A team of UK and USA researchers, led by Prof. Dean Fennell of the Thoracic Medical Oncology Department, University Hospital of Leicester, UK, performed a prospective exploratory liquid biopsy analysis with the aim of identifying somatic mutations in plasma as predictors of clinical outcome with ganetespib in the GALAXY-1 trial. They showed that ultra-deep sequencing of multiple somatic mutations in circulating free DNA is feasible and provides a new potential for understanding the evolution of tumour heterogeneity and mechanisms of resistance. The results were presented in a poster discussion session at 4th European Lung Cancer Conference (26-29 March 2014, Geneva, Switzerland).
Ganetespib is a second generation Hsp90 inhibitor. Inhibition of Hsp90, a key molecular chaperone required for activation of many oncoproteins critical to non-small-cell lung cancer (NSCLC) growth and aggressiveness, can lead to cancer cell death. As a single agent, ganetespib showed activity in patients with tumours harbouring ALK, KRAS, HER2, and BRAF mutations.
A next-generation Hsp90 inhibitor, ganetespib, is well tolerated with an acceptable safety profile. Over 1000 patients have been treated to date. Diarrhoea is a class effect with grade 1 and 2 toxicity manageable with prophylactic treatment.
Signal of activity with ganetespib, as a single agent, is observed in lung and metastatic breast cancer.
In the GALAXY-1 trial, a combination of ganetespib and docetaxel improved progression-fee survival (PFS) and overall survival (OS) in second-line treatment for advanced NSCLC. The PFS in combination arm was 5.3 months vs. 3.4 months in docetaxel arm (p = 0.031), while median OS was 10.7 months vs. 7.4 months (p = 0.065).
A new approach to biomarker discovery in the context of phase II trial
Circulating free DNA is present at low levels in the plasma of healthy individuals. Therefore, its capturing by liquid biopsy allows detection of somatic mutations by deep sequencing. Based on this rationale, the researchers prospectively collected plasma samples from patients with lung adenocarcinoma included in the GALAXY-1 trial. The samples were collected at baseline prior to initiation of treatment with ganetespib, during cycles 1 and 2, and at the end of treatment.
The researchers plan is that evaluation of circulating free DNA comprises three steps: method optimisation, a pilot study with 105 samples and a confirmatory study with approximately 215 samples. They use next-generation sequencing technology for the analysis. Circulating free DNA sequence analysis reached the quality control threshold in 94% of collected samples in pilot phase. By ultra-deep sequencing the investigators surveyed 739 amplicons in 46 cancer genes.
Analysis of the first 105 patients revealed multiple concurrent mutations in Hsp90 client proteins. Mutation presence was correlated with OS to identify mutations that most strongly segregated the two treatment arms. Mutations in PTEN and KIT were associated with changes in OS. However, the results should be considered with caution because the sample size is a subset of patients in the GALAXY-1 trial, and is not representative of the entire patient population. A slight imbalance in prevalence rate of KIT mutations between study arms will be addressed in the final analysis.
A full circulating free DNA analysis on remaining samples, both baseline and post-treatment, is currently in progress. Tissue mutational analysis from the same patients is also ongoing. The study investigators plan to correlate it with plasma circulating free DNA results.
This is one of the largest series of patients to be profiled for somatic mutations in circulating free DNA. One of the main caveats is the continuous evolution of technology used, which in turn necessitates repeating the analyses. The study demonstrated the presence of the commonly prevalent mutations in NSCLC, as well as germ-line single nucleotide polymorphisms. Although the sample size is small for definitive conclusions, the current results suggest that KIT mutations correlate with favourable response to combination treatment; in contrast, PTEN mutations appear to have negative impact.
The authors concluded that ultra-deep re-sequencing of multiple somatic mutations in circulating free DNA is feasible, and could be used to identify biomarkers of response to ganetespib.
Dr Mark Ladanyi, who discussed the poster findings, said that the strong points of the current study are a solid rationale for studying Hsp90 inhibitor in NSCLC given prominent role of mutant kinases in this cancer (many of which require Hsp90 for folding/stabilisation/activity) and prospective collection of blood for isolation of circulating free DNA built into clinical trial design.
Dr Ladanyi suggested that increased amount of blood collected for plasma DNA extraction and collection of data on NSCLC histologic subtypes and tumour mutation status may be additionally useful.
Ultra-deep sequencing without matched normal DNA makes it difficult or impossible to distinguish somatic mutations from germline polymorphisms and sequencing artifacts. Dr Ladanyi questioned if subset analyses should be based on variant allele percentages or to run sequencing analyses in duplicate as low variant allele percentages show lower reproducibility. In addition he said that somatic KIT mutations are very rare in NSCLC and suggested that specific KIT variants associated with responses should be examined.
The study co-authors El-Hariry, Vukovic and Reichert are full-time employees of Synta Pharmaceuticals and own stocks in the company. All other authors have declared no conflicts of interest.
Abstract 39PD: Ultra-Deep Sequencing of Circulating Free DNA to Identify Predictive, Mutated HSP90 Clients in GALAXY-1 (NCT01348126), A Randomized Phase 2b Study of Ganetespib plus Docetaxel Versus Docetaxel Alone in 2nd Line Advanced NSCLC
The European Lung Cancer Conference (ELCC) is organised by the European Society for Medical Oncology (ESMO) and the International Association for the Study of Lung Cancer (IASLC). During the four-day programme, attendees benefit from educational and scientific updates provided by thoracic oncology specialists on different multidisciplinary topics important for research and clinical practice in the field of lung cancer.