Evaluation of Cyclin D1 expression by western blotting methods and immunohistochemistry in breast cancer patients.

PURPOSE: Considering that cyclin D1 had a prognostic and clinical value for breast cancer patients, adequate measurement of cyclin D1 is necessary. METHODS: In this investigation, we detect cyclin D1 expression in tumour and peritumoral tissue of breast cancer patients by Western blotting method and by immunohistochemistry. RESULTS: Cyclin D1 expression decreased significantly with each advanced clinical stage of disease and tumour size. Also, patients without lymph node involvement, with positive hormone receptors and Luminal A type of tumours had significantly increased the expression of cyclin D1. We show that cyclin D1 expression correlates with longer RFS in the entire group of patients, in the group of ER-positive and in the group of HER2-negative patients. Patients who were both ER and cyclin D1 positive had a better prognosis. CONCLUSION: Taken together, our results, showing correlation of cyclin D1 with clinical stage, tumour size and lymph nodes, suggest that cyclin D1 expression, detected by Western blotting, could be considered as an additional marker for the staging of breast cancer, as well as a marker for longer RFS and survival in ER-positive breast cancer patients.

Interleukin-4-induced natural killer cell antitumor activity in metastatic melanoma patients.

NK cells are important effectors of innate immunity that mount the first line of defense toward tumor growth. Interleukin-4 (IL-4) has recently been shown to regulate NK cell function, although its role in the regulation of NK cell function in cancer patients has not been clarified. The aim of this study was to investigate the effect of IL-4 on the function and the receptor characteristics of CD16-defined NK cells and their cytotoxic CD16bright and regulatory CD16dim subsets. Peripheral blood lymphocytes obtained from 36 metastatic melanoma (MM) patients treated for 18 h with 10 ng/mL IL-4 were evaluated for NK cell cytotoxicity using the radioactive 51chromium release assay. Expression of the activating receptors NKG2D and CD161, as well as the inhibitory receptors CD158a and CD158b, was analyzed on CD3-CD16+ NK cells and their subsets by flow cytometry. IL-4 induced significant in vitro enhancement of NK cell activity, as well as increased expression of the CD107a degranulation marker, by CD3-CD16dim NK cells. NKG2D expression was also increased on CD3-CD16+ cells by IL-4 with no alteration of the expression of CD161 and inhibitory KIR receptors. Although in vitro treatment with IL-4 increased both the expression of NKG2D and the cytotoxicity of NK cells, it had no detectable effect on the transcription of the TGF-ß gene in NK cells of MM patients. The IL-4-induced NK cell cytotoxicity and increased activating NKG2D receptor expression may indicate an important antitumor effect of IL-4 with a potential application for immunotherapy of MM patients.

IL-2 And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma Patients.

Regional lymph nodes (LN)s represent important immunological barriers in spreading of malignant tumors. However, they are the most frequent early metastatic site in melanoma. Immunomodulatory agents including cytokines have been included in therapy of melanoma and have shown severe side effects and toxicity. In this sense, there is a growing need for bringing these agents to further in vitro testing that may enlighten aspects of their regional application. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, the two cytokines with similar immune-enhancing effects, on the expression of activating NKG2D, inhibitory CD158a and CD158b receptors on CD8+ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients. In this study, we showed significant effects of IL-2 and IL-15 cytokine treatments on the expression of activating NKG2D and on inhibitory CD158a and CD158b receptors on lymphocytes, CD8+ T, NKT-like and NK cell lymphocyte subsets originating from regional LNs of melanoma patients. Furthermore, IL-2 and IL-15 by inducing the expression of NKG2D activating receptor on innate and on adaptive lymphocyte subsets and by augmenting NK cell antitumor cytotoxicity that correlated with the cytokine-induced NKG2D expression, increased antitumor potential of immune cells in regional LNs of melanoma patients irrespective of LN involvement. These findings indicate the importance of immune cell population from regional LNs of melanoma patients in the development of immune intervention strategies that may if applied locally increase antitumor potential to the level that controls tumor progressions.

The role of cytokines in the regulation of NK cells in the tumor environment.

Natural killer (NK) cells are innate lymphoid cells that are important effectors in the first line of defense toward transformed cells. This is mediated both by direct cytotoxic mechanisms and by production of immunoregulatory cytokines. Recent evidence has shown that NK cells also display memory, similar to the cells of the adaptive immune system. Cytokines are pivotal for the maturation, activation and survival of NK cells. Interleukins (IL)-2, IL-12, IL-15, IL-18, IL-21 and type I interferons positively regulate NK cell function, either independently or in cooperation, whereas other cytokines, such as IL-23 and IL-27, may enhance or suppress NK cell function depending on the context. In the tumor microenvironment, TGFß, IL-10 and IL-6 suppress NK cell activity not only directly, but also indirectly, by affecting immunosuppressive cells and by antagonizing the effect of stimulatory cytokines, thereby dampening the antitumor response of NK cells and promoting subsequent tumor evasion and progression. Increased understanding of the NK cell response to cytokines has provided a better understanding of their impaired function in tumors which may aid in the development of novel immunotherapeutic strategies to enhance NK cell responses in cancer patients.

pSTAT3 expression associated with survival and mammographic density of breast cancer patients.

BACKGROUND: Constitutive activation of STAT3 have been shown in several tumor types including breast cancer. We investigate STAT3 expresion as possible molecular marker for breast cancer early detection, as well as prognostic factor for determination of tumor agressiveness. METHODS: In this study we measure p(Y705)STAT3 expression in tumor and adjacent tissue of breast cancer patients by Western blot. For relapse-free survival (RFS) and overall survival (OS) we used Log-Rank test. RESULTS: We show that average expression of p (Y705) STAT3 in tumor tissue is higher compared to adjacent tissue. Moreover, we found that patients with HER2 positive receptors had significantly higher pSTAT3 expression compared to HER2 negative patients. We showed that patients with high mammographic density had significantly higher tumor expression of pSTAT3 compared to patients with low mammographic density. Also, we show that pSTAT3 expression correlates with longer RFS in the entire group of patients, as well as in the group of ER positive, in lymph node positive and in older group of breast cancer patients (with age over 50). Furthermore, in the entire group of patients, in ER positive, in lymph node positive and in older group of patient, high expression of pSTAT3 showed a better survival than low expression of pSTAT3. CONCLUSION: Considering that the expression of pSTAT3 is associated with longer RFS and survival, it can be used as prognostic tools for determination of group of breast cancer patients with low-risk.

Effect of cytokines on NK cell activity and activating receptor expression in high-risk cutaneous melanoma patients.

OBJECTIVE: Stage II melanoma patients have high risk for regional and distant metastases and may benefit from novel therapeutic strategies. To clarify the role of NK cells in Stage II melanoma, we characterized the cytotoxic activity of NK cells and the expression of various activating and inhibitory receptors in high-risk cutaneous melanoma patients (Stages IIB and IIC) compared to low-risk patients (Stage IA). MATERIALS AND METHODS: Native and cytokine-treated peripheral blood mononuclear cells were used for functional and phenotypical analyses. RESULTS: Compared to Stage IA-B patients, Stage IIB-C patients showed significantly decreased NK cell activity, as well as decreased expression of the activating NKG2D and CD161 receptors, most likely due to increased serum levels of the immunosuppressive cytokine TGF-ß1 in these patients. Interestingly, treatment of periperal blood mononuclear cells with IFN-α, IL-2, IL-12 or the combination of IL-12 and IL-18 significantly induced NK cell activity for both groups of melanoma patients. However, only low-risk patients had a significant increase in the expression of the NKG2D receptor after in vitro treatment with IFN-α, as well as an significant increase in the expression of CD161 after treatment with IFN-α or IL-12. Although IL-2 induced the expression of NKG2D in both groups of patients, this increase was significantly lower in high-risk melanoma. CONCLUSION: NK cell parameters may be useful as biomarkers of disease progression in localized melanoma patients. Our results further suggest that the use of NK cell-activating cytokines in combination with inhibitors of immunosuppressive factors like TGF-ß1 could be a therapeutic option for the treatment of high-risk cutaneous melanoma patients.

Evaluation of the Functional Capacity of NK Cells of Melanoma Patients in an In Vitro Model of NK Cell Contact with K562 and FemX Tumor Cell Lines.

NK cells of metastatic melanoma (MM) patients display impaired function, making them incapable to mount an effective antitumor response. In this study, we evaluated immunophenotypic characteristics and functional capacity of CD3-CD16+ NK cells of MM patients in an in vitro model based on NK cell contact with an NK sensitive, K562, and a tumor-specific, melanoma FemX tumor cell line. Although our results indicate similar NK cell antitumor cytotoxic potential of MM patients in contact with both cell lines based on the expression of CD107a degranulation marker, there is a discrepancy in NK cell IFNγ production, as it is not significantly induced by FemX tumor cells, found to be, contrary to K562, HLA class I positive. Furthermore, we show NKG2D receptor downregulation by K562 tumor cell line, only. This may result from the obtained higher gene expression of TGFß and VEGFA growth factors in K562 tumor cells that can negatively regulate NKG2D expression. Additionally, aside from postcontact downmodulation of activating CD16 receptor, there are no significant changes in the expression of CD161, CD158a, and CD158b NK cell receptors. Therefore, the applied in vitro model shows that, compared to the full NK cell functional capacity of MM patients displayed in a tumor-sensitive setting represented by contact with K562 cells, tumor-specific melanoma setting provided by FemX tumor cells leads to reduced NK functional potential. The obtained insight into NK cell capacity may be of use for evaluation of the state of disease and can help in selecting effective immunotherapeutic agents for MM patients.

Attenuated in vitro effects of IFN-α, IL-2 and IL-12 on functional and receptor characteristics of peripheral blood lymphocytes in metastatic melanoma patients.

Considering tumor-induced suppression of lymphocytes the aim of this study was to investigate in vitro effects of IFN-α, IL-2, IL-12 and IL-18 as immunomodulating agents on the functional and receptor characteristics of peripheral blood lymphocytes (PBL) in metastatic melanoma (MM) patients compared to healthy controls (HC). In HC IFN-α, IL-2 and IL-12 enhanced mRNA level of perforin by inducing pSTAT-1 and pSTAT-5 signaling molecules. Additionally, the expression of NKG2D activating receptor and its DAP10 signaling molecule was upregulated by IL-2. Contrary to this, in MM patients only IL-2 by upregulating pSTAT-5 increased perforin-mediated cytotoxicity of lymphocytes. Furthermore, there was significantly negative correlation between the percentage of CD4+CD25bright+CD27+ regulatory T (Treg) cells and NK cell cytotoxicity, as well as the expression of NKG2D receptor on PBL in HC and MM patients. Therefore, the absence of IL-2 effect on the increase of NKG2D/DAP10 level in MM patients could be the consequence of the increased percentage of immunosuppressive CD4+CD25bright+CD27+ cells after this cytokine treatment in patients. However, in MM IL-12 significantly decreases the percentage of these inhibitory cells. Although IL-2 as a single agent has numerous side effects, it remains the important cytokine for PBL activation in melanoma immunotherapy. Additionally, the removal of Treg cells from patient PBL by IL-12 before in vitro stimulation with IL-2, may lead to the generation of more potent cytotoxic lymphocytes against tumor cells. Therefore, lymphocyte based therapy for MM patients should integrate not only the choice of appropriate immunostimulatory cytokine, but also the removal of inhibitory cells from tumor microenvironment.

Beneficial in-vitro effects of interleukin-2, interleukin-12, and their combination on functional and receptor characteristics of natural killer cells in metastatic melanoma patients with normal serum lactate dehydrogenase levels.

Considering tumor-mediated suppression of natural killer (NK) cells, the aim of this study was to investigate the in-vitro effects of interleukin (IL)-2 and IL-12, as immunostimulatory cytokines, on the functional and receptor characteristics of NK cells and their subsets in healthy control (HC) and metastatic melanoma (MM) patients. Peripheral blood mononuclear cells of 27 HC and 35 MM patients were stimulated in vitro with IL-2, IL-12, and their combination for functional and phenotypic analysis. IL-2, IL-12, and primarily their combination, significantly induced NK cell activity, CD107a degranulation marker, and perforin expression in NK cells and their subsets in HC and MM patients. Furthermore, the combination of IL-2 and IL-12 was significantly more efficient than IL-12 alone in the augmentation of NK cell cytotoxicity and CD107a expression. Also, IL-2 and IL-12 reciprocally upregulated each other's receptors, IL-2Rα and IL-12Rß1/ß2, on NK cells and their subsets in MM and HCs. In addition, the priming of NK cells with IL-2 before IL-12 treatment led to an increase in the expression of both IL-12 receptors. In contrast to IL-12, IL-2 increased activating NKG2D and DNAM-1, as well as inhibitory CD158a and CD158b KIRs. In addition, the cytokines investigated exerted a more potent effect on the increase in NK cell activity and the expression of various NK cell receptors in MM patients with normal lactate dehydrogenase (LDH) serum levels. Therefore, serum LDH could represent a predictor of response to cytokine immunotherapy in MM patients. The optimization of combined IL-2/IL-12 therapy is needed to enhance NK cell functions in MM patients stratified by their LDH levels.

Decreased CD161 activating and increased CD158a inhibitory receptor expression on NK cells underlies impaired NK cell cytotoxicity in patients with multiple myeloma.

AIM: As innate immune cells natural killer (NK), NK-like T and CTLγδ are important in antitumour response in multiple myeloma (MM), the aim of this study was to investigate some functional and phenotypical characteristics of these cells in MM. METHODS: 29 patients with MM prior to therapy, in clinical stage I-III and 15 healthy controls (HCs) were investigated. Percent of immune cells in peripheral blood, NK cell activity, expression of activating (CD161) and inhibitory (CD158a, CD158b) NK cell receptors on CD3-CD16+ NK cells were evaluated using 51-chromium-release assay and by flow cytometry. Production of interleukin (IL) 2 and tumour necrosis factor (TNF)α was analysed in supernatants from in vitro activated peripheral blood mononuclear cells. RESULTS: In patients with MM the percent of NK cells and their two subsets did not differ from controls, while NK-like T and CTLγδ cells were significantly decreased. Significant impairment of NK cell cytotoxicity, CD107a expression and interferon γ intracellular level was also shown. There was a significant decrease in CD161 and an increase in CD158a receptor expression on NK cells in these patients. Also IL-2 production was lowest in clinical stage III. However, TNF-α production did not differ between patients and HCs. CONCLUSIONS: Altered expression of CD161 activating and CD158a KIR inhibitory receptor is responsible for impaired antitumour activity of NK cells in MM patients. These new biomarkers may be helpful for patient selection for immunotherapy with cytokines, and novel KIR blocking monoclonal antibodies that enhance NK cell antimyeloma activity and provide clinical benefit.

Decreased expression of pSTAT, IRF-1 and DAP10 signalling molecules in peripheral blood lymphocytes of patients with metastatic melanoma.

AIMS: As numerous signalling molecules regulate effector functions of peripheral blood lymphocytes (PBLs) that have an important anti-tumour activity, the aim of this study was to analyse their level in patients with metastatic melanoma (MM) compared with healthy controls (HCs). METHODS: Peripheral blood mononuclear cells (PBMCs) of 36 MMs and 28 HCs were analysed for the level of perforin, interferon-regulating transcription factor-1 (IRF-1), DAP10 and Src homology 2 domain-containing tyrosine phosphatase-1 by reverse transcriptase PCR, level of phosphorylated signal transducers and activators of transcription (pSTAT)-1, pSTAT-4, pSTAT-5 by western blot and interferon (IFN)-γ production by ELISA. The expression of activating NKG2D and inhibitory killer immunoglobulin-like receptors (KIR), CD158a and CD158b, on PBL, CD3-CD56+ natural killer (NK) cells and CD3+CD8+ cytotoxic T lymphocytes (CTLs), as well as the percentage of CD14+HLA-DR- cells in PBMC were estimated by flow cytometry. RESULTS: Patients with MM, compared with HCs, had significantly lower level of cytotoxic molecule perforin and decreased IFN-γ production, as well as lower level of pSTAT-1, pSTAT-4, pSTAT-5 and IRF-1 signalling molecules in PBMC. Furthermore, MM had decreased expression of activating NKG2D receptor on PBL and NK cells and low level of its DAP10 signalling molecule contrary to no changes in KIR expression on all investigated cells. These results could be associated with increased percentage of immunosuppressive CD14+HLA-DR- myeloid-derived suppressor cells detected in patients with MM. CONCLUSIONS: The altered signalling molecules of PBL could represent biomarkers of impaired cytotoxic and immunoregulatory function of these cells, indicating melanoma-associated immunosuppression that facilitates tumour progression.

Natural killer cell receptors: alterations and therapeutic targeting in malignancies.

Numerous newly identified activating and inhibitory NK cell receptors and their engagement by cognate ligands on target tumor cells regulate NK cell antitumor activity. Alterations in NK cell receptor expression and signaling underlie diminished cytotoxic NK cell function. Cytokines, IFN-α, IL-2, IL-12, IL-15 and IL-18, applied systemically and for ex vivo activation and expansion of NK cells have improved NK cell antitumor activity by increasing the expression of NK cell activating receptors and by inducing cytotoxic effector molecules. Moreover, it has been recognized that classical and novel pharmacological agents upregulate cognate ligands for activating receptors on tumor cells and provide better NK cell antitumor response. Some other immunotherapeutic approaches in cancer in the setting of donor-recipient KIR/HLA mismatch have evolved with the aim to potentiate NK cell activity in allogeneic hematopoietic stem cell transplantation that lead to beneficial graft vs. tumor effect. Therefore, better understanding of NK cell activating and inhibitory receptor biology is needed to assist in developing novel approaches to effectively manipulate NK cells and create effective NK cell-based immunotherapy for treatment of cancer patients.

The Actual Role of LDH as Tumor Marker, Biochemical and Clinical Aspects.

Lactate dehydrogenase (LDH) among many biochemical parameters represents a very valuable enzyme in patients with cancer with possibility for easy routine measurement in many clinical laboratories. Previous studies where mostly based on investigated LDH in serum of patients with cancer with aims to estimate their clinical significance. The new directions in investigation of LDH where based on the principle that tumor cells release intracellular enzymes trough damaged cell membrane, that is mostly consequence in intracellular mitochondrial machinery alteration, and apoptosis deregulation. This consideration can be used not only in-vitro assays, but also in respect to clinical characteristics of tumor patients. Based on new techniques of molecular biology it is shown that intracellular characteristics of LDH enzyme are very sensitive indicators of the cellular metabolic state, aerobic or anaerobic direction of glycolysis, activation status and malignant transformation. Using different molecular analyses it is very useful to analyzed intracellular LDH activity in different cell line and tumor tissues obtained from patients, not only to understanding complexity in cancer biochemistry but also in early clinical diagnosis. Based on understandings of the LDH altered metabolism, new therapy option is created with aims to blocking certain metabolic pathways and stop tumors growth.

Decreased Interferon γ Production in CD3+ and CD3- CD56+ Lymphocyte Subsets in Metastatic Regional Lymph Nodes of Melanoma Patients.

As lymphogenic dissemination is very common in melanoma, regional lymph nodes (LN)s represent first immunological barriers to tumor invasion and play a complex role in antitumor immune defense. In this sense, their most prominent role is the presentation of tumor-derived antigens to naïve T cells and generation of cell-mediated adaptive immune response. Since tumor micro-environment affects immune cell function in this study we have evaluated the ability of T cells and NK cells in metastatic (involved) and non-metastatic regional LNs to produce interferon γ (IFNγ), a pleiotropic cytokine that regulates adaptive antitumor immune response. Our results show reduced IFNγ production in both T and NK lymphocyte subsets and decreased prevalence of T cells in metastatic regional LNs of melanoma patients. The decrease of IFNγ production in T cells was more pronounced with increased number of involved regional LNs indicating tumor-induced functional impairment of both T and NK cell lymphocyte subsets in involved regional LNs. Therefore, shown low IFNγ production in metastatic LNs may represent an obstacle in adaptive cell-mediated antitumor immune response and hence may enable tumor progression.

Favorable in vitro effects of combined IL-12 and IL-18 treatment on NK cell cytotoxicity and CD25 receptor expression in metastatic melanoma patients.

BACKGROUND: As IL-12 and IL-18 have important immunostimulatory role the aim of this study was to investigate their in vitro effects on functional and receptor characteristics of NK cells and their subsets in healthy controls (HC) and metastatic melanoma patients (MM). METHODS: Peripheral blood mononuclear cells (PBMC) of HC and MM were stimulated with culture medium alone, medium supplemented with IL-12 (10 ng/ml), IL-18 (100 ng/ml) and their combination. NK cell activity was determined using radioactive cytotoxicity assay, while perforin, CD107a and pSTAT-4 expression, IFN-γ production and the expression of NKG2D, DNAM-1, CD161, CD158a/b, CD25, IL-12R beta 1/2 receptors on CD3(-)CD56(+) NK cells and their CD3(-)CD56(dim+) and CD3(-)CD56(bright+) subsets were analyzed by flow cytometry. Cytokine induced level of DAP10 in PBMC was analyzed by reverse transcription polymerase chain reaction. RESULTS: IL-12 alone or in combination with IL-18 significantly induced NK cell activity and CD107a degranulation marker expression in MM and HC, while IL-18 alone did not have any effect in patients. The combination of IL-12 and IL-18 significantly increased mean fluorescence intensity (MFI) of IFN-γ in all NK cell subsets in HC and only in the bright subset in MM. MM that belong to M1c group with metastasis in liver and increased LDH serum values had significantly lower increase in NK cell cytotoxicity after combined IL-12 and IL-18 treatment compared to the patients in M1a and M1b categories. These results could be explained by decreased IL-12R expression and lower increase in pSTAT-4 and perforin expression in NK cells of M1c patients after IL-12 and combined IL-12 and IL-18 treatment. IL-18 alone significantly decreased NKG2D receptor expression and level of DAP10 signaling molecule in MM, while combined IL-12 and IL-18 increased the expression of CD25 on all NK cell subsets in HC and MM. Additionally, MM that belong to M1a + M1b group had significantly higher increase in CD25 receptor expression compared to the patients in M1c group. CONCLUSIONS: The novel data obtained in this study support the use of IL-12 and IL-18 in combination for developing new therapeutic strategies for metastatic melanoma especially for patients with better survival rate and prognosis.

In-vitro activation of natural killer cells from regional lymph nodes of melanoma patients with interleukin-2 and interleukin-15.

Regional lymph nodes (LNs) represent the first barrier in lymphogenic tumor dissemination in melanoma. Natural killer (NK) cells, the effector cell subpopulation of the innate immune system, are in the first line of antitumor immune defense. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, two cytokines with similar immune-enhancing effects, on antitumor cytotoxic function and immunophenotype of NK cells from regional LNs of melanoma patients. Mononuclear cells purified from regional LNs of 50 melanoma patients in clinical stage II-IV were treated in vitro for 72 h and 7 days with 200 IU/ml rhIL-2 and 25 ng/ml IL-15 at 37°C in 5% CO2. Both cytokines significantly augmented NK cell cytotoxic activity, transcription of the cytotoxic molecule perforin, and the level of functionally mature perforin in both nonmetastatic and metastatic regional LNs. IL-2 treatment increased the percentage of CD3CD56 NK cells by increasing the CD56 NK cell subset in both nonmetastatic and metastatic LNs, whereas IL-15 treatment did not affect the percentage of NK cells and their subsets. Both cytokines increased on NK cells from nonmetastatic and metastatic LNs the expression of CD69 early activation antigen, the NKG2D activating receptor, as well as CD16 and inhibitory killer-cell immunoglobulin-like receptor CD158b, both inherent to the mature and the cytotoxic NK cell phenotype. In conclusion, our data may indicate the therapeutic potential of the NK cell population from regional LNs either as immunotherapeutic targets or as adoptively transferred after activation with IL-2 or IL-15.

HER2-positive breast cancer patients: correlation between mammographic and pathological findings.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancers represent a highly aggressive breast cancer subtype and are associated with a worse prognosis. This study was designed to investigate the mammography finding of HER2-positive breast cancer and to compare the results with the characteristics of HER2-negative breast cancer patients. From January 2010 to October 2011, mammography findings of 65 patients with pathologically confirmed HER2-positive breast cancers (n = 22) or HER2-negative breast cancers (n = 43) were retrospectively reviewed. The authors also reviewed pathological reports for information on the histological type and differentiation grade. Among the two types of breast cancer patients, estrogen receptor-negative/PR-negative/HER2-positive breast cancer patients most commonly had associated calcifications (18 of 22) on mammography. On mammography, cases with a cluster of calcifications usually were presented as pleomorphic calcifications (12 of 20) and branching calcifications (4 of 20). Patients with HER2-positive breast cancers showed a histological grade II. HER2-positive breast cancer patients usually had ductal invasive carcinoma (17 of 22). Moreover, postmenopausal patients showed a significantly higher frequency of HER2-positive tumours. Our results suggest that the imaging findings might be useful in diagnosing HER2-positive breast cancer patients.

Decreased expression of NKG2D, NKp46, DNAM-1 receptors, and intracellular perforin and STAT-1 effector molecules in NK cells and their dim and bright subsets in metastatic melanoma patients.

Although natural killer (NK) cells play an important antitumor role, melanoma cells may affect their effector functions. In this study, we analyzed the expression of various receptors and effector molecules in NK cells and their subsets in metastatic melanoma (MM) patients compared with healthy controls (HCs). In HC and MM patients, we analyzed NK cell activity using a chromium release assay and the expression of CD107a degranulation marker, activating NKG2D, NKp46, DNAM-1, and inhibitory CD158a and CD158b receptors, IL-12R beta 1, IL-12R beta 2, intracellular interferon (IFN)-γ, perforin, and STAT-1 in CD3-CD56+ NK cells, and cytotoxic CD3-CD56 and immunoregulatory CD3-CD56 subsets by flow cytometry. MM patients compared with HC not only had significantly decreased NK cell activity, lower expression of CD107a, and impaired IFN-γ production but also had decreased expression of activating NKG2D, NKp46, and DNAM-1 receptors, which was followed by lower expression of perforin, STAT-1, and both IL-12R subunits in NK cells. In MM patients only, there was a positive correlation between NKG2D expression and degranulation capacity, as well as IFN-γ production in NK cells. Analysis of the expression of various parameters of NK cell effector functions between MM patients with different localization of distant metastases showed that patients in the unfavorable M1c subclass had decreased expression of NKG2D and NKp46 on NK cells compared with patients in the M1a+b group. Downregulated NKG2D, NKp46, and DNAM-1 receptors associated with impaired NK cell effector function are important biomarkers of advanced disease with a poor prognosis in melanoma patients.

Values of MMP-2 and MMP-9 in tumor tissue of basal-like breast cancer patients.

Gelatinase A (MMP-2) and gelatinase B (MMP-9) are proteolytic enzymes involved in process of tumor invasion, and they are considered as possible tumor markers in breast cancer patients. In this study, we measured activity of latent and active form of MMP-2 and MMP-9 in tumor and adjacent tissue of 60 breast cancer patients by SDS-PAGE zymography. The activity of both form of gelatinases significantly increased with each advancing clinical stage of disease. ProMMP-9 and aMMP-9 activity in tumor tissue shows a positive association with tumor size. Patients with lymph node involvement have higher proMMP-2, aMMP-2 and aMMP-9 activity than node negative patients. Steroid receptor-negative tumors had enhanced aMMP-2 and aMMP-9 activity. Patients with basal-like cancers had higher proMMP-2 tumor activity and aMMP-2 adjacent tissue activity compared to patients with luminal A tumors. Patients with negative hormone receptors are associated with increased activity of both form of gelatinases in adjacent tissue. Reported increased activity of MMP-2 in tumor and adjacent tissue of basal-like tumors implicates that MMP-2 might have a role in aggressive biology of basal-like cancers. Additional investigations regarding molecular pathways in adjacent tissue could give better insight into aggressive nature of basal-like carcinomas.

Distribution of several activating and inhibitory receptors on CD3(-)CD56(+) NK cells in regional lymph nodes of melanoma patients.

BACKGROUND: Natural killer (NK) cells, as the main effector subpopulation of the innate immune system, play an important role in the control of the rise and spread of malignant tumors. Regional lymph nodes (LN) represent the first immunologic barrier to tumor metastasis. Since there are scarce data on NK cells from regional LN of cancer patients, the aim of this study was to investigate the expression of several activating and inhibitory receptors on the entire NK cell population as well as their CD3(-)CD56(dim) and CD3(-)CD56(bright) functional NK subsets from regional LN of melanoma patients. MATERIALS AND METHODS: Mononuclear cells were isolated from 50 regional LN of melanoma patients. The expression of several receptors on NK cells and their functional subsets was analyzed by flow cytometry. RESULTS: We show increased percentages of CD3(-)CD56(+) NK cells in involved LN compared with uninvolved LN, mostly in favor of the CD56(dim) NK cell subset. NK cells in involved LN express similar levels of activating receptor NKG2D, while the level of another activating receptor, CD16, is increased compared with uninvolved LN. Regarding the expression of inhibitory NK cell receptors, we show increased CD158b, but similar low CD158a, inhibitory killer Ig-like cell receptor expression in involved LN compared with uninvolved LN. Furthermore, NK cells in involved compared with uninvolved LN displayed increased CD69 early activation antigen expression. CONCLUSIONS: Our results indicate that with tumor infiltration into regional LN of melanoma patients, NK cells, mostly of the CD56(dim) subset, are recruited into draining LN. The invading NK cells show counterbalance of the increased expression of CD16 activating receptor and increased CD158b inhibitory killer Ig-like cell receptor.