Phase II Clinical Chemoprevention Trial of Weekly Erlotinib before Bladder Cancer Surgery

Abstract

We performed a clinical trial in non-muscle invasive urothelial cancer (NMIUC) patients randomized (2:1) to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib or placebo (either orally once weekly x 3 doses prior to scheduled surgery) to assess for a difference in EGFR phosphorylation in tumor adjacent normal urothelium <24 hours post-study dose and tolerance of weekly erlotinib. Thirty-seven volunteers (6 female/31 male, mean age 70, 35 white/2 non-white) with confirmed or suspected NMIUC were enrolled into either erlotinib (n=24; 900 mg-13, 600 mg-11) or placebo (n=13). Immunohistochemical assessment of phosphorylated and total EGFR in adjacent normal urothelium (20 erlotinib; 9 placebo) or tumor (21 erlotinib and 11 placebo subjects) at study end observed no significant difference between those receiving erlotinib or placebo. This was also true for other assessed tissue biomarkers (phosphorylated ERK, ERK, e-cadherin, p53 and Ki67). Adverse events were more common, in a dose-related fashion, in participants receiving erlotinib, e.g. 38% experienced Grade 1 with rare grade 2 diarrhea and skin toxicity vs 8% in placebo. Clinically insignificant, but statistically significant (p=0.001) elevations in serum total bilirubin and creatinine were observed in erlotinib participants. Serum erlotinib and metabolite concentrations (OSI-420) confirmed compliance in all erlotinib subjects and did not significantly differ between the 600 and 900 mg doses. Despite compelling pre-clinical and clinical data for targeted EGFR inhibition in bladder cancer prevention, these data do not support the use of weekly erlotinib to prevent progression of NMI bladder cancer.

Introduction

Bladder cancer is the most common malignancy of the urinary tract and the sixth most commonly diagnosed malignancy in the United States with an estimated 80,500 cases in 2019 (1). Urothelial cancer of the bladder (UC) is thought to evolve along two phenotypic pathways: low-grade and high-grade. All UCs arise on the urothelial (luminal) surface. Low-grade UCs rarely invade more deeply, and if so, usually only as far as the next layer (lamina propria). High-grade cancers on the other hand invade quickly into and through the bladder wall as well as metastasize once they reach the deeper bladder layers (muscle invading/MI). Both low and high-grade non-muscle invading (NMI) UCs re-occur elsewhere in the bladder in from 30 – 80% of cases after transurethral resection of the original bladder tumor (TURBT), and recurrent cancers often re-occur again (and again) and can be multi-focal despite treatment with repeat TURBTs and intravesical instillations of chemo - or immunotherapy (2,3).

High-grade (almost always) MI UCs are no longer manageable by TURBT and intravesical therapies and require much more aggressive treatments (e.g. neoadjuvant or adjuvant chemotherapy with cystectomy or radiation therapy) (4,5). Because of the high rates of recurrence and risk of progression to MI disease, NMI bladder cancer patients are routinely reevaluated at frequent intervals by regular cystoscopic examinations and monitoring urine cytology and/or other diagnostic markers.

Molecular studies have demonstrated consistent epidermal growth factor receptor (HER1/EGFR) overexpression in human UCs (6-7). The level of overexpression correlates with tumor grade and stage, tumor recurrence and overall survival (8 - 15). NMI UCs (stages Ta, T1, Tis) that express HER1/EGFR are significantly more likely to progress toward MI disease than those with low or no expression (13). Preclinical prevention studies have found that small molecule inhibitors of HER1/EGFR are effective bladder cancer preventive agents (16,17). Erlotinib HCl (Tarceva) is an orally available, highly selective, reversible inhibitor of HER1/EGFR tyrosine kinase that is currently approved for use in locally advanced or metastatic EGFR-mutant non-small cell lung cancer and locally advanced, unresectable or metastatic pancreatic cancer in combination with gemcitabine (16). Inhibition of tyrosine kinase activity prevents HER1/EGFR phosphorylation, the associated downstream signaling events, and may block tumorigenesis mediated by inappropriate HER1/EGFR signaling. The recommended dosing in adults is an oral dose of 150 mg daily with dose-limiting toxicity being grade 3 or 4 diarrhea or skin toxicity (17,18).

Pharmacokinetic analyses have demonstrated that erlotinib has a relatively long half-life (10-12 hours), predominant metabolism via cytochrome P450 with some correlation to hepatic function, and increasing plasma levels with repeated daily dosing (19-20). Correlation between increasing erlotinib plasma concentrations and human tolerance (skin toxicity) has been inconsistent (21-23). Erlotinib pharmacokinetics have been observed to be markedly affected (plasma levels diminished by approximately 50%) by current cigarette smoking or omeprazole co-administration, with some studies correlating this to patient tolerance or anti-tumor effect (24-30).

Concerns over tolerance of daily erlotinib have led to studies of weekly dosing in advanced lung cancer patients (31-33). Milton et al. administered erlotinib in weekly doses of 1200, 1600 and 2000 mg without dose-limiting toxicity, but grade 1 and 2 diarrhea and rash were common (31) Consistent with the benefit of weekly erlotinib, preclinical bladder and breast cancer prevention models have observed significant anti-cancer effects of intermittent erlotinib or gefitinib exposure (34-35). While there have been no clinical prevention trials in patients at increased risk for bladder cancer, in 20 patients with MI bladder cancer receiving 150 mg of erlotinib once daily for four weeks prior to undergoing radical cystectomy, 25% of patients had a complete response (pT0), and in total, seven (35%) had pathologic down staging at cystectomy (36).

To investigate the concept of EGFR antagonism as a potential means of bladder cancer prevention, we conducted a randomized, placebo-controlled, double-blind trial of weekly erlotinib versus placebo in patients scheduled to undergo bladder cancer surgery. Our primary goals were to determine if erlotinib administered weekly for 3 weeks reduces EGFR phosphorylation in normal appearing bladder urothelium adjacent to tumor and to assess the tolerability of this regimen in patients at increased risk of urothelial cancer.

Materials and Methods

Patient eligibility and recruitment

Adults aged 18 and older with a confirmed or suspected MIBC or NMIBC discovered on cystoscopy or radiologic imaging with Karnofsky performance status ≥60% were eligible for this study. Participants were required to have WBC ≥ 3000/mm³; platelets ≥ 100,000mm³, hemoglobin >10 g/dL as well as normal hepatic and renal function and normal electrolyte levels. Participants were limited in what potentially interacting concomitant medications (e.g. cytochrome P45-3A4/CYP3A4 inhibitors or inducers) were allowed. Enrolled women and the female partners of enrolled men were to use two forms of birth control. Participants were also excluded if they were taking other investigational agents, had a history of allergic reactions attributed to compounds similar in composition to erlotinib or clindamycin, a concurrent skin rash or skin condition requiring treatment with a prescription medication, were required to take daily NSAIDS >81 mg aspirin per day, had an uncontrolled intercurrent illness, were pregnant or nursing, or concurrent suspected upper track or prostatic UC. Use of proton pump inhibitors was initially exclusionary (14 participants), however in later versions of the protocol they were allowed and included as part of the stratification for randomization (23 participants). Use of Coumadin was not initially exclusionary (28 participants) but was excluded in later version of the protocol (9 participants).

Trial Design

This placebo-controlled, double-blind phase II clinical trial (ClinicalTrials.gov ID NCT02169284) was randomized on a 1:2 ratio of placebo vs. active study agent and stratified according to either tumor status (NMI or MI), whether likely diminished erlotinib bioavailability (current smokers and/or concomitant proton pump inhibitor medication) or not and planned surgery (TURBT vs. cystectomy). The trial was conducted (employing CONSORT guidelines) at the University of Wisconsin-Madison, which served as the coordinating site, Lahey Clinic, the University of Rochester Medical Center, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Urology San Antonio Research, and Carolina Urologic Research Center. Participants were recruited from a population with a confirmed or suspected bladder tumor to be treated with TURBT or cystectomy. Eligible participants were initially randomly assigned to either 900 mg erlotinib po or matching placebo (20 participants), and in later versions of the protocol were randomly assigned to either 600 mg erlotinib po or matching placebo (17 participants) once weekly for 3 doses preceding their scheduled bladder surgery. Study recruitment started 06/12/2014 to 03/31/2018.

Treatment plan

Participants took study medication on an empty stomach (≥ two hours after last meal, ≥ one hour before next meal) and were evaluated on Day 8 either by phone or in-person study visit. On day 15 participants in early versions of the protocol took their third dose 24 hours before their planned surgery (20 participants) and in later versions 9-18 hours before their planned surgery (17 participants). To ensure compliance, participants were instructed verbally and in writing. Adverse Events (AE) were graded using the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE version 4.0).

Pharmaceutical Information

Erlotinib was supplied as hydrochloride salt by Astellas Pharma Global Development, Inc (Northbrook, Illinois, United States) in 150-mg tablets. Matching placebo tablets were manufactured containing lactose monohydrate, microcrystalline cellulose, croscarmellose sodium and magnesium stearate.

Management of toxicity and dose reductions

Along with the doses of erlotinib/placebo, participants were also dispensed Hydrocortisone 1% and clindamycin 1% ointments and instructed on its use by a study physician if they experienced an adverse skin reaction. Patients were educated on the appropriate use of loperamide with the development of any treatment-related diarrhea. Reduction/interruption of erlotinib dosing for AEs was allowed at any time during the study. For ≥ grade 2 rash and diarrhea or ≥ grade 3 all other toxicities, study dose was reduced by 50% (900→450 mg or 600→300mg). For grade 1 rash or diarrhea, participants had the option of a 50% dose reduction.

Pretreatment and Follow-up evaluations

Baseline evaluation including medical history and physical examination, review of concomitant medications, routine labs (including pregnancy testing for females of child-bearing potential) and International Prostate Symptom Score (IPSS) (37) for males were obtained within 30 days of the subject's Day 1 dose of study agent to confirm eligibility. All baseline examinations were completed before randomization and dispensing study drug. A blood sample for pharmacokinetics was collected at baseline visit. Eligibility confirming baseline cystoscopy assessing tumor status (MI, NMI) was completed within 60 days of randomization. On Day 8 and 15, participants were evaluated prior to ingesting their study dose. Evaluation consisted of assessment of toxicities, IPSS survey (day 15 only) and concomitant medications along with vitals if an in-person study visit. Blood for safety laboratory analysis (WBC, platelets, hemoglobin, hematocrit, BUN, total bilirubin, AST, ALT, alkaline phosphatase, creatinine, sodium, potassium, chloride and bicarbonate, calcium and INR) and pharmacokinetics was obtained prior to study dose. If dose modifying toxicity occurred, the participants were instructed on their modified study dose for day 8 and 15. Approximately 7-14 days after surgery, participants were contacted via telephone to assess toxicity related to the Day 15 study dose.

Biomarker Sample Schedule

Plasma samples were collected at the baseline visit, Day 8 visit, and Surgery Visit (day 15-16). All samples were stored at between −80 to −70º C. Bladder urothelium and tumor (if available) was collected at the Surgery Visit (day 15-16).

Analysis of Erlotinib and OSI-240 Levels in Plasma

To assess the pharmacokinetics of weekly erlotinib, erlotinib and OSI-420 (dominant erlotinib metabolite) were quantified in plasma by LC-MS/MS using a Waters Acquity ultra-performance liquid chromatography (UPLC) system coupled to an AB Sciex Q-Trap 5500 triple quadrupole mass spectrometer. A ten-point standard curve for both compounds was prepared in plasma. Samples (100 μl) of patient plasma, standards, and quality controls were processed on a Waters’ Ostro 96-well plate, using a positive-pressure manifold after addition of four volumes acetonitrile containing the internal standard midazolam. Samples requiring dilution were mixed 1/10 in blank plasma prior to Ostro plate loading. Following processing, samples were dried under nitrogen and resuspended in 500 μl 25% acetonitrile/75% water/0.1% formic acid.

Samples were separated on an Agilent XDB-C18 Zorbax RRHD column (2.1 x 50 mm, 1.8 μm particle size) using an increasing gradient of solvent B (5% water/95% acetonitrile [vol/vol] with 1 mM ammonium formate and 0.1% formic acid) to solvent A (95% water/5% acetonitrile [vol/vol] with 1 mM ammonium formate and 0.1% formic acid). The gradient started at 27% B and increased to 32% B in 2-min, then to 95% B by 2.25 min, with a 0.55-min hold, then back to 27% B by 2.90 min with a hold for 0.1 min. The column temperature was 28^oC and the flow was 0.3 ml/min. Samples were analyzed with triplicate injections on the LC-MS/MS. Gas flow, gas pressure, temperatures and voltages were optimized for each compound. All dwell times were 50 ms. Transitions for erlotinib were 394 to 278, 336, and 304. Transitions for OSI-420 were 380 to 278, 322, and 304. Transitions for the internal standard midazolam were 326 to 291, 223, and 249. Quantitation was based on 394 to 336 transition for erlotinib, and 380 to 322 transition for OSI-420. Data analysis was performed using MultiQuant version 3.0.1 software (AB Sciex), fitting the data to a quadratic model with 1/x weighting. The lower level of quantification (LOQ) was 0.6 ng/ml for erlotinib and 0.5 ng/ml for OSI-420, with an upper LOQ of 750 ng/ml for both compounds.

Immunohistochemical analysis

In addition to the primary endpoint (EGFR phosphorylation of tumor adjacent normal appearing urothelium), expression of EGFR, ERK, ERK-phosphorylation, e-cadherin, Ki-67, p53 and let-7 in normal and abnormal urothelium were assessed. Tissue blocks or unstained tissue sections from normal and abnormal appearing urothelium from each patient were obtained from participating institutions. Patient tissue samples were stained at the UW Translational Research Initiatives in Pathology (TRIP) Laboratory. Phospho-p44/42 MAPL (Erk1/2)(Thr202/Tyr204)(D13.14.4E) EP (perk) antibody (Cell Signaling #4370), p44/42 MAP kinase (L34F12) (ERK) (Cell Signaling #4696), anti-epidermal growth receptor (3C6) (EGFR) antibody (Ventana #790-2988), Phospho-EGFR receptor (Tyr1068 (D7A5) XP (pEGFR) (Cell Signaling #4696), p53 (Bp53-11) antibody (Ventana #760-2542), Ki-67 (30-9) antibody (Ventana #790-4286), Ecadherin (EP700Y) antibody (Ventana #760-4440) for the detection of the target biomarkers and cytokeratin clone AE1/AE3 antibody (Agilent DAKO # M351501-2) was used as the mask antibody for tissue segmentation. Dual or triple immunohistochemical staining assays were utilized and performed by the automated Ventana Discovery BioMarker platform (38-40) . Biomarkers were visualized with Discovery UltraMap chromogens (DAB, red and purple). The nuclei were counterstained with hematoxylin. The stained slides were scanned at 20X and analyzed with Vectra^™ Imaging System (Perkin Elmer). 3-5 representative images, if available, from each slide were included for analysis. inForm2.4 software was used for biomarker quantification. Using inForm software, the bladder tissues were segmented into epithelium and stroma, and each cell in the cancer and normal epithelia was further segmented into cytoplasmic membrane, cytoplasm and nucleus. Biomarker expression levels in the subcellular compartments and the entire cells of benign and cancer epithelia from the placebo and erlotinib groups were analyzed and compared.

Statistical Analysis

The primary objective was to determine if there was a difference in EGFR phosphorylation in tumor-adjacent normal appearing urothelium ≤ 24 hours post-study dose, between patients randomized to erlotinib weekly as compared to placebo in patients with bladder cancer. The difference in EGFR phosphorylation (expressed as mean optical density) between the placebo arm and the erlotinib arm was tested as-randomized using a two-sample t-test with normalizing transformation if necessary or Wilcoxon rank-sum test. Supporting analysis of the relationship between EGFR phosphorylation and measure of dose received, smoking status and proton inhibitor use was also performed.

Secondary objectives were 1) to assess the tolerance of high dose weekly erlotinib compared to placebo, 2) to assess the expression of EGFR in tumor tissue when available, 3) to assess the expression of markers associated with EGFR and/or carcinogenesis: e-cadherin, Ki-67, phosphorylated ERK, p53 and let-7 micro-RNA in normal vs. abnormal urothelium, 4) to assess plasma concentrations and limited pharmacokinetics of weekly erlotinib, and 5) perform an exploratory assessment of male urinary symptoms on study. The secondary endpoints were summarized by treatment arm with appropriate descriptive statistics and analyzed appropriately (Wilcoxon rank-sum for ordinal data, Fisher’s exact test for dichotomous data, and log rank test for time to event data).

Sample Size Justification

The sample size was based on comparing EGFR phosphorylation in bladder epithelium as measured by quantitative Vectra assay analysis of expression between the erlotinib and placebo arms. The primary endpoint was considered a continuous random variable. The sample size justification was thus based on a two-tailed two-sample t-test of the difference between the two groups at a significance level of 0.05. In order to detect an effect size of 1, i.e. the difference in the mean change between the placebo arm and the erlotinib arm of 1 standard deviation, with power 0.85, the trial required an effective sample size of 14 in the placebo arm and 28 in the erlotinib arm. Assuming a random drop-out of up to 5%, 15 participants in the placebo arm and 30 participants in the erlotinib arm were to be enrolled for a total of 45 participants for this exploratory study.

Ethical statement

The study underwent scientific and protocol review by the University of Wisconsin Carbone Cancer Center (UWCCC) prior to review and approval by Institutional Review Board for protection of Human Subjects at the University of Wisconsin. All participants provided written informed consent as the study was ethically conducted in accordance with Belmont Report and US Common Rule.

Data Availability

The data generated in this study are available upon request from the corresponding author.

Results

Subjects

Thirty-seven participants enrolled between April 2015 and November 2017. Thirteen participants were assigned to placebo and twenty-four participants were assigned to erlotinib (13 at 900 mg and 11 at 600 mg; 1 participant from the 900 mg erlotinib dose group reduced to 450 mg for their 2^nd and 3^rd doses and 2 participants from the 900 mg erlotinib dose group reduced to 450 mg for their 3^rd dose). The baseline participant characteristics are summarized in Table 1. The majority of the participants were non-Hispanic (34 of 37, 92%), white (35 of 37, 95%), male (31 of 37, 84%), with a mean age of 70 years. No participants had received prior intravesicular therapy for NMIUC. Those with pathologically confirmed NMIUC were evenly distributed between low- and high-grade tumors.

Table 1.

Patient characteristics. Data are expressed as number of patients (%) or mean (±SD).

Characteristic	Placebo (n=13)	Erlotinib (n=24)
Age, yrs	69.3 ±8.4	70.3 ±10.8
Height, cm	174 ±9.6	175 ±9.3
Weight, kg	86.8 ±22.7	89.7 ±16.3
BMI	28.3 ±5.9	29.4 ±5.6
Female Sex	3 (23.1)	3 (12.5)
Race
White	13 (100.0)	22 (91.7)
Black	0 (0.0)	2 (8.3)
Ethnicity
Hispanic/Latino	2 (15.4)	1 (4.2)
Non-Hispanic/Latino	11 (84.6)	23 (95.8)
Karnofsky Status
80	0 (0.0)	1 (4.2)
90	3 (23.1)	4 (16.7)
100	10 (76.9)	19 (79.2)
Smoking Status
Never	2 (15.4)	3 (12.5)
Former	8 (61.5)	15 (62.5)
Current	3 (23.1)	6 (25.0)
Tumor Grade at TURBT
No Visible Cancer	1	2
Low-grade	7	9
High-grade	4	10
No Pathology (4)

Twenty-four participants receiving erlotinib and 12 participants receiving placebo completed the study, with one participant assigned to placebo withdrawing from the study between week 2 and 3 (per physician decision). No additional participants withdrew or had study drug interruptions after week 2 and no participants had study drug interruptions for Week 1 or Week 2. There were no significant differences in agent interruption, compliance, and reason for taking off-study between treatment arms.

Safety

Selected toxicities are presented in Table 2. AEs were reported in 8 (62%) subjects on placebo, and 22 (92%) of subjects on erlotinib. The most common treatment-related AEs were hypertension and diarrhea (both 64%). Subjects receiving erlotinib were more likely than subjects on placebo to experience an AE (p=0.025), have multiple AEs (20 (83%) vs 2 (15%); p<0.001) and ultimately have more AEs per subject (p=0.004). Secondary analysis of AEs by erlotinib dose received (900 mg, 600 mg or placebo) also showed a dose effect for any AE or multiple AEs (p<0.05).

Table 2.

Selected toxicities in placebo vs. erlotinib group. Data are expressed as number of patients (%). Adverse events shown here have a frequency of 15% or higher in either treatment arm or are graded as moderate (Common Terminology Criteria for Adverse Events (CTCAE) grade 2) or severe (CTCAE grade 3).

	Placebo (n=13)					PFE (n=24)
	All	(%)	1	2	3	All	(%)	1	2	3
hypertension	4	(27)	3	1	0	9	(64)	4	2	3
diarrhea	1	(7)	1	0	0	9	(64)	8	1	0
rash acneiform	0	(0)	0	0	0	6	(43)	6	0	0
fatigue	2	(13)	2	0	0	2	(14)	2	0	0
pruritus	0	(0)	0	0	0	4	(29)	4	0	0
rash maculo-papular	0	(0)	0	0	0	4	(29)	4	0	0
Bladder Spasms and pain after TURBT	1	(7)	0	0	1	0	(0)	0	0	0
Permanent Pacemaker Implantation	1	(7)	0	0	1	0	(0)	0	0	0
lower gastrointestinal hemorrhage	0	(0)	0	0	0	1	(7)	0	0	1

Examination of specific toxicity, principally gastrointestinal (GI) and skin/subcutaneous AEs, which are usually dose-limiting for erlotinib revealed a significant dose relationship. Any GI AE occurred in 15%, 46% and 54% of subjects receiving placebo, 600 and 900 mg of erlotinib weekly; assessed by occurrence and severity a significant dose relationship was evident (Cochran-Armitage p=0.016). Skin/subcutaneous toxicity was similar with any AE occurring in 0, 55% and 77% of the dose groups and a significant dose relationship (p<0.001) when assessing occurrence and severity of AE. Assessment of baseline and end of study safety labs revealed a modest, but significant increase in serum total bilirubin in erlotinib as compared to placebo subjects (placebo baseline, mean±S.D., 10.8±4.7mmol/l;11.2±5.2, end of study; erlotinib baseline,11.3±3.3, end of study, 21.6±10.4, p=0.001) with no placebo subjects having an abnormal value at baseline or end of study and 1/23 and 12/23 with abnormal values at baseline and end of study in subjects receiving erlotinib. Serum creatinine values also were different between placebo and erlotinib subjects with placebo subjects with baseline and end of study creatinine values of 79.3±19.5 umol/l and 77.7±17.8 as compared to erlotinib baseline and end of study values of 87.1±22.4 and 98.7±26.5, p=0.01. The only other observed difference in safety laboratory values was placebo subjects having a small decline in serum calcium levels from baseline to day 8 as compared to erlotinib subjects (p=0.047).

Erlotinib plasma levels

As expected, neither erlotinib nor OSI-420 were detected at baseline. Prior to erlotinib administration on day 8, erlotinib was detected at low levels in two patients and OSI-420 in four patients. Pre-surgery plasma concentrations for erlotinib and OSI-420 were 2218 ±1096 ng/mL (450 mg dose, N=3, 1823±776; 600 mg dose, N=11, 2351±782; 900 mg dose N=10, 2191±1477) and 44.4 ± 34.6 ng/mL (450 mg – 34.2±17.0;600 mg – 43.4±42.8; 900 mg – 48.5±48.3), respectively and did not significantly differ between the 450, 600 and 900 mg doses.

Urinary symptoms

Twenty-one male subjects (placebo group 6, erlotinib group 15) completed baseline (mean value S.D. 8.0+6.0) and day 15 (7.5+5.7) IPSSS surveys. Three placebo group subjects (50%) had a baseline score of mild (1-7) and 3 (50%) had moderate (8-15) urinary obstructive symptom scores while 15 erlotinib subjects were 7 (47%) with mild, 7 (47%) moderate and 1 (6%) severe (>15). There was no evidence for a significant change from baseline on day 15 for either treatment group with placebo group scoring 4 (67%) mild and 2 (33%) moderate and erlotinib group scoring the same as baseline 7 mild, 7 moderate and 1 severe. There was no evidence for significant change from baseline within subjects in general or by treatment group.

Tissue Biomarkers

Semi-quantified immunohistochemical assessment of phosphorylated EGFR and total EGFR (whether nuclear, cytoplasmic or entire cell) in tumor, tumor adjacent normal urothelium and distant normal urothelium revealed no significant differences (or obvious trends) between tissue from subjects on erlotinib (tumor – 21 subjects, adjacent normal −8, distant normal - 12) as compared to placebo (tumor - 11 subjects, adjacent normal – 2, distant normal - 7) (Figure 1). Evaluation of phosphorylated EGFR in tumor based on erlotinib concentrations on day of surgery (11 placebo participants vs 12 erlotinib participants with erlotinib concentrations >2300 ng/ml) also failed to observe any evidence of a reduction in phosphorylated EGFR in those with the highest erlotinib exposures. Similar findings were observed for ERK, membrane e-cadherin, Ki67 and p53 expression. Stratification by smoking status did not alter results and comparisons of biomarkers in normal vs tumor urothelium showed no significant differences.

Figure 1.

Cytoplasmic phosphorylated EGFR in normal urothelium (1A) and tumor (1B) by semi-quantified immunohistochemical assessment. Presented as optical density per unit area.

Discussion

This study investigated clinical tolerability and potential molecular efficacy of a weekly dose of an EGFR tyrosine kinase inhibitor in a cohort of patients with bladder cancer. Despite obtaining urothelial tissue within 24 hours of a single large erlotinib dose (600 or 900 mg) we did not observe a difference in neoplastic or adjacent normal appearing urothelial tissue EGFR phosphorylation between erlotinib and placebo groups. Prior attempts to assess in vivo EGFR phosphorylation, whether in tumor tissue or normal skin of patients, have been characterized with inconsistent and variable evidence of decreased phosphorylation even in patients with direct evidence of clinical benefit (41-43). Still, it is surprising to observe no evidence of diminished urothelial EGFR phosphorylation given the presumed EGFR inhibition-driven toxicity (skin, gastrointestinal) observed and plasma levels achieved.

Potential explanations are many including: 1) constitutive expression of phosphorylated EGFR alters little or only with extended, repeated daily dosing/EGFR inhibition (e.g., as noted above, prior human studies compared intra-patient pre- to post-exposure after many weeks of daily EGFR inhibitors with inconsistent EGFR phosphorylation inhibition (24, 41-42) while our study compared cumulative data across groups); 2) Our analysis and/or sample size were insensitive to modest changes in EGFR phosphorylation (our assay/antibodies have observed differences in urothelial EGFR phosphorylation in prior clinical trials (43) but our study was likely underpowered enrolling 36 participants (12 placebo:24 erlotinib) rather than our enrolment goal of 45 (15 placebo:30 erlotinib)); 3) erlotinib poorly penetrates into urothelial tissue but prior effectiveness of erlotinib in locally advanced urothelial tumors contradicts this (32-33, 36); or 4) ultimately intermittent weekly erlotinib doesn’t have the same beneficial pharmacodynamic effect as continuous daily exposure. While there are no direct clinical comparisons between daily and intermittent erlotinib, pre-clinical and limited clinical data observe similar positive biological effects including similar toxicity which is attributed to inhibition of EGFR, and when we examined EGFR phosphorylation based on erlotinib pharmacokinetics we still did not observe evidence of urothelial EGFR inhibition (34-35).

While we did not confirm an effect upon urothelial EGFR phosphorylation or other related tissue biomarkers (EGFR, phosphorylated ERK, ERK, Ki67, p53) with weekly erlotinib, we did observe more toxicity in participants receiving weekly erlotinib (600 or 900 mg) than those randomized to placebo. The majority of these observed AEs were toxicities consistent with EGFR inhibition, e.g., mild to moderate skin and GI toxicity. The perception of investigators was the observed toxicity was less than expected for comparable cumulative daily dosing of erlotinib and consistent with toxicity described in the phase 1 weekly erlotinib dose escalation trials (31-33) but whether sufficiently tolerable to allow for widespread acceptance by patients and prescribing non-medical oncologists would only be answered with further, larger scale clinical trials.

Limited assessment of erlotinib plasma levels were done with results consistent with large weekly doses, with some evidence of accumulation and plasma levels approximately 12 hours after a 600 or 900 mg dose comparable to peak levels 2 hours after a 150 mg dose. The observed inter-patient variability is consistent with the known effects of smoking and/or concomitant proton pump inhibitor usage among participants, both known to diminish erlotinib plasma levels by 50% (22, 29-30).

There are compelling data to implicate a role for EGF pathways in bladder carcinogenesis and clinical data observing anti-tumor effects of EGFR antagonists (both small molecule inhibitors like erlotinib or monoclonal antibodies) in MI UC patients, thus supporting the potential ability of well-timed erlotinib administration to prevent bladder neoplastic progression/development. This coupled with preclinical and clinical data supporting the potential effectiveness with reduced toxicity of weekly/intermittent erlotinib led to our exploratory phase 2 clinical prevention trial in participants with superficial bladder neoplasia. We observed mild to moderate usual EGFR antagonist toxicities (skin, GI) over a 3-week span which wouldn’t preclude wider application in this population. However, the lack of a demonstrable biological effect in urothelial tissue either portends limited erlotinib urothelial activity or the lack of a potential surrogate biomarker, which means future clinical considerations in bladder cancer would need to be toward larger trials examining direct tumor or adjuvant therapy effects. This coupled with the growing data for systemic immunomodulatory considerations in high risk NMI and advanced UC have limited our current enthusiasm for future erlotinib bladder cancer prevention trials (44-45).

Prevention Relevance Statement.

We evaluated the potential of erlotinib in preventing cancer by performing a randomized, double-blind, placebo-controlled trial of weekly erlotinib in participants undergoing surgical removal of suspected non-invasive bladder neoplasia. Weekly erlotinib was tolerated with common grade 1 to 2 toxicities but without evidence of beneficial effect upon urothelial tissue.