line standard-of-care therapy for advanced pancreatic cancer in 1997 [6].Since, a number of phase II and III trials in advanced pancreatic cancer have been conducted, but significant improvement in survival has not been demonstrated, except for FOLFIRINOX and a combination of nab-paclitaxel with gemcitabine [7]- [27].Despite these discouraging statistics, there is optimism with the rapid development of molecular targeted approaches for the treatment of this almost uniformly deadly disease [28] [29].
Standard-of-care second-line treatment in pancreatic cancer is not well-established and is only used in approximately 30% of patients, as positive treatment outcome in such patients is considered too low for further therapy [30].
Sodium phenylbutyrate (PB), the salt of an aromatic fatty acid is used to treat urea cycle disorders [31].The drug, a histone deacetylase (HDAC) inhibitor is being explored in combination with cytotoxics and other novel drugs.Derived from its HDAC activity, PB is being investigated for use as a potential differentiation-inducing agent in malignant glioma, acute promyelocytic leukemia and many other disorders [32]- [34].Bortezomib and the HDAC inhibitors vorinostat, valproic acid and PB showed synergistic cell killing activity of GBM stem-like cells [35].In the liver, PB is metabolized to phenylacetylglutaminate (PG) and phenylacetate (PN).The data from the study of the effect of PG and PN on the GBM genome have shown that these compounds affect approximately 100 genes in the cancer genome [36].
This article provides a brief description of results of treatment of 14 cases of advanced pancreatic cancer patients who had failed first-line therapy and discusses a strategy for personalized targeted therapy for advanced recurrent pancreatic cancer.

Patients and Methods
Subjects were diagnosed with metastatic or locally advanced pancreatic ductal adenocarcinoma (PDA), and after recurrence received their treatment in private practice at Burzynski Clinic (BC) in Houston, TX.Fourteen patients were assessed who had failed at least one standard treatment modality, were diagnosed with Stage IVA or IVB pancreatic cancer and received treatment between November 28, 2007 andAugust 26, 2013.They all represent consecutively treated patients who could be evaluated for response.
Pathology and radiological evaluations prior to and during treatment were performed by institutions not associated with BC, whereas laboratory tests were performed by both outside laboratories and the laboratory at BC. Tests included standard blood and urine evaluations, as well as the determination of tumor and genomic markers.Molecular profiling on tumor tissue was performed by Caris Life Sciences, Phoenix, Arizona.Prior to treatment, all patients were provided details of the treatment and were required to sign an informed consent document.Treatment plans were formulated based on molecular profiling of patients and included the use of PB in combination with targeted and/or chemotherapeutic agents.
After an initial two to four weeks of treatment performed on an outpatient basis under the authority of the BC, treatment was continued under the care of a local oncologist.Prior to treatment initiation, a computerized tomography (CT) scan with and without contrast and in some instances positron emission tomography (PET) scans were performed.The products of the two largest perpendicular diameters (LPD) of measurable lesions were calculated and totaled for each subject, which provided a baseline for determining response to treatment.Pretreatment evaluations also included Karnofsky Performance Status (KPS), vital signs, clinical disease status, demographics, medical history and current medications, physical examination, and electrocardiogram (EKG).Toxicity was evaluated according to the Common Toxicity Criteria for Adverse Events v. 3 (CTCAEv.3).Poten-tial responses to treatment included complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD).CR required the disappearance of all lesions confirmed at the end of four weeks, PR required 50% or higher decrease of the LPD of measurable lesions, PD was determined when there was over 25% increase of the lesions or new lesions, and SD was classified as the status between PR and PD.The duration of each response was measured from the date that the criteria of the outcome were first met and until the date that PD was first documented.In the case of SD, response duration was measured from the time that therapy commenced.

Treatment
Details of dosing and duration of drug administration for patients treated with combinations containing PB and erlotinib are described in Table 3(a) and Table 3

(b).
Seven patients received combination treatment with PB, erlotinib, and additional chemotherapeutic and targeted agents.Two patients received treatment with the single agent PB, without any additional targeted agents, whereas one patient each was given capecitabine and oxaliplatin in addition to PB and erlotinib.Patient 11 who received monotherapy with PB returned to the UK, where he was treated with gemcitabine and radiation therapy under the care of a British physician.Patient 12 was treated with PB, gemcitabine and nab-paclitaxel (Table 3(a) and Table 3(b)).Three out of seven patients with PB + erlotinib received additionally pazopanib, and another two patients were given sorafenib and sirolimus.One other patient received PB and pazopanib with everolimus, vorinostat, BVZ, and capecitabine.The single patient treated with PB and sorafenib was also given bevacizumab and vorinostat and another patient had riluzole added to the treatment.Among those patients treated with pazopanib, one received additional trastuzumab and another dasatinib and gemcitabine.In the group of patients who did not receive erlotinib, two were treated with sorafenib and bevacizumab; one was given additional capecitabine and another trastuzumab and lapatinib.A further patient was treated with PB, pazopanib, everolimus, bevacizumab, and vorinostat, while another with PB, sunitinib, dasatinib and irinotecan.Patients treated with pazopanib, sorafenib, sunitinib, dasatinib, and vorinostat received doses that were 50% -80% lower as compared to their respective maximum recommended dose, thereby minimizing possible combined toxicity of these drugs.

Responses and Survival
PR (14.3%) was observed in two patients, SD (42.9%) was determined in six patients, whereas six patients developed PD.PR and SD were observed in five patients who were treated with combinations containing PB and erlotinib, two patients who were treated with PB and chemotherapy (gemcitabine or gemcitabine/nab-paclitaxel) and one patient who was given PB, sorafenib, sirolimus and BVZ.However, one patient treated with PB, erlotinib, gemcitabine, and nab-paclitaxel and another patient treated with a combination of PB, erlotinib, sorafenib, and sirolimus developed PD.The data are not sufficient to draw definite conclusions, but the results suggest that a successful treatment combination could consist of PB, erlotinib, pazopanib, or sorafenib in combination with a mammalian target of rapamycin (mTOR) inhibitor (sirolimus or everolimus), SRC inhibitor (dasatinib) or human epidermal growth factor receptor 2 (HER-2) inhibitor (trastuzumab) for patients with amplification of

HER-2.
The selection of the targeted and chemotherapy agents was supported by molecular profiling based on increased levels of extracellular domains of HER-2, vascular endothelial growth factor (VEGF), and epidermal growth factor receptor (EGFR) and tissue profiling performed by Caris Life Sciences (through increased expression of platelet derived growth factor receptor type A or B (PDGFRA/B) and negative phosphatase and tensin homolog (PTEN).Conversely to not impressive response data, the OS compares favorably to selected clinical studies in advanced pancreatic cancer treated with second-line therapy (Table 4) [37]- [43].
Median OS of 10.5 months appears significantly higher than in other studies (Figure 1).

Safety and Adverse Events
Grade 2 and 3 adverse drug events (ADEs) are described in Table 5 and are compared with other clinical trials.The most common ADEs observed were hematologic toxicities that included leukopenia and thrombocytopenia, and gastrointestinal effects that involved nausea, vomiting, dyspepsia, and diarrhea.Hypertension, Grade 2 occurred in 7% of patients and increased transaminases and alkaline phosphatase each in 7% of patients.ADEs were easily reversible within a short time.

Discussion
This paper describes an emerging strategy for more successful treatment of advanced pancreatic cancer.The development of PDA depends on sequential involvement of numerous signaling pathways comparable to normal development.The pattern of mutations is complex and may involve as many as 12 different signaling pathways [44].Most PDAs are associated with somatic mutations of Kirsten rat sarcoma viral oncogene homolog (KRAS), cyclin-dependent kinase inhibitor (INK4A), tumor suppressor gene p53 (TP53), and Smoothened (SMO) and MAD protein family member 4 (SMAD4) [45]- [49].The most common is, however, the KRAS mutation found in over 95% of cases.It results in the synthesis of protein that is continuously active and transduces signal to downstream effectors [50].Differing to findings with colon or lung cancers in which activation of EGFR receptors is mutually exclusive with KRAS mutation, EGFR signaling is essential for KRAS driven pancreatic cancer [51].The EGFR inhibitor, erlotinib, is approved for treatment of metastatic pancreatic cancer [15].The overex-    pression of the HER-2 receptor contributes to the worst prognosis of pancreatic cancer [52].This finding was further explored in experiments with pancreatic cancer xenografts, where treatment with monoclonal antibodies against EGFR and HER-2, cetuximab and trastuzumab exhibited antitumor effects [53].Direct inhibition of KRAS has failed to produce responses in pancreatic cancer thus far, and for this reason, attention has shifted to the interruption of signal transduction in RAF-MEK-ERK, and PI3K-AKT pathways [54].RAF inhibition antagonizes the inhibition of MEK and should be avoided.On the other hand, MEK inhibition leads to the activation of AKT and for this reason, both of these should be inhibited simultaneously [55].mTOR inhibitors, sirolimus and everolimus, activate phosphorylation of AKT [55].Erlotinib and dasatinib can help to down-regulate phosphorylation of AKT [56].Sorafenib plus lapatinib and trastuzumab decrease phosphorylation of AKT in cancers expressing HER-2 [57].Dasatinib also decreases phosphorylation of ERK and mitogen-activated protein kinase (MAPK) [56].Genomic studies of PDA revealed that frequently there is simultaneous activation of KRAS-RAF-MEK-ERK and PI3K-AKT pathways [58].In preclinical studies, combination treatment of dual PI3K-mTOR inhibitor, NVP-BEZ235, and HDAC inhibitor, panobinostat, effectively blocked the growth of PDA by interfering with signaling of both pathways [59].Angiogenesis plays a critical role in tumor growth and promotion of metastasis of pancreatic cancer.High expression of VEGF is associated with poor prognosis and liver metastasis in pancreatic cancer which provides rationale for therapeutic use of monoclonal antibodies and tyrosine kinase inhibitors (TKI) against VEGF [60].
In cells of PDA, there is frequent deregulation of embryonic signaling pathways, Hedgehog (Hh) and Wntβ-catenin [61].According to recent studies, Hh signaling is activated in the microenvironment through the autocrine mechanism, but alone is not sufficient to drive the development of PDA [62] [63].This may explain the failure of the Smo inhibitor vismodegib in clinical trials in pancreatic cancer [64].On the other hand, Smo activates Hh targeted genes and its inhibition by vismodegib was instrumental in the successful treatment of medulloblastoma [65].Contrary to medulloblastoma and colon cancer in which upregulated Hh or Wnt can provide the starting event, abnormal Hh or Wnt signaling alone is not sufficient to trigger the development of PDA [65].Increased activity of KRAS differentiates acinar pancreatic cells into ductal epithelial neoplastic cells.Hh and Wnt signaling, which was initially at a low level, is then reactivated and helps PDA maintenance [61].The other signal transduction pathways, Notch and TGFβ, modulate Wnt-β-catenin signaling [66] [67].Hh signaling also supports the maintenance of cancerous stem cells (CSC), which is helped by autocrine VEGF and integrin signaling [68] [69].Pazopanib and sorafenib block signaling of vascular endothelial growth factor receptor 2 (VEGFR2) and metastasis oncogene (MET) and affect the maintenance of CSCs [70].
Additional approaches to control PDA may include interference in cell cycle, cancer cell metabolism, angiogenesis and autophagy, and inhibition of apoptosis.We effectively explored these mechanisms in the treatment of recurrent GBM by using PB in combination with targeted agents [4].
Based on research on molecular mechanisms in PDA and our experience with personalized targeted therapy, we propose the following strategy for improving survival of patients with advanced pancreatic cancer who failed standard chemotherapy including gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, docetaxel, and nab-paclitaxel.This strategy is illustrated in Figure 2 and Figure 3 and consists of simultaneous interference in signal transmission in multiple pathways: 1) KRAS-MEK-ERK, 2) PI3K-AKT, 3) angiogenesis, and 4) mTOR and mTOR negative feedback loop.It is also necessary to interfere in cell cycle, cancer cell metabolism, autophagy, and to promote apoptosis, and block maintenance and function of CSCs.
KRAS-MEK-ERK and PI3K-AKT signaling can be disrupted with a combination of erlotinib, everolimus, vorinostat and PB.MEK inhibitors, including trametinib can be considered instead of vorinostat.In cases that overexpress HER-2, trastuzumab or lapatinib can be considered instead of erlotinib.Dasatinib can be also be used in patients who do not tolerate erlotinib.
Angiogenesis can be blocked through application of pazopanib or bevacizumab.PB and other agents can interfere in cell cycle, cancer cell metabolism, and autophagy and can promote apoptosis, whose mechanism is explained in a recent publication [4].Treatment can be wide-ranging and may include a combination of as many as five or six medications.Based on our experience, this would necessitate a reduction of doses by 50% to 75%, as described in Table 3(a) and Table 3

(b).
With appropriate dose reduction, treatment appears reasonably well-tolerated.Additional targeted agents may replace some medications based on molecular profiling.
After reduction of the bulk of the disease, the emphasis may be switched to the destruction of CSCs.Hh, mTOR, and VEGF inhibitors can be considered for this purpose, which would include vismodegib, everolimus, pazopanib, vorinostat, bortezomib, and PB [35].
Treatment with PB in combination with targeted agents and chemotherapy appears to provide another option for improved outcomes in patients who failed first-line chemotherapy for advanced pancreatic cancer.With proper dosage reduction, the treatment appears tolerable and provides a longer OS than other available treatments.With the accessibility of new targeted drugs and with advanced genomic analyses, options for more successful treatment outcomes are a distinct possibility.Though the findings with PB in combination with targeted agents and/or chemotherapy are preliminary, we suggest the validation of our initial findings using a well-designed Phase I/II trial in recurrent advanced pancreatic cancer.We further propose using this principle in a Phase I/II trial for the treatment of PDA in patients who have failed first-line therapy with antineoplastons (ANP), which share ingredients with metabolites of PB and that have shown promise in the treatment of various brain tumors, including recurrent GBM [4] [36] [71]- [77].ANP can offer the advantage of higher anticancer activity since they are available in intravenous dosage form [36].

Conclusion
An established standard-of-care for recurrent, advanced pancreatic cancer is not yet available.Despite numerous clinical trials, progress in this area has been very modest.The use of targeted agents as a single treatment or in combination with chemotherapy did not provide substantial survival benefit.The results reported by us are based on a small series of patients who were consecutively admitted for treatment at BC during the last few years.This is a retrospective evaluation that shows substantial increase of OS and tolerable toxicity compared to the other available treatments.The choice of targeted agents was very limited when this treatment began.Molecular profiling was in its infancy, providing only limited data that were helpful for the design of treatment plans.This group included only evaluable patients, which is typical for a retrospective assessment, but the results of some other studies reported in Table 4 were also limited to evaluable patients.The authors realize that these are Figure 2. Interruption of signal transduction by PB and targeted agents.Abbreviations: AKT-protein kinase B; AKT2protein kinase B 2; ANP-antineoplastons; BVZ-bevacizumab; DUSP1-dual specificity protein phosphatase 1; DUSP6dual specificity protein phosphatase 6; EGFR-epidermal growth factor receptor; ERK-extracellular signal regulated kinase; ERK5-extracellular-signal-regulated kinase 5; HDAC-histone deacetylase; HER-2-human epidermal growth factor receptor 2; IGF-1R-insulin-like growth factor 1 receptor; JUN-proto-oncogene; MEK-MAP kinase; MET-mesenchymal epithelial transition factor; mTOR-mammalian targets of rapamycin; p27-kinase inhibitor protein p27, PB-sodium phenylbutyrate; PDGFR-platelet derived growth factor receptor; PI3K-phosphoinositide 3 kinase; PTEN-phosphatase and tensin homolog; PTPRR-protein phosphatase R; RAS-rat sarcoma gene protein family; SRC-Rous sarcoma inducing oncogene; TP53-tumor protein p53; TXNIP-thioredoxin-interacting protein; VDUP1-vitamin D3 up-regulated protein; VEGF-vascular endothelial growth factor; VEGFR-vascular endothelial growth factor receptor; VEGFR2vascular endothelial growth factor receptor 2.

Figure 1 .
Figure 1.The Kaplan-Meier survival curve.Overall survival from the treatment start.
All authors are employed by Burzynski Clinic.BC was established in 1977 to treat advanced cases of cancer in a private practice setting and to conduct clinical trials.Between November 2007 and August 2013, BC employed
three oncologists/hematologists, three internists, and a family practitioner in addition to medical support staff.Dr. Stanislaw R. Burzynski and Dr. Gregory S. Burzynski are shareholders and directors, and Dr. Tomasz J. Janicki is the Vice-President of Burzynski Research Institute, Inc.Dr. Stanislaw R. Burzynski is President of Burzynski Research Institute, Inc., Dr. Gregory S. Burzynski is Vice-President of Burzynski Clinic and Dr. Sheldon Brookman is Director of Pharmaceutical Development of Burzynski Clinic.

Table 1 .
Patient characteristics are described in Among 14 pancreatic cancer patients, 12 cases were categorized as Stage IVB and two cases were Stage IVA.All patients had recurrent disease, with 50% failing one line of chemotherapy, 36% two lines, and 14% three or more lines of chemotherapy.The majority of patients (64.3%) had multiple liver metastases.Data confirming diagnosis, treatment, recurrence, and response to treatment received are shown in Table2.

Table 1 .
Demographics of patients with recurrent adenocarcinoma of the pancreas, stage IVA and B.

Table 2 .
Confirmation of diagnosis, treatment, recurrence and response.

Table 3 .
Medication dose and duration of treatment of targeted drugs until first response.

Table 4 .
Selected clinical studies in advanced pancreatic cancer with second-line therapy.

Table 5 .
Incidence of adverse drug events (ADEs), grades 2, 3 and 4 in second-line therapies for patients with advanced pancreatic cancer.