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Calais da Silva et al. Systemic humoral responses during BCG treatment

followed by three weekly instillations at month 3, 6, (Ct) of the amplification curve of target gene and that of
9, and 12. All the patients were followed for one year endogenous controls. Reactions with a Ct value higher
and the outcome variables were: tumor progression, than 35 cycles were considered negligible and were
relapse, and disease-specific survival. Patients who not considered further. Efficiency of the amplification
showed no relapses within a year after the beginning reaction for each primer-probe was above 95% (as
of treatment were named “BCG responders” [40 determined by manufacturer).
patients (3 women/37 men, mean age of 68, 3 years)].
Those patients who manifested relapses within that Statistical analysis
year were named “BCG-relapsing” [18 patients (1 The Kruskal Wallis test was used to compare ratios
woman/17 men, mean age of 67.5 years)]. In each in patients with and without recurrence. Fisher’s
case, four samples were collected before and after the exact test was used to identify the factors that were
first instillation and the week 6 instillation. The study significant in the univariate analysis. Multivariate
has been approved by the ethical committee of the logistic regression was used to identify the two
Hospital. factors that were retained in the multivariate analysis.
A significance level of P = 0.05 was used to identify
Isolation of RNA and real-time PCR possible factors for predicting response to BCG
Blood specimens (2.5 mL) were collected in PAXgene™ therapy in the univariate analyses. No correction was
tubes (Qiagen, Manchester, UK), incubated at room made for multiple endpoints or testing.
temperature for 4 h for RNA stabilization and then
stored at -80 °C. RNA was extracted from whole RESULTS
blood using PAXgene™ Blood RNA System Kit
(Qiagen), following manufacturer’s instructions, and BCG treatment induces significant systemic
further purification of RNA was done with on-column fast changes
DNase digestion. RNA concentrations and A 260 -to-A To assess whether BCG treatment induced significant
280
ratios were measured in a spectrophotometer, and changes in the expression of specific cytokines and
only samples with A 260 /A 280 ratios between 1.9 and molecular effectors in peripheral blood cells, we
2.1 were further considered. 1 µg of total RNA was analyzed its expression in blood samples from patients
reverse transcribed with random primers, using High during BCG therapy. The selected cytokines and
Capacity cDNA Archive Kit (Applied Biosystems, molecular effectors depicted in Table 1 represent the
Foster City, CA). reported complex events known to locally accompany
BCG treatment, i.e. to occur in the bladder. Blood
Real time PCR gene quantification analysis was samples were collected before and 24 h after BCG
performed as described, [7,15] using Taqman assays treatment to assess fast changes. To assess prolonged
from Applied Biosystems: Hs00234140_m1 (CCL2), changes, samples were collected at the first instillation
Hs00234142_m1 (CCL3), Hs00271615_m1 (CCL8), (week 1) and at week 6 of BCG treatment. Significant
Hs00171065_m1 (CXCL9), Hs00171042_m1 (IP-10), levels of mRNA were obtained from all blood samples.
Hs00175480_m1 (CTLA4), Hs00181225_m1 (Fas-L), Before BCG treatment (pre-BCG stage), the lowest
Hs01110250_m1 (HMOX-1), Hs00167257_m1 expression levels were observed for IL-4 (0.024‰
(NOS2A), Hs00169473_m1 (Perf), Hs00246266_m1 mRNA) and maximum for GNLY (252.38‰ mRNA)
(GNLY), Hs00174128_m1(TNF-α); Hs00174086 _m1 [Figure 1]. During BCG treatment, we observed
(IL-10); Hs00174143_m1 (IFN-γ), Hs00174097_m1 significant fast changes (24 h after the first treatment
(IL-1β), Hs00174114_m1 (IL-2), Hs00174122_m1 (IL- or after the instillation performed at 6th week) in IL-
4), Hs00174131_m1 (IL-6), and 4352935E (β-actin). 1β, TNF-α, IL-10, GNLY, and Perf [Figure 2]. No
Each reaction was performed in duplicate. All genes, significant prolonged changes, i.e. during treatment,
including endogenous controls, were always analyzed were observed [Figure 2]. In fact, 24 h after the first
in the same run to exclude between-run variations. The BCG instillation, significant expression changes were
number of PCR cycles needed to reach fluorescence observed. IL-1β mRNA increased from 12.94‰ before
threshold in each sample was defined as the cycle treatment to 16.48‰ after treatment (P = 0.033),
threshold (Ct). The mRNA expression was normalized TNF-α mRNA increased from 3.70‰ to 4.23‰ (P =
using β-actin gene expression as a reference according 0.002), and IL-10 mRNA increased from 0.17‰ to
to our previous observations. Relative mRNA levels 0.24‰ (P = 0.026) [Figure 2]. At week 6, changes were
[15]
were calculated using formula 2 -∆Ct × 1,000 which still observed and were sometimes more pronounced
infers the number of mRNA molecules of the gene of [Figure 2]. In fact, IL-1β mRNA increased from 11.46‰
interest per 1000 molecules of endogenous control. to 20.76‰ (P = 0.0001), TNF-α mRNA increased from
∆Ct stands for the difference between cycle threshold 3.12‰ to 4.57‰ (P = 0.0045), and IL-10 increased
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ July 14, 2017 119