Preliminary Findings on the Use of Targeted Therapy in Combination with Sodium Phenylbutyrate in Advanced Malignant Mesothelioma : A Strategy for Improved Survival

Advanced malignant mesothelioma (MM) is among the most aggressive and difficult-to-treat diseases. Industrialization and exposure to asbestos is the main causative factor for the dramatic increase in the incidence of MM, which carries a poor prognosis and a median survival of less than 12 months. Combination chemotherapy offers only palliative results; however, targeted therapy carries more promise for future successful treatment. This paper presents preliminary findings of improved overall survival (OS) using a combination of sodium phenylbutyrate (PB) with various chemotherapeutic and targeted agents in advanced MM. The data suggest using a strategy of simultaneous interruption of signal transduction involving RAS-MEK-ERK, PI3K-AKT, mTOR, Merlin, and angiogenesis pathways and interference in cell cycle and epigenetic processes. Complete response was determined in 15.4% and stable disease in 46.2% in the group of 13 evaluable patients. Median OS for MM was higher compared to other treatments (17 months compared to between 6 and 12.1 months). The longest surviving patient continues to be in complete response and in excellent condition for over 12.5 years from the treatment start. These findings are only preliminary and validation of the results using a well-designed phase I/II trial in advanced MM is proposed.


Introduction
Advanced malignant mesothelioma (MM) is one of the most aggressive and difficult-to-treat neoplasms.It arises from the surface serosal cells of the pleura and peritoneum and in rare instances, the pericardium or gonads [1].Exposure to asbestos fibers is the most common cause of MM.A very rare disease, industrialization and exposure to asbestos caused a dramatic increase in the incidence of MM once [2].Further increase is expected, especially in developing nations [2].The prognosis of MM is poor with the median survival from 9 to 12 months from diagnosis, despite advances in chemotherapy and use of targeted therapy [3].Genetic analysis reveals involvement of a number of different signaling pathways and several important genetic alterations [1], which includes RAS-MEK-ERK, PI3K-AKT, m-TOR, Merlin, Hippo and angiogenesis pathways, interference in cell cycle and epigenetic changes.These mechanisms were described in detail in recent review articles [1] [4]- [7].
A combination of pemetrexed with cisplatin for unresectable pleural MM became the standard-of-care firstline chemotherapy [3].Some targeted agents given as monotherapy or in combination, occasionally stabilize the disease, but none are recommended as standard first-line or second-line therapy [8].
For several years, our team has conducted research with antineoplastons (ANP), a group of anticancer agents consisting of peptides, amino acid derivatives, and carboxylic acids originally isolated from blood and urine of healthy subjects [9]- [11].The anticancer activity of these compounds was confirmed in a number of preclinical and clinical studies [12].After identification of the structure, numerous phase II studies were conducted with synthetic ANP A10 and ANP AS2-1 injections involving primary brain tumors and advanced colorectal cancer [13]- [18].Further research revealed that some ingredients of ANP A10 and AS2-1, phenylacetylglutaminate (PG) and phenylacetate (PN), are the same as metabolites of sodium phenylbutyrate (PB) a histone deacetylase (HDAC) inhibitor with multiple targets of activity, which is approved for urea cycle disorders and indicated for glioma and acute promyelocytic leukemia [13] [19] [20].The study of PG and PN on the glioblastoma multiforme (GBM) genome has shown that they affect approximately 100 genes [21].These data and molecular profiling led to the treatment of a number of patients at Burzynski Clinic (BC) with advanced malignancies, including GBM and pancreatic cancer using combinations of PB and targeted agents [22] [23].
This article provides a brief description of treatment results in patients with advanced malignant mesothelioma and suggests a rationale for conducting clinical trials with PB in combination with targeted agents.

Patients and Methods
Thirteen subjects were diagnosed with advanced malignant mesothelioma following pathology and radiologic evaluations performed by independent institutions.Thereafter, treatment was provided in the private practice of the BC in Houston, TX.The patients included consecutively admitted evaluable patients between November 9, 2000 and August 24, 2012.
Blood and urine tests were done by the BC laboratory and by outside clinical laboratories.Tests were undertaken that included standard blood and urine analysis and determination of genomic markers (when these were available).Tissue molecular profiling was performed by Caris Life Sciences in Phoenix, AZ.All patients were required to read, understand, and sign an informed consent document prior to treatment.Treatment plans were formulated based on molecular profiling when obtained and included PB given alone or in combination with targeted and chemotherapeutic agents.Therapy was undertaken on an outpatient basis.After an initial two to four weeks at BC, treatment continued under the care of a local oncologist.Prior to the treatment start, a computerized tomography (CT) scan with and without contrast and in some patients a positron emission tomography (PET) scan was performed.The product of two of the largest perpendicular diameters (LPD) of the largest measurable lesions were calculated and totaled providing a baseline evaluation for each study subject.This baseline provided the reference for determining response outcome to treatment.Additional pretreatment measurements included vital signs, clinical disease status, demographics, medical history, current medications, physical examination, electrocardiogram (EKG) and Karnofsky Performance Status (KPS).The evaluation of toxicity was performed according to the common terminology criteria for adverse events version 3 (CTCAEv.3).Possible responses to treatment were complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD).CR required the disappearance of all lesions confirmed at the end of four weeks or a negative PET scan.PR required 50% or higher decrease of the LPD of measurable lesions confirmed at four weeks, PD was determined when new lesions appeared or when there was an increase over 25% in the existing lesions and SD represented the status of tumors classified as between PR and PD.

Patient Demographics
The characteristics of the 13 patients studied are described in Table 1.All had involvement of the pleura, and seven patients had additional involvement of the peritoneum.Of all the patients, one had no prior treatment, whereas four patients had undergone surgery as their only treatment.The majority of patients had failed prior chemotherapy and one patient failed surgery and radiation therapy (RT).The data confirming diagnosis, recurrence, and response to treatment received are described in Table 2(a) and Table 2(

Treatment
Details of medication dosing and treatment duration of patients who obtained CR and SD are described in Table 3(a) and Table 3

(b).
Five patients received treatment with the single agent PB.Five additional patients were treated with PB and erlotinib-Genentech/Astellas Pharma US/OSI Pharmaceuticals.In four of these, treatment was combined with multikinase inhibitors (sorafenib-Bayer and Onyx Pharmaceuticals, pazopanib-GlaxoSmithKline, or sunitinib-Pfizer).One of five patients that were treated with a combination that included erlotinib was also treated with bevacizumab (BVZ)-Genentech/Roche and chemotherapy consisting of pemetrexate-Eli Lilly and Company, and cisplatin-Bristol-Myers Squibb Company.Lastly, one patient who received erlotinib for a short period of time along with lapatinib-GlaxoSmithKline was also treated with the chemotherapy agent nab-paclitaxel-Abraxis BioScience/Celgene Corporation.

Responses and Survival
CR, SD and PD was achieved in 15.4% (N = 2), 46.2% (N = 6) and 38.4% (N = 5), respectively.Of the five patients who received treatment with PB as monotherapy, one obtained CR, two exhibited SD and two developed PD.Two patients are currently alive.One of these patients who were receiving PB monotherapy (Patient 1) experienced a remarkably prolonged OS in excess of 12.5 years after having failed three lines of chemotherapy.Currently, she continues to be classified as CR and remains disease-free.The second patient who achieved CR (Patient 3) had received treatment with PB plus erlotinib, BVZ, pemetrexate, trastuzumab-Genentech, and cisplatin (cf. Figure 1).The remaining cases of SD were treated with a combination of PB plus erlotinib, multikinase inhibitors, and BVZ.One of these patients also received chemotherapy with nab-paclitaxel.The median OS in this evaluation was 17 months and is compared to the other studies, as described in Table 4. Survival was measured from the first day of administration of therapy at Burzynski Clinic until death from any cause, and time to treatment failure was likewise measured from the first day of the treatment until the date of first observation of progressive disease or death from any cause whichever came first.The distributions of survival and treatment failure were estimated by Kaplan-Meier analysis.OS compares favorably to the other studies with chemotherapy for advanced malignant mesothelioma.The Kaplan-Meier Survival Curves are presented in Figure 2 and Figure 3 (for all patients and for eight patients with recurrent MM, respectively).The Kaplan-Meier analysis was prepared by using the MedCalc Statistical Software version 13.3 (MedCalc Software bvba, Ostend, Belgium; 2014).

Safety and Adverse Events
Grades 3 and 4 adverse drug events (ADEs) described in Table 5 were compared with results in other trials.The most common ADEs noted according to CTCAEv.3 were infections and diarrhea.Grade 2 ADEs are not included based upon a paucity of data in several of the comparator studies.Among the Grade 2 ADEs that were observed in this evaluation, most were nausea and vomiting, diarrhea, rash, fatigue and anorexia.These ADEs were readily reversible within a short time.

Discussion
For over ten years, standard first-line therapy for advanced MM includes combination chemotherapy with pemetrexate and cisplatin [3].Additional chemotherapy regimens have been proposed including mitomycin-C plus vinblastine and cisplatin, and cisplatin plus gemcitabine, but the response rate and OS was lower compared to       the pemetrexate-cisplatin combination [24] [25].Unfortunately, after failure of first-line chemotherapy, the standard therapy has yet to be recommended [26].Numerous targeted therapy regimens have been studied and results summarized in recent reviews [8].
After some success in treating non-small-cell carcinoma of the lung with tyrosine kinase inhibitors (TKI) gefitinib and erlotinib, use of these agents in MM appeared Most MM tumors (68%) expressed epidermal growth factor receptor (EGFR), but contrary to lung cancer, the activating mutations of EGFR are rare in MM [27] [28].This may explain the failure of erlotinib in phase II trials in MM used as a single agent or in combination with BVZ [29] [30].Platelet derived growth factor (PDGF) plays an important part in MM pathogenesis, but the PDGF inhibitor imatinib failed to produce objective responses in phase II trials [31] [32].PR was shown in 2% of SRC and PDGF inhibitor dasatinib patients, but patients developed serious toxicity [33].Inhibition of angiogenesis was another important aspect of the activity of MM.Monoclonal antibody against vascular endothelial growth factor type 2 (VEGF2) and BVZ in combination with cisplatin and pemetrexate, used as a first-line regimen, produced a 43% response rate [34].Multitargeted TKIs, both sunitinib and sorafenib, produced responses and a median OS of 6.7 and 10.7 months, respectively and pazopanib is currently under investigation [35] [36].Histone deacetylase inhibitors have also been tested in clinical studies in MM.Vorinostat is most advanced of these studies and has shown PRs in an early phase I trial.Based upon these results, a phase III trial has been initiated, but was not successful [37].
The findings reported in this paper are derived from the treatment of consecutively admitted evaluable MM patients in private practice at BC.The majority of these cases had already failed standard treatment and were given little, if any, hope by their prior treating physicians.The choice of targeted agents and molecular profiling was very limited when the initial patients began treatment and for this reason, they were treated only with the HDAC inhibitor, PB.
As indicated by our studies on the effect of PB metabolites on the neoplastic genome, the PB treatment may affect over 100 genes instrumental in the promotion of malignant growth [38].Knowing that the effect of PB is not very strong, we were surprised when one out of five patients on PB monotherapy responded with a long period of complete response and survival in excess of 12.5 years.Also of note is that this patient had disease recurrence after three lines of chemotherapy prior to PB.Another patient who obtained a CR was previously treated with pemetrexed, which was continued under our care in combination with PB, erlotinib, BVZ, and cisplatin.Two additional patients obtained SD on PB monotherapy and two other patients obtained SD as the result of a combination of PB, erlotinib plus the multikinase inhibitor, sorafenib or pazopanib.One additional patient had SD after treatment with the combination of PB, sorafenib, and dasatinib.Following is a discussion of the emerging strategy for the successful treatment of advanced malignant mesothelioma.
Carcinogenesis induced by asbestos fibers in MM is discussed in numerous articles [4]- [7].Through a number of different mechanisms, asbestos fibers induce genetic damage and activate signaling networks in mesothelial and stromal cells and macrophages that support transformation and maintenance of the neoplastic process.RAF-MEK-ERK and PI3K-AKT pathway activation plays an important part in this process and can be triggered upstream by several RTK's including EGFR, PDGF, and metastatic oncogene receptor, (MET) [27] [28] [39].One of the downstream targets of PI3K-AKT pathways is mammalian target of rapamycin (mTOR).Simultaneous activation of PI3K-AKT and mTOR signaling was associated with reduced survival in MM [40].Overactive SRC kinases can also play an important part in progression of MM indicating the therapeutic application of dasatinib [41].As described earlier, angiogenesis is an important mechanism in MM, which suggests the potential therapeutic use of BVZ and multitargeted TK's, pazopanib, or sorafenib [34]- [36].Among the most frequently inactivated tumor suppressor genes in MM is cyclin-dependent kinase inhibitor 2A/alternative reading frame inactivation (CDKN2A/ARF).Loss of activity of this gene inactivates tumor suppressing pathways of p53 and retinoblastoma.PB activates p53 and retinoblastoma pathways through inhibition of cyclin-dependent kinases 2 and 4 (CDK2/4) and cyclins E and D3 [38].
Results with personalized targeted therapy in patients treated for MM and data emanating from research on molecular mechanisms in this cancer permitted us to propose the following strategy for improving survival of patients suffering from advanced MM (Figure 4 and Figure 5).Interruption of two crucial signaling pathways: RAF-MEK-ERK and PI3K-AKT plays a very important part in the successful treatment in MM.This can be accomplished through the combination of erlotinib, pazopanib, BVZ, dasatinib, and PB.Alternatively, pazopanib, lapatinib or trastuzumab can be used in patients with amplification of HER-2.Sorafenib can replace pazopanib and PB may be substituted by ANP.Another important aspect is mTOR and mTOR negative feedback loop signaling.Merlin, a product of oncogene NF2, plays an important part in down-regulation of mTOR, but abnormal integrin α5/β1 and CD44 and mutation on NF2 negate this action.This can be overcome by everolimus or sirolimus.Increased signaling through the pathways leads to dysregulated cell cycle, cancerous cell metabolism, inhibition of apoptosis, and maintenance and functions of CSC.These complex mechanisms can be controlled by PB, everolimus, and vorinostat or alternatively by ANP, sirolimus, and bortezomib (Figure 5).The combination therapy with PB, selected targeted agents and/or chemotherapy appears to provide another option for improved survival in patients with advanced MM.The dose reduction of these medications in combination can help avoid serious adverse drug experiences.We are reporting the results of the treatment of a small series of patients who were consecutively admitted for the treatment at BC over the last few years.The strategy was to construct a treatment plan based on molecular profiling when this was obtainable.This resulted in some patients being treated with PB as monotherapy, which in one case contributed to excellent control of the disease (Patient 1).Results obtained in the retrospective evaluation indicate that it was possible to obtain a complete response, stabilization of the disease, and longer median OS compared to other treatment modalities for such patients.We recognize that our findings are preliminary and should be confirmed by well-designed clinical trials.

Conclusion
Combination chemotherapy with pemetrexed and cisplatin has become the standard-of-care for advanced and  unresectable pleural MM.Targeted therapy carries more promise, but only occasionally stabilizes the disease and is not yet recommended as standard treatment.This retrospective evaluation shows substantial increase of OS and tolerable toxicity compared to other available treatments.Median OS is substantially higher compared to other treatment regimens.The authors realize that these are initial findings and should be validated by a welldesigned phase I/II clinical trial with PB or ANP in combination with targeted agents.We advise caution in the use of these combinations, since clinical trials have not yet been conducted to validate this approach.We an-ticipate that future clinical trials based on molecular profiling will help select a subgroup of cases of advanced MM and correlate the treatment response with genomic changes. b).

Figure 1 .
Figure 1.A complete response of pleural malignant mesothelioma; patient 3 PET/CT scan indicating a complete resolution of the tumor.

Figure 2 .
Figure 2. The Kaplan-Meier survival curve.Overall survival from the treatment start for all patients.

Figure 3 .
Figure 3.The Kaplan-Meier survival curve.Overall survival from the treatment start for patients with recurrent MM.

Figure 4 .
Figure 4. Interruption of signal transduction pathways by PB and targeted agents.

Figure 5 .
Figure 5. Inhibition of cell cycle, metabolism, maintenance and function of CSS and promotion of apoptosis by PB and targeted agents.

Table 1 .
Demographics of patients with advanced malignant mesothelioma.

Table 2 .
(a) Confirmation of diagnosis, recurrence, treatment and response; (b) Confirmation of diagnosis, progression and response-mesothelioma, newly-diagnosed.

Treatment Confirmation of Progression Confirmation of Response to PBT Molecular Profiling Pathology Radiology Patient Place and Date Diagnosis Place and Date Diagnosis Place and Date Assessment
Abbreviations: BC-Burzynski clinic; BVZ-bevacizumab; CT-computed tomography; EGFR-epidermal growth factor receptor; KRAS-Kirsten rat sarcoma viral oncogene homolog; MRI-magnetic resonance imaging; OSD-overall survival from diagnosis; OST-overall diagnosis from treatment start; PB-sodium phenylbutyrate; PBT-PB and other drugs; PD-progressive disease; PET-positron emission tomography; PTENphosphatase and tensin homolog; SD-stable disease; TAH/BSO-total abdominal hysterectomy bilateral saphingo-oophorectomy; VEGF-vascular endothelial growth factor

Table 3 .
(a) Medication dose and duration of treatment until first response; (b) Medication dose and duration of treatment until first response.

Table 4 .
Selected clinical studies in advanced malignant mesothelioma.

Table 5 .
Incidence of adverse drug events (ADEs), grades 3 and 4 in second-line therapy of patients with advanced mesothelioma.