Abstract
Purpose
Sorafenib is a kinase inhibitor targeting Raf and other kinases (ie, vascular endothelial growth factor receptor [VEGFR], platelet-derived growth factor receptor [PDGFR], Flt3, and c-KIT). This study assessed its activity and tolerability in patients with recurrent ovarian cancer (OC) or primary peritoneal carcinomatosis (PPC).
Methods
This open-label, multi-institutional, phase II study used a two-stage design. Eligible patients had persistent or recurrent OC/PPC after one to two prior cytotoxic regimens, and they experienced progression within 12 months of platinum-based therapy. Treatment consisted of sorafenib 400 mg orally twice per day. Primary end points were progression-free survival (PFS) at 6 months and toxicity by National Cancer Institute criteria. Secondary end points were tumor response and duration of PFS and overall survival. Biomarker analyses included measurement of ERK and b-Raf expression in tumors and phosphorylation of ERK (pERK) in peripheral-blood lymphocytes (PBLs) before and after 1 month of treatment.
Results
Seventy-three patients were enrolled, of which 71 were eligible. Fifty-nine eligible patients (83%) had measurable disease, and 12 (17%) had detectable disease. Significant grade 3 or 4 toxicities included the following: rash (n = 7), hand-foot syndrome (n = 9), metabolic (n = 10), GI (n = 3), cardiovascular (n = 2), and pulmonary (n = 2). Only patients with measurable disease were used to assess efficacy. Fourteen survived progression free for at least 6 months (24%; 90% CI, 15% to 35%). Two patients had partial responses (3.4%; 90% CI, 1% to 10%); 20 had stable disease; 30 had progressive disease; and seven could not have their tumor assessed. ERK and b-Raf were expressed in all tumors. Exploratory analyses indicated that pERK in post-treatment PBL specimens was associated with PFS.
Conclusion
Sorafenib has modest antitumor activity in patients with recurrent OC, but the activity was at the expense of substantial toxicity.
INTRODUCTION
Ovarian cancer (OC) is the leading cause of mortality among gynecologic malignancies.1 Treatment relies on surgical debulking and platinum-based therapy. Unfortunately, most patients experience relapse and become resistant to platinum and subsequent chemotherapy.2,3 There is a pressing need for more effective therapies that target biologic mechanisms that drive OC progression.4
Sorafenib is an oral bisaryl urea that inhibits c-Raf and b-Raf, two kinases that function in the mitogen-activated protein kinase (MAPK) pathway. This pathway is activated in OC as a consequence of growth factor stimulation that activates Ras. Constitutive Ras-Raf-MAPK activation is less common, as Ras or Raf mutations are rare in OC.5–10 Interestingly, Ras and b-Raf mutations occur with higher frequency in low malignant potential ovarian tumors than in invasive tumors, and constitutive activation of the Ras-Raf-MAPK pathway through mutation or overexpression is prominent in low-grade serous, mucinous, and clear cell ovarian carcinomas.11–15 Overexpression of c-Raf was reported in greater than half of ovarian tumors and was correlated with unfavorable outcome.16 Inhibition of the Ras-Raf-MAPK pathway through genetic or chemical methods blocks the growth and invasion of OC cell lines, which supports the testing of a Raf inhibitor in OC.17,18
In addition, sorafenib nonspecifically blocks other receptor tyrosine kinases involved in tumor progression and angiogenesis, specifically the vascular endothelial growth factor receptors (VEGFRs) 2 and 3, the platelet-derived growth factor receptor (PDGFR) β, Flt-3, and c-KIT. The VEGFR and PDGFR are overexpressed and activated in ovarian tumors and play an important role in tumor vascularization.19–21 In preclinical models, dual inhibition of VEGF and PDGF pathways has potent antiangiogenic effects through destabilization of pericytes.22 In a hepatocellular carcinoma model, sorafenib inhibited tumor angiogenesis by blocking PDGFR and VEGFR signaling.23 Sorafenib also induced apoptosis of endothelial cells and blocked angiogenesis by targeting Raf-MAPK signaling.24,25 These preclinical findings provide strong support for testing sorafenib in OC for which active VEGF and PDGF autocrine and paracrine networks stimulate tumor growth and angiogenesis.19,21
Here, we studied the effects of sorafenib in women with OC or PPC recurring within 12 months of a platinum-based regimen. The main objectives were to measure progression-free survival (PFS) at 6 months and tolerability. Biologic activity was assessed by measuring the level of phosphorylated extracellular signal-regulated protein kinase (pERK) in peripheral-blood lymphocytes (PBLs) before and 1 month after of sorafenib treatment by using reverse-phase protein microarrays, a quantitative protein microarray format developed for multiplexed cell signaling analysis.26,27 Expression of b-Raf and ERK was determined in archival tumors and correlated with clinical outcome.
PATIENTS AND METHODS
Patient Population
Patients with advanced, histologically documented OC or PPC who experienced recurrence within 12 months after platinum-based chemotherapy were eligible. Eligibility included both measurable and nonmeasurable disease. Measurable disease was defined according to Response Evaluation Criteria in Solid Tumor (RECIST).28 Patients with nonmeasurable disease could enroll if they had ascites or pleural effusions attributable to disease, radiologic abnormalities that did not meet RECIST criteria, and a pretreatment serum CA-125 level higher than twice the upper limit of normal. Only patients with measurable disease were used to formally evaluate the activity of the study agent. Patients with nonmeasurable disease enrolled in parallel with patients who had measurable disease for as long as the trial was open and were assessed descriptively with the intent of gaining insight into the distribution of PFS for this subgroup of patients previously not included in Gynecologic Oncology Group (GOG) trials. All patients were at least 18 years old with a GOG performance status of 0 to 2. Eligibility criteria included the requirement of at least one prior, but no more than two prior, cytotoxic therapy; and adequate hematologic, hepatic, and renal functions. Key exclusion criteria were prior treatment with sorafenib, history of brain metastases, clinical evidence of small bowel obstruction, and use of oral anticoagulation. All patients signed written informed consent, and the protocol was approved by institutional review boards.
Treatment Plan
Treatment consisted of sorafenib orally given as 400 mg orally twice per day continuously. Each cycle was 4 weeks, and treatment was continued until occurrence of disease progression (ie, progressive disease [PD]) or intolerable toxicity.
Efficacy and Toxicity Assessment
When possible, tumor burden was evaluated by clinical examination at baseline and before each cycle. Alternatively, disease was evaluated radiographically at baseline, before each odd cycle, and at the end of treatment. Investigator-determined best overall response was defined by using RECIST 1.0 criteria in patients with measurable tumors.28 No independent outcome review was performed. CA-125 measurements were scheduled for all patients on day 1 of each cycle. Adverse events (AEs) were assessed on day 1 of each cycle and were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.
Translational Analyses
Levels of pERK were measured by lysate arrays constructed as previously described26,27 in pre- and post-treatment PBLs that were collected pretreatment (within 14 days of the start of cycle 1) and post-treatment (within 3 days of the start of cycle 2; Appendix, online only). Total b-Raf and ERK expressions were assessed by immunohistochemistry in paraffin-embedded archival tissue, as previously described29 (Appendix, online only). Immunostaining intensity was scored as 1+, 2+, or 3+, and an H score was calculated as the product between the intensity and the percent of staining cells.
Statistical Analysis
This was an open-label, multicenter, two-stage, phase II study performed through the GOG (protocol GOG170F). The primary objectives were determining the efficacy of sorafenib as estimated from the probability of surviving progression free for at least 6 months (ie, PFS at 6 months) and characterizing the toxicity of sorafenib with the frequency and severity of AEs. The time to progression or death was assessed from the date of entry onto the study. The first stage targeted a sample size of 25 eligible patients with measurable disease but was allowed to range from 22 to 29 patients. If five or more patients of 25 were progression free at 6 months, the study was allowed to proceed to the second stage. If the study continued to the second stage, the targeted cumulative accrual was 56 but was allowed to range from 53 to 60 patients. If 11 or fewer patients of 56 were progression free at 6 months, then the activity of the agent was deemed uninteresting. The full set of decision criteria for deeming an agent interesting for additional study was previously presented.30 These decision criteria limit the probability of falsely declaring inactive agents (true probability of PFS at 6 months equal to 15%) as interesting to 10%, with an average probability of early termination of 59%, and the criteria have a probability of correctly declaring active regimens (true probability of PFS at 6 months equal to 30% or more) as interesting with 90% power.31 Efficacy analysis and calculation of sample size were prospectively defined to include only patients with measurable disease. The a priori exclusion of patients with nonmeasurable detectable disease from the efficacy analysis was based on lack of any historical database on which to judge the agent as being interesting for additional study in this group. The secondary objectives were to measure the proportion of patients with objective responses (ie, partial and complete) to estimate the distribution of PFS and overall survival (OS) and to assess the impact of prognostic variables: platinum-free interval and performance status. An exploratory objective was to assess the effect of measurable disease status on PFS and OS. Translational objectives included evaluation of changes in pERK levels in PBLs before and after treatment and the assessment of b-Raf and ERK expression in archival paraffin-embedded tumors with clinical outcome. The exploratory analyses conducted to evaluate these objectives included paired t tests and survival analyses in which transformed levels of b-Raf and ERK expression were included as covariates in Cox modeling.32 Landmark analyses were occasionally used to help assess the potential prognostic significance of biomarkers obtained after study entry.33,34 The Spearman coefficient was used to measure the correlation between intensity of staining for ERK and b-Raf in the stained specimens.
RESULTS
Patients
Seventy-three consenting patients were enrolled, of which 71 (97%) were eligible. The two ineligible patients had wrong histology (n = 1) or detectable disease with low CA-125 (n = 1). Table 1 indicates that 59 patients (83%) had measurable disease and that 12 patients (17%) had nonmeasurable disease. Data from 71 patients were analyzed for toxicity, and data from 59 patients with measurable disease were utilized for efficacy. The median age was 60 years (range, 33 to 80 years). Fifty-eight patients (82%) had OC, and 13 (18%) had PPC. Serous papillary carcinoma was the most common histology (64 patients [90%]). Fifty patients (70%) had platinum-resistant or refractory OC. Forty patients (56%) received one prior regimen, and 31 (44%) received two prior regimens.
Table 1.
Characteristic | Patients |
|
---|---|---|
No. (N = 71) | % | |
Age, years | ||
Median | 60 | |
Range | 33-80 | |
Performance status | ||
0 | 57 | 80.3 |
1 | 14 | 19.7 |
Ethnicity | ||
White | 65 | 91.5 |
African American | 2 | 2.8 |
Other/unspecified | 4 | 5.6 |
Site | ||
Ovary | 58 | 81.7 |
PPC | 13 | 18.3 |
Platinum sensitive | ||
Yes | 21 | 29.6 |
No | 50 | 70.4 |
Measurable disease | ||
Yes | 59 | 83.1 |
No | 12 | 16.9 |
Histologic type | ||
Serous | 64 | 90.1 |
Endometrioid | 2 | 2.8 |
Clear cell | 1 | 1.4 |
Mixed | 3 | 4.2 |
Adenocarcinoma | 1 | 1.4 |
No. of prior chemotherapy regimens | ||
1 | 40 | 56.3 |
2 | 31 | 43.7 |
Abbreviation: PPC, primary peritoneal carcinomatosis.
Treatment Administration and Safety
Among all patients, 219 cycles were administered. The median number of cycles completed was two (range, one to 24 cycles). Causes for treatment discontinuation were as follows: disease progression (n = 55), toxicity (n = 9), withdrawal of consent (n = 3), death (n = 1 as a result of sepsis while on treatment, although attribution to treatment was considered highly unlikely), and other reasons (n = 3). Table 2 lists treatment related AEs. The most common AEs were GI (79%), constitutional (73%), dermatologic (76%), metabolic (61%), and pain (45%); the majority were grades 1 to 2. Grade 3 to 4 toxicities affecting more than one patient included the following: dermatologic (n = 14), metabolic (n = 10), constitutional (n = 3), GI (n = 3), cardiovascular (n = 2), leukopenia (n = 2), neutropenia (n = 2), and pulmonary (n = 2). Antiangiogenic class-specific AEs were as follows: hypertension (n = 20 occurrences; one was grade 3) and proteinuria (n = 3; grades 1 to 2). Twenty-nine women developed hand-foot syndrome (nine were grade 3). Unexpected serious AEs included a nonfatal cardiopulmonary arrest possibly related to treatment. No treatment-related deaths or GI perforations were recorded.
Table 2.
Adverse Event | No. of Adverse Events by Grade |
|||
---|---|---|---|---|
Grade 1 | Grade 2 | Grade 3 | Grade 4 | |
Leukopenia | 10 | 1 | 1 | 1 |
Thrombocytopenia | 13 | 1 | 0 | 1 |
Neutropenia | 7 | 1 | 1 | 1 |
Anemia | 18 | 3 | 1 | 0 |
Other hematologic | 0 | 0 | 1 | 0 |
Allergy | 0 | 1 | 0 | 0 |
Hearing | 1 | 0 | 0 | 0 |
Cardiovascular | 16 | 6 | 1 | 1 |
Coagulation | 2 | 0 | 1 | 0 |
Constitutional | 38 | 11 | 2 | 1 |
Dermatologic | 18 | 22 | 14 | 0 |
Endocrine | 2 | 0 | 0 | 0 |
GI | 35 | 18 | 3 | 0 |
Genitourinary/renal | 1 | 0 | 1 | 0 |
Hemorrhage | 3 | 1 | 0 | 0 |
Infection | 0 | 3 | 0 | 0 |
Lymphatic | 0 | 1 | 1 | 0 |
Musculoskeletal | 3 | 4 | 0 | 0 |
Metabolic | 28 | 5 | 8 | 2 |
Neuropathy | 14 | 4 | 0 | 0 |
Other neurologic | 10 | 0 | 1 | 0 |
Ocular | 4 | 1 | 0 | 0 |
Pain | 22 | 9 | 1 | 0 |
Pulmonary | 7 | 0 | 1 | 1 |
NOTE. No. evaluated = 71.
Efficacy
Fifty-nine patients (83%) had measurable disease and were therefore included in the analysis of efficacy. At 6 months, 14 patients (23.7%) were without disease progression. There were two partial responses by RECIST (3.4%), and 20 patients (33.9%) had stable disease as best response. Durations of the two responses were 6.77 and 6.14 months, respectively. At a median follow-up of 23.6 months, 18 patients were alive, of which three were without evidence of progression. The median PFS was 2.1 months (95% CI, 1.87 to 3.42 months; Fig 1). The median OS was 16.33 months (95% CI, 11.10 to 22.21 months). Multivariate Cox analysis indicated that neither performance status nor length of the platinum-free interval were predictors for PFS (hazard ratio [HR], 0.94; 95% CI, 0.48 to 1.84) for performance status; and HR, 0.70; 95% CI, 0.36 to 1.38 for platinum sensitivity) or OS (HR, 1.90; 95% CI, 0.93 to 3.89 for performance status; and HR, 0.66; 95% CI, 0.32 to 1.35 for platinum sensitivity).
Because patients with detectable disease had not been included in prior GOG protocols, this subgroup was analyzed separately and only with exploratory intent. Of 12 patients with detectable disease enrolled, 11 had PFS shorter than 6 months. The median PFS was 1.87 months (95% CI, 1.74 to 2.83 months), and the median OS was 22.67 months (Appendix Fig A1, online only). Performance status and platinum sensitivity were not substantially associated with PFS or OS in patients who had unmeasurable disease.
Translational Correlative Analyses
Total ERK and b-Raf expressions were assessed by immunohistochemistry in archived paraffin-embedded tumors from 60 women. Total ERK was expressed in 100% of tumors analyzed; 55% (n = 33) were an intensity of 1+, 25% (n = 15) were 2+, and 20% (n = 12) were 3+. b-Raf was also expressed in 100% of tumors analyzed; 48% (n = 29) were an intensity of 1+, 17% (n = 10) were 2+, and 35% (n = 21) were 3+ (Table 3). Intensity of total ERK and of b-Raf expression in tumors (1+, 2+, or 3+) was positively and notably associated (Appendix Table A1, online only; τ = 0.31). Pre- and post-treatment PBLs were used to explore the pharmacodynamic activity of sorafenib. pERK was measured in pre- and post-treatment PBLs in 37 and 36 women, respectively, by using reverse-phase protein microarray. There was no notable change in pERK levels between pre- and post-treatment PBL specimens (Appendix Table A2, online only; Fig 2).
Table 3.
Biomarker | No. | Analysis |
||
---|---|---|---|---|
Median | Lower Quartile | Upper Quartile | ||
pERK* | ||||
Pretreatment | 37 | 1,553.09 | 1,082.47 | 1,931.40 |
Post-treatment | 36 | 1,238.93 | 874.41 | 1,740.80 |
ΔpERK† | 32 | 23.78 | −756.57 | 442.46 |
ERK (pretreatment)‡ | 60 | 0.89 | 0.16 | 1.89 |
Raf (pretreatment)‡ | 60 | 0.87 | 0.13 | 1.90 |
Abbreviation: pERK, phosphorylated ERK.
pERK as measured by lysate arrays.
Post-treatment pERK minus pretreatment pERK.
ERK and Raf immunohistochemistry H score.
The expression of total ERK and b-Raf in tumors or pERK in PBLs were examined for associations with PFS or OS. ERK and b-Raf expression and pretreatment pERK level in PBLs were not notably associated with tumor response, PFS for at least 6 months, or OS. However, there was an indication of post-treatment pERK levels being associated with tumor response (τ = 0.37) and PFS for at least 6 months (τ = 0.36). Higher levels of post-treatment pERK correlated with a lower risk of progression (Table 4; Fig 3; Appendix Table A3; HR, 0.45; 95% CI, 0.22 to 0.93) but not with OS (HR, 1.41; 95% CI, 0.64 to 3.07; Fig 3). Landmark analysis of PFS on post-treatment pERK (6 weeks after trial entry) for 29 patients yielded similar results (HR, 0.47; 95% CI, 0.21 to 1.07). One of the two responders for whom pre- and post-treatment PBLs were available had high post-treatment pERK levels (Appendix Table A3, online only). Expression levels of the other phosphoproteins measured on the arrays did not vary between pre- and post-treatment and did not correlate with PFS (data not shown).
Table 4.
Biomarker | Progression-Free Survival |
Overall Survival |
||
---|---|---|---|---|
HR | 95% CI | HR | 95%CI | |
pERK | ||||
Pretreatment | 0.79 | 0.40-1.57 | 0.88 | 0.41-1.90 |
Post-treatment | 0.45 | 0.22-0.93 | 1.41 | 0.64-3.07 |
ERK (pretreatment) | 1.09 | 0.64-1.84 | 0.74 | 0.40-1.39 |
Raf (pretreatment) | 0.85 | 0.50-1.45 | 1.02 | 0.55-1.90 |
Abbreviations: HR, hazard ratio; pERK, phosphorylated ERK.
DISCUSSION
In this phase II trial, sorafenib demonstrated modest antitumor activity in patients with recurrent OC; there were two objective, sustained responses, and 14 patients were free of progression at 6 months. Sorafenib targets the Raf kinases and the receptors, VEGFR and PDGFR, and it exerts antitumor activity through direct effects on cancer cells and indirect effects on endothelial cells. The agent has demonstrated clinical benefit in hepatocellular, renal, and thyroid carcinomas, and its study in OC was supported by the knowledge that the Ras-Raf-MAPK pathway is activated in ovarian tumors, mostly through nonconstitutive mechanisms, and that OC progression is heavily dependent on angiogenesis.31–33,35–37
The toxicities observed were substantial and consistent with observations from previous trials.36,38 Notably, dermatologic toxicity and metabolic abnormalities were frequent. There were nine occurrences of grade 3 hand-foot syndrome, and there were seven patients who experienced grade 3 rash, as significant sorafenib toxicities. One patient developed a superficial squamous skin carcinoma in the context of grade 3 rash within 5 months of treatment with sorafenib. Squamous cutaneous carcinomas, keratoachantomas, and flares of actinic keratoses have recently been reported with sorafenib.39–41 Decreased cutaneous immune surveillance caused by impairment of dendritic cell function or compensatory hyperactivation of ERK in keratinocytes induced by selective Raf inhibitors are potential factors in the pathogenesis of proliferative skin lesions induced by sorafenib.39,42–44 In contrast, toxicities specific to antiangiogenic agents (ie, hypertension, proteinuria, or coagulation disturbances) were infrequent. Importantly, grade 3 hypertension occurred only once, and venous or arterial thrombotic events were not recorded. One cardiopulmonary arrest occurred in a patient who developed respiratory failure with wheezing shortly after initiating treatment with sorafenib. GI perforations reported with other anti-VEGF agents in OC were not recorded in this trial.45–47 Myelosuppression was not frequently observed.
Greater than two thirds of patients treated on this study had platinum-resistant OC. Of the two responding patients, one had clear cell carcinoma. This is consistent with prior observations that suggest that the clear cell OC subtype may be more responsive to antiangiogenic agents and that supports testing of such an intervention in this subgroup of tumors.47 There were only two responders to sorafenib, and a total of 14 patients were free of PD for at least 6 months; two patients received treatment for greater than 1 year. This suggests a cytostatic effect of sorafenib, consistent with observations from trials in other tumor types.35,36 Unlike results reported for other anti-VEGF agents (eg, bevacizumab), there was no significant effect of sorafenib in patients with ascites. Interestingly, the outcome of patients who had nonmeasurable disease was not better than that of patients who had measurable disease. Only 12 patients who had detectable disease were enrolled, and only one of these patients remained free of PD for 6 months.
Correlative translational analyses confirmed basal expression of total ERK and b-Raf in all archival ovarian tumors analyzed, of which roughly half demonstrated moderate or intense staining for each protein. Activation of the Ras-Raf-MAPK pathway or b-Raf mutations in ovarian tumors was not analyzed in this study. Pre- and post-treatment PBLs were used to help examine the pharmacodynamic activity of sorafenib. There was no notable change in pERK from pre- to post-treatment in PBLs, consistent with observations from other trials.48,49 Although pretreatment pERK levels were not notably correlated with post-treatment levels in this analysis (Pearson r = 0.33), it is possible that a larger sample size would demonstrate a correlation. Interestingly, there was an indication that higher levels of post-treatment pERK in PBLs were associated with longer PFS. A landmark analysis yielded similar results with a slightly wider CI, perhaps as a result of a smaller sample size.
These findings may be explained by the emerging data suggesting complex regulation of the MAPK pathway through feedback loops. Several reports show that selective Raf or MEK inhibitors hyperactivate the Raf kinase through feedback and induce ERK activation.50,51 A recent study showed that c-Raf inhibits b-Raf in vivo and that pharmacologic inhibition of one Raf protein can cause compensatory activation of the other.43 Because sorafenib is a potent c-Raf inhibitor but is less active against wild-type or mutant b-Raf, selective inhibition of c-Raf by sorafenib could alter the b-Raf/c-Raf interaction that allows b-Raf to activate MEK and ERK. Interestingly, this does not appear to occur in cancer cells, because activated b-Raf does not coexist with mutant Ras or with high levels of inhibitory c-Raf.42 However, in normal cells (ie, PBLs, keratinocytes), b-Raf and c-Raf coexist by controlling, through feedback, the level of ERK activation. Therefore, selective inhibition of c-Raf by sorafenib in PBLs may cause engagement of b-Raf and downstream MEK and ERK activation. Additional evaluation of post-treatment pERK in PBLs as a surrogate marker of sorafenib activity may be warranted.
The results of this trial do not support additional investigation of sorafenib as a single agent in recurrent OC. Evaluation of sorafenib in combination regimens are ongoing.52,53
Acknowledgment
We thank Beverly Kratzel and Meg Colahan from the Gynecologic Oncology Group for assistance with protocol development and management, Sandra Dascomb for her support in data management, the GOG Tissue Bank for their assistance with the banking and distribution of specimens, Kim Blaser for her help with publications, and Frank McCormick of University of California, San Francisco, for helpful discussion.
Appendix
Materials and Methods
Immunohistochemistry.
Paraffin-embedded archival tumors from patients enrolled on this protocol were immunostained for Raf and ERK by using commercially available antibodies against Raf (1:100 dilution; Cell Signaling, Beverly, MA) and ERK (1:100 dilution; Cell Signaling) after antigen retrieval by using sodium citrate. Secondary labeling was based on the Avidin/Biotin system (LSAB2 kit; Dako North America, Carpinteria, CA). Slides were stained with 3 to 3′ diaminobenzidine and were counterstained with hematoxylin. Staining was graded from 0 (no staining) to 3+ (strong staining), and percentage of positively staining cells was recorded for each tumor. An H score was calculated as the product between the intensity and the percent of staining cells.
Isolation of peripheral-blood lymphocytes and reverse-phase protein microarray printing.
Peripheral-blood lymphocytes (PBLs) were collected from patients enrolled on GOG-0170F by using a standard operating procedure. Blood was drawn into sterile Vacutainer tubes (Becton Dickinson and Co, Franklin Lakes, NJ) containing EDTA. Immediately after collection, blood was centrifuged, and the plasma layer was removed. The buffy coat was aliquoted into two fractions and was immediately frozen at −80°C.
PBLs were lysed for 20 minutes in tissue protein extraction reagent (Pierce, Rockford, IL) with 300 mmol/L NaCl, 1 mmol/L orthovanadate, and protease inhibitors. Before reverse-phase protein array printing, lysates were diluted to 1.0 mg/mL and were mixed 1:1 with 2X sodium dodecyl sulfate sample buffer (final concentration, 0.5 mg/mL). Approximately 40 nL of lysate was printed in duplicate onto nitrocellulose-coated glass slides (FAST Slides; Whatman, Keene, NH) with an Aushon 2470 solid pin microarrayer (Aushon Biosystems, Billerica, MA) equipped with 350-μm pins. Samples were printed in duplicate along with a series of positive and negative control lysates that consisted of cell lines treated or untreated with compounds that cause broad phosphoprotein increases (eg, pervanadate, clayculin), and slides were stored, dessicated, at −20°C before antibody staining.
Reverse-phase protein microarray staining.
For estimation of total protein amounts, selected arrays were stained with Sypro Ruby Protein Blot Stain (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and were visualized on a NovaRay CCD fluorescent scanner (Alpha Innotech, San Leandro, CA). Printed slides were prepared for staining by treating with 1X Reblot (Chemicon, Temecula, CA) for 15 minutes followed by two 5-minute washes with phosphate-buffered saline. Slides were treated for at least 5 hours or overnight with blocking solution (1 g I-block [Applied Biosystems, Bedford, MA] and 0.5% Tween-20 in 500 mL phosphate-buffered saline) with constant rocking.
Blocked arrays were stained with antibodies on an automated slide stainer (Dako North America) by using the Catalyzed Signal Amplification System kit according to the manufacturer's recommendation (CSA; Dako North America). Briefly, endogenous biotin was blocked for 10 minutes with the biotin blocking kit (Dako North America), which was followed by application of protein block for 5 minutes; primary antibodies were diluted in antibody diluent and incubated on slides for 30 minutes; biotinylated secondary antibodies were incubated for 15 minutes. Signal amplification involved incubation with a streptavidin-biotin-peroxidase complex provided in the CSA kit for 15 minutes and amplification reagent (ie, biotinyl-tyramide/hydrogen peroxide, streptavidin-peroxidase) for 15 minutes. Signal was generated by using streptavidin-conjugated IRDye680 (LI-COR Biosciences, Lincoln, NE). Slides were allowed to air dry after development.
Slides were stained with a set of 13 antibodies against phosphorylated forms of proteins within the MAPK signaling pathway. All antibodies were subjected to extensive validation for single-band, appropriate molecular weight specificity by Western blot as well as phosphorylation specificity through the use of cell lysate controls (eg, Hela with or without pervandate, Jurkat with or without Calyculin).
Image analysis.
Stained slides were scanned individually on the NovaRay CCD fluorescent scanner (Alpha Innotech). The TIF images for antibody-stained slides and Sypro-stained slide images were analyzed by using MicroVigene version 2.9.9.9 (VigeneTech, Carlisle, MA). Briefly, Microvigene performed spot finding, local background subtraction, replicate averaging, and total protein normalization, which produced a single value for each sample at each end point.
Participating Institutions
The following Gynecologic Oncology Group member institutions participated in this study: Walter Reed Army Medical Center, University of Mississippi Medical Center, Colorado Gynecologic Oncology Group PC, University of Washington, University of Cincinnati, University of Iowa Hospitals and Clinics, Indiana University School of Medicine, University of California Medical Center at Irvine, Washington University School of Medicine, Columbus Cancer Council, University of Virginia Health Sciences Center, University of Chicago, Women and Infants Hospital, and Community Clinical Oncology Program.
Table A1.
ERK | Raf |
|||
---|---|---|---|---|
1+ | 2+ | 3+ | Total | |
1+ | 21 | 5 | 7 | 33 |
2+ | 6 | 3 | 6 | 15 |
3+ | 2 | 2 | 8 | 12 |
Total | 29 | 10 | 21 | 60 |
Table A2.
Pretreatment | Post-Treatment |
||
---|---|---|---|
Low | High | Total | |
Low | 11 | 6 | 17 |
High | 5 | 10 | 15 |
Total | 16 | 16 | 32 |
pERK, phosphorylated ERK; PBL, peripheral-blood lymphocytes.
Table A3.
pERK | Response |
|||
---|---|---|---|---|
Increasing Disease | Stable Disease | Partial Response/ Complete Response | Total | |
Low | 14 | 3 | 0 | 17 |
High | 8 | 8 | 1 | 17 |
Total | 22 | 11 | 1 | 34 |
Abbreviation: pERK, phosphorylated ERK.
Footnotes
Supported by National Cancer Institute grants No. CA 27469 to the Gynecologic Oncology Group Administrative Office and CA 37517 to the Gynecologic Oncology Group Statistical and Data Center and by grant No. MRSG107613 from the American Cancer Society (D.M.), and by the Cancer Therapy and Evaluation Program.
Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
Clinical trial information can be found for the following: NCT00093626.
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Employment or Leadership Position: None Consultant or Advisory Role: Emanuel F. Petricoin, Theranostics Health (U) Stock Ownership: Emanuel F. Petricoin, Theranostics Health Honoraria: Daniela Matei, Bayer; David Mutch, GlaxoSmithKline, Merck, Eli LillyResearch Funding: David Mutch, Eli Lilly, Genentech Expert Testimony: David Mutch, Schroeder, Roger (U) Other Remuneration: None
AUTHOR CONTRIBUTIONS
Conception and design: Daniela Matei, Michael W. Sill, Michael J. Birrer
Financial support: Daniela Matei
Administrative support: David Mutch
Provision of study materials or patients: Daniela Matei, Koen DeGeest, Robert E. Bristow, David Mutch, S. Diane Yamada, David Cohn, Michael J. Birrer
Collection and assembly of data: Daniela Matei, Heather A. Lankes, S. Diane Yamada, Valerie Calvert, John Farley, Emanuel F. Petricoin
Data analysis and interpretation: Daniela Matei, Michael W. Sill, Heather A. Lankes, Valerie Calvert, John Farley, Emanuel F. Petricoin, Michael J. Birrer
Manuscript writing: Daniela Matei, Michael W. Sill, Heather A. Lankes, Koen DeGeest, Robert E. Bristow, David Mutch, S. Diane Yamada, David Cohn, Valerie Calvert, John Farley, Emanuel F. Petricoin, Michael J. Birrer
Final approval of manuscript: Daniela Matei, Michael W. Sill, Heather A. Lankes, Koen DeGeest, Robert E. Bristow, David Mutch, S. Diane Yamada, David Cohn, Valerie Calvert, John Farley, Emanuel F. Petricoin, Michael J. Birrer
REFERENCES
- 1.Jemal A, Siegel R, Ward E, et al. Cancer Statistics, 2009. CA Cancer J Clin. 2009;59:225–249. doi: 10.3322/caac.20006. [DOI] [PubMed] [Google Scholar]
- 2.Ozols R. Cancer, Principles and Practice of Oncology. Philadelphia, PA: Lippincott-Raven; 1997. Ovarian cancer, fallopian tube carcinoma and peritoneal carcinoma, in DeVita VT (ed) pp. 1502–1540. [Google Scholar]
- 3.Gordon AN, Fleagle JT, Guthrie D, et al. Recurrent epithelial ovarian carcinoma: A randomized phase III study of pegylated liposomal doxorubicin versus topotecan. J Clin Oncol. 2001;19:3312–3322. doi: 10.1200/JCO.2001.19.14.3312. [DOI] [PubMed] [Google Scholar]
- 4.Bookman MA, Darcy KM, Clarke-Pearson D, et al. Evaluation of monoclonal humanized anti-HER2 antibody, trastuzumab, in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2: A phase II trial of the Gynecologic Oncology Group. J Clin Oncol. 2003;21:283–290. doi: 10.1200/JCO.2003.10.104. [DOI] [PubMed] [Google Scholar]
- 5.Yang-Feng TL, Li SB, Leung WY, et al. Trisomy 12 and K-ras-2 amplification in human ovarian tumors. Int J Cancer. 1991;48:678–681. doi: 10.1002/ijc.2910480508. [DOI] [PubMed] [Google Scholar]
- 6.van't Veer LJ, Hermens R, van den Berg-Bakker LA, et al. Ras oncogene activation in human ovarian carcinoma. Oncogene. 1988;2:157–165. [PubMed] [Google Scholar]
- 7.Mok SC, Bell DA, Knapp RC, et al. Mutation of K-ras protooncogene in human ovarian epithelial tumors of borderline malignancy. Cancer Res. 1993;53:1489–1492. [PubMed] [Google Scholar]
- 8.Dokianakis DN, Varras MN, Papaefthimiou M, et al. Ras gene activation in malignant cells of human ovarian carcinoma peritoneal fluids. Clin Exp Metastasis. 1999;17:293–297. doi: 10.1023/a:1006611220434. [DOI] [PubMed] [Google Scholar]
- 9.Chien CH, Chow SN. Point mutation of the ras oncogene in human ovarian cancer. DNA Cell Biol. 1993;12:623–627. doi: 10.1089/dna.1993.12.623. [DOI] [PubMed] [Google Scholar]
- 10.Gemignani ML, Schlaerth AC, Bogomolniy F, et al. Role of KRAS and BRAF gene mutations in mucinous ovarian carcinoma. Gynecol Oncol. 2003;90:378–381. doi: 10.1016/s0090-8258(03)00264-6. [DOI] [PubMed] [Google Scholar]
- 11.Kurman RJ, Shih IeM. Pathogenesis of ovarian cancer: Lessons from morphology and molecular biology and their clinical implications. Int J Gynecol Pathol. 2008;27:151–160. doi: 10.1097/PGP.0b013e318161e4f5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Bell DA. Origins and molecular pathology of ovarian cancer. Mod Pathol. 2005;18(suppl 2):S19–S32. doi: 10.1038/modpathol.3800306. [DOI] [PubMed] [Google Scholar]
- 13.Cuatrecasas M, Erill N, Musulen E, et al. K-ras mutations in nonmucinous ovarian epithelial tumors: A molecular analysis and clinicopathologic study of 144 patients. Cancer. 1998;82:1088–1095. [PubMed] [Google Scholar]
- 14.Cuatrecasas M, Villanueva A, Matias-Guiu X, et al. K-ras mutations in mucinous ovarian tumors: A clinicopathologic and molecular study of 95 cases. Cancer. 1997;79:1581–1586. doi: 10.1002/(sici)1097-0142(19970415)79:8<1581::aid-cncr21>3.0.co;2-t. [DOI] [PubMed] [Google Scholar]
- 15.Singer G, Oldt R, 3rd, Cohen Y, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst. 2003;95:484–486. doi: 10.1093/jnci/95.6.484. [DOI] [PubMed] [Google Scholar]
- 16.McPhillips F, Mullen P, Monia BP, et al. Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer. Br J Cancer. 2001;85:1753–1758. doi: 10.1054/bjoc.2001.2139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Mullen P, McPhillips F, MacLeod K, et al. Antisense oligonucleotide targeting of Raf-1: importance of raf-1 mRNA expression levels and raf-1-dependent signaling in determining growth response in ovarian cancer. Clin Cancer Res. 2004;10:2100–2108. doi: 10.1158/1078-0432.ccr-03-0154. [DOI] [PubMed] [Google Scholar]
- 18.Mullen P, McPhillips F, Monia BP, et al. Comparison of strategies targeting Raf-1 mRNA in ovarian cancer. Int J Cancer. 2006;118:1565–1571. doi: 10.1002/ijc.21520. [DOI] [PubMed] [Google Scholar]
- 19.Matei D, Emerson RE, Lai YC, et al. Autocrine activation of PDGFRalpha promotes the progression of ovarian cancer. Oncogene. 2006;25:2060–2069. doi: 10.1038/sj.onc.1209232. [DOI] [PubMed] [Google Scholar]
- 20.Schmandt RE, Broaddus R, Lu KH, et al. Expression of c-ABL, c-KIT, and platelet-derived growth factor receptor-beta in ovarian serous carcinoma and normal ovarian surface epithelium. Cancer. 2003;98:758–764. doi: 10.1002/cncr.11561. [DOI] [PubMed] [Google Scholar]
- 21.Boocock CA, Charnock-Jones DS, Sharkey AM, et al. Expression of vascular endothelial growth factor and its receptors flt and KDR in ovarian carcinoma. J Natl Cancer Inst. 1995;87:506–516. doi: 10.1093/jnci/87.7.506. [DOI] [PubMed] [Google Scholar]
- 22.Erber R, Thurnher A, Katsen AD, et al. Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. Faseb J. 2004;18:338–340. doi: 10.1096/fj.03-0271fje. [DOI] [PubMed] [Google Scholar]
- 23.Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 2006;66:11851–11858. doi: 10.1158/0008-5472.CAN-06-1377. [DOI] [PubMed] [Google Scholar]
- 24.Kim S, Yazici YD, Calzada G, et al. Sorafenib inhibits the angiogenesis and growth of orthotopic anaplastic thyroid carcinoma xenografts in nude mice. Mol Cancer Ther. 2007;6:1785–1792. doi: 10.1158/1535-7163.MCT-06-0595. [DOI] [PubMed] [Google Scholar]
- 25.Murphy DA, Makonnen S, Lassoued W, et al. Inhibition of tumor endothelial ERK activation, angiogenesis, and tumor growth by sorafenib (BAY45-9006) Am J Pathol. 2006;169:1875–1885. doi: 10.2353/ajpath.2006.050711. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Paweletz CP, Charboneau L, Bichsel VE, et al. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene. 2001;20:1981–1989. doi: 10.1038/sj.onc.1204265. [DOI] [PubMed] [Google Scholar]
- 27.Petricoin EF, 3rd, Bichsel VE, Calvert VS, et al. Mapping molecular networks using proteomics: A vision for patient-tailored combination therapy. J Clin Oncol. 2005;23:3614–3621. doi: 10.1200/JCO.2005.02.509. [DOI] [PubMed] [Google Scholar]
- 28.Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the response to treatment in solid tumors: European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92:205–216. doi: 10.1093/jnci/92.3.205. [DOI] [PubMed] [Google Scholar]
- 29.Kondo T, Nakazawa T, Murata S, et al. Enhanced B-Raf protein expression is independent of V600E mutant status in thyroid carcinomas. Hum Pathol. 2007;38:1810–1818. doi: 10.1016/j.humpath.2007.04.014. [DOI] [PubMed] [Google Scholar]
- 30.Schilder RJ, Sill MW, Chen X, et al. Phase II study of gefitinib in patients with relapsed or persistent ovarian or primary peritoneal carcinoma and evaluation of epidermal growth factor receptor mutations and immunohistochemical expression: A Gynecologic Oncology Group Study. Clin Cancer Res. 2005;11:5539–5548. doi: 10.1158/1078-0432.CCR-05-0462. [DOI] [PubMed] [Google Scholar]
- 31.Chen TT, Ng TH. Optimal flexible designs in phase II clinical trials. Stat Med. 1998;17:2301–2312. doi: 10.1002/(sici)1097-0258(19981030)17:20<2301::aid-sim927>3.0.co;2-x. [DOI] [PubMed] [Google Scholar]
- 32.Cox D. Regression models and life tables. J Royal Stat Soc B. 1972;34:187–220. [Google Scholar]
- 33.Anderson JR, Cain KC, Gelber RD. Analysis of survival by tumor response. J Clin Oncol. 1983;1:710–719. doi: 10.1200/JCO.1983.1.11.710. [DOI] [PubMed] [Google Scholar]
- 34.Buyse M, Piedbois P. On the relationship between response to treatment and survival time. Stat Med. 1996;15:2797–2812. doi: 10.1002/(SICI)1097-0258(19961230)15:24<2797::AID-SIM290>3.0.CO;2-V. [DOI] [PubMed] [Google Scholar]
- 35.Ratain MJ, Eisen T, Stadler WM, et al. Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J Clin Oncol. 2006;24:2505–2512. doi: 10.1200/JCO.2005.03.6723. [DOI] [PubMed] [Google Scholar]
- 36.Abou-Alfa GK, Schwartz L, Ricci S, et al. Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol. 2006;24:4293–4300. doi: 10.1200/JCO.2005.01.3441. [DOI] [PubMed] [Google Scholar]
- 37.Gupta-Abramson V, Troxel AB, Nellore A, et al. Phase II trial of sorafenib in advanced thyroid cancer. J Clin Oncol. 2008;26:4714–4719. doi: 10.1200/JCO.2008.16.3279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Elser C, Siu LL, Winquist E, et al. Phase II trial of sorafenib in patients with recurrent or metastatic squamous cell carcinoma of the head and neck or nasopharyngeal carcinoma. J Clin Oncol. 2007;25:3766–3773. doi: 10.1200/JCO.2006.10.2871. [DOI] [PubMed] [Google Scholar]
- 39.Arnault JP, Wechsler J, Escudier B, et al. Keratocanthomas and squamous cell carcinomas in patients receiving sorafenib. J Clin Oncol. 2009;27:e59–61. doi: 10.1200/JCO.2009.23.4823. [DOI] [PubMed] [Google Scholar]
- 40.Dubauskas Z, Kunishige J, Prieto VG, et al. Cutaneous squamous cell carcinoma and inflammation of actinic keratoses associated with sorafenib. Clin Genitourin Cancer. 2009;7:20–23. doi: 10.3816/CGC.2009.n.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Hong DS, Reddy SB, Prieto VG, et al. Multiple squamous cell carcinomas of the skin after therapy with sorafenib combined with tipifarnib. Arch Dermatol. 2008;144:779–782. doi: 10.1001/archderm.144.6.779. [DOI] [PubMed] [Google Scholar]
- 42.Karreth FA, DeNicola GM, Winter SP, et al. C-Raf inhibits MAPK activation and transformation by B-Raf(V600E) Mol Cell. 2009;36:477–486. doi: 10.1016/j.molcel.2009.10.017. [DOI] [PubMed] [Google Scholar]
- 43.Heidorn SJ, Milagre C, Whittaker S, et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010;140:209–221. doi: 10.1016/j.cell.2009.12.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.McCormick F. How blocking Raf activates the MAPK pathway. Pigment Cell Melanoma Res. 2010;23:187–189. doi: 10.1111/j.1755-148X.2010.00680.x. [DOI] [PubMed] [Google Scholar]
- 45.Burger RA. Experience with bevacizumab in the management of epithelial ovarian cancer. J Clin Oncol. 2007;25:2902–2908. doi: 10.1200/JCO.2007.12.1509. [DOI] [PubMed] [Google Scholar]
- 46.Cannistra SA, Matulonis UA, Penson RT, et al. Phase II study of bevacizumab in patients with platinum-resistant ovarian cancer or peritoneal serous cancer. J Clin Oncol. 2007;25:5180–5186. doi: 10.1200/JCO.2007.12.0782. [DOI] [PubMed] [Google Scholar]
- 47.Garcia AA, Hirte H, Fleming G, et al. Phase II clinical trial of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer: A trial of the California, Chicago, and Princess Margaret Hospital phase II consortia. J Clin Oncol. 2008;26:76–82. doi: 10.1200/JCO.2007.12.1939. [DOI] [PubMed] [Google Scholar]
- 48.Escudier B, Lassau N, Angevin E, et al. Phase I trial of sorafenib in combination with IFN alpha-2a in patients with unresectable and/or metastatic renal cell carcinoma or malignant melanoma. Clin Cancer Res. 2007;13:1801–1809. doi: 10.1158/1078-0432.CCR-06-1432. [DOI] [PubMed] [Google Scholar]
- 49.Tong FK, Chow S, Hedley D. Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells. Cytometry B Clin Cytom. 2006;70:107–114. doi: 10.1002/cyto.b.20092. [DOI] [PubMed] [Google Scholar]
- 50.Friday BB, Yu C, Dy GK, et al. BRAF V600E disrupts AZD6244-induced abrogation of negative feedback pathways between extracellular signal-regulated kinase and Raf proteins. Cancer Res. 2008;68:6145–6153. doi: 10.1158/0008-5472.CAN-08-1430. [DOI] [PubMed] [Google Scholar]
- 51.Dougherty MK, Müller J, Ritt DA, et al. Regulation of Raf-1 by direct feedback phosphorylation. Mol Cell. 2005;17:215–224. doi: 10.1016/j.molcel.2004.11.055. [DOI] [PubMed] [Google Scholar]
- 52.Welch S, Hirte H, Elit L, et al. CA-125 response as a marker of clinical benefit in patients with recurrent ovarian cancer treated with gemcitabine and sorafenib: A trial of the PMH Phase II Consortium. J Clin Oncol. 2007;25(suppl 18S):278s. abstr 5519. [Google Scholar]
- 53.Welch S, Hirte H, Schilder RJ, et al. Phase II study of sorafenib (BAY 43-9006) in combination with gemcitabine in recurrent epithelial ovarian cancer: A PMH phase II consortium trial. J Clin Oncol. 2006;24(suppl 18S):276s. abstr 508. [Google Scholar]