ADAM12 is a circulating marker for stromal activation in pancreatic cancer and predicts response to chemotherapy

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma that harbors tumor-promoting properties. No good biomarkers exist to monitor the effect of stromal targeting therapies or to predict response. We set out to identify such non-invasive markers for PDAC stroma and predict response to therapy. Gene expression datasets, co-culture experiments, xenografts, and patient samples were analyzed. Serum samples were measured from a cohort of 58 resected patients, and 87 metastatic or locally advanced PDAC patients. Baseline and follow-up levels were assessed in 372 additional metastatic PDAC patients who received nab-paclitaxel with gemcitabine (n = 184) or gemcitabine monotherapy (n = 188) in the phase III MPACT trial. Increased levels of ADAM12 were found in PDAC patients compared to healthy controls (p < 0.0001, n = 157 and n = 38). High levels of ADAM12 significantly associated with poor outcome in resected PDAC (HR 2.07, p = 0.04). In the MPACT trial survival was significantly longer for patients who received nab-paclitaxel and had undetectable ADAM12 levels before treatment (OS 12.3 m vs 7.9 m p = 0.0046). Consistently undetectable or decreased ADAM12 levels during treatment significantly associated with longer survival as well (OS 14.4 m and 11.2 m, respectively vs 8.3, p = 0.0054). We conclude that ADAM12 is a blood-borne proxy for stromal activation, the levels of which have prognostic significance and correlate with treatment benefit.

Conflict of interest statement

M.F.B. has received research funding from Celgene. H.W.L. has acted as a consultant for Celgene, Eli Lilly and Company, Nordic Pharma Group and Philips, has received research grants from, Amgen, Bayer Schering Pharma AG, Celgene, Eli Lilly and Company, GlaxoSmithKline Pharmaceuticals, Nordic Pharma Group, Philips, Roche Pharmaceuticals. D.W.P., R.J., and J.S.L. are Celgene employees. Other than Celgene, none were involved in drafting of the manuscript.

Figures

Fig. 1. ADAM12 associates with activated pancreatic cancer stroma and poor-prognosis molecular subclasses.

a Boxes indicate median with first and third quartiles of log2 transformed gene expression values from two U133 Plus 2.0 microarray datasets of pancreatic cancer patients comparing normal and tumor tissue. Badea et al. set (GSE15471), n = 36 paired biopsies; Pei et al. set (GSE16515); n = 16 (normal), n = 36 (tumor). ***p < 0.001, statistical testing was by two-tailed Student’s t test. b Kaplan–Meier analysis of patients from the ICGC cohort, dichotomized for median ADAM12 expression. c Log2 transformed ADAM12 expression values from the Pilarksy et al. (E-MEXP-1121) gene expression set obtained from microdissected pancreatic cancer tissue. *p < 0.05, testing by two-tailed Student’s t test. d Transcript levels for indicated Adam/ADAM paralogs relative to Gapdh/GAPDH were measured in xenografts by qPCR using mouse- or human-specific primers. Boxplots show data from 10 individual patient grafts. For each replicate sample measured by qPCR, a technical triplicate was used. Difference between groups was tested by ANOVA for both panels p < 0.0001. e Association of ADAM12 expression with stromal activation markers in the AMC patient cohort was measured by qRT-PCR, n = 15 patients. Size of dots indicates tumor cellularity scored by a pathologist. f Patients from GSE17891 dataset were grouped by published PDAssigner subtype labels and log2 expression of ADAM12 and paralog ADAM10 is shown (n = 27). Patients from GSE17891 were grouped by published PC class labels (n = 96). Difference between groups was tested by ANOVA. g Patients were k-means clustered using the activated stroma gene set and ADAM12 and -10 expression is shown (n = 132). Difference between groups was tested by ANOVA

Fig. 2. ADAM12 expression is induced by tumor cell-derived TGF-β.

a Immortalized stellate cells (PS-1) were starved with 0.5% FCS for 24 h and subsequently treated with the indicated ligands for 48 h. Concentrations used: TGF-β, 5 ng/mL; TGF-β receptor I inhibitor A83-01, 1 µM; ShhN supernatant from 293T cells, 1:4; EGF, 50 ng/ml; bFGF, 10 ng/ml; HGF, 10 ng/ml; IL-1α, 10 ng/ml; IL-1β, 10 ng/ml. In addition to ADAM12, expression analysis of stromal activation marker genes ACTA and SPARC in response to TGF-β is shown. Two-tailed Student’s t test comparing control vs. TGF-β, ***p < 0.001, **p < 0.001. n = 6, across two separate experiments for control and TGF-b, n = 3 in one experiment for other treatments. For each replicate sample measured by qPCR, a technical triplicate was used. Bars show mean ± SEM. b To ascertain that tumor cell-derived TGF-β drives ADAM12 expression, stellate cells were treated as for panel a, or cocultured with tumor cells of the 84 primary culture. Supernatant was harvested and cleared by centrifugation. Soluble ADAM12 levels were measured by ELISA. At least three replicates are shown, statistical testing was by two-tailed Student’s t test comparing control vs. TGF-β, and control vs. coculture, ***p < 0.001. n = 6, across two separate experiments for treatments, n = 3 in one experiment for coculture. For each replicate sample measured by qPCR, a technical triplicate was used. Bars show mean ± SEM

Fig. 3. ADAM12 is elevated in the serum of PDAC patients and predicts poor outcome in patients undergoing resection.

a ADAM12 levels were measured by ELISA in serum of healthy individuals (n = 38), and patients diagnosed with PDAC (n = 157). Boxplots show median with interquartile range. ***p < 0.0001; tested by Mann–Whitney U-test against healthy controls. b Kaplan-Meier survival analysis of AMC PDAC patients who underwent resection of the tumor, dichotomized by baseline serum levels determined by receiver-operator-characteristics (ROC; 316 pg/mL). c As for panel b, for patients who did not undergo resection. Statistical testing was by log-rank test

Fig. 4. Plasma ADAM12 predicts favorable outcome in patients treated with nab-paclitaxel and gemcitabine.

a Baseline recalcified plasma samples from the MPACT cohort were analyzed for ADAM12 and patients were dichotomized for ADAM12 levels above or below detection (2× SD of blanks). Patients in the arm receiving gemcitabine monotherapy are indicated by dashed lines. Numbers in parentheses indicate number of patients in the group (n), and median survival for that group (OS). Analysis of patients in the arm receiving gemcitabine and nab-paclitaxel is shown with solid lines. b Baseline (cycle 1, day 1) and follow-up (cycle 2, day 1) samples were measured and fold change was calculated. Samples with undetectable ADAM12 levels at both baseline and follow-up did not show a fold change and are indicated by the red line. Patients in the arm receiving gemcitabine monotherapy are shown. c As for panel b, analysis of patients in the arm receiving gemcitabine and nab-paclitaxel. Statistical testing in all panels was by log-rank test