Cancer is the second leading cause of death after cardiovascular disease. Considering the entire population, and taking care of non-melanoma skin cancers, the most frequent tumors are those of the udder and rectum, followed by that of the lung and prostate and in the case of skin cancers, the five most frequently diagnosed cancers among men are prostate cancer, lung cancer, colorectal cancer, bladder cancer, kidney cancer, and urinary tract cancer; among women, breast cancer, colorectal cancer, lung cancer, thyroid cancer, and cervical cancer. In cancer patients, there is a serious impairment of the immune system, as cancer cells develop mechanisms to evade immuno-surveillance (Smyth et al., 2001). Immunotherapy has been developed that uses the patient’s immune system, or certain components stimulating the body’s immune system made it more effective in recognizing and destroying cancer cells. This strategy is based on the fact that inflammatory and immune cells play a role in different cancers.

Oncology immunotherapy can be divided into active and passive immunotherapy. Active immunotherapy mainly includes cancer vaccines based on dendritic cells and T-cell transfer: these treatment strategies modify the patient’s dendritic cells or T-cells, and then re-infused and the same later; passive, instead, including monoclonal antibodies, checkpoint inhibitors, cytokines, and bispecific T-cell engagers to influence T-cell proliferation, angiogenesis, and pathways to activation. Inflammatory responses with the involvement of the cell infiltrates can be expected in human breast cancer patients under treatment with anti-HER2 monoclonal antibodies (mabs) combined with chemotherapy. Cardiovascular, inflammatory, and immune-related reactions were associated with the use of anti-HER2 mabs alone or in combination with anthracycline and chemotherapy in interventional clinical trials requiring cardiologist intervention (Martin et al., 2009; Albini et al., 2010; Yousif and Al-Amran, 2011; Bowles et al., 2012; ElZarrad et al., 2013; Zamorano et al., 2016; Lobenwein et al., 2021). Among these, trastuzumab i.v.-s.c. (HERCEPTIN Roche), trastuzumab emtansine i.v. (KADCYLA Baxter), pertuzumab i.v. (PERJETA Roche), and trastuzumab deruxtecan i.-v. (ENHERTU Daiichi Sankyo Europe) cause adverse reactions in an elevated percentage of patients that increase as a function of age, comorbidities, and cancer progression (Bowles et al., 2012; Serrano et al., 2012). Case reports showed the first case of trastuzumab emtansine-associated pleural and pericardial effusions in a patient with breast cancer in the absence of other etiologies was reported that was associated with severe inflammatory reactions due to trastuzumab emtansine (Lombardi et al., 2021). A higher rate of peripheral neuropathy and liver disfunction was also reported following trastuzumab emtansine treatments in the patients that were associated with inflammatory reactions (Battisti et al., 2020).

Five trastuzumab biosimilars have been approved by the F.D.A. and European Medicines Agency: trastuzumab-dkst (Mylan GmbH, Steinhausen, Switzerland), trastuzumab-pkrb (Celltrion Inc., Incheon, Republic of Korea), trastuzumab-dttb (Samsung Bioepis Co., Ltd., Incheon, Republic of Korea), trastuzumab-qyyp (Pfizer Ireland Pharmaceuticals, Cork, Ireland) and trastuzumab-anns (Amgen Inc., Thousand Oaks, CA, United States) (Barbier et al., 2019; Miller and Schwartzberg, 2019).

The role of inflammatory cell infiltrate in cancer is emerging; T-lymphocytes have been associated with breast cancer (Macchetti et al., 2006; Kuroda et al., 2021), seminoma (Hadrup et al., 2006), melanoma (Taylor et al., 2007), colorectal (Galon et al., 2006), cervical (Piersma et al., 2007), and ovarian (Zhang et al., 2003) in human. Biomarkers of inflammation such as the neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR), are possible surrogate markers of outcome in various cancers including breast cancer with low NLR and PLR associated with better outcomes in different cancers (Hee Yeon et al., 2019; Al Jarroudi et al., 2021). Neutrophils play an important role in immune response since they affect both innate immunity and cell signaling in the adaptive immune response, and they are also a major factor in the suppression of cancer progression. Inflammation that begins with the passage of neutrophils from the circulation to tissues is an essential element not only for infections but also for immune response in cancer. Neutrophils act on the elements of the complement system and the adaptive immune system, thereby strengthening the inflammatory response (Hee Yeon et al., 2019; Kaytaz Tekyol et al., 2021). Platelets can also be involved in the inflammatory response by increasing angiogenesis or by providing growth factor release. Substances released from platelets are required for the activation of endothelial cells surrounding the vascular tract in inflammation. The presence of different subtype T lymphocytes in the tumor microenvironment (tumor-infiltrating lymphocytes) has also been associated with a positive prognosis in various malignancies including triple-negative breast cancer (Graziano et al., 2019; Hee Yeon et al., 2019; Kuroda et al., 2021). The role of neutrophils is however controversial; neutrophils have been associated with high breast cancer risk in a population-based case-control study conducted in Spain (Gago-Dominguez et al., 2018). Inflammatory cells have also an emerging role in canine breast cancer with some similarities with human disease in terms of inflammatory response and the cytotoxic role of the CD8⁺ lymphocyte (Carvalho et al., 2014); much less is known about the role of the CD20 + lymphocyte in canine breast cancer that represents a spontaneous animal model of disease. Mammary tumors are the most common neoplasms in female dogs as in humans. It is evident a greater frequency of this tumor in Europe than in the United States (Sorenmo, 2003). Age and race are risk factors in humans and canine breast cancer and the most prevalent case are hormone-related. The familial case were associated with BRCA-1 and BRCA-2 in human whose mutations predispose to the onset of breast and ovarian cancer and which are estimated to be responsible for about 80% of hereditary syndromes affecting these two organs and two-thirds of hereditary breast cancers alone in humans. Similarly, in dogs, these genes were associated with familial cases (Rivera et al., 2009). Despite several similarities with human disease, the incidence of this tumor is three times that in humans suggesting additional risk factors (Pastor et al., 2020).

Tumor-infiltrating lymphocytes, in particular CD8⁺ cells and CD20⁺ cells, were strongly associated with survival in different carcinomas (Nielsen et al., 2012; Edin et al., 2019; Hee Yeon et al., 2019; Kuroda et al., 2021) and canine glioma (Krane et al., 2021), but the role of CD20⁺ B cells was not reported in canine breast cancer. The CD20⁺ lymphocyte is proposed to play a role in the immune response against cancers in association with lymphocytes such as CD8⁺. In addition, inflammatory-based reactions data of these mabs from spontaneous reports and their correlation with the outcome are however poorly described.

In this work, we investigated the relationship between the inflammatory cells response, cancer grade, and progression in the canine breast cancer biopsies collected in our regional area for diagnostic purposes and in the unselected population (18–64 years.o.) under anti-HER2 treatments. The role of the inflammatory cells in breast cancer was validated using for the first time to our knowledge data extracted from the EudraVigilance database that contains reports of inflammatory and immune-related reactions (ADR) of patients under anti-HER2 treatments in the adult unselected population. These patients represent the most prevalent population affected by breast cancer.

The data were expressed as mean ± E.S. unless otherwise specified. The significance between data pairs was calculated by the paired t-student test. Significant differences of p ≤ 0.05 were taken into account. The ADR data extracted from EudraVigilance were collected and analyzed on Excel Software Microsoft.

The PRR was calculated using the following equation: a/ ( a + c ) /b/ ( b  +  d ) where a is the reaction of interest to a given drug of interest, b is the reaction of interest for all other drugs in the class, c are all other reactions to a given drug of interest, d all other reactions to all other drugs in the class (Evans et al., 2001). Signal definition: PRR ≥2, a minimum of three ratios/cases for the reaction of interest, X² ≥ 4. No signal is identified, if PRR is = 1. The duplication of a specific report has been evaluated manually and excluded by the analysis (Mele et al., 2014; Maqoud et al., 2021).

In this work, we investigated the role of inflammatory cells experimentally in a spontaneous canine model of breast cancer. The role of the inflammatory cells was also evaluated using data from the spontaneous report in the human database. Inflammatory and immune-related adverse reactions (ADR) in breast cancer were extracted from EudraVigilance in the unselected adult population (18–64 years.o.) under anti-HER2 treatments.

Trastuzumab i.v.-s.c. showed a low risk of cardiovascular and inflammatory reactions (PRR < 2) and a PRR ≥2 for loss of effectiveness, but unfavorable PRR values > 2 for immune system reactions and intolerances with drug withdrawal, and a PRR >7 for technological problems mostly related to the use of the i.v. device.

Trastuzumab emtansine showed a high risk within the mabs of loss of effectiveness with a PRR >2 and an unfavorable PRR >3 for inflammatory reactions with edema, skin reactions, and extravasation mostly due to the serious grade of reactions (PRR > 3), but a more favorable PRR values <1 for the cardiovascular and immune system within the mabs. The different safety profile of this mab vs. the trastuzumab i.v.-s.c. can be ascribed to the presence of the emtansine inducing inflammatory reactions.

Pertuzumab showed a low risk of loss of efficacy with a PRR <2, favorable PRR values for C.R.S., immunological and inflammatory reactions due to not serious grade of reactions (PRR < 1), and values <1 for cardiovascular and other reactions.

Inflammatory cells have a recognized role in canine breast cancer like in humans (Carvalho et al., 2014). We found that macrophages count was correlated with the grade of severity of the tumor in canine breast cancer and lymphocytes infiltration were observed in all tumor sections however showing an inverse correlation with the grade of severity of the tumor thereby increasing in density in the G1 supporting respectively the negative and positive prognostic role of these cells in breast cancer also in the dog. B lymphocytes do produce cytokines that support the T cell response promoting the CD8⁺ lymphocytes cytotoxic activity. The B cells may also act as antigen-presenting cells to T cells, and produce antibodies directed against tumor antigens. B lymphocyte-derived antibodies have been shown to recognize tumor antigens per se.

Inflammatory and immune-related adverse reactions were associated with the use of mabs with pertuzumab showing a low risk of loss of efficacy and C.R.S., immunological, inflammatory reactions and cardiovascular reactions, and low risk of reduced inflammatory cell counts. Inflammatory reactions with edema, skin reactions, and extravasation mostly due to serious grade of reactions and a high number of platelet count and lymphocyte count decrease were associated with trastuzumab emtansine that showed a high risk within the mabs of loss of effectiveness, but a favorable profile for the cardiovascular and immune system within the mabs. A case report showed the first case of trastuzumab emtansine-associated pleural and pericardial effusions in a patient with breast cancer in the absence of other etiologies (Lombardi et al., 2021), and higher rates of peripheral neuropathy and liver disfunction was also reported following trastuzumab emtansine treatments in the patient’s population (Battisti et al., 2020). Trastuzumab i.v.-s.c. showed however a low risk of cardiovascular and inflammatory reactions as well as of loss of effectiveness, but unfavorable risk profile for immune system reactions and intolerances and technological problems. This is associated with platelet count and neutrophil count decrease reports.

Trastuzumab i.v.-s.c. and trastuzumab emtansine respectively showed a high neutrophil count decrease reports and a high platelet count reports of serious grade in the absence of lymphocytes changes not associated with disease progression as by case report analysis thereby indicating a better outcome.

Analyzing, moreover, the ADR for all 4 drugs including the different trastuzumab i.v and s.c. formulations in the 2 years 2019–2020, among adults (18–64), we have noticed the absence of cases of myocarditis for trastuzumab i.v. and s.c., 2 cases of myocarditis for trastuzumab emtansine and pertuzumab, and no case of vasculitis for trastuzumab emtansine and 1 case for pertuzumab. Therefore, rare cases of immunological reactions affecting cardiovascular apparatus were reported with the anti-HER2 mabs in the 2 years of observation.

We further investigated the reactions that may also have an inflammatory pathophysiological basis reported for the s.c and i.v formulations of trastuzumab to further evaluate the contribution of the administration route, and we found that trastuzumab s.c. showed additional risk related to infection and infestation, musculoskeletal and connective tissue disorders with arthralgia, and psychiatric disorders. Clinical Trials (C.T.) showed no differences in terms of efficacy between the two formulations of trastuzumab but safety was in favor of i.v. formulation vs s.c. formulation to date with an unbalance of serious A.E. of the s.c. formulation (Ismael et al., 2012; Gligorov et al., 2017; Jackisch et al., 2019).

We found several similarities in our investigation using pharmacovigilance data with C.T. data; severe cases of arthralgia, asthenia, and fatigue were more frequently reported in the patients treated with the s.c. formulation of trastuzumab. Arthralgia, diarrhea, and fatigue were indeed the most commonly reported AEs in HER2-positive breast cancer patients under treatment with s.c trastuzumab in combination with chemotherapy (Ismael et al., 2012; Gligorov et al., 2017). In addition, we found an unbalance related to the infections and infestations again in favor of i.v formulation like in C.T. trials. No differences were found in the cardiovascular safety between the two formulations in our investigations in agreement with C.T. data. Differently from C.T. data, we did not find reports on anti-drug antibody production or PH-20 antibodies against the human hyaluronidase as instead demonstrated in the open-label Phase 3 HannaH trial (Jackisch et al., 2019).

We additionally found an imbalance of psychiatric disorders in the patients treated with the trastuzumab s.c. vs. the i.v. with concomitant use of antipsychotic and anxiolytic drugs that were not reported in C.T. that can be related with the use of a novel unknown formulation. Depressive disorders were on the other hand observed in these patient populations (Chang et al., 2015).

Despite these reports and the not favorable cost (Inotai et al., 2019), it is of note the fact that several patients have a great benefit from the trastuzumab s.c. the formulation in terms of quality of life and tolerance particularly those under long-term maintenance therapy that are not anymore exposed to chemotherapy. It is of note the fact that the FDA’s approved in 2020 following the FeDeriCa study (https://www.notiziariochimicofarmaceutico.it/2019/12/20/formulazione-sottocutanea-di-pertuzumab-e-trastuzumab-per-il-tumore-al-seno-her2-positivo) the combo pertuzumab + trastuzumab s.c. that is, now available.

Pharmacovigilance data combined with experimental data using a spontaneous animal model of disease sharing the same environment of human patients offered the advantage of evaluating the role of the inflammatory reactions and related cells in breast cancer patients under mabs treatment in uncontrolled conditions avoiding the possible bias of data selection. The finding that trastuzumab i.v-s.c. and trastuzumab emtansine showed, respectively, high neutrophil and platelet count decrease with no cancer progression in the absence of reports of lymphocyte decrease, and that a high density of CD20⁺ lymphocytes was observed in the less severe canine breast cancer samples in parallel with high neutrophil and platelet count decrease support the idea that these cells play a favorable role either in breast cancer in human and in this spontaneous model of disease.