Parames Thavasu

Phase I and Pharmacokinetic Study of PKC412, an Inhibitor of Protein Kinase C

PURPOSE N-Benzoyl staurosporine (PKC412) is a protein kinase C inhibitor with antitumor activity in laboratory models. We determined the toxicity of oral PKC412 administered daily for repeat cycles of 28 days. PATIENTS AND METHODS Thirty-two patients with advanced solid cancers were treated at seven dose levels (12.5 to 300 mg daily) for a total of 68 cycles. RESULTS The most frequent treatment-related toxicities were nausea, vomiting, fatigue, and diarrhea. At the two top dose levels (225 and 300 mg/d), 15 of 16 patients experienced nausea/vomiting (common toxicity criteria [CTC], version 1), grade 2 in nine of 16 and grade 3 in three of 16 patients; and six of 16 patients developed CTC grade 2 diarrhea. After 1 month of treatment, there were significant reductions in circulating lymphocyte (P <.02) and monocyte (P <.01) counts in patients receiving doses > or = 100 mg/d. Nevertheless, only two patients developed myelosuppression (both grade 2). Of two patients with progressive cholangiocarcinoma, one attained stable disease lasting 4.5 months and one a partial response lasting 4 months. There was a linear relationship between PKC412 dose and area under the curve (0-24 hours) and maximum plasma concentration with marked interpatient variability. The estimated median elimination half-life was 1.6 days (range, 0.9 to 4.0 days), and a metabolite with a median half-life of 36 days was detected. Steady-state PKC412 plasma levels at the top three dose cohorts (150 to 300 mg) were five to 10 times the cellular 50% inhibitory concentration for PKC412 of 0.2 to 0.7 micromol/L. CONCLUSION PKC412 can be safely administered by chronic oral therapy, and 150 mg/d is suitable for phase II studies. The pharmacokinetics and lack of conventional toxicity indicate that pharmacodynamic measures may be additionally needed to optimize the drug dose and schedule.

Measuring cytokine levels in blood: Importance of anticoagulants, processing, and storage conditions

The stability and recovery of six human recombinant cytokines (tumour necrosis factor (TNF), interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6) from whole blood was investigated with a view to optimizing blood collection and storage procedures prior to performing immunoassays. Blood from healthy volunteers was subjected to various processing and storage procedures. Blood samples were treated with either: ethylenediamine tetraacetic acid (EDTA) (1.5 mg/ml blood) (E); EDTA/Trasylol (1.5 mg and 1000 KIU/ml blood) (ET); heparin (30 IU/ml) (H) or allowed to clot (serum). The bloods were spiked with individual cytokines, split into aliquots and kept at 4 degrees C or RT. In the first instance spiked bloods from healthy volunteers (n = 5 per cytokine) were processed using sterile and non-pyrogenic materials and procedures. At regular time intervals, samples were cold spun, separated, flash frozen and assayed for the appropriate cytokine using RIA/IRMA methods. In a further study, timed separation was repeated with spiked blood from healthy volunteers (n = 5 per cytokine) using normal commercially available blood collection materials and procedures. In a third study, spiked blood from healthy volunteers (n = 3 per cytokine) was processed under sterile and non-pyrogenic conditions, and the blood samples separated, aliquoted and flash frozen within half hour of collection. These were then subjected to repeated cycles of freeze thawing at 4 degrees C or RT before assaying. In general, the stability of cytokines in whole blood was improved by storage at 4 degrees C and/or rapid separation. There was no significant difference between samples handled under sterile, non-pyrogenic conditions and those collected using normal blood collection procedures. The blood collection procedures described in this paper did not induce any of the six cytokines in the unspiked blood. Overall, EDTA-treated samples performed most consistently. The addition of trasylol did not significantly affect the results. Most of the cytokines appeared unaffected by up to three freeze thaw cycles. The stability and recovery of the spiked cytokines varied from least stable to most stable spiked cytokine as follows; TNF-alpha less than IL-6 less than IFN-gamma less than IL-1 alpha less than IFN-alpha less than IL-1 beta. The recovery of spiked IFN-gamma from heparinized plasma samples was considerably higher than any other plasma or serum samples. The recovery of spiked TNF-alpha and IL-6 from serum samples was consistently lower than amounts recovered from plasma samples (anticoagulant treated).(ABSTRACT TRUNCATED AT 400 WORDS)

The Association of PI3 Kinase Signaling and Chemoresistance in Advanced Ovarian Cancer

Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN. Mol Cancer Ther; 11(7); 1609–17. ©2012 AACR.

Titration of Signalling Output: Insights into Clinical Combinations of MEK and AKT Inhibitors

Our results show that sub-optimal inhibition of MEK in combination with AKT is less efficient in inhibiting cellular growth compared to completely inhibiting MEK and AKT alone. The results also show that KRAS mutant cells are more sensitive to a combination of MEK and AKT inhibitors compared to BRAF and PIK3CA mutant cells. Both these findings have clinical implications.

Association of creatine kinase and skin toxicity in phase I trials of anticancer agents.

Background:We investigated the association between skin rash and plasma creatine kinase (CK) levels in oncology phase I trials.Methods:We analysed data from 295 patients treated at our institution within 25 phase I trials which included CK measurements in the protocol. Trials involved drugs targeting EGFR/HER2, m-TOR, VEGFR, SRC/ABL, aurora kinase, BRAF/MEK, PARP, CDK, A5B1 integrin, as well as oncolytic viruses and vascular disrupting agents.Results:Creatine kinase measurements were available for 278 patients. The highest levels of plasma CK during the trial were seen among patients with Grade (G) 2/3 rash (median 249 U l−1) compared with G1 (median 81 U l−1) and no rash (median 55 U l−1) (P<0.001). There was a significant reduction in CK after the rash resolved (mean 264.2 vs 100.1; P=0.012) in 25 patients, where serial CK values were available. In vitro exposure of human keratinocytes to EGFR, MEK and a PI3Kinase/m-TOR inhibitor led to the increased expression of CK-brain and not CK-muscle or mitochondrial-CK.Conclusion:Plasma CK elevation is associated with development of skin rash caused by novel anticancer agents. This should be studied further to characterise different isoforms as this will change the way we report adverse events in oncology phase I clinical trials.

A study of PD-L1 expression in KRAS mutant non-small cell lung cancer cell lines exposed to relevant targeted treatments

We investigated PD-L1 changes in response to MEK and AKT inhibitors in KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression (H441, H2291, H23, H2030 and A549) were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5 (H23, H2030 and A549) and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.

Characterisation of the Phosphatidylinositol 3-Kinase Pathway in Non-Small Cell Lung Cancer Cells Isolated from Pleural Effusions

Objectives: We hypothesised that it was possible to quantify phosphorylation of important nodes in the phosphatidylinositol 3-kinase (PI3K) pathway in cancer cells isolated from pleural effusions of patients with non-small cell lung cancer (NSCLC) and study their correlation to somatic mutations and clinical outcomes. Materials and Methods: Cells were immunomagnetically separated from samples of pleural effusion in patients with NSCLC. p-AKT, p-S6K and p-GSK3β levels were quantified by ELISA; targeted next-generation sequencing was used to characterise mutations in 26 genes. Results: It was possible to quantify phosphoproteins in cells isolated from 38/43 pleural effusions. There was a significant correlation between p-AKT and p-S6K levels [r = 0.85 (95% confidence interval 0.73-0.92), p < 0.0001], but not p-AKT and p-GSK3β levels [r = 0.19 (95% confidence interval -0.16 to 0.5), p = 0.3]. A wide range of mutations was described and p-S6K was higher in samples that harboured at least one mutation compared to those that did not (p = 0.03). On multivariate analysis, p-S6K levels were significantly associated with poor survival (p < 0.01). Conclusion: Our study has shown a correlation between p-AKT levels and p-S6K, but not GSK3β, suggesting differences in regulation of the distal PI3K pathway by AKT. Higher p-S6K levels were associated with adverse survival, making it a critically important target in NSCLC.

Insights into significance of combined inhibition of MEK and m-TOR signalling output in KRAS mutant non-small-cell lung cancer

Background:We aimed to understand the dependence of MEK and m-TOR inhibition in EGFRWT/ALKnon-rearranged NSCLC cell lines.Methods:In a panel of KRASM and KRASWT NSCLC cell lines, we determined growth inhibition (GI) following maximal reduction in p-ERK and p-S6RP caused by trametinib (MEK inhibitor) and AZD2014 (m-TOR inhibitor), respectively.Results:GI caused by maximal m-TOR inhibition was significantly greater than GI caused by maximal MEK inhibition in the cell line panel (52% vs 18%, P<10−4). There was no significant difference in GI caused by maximal m-TOR compared with maximal m-TOR+MEK inhibition. However, GI caused by the combination was significantly greater in the KRASM cell lines (79% vs 61%, P=0.017).Conclusions:m-TOR inhibition was more critical to GI than MEK inhibition in EGFRWT/ALKnon-rearranged NSCLC cells. The combination of MEK and m-TOR inhibition was most effective in KRASM cells.

Evaluation of the combination of the dual m-TORC1/2 inhibitor vistusertib (AZD2014) and paclitaxel in ovarian cancer models

Activation of the PI3K/mTOR pathway has been shown to be correlated with resistance to chemotherapy in ovarian cancer. We aimed to investigate the effects of combining inhibition of mTORC1 and 2 using the mTOR kinase inhibitor vistusertib (AZD2014) with paclitaxel in in vitro and in vivo ovarian cancer models. The combination of vistusertib and paclitaxel on cell growth was additive in a majority of cell lines in the panel (n = 12) studied. A cisplatin- resistant model (A2780Cis) was studied in vitro and in vivo. We demonstrated inhibition of mTORC1 and mTORC2 by vistusertib and the combination by showing reduction in p-S6 and p-AKT levels, respectively. In the A2780CisR xenograft model compared to control, there was a significant reduction in tumor volumes (p = 0.03) caused by the combination and not paclitaxel or vistusertib alone. In vivo, we observed a significant increase in apoptosis (cleaved PARP measured by immunohistochemistry; p = 0.0003). Decreases in phospholipid and bioenergetic metabolites were studied using magnetic resonance spectroscopy and significant changes in phosphocholine (p = 0.01), and ATP (p = 0.04) were seen in tumors treated with the combination when compared to vehicle-control. Based on this data, a clinical trial evaluating the combination of paclitaxel and vistusertib has been initiated (NCT02193633). Interestingly, treatment of ovarian cancer patients with paclitaxel caused an increase in p-AKT levels in platelet-rich plasma and it was possible to abrogate this increase with the co-treatment with vistusertib in 4/5 patients: we believe this combination will benefit patients with ovarian cancer.

Percentage Change in Plasma Cytokeratin 18 Is Associated with Clinical Outcomes in Patients Receiving Pemetrexed and Carboplatin for the Adenocarcinoma Subtype of NSCLC

Background: The adenocarcinoma subtype of non-small cell lung cancer (adeno-NSCLC) is routinely treated with chemotherapy if patients do not have molecular aberrations such as epidermal growth factor receptor mutations or anaplastic lymphoma kinase rearrangements. There are currently no validated biomarkers that can predict if patients will gain clinical benefit from chemotherapy, leading to a majority of patients receiving many cycles of unnecessary chemotherapy. We hypothesized that the percentage rise in plasma caspase-cleaved cytokeratin 18 (cCK18) and total cytokeratin 18 (tCK18) assessed before and after chemotherapy correlates with the radiological response to chemotherapy. Methods: Plasma samples from 40 patients with stage IV adeno-NSCLC, treated with first-line chemotherapy with carboplatin (AUC5) plus pemetrexed (500 mg/m2), were collected prior to chemotherapy and 48 h after treatment. ELISA was used to quantify cCK18 and tCK18. Results: The male-to-female ratio was 3:1, and the median age of patients was 63 years. Patients who had a clinical benefit (complete response, partial response or stable disease) at the first radiological assessment following chemotherapy had a significantly higher percentage change in plasma tCK18 levels compared to those who had no clinical benefit, i.e. progressive disease (69.5 ± 75.1 vs. 25.3 ± 30.9%, respectively; p = 0.042). The receiver operating characteristic area was 0.712 (p = 0.039). There was an increase in the percentage change in cCK18 in patients with clinical benefit compared to those without clinical benefit but this was not statistically significant (57.6 ± 112.8 vs. 24.38 ± 45.1%, respectively; p = 0.85). Conclusions: The percentage change in plasma tCK18 levels before and after the first cycle of pemetrexed and carboplatin chemotherapy is associated with clinical benefit. If validated in larger cohorts, this test can be used to identify patients unlikely to respond to treatment who can thus be offered alternative treatments or entry into clinical trials.