Immune Checkpoint Inhibitors and Other Immune Therapies in Breast Cancer: A New Paradigm for Prolonged Adjuvant Immunotherapy.

Background: Breast cancer is the most common form of cancer in women worldwide. Advances in the early diagnosis and treatment of cancer in the last decade have progressively decreased the cancer mortality rate, and in recent years, immunotherapy has emerged as a relevant tool against cancer. HER2+ and triple-negative breast cancers (TNBCs) are considered more immunogenic and suitable for this kind of treatment due to the higher rate of tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression. In TNBC, genetic aberrations further favor immunogenicity due to more neo-antigens in cancer cells. Methods: This review summarizes the principal ongoing conventional and investigational immunotherapies in breast cancer. Particularly, immune checkpoint inhibitors (ICIs) and their use alone or combined with DNA damage repair inhibitors (DDRis) are described. Then, the issue on immunotherapy with monoclonal antibodies against HER-2 family receptors is updated. Other investigational immunotherapies include a new schedule based on the interferon beta-interleukin-2 sequence that was given in ER+ metastatic breast cancer patients concomitant with anti-estrogen therapy, which surprisingly showed promising results. Results: Based on the scientific literature and our own findings, the current evaluation of tumor immunogenicity and the conventional model of adjuvant chemotherapy (CT) are questioned. Conclusions: A novel strategy based on additional prolonged adjuvant immunotherapy combined with hormone therapy or alternated with CT is proposed.

Minimal residual disease in advanced or metastatic solid cancers: The G0-G1 state and immunotherapy are key to unwinding cancer complexity.

In the last decade, a large amount of research has focused on elucidating the mechanisms that account for homing disseminated cancer cells (DCCs) from solid tumours to distant organs, which successively progress to overt metastatic disease; this is currently incurable. A better understanding of DCC behaviour is expected to allow detectable metastasis prevention by more effectively targeting 'metastatic seeds before they sprout'. As DCC biology co-evolved with that of the primary tumour, and due to the many similarities between them, the term 'niche' has been borrowed from normal adult stem cells (ASCs) to define the site of DCC metastatic colonisation. Moreover, heterogeneity, survival, protection, stemness and plasticity as well as the prolonged G0-G1 dormant state in the metastatic niche have been the main aspects of intense investigation. Consistent with these findings, in solid cancers with minimal residual disease (MRD), it has been proposed to prolong adjuvant therapy by targeting specific molecular pathway(s) involving DCC dormancy. However, so far, few disappointing clinical data have been reported. As an alternative strategy, because immune-surveillance contributes to the steady state of the DCC population and likely to the G0-G1 state of cancer cells, we have used prolonged immune-modulatory cytostatic chemotherapy, active immune stimulation with an INF-ß/IL-2 sequence or drugs inhibiting myeloid-derived suppressor cell (MDSC)/Treg-mediated immune suppression. This strategy, mainly aimed at boosting the immune response, is based on recent findings suggesting the downregulation of immune escape mechanisms as well as other principal hallmarks during the G0-G1 state and/or in MRD. Preliminary clinical and/or laboratory data suggest the efficacy of this strategy in gastrointestinal and some endocrine-dependent cancers. Following this, we propose therapeutic schedules to prevent DCC activation and proliferation in solid cancers at a high risk of relapse or as maintenance therapy in metastatic patients after complete response (CR) to conventional treatment.

A new immunotherapy schedule in addition to first-line hormone therapy for metastatic breast cancer patients in a state of clinical benefit during hormone therapy.

Acquired resistance occurs in metastatic hormone receptor-positive breast cancer patients. The addition of interferon beta/interleukin-2 immunotherapy to first-line salvage hormone therapy (HT) prolonged progression-free (PFS) and overall survivals (OS) in 26 patients, as compared with 30 historical controls and literature data. This was a 2 : 1 ratio case-control retrospective observational study. The cases were from an open pilot study, started in 1992, and controls were recruited in 2006. The planned mean follow-up time was the time at which more than 80% of controls progressed. The median PFS was significantly longer in the cases than that in controls, 33.1 (95% CI 24.5-41.8) vs 18 (95% CI 12.1-23.8) months (p < 0.0001). Also, median OS was significantly longer in the cases, 81 vs 62 (95% CI 48.1-75.9) months (p < 0.0029). When analysis of the 2 groups was adjusted for the disease-free interval (DFI), hormone receptor status, HER2, site of metastases and molecular-targeted therapies, the hazard ratio for PFS and for OS in the cases increased from 2.347 to 3.090 and from 1.874 to 2.147, respectively. This occurred in spite of the higher proportion of controls (82% vs 7.1%) treated with aromatase inhibitors (AIs), while selective oestrogen receptor modulators (SERMs) were given to 92.9% of the cases and to 18% of the control group (p < 0.0001). Immunotherapy significantly prolonged PFS and OS during conventional first-line HT. A multi-centre randomised clinical trial is advised to enter this immunotherapy into clinical practice. KEY MESSAGES: • Acquired resistance occurs in metastatic endocrine-dependent breast cancer patients. • Interferon beta-interleukin-2 immunotherapy added to first-line salvage hormone therapy prolonged progression-free (PFS) and overall (OS) survivals in 26 patients of a pilot study as compared with 30 historical controls. • In this 2:1 ratio case-control prospective observational study, the PFS median time was significantly longer in the study group than that in controls, 33.1 (95% CI 24.5-41.8) vs 18 (95% CI 12.1-23.8) months (p < 0.0001). • Also, the OS median time was significantly longer in the study group, 81 vs 62 (95% CI 48.1-75.9) months (p < 0.0029).

Treatment of Metastatic or High-Risk Solid Cancer Patients by Targeting the Immune System and/or Tumor Burden: Six Cases Reports.

This article summarizes the histories of six patients with different solid tumors treated with a new strategy based on tumor burden reduction and immune evasion as potential targets. All six patients were at a high risk of relapse and were likely to have a minimal residual disease following conventional therapy: biochemical recurrence (BCR) following radical prostatectomy (RP) (two prostate cancers patients), removal of distant metastases (one colorectal and one breast cancer), and complete response (CR) of distant metastases to conventional therapy (one breast cancer and one esophageal-gastric junction cancer). Four of the patients, two after RP and BCR, one after removal of a single pulmonary metastasis from breast cancer, and one after CR to chemotherapy of peritoneal metastases and ascites from an esophageal-gastric junction primary cancer, regularly received cycles of a new drug schedule with the aim of inhibiting immune suppression (IT). In these four patients, preliminary laboratory tests of peripheral blood suggested an interleukin (IL)-2/IL-12 mediated stimulation of cellular immune response with a concomitant decrease in vascular endothelial growth factor (VEGF) immune suppression. The fifth case was a breast cancer patient with distant metastases in CR, while receiving beta-interferon and interleukin-2 in addition to conventional hormone therapy. To date, all five patients are alive and doing well and they have been unexpectedly disease-free for 201 and 78 months following BCR, 28 months following the removal of a single pulmonary metastases, 32 months following CR to chemotherapy of peritoneal metastases and ascites, and 140 months following diagnosis of multiple bone metastases, respectively. The sixth patient, who had colorectal cancer and multiple synchronous liver metastases and underwent nine surgical interventions for metastatic disease, although not disease-free, is doing well 98 months after primary surgery. Our six cases reports can be interpreted with the hypothesis that immune manipulation and/or a concomitant low tumor burden favored their clinical outcome.

The Use of Immunotherapy to Treat Metastatic Breast Cancer.

This article reviews the principal attempts of immune-modulation or immune therapy in metastatic breast cancer. It considers their rationale and reports on results from the relevant key clinical trials. Immune-modulatory or immune-stimulating cytokines used alone or combined with conventional therapies is among the principal approaches of immune manipulation in breast cancer. As this issue has recently been reviewed by us, the aim of the current article is to discuss our updated and unpublished data on this topic. Overall survival in luminal (28 patients) and non-luminal (9 patients) molecular subtypes is 91 and 59 months respectively that is about two and half or three times longer than expected. Thereafter, we focus on monoclonal antibodies (mAb) based-therapies including novel strategies to overcome resistance to anti-HER2 mAb. The main vaccine platforms in different molecular subtypes and immune therapies in triple negative metastatic breast cancer (m-TNBC) are discussed in the last sections. Some phase III investigations have already changed the current clinical practice. In fact, pertuzumab plus trastuzumab and docetaxel is the recommended first line regimen in HER2 positive locally recurrent or metastatic breast cancer and bevacizumab plus paclitaxel or docetaxel is a reasonable option for m-TNBC. In some other observational or phase I/II studies on first-line trastuzumab plus chemotherapy and hormonal therapy and in that on HER2 peptide/protein vaccines promising although preliminary findings have been reported to be further validated. In the remaining studies, results were disappointing. In the future, finding new predictive biomarkers and exploring more suitable synergizing combinations, time and dose-dependent-scheduled sequences of currently and further investigated immunological approaches are main challenges.

An individual reference limit of the serum CEA-TPA-CA 15-3 tumor marker panel in the surveillance of asymptomatic women following surgery for primary breast cancer.

PURPOSE: The purpose of this study was to evaluate the combined measurement of serum CEA, TPA, and CA 15-3, using an individual reference limit (IRL), for predicting distant metastases in asymptomatic women following a diagnosis of primary breast cancer. METHODS: A total of 231 patients were followed up for a mean of 5.5±1.6 years. An IRL for defining critical changes (CCs) in marker levels was used as a warning signal of pending distant metastases. RESULTS: Sensitivity, specificity, and accuracy of the combined CEA-TPA-CA 15-3 marker panel for predicting patient outcome were 95.2%, 97.8%, and 97.9%, respectively. In all, 19 (8.3%) patients relapsed with a mean lead time to radiological evidence of metastases of 11.7±13.8 months. CONCLUSION: We concluded that the combined measurement of CA 15-3, CEA, and TPA using an IRL for determining the CC in markers levels is an accurate strategy for predicting outcome during postoperative monitoring of asymptomatic breast cancer patients. Whether the early prediction of metastasis and subsequent administration of therapy impacts on patient outcome should now be the objective of a prospective clinical trial. The marker panel described here could serve as the basis for such a trial.

Tumour growth and immune evasion as targets for a new strategy in advanced cancer

It has become clearer that advanced cancer, especially advanced breast cancer, is an entirely displayed pathological system that is much more complex than previously considered. However, the direct relationship between tumour growth and immune evasion can represent a general rule governing the pathological cancer system from the initial cancer cells to when the system is entirely displayed. Accordingly, a refined pathobiological model and a novel therapeutic strategy are proposed. The novel therapeutic strategy is based on therapeutically induced conditions (undetectable tumour burden and/or a prolonged tumour 'resting state'), which enable an efficacious immune response in advanced breast and other types of solid cancers.

Alterations of Signaling Pathways Related to the Immune System in Breast Cancer: New Perspectives in Patient Management.

In recent years, immune manipulation for cancer treatment, including breast cancer, has been increasingly gaining consent, and many attempts have been made, mainly by either strengthening the immune response (IR) or by inhibiting immune evasion. Therefore, elucidating the related mechanisms is of importance due to the potential to improve the management of cancer patients by immunotherapy. This review article summarized some recent experimental studies, which have discovered novel alterations of signaling pathways related to the immune system in breast cancer. These altered signaling pathways have been grouped according to the general biological mechanism involved: tumor-initiating cells (TICs), cancer stem cells (CSCs), immune evasion, tumor growth and progression, prediction of clinical outcome and prediction of response, or resistance to chemotherapy. These altered pathways related to the immune system open clinical opportunities for the prognosis or treatment of patients. Many of these pathways are related to the origin of breast cancer and immune evasion. We recommended development of new drugs which act on these molecular pathways, and the designing of clinical trials to be carried out mainly in breast cancer patients who required adjuvant treatment.

Recent Advances in Comprehending the Signaling Pathways Involved in the Progression of Breast Cancer.

This review describes recent advances in the comprehension of signaling pathways involved in breast cancer progression. Calcium sensing receptor (CaSR), caveolae signaling, signaling referred to hypoxia-inducing factors and disturbances in the apoptotic machinery are related to more general biological mechanisms and are considered first. The others refer to signaling pathways of more specific biological mechanisms, namely the heparin/heparin-sulfate interactome, over-expression of miRNA-378a-5p, restriction of luminal and basal epithelial cells, fatty-acid synthesis, molecular pathways related to epithelial to mesenchimal transition (EMT), HER-2/neu gene amplification and protein expression, and the expression of other members of the epithelial growth factor receptor family. This progress in basic research is fundamental to foster the ongoing efforts that use the new genotyping technologies, and aim at defining new prognostic and predictive biomarkers for a better personalized management of breast cancer disease.

Immunotherapy and Hormone-therapy in Metastatic Breast Cancer: A Review and an Update.

Historically, antiestrogen is the first targeted therapy used in breast cancer treatment. In fact, its rationale lies in the molecular pathways elucidated by basic research. In estrogen receptor (ER)-alpha positive metastatic breast cancer patients, hormone-therapy remains the first option of treatment. While tamoxifen concomitant with suppression of ovarian function with luteinizing hormone releasing hormone (LHRH) agonists is the standard first line treatment in premenopausal, third generation aromatase inhibitors (AIs) are the first line standard hormone therapy in postmenopausal. However, the development of acquired resistance during antiestrogen therapy continues to be a central clinical problem. This review provides an update on the antiestrogen action and report on immunological treatment of the advanced disease by some cytokines. Interleukin-2, interleukin-12 and interferons used alone or in combination demonstrated an anti-tumor action directly and/or through synergism with antiestrogens. A rationale for the addition of interferon-beta and interleukin-2 to antiestrogens is described. Furthermore, we summarize and interpret the clinical and laboratory data of a recent long-term hormone- immunotherapy study in metastatic endocrine dependent breast cancer patients. Prospective randomized trials are necessary to confirm some recent promising results based on an immunological approach in addition to antiestrogens to overcome or delay acquired hormone resistance.

An individual reference limit for 'early' diagnosis of metastatic breast cancer during postoperative follow-up.

AIMS: This study is a clinical pilot study with the principal aim to investigate the accuracy of a panel of serum tumor markers for the early diagnosis of relapses. We propose a systematic use of serum CEA-TPA-CA15.3 tumor marker panel and criteria in order to make it an accurate tool for a postoperative breast cancer monitoring. MATERIALS & METHODS: 204 disease free breast cancer patients after mastectomy were intensively monitored with serial serum determination of CEA, CA15.3 and TPA. RESULTS: During a mean follow-up of 3.7 years the sensitivity of the CEA-TPA-CA15.3 tumor marker panel was 93%, the specificity was 97.6% and the rate of false 'warning signals' per year of follow-up was 9 per 100 patients. CONCLUSIONS: Our results show that the proposed tool is promising for a postoperative monitoring of breast cancer patients.

Clinical and laboratory patterns during immune stimulation in hormone responsive metastatic breast cancer.

This study clarifies the relationship between clinical and laboratory patterns, in endocrine-responsive metastatic breast cancer patients treated with a cyclic beta-interferon and interleukin-2 sequence added to anti-estrogens. In 31 patients, a regular laboratory and immunological assessment was made. During clinical benefit, as opposed to progression, a significant increase in the total number of lymphocytes, CD4+, CD8+, NK cells, CRP and IL-12 was confirmed. Also, a significant CEA, TPA, CA15.3 decrease occurred 24-72h after interleukin-2 administration. At the progression, both basally and after interleukin-2 stimulation, the mean values of CD4+ plus CD25+ cells were more than twice higher than during clinical benefit, with a decrease of CD4+ plus CD8+ (Teffector)/CD4+CD25+ (Treg) ratio. Moreover, a significant increase for CEA and for all 3 markers (standardized values) was found 24-72h after interleukin-2 administration. In patients who survived less than 5years, the Treg cell increase occurred at a significantly shorter time interval than in those who survived longer than 5years (20 vs 45.5months, respectively; P=0.001). These data show laboratory evidence of the effect of immunotherapy as well as that of hormone resistance occuring concomitantly with a laboratory pattern compatible with immune inhibition.

Bridging hypoxia, inflammation and estrogen receptors in thyroid cancer progression.

Thyroid cancer is a common endocrine-related cancer with a higher incidence in women than in men. Thyroid tumors are classified on the basis of their histopathology as papillary, follicular, medullary, and undifferentiated or anaplastic. Epidemiological and in vitro or in vivo studies have suggested a correlation between incidence of thyroid malignancies and hormones. In particular, growing evidence indicates a role of estrogens and estrogen receptors (ERs) in thyroid tumorigenesis, reprogramming and progression. In this scenario, estrogens are hypothesized to contribute to the observed female predominance of thyroid cancer in reproductive years. However, the precise contribution of estrogens in thyroid proliferative disease initiation and progression is not well understood. HIF-1α and NF-κB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression and resistance to chemotherapy. In fact, HIF-1α and NF-κB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic and differentiation reprogramming, inflammatory-reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement, they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7 and some TLRs are activated by HIF-1α; and that, in turn, alarmin receptors strongly activate NF-κB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these inhibitors are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors to achieve a reduction of metastasis formation and, more importantly, a net increase in survival. This review highlights the central role of HIF-1α activated in hypoxic regions of the tumor, of NF-κB activation and proinflammatory gene expression in transformed thyroid cells to understand their progression toward malignancy. The role of ER-α will be underlined, considering also its role in reprogramming cancer cells.

Malnutrition, anorexia and cachexia in cancer patients: A mini-review on pathogenesis and treatment.

Malnutrition, anorexia and cachexia are a common finding in cancer patients. They become more evident with tumor growth and spread. However, the mechanisms by which they are sustained often arise early in the history of cancer. For malnutrition, these mechanisms can involve primary tumor or damage by specific treatment such as anticancer therapies (surgery, chemotherapy, radiotherapy) also in cancers that usually are not directly responsible for nutritional and metabolic status alterations (i.e. bone tumors). For anorexia, meal-related neural or hormonal signals and humoral signals related to body fat or energy storage and the interaction of these signals with the hypothalamus or the hypothalamic inappropriate response play a pathogenetic role. Some cytokines are probably involved in these mechanisms. For cachexia, the production of proinflammatory cytokines by tumour cells is the initial mechanism; the main biochemical mechanisms involved include the ubiquitine proteasome-dependent proteolysis and heat shock proteins. Treatment includes pharmaceutical and nutritional interventions.

Targeted therapies of metastatic breast cancer: relationships with cancer stem cells.

In the last years, many targeted agents have been developed for metastatic breast cancer (MBC) treatment and are being tested in clinical trials. In spite of this, apart from epidermal growth factor receptor 2 (HER2) positive subset, no significant increase in the median overall survival (OS) has been reported. Similarly to conventional chemo- and radiotherapy, the cancer stem cell theory has been evoked to explain the frustrating results often obtained with this emerging category of drugs. This review examines the results in MBC of the approved targeted therapies or those currently under evaluation in experimental studies or in clinical trials, in the light of their relationships with breast CSCs and of the efforts to circumvent the development of resistance. In the next, there is the principal need to investigate if the effects on CSCs may be used to overcome cancer resistance and it will be opportune to consider whether molecular targeted therapies should be used alone or combined with conventional therapy, or with a different target drug specific for CSCs.

Testosterone and cardiovascular risk.

Cardiovascular (CV) disease is one of the most common causes of death in the western populations and, nowadays, its incidence is increasing even in the developing countries; although CV disease affects both sexes, it is more frequent in males in whom it shortens the average life expectancy. In this regard, this difference has been wrongly attributed for many years to the negative effects of testosterone (T); however, nowadays, a large amount of evidence suggests that this hormone may have protective effects on the CV system and that, indeed, the low levels of T could be associated with an increased CV risk and with an augmentation of morbidity and mortality in males. Such an aspect gains great relevance in light of the consideration that T decrease, besides occurring as a consequence of rare pathological conditions, can often take place with natural aging, causing a state of "male menopause", also called late-onset hypogonadism. In this review, we aimed to summarize the present state of the art concerning the association between T deficit and CV disease by analyzing the protective role of T on CV system and the relationship of this hormonal lack with metabolic syndrome, CV morbidity and mortality, and with the CV complications, such as ischemic heart disease, heart failure and stroke, that frequently occur in T deficiency.

Effect of physical activity on heart rate variability and carotid intima-media thickness in older people.

We investigated the effect of physical activity on heart rate variability (HRV) and carotid intima-media thickness (IMT) in elderly subjects and the relationship between HRV and IMT. Thirty-two elderly sedentary subjects and 32 age-matched endurance athletes underwent ultrasonography of the carotid wall for measuring IMT, and 24-h ECG monitoring for measuring HRV. Elderly athletes had evidence of increased vagal activity in the time (SDANN, rMSSD, and pNN50; p < 0.01) and frequency domain (HF and LF/HF ratio, p < 0.01) with respect to sedentary subjects. Moreover, athletes showed lower IMT than control subjects (p < 0.01). In the whole population SDNN was inversely related to IMT, respectively (r = -0.60 and r = -0.58, p < 0.0001), while LF/HF ratio related positively to IMT. In conclusion, this study demonstrated that in aging HRV is negatively associated with IMT, a putative index of atherosclerosis, confirming cardiac autonomic neuropathy as part of the pathophysiological pathway for atherosclerosis. It confirms that the regular physical activity represents a valuable strategy to counter age-related impairments of cardiac autonomic activity and artery structural changes.

Subclinical hyperthyroidism and cardiovascular manifestations: a reevaluation of the association.

Subclinical hyperthyroidism (SH) has been reported associated with atrial fibrillation (AF), heart failure (HF) and coronary heart disease events, including mortality. An expert opinion indicates that AF is the possible link between SH and the other important cardiovascular (CV) manifestations. We analyzed the data of three recent studies including 60,883 subjects of whom 2,284 SH patients. In these subjects, the ratio between the AF events and each of the other above reported CV events varied from 0.14 to 0.4 in SH and from 0.2 to 2.4 in euthyroidism (ET). The general pattern of this ratio in 6 comparisons performed was not significantly higher for SH than ET. This data suggest that AF is not the major link between SH and the related CV manifestations. We suggest that a further link to be considered is the higher frequency of the early atherosclerosis manifestations such as carotid intima media thickness or carotid integrated back scatter, observed in SH. This atherogenic effect of SH can affect the occurrence of all the above clinical CV manifestations.

Sirtuins: the molecular basis of beneficial effects of physical activity.

The research of the last decade highlighted the existence of a family of genes activated by cellular stresses that allow the cells to reactivate defense and repair activities regardless of age. The prolonged activation of these genes enhances the organism health and lifespan. Members of this gene family are called sirtuins (SIRT). The founding member of the SIRT protein family, Sir2 is a limiting component of yeast longevity. Many members of this family have been also identified as key longevity regulators in species ranging from yeast to fly. On the other hand, the role of SIRTs in the regulation of mammalian ageing has been questioned. While SIRTs' effects on lifespan are still a matter of scientific debate, the beneficial effects of SIRTs in terms of physical health and quality of aging are widely accepted. Increasing evidence suggests a pivotal role for SIRTs in mediating the adaptive response to physical exercise. The following review summarizes the knowledge so far acquired on sirtuins' role in mediating beneficial effects of physical exercise. In particular, the first paragraph gives an overture on mammalian sirtuins defining their localization, function when possible, and substrates. In the second paragraph, we discuss recent data regarding alteration of sirtuins expression and activity after physical exercise collected by our laboratory and others'.