Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform.

RNA interference (RNAi) represents a powerful, new tool for scientific investigation as well as a promising new form of targeted gene therapy, with applications currently in clinical trials. Bifunctional short hairpin RNA (shRNA) are synthetic RNAi molecules, engineered to utilize multiple endogenous RNAi pathways to specifically silence target genes. Pancreatic and duodenal homeobox 1 (PDX1) is a key regulator of pancreatic development, ß-cell differentiation, normal ß-cell function and pancreatic cancer. Our aim is to review the process of identifying PDX1 as a specific, potential RNAi target in pancreatic cancer, as well as the underlying mechanisms and various forms of RNAi, with subsequent testing and development of PDX1-targeted bifunctional shRNA therapy.

Assessment of intravenous pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) in yucatan swine.

PDX1 (pancreatic and duodenal homeobox 1) is overexpressed in pancreatic cancer, and its reduction results in tumor regression. Bi-functional pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) utilizes the endogenous micro-RNA biogenesis pathway to effect cleavage- and non-cleavage-dependent degradation of PDX1 mRNA. We have shown that OFHIRNA-PDX1 reduces pancreatic tumor volume in xenograft models. Thus, we are now exploring biorelevant large animal safety of OFHIRNA-PDX1. Mini pigs were chosen as the biorelevant species based on the similarity of human and pig PDX1 target sequence. In the initial study, animals developed fever, lethargy, hyporexia and cutaneous hyperemia following administration of OFHIRNA-PDX1. Twenty-one days later, the same animals demonstrated less toxicity with a second OFHIRNA-PDX1 infusion in conjunction with a prophylactic regimen involving dexamethasone, diphenhydramine, Indocin and ranitidine. In a new group of animals, PDX1 protein (31 kDa) expression in the pancreas was significantly repressed at 48 and 72 h (85%, P=0.018 and 88%, P=0.013; respectively) following a single infusion of OFHIRNA-PDX1 but recovered to normal state within 7 days. In conclusion, a single intravenous infusion of OFHIRNA-PDX1 in conjunction with premedication in pigs was well tolerated and demonstrated significant PDX1 knockdown.

A multicenter phase 1 study of PX-866 in combination with docetaxel in patients with advanced solid tumours.

BACKGROUND: This phase I, dose-finding study determined the safety, maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), pharmacokinetics, and antitumour activity of PX-866, a phosphatidylinositol 3-kinase inhibitor, combined with docetaxel in patients with incurable solid tumours. METHODS: PX-866 was administered at escalating doses (4-8 mg daily) with docetaxel 75 mg m⁻² intravenously every 21 days. Archived tumour tissue was assessed for potential predictive biomarkers. RESULTS: Forty-three patients were enrolled. Most adverse events (AEs) were grade 1 or 2. The most frequent study drug-related AE was diarrhoea (76.7%), with gastrointestinal disorders occurring in 79.1% (docetaxel-related) and 83.7% (PX-866-related). No dose-limiting toxicities were observed. The RP2D was 8 mg, the same as the single-agent MTD. Co-administration of PX-866 and docetaxel did not affect either drug's PKs. Best responses in 35 evaluable patients were: 2 partial responses (6%), 22 stable disease (63%), and 11 disease progression (31%). Eleven patients remained on study for >180 days, including 8 who maintained disease control on single-agent PX-866. Overall median progression-free survival (PFS) was 73.5 days (range: 1-569). A non-significant association between longer PFS for PIK3CA-MUT/KRAS-WT vs PIK3CA-WT/KRAS-WT was observed. CONCLUSION: Treatment with PX-866 and docetaxel was well tolerated, without evidence of overlapping/cumulative toxicity. Further investigation with this combination is justified.

Immune response and survival of refractory cancer patients who received TGF-ß2 antisense/GM-CSF gene modified autologous tumor cell (TAG) vaccine.

TAG vaccine is a novel 'triad vaccine' that involves transfection of autologous tumor with a dual plasmid, TGFß2 antisense gene and GM-CSF gene. Patients with advanced cancer who failed standard therapy were treated. IFN-γ ELISPOT analysis (Enzyme-Linked Immunospot Assay for Interferon Gamma) using TAG autologous vaccine target cells was performed prior to vaccination and at week 12 after the third vaccination. The purpose of this assessment was to correlate the IFN-γ ELISPOT immune response with long-term survival of advanced cancer patients who received TAG vaccination. Twenty-three of 28 patients received ≥ 3 TAG vaccinations (two patients withdrew consent and three had disease progression prior to the third vaccination). Eleven patients demonstrated a positive ELISPOT response (>10 spots and ≥ 2 × baseline) at week 12 and 12 patients did not (P=0.002). Median survival from time of treatment between ELISPOT-positive and -negative groups was significantly different (550 vs 159 days, P=0.036), as was median survival from the time of procurement (627 vs 257 days, respectively, P=0.043). In conclusion, the IFN-γ ELISPOT assay may provide an effective measure of immune response following treatment with 'triad vaccines', but additional patient numbers and/or other immune modulatory parameters are necessary for future testing.

A Phase-1b study of tivantinib (ARQ 197) in adult patients with hepatocellular carcinoma and cirrhosis.

BACKGROUND: The mesenchymal-epithelial transition factor (MET) receptor is dysregulated in hepatocellular carcinoma (HCC), and tivantinib (ARQ 197) is an oral, selective, MET inhibitor. METHODS: This Phase-1b study assessed tivantinib safety as primary objective in patients with previously treated HCC and Child-Pugh A or B liver cirrhosis. Patients received oral tivantinib 360 mg twice daily until disease progression or unacceptable toxicity. RESULTS: Among 21 HCC patients, common drug-related adverse events (AEs) were neutropaenia, anaemia, asthenia, leucopaenia, anorexia, diarrhoea, and fatigue. No drug-related worsening of liver function or performance status occurred, but one Child-Pugh B patient experienced drug-related bilirubin increase. Four patients had drug-related serious AEs, including one neutropaenia-related death. Haematologic toxicities were more frequent than in previous tivantinib studies but were manageable with prompt therapy. Best response was stable disease (median, 5.3 months) in 9 of 16 evaluable patients (56%). Median time to progression was 3.3 months. CONCLUSION: Tivantinib demonstrated a manageable safety profile and preliminary antitumour activity in patients with HCC and Child-Pugh A or B cirrhosis.

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference.

RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

Comparative assessment of siRNA and shRNA off target effects: what is slowing clinical development.

This review considers comparisons of the off-target effects of siRNA to shRNA and their potential impact on the efficacy and toxicity of RNAi based therapeutics.

Phase II trial of talabostat and docetaxel in advanced non-small cell lung cancer.

AIMS: Currently available therapies do improve survival in advanced stage non-small cell lung cancer (NSCLC), but only to a limited degree. Talabostat mesilate (PT-100) is an orally available amino boronic dipeptide that specifically inhibits dipeptidyl peptidases (including fibroblast activation protein) and enhances an immune response. The aim of this study was to determine the efficacy and safety of talabostat in NSCLC patients. MATERIALS AND METHODS: A phase II trial was conducted to evaluate talabostat in combination with docetaxel in patients with advanced NSCLC after failure of previous platinum-based chemotherapy. In total, 42 patients were enrolled. RESULTS: Talabostat was well tolerated. Two patients achieved a partial response and one achieved a complete response. CONCLUSION: There was no evidence that talabostat enhanced the clinical activity of docetaxel in patients with NSCLC.

Phase II trial of Belagenpumatucel-L, a TGF-beta2 antisense gene modified allogeneic tumor vaccine in advanced non small cell lung cancer (NSCLC) patients.

In a previous dose escalation trial we demonstrated dose related survival correlation to Belagenpumatucel-L. In order to further evaluate safety and response at the previously defined optimal dose and schedule and to gain preliminary evidence on a hypothesis that the level of circulating tumor cells (CTCs) in blood may correlate with the overall survival of patients with stage IV NSCLC, we initiated a phase II trial. Patients received intradermal immunization of 2.5 x 10(7) transfected allogeneic tumor cells (Belagenpumatucel-L, supplied by NovaRx) 1 x every month for a total of 16 months. Circulating tumor cells (Veridex, Raritan, NJ) were measured every 4 weeks. Twenty-one advanced NSCLC patients were enrolled on this study. No significant toxic effect was observed. Overall survival was 562 days. The median survival was 660 days in patients having less than 2 CTCs at baseline compared to 150 days in patients with 2 or more CTCs (P=0.025). Phase II results of safety and response are consistent with prior experience following treatment with Belagenpumatucel-L and there is a suggestion that the number of circulating tumor cells at baseline appears to correlate with overall survival. A larger clinical trial is warranted to further explore this observation.

MicroRNA profile analysis of human prostate cancers.

We examined the microRNA (miRNA) expression profile of 40 prostatectomy specimens from stage T2a/b, early relapse and non-relapse cancer patients, to better understand the relationship between miRNA dysregulation and prostate oncogenesis. Paired analysis was carried out with microdissected, malignant and non-involved areas of each specimen, using high-throughput liquid-phase hybridization (mirMASA) reactions and 114 miRNA probes. Five miRNAs (miR-23b, -100, -145, -221 and -222) were significantly downregulated in malignant tissues, according to significance analysis of microarrays and paired t-test with Bonferroni correction. Lowered expression of miR-23b, -145, -221 and -222 in malignant tissues was validated by quantitative reverse transcription (qRT)-PCR analyses. Ectopic expression of these miRNAs significantly reduced LNCaP cancer cell growth, suggesting growth modulatory roles for these miRNAs. Patient subset analysis showed that those with post-surgery elevation of prostate-specific antigen (chemical relapse) displayed a distinct expression profile of 16 miRNAs, as compared with patients with non-relapse disease. A trend of increased expression (>40%) of miR-135b and miR-194 was observed by qRT-PCR confirmatory analysis of 11 patients from each clinical subset. These findings indicate that an altered miRNA expression signature accompanied the prostate oncogenic process. Additional, aberrant miRNA expression features may reflect a tendency for early disease relapse. Growth inhibition through the reconstitution of miRNAs is potentially applicable for experimental therapy of prostate cancer, pending molecular validation of targeted genes.

Transcription factors: their potential as targets for an individualized therapeutic approach to cancer.

Pro-cancer signals are controlled by the expression and transcription of oncogenes. Transcription of DNA is dependent on the spatially and temporally coordinated interaction between transcriptional machinery (RNA polymerase II, transcription factors (TFs)) and transcriptional regulatory components (promoter elements, enhancers, silencers and locus control regions). Unique TFs have been identified in association with cancer. This review summarizes key oncogene-related TFs and organizes their pro-cancer activity according to the six hallmark functions (self sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death, limitless replicative potential, sustained angiogenesis and metastatic spread) proposed as constituting the infrastructure of the malignant process.

A phase I trial of intravenous infusion of ONYX-015 and enbrel in solid tumor patients.

ONYX-015 is an attenuated chimeric human group C adenovirus, which preferentially replicates in and lyses tumor cells that are p53 negative. The purpose of this phase I, dose-escalation study was to determine the safety and feasibility of intravenous infusion with ONYX-015 in combination with enbrel in patients with advanced carcinoma. Enbrel is a recombinant dimer of human tumor-necrosis factor (TNF)-alpha receptor, previously shown to reduce the level of functional TNF. Nine patients, three in each cohort received multiple cycles of ONYX-015 infusion (1 x 10(10), 1 x 10(11) and 1 x 10(12) vp weekly for 4 weeks/cycle) in addition to subcutaneous enbrel (only during cycle 1) injections per FDA-indicated dosing. Of the nine patients, four had stable disease. No significant adverse events were attributed to the experimental regimen, confirming that enbrel can be safely administered along with oncolytic virotherapy. Two of the three patients in cohort 3 had detectable viral DNA at days 3 and 8 post-ONYX-015 infusion. Their detectable circulating viral DNA was markedly higher during cycle 1 (with enbrel coadministration) as compared with cycle 2 (without enbrel) at the same time points. Area under the curve determinations indicate a marked higher level of TNF-alpha induction and accelerated clearance at cycle 2 in the absence of enbrel. Further assessment is recommended.

Proof concept for clinical justification of network mapping for personalized cancer therapeutics.

To identify signature targets associated with patient-specific cancer lesions based on tumor versus normal tissue differential protein and mRNA coexpression patterns for the purpose of synthesizing cancer-specific customized RNA interference knockdown therapeutics. Analysis of biopsied tissue involved two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis coupled with MALDI-TOF/TOF mass spectrometry for proteomic assessment. Standard microarray techniques were utilized for mRNA analysis. Priority was assigned to overexpressed protein targets with co-overexpressed genes with a high likelihood of functional nodal centrality in the cancer network as defined by the interactive databases BIND, HPRD and ResNet. HPLC-grade small interfering RNA (siRNA) duplexes were utilized to assess knockdown of target proteins in expressive cell lines as measured by western blot. Seven patients with metastatic cancer underwent biopsy. One patient (RW001) had biopsies from two disease sites 10 months apart. Seven priority proteins were identified, one for each patient (RACK 1, Ras related nuclear protein, heat-shock 27 kDa protein 1, superoxide dismutase, enolase1, stathmin1 and cofilin1). Prioritized proteins in RW001 from the two disease sites over time were the same. We demonstrated >80% siRNA inhibition of RACK 1 and stathmin1 of inexpressive malignant cell lines with correlated cell kill. Identification of functionally relevant target gene fingerprints, unique to an individual's cancer, is feasible 'at the bedside' and can be utilized to synthesize siRNA knockdown therapeutics. Further animal safety testing followed by clinical study is recommended.

Ionizing radiation: a genetic switch for cancer therapy.

Gene therapy of cancer represents a promising but challenging area of therapeutic research. The discovery of radiation-inducible genes led to the concept and development of radiation-targeted gene therapy. In this approach, promoters of radiation-inducible genes are used to drive transcription of transgenes in the response to radiation. Constructs in which the radiation-inducible promoter elements activate a transgene encoding a cytotoxic protein are delivered to tumors by adenoviral vectors. The tumoricidal effects are then localized temporally and spatially by X-rays. We review the conceptual development of TNFerade, an adenoviral vector containing radiation-inducible elements of the early growth response-1 promoter upstream of a cDNA encoding human tumor necrosis factor-alpha. We also summarize the preclinical work and clinical trials utilizing this vector as a treatment for diverse solid tumors.

Prostate cancer: multimodality approaches with docetaxel.

The trend toward earlier diagnosis of prostate cancer and technological advances in radiotherapeutics (eg, imaging enhancement, planning optimization, refinement of calculation algorithms, and computerized delivery systems) have led to increased use of radiation therapy (RT) as primary treatment for presumed localized disease. However, monomodal local therapy fails to achieve consistently successful long-term disease control, especially in patients with intermediate- and high-grade risk factors. Local-regional factors, such as absolute and relative resistance mechanisms, epigenetic influences, and clonogenic heterogeneity, and probable micrometastatic disease require consideration, evaluation, and potentially the implementation of combined modality approaches. Patients receiving combined RT and androgen suppression (AS) in various sequences (AS --> AS + RT, AS + RT, AS --> AS + RT --> AS, and RT --> AS) have shown enhanced disease-free survival, increased pathologic local control related to the duration of AS treatment, and improved overall survival with prolonged AS. Furthermore, limited but provocative trials suggest that multimodality chemoradiotherapy may also enhance tumor control in patients with locally advanced disease with acceptable toxicity. Several new trials that will test the efficacy and safety of docetaxel combined with radiotherapy as well as biologic modifiers are described.

Benzydamine HCl for prophylaxis of radiation-induced oral mucositis: results from a multicenter, randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Benzydamine was evaluated in patients with head and neck carcinoma for treatment of radiation-induced oral mucositis, a frequent complication of radiation therapy (RT) for which there is no predictable therapy or preventive treatment currently available. METHODS: The safety and efficacy of 0.15% benzydamine oral rinse in preventing or decreasing erythema, ulceration, and pain associated with oral mucositis during RT were evaluated in a randomized, placebo-controlled trial conducted in patients with head and neck carcinoma. Subjects were to rinse with 15 mL for 2 minutes, 4-8 times daily before and during RT, and for 2 weeks after completion of RT; study evaluations were conducted before RT and routinely thereafter up to 3 weeks after RT. RESULTS: During conventional RT, regimens up to cumulative doses of 5000 centigrays (cGy) benzydamine (n = 69) significantly (P = 0.006) reduced erythema and ulceration by approximately 30% compared with the placebo (n = 76); greater than 33% of benzydamine subjects remained ulcer free compared with 18% of placebo subjects (P = 0.037), and benzydamine significantly delayed the use of systemic analgesics compared with placebo (P < 0.05). Benzydamine was not effective in subjects (n = 20) receiving accelerated RT doses (> or = 220 cGy/day). The incidence of adverse events between treatment groups was comparable without significant differences. Early discontinuation because of adverse events occurred in 6% of benzydamine subjects and 5% of placebo subjects, and there was 1 death (related to the primary diagnosis) in a placebo subject. CONCLUSIONS: Benzydamine oral rinse was effective, safe, and well tolerated for prophylactic treatment of radiation-induced oral mucositis.

Guidelines for systemic therapy of early stage breast cancer.

Optimal treatment of early stage breast cancer remains an active area of study. An expert multidisciplinary committee reviewed clinical data on systemic therapy for early stage, stage I and II breast cancer. Guidelines for treatment were developed for Texas Oncology. P.A., the largest private practice group of oncologists in the United States. This group of physicians treats approximately 5000 new breast cancer patients each year and has a major impact on oncology care in the state of Texas. These guidelines identify prognostic factors which help the practitioner in choosing treatment for patients. Subsets of patients are identified for whom no systemic therapy is warrented. Standard chemotherapy and hormonal therapy regimens are outlined for patients with early stage disease at increased risk for relapse. Dose intensification for high risk stage II patients is reviewed. Timing of therapy and the sequencing of chemotherapy and radiation therapy is addressed. Strategies for the follow-up of patients with a history of breast cancer are outlined.

Is liver transplantation indicated for cholangiocarcinoma?

Liver transplantation for unresectable primary liver cancer has met with skepticism due to early aggressive recurrences occurring in early series from many centers. The primary tumors for which transplantation has been performed have been hepatocellular carcinoma and cholangiocarcinoma. In an attempt to improve these poor results, we instituted a protocol with adjuvant chemotherapy and radiotherapy after liver transplantation for cholangiocarcinoma. Between December 1984 and December 1992, 701 patients underwent 795 liver transplants. Seventeen patients had a diagnosis of cholangiocarcinoma and form the basis of this review. Three patients were excluded from the study, two because of early postoperative deaths, and one because of an unknown bony metastasis present at the time of transplant. Eleven patients have experienced a recurrence, and 7 of these died secondary to their recurrence. Three patients are alive with no evidence of disease 44 months, 31 months, and 28 months, respectively, after transplant. The 1-year survival rate in these patients is 53%, and the disease-free survival rate is 40%. Patients were able to tolerate the adjuvant chemotherapy and radiotherapy without apparent morbidity or mortality. Unfortunately, this protocol does not appear to provide significant benefit. Until a better adjuvant therapy protocol is developed, it is questionable whether unresectable cholangiocarcinoma should be considered an indication for liver transplantation.