Translational Predictive Biomarker Analysis of the Phase 1b Sorafenib and Bevacizumab Study Expansion Cohort
2013; Elsevier BV; Volume: 12; Issue: 6 Linguagem: Inglês
10.1074/mcp.m112.026427
ISSN1535-9484
AutoresNilofer S. Azad, Minshu Yu, Ben Davidson, Peter Choyke, Clara C. Chen, Bradford J. Wood, Aradhana M. Venkatesan, Ryan K. Henning, Katherine R. Calvo, Lori M. Minasian, Daniel C. Edelman, Paul S. Meltzer, Seth M. Steinberg, Christina M. Annunziata, Elise C. Kohn,
Tópico(s)Lung Cancer Treatments and Mutations
ResumoPredictive biomarkers are needed to triage patients to the best therapy. We prospectively planned examination of sequential blood, biopsy, and functional imaging with which to confirm the mechanism and to identify potential predictive biomarkers in a phase Ib clinical trial expansion of patients with solid tumors receiving sorafenib/bevacizumab. The maximally tolerated doses of sorafenib at 200 mg twice daily with bevacizumab at 5 mg/kg every other week were given to biopsiable patients. Patients were randomized to receive either sorafenib or bevacizumab monotherapy for the first 28-day cycle with the second drug added with cycle 2. Biopsies, dynamic contrast-enhanced MRI, and fluorodeoxyglucose-proton emission tomography were done pre-therapy and at 2 and 6 weeks (2 weeks into combination therapy). Tumor and serum proteomics, Ras/Raf mutational analysis, and functional imaging results were examined individually and across the dataset to identify potential changes predictive of response to therapy and those that confirm the biochemical drug mechanism(s). Therapy with sorafenib/bevacizumab resulted in clinical benefit in 45% of this mixed solid tumor group. ERK activation and microvessel density were decreased with monotherapy treatment with sorafenib or bevacizumab, respectively; whereas a decreased signal over the group of total AKT, phospho(p)-VEGF receptor2, p-endothelial nitric-oxide synthase, b-RAF, and cleaved poly(ADP-ribose) polymerase was associated with earlier progression of disease. Tumor metabolic activity decreased in those patients with clinical benefits lasting longer than 4 months, and activity increased with progression of disease. Cleavage of caspase 3 and poly(ADP-ribose) polymerase was increased, and Ki67 expression decreased in patients with prolonged clinical benefits, consistent with decreased proliferation and increased apoptosis. The conglomerate analysis, incorporating pharmacodynamic and tumor biochemistry, demonstrated sorafenib/bevacizumab-targeted vascular activity in the tumor. Results suggest potential biomarkers for which changes, as a group, during early therapeutic exposure may predict clinical benefit. Predictive biomarkers are needed to triage patients to the best therapy. We prospectively planned examination of sequential blood, biopsy, and functional imaging with which to confirm the mechanism and to identify potential predictive biomarkers in a phase Ib clinical trial expansion of patients with solid tumors receiving sorafenib/bevacizumab. The maximally tolerated doses of sorafenib at 200 mg twice daily with bevacizumab at 5 mg/kg every other week were given to biopsiable patients. Patients were randomized to receive either sorafenib or bevacizumab monotherapy for the first 28-day cycle with the second drug added with cycle 2. Biopsies, dynamic contrast-enhanced MRI, and fluorodeoxyglucose-proton emission tomography were done pre-therapy and at 2 and 6 weeks (2 weeks into combination therapy). Tumor and serum proteomics, Ras/Raf mutational analysis, and functional imaging results were examined individually and across the dataset to identify potential changes predictive of response to therapy and those that confirm the biochemical drug mechanism(s). Therapy with sorafenib/bevacizumab resulted in clinical benefit in 45% of this mixed solid tumor group. ERK activation and microvessel density were decreased with monotherapy treatment with sorafenib or bevacizumab, respectively; whereas a decreased signal over the group of total AKT, phospho(p)-VEGF receptor2, p-endothelial nitric-oxide synthase, b-RAF, and cleaved poly(ADP-ribose) polymerase was associated with earlier progression of disease. Tumor metabolic activity decreased in those patients with clinical benefits lasting longer than 4 months, and activity increased with progression of disease. Cleavage of caspase 3 and poly(ADP-ribose) polymerase was increased, and Ki67 expression decreased in patients with prolonged clinical benefits, consistent with decreased proliferation and increased apoptosis. The conglomerate analysis, incorporating pharmacodynamic and tumor biochemistry, demonstrated sorafenib/bevacizumab-targeted vascular activity in the tumor. Results suggest potential biomarkers for which changes, as a group, during early therapeutic exposure may predict clinical benefit. Sorafenib and bevacizumab have demonstrated clinical utility as single agents or in combination with chemotherapy for solid tumors. Sorafenib, initially developed as a c-Raf kinase inhibitor, also has potent inhibitory activity against the vascular endothelial growth factor receptor-2 (VEGFR2). 1The abbreviations used are:VEGFR2vascular endothelial growth factor receptor-2RPPAreverse phase proteomic arrayFDG-PETfluorodeoxyglucose-proton emission tomographyDCE-MRIdynamic contrast-enhanced magnetic resonance imagingPARPpoly(ADP-ribose) polymeraseMRmagnetic resonanceGKMgeneral kinetic Kety modelSUVsmall unilamellar vesicleHRhazard ratioPRpartial responsePDprogressive diseaseSDstable diseasePFSprogression free survival. 1The abbreviations used are:VEGFR2vascular endothelial growth factor receptor-2RPPAreverse phase proteomic arrayFDG-PETfluorodeoxyglucose-proton emission tomographyDCE-MRIdynamic contrast-enhanced magnetic resonance imagingPARPpoly(ADP-ribose) polymeraseMRmagnetic resonanceGKMgeneral kinetic Kety modelSUVsmall unilamellar vesicleHRhazard ratioPRpartial responsePDprogressive diseaseSDstable diseasePFSprogression free survival. Clinical activity has been shown for bevacizumab, the humanized neutralizing monoclonal antibody against VEGF, also alone and in chemotherapy combinations (1Wilson P.M. LaBonte M.J. Lenz H.J. Assessing the in vivo efficacy of biologic antiangiogenic therapies.Cancer Chemother. Pharmacol. 2013; 71: 1-12Crossref PubMed Scopus (18) Google Scholar, 2Chen H.X. Expanding the clinical development of bevacizumab.Oncologist. 2004; 9: 27-35Crossref PubMed Scopus (35) Google Scholar, 3Heath V.L. Bicknell R. Anticancer strategies involving the vasculature.Nat. Rev. Clin. Oncol. 2009; 6: 395-404Crossref PubMed Scopus (230) Google Scholar, 4Teoh D.G. Secord A.A. Antiangiogenic therapies in epithelial ovarian cancer.Cancer Control. 2011; 18: 31-43Crossref PubMed Scopus (30) Google Scholar, 5Kelly R.J. Sharon E. Hassan R. Chemotherapy and targeted therapies for unresectable malignant mesothelioma.Lung Cancer. 2011; 73: 256-263Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The role of combining two agents with overlapping target biology had not yet been studied. vascular endothelial growth factor receptor-2 reverse phase proteomic array fluorodeoxyglucose-proton emission tomography dynamic contrast-enhanced magnetic resonance imaging poly(ADP-ribose) polymerase magnetic resonance general kinetic Kety model small unilamellar vesicle hazard ratio partial response progressive disease stable disease progression free survival. vascular endothelial growth factor receptor-2 reverse phase proteomic array fluorodeoxyglucose-proton emission tomography dynamic contrast-enhanced magnetic resonance imaging poly(ADP-ribose) polymerase magnetic resonance general kinetic Kety model small unilamellar vesicle hazard ratio partial response progressive disease stable disease progression free survival. We tested the clinical hypothesis that signal interruption at collaborative pathway points, both vertical and horizontal interactions, may yield equal or greater effect than the agents in isolation in a phase I trial combining bevacizumab and sorafenib (NCT00095459), and we now report the translational analyses (6Azad N.S. Posadas E.M. Kwitkowski V.E. Steinberg S.M. Jain L. Annunziata C.M. Minasian L. Sarosy G. Kotz H.L. Premkumar A. Cao L. McNally D. Chow C. Chen H.X. Wright J.J. Figg W.D. Kohn E.C. Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and antitumor activity.J. Clin. Oncol. 2008; 26: 3709-3714Crossref PubMed Scopus (292) Google Scholar). Sorafenib was selected for its ability to target both receptor and cytosolic kinases important in a variety of activated cells in the tumor microenvironment, including stromal, endothelial, and malignant cells. Because such kinase inhibitor treatment has been shown to up-regulate production of proangiogenic cytokines, we added bevacizumab to reduce VEGF ligand availability and augment inhibition of endothelial cells. We observed the clinical benefit, including partial response and prolonged disease stabilization, using attenuated doses of the individual agents as determined by safety assessments during the trial; partial response or disease stabilization of at least 4 months occurred in 59% of the daily sorafenib cohort and in 55% of those on the intermittent, 5 of 7 days, sorafenib schedule (6Azad N.S. Posadas E.M. Kwitkowski V.E. Steinberg S.M. Jain L. Annunziata C.M. Minasian L. Sarosy G. Kotz H.L. Premkumar A. Cao L. McNally D. Chow C. Chen H.X. Wright J.J. Figg W.D. Kohn E.C. Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and antitumor activity.J. Clin. Oncol. 2008; 26: 3709-3714Crossref PubMed Scopus (292) Google Scholar, 7Lee J.M. Sarosy G.A. Annunziata C.M. Azad N. Minasian L. Kotz H. Squires J. Houston N. Kohn E.C. Combination therapy: intermittent sorafenib with bevacizumab yields activity and decreased toxicity.Br. J. Cancer. 2010; 102: 495-499Crossref PubMed Scopus (59) Google Scholar). These benefits lasted up to 37+ months with over 25% of patients receiving 12 or more months of therapy. The trial prospectively planned comprehensive translational assessment using a randomized drug addition design (Fig. 1A) to evaluate individual drug target specificity and combination drug effects to identify potential predictive biomarkers to examine in the ongoing phase II study of sorafenib/bevacizumab in ovarian cancer. Predictive biomarkers are increasingly important for the advancement of targeted therapies. Such knowledge should allow more effective triage of patients to interventions more likely to provide clinical benefit. Biomarkers that predict drug response may consist of direct measures of activity, such as modulation of biochemical signals in the tumor (8Lee J.-M. Han J.J. Altwerger G. Kohn E.C. Proteomics and biomarkers in clinical trials for drug development.J. Proteomics. 2011; 74: 2632-2641Crossref PubMed Scopus (56) Google Scholar) or those that yield pharmacodynamic measures, such as functional imaging (9McDonald D.M. Choyke P.L. Imaging of angiogenesis: from microscope to clinic.Nat. Med. 2003; 9: 713-725Crossref PubMed Scopus (846) Google Scholar, 10Turkbey B. Kobayashi H. Ogawa M. Bernardo M. Choyke P.L. Imaging of tumor angiogenesis: functional or targeted?.AJR Am. J. Roentgenol. 2009; 193: 304-313Crossref PubMed Scopus (83) Google Scholar). Changes in metabolic activity and/or blood flow using dynamic imaging may fall into both categories with decreased glucose uptake due to reduced glucose delivery and/or reduced glucose metabolism and altered vascular permeability in response to attenuation of the VEGF drive. Aggregate analysis of these varied translational measures may yield a more detailed view of the cancer and the drug combination, allowing broader dissection into potential predictive biomarkers. Linking modulation of activity with clinical benefit is a first step in validating prospective biomarkers. We designed a novel drug administration schema from which to examine the contribution of both sorafenib and bevacizumab on the modulation of tumor and the tumor microenvironment behavior. The biochemical and imaging data demonstrate changes consistent with alteration of tumor vascularity, demonstrate direct association of target effect with clinical outcome across solid tumor types, and confirm the benefit of complementary pathway targeting. Reduction in blood flow, up-regulation of cytokine production, and inhibition of a set of anti-apoptotic anti-proliferative signaling events together may define potentially predictive changes to examine in early drug administration in subsequent trials. —Details of the NCI IRB-approved phase I study and expansion cohort have been published previously (6Azad N.S. Posadas E.M. Kwitkowski V.E. Steinberg S.M. Jain L. Annunziata C.M. Minasian L. Sarosy G. Kotz H.L. Premkumar A. Cao L. McNally D. Chow C. Chen H.X. Wright J.J. Figg W.D. Kohn E.C. Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and antitumor activity.J. Clin. Oncol. 2008; 26: 3709-3714Crossref PubMed Scopus (292) Google Scholar). The maximally tolerated dose, 200 mg of sorafenib twice daily and 5 mg/kg bevacizumab every 2 weeks in 28-day cycles, was administered in this expansion cohort in a novel randomization schema (Fig. 1A; one additional patient was enrolled after submission of the phase I report). Blood samples were obtained, processed, and stored within 4 h of sampling (6Azad N.S. Posadas E.M. Kwitkowski V.E. Steinberg S.M. Jain L. Annunziata C.M. Minasian L. Sarosy G. Kotz H.L. Premkumar A. Cao L. McNally D. Chow C. Chen H.X. Wright J.J. Figg W.D. Kohn E.C. Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and antitumor activity.J. Clin. Oncol. 2008; 26: 3709-3714Crossref PubMed Scopus (292) Google Scholar). Peripheral blood mononuclear cells were collected once for mutational analyses, and serum and plasma were collected monthly. Patients underwent elective image-guided percutaneous 18-gauge core needle biopsy, under separate informed consent and local anesthesia, by an interventional radiologist. Tissue samples were frozen immediately in OCT in the interventional radiologist suite and stored at −80 °C until analysis. If the quality of the initial biopsy was poor or tumor was lacking, subsequent biopsies were aborted per protocol. All MR images were obtained at base line and at protocol-defined points. Region-of-interest MR measurements were obtained from one selected target lesion, independent of the biopsy site, from scans within 48 h prior to each of the three biopsies. Imaging was performed on a 1.5-T MR system (GE Healthcare or Philips Achieva, Best, The Netherlands) using dedicated receive-only phased array coils. Continuous 30-s imaging data sets were obtained before, during, and after administration of the contrast medium for a total of 8 min, resulting in 23 repeated datasets. T2-weighted images (time of repetition (TR)/time of echo (TE) 4600/100 ms, a section thickness of 6 mm, 400-mm field of view, and a matrix of 320 × 320) were used to locate the target tumor. Next, unenhanced T1-weighted images (TR/TE 9/3.6 ms, a 5° flip angle, 5-mm-thick sections, 400-mm field of view, and a matrix of 256 × 256) were obtained with a three-dimensional spoiled gradient-echo sequence to determine the tissue T1 map. Finally, DCE-MR images (TR/TE 9/3.6 ms, a 15° flip angle, 5-mm-thick sections through the entire target lesion, 400-mm field of view, an acquisition time of 30 s per data set, and a matrix of 256 × 256) were obtained with a three-dimensional spoiled gradient-echo sequence. After three base-line unenhanced image acquisitions, an automatic injector (Medrad Spectris, Indianola, PA) was used to intravenously infuse gadopentetate dimeglumine (Magnevist; Bayer Healthcare Pharmaceuticals, Wayne, NJ) at 0.3 ml/s, for a total of 0.1 mmol/kg of body weight (typically 15–20 ml), followed by a 50-ml normal saline flush. MR data were analyzed using a two-compartment model based on the general kinetic (GKM) Kety model (11Choyke P.L. Dwyer A.J. Knopp M.V. Functional tumor imaging with dynamic contrast-enhanced magnetic resonance imaging.J. Magn. Reson. Imaging. 2003; 17: 509-520Crossref PubMed Scopus (365) Google Scholar, 12Kety S.S. The theory and applications of the exchange of inert gas at the lungs and tissues.Pharmacol. Rev. 1951; 3: 1-41PubMed Google Scholar). Three parameters derived from the curve-fitting GKM algorithm were used to generate quantitative parameters as follows: Ktrans, the forward contrast transfer rate; kep, the reverse contrast transfer rate; Ve, extravascular, extracellular volume fraction of the tumor. The DCE-MRI model incorporates an arterial input function derived from large arteries (e.g. aorta) and a T1 map for converting signal intensity to gadolinium concentration. It is based on a two-compartment model that assumes that the vascular space is in rapid equilibrium with the extravascular, extracellular space, and it further assumes a rapid water exchange between intra- and extracellular water. The GKM model was programmed in an IDL-based (Interactive Data Language; Research Systems Inc., Boulder, CO) research tool (Cine Tool, GE Healthcare). Patients fasted at least 6 h prior to the intravenous injection of [18F]fluorodeoxyglucose (15 mCi). Emission images (8 min) were obtained in two-dimensional mode from the upper thigh to the base of skull starting ∼60 min after injection. Transmission scans (3 min) were obtained for attenuation correction. Scans were performed using a GE Advance scanner (General Electric Medical Systems) with a 15-cm axial field of view. PET images were reconstructed on a 256 × 256 matrix using an iterative algorithm provided by the manufacturer. SUV values corrected for lean body mass were obtained using the maximum pixel activity value within a region of interest drawn over index lesions. Index lesions assessed on MR were identified and SUV measurements performed. Serial frozen sections for the set of three time points were fixed in acetone and incubated with primary antibody (Table I). CD31-stained slides were pre-heated in a microwave oven for 5 min; no other antigen retrieval was done. Visualization was achieved using the Dako EnVisionTM+ peroxidase system. Appropriate positive and negative controls were included in each staining experiment, and all specimens containing 0.70, moderate if 0.5 < |r| <0.7, and of decreasing strength if below 0.5. Protein intensity fold-change ratios were calculated from the RPPA intensity values. Ratios were calculated between tumors sampled prior to initiating single agent administration, after 2 weeks of single agent (2 weeks/0 weeks ratio), and after 2 weeks of dual agent administration (6 weeks/2 weeks for second drug addition and 6 weeks/0 weeks for total change over time). These ratios were median-centered and clustered using Cluster 2.0 software. Student's t test for differences between average protein intensity ratios at these intervals between patients in cluster 1 versus cluster 2 was calculated using Microsoft Excel by grouping all the ratios for individual clusters. A second analysis using Student's t test evaluated the ratio of signal at 6 weeks versus on-study to identify a core group of putative biomarkers of treatment interval. Time-to-event end points, such as progression-free survival, were computed using Kaplan-Meier statistics, with comparisons made using a log-rank test. Twenty eight patients were enrolled in the expansion translational cohort to get 10 patients with triplet biopsies (Consort diagram, Fig. 1B). Patient demographics, tumor characteristics, and clinical activity are shown in Table II. After final analysis, only 19 patients had three serial biopsies with at least one core per time point of adequate tissue quality and quantity for use. Reasons for incomplete biopsy sets included poor quality, fluid only, or ≤50% tumor cells (1 patient each), refusal (1Wilson P.M. LaBonte M.J. Lenz H.J. Assessing the in vivo efficacy of biologic antiangiogenic therapies.Cancer Chemother. Pharmacol. 2013; 71: 1-12Crossref PubMed Scopus (18) Google Scholar), safety (2Chen H.X. Expanding the clinical development of bevacizumab.Oncologist. 2004; 9: 27-35Crossref PubMed Scopus (35) Google Scholar), or study removal due to toxicity or disease progression (3Heath V.L. Bicknell R. Anticancer strategies involving the vasculature.Nat. Rev. Clin. Oncol. 2009; 6: 395-404Crossref PubMed Scopus (230) Google Scholar). Twenty six patients had adequate peripheral blood mononuclear cell ascertainment and serial monthly blood sampling (aggregate VEGF concentration results were reported (6Azad N.S. Posadas E.M. Kwitkowski V.E. Steinberg S.M. Jain L. Annunziata C.M. Minasian L. Sarosy G. Kotz H.L. Premkumar A. Cao L. McNally D. Chow C. Chen H.X. Wright J.J. Figg W.D. Kohn E.C. Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and antitumor activity.J. Clin. Oncol. 2008; 26: 3709-3714Crossref PubMed Scopus (292) Google Scholar)). Twenty three patients had all three planned [18F]FDG-PET scans, and 16 patients had all planned MRs; most common causes for incomplete imaging series were obesity and claustrophobia for MR, and scheduling for out-of-town patients.Table IIPatient demographicsMale/Female10:18Median age58.4 (30.3–76)Tumor typesOvarian/fallopian/peritoneal7Melanoma5Sarcoma5OtheraOther includes the following: adrenocortical, papillary renal cell, cervical, colon, peritoneal mesothelioma, uterine papillary serous, unknown primary, papillary thyroid, rectal squamous, adenoid cystic breast, and granulosa cell cancers (one each).11Median 28 day cycles on therapy4.75 (0.5–36.5)Best responsePartial response5Stable disease19Progressive disease3Not evaluable1a Other includes the following: adrenocortical, papillary renal cell, cervical, colon, peritoneal mesothelioma, uterine papillary serous, unknown primary, papillary thyroid, rectal squamous, adenoid cystic breast, and granulosa cell cancers (one each). Open table in a new tab RPPA was used to confirm the presence, activation, and inhibition of select putative biochemical/predictive targets. ERK activation, the Raf kinase downstream event, was significantly reduced with 2 weeks of sorafenib but not in patients receiving bevacizumab (p = 0.02; Fig. 3A). Patients randomized to single agent bevacizumab had a lower CD31 vessel count at 2 weeks (p = 0.05; Fig. 3B), consistent with the greater activity of ligand neutralization. Increased circulating VEGF concentrations have been observed with multiple inhibitors of angiogenic signaling pathways (5Kelly R.J. Sharon E. Hassan R. Chemotherapy and targeted therapies for unresectable malignant mesothelioma.Lung Cancer. 2011; 73: 256-263Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 16Zurita A.J. Jonasch E. Wang X. Khajavi M. Yan S. Du D.Z. Xu L. Herynk M.H. McKee K.S. Tran H.T. Logothetis C.J. Tannir N.M. Heymach J.V. A cytokine and angiogenic factor (CAF) analysis in plasma for selection of sorafenib therapy in patients with metastatic renal cell carcinoma.Ann. Oncol. 2012; 23: 46-52Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and were confirmed in this patient cohort (6Azad N.S. Posadas E.M. Kwitkowski V.E. Steinberg S.M. Jain L. Annunziata C.M. Minasian L. Sarosy G. Kotz H.L. Premkumar A. Cao L. McNally D. Chow C. Chen H.X. Wright J.J. Figg W.D. Kohn E.C. Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and antitumor activity.J. Clin. Oncol. 2008; 26: 3709-3714Crossref PubMed Scopus (292) Google Scholar). Consistent with this, we found decreased tissue pERK at 2 weeks correlated with higher circulating VEGF concentration at both 2 and 6 weeks (r = −0.60, p = 0.024; r = −0.63, p = 0
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