High mobility group box 1 (HMGB1) is an extremely versatile protein that is located predominantly in the nucleus of quiescent eukaryotic cells, where it is critically involved in maintaining genomic structure and function. During cellular stress, however, this multifaceted, cytokine-like protein undergoes posttranslational modifications that promote its translocation to the cytosol, from where it is released extracellularly, either actively or passively, according to cell type and stressor. In the extracellular milieu, HMGB1 triggers innate inflammatory responses that may be beneficial or harmful, depending on the magnitude and duration of release of this pro-inflammatory protein at sites of tissue injury. Heightened awareness of the potentially harmful activities of HMGB1, together with a considerable body of innovative, recent research, have revealed that excessive production of HMGB1, resulting from misdirected, chronic inflammatory responses, appears to contribute to all the stages of tumorigenesis. In the setting of established cancers, the production of HMGB1 by tumor cells per se may also exacerbate inflammation-related immunosuppression. These pro-inflammatory mechanisms of HMGB1-orchestrated tumorigenesis, as well as the prognostic potential of detection of elevated expression of this protein in the tumor microenvironment, represent the major thrusts of this review.

Background: Tumor-infiltrating lymphocytes are associated with a better prognosis in early triple-negative breast cancer (TNBC). These cells can be enumerated in situ by the “Immunoscore Clinical Research” (ISCR). The original Immunoscore^® is a prognostic tool that categorizes the densities of CD3⁺ and CD8⁺ cells in both the invasive margin (IM) and center of the tumor (CT) in localized colon cancer, yielding a five-tiered classification (0–4). We evaluated the prognostic potential of ISCR and pathological complete response (pCR) following neoadjuvant chemotherapy (NACT). Methods: The cohort included 53 TNBC, 32 luminal BC, and 18 HER2-positive BC patients undergoing NACT. Pre-treatment tumor biopsies were immune-stained for CD3⁺ and CD8⁺ T-cell markers. Quantitative analysis of these cells in different tumor locations was performed using computer-assisted image analysis. Results: The pCR rate was 44%. Univariate analysis showed that primary tumor size, estrogen-receptor negative, progesterone-receptor negative, luminal vs. HER2-positive vs. TNBC, high Ki-67, high densities (cells/mm²) of CD3 CT, CD8⁺ CT, CD3⁺ IM, and CD8⁺ IM cells were associated with a high pCR. ISCR was associated with pCR following NACT. A multivariate model consisting of ISCR and the significant variables from the univariate analysis showed a significant trend for ISCR; however, the low sample size did not provide enough power for the model to be included in this study. Conclusions: These results revealed a significant prognostic role for the spatial distributions of the CD3⁺, and CD8⁺ lymphocytes, as well as the ISCR in relation to pCR following NACT.

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.

Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells can up-regulate or down-regulate these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICMs) can be measured in human plasma. The study aimed to measure the systemic levels of a series of positive and negative ICMs at diagnosis, post-neo-adjuvant chemotherapy (NAC) and post-surgery in newly- diagnosed BC patients (pts) relative to those of a healthy control group. Method: Soluble ICMs were measured using Multiplex® bead array technology in plasma from 72 BC patients and 45 healthy controls. Data was prospectively obtained and levels compared between pre-treatment, post-NAC, and post-surgery using non-parametric tests (Mann-Whitney & Kruskal-Wallis). Results: Pre-treatment, soluble stimulatory molecules viz. GITR (p<0.0000), GITRL (p< 0.0199), CD27 (p< 0.0243), CD40 (p< 0.0209), ICOS (p< 0.0087), as well as the inhibitory molecules PD-L1 (p< 0.0000), CTLA-4 (p< 0.005), TIM-3 (p< 0.0004), HVEM (p< 0.0004) levels were significantly lower in early BC pts compared to controls. Post treatment, there were significant increases in most ICM levels (Table 1), with the exception of CTLA-4 which decreased significantly following treatment. A pCR was documented in 65% of patients (mostly TNBC). There was no correlation between pre-treatment ICM levels and pCR. Conclusion: We identified low levels of stimulatory and inhibitory ICMs in newly-diagnosed, non-metastatic BC patients compared to healthy controls. Following treatment, with the exception of CTLA-4, most of these pre-treatment abnormalities of systemic ICM levels corrected. NAC is associated with upregulation of sPD-L1 and most other ICMs. These results indicate that early BC is associated with down-regulation of soluble stimulatory and inhibitory ICMs. Newly-diagnosed early BC patients appear to have generalized immune-suppression independent of subtype and stage. To our knowledge, this is the first study to describe the effect of treatment on systemic ICMs in early BC patients. Table 1.The effect of treatment on soluble, systemic ICMsICMControlDiagnosis (Group A)Post-NAC (Group B)Post-surgery (Group C) Group A vs Group BComparing pre-treatment levels vs post-treatment levelsMedian (pg/ml)p-valueBTLA18147130229987127770,0366CD8023291678304836110,0000CD8614297115859922124390,2789CTLA-4261815665986870,0000LAG31504161312754648805001330,0000PD-L133421647479452150,0000PD-1149171230513350150760,8888TIM350473897997596150,0000CD2745773342535154270,0000CD28461353291444277500580,0415CD4019771523203020540,0002GITR37971497403544340,0000GITRL71515886533959270,4938ICOS265061512326586297460,0001HVEM22901865404739500,0000TLR2304772683133837370420,0257 Citation Format: Bernardo Leon Rapoport, Helen C Steel, Carol Ann Benn, Simon Nayler, Teresa Smit, Liezl Heyman, Annette Theron, Nomsa Hlatswayo, Luyanda Kwofie, Pieter Meyer, Ronald Anderson. Dysregulation of immune checkpoint proteins in newly- diagnosed early breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-11.