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

Table 3: Cut-off values of the relevant molecules with a predictive meaning for patients’ response to BCG.
Multivariate logistic regression was used to identify the two factors retained in the multivariate analysis
Good factor Prejudicial factor
Univariate analysis IL-1β at 6WAT LE 12.5 GT 12.5
IFN-γ at 6WBT LE 0.17 GT 0.17
GNLY at 6WBT LE 110.0 GT 110.0
HMOX-1 at 6WBT LE 13.0 GT 13.0
Multivariate analysis GNLY at 6WBT LE 110.0 GT 110.0
HMOX-1 at 6WBT LE 13.0 GT 13.0
Values are relative mRNA molecules calculated by formula 2 -∆Ct *1,000, which infers the number of mRNA molecules of each gene per 1,000
molecules of the endogenous control (β-actin). The designation of “good factor” and “prejudical factor” is correlated with patients’ response
to BCG, namely no relapse or relapse in less than one year after treatment. BCG: Bacille Calmette-Guérin; 6WAT: sample collected 24 h
after BCG instillation at week 6; 6WBT: sample collected before BCG instillation at week 6; LE: less than or equal to; GT: greater than
response (≤ 12.5‰ to IL-1β; ≤ 0.17‰ to IFN-γ, ≤ 110‰ DISCUSSION
to GNLY, and ≤ 13.0‰ to HMOX-1).
Immunotherapies boost patient’s immune response
In the multivariate analysis, only HMOX-1 and GNLY to improve its capacity to eliminate tumor. BCG is an
were shown to be independent predictive biomarkers. immunotherapy, used as a standard of care to treat
As shown in Table 3, we established a cut-off, above NMIBC patients to reduce cancer relapses. BCG
which these biomarkers are considered prejudicial instillations into the bladder attract antitumor effector
factors. We then subdivided patients according immune cells to the tumor site, [8,9] and three months
to whether they show no prejudicial factors, one after the BCG treatment course, the cellular infiltrate
prejudicial factor, or two prejudicial factors (see of T and B cells is concentrated in the persisting
[16]
supplementary data). One in twelve patients with no granulomas, focused on elimination of cancer cells.
However, BCG treatment remains suboptimal because
prejudicial factors relapsed (8.3%). Four in twenty- 30% to 50% of the patients show no response and/or
three patients showing one prejudicial factor relapsed relapse within the first year of treatment. [17]
(17.4%) and finally, ten of fifteen patients showing two
prejudicial factors relapsed (66.7%). This allowed us Although the underlying mechanisms of BCG therapy
to establish predictive cut-off values and a predictive are not fully elucidated, it is known that a Th1 response
grouping system, thus identifying the probability of is required to stimulate cell-mediated tumoricidal
relapse after BCG treatment. activity. The level of the Th1 cytokine, IL-2, expressed
[18]

Figure 1: All the analyzed genes were significantly expressed at pre-BCG state, with the lowest mRNA levels observed for IL-4 and
maximum for GNLY. Relative mRNA levels of IL-4, NOS2A, IL-2, IL-6, CCL8, CCL2, IL-10, CXCL9, IFN-γ, IP-10, CCL3, CTLA4, TNF-α,
Fas-L, IL-1β, HMOX-1, Perf and GNLY were evaluated by real time PCR, as described in the Methods section. mRNA was obtained from
blood samples of 58 patients, collected before any BCG treatment. Values were calculated, as referred to in the Methods section, by
formula 2 -∆Ct *1,000 and infers the number of mRNA molecules of a certain gene per 1,000 molecules of the endogenous control (β-actin).
BCG: Bacille Calmette-Guérin; PCR: polymerase chain reaction
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ July 14, 2017 121