Insights into the Tumor Microenvironment-Components, Functions and Therapeutics.

Similarly to our healthy organs, the tumor tissue also constitutes an ecosystem. This implies that stromal cells acquire an altered phenotype in tandem with tumor cells, thereby promoting tumor survival. Cancer cells are fueled by abnormal blood vessels, allowing them to develop and proliferate. Tumor-associated fibroblasts adapt their cytokine and chemokine production to the needs of tumor cells and alter the peritumoral stroma by generating more collagen, thereby stiffening the matrix; these processes promote epithelial-mesenchymal transition and tumor cell invasion. Chronic inflammation and the mobilization of pro-tumorigenic inflammatory cells further facilitate tumor expansion. All of these events can impede the effective administration of tumor treatment; so, the successful inhibition of tumorous matrix remodeling could further enhance the success of antitumor therapy. Over the last decade, significant progress has been made with the introduction of novel immunotherapy that targets the inhibitory mechanisms of T cell activation. However, extensive research is also being conducted on the stromal components and other cell types of the tumor microenvironment (TME) that may serve as potential therapeutic targets.

Molecular Pathology of Skin Melanoma: Epidemiology, Differential Diagnostics, Prognosis and Therapy Prediction.

Similar to other malignancies, TCGA network efforts identified the detailed genomic picture of skin melanoma, laying down the basis of molecular classification. On the other hand, genome-wide association studies discovered the genetic background of the hereditary melanomas and the susceptibility genes. These genetic studies helped to fine-tune the differential diagnostics of malignant melanocytic lesions, using either FISH tests or the myPath gene expression signature. Although the original genomic studies on skin melanoma were mostly based on primary tumors, data started to accumulate on the genetic diversity of the progressing disease. The prognostication of skin melanoma is still based on staging but can be completed with gene expression analysis (DecisionDx). Meanwhile, this genetic knowledge base of skin melanoma did not turn to the expected wide array of target therapies, except the BRAF inhibitors. The major breakthrough of melanoma therapy was the introduction of immune checkpoint inhibitors, which showed outstanding efficacy in skin melanoma, probably due to their high immunogenicity. Unfortunately, beyond BRAF, KIT mutations and tumor mutation burden, no clinically validated predictive markers exist in melanoma, although several promising biomarkers have been described, such as the expression of immune-related genes or mutations in the IFN-signaling pathway. After the initial success of either target or immunotherapies, sooner or later, relapses occur in the majority of patients, due to various induced genetic alterations, the diagnosis of which could be developed to novel predictive genetic markers.

Identification of a Tumor Cell Associated Type I IFN Resistance Gene Expression Signature of Human Melanoma, the Components of Which Have a Predictive Potential for Immunotherapy.

We developed a human melanoma model using the HT168-M1 cell line to induce IFN-α2 resistance in vitro (HT168-M1res), which was proven to be maintained in vivo in SCID mice. Comparing the mRNA profile of in vitro cultured HT168-M1res cells to its sensitive counterpart, we found 79 differentially expressed genes (DEGs). We found that only a 13-gene core of the DEGs was stable in vitro and only a 4-gene core was stable in vivo. Using an in silico cohort of IFN-treated melanoma tissues, we validated a differentially expressed 9-gene core of the DEGs. Furthermore, using an in silico cohort of immune checkpoint inhibitor (ICI)-treated melanoma tissues, we tested the predictive power of the DEGs for the response rate. Analysis of the top four upregulated and top four downregulated genes of the DEGs identified WFDC1, EFNA3, DDX10, and PTBP1 as predictive genes, and analysis of the "stable" genes of DEGs for predictive potential of ICI response revealed another 13 genes, out of which CDCA4, SOX4, DEK, and HSPA1B were identified as IFN-regulated genes. Interestingly, the IFN treatment associated genes and the ICI-therapy predictive genes overlapped by three genes: WFDC1, BCAN, and MT2A, suggesting a connection between the two biological processes.

Immunologic and immunogenomic aspects of tumor progression.

Tumor progression to metastatic disease is characterized by continuous genetic alterations due to instability of the genome. Immune sensitivity was found to be linked to tumor mutational burden (TMB) and the resulting amount of neoantigens. However, APOBEC activity resulting in increase in TMB causes immune evasion. On the other hand, clonal or acquired genetic loss of HLA class I also hampers immune sensitivity of tumors. Rare amplification of the PD-L1 gene in cancers may render them sensitive to immune checkpoint inhibitors but involvement of broader regions of chromosome 9p may ultimately lead again to immune evasion due to inactivation of the IFN-γ signaling pathway. Such genetic changes may occur not only in the primary tumor but at any phase of progression: in lymphatic as well as in visceral metastases. Accordingly, it is rational to monitor these changes continuously during disease progression similar to target therapies. Moreover, beside temporal variability, genomic features of tumors such as mutation profiles, as well as the tumor immune microenvironment also show considerable inter- and intratumoral spatial heterogeneity, suggesting the necessity of multiple sampling in biomarker studies.

Low socioeconomic position is a risk factor for delay to treatment and mortality of testicular cancer patients in Hungary, a prospective study.

BACKGROUND: In Hungary, the mortality rate for testicular germ cell cancer (TGCC) is 0,9/100000 which is significantly higher than the EU average. We prospectively evaluated the effect of socioeconomic position on patient delay and therapy outcomes. METHODS: Questionnaires on subjective social status (MacArthur Subjective Status Scale), objective socioeconomic position (wealth, education, and housing data), and on patient's delay were completed by newly diagnosed TGCC patients. RESULTS: Patients belonged to a relatively high socioeconomic class, a university degree was double the Hungarian average, Cancer-specific mortality in the highest social quartile was 1.56% while in the lowest social quartile 13.09% (p = 0.02). In terms of patient delay, 57.2% of deceased patients waited more than a year before seeking help, while this number for the surviving patients was 8.0% (p = 0.0000). Longer patient delay was associated with a more advanced stage in non-seminoma but not in seminoma, the correlation coefficient for non-seminoma was 0.321 (p < 0.001). For patient delay, the most important variables were the mother's and patient's education levels (r = - 0.21, p = 0.0003, and r = - 0.20, p = 0.0005), respectively. Since the patient delay was correlated with the social quartile and resulted in a more advanced stage in non-seminoma, the lower social quartile resulted in higher mortality in non-seminoma patients (p = 0.005) but not in seminoma patients (p = 0.36) where the patient delay was not associated with a more advanced stage. CONCLUSIONS: Based on our result, we conclude that to improve survival, we should promote testicular cancer awareness, especially among the most deprived populations, and their health care providers.

Tumor-infiltrating immune cells as potential biomarkers predicting response to treatment and survival in patients with metastatic melanoma receiving ipilimumab therapy.

Monoclonal antibodies targeting immune checkpoints are gaining ground in the treatment of melanoma and other cancers, and considerable effort is made to identify biomarkers predicting the efficacy of these therapies. Our retrospective study was performed on surgical tissue samples (52 lymph nodes and 34 cutaneous/subcutaneous metastases) from 30 patients with metastatic melanoma treated with ipilimumab. Using a panel of 11 antibodies against different immune cell types, intratumoral immune cell densities were determined and evaluated in relation to response to ipilimumab treatment and disease outcome. For most markers studied, median immune cell densities were at least two times higher in lymph node metastases compared to skin/subcutaneous ones; therefore, the prognostic and predictive associations of immune cell infiltration were evaluated separately in the two groups of metastases as well as in all samples as a whole. Higher prevalence of several immune cell types was seen in lymph node metastases of the responders compared to non-responders, particularly FOXP3+ cells and CD8+ T lymphocytes. In subcutaneous or cutaneous metastases, on the other hand, significant difference could be observed only in the case of CD16 and CD68. Associations of labeled cell densities with survival were also found for most cell types studied in nodal metastases, and for CD16+ and CD68+ cells in skin/s.c. metastatic cases. Our results corroborate the previous findings suggesting an association between an immunologically active tumor microenvironment and response to ipilimumab treatment, and propose new potential biomarkers for predicting treatment efficacy and disease outcome.

[Predictive markers of immunotherapy of cancer, practical issues of PD-L1 testing]. / A daganatok immunterápiájának prediktív markerei, a PD-L1-meghatározás gyakorlati kérdései.

Pathologists have detected signs of antitumor immune reactions for a long time but only in case of a few cancer types became this part of the report. The advent of immunotherapy of cancers, however, radically alters this routine and promotes the development of clinically valid prognostic and predictive immunological makers. The most advanced immunological markers are the Immunoscore (density of T-cell subpopulations), and PD-L1 protein expression on tumor or immune cells. PD-L1 testing of cancers raises new issues since almost all novel therapies developed its own in vitro diagnostics. Due to the incompatibility of these assays it is suggested to use the companion diagnostic of the given immunotherapeutic drug defined in its label.

[Immunologic biomarkers in predicting the efficacy of cancer therapy]. / Immunológiai biomarkerek a rákellenes kezelés hatásának megjóslásában.

Immune checkpoint agents, representing a new class of immunotherapeutic modalities, have proven their beneficial effect inducing durable clinical response in a widening spectrum of tumor types. To enhance their efficacy it is of primary importance to search for biomarkers that could help predict the likelihood of therapeutic effect. Results of studies on potential predictive immunological parameters suggest that the existing antitumor immune activity detectable in the patients, although not sufficient to control tumor progression in itself, could increase the efficacy of different immunotherapies. Moreover, the contribution of immune reactions to the effect of other antitumor treatment modalities as chemo-, radio-, and targeted therapy has also been demonstrated. Combinations of immunotherapies with these and with each other will probably represent treatment approaches resulting in the highest therapeutic effect, which may necessitate the development of biomarker panels with multiple components characterizing the immune status of the patient and the tumor as well.

[Immunotherapy opportunities in breast cancer]. / Új immunterápiás lehetõségek az emlõrák kezelésében.

The prognostic value of tumor infiltrating lymphocytes in breast cancer has long been recognized by histopathologists. These observations were reaffirmed by recent immunohistochemistry and gene expression profiling studies that also revealed an association between greater chemotherapy sensitivity and extensive lymphocytic infiltration in early stage breast cancers treated with neoadjuvant chemotherapy. These results suggest that local anti-tumor immune response can at least partially control cancer growth and may mediate the antitumor effects of chemotherapy. However, until recently, there was no direct clinical evidence to demonstrate that enhancing anti-tumor immune response could lead to clinical benefit in breast cancer patients. The recent development of clinically effective immune checkpoint inhibitors made it possible to test the therapeutic impact of augmenting the local anti-tumor immune response. Two Phase I clinical trials using single agent anti-PD-1 (MK-3475, pembrolizumab) and anti-PD-L1 (MPDL3280A, atezolizumab) antibodies demonstrated close to 20% tumor response rates in heavily pretreated, metastatic, triple negative breast cancers. The most remarkable feature of the responses was their long duration. Several patients had disease control close to a year, or longer, which has not previously been seen with chemotherapy regimens in this patient population. A large number of clinical trials are currently underway with these and similar drugs in the neoadjuvant, adjuvant and metastatic settings to define the role of this new treatment modality in breast cancer.

[Immunotherapy in the treatment of genitourinary cancers]. / Immunterápia az urológiai daganatok kezelésében.

In this clinical review we provide information regarding advance and main achievements in the immunotherapy of genitourinary, particularly renal cell and prostate cancer. Nivolumab treatment became the new standard of care in locally advanced or metastatic renal cell cancer after failure on tyrosine kinase inhibitor treatment. Sipuleucel-T prolonged survival in patients with asymptomatic or minimally symptomatic metastatic castration resistant prostate cancer but had no effect on progression-free survival. Based on the results of phase I/II trials anti-PD-1/PD-L1 monoclonal antibodies are a new hope in the treatment of urothelial bladder cancer. Regarding germ cell tumors basic research is ongoing.

[Melanoma research in Hungary: promising results in a previously orphan tumor]. / Hazai melanomakutatások: reményt keltõ eredmények egy korábban reménytelen daganatban.

Melanoma research has a two decade history in Hungary and is based on three groups located at the National Institute of Oncology (NIO), the University of Debrecen (DU) and Semmelweis University (SU). Previously we have summarized the achievements of the NIO group in this Journal, now this paper summarizes the recent results of their collaborations. The research group of DU revealed several novel genetic alterations in the melanoma genome which might have clinical relevance as prognosticators or predictors in light of the novel target therapies. Data indicating unique, perhaps melanoma-specific epigenetic changes during progression might be even more important, identifying novel genes otherwise not detected as genetically altered ones. The research group in Budapest extensively used experimental human melanoma models and demonstrated the host sex as a key factor in progression due to the specific function of NK cells. Identification of functional glucocorticoid receptor in human melanoma might lead to therapeutic exploitation similar to certain leukemias. Studies on extracellular matrix revealed collagen XVII and CD44 splice variants as progression associated factors of melanoma. Since the double wild type genotype of melanoma is lacking effective therapy, data on the use of FGFR2, c-met or cannabinoid receptor as target can be important. On the other hand, experimental data on the antitumoral effects of heparin derivatives or bisphosphonate in melanoma models can also be encouraging.

Density of tumor-infiltrating NK and Treg cells is associated with 5 years progression-free and overall survival in resected lung adenocarcinoma.

Surgical resection of pulmonary adenocarcinoma is considered to be curative but progression-free survival (PFS) has remained highly variable. Antitumor immune response may be important, however, the prognostic significance of tumor-infiltrating natural killer (NK) and regulatory T (Treg) lymphocytes is uncertain. Resected pulmonary adenocarcinoma tissues (n = 115) were studied by immunohistochemical detection of NKp46 and FoxP3 positivity to identify NK and Treg cells, respectively. Association of cell densities with clinicopathological features and progression-free survival (PFS) as well as overall survival (OS) were analyzed with a follow-up time of 60 months. Both types of immune cells were accumulated predominantly in tumor stroma. NK cell density showed association with female gender, non-smoking and KRAS wild-type status. According to Kaplan-Meier analysis, PFS and OS proved to be longer in patients with high NK or Treg cell densities (p = 0.0293 and p = 0.0375 for PFS, p = 0.0310 and p = 0.0448 for OS, respectively). Evaluating the prognostic effect of the combination of NK and Treg cell density values revealed that PFS and OS were significantly longer in NKhigh/Treghigh cases compared to the other groups combined (p = 0.0223 and p = 0.0325, respectively). Multivariate Cox regression analysis indicated that high NK cell density was independent predictor of longer PFS while high NK and high Treg cell densities both proved significant predictors of longer OS. The NKhigh/Treghigh combination also proved to be an independent prognostic factor for both PFS and OS. In conclusion, NK and Treg cells can be components of the innate and adaptive immune response at action against progression of pulmonary adenocarcinoma.

Immune cell profile of sentinel lymph nodes in patients with malignant melanoma - FOXP3+ cell density in cases with positive sentinel node status is associated with unfavorable clinical outcome.

BACKGROUND: Besides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions. METHODS: In this retrospective study we determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ (forkhead box P3) regulatory T cells, DC-LAMP+ (dendritic cell-lysosomal associated membrane protein) mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients. Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n = 37), and analyzed with regard to associations with clinicopathological parameters and disease outcome. RESULTS: Sentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes. Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied. In patients with positive sentinel node status, high amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P = 0.0011) and overall survival (P = 0.0014), while no significant correlation was found in the case of sentinel-negative patients. The density of OX40+, CD123+ or DC-LAMP+ cells did not show significant association with the outcome of the disease. CONCLUSIONS: Taken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells. The density of FOXP3+ lymphocytes showed association with progression and survival in patients with positive SLN status, while the other immune markers studied did not prove of prognostic importance. These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of the patients. On the other hand, FOXP3+ cell density in SLNs, but not in the primary tumor, was found predictive of disease outcome in melanoma patients.

[Prognostic value of tumor-infiltrating immune cells in melanoma]. / A tumort infiltráló immunsejtek prognosztikai értéke melanómában.

Host cells representing an integral component of solid tumors, among them cells contributing to the development of native and adaptive immune responses, can exert both positive and negative effects on the outcome of the disease. Infiltration of T lymphocyte subsets and antigen presenting dendritic cells, crucial in mounting an efficient antitumor immune response, is generally associated with favorable prognosis. On the contrary, accumulation of tumor-associated macrophages, mast cells and neutrophils, playing important roles in chronic inflammatory processes promoting tumor progression, predicts unfavorable disease outcome in most cases. In melanoma, studies on the prognostic impact of the lymphoid infiltrate in general, and that of T cells, yielded controversial results. In our studies, density of activated T lymphocytes correlated with patients' survival, and we obtained similar results in the case of B cells and mature dendritic cells. The amount of both B cells and dendritic cells showed correlation with that of activated T lymphocytes, and their combined analysis identified patient subgroups with different prognosis. According to data from the literature, intense infiltration by neutrophil granulocytes could be associated with shorter survival, while no unambiguous conclusions can be drawn from studies on the prognostic value of tumor-associated macrophages and other immune cell types. Our results indicate that a prominent infiltration of dendritic cells, B cells and activated T lymphocytes can be considered as a favorable prognostic factor in malignant melanoma.

[Unblocking antitumor immune response: novel possibilities for the immunotherapy of melanoma]. / Az immunválasz "akadálymentesítése": újabb lehetõségek a melanóma immunterápiájában.

Recent advances in tumor immunology, a better understanding of mechanisms regulating the immune response has led to experimental and clinical testing of a novel type of immunotherapeutics: antibodies blocking negative regulatory mechanisms of T-cell activation [corrected]. The application of the CTLA-4 antagonist ipilimumab, the prototype of this new class of immune stimulating agents, represents the first treatment that resulted in significant prolongation of the survival of metastatic melanoma patients in randomized, controlled trial, leading to the approval of its use for the therapy of these patients in 2011. Together with the BRAF inhibitor vemurafenib, which was also approved in 2011, ipilimumab has changed the standard therapy of metastatic melanoma, and also paved the way for other agents aiming at influencing immune regulating molecules, of which antibodies blocking the PD-1 pathway also showed promising clinical activity. According to clinical experience collected so far, these agents induce objective tumor response in a relatively small proportion of patients, with a characteristic response kinetics frequently showing delayed activity, but resulting in durable remission in a considerable proportion of the responding patients. On the other hand, antitumor activity is frequently accompanied by significant toxicity. The spectrum of side effects is different from that of conventional therapies, and a large part of them is caused by the enhanced systemic immune activity. In order to spare non-responding patients of the severe side effects and to increase response rate, the search for biomarkers that could help in identifying patients likely to react to the treatment represents an important focus of studies. Furthermore, development of combinations with other immunotherapeutic modalities, chemo- or targeted therapies may further increase the efficiency of immunomodulatory antibodies.

[New TNM classification (AJCC 2009) and the pathological significance of sentinel lymph node biopsy in malignant melanoma]. / A malignus melanóma új TNM-klasszifikációja (AJCC, 2009) és az õrszemnyirokcsomó-biopszia patológiai jelentõsége.

The novel TNM staging system (AJCC, 2009) has some new aspects on pathological microstaging of malignant melanoma. It highlights and reflects the importance of vertical growth phase of these tumors. Furthermore, the morphometrical evaluation of sentinel lymph nodes (SLNs) is more important than ever since, according to the new classification, even the presence of isolated tumor cells means more advanced stage.

Metastatic Progression of Human Melanoma.

This Topical Collection, comprising 13 papers (10 original articles and 3 reviews), addresses various aspects of the field of melanoma progression: genomic and proteomic approaches, experimental studies, the questions of sentinel lymph node dissection, and metastasis formation of uveal and conjunctival melanomas is also discussed [...].

p38 Mitogen-Activated Protein Kinase Inhibition of Mesenchymal Transdifferentiated Tumor Cells in Head and Neck Squamous Cell Carcinoma.

High mortality in head and neck squamous cell carcinoma (HNSCC) is due to recurrence, metastasis, and radiochemotherapy (RCT) resistance. These phenomena are related to the tumor cell subpopulation undergoing partial epithelial to mesenchymal transition (pEMT). Repeated transforming growth factor-beta (TGF-beta-1) treatment via the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway induces pEMT in SCC-25 HNSCC cells, and activates and stabilizes the pro-EMT transcription factor Slug. We investigated the growth inhibitory, cisplatin-sensitizing, and pro-apoptotic effects of p38 MAPK inhibition in cisplatin-resistant (SCC-25) and -sensitive (UPCI-SCC090) HNSCC cell lines, using two specific p38 MAPK inhibitors, SB202190 and ralimetinib. Cell viability was measured by MTT assay; cell cycle distribution and cell death were evaluated by flow cytometry; p38 MAPK phosphorylation, Slug protein stabilization, and p38 MAPK downstream targets were investigated by Western blot. p-p38 inhibitors achieved sustained phosphorylation of p38 MAPK (Thr180/Tyr182) and inhibition of its function, which resulted in decreased phosphorylation (Thr69/71) of the downstream target pATF2 in pEMT cells. Subsequently, the p-p38 inhibition resulted in reduced Slug protein levels. In accordance, p-p38 inhibition led to sensitization of pEMT cells to cisplatin-induced cell death; moreover, p-p38 inhibitor treatment cycles significantly decreased the viability of cisplatin-surviving cells. In conclusion, clinically relevant p38 inhibitors might be effective for RCT-resistant pEMT cells in HNSCC patients.

Evolving Acquired Vemurafenib Resistance in a BRAF V600E Mutant Melanoma PDTX Model to Reveal New Potential Targets.

Malignant melanoma is challenging to treat, and metastatic cases need chemotherapy strategies. Targeted inhibition of commonly mutant BRAF V600E by inhibitors is efficient but eventually leads to resistance and progression in the vast majority of cases. Numerous studies investigated the mechanisms of resistance in melanoma cell lines, and an increasing number of in vivo or clinical data are accumulating. In most cases, bypassing BRAF and resulting reactivation of the MAPK signaling, as well as alternative PI3K-AKT signaling activation are reported. However, several unique changes were also shown. We developed and used a patient-derived tumor xenograft (PDTX) model to screen resistance evolution in mice in vivo, maintaining tumor heterogeneity. Our results showed no substantial activation of the canonical pathways; however, RNAseq and qPCR data revealed several altered genes, such as GPR39, CD27, SLC15A3, IFI27, PDGFA, and ABCB1. Surprisingly, p53 activity, leading to apoptotic cell death, was unchanged. The found biomarkers can confer resistance in a subset of melanoma patients via immune modulation, microenvironment changes, or drug elimination. Our resistance model can be further used in testing specific inhibitors that could be used in future drug development, and combination therapy testing that can overcome inhibitor resistance in melanoma.

[Molecular pathology of skin melanoma]. / A bor melanómájának molekuláris patológiája: diagnosztikus, prognosztikus és prediktív markerek.

Skin melanoma became one of the most frequent malignancies of the skin mainly due to the increased exposure to environmental UV irradiation. However, this did not lead to the increase of mortality, mainly due to the efficient early diagnostics as well as to the development of new therapies which were based on the identification of the mutation patterns and understanding of the immunobiology. Molecular markers are important to differentiate malignant tumors from precancerous ones but also routinely available to define prognosis. Immunotherapy can be used in any molecular class of melanoma, while target therapy is only available in case of BRAF mutant form. Meanwhile these novel therapies induce new resistance mechanisms based on the development of acquired genetic alterations. Due to these facts the initial molecular profiling of the resected primary tumors must gradually be replaced by continuous monitoring.