The prognostic significance of peritumoral tertiary lymphoid structures in breast cancer.

Tumors and their surrounding area represent spatially organized "ecosystems", where tumor cells and the immune contextures of the different compartments are in a dynamic interplay, with potential clinical impact. Here, we aimed to investigate the prognostic significance of peritumoral tertiary lymphoid structures (TLS) either alone or jointly with the intratumoral densities and spatial distribution of CD8 + and CD163 + cells in breast cancer (BCa) patients. TLS were identified peritumorally, within the area distancing up to 5 mm from the infiltrative tumor border, counted and further characterized as adjacent or distal, in formalin-fixed, paraffin-embedded tumor tissue samples from a cohort of 167 patients, with histologically confirmed invasive ductal BCa. TLS and tumor-infiltrating immune cells were determined by H&E and immunohistochemistry. Clinical follow-up was available for 112 of these patients. Patients with peritumoral TLS exhibited worse disease-free survival (DFS) and overall survival (OS) as compared to patients lacking TLS. Moreover, the density of peritumoral TLS was found to be crucial for prognosis, since patients with abundant TLS exhibited the worst DFS and OS. By combining the density of adjacent TLS (aTLS) with our recently published intratumoral signatures based on the differential distribution of CD8 + and CD163 + in the tumor center and invasive margin, we created two improved immune signatures with superior prognostic strength and higher patient population coverage. Our observations strengthen the notion for the fundamental role of the dynamic interplay between the immune cells within the tumor microenvironment (center/invasive margin) and the tumor surrounding area (peritumoral TLS) on the clinical outcome of BCa patients.

Serum miRNA-based distinct clusters define three groups of breast cancer patients with different clinicopathological and immune characteristics.

Breast cancer (BCa) is a heterogeneous disease with different histological, prognostic and clinical aspects. Therefore, the need for identification of novel biomarkers for diagnosis, prognosis and monitoring of disease, as well as treatment outcome prediction remains at the forefront of research. The search for circulating elements, obtainable by simple peripheral blood withdrawal, which may serve as possible biomarkers, constitutes still a challenge. In the present study, we have evaluated the expression of 6 circulating miRNAs, (miR-16, miR-21, miR-23α, miR-146α, miR-155 and miR-181α), in operable BCa patients, with non-metastatic, invasive ductal carcinoma, not receiving neoadjuvant chemotherapy. These miRNAs, known to be involved in both tumor cell progression and immune pathways regulation, were analyzed in relation to circulating cytokines, tumor immune-cell infiltration and established prognostic clinicopathological characteristics. We have identified three different clusters, with overall low (C1), moderate (C2) or high (C3) expression levels of these six circulating miRNAs, which define three distinct groups of non-metastatic BCa patients characterized by different clinicopathological and immune-related characteristics, with possibly different clinical outcomes. Our data provide the proof-of-principle to support the notion that, up- or down-regulation of the same circulating miRNA may reflect different prognosis in BCa. Nonetheless, the prognostic and/or predictive potential of these three "signatures" needs to be further evaluated in larger cohorts of BCa patients with an, at least, 5-year clinical follow-up.

Peripheral T cell responses to tumour antigens are associated with molecular, immunogenetic and cellular features of breast cancer patients.

PURPOSE: Breast cancer is a leading cause of cancer deaths in women, but despite steady improvements in therapies, treatment is still suboptimal. Immunotherapy holds promise as a more effective therapy for breast cancer; supporting this, our prior study showed that patients possessing HER2-reactive CD8+ T cells in blood experience survival superior to patients without these cells. Here, we define a composite set of biomarkers that identify patients with T cell responses to tumour antigens. METHODS: We assessed T cell responses following in vitro stimulation with the HER2, MUC1 and SUR tumour-associated antigens (TAA) by flow cytometry and intracellular cytokine staining in 50 breast cancer patients. We also measured HLA type, serum cytokines, tumour-infiltrating leukocytes and blood leukocyte populations. RESULTS: We found few correlations between TAA-reactive T cells and HLA type, serum cytokines and tumour-infiltrating leukocytes, whereas blood leukocyte phenotypes broadly correlated with TAA responses. This showed monocytes, natural killer cells, dendritic cells and T cells to be inversely associated with both CD4+ and CD8+ T cells reactive to tumour antigens. Moreover, combining multiple parameters improved the accuracy in predicting patients with TAA-responsive T cells. CONCLUSION: This study therefore defines composite immune profiles that identify patients responding to TAAs which may allow better personalisation of cancer therapies.

CD4+CD25+ regulatory T-cell frequency in HER-2/neu (HER)-positive and HER-negative advanced-stage breast cancer patients.

PURPOSE: CD4(+)CD25(bright) regulatory T cells (Tregs) are increased in patients with several malignancies and correlate with disease stage and prognosis. Breast cancer patients represent a heterogeneous population with unpredictable disease progression even at advanced stages. Circulating Tregs in correlation with HER-2/neu (HER) status and treatment with chemotherapy, either alone or in combination with trastuzumab therapy, were monitored in advanced-stage breast cancer patients. EXPERIMENTAL DESIGN: Circulating Treg frequency and absolute counts of 46 HER(+) and 28 HER(-), stage III and IV, breast cancer patients before therapy and during trastuzumab therapy and/or chemotherapy have been compared with 24 healthy donors and correlated with plasma HER extracellular domain concentration and clinical outcome. RESULTS: Treg frequency in HER(+) patients was significantly increased compared with both HER(-) patients and healthy donors. Trastuzumab therapy, with or without combined chemotherapy, resulted in a progressive decrease of circulating Tregs. Percentage change in Tregs statistically correlated with percentage change in plasma HER extracellular domain. Furthermore, decrease in Tregs correlated with either objective clinical response or stable disease, whereas increased Treg frequency during trastuzumab therapy coincided with disease progression. No statistically significant change in Treg frequency following chemotherapy was observed in HER(-) patients. CONCLUSIONS: Treg cell frequency does not directly correlate with clinical stage in breast cancer, as stage III and IV HER(+) and HER(-) patients exhibit significantly different Treg profiles. Trastuzumab therapy, either alone or combined with chemotherapy, results in decreased Treg frequency in HER(+) advanced patients with an objective clinical response.

Increased frequency of CD4+ cells expressing CD161 in cancer patients.

PURPOSE: Although the function of natural killer receptors on T cells infiltrating tumors and their potential effect on antitumor immunity has been investigated, little is known about T cells expressing NKR-P1A (CD161) in cancer patients. In the present study, we examined T cells expressing CD161 in the peripheral blood, the tumor tissue and in malignant effusions of patients with several types of malignancies. EXPERIMENTAL DESIGN: Expression of CD161 in CD4(+) or CD8(+) (lacking CD56) T cells isolated from peripheral blood (n = 61), tumor specimens (n = 8), and malignant effusions (n = 37) of cancer patients was examined using four-color flow cytometry. Proliferative capacity and cytokine production of purified CD4(+)CD161(+)CD56(-) cells were studied after weak or strong stimulation, with or without costimulation, in the presence or absence of interleukin 2. The possible regulatory function of activated CD4(+)CD161(+)CD56(-) cells on T-cell alloresponses was also investigated. RESULTS: CD4(+) cells expressing CD161 were increased in cancer patients, compared with healthy individuals. This increase in the peripheral blood of cancer patients positively correlated with disease stage and was augmented at the tumor site. Phenotypic analysis revealed that CD4(+)CD161(+) cells are memory T cells, with low expression of activation markers. CD4(+)CD161(+) cells play an immunoregulatory role through cytokine production, because upon receiving costimulatory signals via CD28, they exert suppressive activity on autologous peripheral blood mononuclear cell alloresponses. CONCLUSIONS: CD4(+)CD161(+)CD56(-) cells represent a distinct memory T-cell population significantly increased in cancer patients. Depending on the type of signals provided by the tumor microenvironment, CD4(+)CD161(+) cells may regulate the immune response.

Differential intratumoral distributions of CD8 and CD163 immune cells as prognostic biomarkers in breast cancer.

BACKGROUND: Tumor immune cell infiltrates are essential in hindering cancer progression and may complement the TNM classification. CD8+ and CD163+ cells have prognostic impact in breast cancer but their spatial heterogeneity has not been extensively explored in this type of cancer. Here, their potential as prognostic biomarkers was evaluated, depending on their combined densities in the tumor center (TC) and the tumor invasive margin (IM). METHODS: CD8+ and CD163+ cells were quantified by immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples from a cohort totaling 162 patients with histologically-confirmed primary invasive non-metastatic ductal breast cancer diagnosed between 2000 and 2015. Clinical follow-up (median 6.9 years) was available for 97 of these patients. RESULTS: Differential densities of CD8+ and CD163+ cells in the combined TC and IM compartments (i.e., high(H)/low(L), respectively for CD8+ cells and the reverse L/H combination for CD163+ cells) were found to have significant prognostic value for survival, and allowed better patient stratification than TNM stage, tumor size, lymph node invasion and histological grade. The combined evaluation of CD8+ and CD163+ cell densities jointly in TC and IM further improves prediction of clinical outcomes based on disease-free and overall survival. Patients having the favorable immune signatures had favorable clinical outcomes despite poor clinicopathological parameters. CONCLUSIONS: Given the important roles of CD8+ and CD163+ cells in regulating opposing immune circuits, adding an assessment of their differential densities to the prognostic biomarker armamentarium in breast cancer would be valuable. Larger validation studies are necessary to confirm these findings. TRIAL REGISTRATIONS: Study code: IRB-ID 6079/448/10-6-13 Date of approval: 10/06/2013 Retrospective study (2000-2010) First patient prospectively enrolled 14/2/2014.

Erratum to: Differential intratumoral distributions of CD8 and CD163 immune cells as prognostic biomarkers in breast cancer.

[This corrects the article DOI: 10.1186/s40425-017-0240-7.].

Ii-Key/HER-2/neu(776-790) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/neu+ tumors.

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.

Immunogenic HER-2/neu peptides as tumor vaccines.

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination.

Effect of IL-21 on NK cells derived from different umbilical cord blood populations.

IL-21 plays a role in the proliferation and maturation of NK cells developed from hematopoietic stem cells. In this study, we found that IL-21, in the presence of physiological concentration of hydrocortisone (HC), has a significant impact on the functions of NK cells derived from umbilical cord blood (CB) populations. We demonstrate that IL-21, in combination with Flt3-ligand, IL-15 and HC, induces high proliferative responses and, apart from enhancing NK-mediated cytotoxicity, it also induces a significant increase in lymphokine-activated killer activity of CB/CD34+-derived CD56+ cells. In addition, IL-21 induced changes in the CD56+ cell cytokine secretion profile. Thus, we observed increased levels of IL-10 and granulocyte macrophage colony-stimulating factor, whereas tumor necrosis factor-alpha levels decreased. IFN-gamma production was also modified by IL-21, depending on the presence or absence of IL-18. CB/CD34+ cells did not express the IL-21R ex vivo, but receptor expression was induced during their commitment to differentiation into CD56+ cells. Our data ascribe to IL-21 an essential role on NK cell development and function under conditions similar to the in vivo CB microenvironment.

Beneficial effect of short-term exposure of human NK cells to IL15/IL12 and IL15/IL18 on cell apoptosis and function.

Monokines IL12, IL15, and IL18 have been shown to activate NK cell function, however with high apoptosis induced by their combination within 48 h. Here, we demonstrate for the first time that CD56+ cells incubated for only 18 h with the combination of IL15/IL12 or IL15/IL18, then washed, and further cultured in plain medium, exhibit low levels of apoptosis. These shortly activated CD56+ cells show high killer activity against NK- and LAK-sensitive tumor targets that persists over a culture period of 18 days after two additional 6 h cycles of exposure to the monokines applied every 8 days and also retain their ability for high cytokine production during each exposure. Moreover, these repetitive short-term exposures of CD56+ cells to the monokine combinations result in long-lived CD56+ cells with slower rates of FcgammaRIII receptor (CD16) decline, therefore exhibiting higher antibody depended cytotoxicity, as opposed to the continuous incubation with the monokine combinations. In conclusion, short-term exposure of CD56+ cells to IL15/IL12 or IL15/IL18 at 8-day intervals may hold a promise for improved clinical results in cellular adoptive cancer immunotherapy and for the in vivo injections of the monokines.

A potential role for hydrocortisone in the positive regulation of IL-15-activated NK-cell proliferation and survival.

Although glucocorticoids (GCs) have been described as acting mainly as anti-inflammatory and immunosuppressive drugs, they may also positively influence the immune system. In the present study, we demonstrate for the first time that hydrocortisone (HC), in synergy with interleukin-15 (IL-15), induces a dramatic increase in the expansion of peripheral blood-derived CD56+ cells, favoring the preferential outgrowth of classical natural killer (CD56+CD3- NK) over CD56+CD3+ natural killer T (NKT) cells. HC plus IL-15-driven CD56+ cells exhibited an increased potential for cytokine production with no impairment in their NK- and lymphokine-activated killer (LAK) activities. Elevated levels of GC-induced leucine zipper protein (GILZ) messenger RNA (mRNA) were detected in both NK and NKT cells cultured with HC and IL-15, in comparison to IL-15 alone. Phosphorylation status of signal transducer and activator of transcription 5 (STAT5) was not affected by the presence of HC in either of the populations. On the contrary, HC differentially affected the IL-2/IL-15R beta- and gamma-chain surface expression and the phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) in IL-15-activated NK and NKT cells. Our data ascribe a novel role to GCs on mature NK-cell expansion and function and open new perspectives for their use in cellular adoptive cancer immunotherapy.

Immunobiology of HER-2/neu oncoprotein and its potential application in cancer immunotherapy.

HER-2/neu (also known as HER2 or c-erb-B2) is a 185-kDa protein receptor with tyrosine kinase activity and extensive homology to the epidermal growth factor (EGF) receptor. HER-2/neu is expressed in many epithelial tumors and known to be overexpressed in approximately 20-25% of all ovarian and breast cancers, 35-45% of all pancreatic adenocarcinomas, and up to 90% of colorectal carcinomas. HER-2/neu overexpression represents a marker of poor prognosis. HER-2/neu-positive tumor cells are potentially good targets for tumor-reactive cytotoxic T lymphocytes which have been utilized in immunotherapeutic trials. In addition, the "humanized" monoclonal antibody Herceptin has been tested in several clinical trials and proved to be an effective adjuvant therapy for HER-2/neu-positive breast and ovarian cancers. Vaccinations aiming at generating T-cell responses are being examined in both experimental and clinical trials. Natural immunity at the level of T and B cells has been observed in patients with HER-2/neu-positive tumors confirming the immunogenicity of HER-2/neu and encouraging vaccination trials with HER-2 protein-derived subunits or synthetic peptides. This review summarizes recent data from patients with various types of HER-2/neu-overexpressing cancers carrying different HLA alleles and exhibiting preexistent immunity to HER-2/neu-derived synthetic peptides. It also discusses potential advantages of the various vaccination approaches to immunotherapy targeting the HER-2/neu molecule.

Generation of human tumor-specific CTLs in HLA-A2.1-transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors.

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu+ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201+ HER-2/neu+ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu+ tumor cell lines (also including the original HER-2/neu+ primary tumor cells from which the ACEs were derived) in an HLA-A*0201-restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201+ HER-2/ neu+ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/ neu peptides HER-2 (9(369)) and HER-2 (9(435)) demonstrating the immunodominance of these epitopes. HER-2 peptide-specific CTLs generated in the HLA-A*0201-transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu+ human tumor cell lines in an HLA-A*0201-restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide-specific CTLs (i.e., HER-2 [9(369)] or HER-2 [9(435)]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu+ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu+ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu+ malignancies.

HER-2/neu-derived peptide 884-899 is expressed by human breast, colorectal and pancreatic adenocarcinomas and is recognized by in-vitro-induced specific CD4(+) T cell clones.

HER-2/neu peptides recognized in the context of HLA-DR molecules by CD4(+) Th lymphocytes on antigen-presenting cells have been identified. In this report, we demonstrate for the first time that HER-2/neu helper epitopes are also expressed on the surface of metastatic breast, colorectal and pancreatic carcinomas. Peripheral blood mononuclear cells from an HLA-DR4 healthy donor were used to induce HER-2/neu peptide-specific CD4(+) T cell clones by in vitro immunization with HER-2/neu peptide (884-899)-pulsed autologous dendritic cells (DCs). Strong proliferation and significant levels of IFN-gamma were induced by the CD4(+) T cell clones in response to specific stimulation with autologous DCs loaded with HER-2(884-899). Furthermore, these clones also recognized HER-2/neu(+) tumor cell lines, and tumor cells from breast, colorectal and pancreatic adenocarcinomas induced to express HLA-DR4, but also the HLA-DR4(+) melanoma cell line FM3 transfected to express HER-2/neu. The recognition of tumor cells was strongly inhibited by an anti-HLA-DR mAb. Taken altogether, we provide novel information for the role of HER-2(884-899) as a naturally processed epitope expressed by breast, colorectal and pancreatic carcinomas and the capacity of HER-2/neu protein to follow the endogenous class II processing pathway. Our results suggest that HER-2(884-899) might be attractive for broadly applicable vaccines and may prove useful for adoptive immunotherapy designed for breast, colorectal and pancreatic carcinomas.