Previously, we have demonstrated in two independent cohorts impaired IFN signaling and downstream functional consequences in PBLs from patients with minimally metastatic stage III and widely metastatic stage IV melanoma as compared to healthy controls [15, 16]. In the current study, we analyzed serial PBMC samples obtained from patients before and after the induction phase of HDI in a new independent cohort of minimally metastatic stage IIIb and IIIc melanoma patients to advance our current understanding of immune dysfunction in cancer. This exploratory study demonstrated that patients with high-risk operable nodal involvement with melanoma who had a clinical response to high dose IFN-α2b therapy over the 4-week induction phase of neoadjuvant therapy had a significant increase in STAT1 activation in peripheral blood T cells, but not B cells, upon IFN-α stimulation from Day 0 to Day 29. Moreover, this increase in pSTAT1 in peripheral blood T cells also correlated with good clinical outcome suggesting the efficacy of HDI in the clinical responders may be due, as least in part, to augmentation of their pSTAT1 responsiveness.
Moreover, the differences in STAT1 activation from pre- to post- HDI treatment could distinguish patients who were inclined to have a favorable or unfavorable outcome. As expected, patients who had minimal or negative changes in pSTAT1 (median ratio < 1.15) had poor outcome. Of patients who showed increased pSTAT1 signaling after HDI therapy, only patients who displayed modest augmentation (median ratio 1.15 - 1.35) had good outcome. Interestingly, patients who had 'hyper' IFN signaling responses (median ratio >1.35) of pSTAT1 pre- to post- HDI therapy had poor outcome, similar to those who has minimal or negative changes. These results warrant further confirmation in a larger patient cohort to investigate the underlying mechanisms by which HDI alters IFN signaling patterns in patients with different clinical outcomes.
Assessing the IFN signaling patterns in peripheral blood T cells from melanoma patients from Day 0 to Day 29 HDI therapy may be a clinically useful approach to select patients who would be more inclined to benefit from further treatment, and hence should be maintained on HDI. A trend was observed which showed that responding patients, prior to HDI therapy, have a lower response to IFN-α induced pSTAT1 compared to those of the non-responding patients. We have previously found two subsets of IFN responses in melanoma patients, IFN low-responders and IFN high-responders . We showed that in IFN low-responders, prolonged in vitro stimulation with high doses of IFN partially restored IFN signaling, suggesting a possible mechanism for the beneficial effect of HDI therapy in these melanoma patients. Prior to initiation with HDI (pre), reduced activation of STAT1 in the responding patients compared to the non-responding patients may be explained in that this patient subset exhibited a severe impairment in IFN signaling which was restored during the initiation phase of HDI therapy. In contrast, patients who had higher levels of STAT1 activation prior to the HDI initiation phase may not have had an IFN signaling defect and therefore, would not have benefited from HDI therapy. In our previous study , the differences in the median activation of pSTAT1 between melanoma patients and healthy controls were 1.6 fold were as, in the current study, the median differences in STAT1 activation between the responders and non-responders were 1.4 fold.
Though B cells showed a significantly lower trend in overall STAT1 activation in responders compared to non-responders before HDI, there was no significant increase in STAT1 activation during the induction phase of HDI in the responding group, and interestingly, non-responders exhibited decreased overall STAT1 activation. It has been reported that subsets of immune cells respond differently to IFNs [28, 29] and that in the presence of high amounts of exogenous IFNs, downregulation of the receptors may occur as a negative feedback mechanism , thereby reducing responsiveness to IFNs. Additionally, reduced responsiveness in leukocytes may reflect the effects of a high tumor burden whereby tumor cells secreting immunosuppressive cytokines, such as IL-10 and TGF-β, have been shown to induce expression of STAT1 negative regulators such as the suppressors of cytokine signaling (SOCS) proteins and Src homology 2 (SH2)-containing phosphatase-1 and -2 and CD45 [31–33], thereby inhibiting the anti-tumor activity of immune effector cells . Tregs and myeloid-derived suppressor cells, known for their suppressive roles on immune cells [35–37] in cancer, may also contribute to reduced responsiveness to HDI. Altered plasma or serum cytokine profiles in cancer patients [38, 39] may predispose peripheral blood leukocytes to impaired IFN signaling.
There was variation and overlap between the responder and non-responder groups. We and others have previously described variation of signaling responses in melanoma patients' PBMCs using phosflow [15, 40, 41], suggesting that signaling abnormalities may not arise in all patients, but rather in a subset of patients. Variable responsiveness may explain the small differences observed between the clinical responders and non-responders. The sample size of this study was too small (14 patients) to be conclusive, but these results warrant a larger confirmatory study.
The importance of STAT1 in IFN signaling has been demonstrated in STAT1 knockout mice where STAT1 deficient mice were more likely to develop spontaneous tumors than wild type mice , and were more susceptible to viruses and pathogens showing an IFN-dependent link to STAT1 . Previous studies have attempted to link pSTAT1 (and pSTAT3) levels in tumor cells and lymphocytes to clinical outcome of melanoma patients receiving interferon treatment [44, 45]. Patients with higher pSTAT1/pSTAT3 ratios in pretreated lymph node biopsy tissues had better clinical outcome ; however, these studies did not find a correlation between pSTAT1/pSTAT3 ratios among lymphocytes of regional lymph nodes and survival. One novelty in the present study is to consider pSTAT1 levels in different subsets of peripheral blood lymphocytes.
The use of immune profiles as a prognostic tool to determine melanoma patient survival has been studied, such as using quantification of tumor infiltrating lymphocytes (TILs) in metastatic lesions , as well as gene expression profiling of TILs and CD3 T cells from primary cutaneous melanoma where the genes that were positively associated with survival were mainly related to the immune response . Beyond these prognostic implications, assessing STAT1 activation in PBL of melanoma patients may provide an additional predictive tool to guide the application of HDI therapy.