II Personalized Therapy Trial for Metastatic Colorectal

Article pubs.acs.org/jpr

Pilot Phase I/II Personalized Therapy Trial for Metastatic Colorectal Cancer: Evaluating the Feasibility of Protein Pathway Activation Mapping for Stratifying Patients to Therapy with Imatinib and Panitumumab M. Pierobon,† A. Silvestri,†,‡ A. Spira,§ A. Reeder,† E. Pin,†,‡ S. Banks,§ Erika Parasido,†,‡ K. Edmiston,∥ L. Liotta,† and E. Petricoin*,† †

Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, United States ‡ Division of Experimental Oncology 2, CRO-IRCCS, National Cancer Institute, via F. Gallini 2, 33081 Aviano, Italy § Virginia Cancer Specialists, 8503 Arlington Boulevard, Room 400, Fairfax, Virginia 22031, United States ∥ Department of Surgery, Inova Fairfax Hospital, Inova Health System, 8110 Gatehouse Road, Falls Church, Virginia 22042, United States S Supporting Information *

ABSTRACT: This nonrandomized phase I/II trial assessed the efficacy/tolerability of imatinib plus panitumumab in patients affected by metastatic colorectal cancer (mCRC) after stratification to treatment by selection of activated imatinib drug targets using reverse-phase protein array (RPPA). mCRC patients presenting with a biopsiable liver metastasis were enrolled. Allocation to the experimental and control arms was established using functional pathway activation mapping of cKit, PDGFR, and c-Abl phosphorylation by RPPA. The experimental arm received run-in escalation therapy with imatinib followed by panitumumab. The control arm received panitumumab alone. Seven patients were enrolled in the study. For three of the seven patients, sequential pre- and post-treatment biopsies were used to evaluate the effect of the therapeutic compounds on the drug targets and substrates. A decrease in the activation level of the drug targets and downstream substrates was observed in two of three patients. Combination therapy increased the activation of the AKT−mTOR pathway and several receptor tyrosine kinases. This study proposes a novel methodology for stratifying patients to personalized treatment based on the activation level of the drug targets. This workflow provides the ability to monitor changes in the signaling pathways after the administration of targeted therapies and to identify compensatory mechanisms. KEYWORDS: Colorectal cancer, liver metastasis, clinical trial, reverse-phase protein microarray, individualized treatment, chemoresistance

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INTRODUCTION Colorectal cancer (CRC) is the third leading cause of cancer related death in the U.S.1 In 2013, approximately 51 000 patients will die of colorectal cancer, mostly because of the development of distant metastasis.2 Although in recent decades the therapeutic options for CRC patients have positively impacted disease-free and overall survival, the proportion of metastatic patients that reach complete response still needs substantial improvement.2 Recent findings have shown that the molecular profiles of CRC patients are highly heterogeneous and that the patient-specific genomic variability greatly influences response to therapy, especially in patients treated with target-specific drugs.3,4 Significant alterations of cellular networks appear to be necessary for primary tumors to migrate and adapt to a secondary organ, suggesting that treatment of © 2014 American Chemical Society

metastatic patients should be driven by the molecular profile of the secondary lesion.5−8 The manifestation of these genomic events is most often hyperactivation of protein signaling networks, based largely on alterations in the protein kinase enzymatic activity that comprises the cancer pathway architecture. The ongoing development and clinical utilization of pharmacological compounds able to selectively modulate protein signaling pathways within the malignant cells is the central nexus of personalized medicine.9,10 Although new approaches are needed to generate actionable molecular profiling for patientspecific treatment, implementation of these approaches in the Received: December 23, 2013 Published: May 1, 2014 2846

dx.doi.org/10.1021/pr401267m | J. Proteome Res. 2014, 13, 2846−2855

Journal of Proteome Research

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Figure 1. Cut point determination and development of a calibrated assay. (A) Distribution of pc-Abl, pc-Kit, and p-PDGFR within the primary tumor and liver metastasis. On the basis of the activation status of c-Abl, c-Kit, and PDGFR, a “phospho-Gleevec® drug target (PGDT) score” was created for each patient. The PGDT score was generated by combining the individual quantitative RPPA intensity values of the three drug targets in the metastatic lesions. Cut points were established so that only the top tertile of the population would be selected for the experimental arm. (B) Representation of the calibrators and controls that were used to standardize the RPPA platform.

metastasis (p values = 0.0037, 0.0055, and 0.0181 for pc-Abl, pc-Kit, and p-PDGR, respectively).12 This data provided the opportunity to directly investigate the significance of this finding through the implementation of a prospective translational clinical trial for a potential new indication to an existing highly tolerated FDA approved targeted therapy and to evaluate the technical feasibility of generating robust protein pathway activation signatures under an LCM−RPPA-based workflow. The clinical aim of the trial was to investigate the efficacy of imatinib in association with standard of care in recurrent and previously treated CRC patients presenting with hepatic lesions. Patient selection for targeted treatment was based on the activation/phosphorylation status of all three drug targets (c-Abl, c-Kit, and PDGFR) measured by LCM−RPPA.

metastatic setting represents an even greater challenge. Until recently, the motivation to perform an invasive biopsy of the metastatic lesion as the input for molecular analysis has not been standard practice. Only in the past few years has data revealed that the metastatic tumor bears little molecular resemblance to the primary tumor and that accurate molecular profiling can be achieved only by direct analysis of the metastasis.3,6−8,11,12 Development and clinical implementation of technological approaches that could quantitatively measure in a timely manner multiplexed derangements in the activated protein machinery using microscopic quantities of cells produced by biopsy-based procedures had not existed. To meet this new medical challenge, we have developed a workflow for personalized therapy-based applications that couples laser capture microdissection (LCM) to the reversephase protein microarray (RPPA) platform for the analysis of clinical samples. The RPPA is considered a powerful and unparalleled tool for providing a highly multiplexed snapshot of the drug target signaling architecture from very small amounts of biological material with accuracy and precision comparable to FDA approved methods.13−17 A recent LCM−RPPA study conducted by our group evaluated the changes of the phosphorylation status of dozens of key kinases in primary human CRCs and patient-matched liver metastases from newly diagnosed patients presenting with metastatic synchronous disease.12 The study confirmed significant heterogeneity in the signaling architecture of primary tumors compared to the matched liver metastases. Unexpectedly, we found that the entire repertoire of the targets for the FDA approved drugs imatinib mesylate and niltonib, namely, PDGFR, c-Kit, and c-Abl, were highly activated/phosphorylated in approximately one-third of the patients with hepatic

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METHODS

Collection of Baseline Data

Distribution of the three drug targets (phosphorylated c-Abl, cKit, and PDGFR) was assessed in a unique study set of 34 primary tumors and matched synchronous liver metastases obtained at surgery from newly diagnosed patients.12 On the basis of the activation status of c-Abl, c-Kit, and PDGFR, a “phospho-Gleevec® drug target (PGDT) score” was created for each patient by combining the individual quantitative relative intensity values (RU) of the three drug targets in the metastatic lesions based upon the population distribution of patient-matched tumors we previously described.12 The PGDT score is a unitless numerical value that is derived from the simple addition of RU phosphorylated c-Abl + RU phosphorylated c-Kit + RU phosphorylated PDGFR obtained in the initial 2847



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Figure 2. Diagram of the trial design and accrual.

independent calibrators that spanned through the cut-point value. Calibrators were made using commercially available cell lysates characterized by low/absent signal for the analyte of interest. Specifically, HeLa cells (BD Biosciences, San Jose, CA) were used to create PDGFR and c-Kit calibrators, whereas NIH 3T3 cells (Santa Cruz Biotechnology, Santa Cruz, CA) were used for the c-Abl calibrator. Decreasing amounts of c-Abl (Abcam, Cambridge, MA), c-Kit (Cell Signaling Technology, Danvers, MA), and PDGFR (Cell Signaling Technology) phospho-peptides were spiked into the cell lines to create a fivepoint linear dilution curve. Positive and negative controls were identified for each analyte by screening a large number of cell lines (Figure 1B). To evaluate the adequateness of the calibrators and the performance of the assay over time, a subgroup of samples included in the discovery set was tested against the calibrators in multiple independent runs. Calibrators and controls were printed in triplicate and were tested regularly throughout the course of the trial.

34 patients. A cut point of 2.3 relative units for the PGDT score was identified so that only the top tertile of the population would be selected for treatment. As shown in Figure 1A, this cut point would select nearly exclusively metastatic lesions. By using an aggregate score, high activation of one or more of the imatimib drug targets could drive the determination of a score to greater than 2.3 because it was not possible to know ahead of time which imatinib target would be activated. The numeric value of the PDGDT score itself was routinely measured by bridging to a calibration curve that was printed along with the lysates derived from the primary and metastasis pairs (described below). Calibration of the RPPA Assay

For each of the three drug targets, a reference calibrator was prepared to span the distribution of the population where the PDGT score was derived. Consequently, we were effectively able to bridge the PGDT score from the population data to 2848



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Clinical Trial Design

Signaling Technology, Danvers, MA). Data were analyzed as previously described.12 After qualification of the within-array QA/QC, the PGDT score was provided to the treating physician within 72 h from sample arrival at George Mason University (Figure 3).

The study was designed as a two arm, prospective, nonrandomized, phase I/II clinical trial investigating the safety and efficacy of imatinib and panitumumab in mCRC patients presenting with hepatic metastases that progressed on at least one prior line of therapy including a fluoropyrimidine and oxaliplatin or irinotecan. All patients were enrolled at Inova Fairfax Hospital, Fairfax, VA. The study was carried out in accordance with The Code of Ethics of the World Medical Association. Study protocol, medical, and therapeutic procedures utilized in the trial were approved by the Institutional Review Board. All patients enrolled in the study presented with stage IV metastatic adenocarcimonas of the colon and liver metastasis at the time of diagnosis. Participants had already received either FOLFOX with bevacizumab or FOLFIRI with bevacizumab. Some patients had received cetuximab as a single agent as firstline therapy before entering the trial. All patients initially responded to first-line therapy and then progressed. Additional eligibility criteria are described in Supporting Information Table 1. After enrollment and collection of voluntary signed informed consent, patients received a CT-guided core needle biopsy of the liver metastasis. Patient allocation in the experimental or control arm was established on the bases of the PGDT score and K-Ras status of the metastatic lesion (Figure 2). Patients with a PGDT score greater than 2.3 were allocated in the experimental arm (Arm 1). Patients presenting with a PGDT score lower than 2.3 entered the control arm (Arm 2). Subjects presenting with K-Ras mutation were ineligible for the study. Patients enrolled in the experimental arm entered into a sequential cohort treated with escalating doses of imatinib (Supporting Information Table 2). Because of the low enrollment numbers, all patients received only the lowest dose of imatinib (300 mg/day). Follow-up imaging and additional liver biopsy, when feasible, were collected after each round of treatment (Figure 2). Patients entering the control arm (Arm 2) received standard-of-care therapy with panitumumab (6 mg/kg every 2 weeks) until tumor progression. Follow-up imaging and biopsy were collected 2 to 3 months from the beginning of treatment. Treatment efficacy was evaluated as the objective tumor response based on the response evaluation criteria in solid tumors (RECIST).18 Safety assessment for the combination therapy (imatinib plus panitumumab) was conducted throughout the trial. All adverse events were appropriately recorded.

Figure 3. LCM−RPPA workflow from sample receipt at GMU to data delivery.

For the three patients from whom two or more biopsies were collected, in vivo target inhibition evaluation was performed by comparing the activation status of the drug targets and downstream substrates before and after treatment (Supporting Information Table 3). For each patient, data were analyzed as fold change compared to the baseline values, and changes were represented using bar graphs.

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RESULTS

Population Characteristics and Arm Allocation

Of the 39 subjects screened, nine met the eligibility criteria (Figure 2). Of those, an additional two patients did not participate in the study because they presented with a K-Ras mutation of the metastatic lesion. None of the patients subjected to CT-guided needle biopsy experienced adverse effects because of the procedure. The interassay coefficient of variation for the calibrators was 24, 22, and 17 for phospho-c-Abl, phospho-c-Kit, and phosphoPDGFR measurments, respectively (using data collected over 22 weekly runs). Using these calibrators, positive and negative controls passed QA/QC in 6/7 runs, with the negative control failing in one of the 7 independent runs. Sample and controls were immediately rerun, and the QA/QC metrics were met. Figure 4 shows the interassay variance and overall process reproducibility for calibrators, controls, and patient samples. A total of six patients were allocated to Arm 1, and only one patient was allocated to Arm 2. One of the patients enrolled in

Patient Stratification

CT-guided core needle biopsies were collected from each patient before and during treatment. All samples were frozen within 20 min of extraction. Colonic malignant cells were isolated from the surrounding hepatic microenvironment using LCM.19 To preserve the phospho-proteomic signal during LCM, complete miniprotease inhibitor tablets (Roche Applied Science, Indianapolis, IN) were added to 70% ethanol, deionized water, and hematoxylin. Samples were microdissected for 30 min or less to ensure that the phosphoproteome was preserved overtime, a process and time period that has been shown to minimize phosphoprotein degredation ex vivo.20 Captured cells were lysed and printed onto nitrocellulose-coated slides along with calibrators and controls.16,21 Arrays were stained with antibodies binding selectively to the phosphorylated sites of c-Abl, c-Kit, and PDGFR (Cell 2849



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post-treatment biopsies. The remaining four patients either refused the procedure or were hospitalized in an end-of-life medical facility before they were schedule for follow-up images. Nevertheless, the three patients that presented with multiple hepatic lesions at the time of enrollment showed a trend of decreased growth in the lesion that was subjected to pathway mapping (target lesion) compared to the nonbiopsied lesions (nontarget lesion) (Figure 5). One patient showed stable disease of the biopsied lesion, but was withdrawn from the study because of the development of a new metastasis (Figure 5). Imatinib Effects on PDGFR, c-Kit, and c-Abl Phosphorylation

Given the lack of objective clinical outcome, we sought to understand, for the three patients where pre- and postmatched biopsies were procured, if phosphorylation of the imatinib drug targets was modulated by the treatment. Two of the three patients evaluated showed decrease in the activation/ phosphorylation level of all drug targets and several biochemically linked downstream substrates (Figure 6). For the patient that received both imatinib and panitumumab, an increase in the activation of the AKT−mTOR pathway was observed between the first and second post-treatment biopsy (Figure 7). In addition, an increase in the activation of two receptor tyrosine kinases (IGF1R and HER3) was revealed (Figure 7).

Figure 4. Scatter plots representing the variability of the high and low controls run along with patient samples and patient PGDT scores. Mean and standard error for the mean (SEM) are reported as a measure of the central tendency and spread.

Arm 1 was lost at follow up and was not included in this analysis. Because none of the patients enrolled in the trial showed objective response/confirmed stable disease by RECIST after 28 days of treatment with imatinib alone, accrual was prematurely terminated (Figure 5). After the first round of treatment, only three of the seven patients agreed to receive

Evaluation of Intratumor Heterogeneity

Although the intratumoral heterogeneity was postulated to be minimal based on the data obtained from the discovery set

Figure 5. Representation to the response to therapy of the profiled lesion (target lesion) and of the other lesion(s) (nontarget lesion). (A) Percent change of the largest diameter of the target lesion across time. (B) Percent change of the largest diameter of the profiled lesion compared that of the nontargeted lesions. 2850



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Figure 6. Changes in phosphorylation of the drug targets and downstream substrates after administration of imatinib and, when possible, panitumumab. Changes in the signaling networks are represented using bar graphs. White, gray, and black bars were used for patients one, two, and three, respectively.

architecture during sample handling and LCM processing is critical. Fortunately, through the use of rigorous standard operating procedures developed in our laboratory to ensure timely fixation and microdissection of the tissue, we have shown that the LCM−RPPA-based workflow provides superior concordance to FDA approved HER2 testing methods and much better concordance with known genomic mutational effects (e.g., PTEN loss) than undissected and immediately processed whole-tissue preparations.12,22,23 Moreover, this study provided the opportunity for an initial evaluation of the interpatient and intratumoral heterogeneity of the phosphoprotein data generated over the course of targeted treatment with biopsies collected from different regions of the same lesion in a 4 weeks interval. Overall, the RPPA-generated protein signaling architecture obtained from this small pilot set showed that the interpatient variability is more pronounced than the intrapatient variability, as 3/3 of the patients’ tumor biopsies clustered together. These results are in keeping with recently published proteomics data that also found intratumoral heterogeneity to be significantly smaller than the interpatient protein signatures.24 Although many molecular profiling technologies such as targeted exome analysis and whole-genome sequencing are being evaluated for companion diagnostic utility, the average turn-around time for these assays is 2−4 weeks. The utilization of newer molecular technologies like RPPA in the clinical

previously described that showed a common elevation of the PDGFR−cKIT−c-ABL signaling pathways across patients with hepatic metastasis, the consistency of the signaling network within any given patient was evaluated. Using the patientmatched pre- and post-treatment biopsies collected from different regions of the same metastasis across time, the activation level of 18 key signaling proteins that are not direct targets of imatinib and panitumumab was analyzed. As shown in Figure 8, unsupervised hierarchical clustering analysis revealed that the intratumoral differences were minimal compared to interpatient differences, with all patients clustering together.

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DISCUSSION This is the first study in which LCM−RPPA was used as a molecular profiling tool to stratify patients for personalized therapy. Although imatinib is FDA approved and could be prescribed for patients as an off-label treatment without RPPAbased analysis and despite the lack of clinical benefit reported here, this study introduced innovative elements for implementing the delivery of targeted therapies. The trial itself revealed the technical feasibility of an LCM−RPPA worflow to deliver robust, quantitative pathway activation measurments in a timely manner (within 72 h from the collection of the specimen). Owing to the known lability of the phosphoproteome, maintenance of the in vivo phosphoprotein signaling 2851



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Figure 7. Broad protein activation mapping allowed for identifying possible compensatory mechanisms that might be involved in tumor progression after administration of imatinib. Black bars refer to pretreatment specimens, and white bars, to post-treatment ones.

Figure 8. Unsupervised hierarchical clustering analysis of patient-matched pre- and post-treatment biopsies of 18 analytes that are not directly affected by the administration of imatinib and/or panitumumab.

setting presents unique potential for identifying patients that might benefit from specific drugs and for monitoring therapeutic response over time. The RPPA platform has the capability to deliver key information that is becoming increasingly critical for personalized medicine efforts by monitoring the in vivo effects of a drug on its target(s) and

downstream signaling networks. Indeed, our analysis of patientmatched pre- and post-imatinib therapy revealed decreased phosphorylation of the three drug targets in all (3/3) patients along with decreased activation of downstream AKT and ERK signaling (Figure 6). Moreover, using serial biopsies, we were able to detect increases in signaling activation of other receptor 2852



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ylation represents a key component of kinase-driven signaling networks, the ability to directly measure these drug targets in patient biopsies provides a direct link to the mechanism of action of the drug and to the mechanism of resistance. RPPA appears to be a powerful, multiplexed pathway-oriented companion diagnostic tool that can be utilized in a rapid turn around-based clinical setting for patient stratification, and it has the potential to impact the therapeutic decision process. We propose that the technique be further evaluated in rigorous clinical trial applications.

tyrosine kinases (IGF1R and HER3) as patients received imatinib. It is not known if these changes are causally associated with tumor growth, are adopted by the tumor to overcome the effect of treatment, or are the consequence of the expansion of a different subclone. Further investigation in larger prospective studies is required to form a testable hypothesis. Despite the low efficacy of imatinib in this population that led to premature termination of the trial, we were able to observe differences in the response rate to therapy of the lesion that was analyzed compared to the other hepatic lesions in three of the patients. This data reinforces previously reported findings concerning the intertumoral heterogeneitry within the metastatic lesions from the same patient.11,25 Given the small number of tumor samples analyzed, the biopsied lesion trend effects are intriguing only as a discussion point. Although the design of the trial has unique aspects and strengths, some limitations need to be discussed. One important aspect of the trial that could have impacted the clinical outcome is that while the baseline data used to establish the PGDT cut point were collected on newly diagnosed chemonaive patients, the trial described herein targeted patients that had already been treated with standard of care. Previous results revealed that mRNA levels of PDGFR-β and c-Kit in patients affected by rectal cancer significantly increase after chemotherapy and radiation.26 These results suggest that the treatment received by the patients enrolled in this trial might have significantly altered the expression and activation of the drug targets of interest. As a consequence, the establishment of the cut point on a chemonaive population might have affected the accuracy of the stratification process. Of course, it would be nearly impossible to obtained population data on the signaling architecture of patient-matched primary and metatastatic CRC tumors from patients who had progressed on standard-of-care therapy because this group of patients would most likely not be eligible for surgical treatment. Morover, previous trials evaluating the efficacy of imatinib in the treatment of refractory c-Kit and PDGFR expressing solid tumors have shown limited efficacy.27−33 Although these trials did not utilize a functional phosphoprotein measurement for patient selection, the lack of efficacy of imatinib may point to a biological clue concerning the secondary importance of these specific drug targets as a driving aspect of these specific tumors. Nevertheless, because of early termination of the trial and the dose escalation study design, all patients received the lowest possible dose of imatinib. It would have been interesting to see if higher doses of the drug would have produced any durable response. Lastly, because the molecular network driving cancer progression is complex and often requires the interaction among a myriad of signaling pathways, by focusing only on the molecular targets of imatinib, the signaling architecure of many other potential driving derangemtents that underpin progression of the metastatic lesions was not evaluated.34−36 In this instance, the concomitant evaluation of additional key targetable substrates, such as combination of imatinib with AKT−mTOR signaling inhibitors, might have significantly improved the selection of the most appropriate therapy for this cohort of patients.37

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ASSOCIATED CONTENT

S Supporting Information *

Clinical trial inclusion/exclusion criteria, dose escalation schema of imatinib monotherapy followed by combination therapy of imatinib plus panitumumab, and list of antibodies and phosphorylation sites used for the RPPA analysis. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 571-830-4166; Fax: 703-993-8606; E-mail: epetrico@ gmu.edu. Notes

The authors declare the following competing financial interest(s): The authors are inventors on U.S. Government and University assigned patents and patent applications that cover aspects of the technologies discussed. As inventors, they are entitled to receive royalties as provided by U.S. Law and George Mason University policy. Mariaelena Pierobon, Lance Liotta, and Emanuel Petricoin are consultants to and shareholders of Theranostics Health, Inc.

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ACKNOWLEDGMENTS This work was supported by Novartis as well as the generous support of the College of Science, George Mason University. Neither of the funding sources was involved in the preparation/ conduct of the study described. We thank Heather Huryk for the technical support offered during the trial.

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REFERENCES

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CONCLUSIONS This study describes a novel approach for delivering unique phosphoprotein-driven molecular information to physicians to be used for personalized cancer treatment. Because phosphor2853



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