Article Figures & Data
Figures
- Figure 1.
Tuberculin-inducible IFNγ and IL2 may predict outcome of intravesical BCG therapy in about two thirds of patients. Freshly isolated PBMCs were stimulated in vitro with tuberculin for 16 hours. IFNγ, IL2, and TNF were measured by flow cytometry using intracellular cytokine staining. Alternatively, secreted cytokines were measured in the supernatant by electrochemiluminescence. CD4+ T-cell frequencies are expressed as fractions (0.01 = 1%). A, Patients without recurrence of cancer at 6 months show significantly higher numbers of cytokine-producing CD4+ T cells in response to tuberculin stimulation (left). B, Secreted IFNγ and IL2 but not TNF seem to discriminate better between treatment success and recurrence than the percentage of CD4+ T cells displaying the corresponding cytokine under (A). C, ROC analysis for cytokine-positive CD4+ T cells. The 95% CI for the AUCs were, IFNγ+: 0.555–0.874, IL2+: 0.520–0.922, TNF+ CD4+ T-cells: 0.590–0.900. D, ROC analysis for secreted cytokines. The 95% CI for the AUCs were, secreted IFNγ: 0.654–0.938, secreted IL2: 0.715–0.989, and secreted TNF: 0.336–0.741. E, Frequencies of CD154+ and IL17+ CD4+ T cells in individuals with and without recurrence at 6 months. A, B, and E, Error bars show median and interquartile range.
- Figure 2.
Following induction therapy, differences between recurrence-free patients and those with recurrence were less conspicuous. A, Plots show changes between time points one (before induction therapy) and two (after induction therapy) in regards to IFNγ-producing tuberculin-inducible CD4+ T cells (left) and secreted IFNγ (right). CD4+ T cell frequencies are expressed as fractions (0.01 = 1%). B, ROC analysis shows discrimination between the outcome groups by age. C, Scatterplots show associations between age and secreted IFNγ (left) or IL2 (right). Patients with remission are shown as empty squares, patients with recurrence as filled circles. D, The scatter plot illustrates how recurrence-free patients and those with recurrence may be discriminated based on tuberculin-induced IFNγ and IL2 secretion prior to therapy using the indicated, tentative thresholds for IFNγ and IL2. Open squares indicate recurrence-free survival, filled circles recurrence. A group of seven patients with recurrence whose tuberculin-induced IL2 levels were below 120 pg/mL are highlighted (dotted oval).
- Figure 3.
T-cell polyfunctionality is increased in recurrence-free survivors. A, Scatter plots show four functional CD4+ T-cell subsets that were significantly different between recurrence-free survival (empty squares) and recurrence (filled circles). Note that CD4+ T cell frequencies are expressed as fractions (0.01 = 1%). Error bars show median and interquartile range. B, ROC curves show the discrimination between the outcome groups based on the two most promising subsets, producing IFNγ and TNF but not IL17 (95% CI for the AUC: 0.673–0.932), or, IFNγ, TNF, and IL2 (95% CI for the AUC: 0.661–941).
Tables
- Table 1.
Patient characteristics
Total number 42 Age, median (range) 71.2 years (47–89) Gender Male 34 Female 8 Grade G3 37 (88.1%) Carcinoma in situ 18 (42.8%) Grade G3 + carcinoma in situ 13 (30.9%) Carcinoma in situ alone 5 (11.9%) Stage Ta 12 (28.6%) T1 24 (57.1%) Lymphovascular invasion 0 Focality Single 30 Multiple 12 Follow-up time in months median (range) 16 (15–33) History of BCG vaccination 24 (57.1%) - Table 2.
Patient outcomes at 6 months by tumor characteristics
No recurrence Recurrence P Number 28 14 Age, mean (±STD) 71 (±10.7) 74 (±9.2) n.s. Male:Female 23:5 11:3 n.s. Original histology (Grade) G3 24 82.7% 13 92.9% n.s. CIS 14 50.0% 4 28.6% n.s. Stage Ta 9 32.1% 3 21.4% n.s. T1 14 50.0% 10 71.4% n.s. Multifocal 5 17.8% 7 50.0% n.s. History of BCG vaccination 16 57.1% 8 57.1% n.s. - Table 3.
The effect of tuberculin-induced secreted IL2 at baseline and patient age on BCG immunotherapy outcome
Parameter P OR (95% CI) IL2 secretion (Log10) 0.003 0.018 (0.001–0.274) Age 0.832 1.009 (0.932–1.109) Constant 0.064 4,144.925 NOTE: A 10-fold increase in IL2 is associated with a 98.2% reduction in the odds of developing recurrence by 6 months. The model provided correct classification of individuals into remission and recurrence groups of 87.6%.
Supplementary Data
- Figure S1 - Study time line
- Figure S2 - Flow-cytometry gating strategy
- Figure S3 - Recurrence free survival and effect of BCG-inducible IL2 secretion
- Figure S4 - Effect of BCG vaccination on secreted cytokines
- Figures S5 and S6 - Polyfunctional T-cell profiles
- Table S1 - Urine cytokines before and after BCG induction therapy
Volume 6, Issue 10
