The immune system plays a crucial role in controlling the development of cancer.
The concept of immunosurveillance proposed in the 1950s by Burnet provides that cancer cells can be recognized and eliminated by the effector mechanisms of humoral and cellular immunity, innate (macrophages, Natural Killer) and adaptive (T and B lymphocytes).
As is known, most tumors still develop strategies that allow them to escape immune responses. It is now also established, that the immune system itself sometimes promotes tumor growth, through cells that mediate the suppression of antitumor immunity. Among these, a key role is played by regulatory T cells. Multiple studies conducted on mouse models and cancer patients have shown the presence of a high number of Treg lymphocytes in the peripheral blood of patients with cancer and in the leukocyte infiltrate of many neoplasms and that this correlates with the progression of the disease and with a worse prognosis. Recently, the identification of a unique lineage of CD4+ T cells, named Treg, with the capacity of suppressing T cell effector function has put a new light on the mechanisms of tumor escape [18]. Treg are also able to inhibit the proliferation of CD4+/CD25- and CD8+ lymphocytes: these two properties, taken together, represent a big risk for the equilibrium between formation and destruction of cancer cells: so far, it has become more and more important to investigate whether some drugs, or some schedules could exert an inhibitory effect on Treg population.
Preliminary preclinical and clinical studies suggested that CHT administered at the Maximum-Tolerated-Dose (MTD) on one side ablate cancer cells, but on the other side also could compromise the immune system [19], whereas mCHT can increase the ablation of immunosuppressive Treg cells [20,21,22].
Based on these preclinical data, Ge et Al. conducted a pilot study in 12 MBC patients, who received single agent CTX at the dose of 50 mg per day, with the aim of testing if a metronomic regimen of CTX was able to affect Treg population [10]. They showed that, after an initial substantial depletion of Treg in the first 14 days of treatment, Treg numbers completely recovered during the therapy. Despite the complete recovery of Treg at the end of observation, the increase in breast-tumor reactive T cells remained at high levels for the whole treatment. The Treg suppression exerted by CTX in almost all patients was maintained until Day 42, when Tregs gradually recovered until their complete restoration on Day 84. These data showed for the first time that a metronomic schedule of CTX was able to deplete Tregs, even if not in the longer period.
Orecchioni et Al. studied the effects of different doses of three orally active drugs (VRL, CTX and 5-FU) over the landscape of circulating and tumor-infiltrating immune cells involved in checkpoint inhibitors activity [23], as well as on local and metastatic tumor growth [24].
The Authors showed that VRL and CTX (at medium/high doses) reduced circulating Tregs. Cyclophosphamide (at low doses) and 5-FU (at medium doses) slightly increased circulating Tregs.
In the present study, we present data regarding the immunomodulatory effects of mVRL on Treg.
To our knowledge, this is the first study regarding the immune effects of mVRL, alone, or in combination with mCAPE.
In our series, mVRL did not seem to reduce Tregs during treatment: mean values of Tregs (total, memory, naïve and activated), expressed both as a percentage and as absolute value, were stable without statistically significant increases or reductions.
It is therefore potentially possible that VRL, albeit in a metronomic schedule, has a marginal immunomodulatory effect compared to other drugs such as cyclophosphamide, exerting its effects mainly on neo-angiogenesis and on the reduction in colony formation, as suggested by some pre-clinical data [23]. The increase in the pool of regulatory T lymphocytes after the first 14 days of therapy could be justified both by the increase in the naïve and activated Treg subpopulations and by the slight increase in the CD4+ T population in the group of depleted patients. In fact, tumor-related CD4+ T cells spontaneously produce IL-2, a cytokine essential for the development and activation of Tregs.
Orecchioni et Al. demonstrated that VRL at medium doses (6–9 mg/kg) and CTX at medium (20 mg/kg/day) and high (40 mg/kg/day) doses can reduce the number of circulating Treg in a tumor-bearing mouse model [23, 24]. It is possible therefore possible to argue that the doses of VRL used in our population (40 mg thrice a week in combination and 50 mg thrice a week as single agent) were too low to observe an immune effect on Tregs. In fact, the Optimal Biological Dose (OBD) of VRL was established at 9 mg/kg based on the capability of inhibiting CEC by 50%; so far, the OBD for a immunomodulatory effect could be different, with a clear consequence on the results observed in our population.
In fact, if the apoptotic action induced by VRL on endothelial cells with consequent inhibition of angiogenesis is rather known, there are no data that attest to the role played on immune suppression nor the possible dosage of drug capable of determining this effect. The timing of action in terms of immunosuppression of the two drugs may also be different and further studies are needed in this regard. It could also be useful to perform evaluations on the activity of the immune system in combination metronomic schemes such as VEX (Vinorelbine - Cyclophosphamide - Capecitabine) to observe and analyze the possible synergistic effect of chemotherapy drugs on different lymphocyte populations.
Finally, the analysis of total lymphocytes and of T lymphocytes and NK populations did not show a significant decrease over the time, confirming the literature data according to which metronomic chemotherapy does not exert a depletion of lymphocytes [25].
Interestingly, our data show that Treg memory trends are completely different in depleted vs non depleted groups of patients.
Recently, Treg cells have been subgrouped as either naive or effector/memory by their developmental stage and phenotype [26, 27]. Memory Treg cells are characterized by CD103 expression and the ability to home into inflammation sites. They have been proposed to be sentinels of tolerance, providing a first line of defense against potentially harmful inflammatory reactions.
Lin et al. [28] recently showed, using a concomitant immunity tumor model, that memory Treg cells increased as the progression of the primary tumor was observed [29]. Notably, these cells more greatly express killing molecules and suppress the functions of tumor-bearing CD8+ T cells in vitro and in vivo.
Moreover, other Authors [30] showed results like ours treating 23 MBC HR+ patients with exemestane + mCTX, they did not observe any treatment-related decrease in Tregs. Notably, in their study baseline Naïve Tregs were associated with 3-month PFS.
As expected, considering the small sample size, we didn’t observe any difference in terms PFS between depleted and not depleted patients.
