Torveig Weum Abrahamsen

Vorinostat, a histone deacetylase inhibitor, combined with pelvic palliative radiotherapy for gastrointestinal carcinoma: the Pelvic Radiation and Vorinostat (PRAVO) phase 1 study.

BACKGROUND Histone deacetylase (HDAC) inhibitors have shown radiosensitising activity in preclinical tumour models. This phase 1 study assessed the use of vorinostat combined with pelvic palliative radiotherapy for gastrointestinal carcinoma. METHODS Between Feb 14, 2007, and May 18, 2009, eligible patients with histologically confirmed carcinoma, scheduled to receive pelvic palliative radiation to 30 Gy in 3 Gy daily fractions over 2 weeks, were enrolled into cohorts of escalating vorinostat dose. Vorinostat was administered orally once daily, 3 h before each radiotherapy fraction, at the following dose levels: 100 mg (n=1), 200 mg (n=4), 300 mg (n=6), and 400 mg (n=6). Endpoints included safety, tolerability, and biological activity (tumour histone acetylation). This study is registered with ClinicalTrials.gov, number NCT00455351. FINDINGS One patient withdrew consent after one treatment day, leaving 16 patients evaluable for tolerability. Most recorded adverse events were grade 1 and 2, among which fatigue (all patients) and gastrointestinal events (all patients) were most common. Grade 3 adverse events included fatigue (n=5), anorexia (n=3), diarrhoea (n=2), hyponatraemia (n=1), hypokalaemia (n=1), and acneiform rash (n=1). Of these, treatment-related grade 3 events (ie, dose-limiting toxicities) were observed in one of six patients at vorinostat 300 mg once daily (fatigue and anorexia), and in two of six patients at vorinostat 400 mg once daily (two events of diarrhoea and one each of fatigue, anorexia, hyponatraemia, and hypokalaemia). The maximum-tolerated dose of vorinostat in combination with palliative radiotherapy was thus determined to be 300 mg once daily. Histone hyperacetylation was detected, indicating biological activity of vorinostat. INTERPRETATION Vorinostat can be safely combined with short-term pelvic palliative radiotherapy. This study highlights the potential use of HDAC inhibitors with radiation, and suggests investigation of vorinostat in long-term curative pelvic radiotherapy--eg, as a component of preoperative chemoradiotherapy for rectal cancer. FUNDING Merck & Co, Inc, Norwegian Cancer Society, Norwegian Health and Rehabilitation Foundation.

Clinical relevance of microRNA miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145 in colorectal cancer

BackgroundMicroRNAs (miRNAs) regulate gene expression by binding to mRNA, and can function as oncogenes or tumor suppressors depending on the target. In this study, using qRT-PCR, we examined the expression of six miRNAs (miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145) in tumors from 193 prospectively recruited patients with colorectal cancer, and associations with clinicopathological parameters and patient outcome were analyzed. The miRNAs were chosen based on previous studies for their biomarker potential and suggested biological relevance in colorectal cancer.MethodsThe miRNA expression was examined by qRT-PCR. Associations between miRNA expression and clinicopathological variables were explored using Mann–Whitney U and Kruskal-Wallis test while survival was estimated using the Kaplan-Meier method and compared using the log-rank test.ResultsMiR-101 was hardly expressed in the tumor samples, while for the other miRNAs, variable expression levels and expression ranges were observed, with miR-21 being most abundantly expressed relative to the reference (RNU44). In our study cohort, major clinical significance was demonstrated only for miR-31, as high expression was associated with advanced tumor stage and poor differentiation. No significant associations were found between expression of the investigated miRNAs and metastasis-free or overall survival.ConclusionsInvestigating the expression of six miRNAs previously identified as candidate biomarkers in colorectal cancer, few clinically relevant associations were detected in our patient cohort. Our results emphasize the importance of validating potential tumor markers in independent patient cohorts, and indicate that the role of miRNAs as colorectal cancer biomarkers is still undetermined.

Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.

Tumor Phosphatidylinositol-3-Kinase Signaling and Development of Metastatic Disease in Locally Advanced Rectal Cancer

Background Recognizing EGFR as key orchestrator of the metastatic process in colorectal cancer, but also the substantial heterogeneity of responses to anti-EGFR therapy, we examined the pattern of composite tumor kinase activities governed by EGFR-mediated signaling that might be implicated in development of metastatic disease. Patients and Methods Point mutations in KRAS, BRAF, and PIK3CA and ERBB2 amplification were determined in primary tumors from 63 patients with locally advanced rectal cancer scheduled for radical treatment. Using peptide arrays with tyrosine kinase substrates, ex vivo phosphopeptide profiles were generated from the same baseline tumor samples and correlated to metastasis-free survival. Results Unsupervised clustering analysis of the resulting phosphorylation of 102 array substrates defined two tumor classes, both consisting of cases with and without KRAS/BRAF mutations. The smaller cluster group of patients, with tumors generating high ex vivo phosphorylation of phosphatidylinositol-3-kinase-related substrates, had a particularly aggressive disease course, with almost a half of patients developing metastatic disease within one year of follow-up. Conclusion High phosphatidylinositol-3-kinase-mediated signaling activity of the primary tumor, rather than KRAS/BRAF mutation status, was identified as a hallmark of poor metastasis-free survival in patients with locally advanced rectal cancer undergoing radical treatment of the pelvic cavity.

Individual tumor volume responses to short-course oxaliplatin-containing induction chemotherapy in locally advanced rectal cancer – Targeting the tumor for radiation sensitivity?

BACKGROUND Neoadjuvant treatment of locally advanced rectal cancer (LARC) involves chemoradiotherapy (CRT), which may cause significant toxicity, and the potential role and sequential placement of neoadjuvant chemotherapy (NACT) relative to CRT is under debate. PATIENTS AND METHODS In a non-randomized study of 72 LARC patients, short-course oxaliplatin-containing NACT was administered prior to CRT. Tumor volumes were calculated from magnetic resonance images before and after NACT, and four weeks after CRT, and associations between tumor volume responses and outcome were analyzed. Additionally, the impact of oxaliplatin exposure on radiosensitivity was examined in colorectal carcinoma cell lines. RESULTS All tumors except one responded to NACT, with better responses in T3 than T4 cases, and 69/72 patients obtained additional tumor volume reduction after subsequent CRT. However, no associations were found between tumor volume reduction and long-term outcome. Of note, oxaliplatin-resistant cells were significantly more radiosensitive than the oxaliplatin-sensitive counterparts. CONCLUSIONS Oxaliplatin-containing NACT led to substantial tumor volume reduction with particularly good responses in T3 cases. NACT did not impede subsequent CRT response, and experimental results rather suggested enhanced radiosensitivity in oxaliplatin-exposed cells, encouraging studies to explore the administration of NACT prior to CRT. Data are still lacking to support omitting radiation in LARC management.

Abstract C63: The Pelvic Radiation and Vorinostat (PRAVO) phase 1 study identifying MYC repression as biomarker of histone deacetylase inhibitor activity.

In modern radiation oncology, new insights into molecular radiobiology provide an opportunity for the rational integration of molecularly targeted therapeutics to optimize clinical radiation effects. One example is the use of histone deacetylase (HDAC) inhibitors as potentially radiosensitizing drugs. Conveyed by histone acetylation, HDAC inhibition causes perturbations in gene regulation implicated in cell cycle progression, DNA damage signaling and repair, and apoptosis. Following the demonstration that HDAC inhibitors enhanced radiation-induced clonogenic suppression in human colorectal carcinoma cell lines and xenograft models [1-3], the PRAVO study was conducted [4-5]. This trial, undertaken in patients treated with pelvic palliative radiotherapy (30 Gy in 3-Gy daily fractions) combined with the HDAC inhibitor vorinostat (administered once daily, three hours before radiation) for advanced gastrointestinal malignancy, was the first to report on the use of an HDAC inhibitor in clinical radiotherapy. It was designed to demonstrate that vorinostat reached the specific target (detection of tumor histone acetylation), the applicability of non-invasive tumor response assessment (using functional imaging), and importantly, that this combined-modality therapy was safe and tolerable. In the present report, potential biomarkers of vorinostat radiosensitizing action, not simultaneously manifesting molecular perturbations elicited by the radiation itself, were explored by gene expression array analysis of the PRAVO study patients’ peripheral blood mononuclear cells (PBMC), sampled at baseline (T0) and on-treatment two and 24 hours (T2 and T24) after the patients had received vorinostat. This strategy revealed 1,600 array probes that were common for the comparisons T2 versus T0 and T24 versus T2 across all of the patients, and furthermore, that no significantly differential expression was observed between the T0 and T24 groups. Functional annotation analysis of the array data showed that a significant number of the identified genes were implicated in biological processes and pathways comprising gene regulation (transcription, RNA processing), cell cycle progression (including p53 signaling, commonly involved in the DNA damage response), and chromatin biology. Of five genes that were selected both for verification of patients’ PBMC expression and for validation of vorinostat-regulated expression in human colorectal carcinoma xenograft models, transient repression of MYC was consistently observed in all conditions. In conclusion, within the design of the PRAVO study, all of the identified genes showed rapid and transient induction or repression and therefore, in principle, fulfilled the requirement of being pharmacodynamic biomarkers of vorinostat activity in fractionated radiotherapy, possibly underscoring the regulatory role of myc in this therapeutic setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C63. Citation Format: Anne Hansen Ree, Marie G. Saelen, Erta Kalanxhi, Ingrid H. G. Ostensen, Kristina Schee, Kathrine Roe, Torveig W. Abrahamsen, Svein Dueland, Kjersti Flatmark. The Pelvic Radiation and Vorinostat (PRAVO) phase 1 study identifying MYC repression as biomarker of histone deacetylase inhibitor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C63.

Abstract C8: Ex vivo kinome profiling of tumors from rectal cancer patients for possible identification of functional biomarkers of EGFR signaling pathway druggability.

In order to optimize and individualize therapeutic efficacy of tyrosine kinase inhibiting agents, it seems rational to exploit the specific pattern of tyrosine kinase activity of the patient9s tumor as functional biomarker of druggability. Ample research has focused on identifying biomarkers for the optimum selection of patients with metastatic colorectal cancer to treatment with drugs targeting tyrosine kinase signaling mediated by EGFR. The present study examined to which degree tumor kinase activity might reflect mutations within KRAS, BRAF, and PIK3CA genes, encoding effector proteins downstream of EGFR in the signaling cascade, which have been shown to correlate with intrinsic therapeutic resistance to anti-EGFR monoclonal antibodies. Primary tumors from 63 patients with locally advanced rectal cancer, enrolled onto a prospective study of chemoradiotherapy followed by radical surgery, were biopsied at the time of diagnosis. Mutations in KRAS exon 2, BRAF exon 15, and PIK3CA exons 9 and 20 were determined by denaturant capillary electrophoresis of PCR-amplified gene sequences. Using peptide arrays with tyrosine kinase substrates, phosphopeptide signatures were generated from the tumor biopsies. A model for predicting tumor KRAS/BRAF mutation status from the tyrosine kinase activity profile was obtained by partial-least-squares discriminant analysis and evaluated by 20-fold cross validation. Distribution of parameters between different groups was compared using Pearson9s Chi-square exact two-sided test, and metastasis-free survival was estimated by the Kaplan-Meier method. Mutation in KRAS (p.G12D, p.G12V, p.G13D, p.G12C, p.G12S, or p.G13S), BRAF (p.D594G or p.V600E), and PIK3CA was detected in 35%, 6.3%, and 9.5% of cases, respectively, and with the exception of one case, single tumor mutations were found. No differences were observed between patients harboring mutated and non-mutated tumors regarding radiological TNM stage at diagnosis, histological ypTN stage or histomorphologic tumor regression grade of the surgical specimens following the chemoradiotherapy, or development of metastatic disease at median follow-up of 43 months (range 7–65). Ex vivo tumor kinase activity profiles were derived from 102 peptide array substrates after substrate signal intensity had been normalized to the mean signal intensity of KRAS/BRAF wild-type samples. Using the generated phosphopeptide profiles, correct prediction of tumor KRAS/BRAF mutation status was obtained in 67% of cases. No improvement of prediction accuracy was achieved on inclusion of another layer of information, namely PIK3CA mutations, to the group of mutated tumor samples. Phosphorylation of 11 peptide array substrates by tumor samples was significantly higher in the KRAS/BRAF wild-type cases, with a majority of the phosphosubstrates representing factors involved in the EGFR signaling pathway. In conclusion, tumor tyrosine kinase activity did not strongly reflect KRAS/BRAF mutation status in rectal cancer. However, our findings may add to the ongoing debate on the current practice of selecting patients with metastatic colorectal cancer for anti-EGFR antibody treatment solely from the tumor KRAS/BRAF mutation status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C8.