SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1 Quantification of soluble Endo180 ectodomain in BCa
patient plasma samples. (A) The blot presented includes plasma samples from
eight out of the eighty-eight BCa patients included in the study resolved on an
8% w/v sodium dodecyl sulfate polyacrylamide gel, transferred to a
polyvinylidene fluoride membrane and immunoblotted with 1 mg/ml antiEndo180 39.10 monoclonal antibody and 1/4000 dilution of horse radish
peroxidase -conjugated goat anti-mouse IgG. The immunoblot was exposed to
Hyperfilm™ for 10 seconds and band intensities quantified using ImageJ
software. (B) The graph shows relative Endo180 levels calculated by
densitometric analysis of the 180 kDa immunoreactive bands corresponding to
soluble Endo180 ectodomain levels in patient samples (lanes 2-8) normalised
against the reference plasma sample in lane 1 (relative Endo180 level = 1.0).
Molecular weight markers are indicated in kilodaltons (kDa) with black
arrowheads.
Supplementary Figure 2 Calculation of the range of sensitivity and specificity
for Endo180 in the detection of metastatic BCa. Plasma samples from BCa
patients (n = 85a) were resolved on 8% w/v sodium dodecyl sulfate
polyacrylamide gels, transferred to polyvinylidene fluoride membranes and
immunoblot analysis was performed using 1 mg/ml anti-Endo180 39.10
monoclonal antibody and 1/4000 dilution of horse radish peroxidase-conjugated
goat anti-mouse (a representative blot is shown in Supplementary Figure 1).
Relative plasma Endo180 levels were calculated from the mean measurement
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obtained from three analytical repeats of Endo180 immunodetection using
39.10 monoclonal antibody, as described above. Plasma from the same early
BCa patient was used as a reference control (relative Endo180 level = 1.0) and
the normalisation of densitometric measurements on all analytical gels. CA 15-3
antigen concentrations (U/mL) were measured using a non-competitive twostep immunoassay in plasma samples (n = 86a) collected in parallel. The
graphs show the sensitivity (% true positive rate) and specificity (% false
positive rate) for the classification of metastasis. A selected range of Endo180
cut-offs (relative plasma levels: 0.95 – 1.65) combined with CA 15-3 antigen
(cut-off: 28 U/mL) (solid and hatched purple bars) or alone (orange and hatched
orange bars) are shown for all eighty-six patients (solid bars) or all fifty-seven
bisphosphonate naïve patients (hatched bars) included in the study. aData for
CA 15-3 antigen was missing for two out of the twenty-nine early BCa patients
included in the study.
Supplementary Figure 3. Linear regression correlation of Endo180 and CA 153 antigen in BCa patients. Plasma samples from BCa patients (n = 86a) were
resolved on 8% w/v sodium dodecyl sulfate polyacrylamide gels, transferred to
polyvinylidene fluoride membranes and immunoblot analysis was performed
using 1 mg/ml anti-Endo180 39.10 monoclonal antibody and 1/4000 dilution of
horse radish peroxidase-conjugated goat anti-mouse (a representative blot is
shown in Supplementary Figure 1). Relative plasma Endo180 levels were
calculated from the mean measurement obtained from three analytical repeats
of Endo180 immunodetection using 39.10 monoclonal antibody, as described
2
above. Plasma from the same early BCa patient was used as a reference
control (relative Endo180 level = 1.0) and the normalisation of densitometric
measurements on all analytical gels. CA 15-3 antigen concentrations (U/mL)
were measured using a non-competitive two-step immunoassay in plasma
samples (n = 86a) collected in parallel. The graph shows relative plasma
Endo180 levels versus CA 15-3 antigen (Log10 [U/mL]). aData for CA 15-3
antigen was missing for two out of the twenty-nine early BCa patients included
in the study. The formula for the line of best fit and the corresponding Pearson’s
Correlation Coefficient (r) are both shown and indicate a weak linearity between
the two markers.
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SUPPLEMENTARY TABLE FOOTNOTES
Supplementary Table 1 Mean ± S.D relative Endo180 plasma levels are
shown; n, number of patients; % of patients indicated in parentheses. The Pvalues shown are for comparisons between early and metastatic BCa cases
using two-sided Mann Whitney U test.
Supplementary Table 2 Mean ± S.D relative Endo180 plasma levels shown; n,
number of patients; % patients shown in parentheses calculated using patients
where data is known. The P-values calculated for comparisons between known
variables only using two-sided Mann Whitney U test for two group comparisons
or Kruskal-Wallis one-way analysis of variance for more than two group
comparisons.
Supplementary Table 3 Mean ± S.D relative Endo180 plasma levels shown; n,
number of patients; % patients shown in parentheses calculated using patients
where data is known. The P-values calculated for comparisons between two
variables using two-sided Mann Whitney U test. Receptor status confirmed at
diagnosis and treatment at time of sample collection.
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