Figure S1. CXCR3 rs2280963 polymorphism
Electopherograms showing representative G, GT, and T carriers and the four G carriers
Figure S2. CXCR3 and CCR5 expression by tumor infiltrating lymphocytes (TILs).
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A) Scatter plot of CXCR3 and CCR5 expression in TILs. X axis: CCR5 Log2 intensity; Y
axis: CXCR3 Log2 intensity, Vertical and horizontal dotted lines represent the median
values of the X and Y axes, respectively. CR (complete response) samples are
represented with red dots, PR (partial response) samples with green dots and NR (non
response) samples with blue dots.
Figure S3. CXCR3 and CCR5 expression in TILs and pre-treatment tumor biopsies
according to the corresponding polymorphism.
2
Scatter plots showing CCR5 and CXCR3 expression in TILs (A-B) and pre-treatment
tumor biopsies (C-D) according to CCR5-Δ32 polymorphism and CXCR3 rs2280964 C/T
polymorphism, respectively. Horizontal lines indicate mean and standard deviation. X axis:
CCR5 (A, C) and CXCR3 rs2280964 (B,D) genotypes. CXCR3 expression for G carriers
(not represented in the figure) were 8.35, 9.02, 8.47, 8.42 Log2intensity in the four TIL
samples and 5.25 and 5.51 Log2intensity in the two tumor samples.
Y axis: CCR5 (A,C) and CXCR3 (B,D) gene expression expressed as Log2intensity.
In the CCR5 panels, the gray arrow indicates the CCR5-Δ32 homozygous sample. The
association between TIL (A, B) and tumor (C, D) gene-expression and gene
polymorphisms was assessed by Student t-test, after checking for normal distribution
(D’Agostino-Pearson
normality
test).
Correlation
between
CXCR3
rs2280964
3
polymorphisms (treated as ordered categorical variables) was assessed by Spearman
rank correlation coefficient (ρ) test).
Figure S4. CCL3, CCL4, CXCR3, and CCR5 expression in pre-treatment biopsies.
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Hierarchical clustering of CCL3, CCL4,CCL5, CXCR3 and CCR5 based on the meancentered gene expression values. OR and CR enrichment was calculated by comparing
C1+C2 vs C3, C1 vs C2+C3, and C2 vs C1+C3 by Chi-square test or Fisher’s exact test if
any category has a cell count less than 5. Patients with clinical complete responses (CR)
are shown in red, patients with partial remission (PR) are shown in green and non
responders (NR) are shown in blue. OR: objective response (CR+PR); non-CR: PR+NR.
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Table S1 . Association between TIL CXCR3, CCR5 expression, CCR5-Δ32
polymorphism, and their interaction with clinical outcome.
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Model A (gene-expression model): expression of CXCR3 and CCR5, and their
interactions.
Model definition
Model comparisons
P values
Model A0 (null model):
Y~u
Model A1:
Y ~ u + x1 + x2
Model A2 (full model A):
Y ~ u + x1 + x2 + x1*x2
A2 vs A0
A2 vs A1
CR
OR
.032
.14
.15
.040
Model B (gene-expression/polymorphism model): expression of CXCR3 and
CCR5, and CCR5-Δ32 polymorphism.
Model definition
Model comparisons
P values
Model B0 (null model):
Y~u
Model B1:
Y ~ u + x1 + x2 + x3
Model B2:
Y ~ u + x1 + x2 + x3 + x1*x2 + x1*x3 +
x2*x3
Model B3 (full model B):
Y ~ u + x1 + x2 + x3 + x1*x2 + x1*x3 +
x2*x3 + x1*x2*x3
B3 vs B0
B3 vs B1
B3 vs B2
B3 vs A2
CR
OR
.0009
.003
.99
.003
.007
.015
.056
.007
Model A comparisons. By comparing model A2 and A0, model A2 (full model, which
includes CXCR3 and CCR5 expression and their interaction) fitted significantly better than
model A0 for CR but not significantly better for OR. By comparing model A2 and A1, the
two-way interaction between CXCR3 and CCR5 expression was significantly associated
with OR but not significantly associated with CR.
Model B comparisons. By comparing model B3 and B0, model B3 (the full model, which
includes CXCR3 and CCR5 expression, CCR5-Δ32 polymorphism and their interaction)
fitted significantly better than model B0 for both CR and OR. By comparing model B3 and
B1, the two-way and three-way interactions among the three variables (i.e., CXCR3 and
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CCR5 expression and CCR5-Δ32 polymorphism) were significantly associated with both
CR and OR. When model B3 was compared with model B2, three-way interaction was
only borderline significantly associated with OR but not significantly associated with CR.
Moreover, model B3 was significantly better than model A2, both for OR and CR.
X1, expression of CCR5; X2, expression of CXCR3; X3, CCR5-Δ32 polymorphism; Y,
Endpoint (CR or OR); CR, complete response; OR, overall response.
Table S2 . Association between treatment, TIL CXCR3, CCR5 gene expression,
CCR5-Δ32 polymorphism, with clinical outcome.
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Model C (gene-expression/treatment model): treatment, expression of
CCR5 and CXCR3, and their interactions.
Model definition
Model fitting
P values
Model C1
Y ~ u + Rx
Model C2
Y ~ u + x1 + x2 + x1*x2
Model C3 (full model C)
Y ~ u + Rx + x1 + x2 + x1*x2
C3 vs C1
C3 vs C2
CR
OR
.002
.002
.010
.0009
Model D (gene-expression/polymorphism and treatment model): treatment,
expression of CXCR3 and CCR5, CCR5-Δ32 polymorphism, and their
interactions.
Model definition
Model fitting
P values
Model D1:
Y ~ u + Rx
Model D2:
Y ~ u + x1 + x2 + x3 + x1*x2 + x1*x3 +
x2*x3 + x1*x2*x3
Model D3 (full model D):
Y ~ u + Rx + x1 + x2 + x3 + x1*x2 + x1*x3
+ x2*x3 + x1*x2*x3
D3 vs D1
D3 vs D2
D3 vs C3
CR
OR
.0001
.003
.005
.003
.004
.036
Model C comparisons. By comparing model C3 and model C1, model C3 (full model,
which includes expression of CXCR3 and CCR5 and their interaction) was significantly
associated with clinical outcomes, either CR or OR, when controlling for treatment effect.
By comparing model C3 and C2, the treatment effect was significantly associated with
clinical outcomes when the treatment effect was included in the gene-expression model.
Model D comparisons. By comparing model D3 and D1, model D3 (full model, which
includes CXCR3 and CCR5 expression, CCR5-Δ32 polymorphism and their interaction
which includes with three variables and their interactions) was significantly associated with
CR and OR when controlling for treatment effect. By comparing model D3 and model D2,
the treatment effect was significantly associated with clinical outcomes, either CR or OR.
Moreover, model D3 was significantly better than model C3 both for OR and CR.
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X1, expression value of CCR5; X2, expression value of CXCR3; X3, CCR5-Δ32
polymorphism; Rx, treatment (TNMA, T200, T1200, TYT, and TCD8); Y, Endpoint (CR or
OR); CR, complete response; OR, overall response.
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