Dysregulation of systemic soluble immune checkpoints in early breast cancer is attenuated following administration of neoadjuvant chemotherapy and is associated with recovery of CD27, CD28, CD40, CD80, ICOS and GITR and substantially increased levels of PD-L1, LAG-3 and TIM-3

Neoadjuvant chemotherapy (NAC) may alter the immune landscape of patients with early breast cancer (BC), potentially setting the scene for more effective implementation of checkpoint-targeted immunotherapy. This issue has been investigated in the current study in which alterations in the plasma concentrations of 16 soluble co-stimulatory and co-inhibitory, immune checkpoints were measured sequentially in a cohort of newly diagnosed, early BC patients (n=72), pre-treatment, post-NAC and post-surgery using a Multiplex® bead array platform. Relative to a group of healthy control subjects (n=45), the median pre-treatment levels of five co-stimulatory (CD27, CD40, GITRL, ICOS, GITR) and three co-inhibitory (TIM-3, CTLA-4, PD-L1) soluble checkpoints were significantly lower in the BC patients vs. controls (p<0.021-p<0.0001; and p<0.008-p<0.00001, respectively). Following NAC, the plasma levels of six soluble co-stimulatory checkpoints (CD28, CD40, ICOS, CD27, CD80, GITR), all involved in activation of CD8+ cytotoxic T cells, were significantly increased (p<0.04-p<0.00001), comparable with control values and remained at these levels post-surgery. Of the soluble co-inhibitory checkpoints, three (LAG-3, PD-L1, TIM-3) increased significantly post-NAC, reaching levels significantly greater than those of the control group. PD-1 remained unchanged, while BTLA and CTLA-4 decreased significantly (p<0.03 and p<0.00001, respectively). Normalization of soluble co-stimulatory immune checkpoints is seemingly indicative of reversal of systemic immune dysregulation following administration of NAC in early BC, while recovery of immune homeostasis may explain the increased levels of several negative checkpoint proteins, albeit with the exceptions of CTLA-4 and PD-1. Although a pathological complete response (pCR) was documented in 61% of patients (mostly triple-negative BC), surprisingly, none of the soluble immune checkpoints correlated with the pCR, either pre-treatment or post-NAC. Nevertheless, in the case of the co-stimulatory ICMs, these novel findings are indicative of the immune-restorative potential of NAC in early BC, while in the case of the co-inhibitory ICMs, elevated levels of soluble PD-L1, LAG-3 and TIM-3 post-NAC underscore the augmentative immunotherapeutic promise of targeting these molecules, either individually or in combination, as a strategy, which may contribute to the improved management of early BC

Dysregulation of systemic soluble immune checkpoints in early breast cancer is attenuated following administration of neoadjuvant chemotherapy and is associated with recovery of CD27, CD28, CD40, CD80, ICOS and GITR and substantially increased levels of PD-L1, LAG-3 and TIM-3

DATA AVAILABILITY STATEMENT : The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SUPPLEMETARY MATERIAL : SUPPLEMENTARY FIGURE 1 Box and whisker plots depicting the progressive changes in the median plasma concentrations (with 95% confidence limits) of three co-inhibitory immune checkpoints (BTLA, CTLA-4 and PD-1) throughout the course of neoadjuvant chemotherapy (NAC) (pre-treatment/diagnosis, post-NAC and post-surgery) in relation to the corresponding median values of the control subjects. The p values represent the comparison between pre-treatment/ diagnosis and post-NAC values.SUPPLEMENTARY FIGURE 2 Box and whisker plots depicting the progressive changes in the median plasma concentrations (with 95% confidence limits) of the remaining four co-stimulatory immune checkpoints (CD28, CD40, CD86 and GITRL) throughout the course of neoadjuvant chemotherapy (NAC) (pretreatment/ diagnosis, post-NAC and post-surgery) in relation to the corresponding median values of the control subjects. The p values represent the comparison between the pre-treatment/diagnosis and post- NAC values.SUPPLEMENTARY FIGURE 3 Box and whisker plots depicting the progressive changes in the median plasma concentrations (with 95% confidence limits) of the two dual-activity immune checkpoints (TLR-2 and HVEM) throughout the course of neoadjuvant chemotherapy (NAC) (pre-treatment/diagnosis, post-NAC and post-surgery) in relation to the corresponding median values of the control subjects. The p values represent the comparison between the pre-treatment/ diagnosis and post-NAC values.SUPPLEMENTARY FIGURE 4 Histological photomicrographs of pre-treatment tissue of a patient who attained a pathological complete response. (A) x20 Magnification: Core biopsy hematoxylin and eosin (H&E) stained slide breast carcinoma no special type (NST), prior to therapy. (B) X10 Magnification: Positive ECadherin immunoperoxidase stain of tumor confirming ductal differentiation. (C) x20 Magnification: Estrogen receptor immunoperoxidase stain of tumor, showing no staining (ER negative).SUPPLEMENTARY FIGURE 5 Histological photomicrographs of pre-treatment tissue of a patient who attained a pathological complete response. (A) x20 Magnification: Progesterone receptor immunoperoxidase stain of tumor (PR negative). (B) x20 Magnification: HER2 immunoperoxidase stain of tumor (HER2 negative). (C) x20 Magnification :Ki67 immunoperoxidase stain of tumor (90% of tumor cells staining positive).SUPPLEMENTARY FIGURE 6 Histological photomicrographs of post-surgery tissue obtained during surgery of a patient who attained a pathological complete response. (A) X10 Magnification: Tumor bed post chemotherapy showing stromal fibrosis and dystrophic calcification with NO tumor cells H&E. (B) X10 Magnification: Tumor bed post chemotherapy showing loose fibrovascular response and elastosis with NO tumor cells H&E. (C) x20 Magnification: MNF116 (broad pancytokeratin) immunoperoxidase stain of tumor bed post chemotherapy showing NO residual staining tumor cells.Neoadjuvant chemotherapy (NAC) may alter the immune landscape of patients with early breast cancer (BC), potentially setting the scene for more effective implementation of checkpoint-targeted immunotherapy. This issue has been investigated in the current study in which alterations in the plasma concentrations of 16 soluble co-stimulatory and co-inhibitory, immune checkpoints were measured sequentially in a cohort of newly diagnosed, early BC patients (n=72), pre-treatment, post-NAC and post-surgery using a Multiplex® bead array platform. Relative to a group of healthy control subjects (n=45), the median pre-treatment levels of five co-stimulatory (CD27, CD40, GITRL, ICOS, GITR) and three co-inhibitory (TIM-3, CTLA-4, PD-L1) soluble checkpoints were significantly lower in the BC patients vs. controls (p<0.021-p<0.0001; and p<0.008-p<0.00001, respectively). Following NAC, the plasma levels of six soluble co-stimulatory checkpoints (CD28, CD40, ICOS, CD27, CD80, GITR), all involved in activation of CD8+ cytotoxic T cells, were significantly increased (p<0.04-p<0.00001), comparable with control values and remained at these levels post-surgery. Of the soluble co-inhibitory checkpoints, three (LAG-3, PDL1, TIM-3) increased significantly post-NAC, reaching levels significantly greater than those of the control group. PD-1 remained unchanged, while BTLA and CTLA-4 decreased significantly (p<0.03 and p<0.00001, respectively). Normalization of soluble co-stimulatory immune checkpoints is seemingly indicative of reversal of systemic immune dysregulation following administration of NAC in early BC, while recovery of immune homeostasis may explain the increased levels of several negative checkpoint proteins, albeit with the exceptions of CTLA-4 and PD-1. Although a pathological complete response (pCR) was documented in 61% of patients (mostly triple-negative BC), surprisingly, none of the soluble immune checkpoints correlated with the pCR, either pre-treatment or post-NAC. Nevertheless, in the case of the co-stimulatory ICMs, these novel findings are indicative of the immune-restorative potential of NAC in early BC, while in the case of the co-inhibitory ICMs, elevated levels of soluble PD-L1, LAG-3 and TIM-3 post-NAC underscore the augmentative immunotherapeutic promise of targeting these molecules, either individually or in combination, as a strategy, which may contribute to the improved management of early BC.The Cancer Association of South Africa (CANSA).http://www.frontiersin.org/Oncologyam2024ImmunologySDG-03:Good heatlh and well-bein

Systemic immune dysregulation in early breast cancer is associated with decreased plasma levels of both soluble co-inhibitory and co-stimulatory immune checkpoint molecules

Breast cancer cells exploit the up-regulation or down-regulation of immune checkpoint proteins to evade anti-tumor immune responses. To explore the possible involvement of this mechanism in promoting systemic immunosuppression, the pre-treatment levels of soluble co-inhibitory and co-stimulatory immune checkpoint molecules, as well as those of cytokines, chemokines, and growth factors were measured in 98 newly diagnosed breast cancer patients and compared with those of 45 healthy controls using multiplex bead array and ELISA technologies. Plasma concentrations of the co-stimulatory immune checkpoints, GITR, GITRL, CD27, CD28, CD40, CD80, CD86 and ICOS, as well as the co-inhibitory molecules, PD-L1, CTLA-4 and TIM-3, were all significantly lower in early breast cancer patients compared to healthy controls, as were those of HVEM and sTLR-2, whereas the plasma concentrations of CX3CL1 (fractalkine), CCL5 (RANTES) and those of the growth factors, M-CSF, FGF-21 and GDF-15 were significantly increased. However, when analyzed according to the patients’ breast cancer characteristics, these being triple negative breast cancer (TNBC) vs. non-TNBC, tumor size, stage, nodal status and age, no significant differences were detected between the plasma levels of the various immune checkpoint molecules, cytokines, chemokines and growth factors. Additionally, none of these biomarkers correlated with pathological complete response. This study has identified low plasma levels of soluble co-stimulatory and co-inhibitory immune checkpoint molecules in newly diagnosed, non-metastatic breast cancer patients compared to healthy controls, which is a novel finding seemingly consistent with a state of systemic immune dysregulation. Plausible mechanisms include an association with elevated levels of M-CSF and CCL5, implicating the involvement of immune suppressor cells of the M2-macrophage/monocyte phenotype as possible drivers of this state of systemic immune quiescence/dysregulation.The Cancer Association of South Africa (CANSA).https://www.frontiersin.org/journals/immunologydm2022Immunolog