Research ArticleCancer

Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers

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Science Translational Medicine  12 Jul 2017:
Vol. 9, Issue 398, eaah5583
DOI: 10.1126/scitranslmed.aah5583
  • Fig. 1. Phase 1 validation studies and THBS2 expression in PDAC and other human tumors.

    (A) AUC analysis of blinded ELISA data for the proteins MMP2, MMP10, and THBS2 in plasma samples from 10 patients with PDAC at various stages of disease compared to 10 healthy controls. CI, confidence interval. (B) Box plots of THBS2 mRNA expression measured in various human tumors (sample sizes in parentheses) assessed by RNA-seq. Tumors are sorted in order of decreasing median expression of THBS2 mRNA. Of the pancreatic cancer samples from the TCGA database (n = 179), we analyzed only PDAC (n = 134). All expression values are log2(RSEM values = 1)–transformed.

  • Fig. 2. THBS2 and CA19-9 concentrations in plasma samples from patients with PDAC versus healthy controls.

    (A and B) Scatter plots of THBS2 concentrations in plasma samples from patients at all stages of PDAC versus controls for the phase 2a (A) and phase 2b (B) validation studies. (C and D) ROC curves for THBS2, CA19-9, and THBS2 + CA19-9 concentrations in plasma samples from patients with all stages of PDAC versus healthy controls for phase 2a (PDAC, n = 81; controls, n = 80) (C) and phase 2b (PDAC, n = 197; controls, n = 140) (D) studies. (E and F) Scatter plots showing THBS2 and CA19-9 concentrations in plasma samples from patients with all stages of PDAC versus healthy controls for phase 2a (E) and phase 2b (F) studies.

  • Fig. 3. THBS2 and CA19-9 concentrations in plasma samples from all-stage PDAC cases versus benign pancreatic disease cases.

    (A to D) ROC curves for THBS2, CA19-9, and THBS2 + CA19-9 concentrations in plasma samples from patients with PDAC in the phase 2b study (n = 197) versus pancreatitis (n = 55) (A), PDAC versus intraductal papillary mucinous neoplasm (IPMN) (n = 115) (B), PDAC versus PNET (n = 30) (C), and PNET (n = 30) versus healthy controls (n = 140) (D) are shown.

  • Fig. 4. Expression of THBS2 in human PanIN tissue and PDAC tumor tissue.

    (A and B) Representative THBS2 immunohistochemistry analysis of incidental PanIN1/2 tissue derived from the head and neck of a pancreas from a patient with pancreatic periampullary cancer using two different antibodies (Ab). The arrows indicate that PanIN2 tissue stained positive for THBS2; dotted arrows indicate weak or negative staining of PanIN1 tissue. (C to K) THBS2 expression, designated by arrows, was also confirmed in stage II (C to E) and stage III (F to K) PDAC pancreatic cancer tissue arrays. Competitive assays were performed for antibody #2 by preincubating the antibody with a 10-fold excess of antigen peptide (E, H, and K) to confirm target specificity. Brown color indicates THBS2 staining, and blue color indicates hematoxylin nuclear staining. THBS2 was detected in the epithelial cells of noninvasive lesions (PanINs and intraductal papillary mucinous neoplasms) and poorly differentiated PDAC tissue as well as in fibroblasts in invasive PDAC tissue (see table S8 and fig. S6).

  • Table 1. Demographic and clinical characteristics of patients.

    Continuous variables (age, body mass index, and CA19-9 concentration) are presented as mean (SD); categorical variables (male gender, personal history of diabetes, and stage of disease) are presented as frequency (%). IPMN, intraductal papillary mucinous neoplasm; PNET, pancreatic neuroendocrine tumor.

    Adenocarcinoma stage I/IIAdenocarcinoma stage III/IVControlsIPMN, no
    adenocarcinoma
    PNETPancreatitis
    Discovery phase 1n = 6n = 4n = 10
    Age56.8 (7.5)65.0 (13.0)62.2 (15.4)
    Male gender4 (66.7%)2 (50.0%)5 (50.0%)
    Body mass index (kg/m2)31.2 (8.6)26.3 (5.4)26.0 (3.9)
    Personal history of diabetes1 (16.7%)02 (20.0%)
    CA19-920770.8 (47060.9)111224.0 (217927.2)12.0 (6.4)
    Stage of disease
      II1 (16.7%)
      IIA1 (16.7%)
      IIB4 (66.7%)
      III1 (25.0%)
      IV3 (75.0%)
    Validation phase 2an = 58n = 23n = 80n = 28
    Age67.6 (9.4)68.6 (10.9)67.4 (9.8)61.5 (9.4)
    Male gender43 (74.1%)12 (52.2%)54 (67.5%)19 (67.9%)
    Body mass index (kg/m2)28.9 (5.2)28.5 (5.7)27.2 (4.7)26.3 (4.8)
    Personal history of diabetes15 (25.9%)3 (13.0%)12 (15.0%)3 (10.7%)
    CA19-9305.3 (411.1)2137.5 (2983.7)10.6 (6.9)68.9 (165.5)
    Stage of disease
      I1 (1.7%)
      IA1 (1.7%)
      IB6 (10.3%)
      II16 (27.6%)
      IIA13 (22.4%)
      IIB21 (36.2%)
      III10 (43.5%)
      IV13 (56.5%)
    Validation phase 2bn = 88n = 109n = 140n = 115n = 30n = 55
    Age66.5 (11.3)64.4 (11.0)65.8 (10.8)68.8 (8.7)63.2 (7.1)55.9 (17.7)
    Male gender45 (51.1%)62 (56.9%)70 (50.0%)58 (50.4%)22 (73.3%)27 (50.0%)
    Body mass index (kg/m2)28.4 (5.4)29.1 (6.0)27.1 (4.4)26.5 (4.0)29.0 (4.9)27.8 (5.0)
    Personal history of diabetes34 (38.6%)25 (22.9%)15 (10.7%)20 (17.4%)10 (33.3%)7 (13.0%)
    CA19-9633.8 (1665.9)2399.3 (3481.1)12.0 (14.5)15.4 (12.3)45.3 (98.0)35.9 (66.0)
    Stage of disease
      I4 (4.5%)
      IA2 (2.3%)
      IB4 (4.5%)
      II37 (42.0%)
      IIA15 (17.0%)
      IIB26 (29.5%)
      III41 (37.6%)
      IV68 (62.4%)
  • Table 2. AUC calculations of ELISA results for PDAC at different stages, bootstrapped (1000 repetitions) at 95% CIs.
    PDAC versus healthy controlsCA19-9 (≥55)THBS2CA19-9 (≥55) + THBS2
    nAUC95% CIAUC95% CIAUC95% CIP
    Validation phase 2a
    All stages81/800.8460.810.890.8420.800.900.9560.930.980.0003
    Stage I/II58/800.8450.800.890.8320.780.890.9460.920.980.0067
    Stage I8/800.9380.831.000.8390.690.980.9770.931.000.5038
    Stage II50/800.8300.780.880.8300.780.890.9400.910.980.0080
    Stage III/IV23/800.8460.790.900.8420.780.900.9560.920.990.0003
    Validation phase 2b
    All stages197/1400.8810.860.910.8750.850.900.9700.960.98<0.0001
    Stage I/II88/1400.8340.790.870.8870.850.920.9600.940.98<0.0001
    Stage I10/1400.7930.660.930.8960.830.970.9520.920.990.0574
    Stage II78/1400.8390.800.880.8850.850.920.9610.940.98<0.0001
    Stage III/IV109/1400.9190.890.950.8660.830.900.9800.970.990.0028
  • Table 3. THBS2 concentration cutoff points based on percentiles of distribution in control plasma samples.
    MarkerPhase 2aPhase 2b
    CutoffSensitivitySpecificitySensitivitySpecificity
    CA19-9 (≥55)69.1410077.6698.57
    THBS2 (ng/ml)
      95%3633.3396.2558.3893.57
      96%3733.3397.5057.3695.00
      97%3830.8697.5053.8196.43
      98%4028.4097.5053.3097.86
      99%4224.6997.5051.7899.29
      100%73.47.4110023.35100
    CA19-9 (≥55) and THBS2 (ng/ml)
      95%3674.0796.2588.3292.86
      96%3774.0797.5088.3294.29
      97%3874.0797.5087.8295.71
      98%4074.0797.5087.8297.14
      99%4272.8497.5087.3197.86
      100%73.469.1410081.7398.57
  • Table 4. AUC calculations of ELISA results for patients with all-stage PDAC versus patients with benign pancreatic diseases, bootstrapped (1000 repetitions) at 95% CIs.
    PDAC versus benign pancreatic diseasesCA19-9 (≥55)THBS2CA19-9 (≥55) + THBS2
    nAUC95% CIAUC95% CIAUC95% CIP
    Validation phase 2a
    PDAC versus pancreatitis81/280.7740.710.840.7270.640.810.8420.760.920.2740
    Validation phase 2b
    PDAC versus pancreatitis197/550.8160.770.860.7310.670.790.8670.820.920.2450
    PDAC versus IPMN197/1150.8840.860.910.7840.740.820.9520.930.970.0003
    PDAC versus PNET197/300.8050.750.860.4440.360.710.7850.720.890.6502
    PNET versus controls30/1400.5760.520.630.7510.670.840.7550.670.850.0089

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/398/eaah5583/DC1

    Fig. S1. Validation of cross-reactivity of antibodies enclosed in THBS2 ELISA with THBS1 by Western blot.

    Fig. S2. Validation of cross-reactivity and interference of THBS1 in THBS2 ELISA.

    Fig. S3. Reproducibility of the ELISA for THBS2.

    Fig. S4. Distribution of THBS2 values in phase 2 samples.

    Fig. S5. Cross-validation tests, performed 1 year apart, of THBS2 concentrations in the same set of plasmas as determined in different laboratories.

    Fig. S6. Representative immunohistochemistry images of THBS2 in human normal pancreas, pancreatitis, and PDAC tissue.

    Table S1. List of 53 proteins secreted or released from 10-22 cell, PanIN-stage lesions that are at low abundance in healthy human plasma proteome and RNA-seq databases.

    Table S2. Mass spectrometry assessment of THBS2 concentrations in phase 1 plasma samples.

    Table S3. Range and median values of THBS2 and CA19-9 in this study.

    Table S4. Impact of excluding stage IIB (and unspecified stage II) subjects.

    Table S5A. THBS2 values by sex and diabetes mellitus status.

    Table S5B. Spearman correlation analysis of age and THBS2 values.

    Table S6A. Obstructive jaundice cases in the PDAC cohorts.

    Table S6B. THBS2 and CA19-9 values and obstructive jaundice status.

    Table S6C. AUC values for CA19-9, THBS2, and combined markers by jaundice status in phases 2a and 2b of PDAC cases versus controls.

    Table S7A. Cross-tabulation of normal versus elevated THBS2 values, given a cutoff of 42 ng/ml, for the original cross-validation THBS2 assays (κ = 0.786).

    Table S7B. Cross-tabulation of normal versus elevated scaled THBS2 values, given a cutoff of 2.47, for the original and cross-validation THBS2 assays (κ = 0.895).

    Table S8. Summary of THBS2 immunohistochemistry in a total of 42 human PDAC and 4 cases of incidental PanIN and intraductal papillary mucinous neoplasm by immunohistochemistry.

  • Supplementary Material for:

    Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers

    Jungsun Kim, William R. Bamlet, Ann L. Oberg, Kari G. Chaffee, Greg Donahue, Xing-Jun Cao, Suresh Chari, Benjamin A. Garcia, Gloria M. Petersen,* Kenneth S. Zaret*

    *Corresponding author. Email: zaret{at}upenn.edu

    Published 12 July 2017, Sci. Transl. Med. 9, eaah5583 (2017)
    DOI: 10.1126/scitranslmed.aah5583

    This PDF file includes:

    • Fig. S1. Validation of cross-reactivity of antibodies enclosed in THBS2 ELISA with THBS1 by Western blot.
    • Fig. S2. Validation of cross-reactivity and interference of THBS1 in THBS2 ELISA.
    • Fig. S3. Reproducibility of the ELISA for THBS2.
    • Fig. S4. Distribution of THBS2 values in phase 2 samples.
    • Fig. S5. Cross-validation tests, performed 1 year apart, of THBS2 concentrations in the same set of plasmas as determined in different laboratories.
    • Fig. S6. Representative immunohistochemistry images of THBS2 in human normal pancreas, pancreatitis, and PDAC tissue.
    • Table S1. List of 53 proteins secreted or released from 10-22 cell, PanIN-stage lesions that are at low abundance in healthy human plasma proteome and RNA-seq databases.
    • Table S2. Mass spectrometry assessment of THBS2 concentrations in phase 1 plasma samples.
    • Table S3. Range and median values of THBS2 and CA19-9 in this study.
    • Table S4. Impact of excluding stage IIB (and unspecified stage II) subjects.
    • Table S5A. THBS2 values by sex and diabetes mellitus status.
    • Table S5B. Spearman correlation analysis of age and THBS2 values.
    • Table S6A. Obstructive jaundice cases in the PDAC cohorts.
    • Table S6B. THBS2 and CA19-9 values and obstructive jaundice status.
    • Table S6C. AUC values for CA19-9, THBS2, and combined markers by jaundice status in phases 2a and 2b of PDAC cases versus controls.
    • Table S7A. Cross-tabulation of normal versus elevated THBS2 values, given a cutoff of 42 ng/ml, for the original cross-validation THBS2 assays (κ = 0.786).
    • Table S7B. Cross-tabulation of normal versus elevated scaled THBS2 values, given a cutoff of 2.47, for the original and cross-validation THBS2 assays (κ = 0.895).
    • Table S8. Summary of THBS2 immunohistochemistry in a total of 42 human PDAC and 4 cases of incidental PanIN and intraductal papillary mucinous neoplasm by immunohistochemistry.

    [Download PDF]

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