# Abstract

 

## Purpose Sonic hedgehog (SHH), an activating ligand of smoothened (SMO), is overexpressed in > 70% of pancreatic cancers (PCs). We investigated the impact of vismodegib, an SHH antagonist, plus gemcitabine (GV) or gemcitabine plus placebo (GP) in a multicenter phase Ib/randomized phase II trial and preclinical PC models. 

## Patients and Methods Patients with PC not amenable to curative therapy who had received no prior therapy for metastatic disease and had Karnofsky performance score ≥ 80 were enrolled. Patients were randomly assigned in a one-to-one ratio to GV or GP. The primary end point was progression-free-survival (PFS). Exploratory correlative studies included serial SHH serum levels and contrast perfusion computed tomography imaging. To further investigate putative biologic mechanisms of SMO inhibition, two autochthonous pancreatic cancer models (Kras^G12D^; p16/p19^fl/fl^; Pdx1-Cre and Kras^G12D^; p53^R270H/wt^; Pdx1-Cre) were studied. 

## Results No safety issues were identified in the phase Ib portion (n = 7), and the phase II study enrolled 106 evaluable patients (n = 53 in each arm). Median PFS was 4.0 and 2.5 months for GV and GP arms, respectively (95% CI, 2.5 to 5.3 and 1.9 to 3.8, respectively; adjusted hazard ratio, 0.81; 95% CI, 0.54 to 1.21; *P* = .30). Median overall survival (OS) was 6.9 and 6.1 months for GV and GP arms, respectively (95% CI, 5.8 to 8.0 and 5.0 to 8.0, respectively; adjusted hazard ratio, 1.04; 95% CI, 0.69 to 1.58; *P* = .84). Response rates were not significantly different. There were no significant associations between correlative markers and overall response rate, PFS, or OS. Preclinical trials revealed no significant differences with vismodegib in drug delivery, tumor growth rate, or OS in either model. 

## Conclusion The addition of vismodegib to gemcitabine in an unselected cohort did not improve overall response rate, PFS, or OS in patients with metastatic PC. Our preclinical and clinical results revealed no statistically significant differences with respect to drug delivery or treatment efficacy using vismodegib. # RESULTS 

## Clinical Trial 

### Patient characteristics. Seven patients were enrolled onto the phase Ib open-label GV portion, and no safety issues were identified. For the randomized phase II part of the trial, 111 patients were enrolled at 13 sites between February 2010 and June 2012, stratified by Karnofsky performance score (80 *v* 90 or 100) and disease status (newly diagnosed *v* recurrent; [Fig 1](#)). Of these, four patients withdrew consent before starting treatment (two from each arm), and one patient (randomly assigned to GV) was subsequently found to have been ineligible and never started therapy. Analyses were based on the remaining 106 patients. Patient characteristics were similar between treatment arms, except for the incidence of peritoneal metastases, which were higher in the GP arm (9% *v* 23%; [Table 1](#)). <figure> <p><img src="" /></p> <figcaption>CONSORT diagram of clinical trial enrollment and treatment in phase II trial. gem, gemcitabine.</figcaption> </figure> ::::table-wrap

::: caption
Baseline Patient Demographic and Clinical Characteristics
:::

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Characteristic No. (%)

______________________________________________________________________

GV (n = 53) GP (n = 53) Total (N = 106)\
Age, years\
Median 64 64 64
Range 49-82 39-84 39-84
Sex\
Male 31 (58) 27 (51) 58 (55)
Female 22 (42) 26 (49) 48 (45)
Race\
White 40 (77) 45 (88) 85 (83)
African American 10 (19) 6 (12) 16 (16)
Asian 1 (2) 0 (0) 1 (1)
Other 1 (2) 0 (0) 1 (1)
Missing 1 2 3
Karnofsky performance score\
100 19 (36) 17 (32) 36 (34)
90 14 (26) 20 (38) 34 (32)
80 20 (38) 16 (30) 36 (34)
Disease status at enrollment\
Newly diagnosed 48 (91) 48 (91) 96 (91)
Recurrent metastatic 5 (9) 5 (9) 10 (9)
Primary tumor location\
Head 23 (43) 24 (46) 47 (45)
Neck/uncinate 1 (2) 2 (4) 3 (3)
Body 16 (30) 11 (21) 26 (25)
Tail 13 (25) 13 (25) 26 (25)
Site of metastasis[\*](#)\
Liver 41 (77) 44 (83) 85 (80)
Lung 12 (23) 14 (26) 26 (25)
Peritoneum 5 (9) 12 (23) 17 (16)
Other 8 (15) 10 (19) 18 (17)
Crossover to GV 22 (42)

Abbreviations: GP, gemcitabine plus placebo; GV, gemcitabine plus
vismodegib.

Patients may have > one primary or metastatic site.:::: 

### Safety. Median number of cycles was four (range, one to 12 cycles) in the GV arm and three (range, zero to 14 cycles) in the GP arm. Combination therapy with GV was generally well tolerated and did not result in unexpected toxicities ([Table 2](#)). There were no statistically significant differences in the rate of adverse events (AEs) between the arms. Four patients in the GV arm and two in the GP arm withdrew from treatment as a result of AEs. Sixteen patients (GV, n = 10; GP, n = 6) died while receiving treatment. ::::table-wrap
::: caption
Grade 3 to 5 Toxicities at Least Possibly Related to Treatment
:::

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Toxicity No. (%) *P*

______________________________________________________________________

GV (n = 53) GP (n = 53)\
Neutropenia 15 (28) 12 (23) .66
Fatigue 7 (13) 4 (8) .53
Anorexia 5 (9) 2 (4) .44
Vomiting 5 (9) 2 (4) .44
WBC decreased 5 (9) 7 (13) .76
Platelet count decreased 6 (11) 5 (9) 1.0
Anemia 4 (8) 7 (13) .53
Nausea 4 (8) 3 (6) 1.0
Elevated AST 5 (9) 3 (6) .72
Elevated ALT 3 (6) 2 (4) 1.0
Blood bilirubin increased 3 (6) 1 (2) .62
Hyperglycemia 3 (6) 3 (6) 1.0
Hypokalemia 4 (8) 2 (4) .68
Alkaline phosphatase increased 2 (4) 2 (4) 1.0
Lymphocyte count decreased 2 (4) 5 (9) .44
Hyponatremia 1 (2) 5 (9) .20

Abbreviations: GP, gemcitabine plus placebo; GV, gemcitabine plus
vismodegib.:::: 

### Efficacy. ORRs were similar in the two arms (GV: complete response [CR], 0 [0%]; partial response [PR], 4 [8%]; stable disease [SD], 27 [51%]; disease control [ie, CR + PR + SD], 31 [58%]; GP: CR, 1 [2%]; PR, 6 [11%]; SD, 20 [38%]; disease control, 27 [51%]). The difference in response rates (8% *v* 13%) was not significant (*P* = .53; Data Supplement). The primary end point of the study was PFS. At the final analysis, events (progression or death) occurred in 48 patients (91%) receiving GV and 51 (95%) receiving GP. Median PFS was 4.0 months for GV and 2.5 months for GP (adjusted hazard ratio [HR], 0.83; 95% CI, 0.55 to 1.23; [Fig 2](#)A; Data Supplement). <figure> <p><img src="" /></p> <figcaption>(A) Progression-free and (B) overall survival by treatment arm. Blue, gemcitabine plus vismodegib; gold, gemcitabine plus placebo. Hazard ratio (HR) after adjusting (adj) for Karnofsky performance score and disease status (newly diagnosed <em>v</em> recurrent).</figcaption> </figure> Median OS was 6.9 months for GV and 6.1 month for GP (adjusted HR, 0.96; 95% CI, 0.64 to 1.44; [Fig 2](#)B; Data Supplement). No survival differences were noted in a preplanned secondary analysis of OS that censored patients receiving GP at first progression, before crossover to GV (*P* = .69). Note that patient crossover did not affect the primary end point (ie, PFS), because crossover happened after the event occurred. For patients receiving GP who crossed over at progression (n = 22 [42%]), median PFS was 1.8 months, and median OS was 2.9 months (Data Supplement). One-year survival rates in the GV and GP arms were 15% and 25%, respectively (*P* = .3). OS and PFS did not differ significantly by Karnofsky performance score (*P* = .66 and .42, respectively; Data Supplement). Mortality and disease progression rates were consistent and uniformly high across all centers. 

## Clinical Trial Correlative Results 

### SHH serum levels. Median pretreatment plasma SHH level pooled for both treatment arms was 1.01 ng/mL (GV arm, 1.01 ng/mL; GP arm, 1.06 ng/mL). SHH levels did not change significantly with subsequent cycles (*P* = .087), nor was there a difference between treatment groups (*P* = .85) or patients with cancer (n = 89) and normal controls (n = 40; *P* = .4) ([Figs 3](#)A and [3](#)B). SHH serum levels did not correlate with age in either patients with cancer (*r* = 0.17; *P* = .13) or controls (*r* = 0.03; *P* = .87; [Figs 3](#)C and [3](#)D). <figure> <p><img src="" /></p> <figcaption>Clinical trial translational correlatives. Serum SHH levels (A) comparing controls (n = 40) with patients with pancreatic cancer enrolled onto trial (n = 89), (B) by treatment group (gemcitabine plus vismodegib [GV] or gemcitabine plus placebo [GP]) with increasing treatment (TX) cycle, and association with age in (C) patients with cancer and (D) controls. (E) Radiologic correlatives evaluating association of baseline tumor perfusion with tumor response to therapy.</figcaption> </figure> 

### Radiologic tumor perfusion. By univariable analysis, baseline computed tomography (CT) perfusion of the primary tumor was not associated with response to therapy, expressed as percent change in tumor size (*r* = −0.09; *P* = .77; [Fig 3](#)E; Data Supplement). 

## Murine Translational Correlatives 

### SMO inhibition does not reveal quantifiable changes to tumor stroma in vivo. Previous preclinical studies have suggested that SHH is abnormally expressed in genetically engineered models of PC.^[29](#),[37](#)^ SHH expression and pathway activation were confirmed in the KPP genetically engineered mouse model of PC by transcriptional gene analysis and immunohistochemistry. SHH immunoreactivity exhibited focal staining throughout tumors, predominantly in mucinous and well-defined ductal epithelial cells (data not shown), consistent with previous reports.^[26](#)^ Comparative transcriptional analysis of normal pancreas and tumor revealed significantly elevated expression of Hh ligands *SHH* and Indian Hh (*IHH*), as well as *SMO*, glioma-associated oncogene family zinc finger 1 (*GLI1*), *GLI2*, Hh Interacting Protein (*HHIP*), and *PTCH*1 (Data Supplement). To determine whether inhibition of SMO leads to changes in tumor stroma, we used a small molecule, HhAntag,^[38](#)^ a potent orally available preclinical surrogate of the US Food and Drug Administration--approved vismodegib (GDC-0449).^[10](#)^ Previous reports have shown that antagonism of Hh signaling resulted in increased mean microvessel density (MVD).^[29](#)^ HhAntag treatment on tumor-bearing KPP animals revealed no significant change in MVD ([Fig 4](#)A; *P* = .54; Data Supplement), despite observing pathway inhibition (Data Supplement). Moreover, HhAntag treatment did not affect intratumoral extracellular matrix deposition assessed through trichrome staining ([Fig 4](#)B; *P* = .29). <figure> <p><img src="" /></p> <figcaption>Effects of hedgehog pathway antagonism (HhAntag) on vasculature, stromal content, and intratumoral gemcitabine metabolites in Kras<sup>G12D</sup>; p16/p19<sup>fl/fl</sup>; Pdx1-Cre (KPP) tumors. (A) Quantitation of immunohistochemical staining for meca-32 expression in pancreatic tumors of KPP mice treated with vehicle (circles; n = 13) or smoothened (SMO) inhibitor (squares; n = 13) for 10 days. Data presented as percentages of meca-32–positive areas over analyzed tumor areas for each tumor (Mann-Whitney <em>P</em> = .54; scale bar, 200 um). (B) Quantitative analysis of stromal content by trichromatic stain in pancreatic tumors of KPP mice treated with vehicle or SMO inhibitor for 10 days. Data presented as percentages of positive stain areas over analyzed tissue areas (Mann-Whitney <em>P</em> = .29). (C) Mass spectromic quantitation of intratumoral concentration of 2′,2′-difluorodeoxycytidine triphosphate (dFdCTP; active form of gemcitabine) from each tumor after treatment for 10 consecutive days with vehicle or SMO inhibitor and gemcitabine 50 mg/kg 30 minutes before tumor collection (Mann-Whitney <em>P</em> = 1.000). (D) Ratios of 2′,2′-difluoro 2′-deoxycytidine (dFdC) to diflurodeoxyuridine (dFdU) in pancreatic tumors from each tumor (Mann-Whitney <em>P</em> = .48). NS, not significant.</figcaption> </figure> 

### SMO inhibition does not affect gemcitabine drug delivery in vivo. The modest response rates observed in patients with PC treated with gemcitabine has, in part, been attributed to poor drug delivery associated with a prohibitive tumor microenvironment. Gemcitabine undergoes intracellular conversion, from 2′,2′-difluoro 2′-deoxycytidine (dFdC) to the active triphosphate form 2′,2′-difluorodeoxycytidine triphosphate (dFdCTP), responsible for inhibition of DNA synthesis and repair. To directly measure drug delivery, dFdCTP levels were analyzed in PC tumors of KPP mice after continuous 10-day HhAntag treatment and a single dose of gemcitabine. Liquid chromatography--tandem mass spectrometry analysis revealed that dFdCTP concentrations were similar in both vehicle- and HhAntag-treated KPP mice ([Fig 4](#)C; Mann-Whitney *P* = 1.00). Intracellular gemcitabine is also converted to an inactivated form, diflurodeoxyuridine (dFdU). The ratio of the unprocessed form (dFdC) to inactive form (dFdU) estimates intratumoral gemcitabine exposure. The average ratio of dFdC to dFdU in KPP tumors of HhAntag-treated mice was not significantly different between vehicle and SMO inhibitor groups ([Fig 4](#)D; *P* = .48). Together, these results indicated that HhAntag did not increase gemcitabine delivery in this genetically engineered PC mouse model. 

### Pancreatic tumor progression and OS remain unaltered by Hh pathway inhibition. Next, we investigated the therapeutic impact of HhAntag in KPP mice. Growth rates of tumors, as determined by serial ultrasound imaging, were not significantly different when comparing HhAntag and vehicle arms, indicating no single-agent effect of Hh pathway inhibition ([Figs 5](#)A and [5](#)B). Although the combination of gemcitabine plus HhAntag significantly decreased tumor growth relative to control treatment (log-rank *P* = .0052), the growth rate was not different from that of gemcitabine alone, known to affect tumor growth in this model.^[39](#)^ Consistent with the lack of tumor growth effect, the HhAntag plus gemcitabine combination did not provide significant improvement in OS when compared with gemcitabine alone ([Fig 5](#)C). In summary, the addition of HhAntag did not affect tumor growth or animal longevity in the KPP model. <figure> <p><img src="" /></p> <figcaption>Smoothened (SMO) inhibitor does not affect tumor progression or overall survival in Kras <sup>LSL-G12D</sup>; p16/p19 <sup>fl/fl</sup>; Pdx1-Cre (KPP) mice. (A) Individual tumor growth rates plotted by from serial ultrasound images as volumes depicted longitudinally by animal within each regimen. (B) Antilogged values of slopes in each longitudinal plot are graphed, and average tumor burden fold changes per day in each study group of KPP mice are shown, with approximate 95% CIs (vehicle <em>v</em> SMO inhibitor, <em>P</em> = .86; vehicle <em>v</em> combination, <em>P</em> = .0156; gemcitabine <em>v</em> combination, <em>P</em> = .18) (C) Kaplan-Meier plots of KPP mice treated with vehicle (blue, n = 15; median, 1.9 weeks), SMO inhibitor (gold, n = 16; median, 1.2 weeks), gemcitabine (gray, n = 14; median, 3.8 weeks), and gemcitabine plus SMO inhibitor combination (red, n = 12; median, 3.4 weeks; gemcitabine <em>v</em> vehicle, <em>P</em> = .0059; combination <em>v</em> vehicle, <em>P</em> = .0179; gemcitabine <em>v</em> combination, <em>P</em> = .10 \[all <em>P</em> values from log-rank test\]). HhAntag, hedgehog pathway antagonism.</figcaption> </figure> 

### Analysis of SMO inhibition in KR mice. Although the KPP model represents the genetics of a subset of the PC population, previous findings were generated in a model composed of mutant *KRAS* and mutant *p53* expression in the pancreas.^[40](#)^ Therefore, we reproduced all in vivo studies in the KR model. HhAntag treatment on tumor-bearing KR animals revealed a significant decrease in MVD using meca-32 (Data Supplement; *P* = .0418) and trend toward decrease using CD31 (Data Supplement; *P* = .29). Trichrome staining revealed no significant changes to the stroma of HhAntag-treated tumors (Data Supplement; *P* = .83). Together these findings indicated that as in the previous KPP model, the stroma of the developed KR tumors was not significantly affected by HhAntag treatment. Finally, we observed no significant change in concentration of the gemcitabine metabolite dFdCTP in tumors after HhAntag (Data Supplement; *P* = .25) and no significant change in the ratio of dFdC to dFdU (Data Supplement). Furthermore, neither HhAntag nor the gemcitabine plus HhAntag combination affected tumor growth rates in KR animals compared with gemcitabine alone (Data Supplement). Consistent with the tumor growth study, HhAntag did not lead to survival benefit over vehicle or gemcitabine treatment (Data Supplement). Taken together, neither of the murine PC model genotypes (KPP or KR) demonstrated any measurable differences between gemcitabine delivery to tumors, tumor growth rate, or OS with HhAntag treatment. A significant decrease in microvessel density, consistent with previous work in transplantable models using vismodegib, was observed.^[41](#)^


Extracted trial result:
Outcome: Response rates (continuous)
Intervention: vismodegib plus gemcitabine (GV)
Comparator: gemcitabine plus placebo (GP)
Intervention group size: 53
Intervention mean: 8
Comparator group size: 53
Comparator mean: 13
Notes: Only difference in response rates as percent is given.