Increased circulating TGF-ß1 is associated with impairment in NK cell effector functions in metastatic melanoma patients.

Transforming growth factor beta (TGF-ß) plays a complex role in carcinogenesis. In 30 melanoma patients and 20 healthy controls (HC) we analysed functional and phenotypic characteristics of NK cells by Flow cytometry, gene expression of TGF-ß1 in peripheral blood mononuclear cells by qPCR and serum and supernatant level of free TGF-ß1 by ELISA. Melanoma patients had significantly higher serum level of circulatingTGF-ß1 compared to HC, especially those with metastasis into the central nervous system (subclass M1d) and high LDH serum values. Melanoma patients compared to HC had significantly higher level of TGF-ß1 gene in PBMC. TGF-ß1 serum values negatively correlate with NK cell activity analysed by CD107a (degranulation marker), IFN-γ, NKG2D, and NKp46 in patients. Study shows the association of high level of TGF-ß1 with NK cell inhibition in patients represents the main mechanism of tumour immune evasion. Targeting TGF-ß may become an important cancer treatment for improving antitumor immunity.

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.

Ombrabulin plus cisplatin versus placebo plus cisplatin in patients with advanced soft-tissue sarcomas after failure of anthracycline and ifosfamide chemotherapy: a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Ombrabulin (AVE8062) disrupts the vasculature of established tumours and has shown preclinical synergistic anti-tumour activity when combined with cisplatin. In this phase 3 trial, we aimed to assess the efficacy and safety of ombrabulin plus cisplatin compared with placebo plus cisplatin in patients with advanced soft-tissue sarcomas. METHODS: We did this multinational, randomised, double-blind, placebo-controlled phase 3 study at 44 centres in ten countries. Patients aged 18 years and older with metastatic soft-tissue sarcomas, an Eastern Cooperative Oncology Group performance status of 0-2, and who had previously received treatment with anthracycline and ifosfamide were randomly assigned (1:1) to intravenous infusion of ombrabulin 25 mg/m(2) plus cisplatin 75 mg/m(2) or intravenous infusion of placebo plus cisplatin 75 mg/m(2) every 3 weeks. Patients were allocated to treatment using a permuted blocks randomisation scheme (block size of four) via an interactive voice-response system, and stratified by histological subtype. Patients, medical staff, study investigators, and individuals who handled and analysed the data were masked to treatment assignment. Our primary endpoint was median progression-free survival in the intention-to-treat population. Safety analyses were done on all randomised patients who received at least one dose of study drug. This trial is now closed, and is registered with ClinicalTrials.gov, number NCT00699517. FINDINGS: Between June 13, 2008, and April 26, 2012, we randomly assigned 355 patients to ombrabulin plus cisplatin (n=176) or placebo plus cisplatin (n=179). Median duration of follow-up was 27·9 (IQR 20·9-33·2) in the placebo group and 30·5 months (20·7-37·6) in the ombrabulin group. Progression-free survival was slightly, but significantly, improved in the ombrabulin group compared with the placebo group (median 1·54 months [95% CI 1·45-2·69] vs 1·41 [1·38-1·58] months; hazard ratio 0·76 [95% CI 0·59-0·98]; p=0·0302). Grade 3 or 4 adverse events occurred more frequently in individuals in the ombrabulin group than in those in the placebo group and included neutropenia (34 [19%] in the ombrabulin group vs 14 [8%] in the placebo group) and thrombocytopenia (15 [8%] vs six [3%] for placebo). Adverse events leading to death occurred in 18 patients in the ombrabulin group and 10 patients in the placebo group. INTERPRETATION: The combination of ombrabulin and cisplatin significantly improved progression-free survival; however, it did not show a sufficient clinical benefit in patients with advanced soft-tissue sarcomas to support its use as a therapeutic option. Predictive biomarkers are needed for the rational clinical development of tumour vascular-disrupting drugs for soft-tissue sarcomas. FUNDING: Sanofi.

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.

Circulating IL-6 is associated with disease progression in BRAFwt metastatic melanoma patients receiving anti-PD-1 therapy.

AIMS: Despite efficacy of anti-PD-1 blockade in treatment of metastatic melanoma (MM), many patients achieve rapid disease progression (DP). Therefore, the aim of this study is to better define biomarkers for DP by analysing levels of circulating cytokines TGF-ß, IFN-γ, IL-6, IL-8 and IL-10 in MM patients prior to anti-PD-1 therapy. METHODS: Cytokine levels were evaluated before therapy with pembrolizumab in peripheral blood of BRAF wild-type (wt) MM patients by ELISA method. RESULTS: In this study, we give pretherapy levels for circulating TGF-ß, IFN-γ, IL-6, IL-8 and IL-10 in BRAFwt MM patients and analyse them according to metastasis stage (M1a+M1 b, M1c, M1d groups), lactate dehydrogenase (LDH) level and occurrence of DP. Increased IL-6 level was found in M1d group (central nervous system metastasis), while LDH+patients (LDH ≥460 IU/L) have increased IL-6 and IL-8 values that correlate with LDH level. Also, IL-6 correlates with C reactive protein values. Furthermore, patients with DP have significantly higher IL-6 level compared with non-DP patients. Conversely, the other analysed cytokines are similar in investigated groups of MM patients. By receiver operating characteristics curve analysis, pretherapy IL-6 level was found to be a biomarker for the occurrence of DP with cut-off value of 3.02 pg/mL. Patients in M1d stage are prevalent in the group with IL-6 ≥3.02 pg/mL that is characterised with reduced progression-free survival and higher pretherapy IL-8 and LDH. CONCLUSION: The evidence in this study implies that baseline IL-6 could be a biomarker of DP and poor prognosis in BRAFwt MM patients treated with pembrolizumab.

Increased circulating monocyte MDSCs positively correlate with serum Interleukin-10 in metastatic melanoma patients.

Numerous immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and inhibitory cytokines identified in melanoma microenvironment have the important role in immune escape. Therefore, in this study we analyzed monocytic (m)MDSCs in peripheral blood of metastatic melanoma (MM) patients. In peripheral blood of 35 MM patients and 30 healthy controls we analyzed percentage of CD14 + HLA-DR- mMDSCs in monocyte gate and the mean fluorescence intensity of Foxp3 in CD25 + CD4 + regulatory T cells by Flow cytometry. Serum levels of transforming growth factor beta, interferon-gamma, interleukin (IL)-6, IL-8, IL-10 are measured by ELISA assays. In this study MM patients have significantly higher percentage of CD14 + HLA-DR- mMDSCs, as well as increased the baseline mMDSC/PBMC subset (NK, T, B cells, monocytes) ratio. Although there is no significant difference in the percentage of mMDSCs between groups of MM patients with different localization of distant metastasis, patients with elevated serum lactate dehydrogenase (LDH) have statistically significant higher percentage of these cells compared to LDH negative patients. Furthermore, in MM patients there is statistically significant positive correlation between values of IL-10 and the percentage of mMDSCs, only. Therefore, therapeutics that target circulating mMDSCs and IL-10 may have a big importance in the improvement of antitumor immunity in MM patients.

Real World Overall Survival of Patients With Metastatic Renal Cell Carcinoma Treated With Only Available Sunitinib and Pazopanib in First-Line Setting.

Background: The emerging new standard of care for metastatic clear cell renal carcinoma (mRCC) becomes a challenge when access to new drugs is limited. In Serbia, sunitinib and pazopanib are the only available first-line therapies. The second-line treatment for mRCC has never been and is still not available. We aimed to assess overall survival (OS) in patients with mRCC who received first-line sunitinib or pazopanib when access to second-line treatment was not available. Methods: This retrospective observational study analyzed data from a nationally representative cohort of 759 patients who started on first-line sunitinib or pazopanib between 1 January 2012 and 30 June 2019, in 4 centers in Serbia. The data cut-off date was 31 December 2019. Key eligibility criteria were clear cell RCC histology, measurable metastatic disease, performance status 0 or 1, and the Memorial Sloan Kettering Cancer Center favorable or intermediate prognosis. The primary outcome was OS from the start of first-line treatment to death or data cut-off date. Results: The study population included 759 patients with mRCC who started with first-line sunitinib (n = 673; [88.7%]) or pazopanib (n = 86; [11.3%]). Overall, the mean age was 61.0 ± 9.7 years at treatment baseline, and 547 (72%) were men. mRCC was primarily diagnosed in 230 (30%) patients, and most of them underwent cytoreductive nephrectomy prior to systemic therapy (n = 181 [79%]). Additional treatment of metastases prior to and/or during treatment was used in 169 patients (22.3%). Grade 3 and 4 adverse events occurred in 168 (22.1%) and 47 patients (6.2%), respectively, and treatment was permanently stopped because of toxicity in 41 (6.9%). The OS was calculated from the start of first-line treatment, and the median follow-up was 14 months (range, 0-97). The median OS in the entire cohort was 17 months (95% CI, 14.6-19.4). Conclusions: With only available sunitinib and pazopanib in first-line treatment, modest improvements are seen in the overall survival of patients with mRCC in real world clinical practice. In circumstances of limited availability of cancer medicines, our results can contribute to accelerating patient access to novel cancer therapies that have been shown to prolong survival in mRCC.

The stage dependent changes in NK cell activity and the expression of activating and inhibitory NK cell receptors in melanoma patients.

BACKGROUND: As NK cell antitumor activity is regulated by the balance between numerous activating and inhibitory receptors the aim of this study was to analyze the changes in these receptors expression as well as the differences in their association with NK cell activity in melanoma patients in different clinical stages. MATERIALS AND METHODS: Forty-three melanoma patients in the three different clinical stages and 17 healthy controls were analyzed for NK cell activity by the standard (51)Cr radioactive assay, as well as for the percentage and absolute number of CD3-CD56+ NK cells and their subsets and the expression of degranulation marker CD107a, activating NKG2D and CD161, and inhibitory KIR, CD158a, and CD158b receptors on CD3-CD56+ NK cells by flow cytometry. IFN-γ and TNF-α PBL production were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: We show that contrary to nonmetastatic, metastatic melanoma patients have significantly impaired NK cell activity, lower CD107a expression, as well as decreased production of IFN-γ and TNF-α. Furthermore, these melanoma patients have a significant decrease in the expression of activating NKG2D receptor, which positively correlates with NK cytotoxicity, and a significant increase in the expression of inhibitory CD158b on CD3-CD56+ NK cells compared with healthy controls. CONCLUSIONS: In this study, we show that metastatic melanoma patients have significant changes in NK cell activity and the expression of activating as well as inhibitory NK cell receptors. These results indicate that only in this patient group, the blocking of inhibitory or enhancing of activating NK cell receptor expression may promote NK cell-mediated cytolysis.

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.

Biomarkers of suppressed natural killer (NK) cell function in metastatic melanoma: decreased NKG2D and increased CD158a receptors on CD3-CD16+ NK cells.

In metastatic melanoma (MM) patients we evaluated natural killer (NK)-cell activity, distribution of several NK receptors and their correlation with NK function. Peripheral blood lymphocytes (PBL) of MM patients and controls were analysed for NK activity and expression of activating NKG2D, CD161 and KIR, CD158a and CD158b receptors on CD3-CD16+ NK cells. MM patients not only had significantly decreased NK activity and NK-cell interferon (IFN)-gamma production, a redistribution of NK-cell subsets with an increase in CD16(dim) and a reduction in CD16(bright) NK subsets. There was a decreased CD161 and NKG2D and an increased CD158a NK-cell expression in MM patients, with a positive correlation between NKG2D expression and NK cytotoxicity and an inverse correlation between CD158b expression and NK-cell cytotoxicity in patients. Furthermore, patients' CD3-CD16(bright) NK subset showed lower expression of CD161 and CD158a. Therefore, NKG2D, CD158a and CD158b expression in MM patients may represent several clinically useful 'biomarkers' of suppressed NK function.

AROME-ESO Oncology Consensus Conference: access to cancer care innovations in countries with limited resources. Association of Radiotherapy and Oncology of the Mediterranean Area (AROME-Paris) and European School of Oncology (ESO - Milan).

PURPOSE: Cancer is a leading cause of mortality worldwide. Its incidence is still increasing, particularly in developing countries. Recent progresses further strengthen the differences between low/middle and high-income countries. This situation calls for joint action to reduce inequities in cancer outcomes among the patients. The Association of Radiotherapy and Oncology of the Mediterranean Area (AROME) and the European School of Oncology (ESO), have initiated joint conferences devoted to access to innovations in oncology in the Mediterranean area. The heterogeneity of the economic, political and cultural situations of the different participating countries, offers the opportunity to develop consensus conference. METHODS: Cancer prevention and treatment strategies were discussed according to existing international guidelines. The Scientific committee prepared 111 questions with an objective to prioritize the access to treatments and innovations in low/middle-income Mediterranean countries. The results from the votes of 65 oncology experts, coming from 16 countries and 33 institutions have been analysed and access priorities classified accordingly. RESULTS: Ninety six percent of the proposed general recommendations concerning national health care strategies, oncology education, and treatment organization were considered to be high priorities. Regarding access to systemic treatments, 41% of the drugs without validated predictive markers and 53% of those with validated predictive markers were considered to be 1st level priority. Only 4 biological tests were considered to be 1st level priority to access to innovation. CONCLUSIONS: AROME-ESO consensus offers to cancer specialists from developing countries a basis for discussion with health authorities and payers on the prioritization of access to innovations in cancer care.

Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients.

Natural killer (NK) cells play a role in the innate and adaptive antitumor immune responses. The activity of NK cells is regulated by functionally opposing, activating and inhibitory receptors whose balance ultimately determines whether target cells will be susceptible to NK cell mediated lysis. As melanoma is an immunogenic tumor, the effect of immunomodulating agents is consistently investigated. In this study in 79 metastatic melanoma (MM) patients and 52 controls NK activity, expression of activating NKG2D and CD161 receptors and KIR receptors, CD158a and CD158b, on freshly isolated PBL and NK cells were evaluated. Native NK cell activity of melanoma patients in clinical stage I-III and MM patients was determined against NK sensitive K562, NK resistant Daudi, human melanoma FemX, HeLa and HL 60 target tumor cell lines. In addition, predictive pretherapy immunomodulating effect after 18 h in vitro treatments of PBL of MM patients with rh IL-2, IFN-alpha (IFN), 13-cis retinoic acid (RA) and combination IFN-alpha and RA was evaluated with respect to NK cell lyses against K562 and FemX cell lines. In this study we show for the first time that low expression of CD161 and activating NKG2D receptors, without increased expression of KIR receptors CD158a and CD158b, as well as a decrease in the cytotoxic, CD16(bright) NK cell subset, is associated with a significant impairment in NK cell activity in MM patients. Furthermore, the predictive pretherapy finding that IL-2, IFN, IFN and RA, unlike RA alone, can enhance NK cell activity of MM patients against FemX melanoma tumor cell line can be of help in the design and development of therapeutic regimens, considering that it has recently been shown that low-dose combination of different immunomodulators represents the most promising approach in the therapy of MM.

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.

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.

Clinicopathological characteristics, diagnosis and treatment of melanoma in Serbia--the Melanoma Focus Study.

BACKGROUND/AIM: Treatment options for metastatic melanoma in Serbia are limited due to the lack of newly approved biologic agents and the lack of clinical studies. Also, there is a paucity of data regarding the treatment approaches in different tertiary centers and efficacy of available chemotherapy protocols. The aim of this study was to obtain more detailed data about treatment protocols in Serbia based on structured survey in tertiary oncology centers. METHODS: Data about the melanoma patients treated in 2011 were analyzed from hospital databases in 6 referent oncology centers in Serbia, based on the structured survey, with the focus on metastatic melanoma patients (unresectable stage IIIC and IV). RESULTS: A total of 986 (79-315 in different centers) patients were treated, with 320 (32.45%) newly diagnosed patients. There were 317 patients in stage IIIC/IV, 77/317 aged < 50 years. At the time of diagnosis 47.3% of patients were < 60 years of age (24.2% < 40 years, 23% 50-59 years, 52.6% > 60 years). At initial diagnosis 12.5% of patients were in stage III and 4.5% in stage IV. The most common type was superficial spreading melanoma (50-660), followed by nodular melanoma (23.5-50%). Apart from the regional and distant lymph node metastases, the most frequent organs involved in stage IV disease were distant skin and soft tissues (12-55%), lungs (19-55.5%), liver (10-60%), and bones (3-10%). The first line therapy in stage IV metastatic melanoma was dacarbazine (DTIC) dimethyl-triazenoimidozole-carboxamide in 61-93% of the patients, while the second line varied between the centers. Disease control (complete response + partial response + stable disease) was achieved in 25.7% of the patients treated with the first line chemotherapy and 23.1% of the patients treated with the second line therapy, but the duration of response was short, in first-line therapy 6.66 +/- 3.36 months (median 6.75 months). More than 90% of patients were treated outside the clinical trials. CONCLUSION: Based on this survey, there is a large unmet need for the new treatment options for metastatic melanoma in Serbia. The development of national guidelines, and greater involvement in international clinical studies could lead to widening of treatment options for this chemotherapy resistant disease.

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.

In-vitro IL-2 or IFN-α-induced NKG2D and CD161 NK cell receptor expression indicates novel aspects of NK cell activation in metastatic melanoma patients.

In metastatic melanoma (MM) immunomodulating agents, such as interleukin-2 (IL-2) and interferon-α (IFN-α), have shown therapeutic benefit as they enhance antitumor immune response. Considering tumor-induced suppression of natural killer (NK) cell activity, it is of interest to study the affect of these cytokines on the functional and receptor characteristics of CD16-defined NK cells and their dim and bright subsets. Peripheral blood lymphocytes of MM patients in clinical stage IV were treated in vitro for 18 h with IFN-α (250 U/ml) and rhIL-2 (200 U/ml) at 37°C. Both the cytokines induced significant in-vitro enhancement of NK cell activity. NKG2D receptor was induced by IL-2, whereas both the NKG2D and CD161 receptor expression was induced by IFN-α on NK cells and CD16(bright) NK cell subset. However, only IL-2 mediated induction of NKG2D on CD3⁻CD16(+) NK cells correlates with enhanced NK cytotoxicity by this cytokine, whereas, on the cytotoxic CD16(bright) subset NKG2D induction by both cytokines correlates with their induction of NK cell activity. In contrast, the observed induction of these receptors on the regulatory CD16(dim) subset shows no correlation with the obtained augmentation of cytotoxicity. We found substantial specific inducibility of pSTAT1 and pSTAT5, as well as induction of interferon-regulatory transcription factor-1 transcription by investigated cytokines in peripheral blood lymphocytes of MM patients. As NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling, induction of activating NKG2D receptor by IL-2 and IFN-α, especially in CD16(bright) NK cell subset, gives insight to novel aspects of NK cell activation by these cytokines that are applied in immunotherapy.

Molecular targeting agents in renal cell carcinoma: present strategies and future perspectives.

Treatment options for metastatic renal cell carcinoma (RCC) have been limited due to its resistance to chemotherapy and radiotherapy. Benefits from immunotherapeutic agents provide only a small subset of patients. During the past decade major advances have been made toward understanding the molecular basis of RCC development. Such acquired knowledge has offered unique opportunities for the development of molecular targeting agents. These agents are predominately small molecules or monoclonal antibodies that exert their action through modulation of protein activity or inhibition of amplified signals directly implicated in disease mechanism. To date, some of newly molecular targeted agents have entered advanced phases of clinical development, received marketing authorization by regulatory agencies and have opened a possibility of multiple treatment options. This article overviews current knowledge in RCC molecular pathology with recent clinical data, and discuss present strategies for future development of targeted therapies.