Phase II study of saracatinib (AZD0530) in patients with previously treated metastatic colorectal cancer

S. M. Reddy 1 & S. Kopetz 2 & J. Morris 3 & N. Parikh 4 & W. Qiao 3 &
M. J. Overman 2 & D. Fogelman 2 & I. Shureiqi 2 & C. Jacobs 2 & Z. Malik 5 &
C. A. Jimenez 6 & R. A. Wolff 2 & J. L. Abbruzzese 7 & G. Gallick 8 & C. Eng 2

Received: 7 April 2015 /Accepted: 2 June 2015 /Published online: 12 June 2015 # Springer Science+Business Media New York 2015

Summary Background Src has a critical role in tumor cell migration and invasion. Increased Src activity has been shown to correlate with disease progression and poor prognosis, sug- gesting Src could serve as a therapeutic target for kinase inhi- bition. Saracatinib (AZD0530) is a novel selective oral Src kinase inhibitor. Methods Metastatic colorectal cancer patients who had received one prior treatment and had measurable
disease were enrolled in this phase 2 study. Saracatinib was administered at 175 mg by mouth daily for 28 day cycles until dose-limiting toxicity or progression as determined by staging every 2 cycles. The primary endpoint was improvement in 4 month progression-free survival. Design of Thall, Simon, and Estey was used to monitor proportion of patients that were progression free at 4 months. The trial was opened with plan

* S. M. Reddy [email protected]
S. Kopetz [email protected]
J. Morris [email protected]
N. Parikh [email protected]
W. Qiao [email protected]
M. J. Overman [email protected]
D. Fogelman [email protected]
I.Shureiqi [email protected]
C. Jacobs [email protected]
Z. Malik [email protected]

1

2

3

4

5

6

G. Gallick [email protected]
C. Eng [email protected]

Hematology-Oncology Fellow, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 463, Houston, TX 77030, USA
Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Unit 426, Houston, TX 77030, USA
Department of Biostatistics & Applied Math, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 0447, Houston, TX 77030, USA
Department of Genitourinary Medical Oncology-Research, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
Norton Cancer Institute, 4950 Norton Healthcare Blvd, Ste 300, Louisville, KY 40241, USA
Department of Pulmonary Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 403, Houston, TX 77030, USA

C. A. Jimenez [email protected]
R. A. Wolff
7 Duke Cancer Institute, DUMC Box 3917, 10 Bryan Searle Drive, Seeley Mudd Building, 2nd floor, Durham, NC 27710, USA

[email protected]
J.L. Abbruzzese [email protected]
8
Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Unit 173, Houston, TX 77030, USA

to enroll maximum of 35 patients, with futility assessment every 10 patients. Results A total of 10 patients were enrolled between January and November 2007. Further enrollment was stopped due to futility. Median progression-free survival was 7.9 weeks, with all 10 patients showing disease progression following radiographic imaging. Median overall survival was 13.5 months. All patients were deceased by time of analysis. Observed adverse events were notable for a higher than ex- pected number of patients with grade 3 hypophosphatemia (n =5). Conclusion Saracatinib is a novel oral Src kinase in- hibitor that was well tolerated but failed to meet its primary endpoint of improvement in 4 month progression-free survival as a single agent in previously treated metastatic colorectal cancer patients.

Keywords Saracatinib . Src inhibitor . Colorectal cancer . Phase 2 . AZD0530 . Colon cancer . Src kinase

Introduction

Metastatic colorectal cancer remains one of the most common cancers in worldwide incidence and mortality. For individuals with surgically unresectable disease, the expected 5-year rel- ative survival is less than 15 % [1]. Despite significant ad- vances in treatment of metastatic colorectal cancer with cur- rently available regimens of FOLFOX and FOLFIRI plus or minus bevacizumab or cetuximab, response rates for those that progress after first line treatment is only 4–17 % [2, 3]. Dual inhibition of the vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) path- ways has been shown in preclinical models to counteract treat- ment resistance by targeting both the tumor biology as well as microenvironment [4]. Despite inferior results with the com- bination of bevacizumab and cetuximab in the phase III PACCE [5] and CAIRO2 [6] trials, preclinical studies in mice [7] show synergistic activity when small molecule inhibitors of VEGF receptors and EGFR were used instead of antibodies, implying importance of inhibiting intracellular signaling.
Src, a non-receptor tyrosine kinase that is activated “down- stream” of both VEGF and EGF signaling pathways, presents a possible single therapeutic target for dual EGFR and VEGF intracellular pathway inhibition. Src activates the Ras-Raf- MEK-ERK1/2 pathway, which is a key downstream effector of the epidermal growth factor receptor [8, 9], as well as the signal transducer and activator of transcription 3 (STAT-3), a mediator of angiogenesis through VEGF and interleukin-8 [10–12]. By additionally interacting with the PI3K/Akt pathway [11], other receptor tyrosine kinases in addition to EGFR [13], and cytoskeletal components including FAK, paxillin, and E-cadherin, Src is a key regulator of tumor cell adhesion, migration, and invasiveness.

Notably, Src expression and activity has been shown to be higher in malignant than benign colon polyps [14] and in metastatic than nonmetastatic colon cancer [15, 16], highlight- ing its role in colorectal carcinogenesis.
AZD0530, also known as saracatinib, is an orally available dual Src/Abl kinase inhibitor with high selec- tivity for c-Src and Abl relative to other tyrosine and serine-threonine kinases [17]. It has been shown in pre- clinical models to inhibit tumor invasion and migration [18, 19]. A Phase 1 study in 30 solid tumor patients showed that it was relatively well tolerated with maxi- mum tolerated dose of 175 mg with most common grade ≥ 3 events of anemia, diarrhea, and asthenia [20]. We conducted a phase 2 study of AZD0530 in patients with previously treated metastatic colorectal cancer to evaluate its efficacy as a single agent as well as its toxicity profile in this patient population. To better un- derstand the underlying mechanism of action and to isolate potential biomarkers for patient selection, we planned to study various downstream effectors of Src, including proteins in- volved in cell adhesions, angiogenesis, and intracellular signaling.

Patients and methods

Patient selection

Eligible patients were required to be ≥ 18 years of age with histologically or cytologically confirmed adenocar- cinoma of the colon or rectum with metastatic, measur- able disease as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria 1.0, and must have received one prior chemotherapy regimen for metastatic colorectal cancer; prior biologic therapy such as bevacizumab was allowed. Patients must have recovered from prior treatment related toxicities (> grade 2 neuropathy or>grade 1 non-neuropathy treatment related adverse events) by study registration. Study subjects were required to have an Eastern Cooperative Oncology Group (ECOG) performance status 0–2. All laboratory tests including complete blood count, complete metabolic panel, and urine protein were to be within acceptable limits as specified in study protocol. The use of CYP3A4-active agents was prohibited. Patients were excluded if they had uncontrolled concurrent illness such as ongoing infection or class III/IV NYHA cardiac disease, known brain metastases, history of another primary malignan- cy within 5 years with exception of non-melanoma skin can- cer and carcinoma in situ of uterine cervix, and HIV positivity (given interaction between AZD0530 and highly active anti- retroviral therapy). Patients were required to provide informed consent as approved by the MD Anderson Institutional Review Board.

Pretreatment evaluation

Baseline evaluations were completed within 1 week prior to administration of AZD0530. Patients underwent a history and physical examination, assessment of performance status, mea- surement of serum β-human chorionic gonadotropin in wom- en of childbearing potential, serum complete blood count with differential and platelets, serum chemistry, adrenocorticotro- pic hormone stimulation test, serum creatinine phosphokinase and troponin levels, serum carcinoembryonic antigen level, urine protein, electrocardiogram, pulmonary function testing, computed tomography (CT) or magnetic resonance imaging (MRI) of chest/abdomen/pelvis, and optional correlative studies.

Treatment

AZD0530 was administered orally at 175 mg daily; one cycle being defined as 28 days. AZD05230 was administered con- tinuously until either disease progression, concurrent illness that prevents further administration of treatment, unacceptable adverse event(s), dose delay for >14 days, patient withdrawal from study, or general or specific changes in patient’s condi- tion that rendered the patient unacceptable for further treat- ment in the judgment of the investigator.

Dose modifications

Adverse event grading was performed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. Up to 2 dose reduc- tions were allowed before study discontinuation. For grades 3 and 4 hematologic toxicity, the dose was held until recovery to ≤ grade 2 toxicity and dose reduced to levels -1 and -2, respectively. For grades 2–3 nausea or vomiting refractory to anti-emetic treatment, the treatment was held until recovery to grade ≤ 1 and dose reduced by -1 dose level. For grade 3 fatigue or neuropathy, treatment was held until recovery to ≤ grade 2 and then dose reduced to level -1. For grades 3 and 4 hypophosphatemia, treatment was held until recovery to ≤grade 2 and then dose reduced to levels -1 and -2, respectively. For all other grades 2–4 non-hematologic toxicities, treatment was held until recovery to≤grade 1 and then restarted at level -1 for grades 2–3 and level -2 for grade 4 non-hematologic toxic- ities. Patients were removed from the study for≥grade 2 adrenal insufficiency or if≥30 % decrease in DLCO relative to baseline with radiographic evidence of pulmonary toxicity on high reso- lution computed tomography scan.

Evaluation of response and toxicities

Patients were evaluated for response according to RECIST 1.0 criteria. Patients were reevaluated for radiologic response by

CT or MRI every 8 consecutive weeks on treatment. Confirmatory CT or MRI scans were required 4 weeks after initial documentation of objective complete or partial re- sponse. Patients remained on study until progression, unac- ceptable toxicity to the patient or as defined by the protocol, or patient withdrawal from study. Toxicity assessment included clinical evaluation every 2 weeks with serum complete blood count and chemistries, weekly urine protein, DLCO at end of cycle 1 and then at end of study treatment, EKG and cardiac serum markers every 2 weeks for first cycle and then every 8 weeks thereafter, and ACTH stimulation test every 2 cycles.

Exploratory correlative studies

All correlative studies were optional under informed consent. Immunohistochemical (IHC) staining on paraf- fin embedded tissue samples was to be completed pre- and post-treatment (2 weeks±2 days after start of treatment of AZD0530) according to manufacturer’s instructions for FAK (Upstate Biotechnology, Lake Placid, NY), FAK Tyr861 (Biosource International, Camarillo, CA), paxillin (US Biological, Swampscott, MA), paxillin Tyr118 (US Biological, Swampscott, MA), E-cadherin (AbCam, Cambridge, MA), Stat3 (US Biological, Swampscott, MA), Stat3 Tyr705 (US Biological, Swampscott, MA), and CD31 (AbCam, Cambridge, MA).
Whole blood was obtained at baseline and 2 weeks (±2 days) after start of treatment to measure serum markers of vascular endothelial growth factor, an indicator of angio- genesis. Serum VEGF-A level was determined by commercial enzyme-linked immunosorbent assay (ELISA) kits following the manufacturers’ instructions (Human VEGF Immunoassay kit, Biosource International, Inc. Camarillo, CA).

Statistical methods

The primary endpoint for this single arm phase II trial was progression free survival (PFS) at 4 months. The design of Thall, Simon, and Estey (1995) was used to monitor the pro- portion of patients that were progression free at 4 months. We defined the study as being positive if the true rate of 4 month PFS on AZD0530 was at least 0.05 higher than true rate of
4month PFS on bevacizumab which was estimated based on prior studies to be 0.358, with one-sided alpha significance level of 0.05. The trial was opened with plan to enroll maxi- mum of 35 patients, with futility assessment every 10 patients. Secondary endpoints included overall survival, time to pro- gression, and biologic correlative studies on pre and post treat- ment blood and tissue blood samples. Kaplan-Meier product- limit survival probability estimates were calculated for overall survival and time to progression. VEGF-A change, defined as the difference between the post-treatment and pre-treatment

levels of VEGF-A, was assessed using the signed-rank test. All computations were carried out in SAS 9.3 and TIBCO Spotfire S+ Version 8.2.

Results

Patient characteristics

Between January and November 2007, 10 patients were enrolled. Table 1 shows the baseline patient characteris- tics of these 10 patients. The mean patient age was 60 years (range 46–70 years). Primary tumor was colon in 9 patients, and sites of metastases included liver (9 patients), lung (3 patients), peritoneum (2 patients), and pericolonic tissue (1 patient). The colorectal adenocarci- nomas showed moderate histology in 8 patients and poor histology in the other 2. Prior chemotherapy regimens includ- ed FOLFOX and bevacizumab (4 patients), FOLFIRI and bevacizumab (4 patients), CapeOx and bevacizumab (1 patient), and FOLFOX alone (1 patient).

Survival

The median progression free survival was 7.6 weeks, as seen in Fig. 1. The study was terminated early with 10 enrolled patients after interim analysis for futility. All 10 patients showed evidence of progression by the 8 week time point as determined by RECIST 1.0 criteria on CT imaging, including one patient who was taken off study at 2 weeks and another at
5weeks for clinical evidence of disease progression. After disease progression, 7 patients proceeded to receive third line treatment. Figure 2 shows the overall survival of patients in our study. By the time of analysis, all patients were confirmed to have died. Median overall survival was 13.5 months.

Treatment-related toxicities

The median time on treatment was 8 weeks, or 2 cycles. As seen in Table 2, 7 patients suffered grade 3 or 4 toxicities, of whom 4 required dose reductions. One patient was removed from the study at 6 weeks due to persistent hypophosphatemia despite 2 dose reductions. Another patient was removed from study at 5 weeks due to a grade 3 shoulder tendon tear, unre- lated to study drug. All other patients continued study drug until disease progression. Table 2 shows grades 1–3 toxicities suffered while on study drug. Grade 3 toxicities included

Table 1 Baseline patient, treatment, and tumor characteristics
hypophosphatemia (N =5), hyponatremia (N =2), hypoalbu- minemia (N =2), nausea (N =2) and one patient each with lym-

Mean Age in Years (Range) Sex N (%)
Male Female
Performance Status N (%) 0
1
2
60 (46–70)

6 (60) 4 (40)

6 (60) 3 (30) 1 (10)
phopenia, leukopenia, dehydration, and hypocalcemia. No pa- tient had grade 4 toxicity related to study drug. Less severe toxicities included nausea, dyspnea, fatigue, proteinuria, and transaminitis. Of note, one patient had a grade 4 bowel perfo- ration resulting in sepsis but this was attributed to disease progression rather than study drug. Another patient died with- in 28 days of study treatment but was found to have progres- sion of disease on radiographic imaging.

Histology N (%)

Poor 2 (20)
Moderate 8 (80) Prior Therapy N (%)
FOLFOX+bevaciziumab 4 (40)
FOLFIRI+bevacizumab 4 (40)
CapeOx+bevacizumab 1 (10)
FOLFOX 1 (10) Site of Primary Disease N (%)
Colon 9 (90)
Rectal 1 (10) Site of Metastatic Disease N (%)
Liver 9 (90)
Lung 3 (30)
Peritoneum 2 (20)
Pericolonic 1 (10)
Mean serum CEA level ng/mL (range) 140.0 (1.8–576.4)
Exploratory correlative studies

6of the 10 patients in this study consented to have serum collected. Figure 3 shows VEGF-A levels at baseline and at 14 days post-treatment in these patients. No statistically sig- nificant trend was seen between these two time points. No patients consented to optional collection of tumor tissue for correlative studies of the cell adhesion markers FAK, paxillin, and E-cadherin, or of angiogenesis markers STAT3 and CD31.

Discussion

This is the first clinical trial of single agent saracatinib (AZD0530), a selective oral Src kinase inhibitor, in colorectal cancer patients. As a key regulator of both the EGFR and

Fig. 1 Kaplan-Meier curve for progression free survival (weeks) in metastatic colorectal patients treated with single agent saracatinib in the second line setting. Median progression free survival was 7.6 weeks

Median TTP (95% CI)=7.6 (5.1,NA)

0 2 4 6 8
Time (week)

angiogenesis pathways and preclinical work suggesting dual inhibition of these pathways can overcome treatment resis- tance, Src inhibition is theoretically an ideal therapeutic strat- egy. We conducted a phase II trial of saracatinib in previously treated patients with metastatic colorectal cancer and found no benefit in our primary endpoint of 4 month progression free survival. The study was stopped early after interim analysis of the first 10 patients demonstrated lack of efficacy and progres- sive disease in all patients at 8 week follow-up.
Toxicities in our patient population consisted of cytopenias, gastrointestinal symptoms, and electrolyte abnormalities, con- sistent with previously published reports [20, 21]. In contrast to other studies, however, our patients exhibited a higher de- gree of hypophosphatemia, with 5 patients having grade 3
hypophosphatemia. It is possible that the presence of a cancer of the gastrointestinal tract with this agent makes this a unique problem for this patient population. Future studies of this agent in colorectal cancer patients and/or other Src kinase inhibitors will need to closely monitor this. Our patients re- ceived phosphate repletion per protocol so clinically signifi- cant hypophosphatemia did not occur.
A significant weakness of this clinical trial is the limited correlative studies. Despite our efforts, no patient consented for a fresh tissue biopsy that would allow analysis of the src kinase pathway on cell adhesion markers and angiogenesis. Furthermore, only 6 patients provided serum samples for VEGF-A analysis, with no significant change noted between levels pre-treatment and 14 days after starting saracatinib.

Fig. 2 Kaplan-Meier curve for overall survival (months) in metastatic colorectal patients treated with single agent saracatinib in second line setting. Median overall survival was
13.5 months

Median OS (95% CI)=13.5 (6.1,NA)

0 5 10 15 20 25
Time (month)

Table 2 Treatment related toxicities

Toxicity

Hematologic Lymphopenia Leukopenia

Grade

1 2 3

1
1

paclitaxel in platinum-resistant ovarian, fallopian and primary peritoneal cancer [30].
Despite the poor single agent activity of saracatinib, scien- tific rationale supports combination treatment of Src inhibitors with currently established agents for metastatic colorectal can- cer. Increased Src expression has been shown in preclinical studies to be associated with acquired resistance to oxaliplatin,

Thrombocytopenia 4 Nonhematologic
Nausea 3

2
with Src mutated cell lines showing increased oxaliplatin in- duced apoptosis [31, 32]. Further, synergistic activity has been demonstrated in colorectal cancer cell lines with addition of

Dyspnea Fatigue
1
4 3
another Src inhibitor, dasatinib, to oxaliplatin. Similarly, addi- tion of dasatinib to cetuximab, an anti-EGFR monoclonal an-

Dehydration 1 tibody used in treatment of metastatic colorectal cancer, has

Proteinuria Transaminitis
4
2
been shown to have antiproliferative effect in vitro and in- creased apoptosis in vivo [33]. This was notably seen in

Hypophosphatemia Hyponatremia Hypoalbuminemia Hypocalcemia
1 5 2 2 1
Kras mutant cell lines not traditionally thought to be sensitive to cetuximab. Saracatinib itself has been tested in head and neck squamous cell cancer cells, where cetuximab has also been clinically effective, and shown to have additive effects when combined with cetuximab [34]. Lastly, saracatinib com- bination with bevacizumab is supported by preclinical data

Given the reduced sample size, the impact of saracatinib on VEGF-A levels could not be adequately ascertained. Thus, it is unknown whether saracatinib was effective in inhibiting its target (Src family kinases) or signaling pathways regulated by these kinases.
As in our trial, other solid tumor studies have been pub- lished also showing the lack of benefit of this agent as mono- therapy in gastric and gastroesophageal junction cancer, recur- rent or metastatic head and neck squamous cell cancer, castration-resistant prostate cancer, metastatic melanoma, hor- mone receptor negative breast cancer, extensive stage small cell lung cancer, and pancreatic cancer [21–27]. A phase II trial in 37 patients with previously-treated advanced non- small-cell lung cancer, however, showed some signal of ben- efit, with 2 of 31 evaluable patients having partial responses lasting 3.7 and 14.6 months, and 6 patients being progression free at 16 weeks (primary endpoint) [28]. Saracatinib has not been studied in combination with other therapeutic agents in colorectal cancer but has not improved outcomes in combina- tion studies with gemcitabine in pancreatic cancer [29] or with
showing that Src inhibition decreases plasma VEGF expres- sion [35]. Src inhibitors may therefore work synergistically with bevacizumab to prevent compensatory increases in VEGF expression to maximize benefit from anti-angiogenic therapy. Based on the above data, further study is warranted of saracatinib in combination with oxaliplatin, cetuximab, and/or bevacizumab in metastatic colorectal cancer patients.
Dasatinib, another selective Src kinase inhibitor, is ap- proved for first-line use in chronic myelogenous leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia due to its inhibition of Bcr-Abl. Dasatinib has also been investigated in phase I/II studies of colorectal cancer. No activity was seen when dasatinib was used as a single agent in a multicenter phase II study of 19 patients with previously treated metastatic colorectal cancer [36]. However, in a recent- ly published phase 1 study of dasatinib in combination with capecitabine, oxaliplatin, and bevacizumab, an expansion co- hort of 10 untreated colorectal cancer patients demonstrated an ORR of 70 % with median PFS of 13.4 mo [37]. Selecting for a subset of patients likely to respond to Src inhibition,

Fig. 3 VEGF-A levels in 6 patients pre- and 14 days post- treatment with saracatinib. No significant difference was seen between the two time points

ORR was 83 % among the 6 patients with high expression of activated Src and 0 % among the 2 patients with low expres- sion of activated Src [37]. Another combination phase 1 study of dasatinib with FOLFOX and cetuximab in refractory colo- rectal cancer patients [38] demonstrated a 24 % overall partial response rate, including 17 % of patients previously refractory to FOLFOX and cetuximab, and median PFS of 4.6 months. The phase 2 portion of this study has been completed with final results pending. Other Src kinase inhibitors under inves- tigation in solid tumors such as bosutinib have not yet been studied clinically in colorectal cancer [39, 40].
Though this phase II study of saracatinib as a single agent in previously treated metastatic colorectal cancer showed no benefit, scientific rationale for Src inhibition and experience from dasatinib should inform future studies of saracatinib. First, as a more selective Src inhibitor than dasatinib, saracatinib presents an alternative agent with potential for en- hanced efficacy and toxicity profile compared to dasatinib and should therefore be further investigated in combination stud- ies. Second, it is important to identify predictive markers such as expression of activated Src [37] to select patients likely to benefit from Src inhibition across all solid tumors, and such biomarkers should be incorporated into future trials.

Compliance with Ethical Standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Funding This study was presented at AACR 2008 and funded by the National Cancer Institute (NCI Protocol #7565, NSC735464).

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