There is far less information available about the tumor infiltrating B (TIL-B) cells, than about the tumor infiltrating T cells. We focused on discovering the features and potential role of B lymphocytes in solid tumors. Our project aimed to develop innovative strategies to define cancer membrane structures. We chose two solid tumor types, with variable to considerable B cell infiltration. The strategy we set up with invasive breast carcinoma, showing medullary features, has been introduced and standardized in metastatic melanoma. After detecting B lymphocytes by immunohistochemistry, VH-JH, Vκ-Jκ immunoglobulin rearranged V region genes were amplified by RT-PCR, from TIL-B cDNA. Immunoglobulin variable-region genes of interest were cloned, sequenced, and subjected to a comparative DNA analysis. Single-chain variable (scFv) antibody construction was performed in selected cases to generate a scFv library and to test tumor binding capacity. DNA sequence analysis revealed an overrepresented VH3-1 cluster, represented both in the breast cancer and the melanoma TIL-B immunoglobulin repertoire. We observed that our previously defined anti GD3 ganglioside-binder antibody-variable region genes were present in melanoma as well. Our antibody fragments showed binding potential to disialylated glycosphingolipids (GD3 ganglioside) and their O acetylated forms on melanoma cancer cells. We conclude that our results have a considerable tumor immunological impact, as they reveal the power of TIL-B cells to recognize strong tumor-associated glycosphingolipid structures on melanomas and other solid tumors. As tumor-derived gangliosides affect immune cell functions and reduce the B lymphocytes' antibody production, we suspect an important B lymphocyte and cancer cell crosstalk mechanism. We not only described the isolation and specificity testing of the tumor infiltrating B cells, but also showed the TIL-B cells' highly tumor-associated GD3 ganglioside-revealing potential in melanomas. The present data help to identify new cancer-associated biomarkers that may serve for novel cancer diagnostics. The two-direction regulation mechanism between immune B cells and the tumor could eventually be developed into an innovative cancer treatment strategy.

Brain metastasis is a frequent complication of the progression of malignant melanoma. In a previous study aquaporin 1 (AQP1) protein expression was found to be associated with increased mortality and decreased progression free survival in cutaneous melanoma. To explore further the potential of this marker we studied the AQP1 protein expression in 67 metastatic melanoma patients using immunohistochemistry. Primary tumor samples were acquired from patients with brain (BR) ( n = 44) and extra-cranial (EC) ( n = 23) metastases, while brain metastatic samples were collected during neurosurgical resection ( n = 5). Patients with brain metastases had shorter overall survival ( p = 0.02) and significantly higher AQP1 expression in the primary tumors (median H-score = 250 vs. 140, p = 0.044) as compared to patients of the EC metastasis group. AQP1 expression was found to be significantly lower in the brain metastases compared to the corresponding primary tumors (median H-score = 35 vs. 300 p = 0.01). However, in brain metastases AQP1 expression was heterogenous, AQP1 protein was more abundant in the melanoma cells far away from the capillaries as compared to tumor cells adjacent to vessels indicating a hypoxia-driven expression of AQP1. We suggest that AQP1 expression could well be a prognostic marker of brain metastatic potential of human cutaneous melanoma.

Ipilimumab was the first immunotherapy approved for metastatic melanoma in decades and is currently registered as a second-line treatment. However, new immunotherapies, in combination with ipilimumab, offer even better clinical outcomes for patients compared with single-agent treatments, at the expense of improved toxicity. The aim of this study was to evaluate the feasibility of ipilimumab outside the clinical trials and to identify survival predictors for treatment benefit. Data were collected on 47 advanced melanoma patients treated with ipilimumab between 2010 and 2015 at a single center. Association of clinical characteristics (including primary tumor characteristics), serum lactate dehydrogenase (LDH), erythrocyte sedimentation rate, absolute eosinophil, lymphocyte, and neutrophil count, neutrophil/lymphocyte and eosinophil/lymphocyte ratio with toxicity and clinical outcome were assessed using univariate and multivariate analysis. Median progression-free survival at a median follow-up of 10 months was 2.7 months and median overall survival was 9.8 months. Objective response was observed in 17% of patients and the disease control rate at week 24 was 40%. The 1- and 2-year survival rates documented were 40 and 28%, respectively. Significant association between high LDH level (>1.5× upper limit of normal) and decreased overall survival was demonstrated in uni- and multivariate analysis (hazard ratio [HR]: 3.554, 95% CI: 1.225–10.306, p = 0.019). Neither biomarkers nor clinical outcome were associated with toxicity. Using baseline serum LDH to identify patients most likely to benefit from ipilimumab therapy could serve as a simple and inexpensive biomarker of clinical outcome.

Absztrakt: A melanoma előfordulasi gyakorisaga az eletkorral nő, legnagyobb aranyban a nem hispaniai feherekben fordul elő. Bar az ocularis melanoma gyakorisaga a toredeke a cutan melanomaenak – az osszes melanomas eset mintegy 4%-a, eves incidenciaja 0,6 : 100 000 –, a szemtumorok kozott a leggyakrabban előfordulo malignitas. Az ocularis es a cutan melanoma egyuttes előfordulasa irodalmi ritkasagnak szamit. Kozlemenyunkben egy 80 eves ferfi esetet prezentaljuk, akinel 2008-ban cutan nodularis melanomat excindaltak. Szemeszeten 2013-ban fokozodo visuscsokkenes miatt uvealis melanomat diagnosztizaltak, amelyet brachytherapiaval kezeltek. Kepalkoto vizsgalatokkal es biopsziaval 2015-ben melanoma hepaticus propagatioja igazolodott. A primer cutan laesio mutacioanalizise BRAF V600 K tipusu funkcionyerő mutaciot igazolt, mig az attetben a vad tipusu gen jelenletet mutattuk ki. Onkoteam javaslata alapjan 2015 augusztusaban intraarterialis majkemoterapia kezdődott, melyből 11 ciklust kapott meg, 21 napos időintervallumokkal. A beteg a kezelest jol toleralta, mellekhatas nem jelentkezett. A 2016. februari CT a majban levő laesio parcialis regressziojat igazolta; a tizenegyedik ciklus intraarterialis majkemoterapiajat kovetően a beteg komplett remisszioba kerult, amely tobb mint egy evig tartott. Az ocularis es a cutan melanoma szinkron előfordulasa igen ritka, metasztatikus progresszio eseten ugyanakkor az optimalis onkoterapia kivalasztasa komoly kihivast jelent. A ket melanomatipus molekularis patologiai hattere elterő, amely segitheti a metasztatikus laesiok eredetenek azonositasat es az optimalis, szemelyre szabott kezeles megvalasztasat. Orv Hetil. 2018; 159(16): 642–647. | Abstract: The incidence rates of cutaneous melanoma in non-Hispanic whites show an increasing tendency with age. While uveal melanoma in general is a rare disease, representing only 4% of all melanomas with an incidence rate of 0.6 per 100 000, it is still the most frequent malignancy of the eye. Synchronous occurrence of ocular and cutaneous melanoma is an exceptional rarity, due to the distinct genetic background of the diseases. We report the case of a 80-year-old man who underwent total excision of a cutaneous melanoma in 2008. In 2013, he was diagnosed with uveal melanoma as part of a routine work-up for reduced vision. The uveal melanoma was treated by brachytherapy. In 2015, liver metastases were suspected by routine ultrasonography. Core biopsy was carried out, and the histology confirmed melanoma metastases. The molecular analysis of the cutaneous lesion showed gain of function mutation of the BRAF V600 K gene, while we found a wild-type BRAF gene in the metastatic lesion. Based on the recommendation of the oncoteam, hepatic intra-arterial Epirubicin-Platidiam therapy was introduced. He received 11 doses of intra-arterial chemotherapy (IAC), in 21 cycles. IAC was well tolerated without any catheter-related complications or toxicities. Partial regression of the hepatic metastases were documented in February 2016. After completing the eleventh cycle of intrahepatic chemotherapy, the disease remained in complete remission for over a year. The parallel occurrence of cutaneous and ocular melanoma is rare, however, the metastatic progression in such cases make the selection of optimal medical therapy challenging. The distinct genetic background of two melanoma types may help the identification of the source of the metastatic lesions, in order to guide the treatment decisions. Orv Hetil. 2018; 159(16): 642–647.

The incidence rates of cutaneous melanoma in non-Hispanic whites show an increasing tendency with age. While uveal melanoma in general is a rare disease, representing only 4% of all melanomas with an incidence rate of 0.6 per 100 000, it is still the most frequent malignancy of the eye. Synchronous occurrence of ocular and cutaneous melanoma is an exceptional rarity, due to the distinct genetic background of the diseases. We report the case of a 80-year-old man who underwent total excision of a cutaneous melanoma in 2008. In 2013, he was diagnosed with uveal melanoma as part of a routine work-up for reduced vision. The uveal melanoma was treated by brachytherapy. In 2015, liver metastases were suspected by routine ultrasonography. Core biopsy was carried out, and the histology confirmed melanoma metastases. The molecular analysis of the cutaneous lesion showed gain of function mutation of the BRAF V600 K gene, while we found a wild-type BRAF gene in the metastatic lesion. Based on the recommendation of the oncoteam, hepatic intra-arterial Epirubicin-Platidiam therapy was introduced. He received 11 doses of intra-arterial chemotherapy (IAC), in 21 cycles. IAC was well tolerated without any catheter-related complications or toxicities. Partial regression of the hepatic metastases were documented in February 2016. After completing the eleventh cycle of intrahepatic chemotherapy, the disease remained in complete remission for over a year. The parallel occurrence of cutaneous and ocular melanoma is rare, however, the metastatic progression in such cases make the selection of optimal medical therapy challenging. The distinct genetic background of two melanoma types may help the identification of the source of the metastatic lesions, in order to guide the treatment decisions. Orv Hetil. 2018; 159(16): 642-647.

Abstract: The incidence rates of cutaneous melanoma in non-Hispanic whites show an increasing tendency with age. While uveal melanoma in general is a rare disease, representing only 4% of all melanomas with an incidence rate of 0.6 per 100 000, it is still the most frequent malignancy of the eye. Synchronous occurrence of ocular and cutaneous melanoma is an exceptional rarity, due to the distinct genetic background of the diseases. We report the case of a 80-year-old man who underwent total excision of a cutaneous melanoma in 2008. In 2013, he was diagnosed with uveal melanoma as part of a routine work-up for reduced vision. The uveal melanoma was treated by brachytherapy. In 2015, liver metastases were suspected by routine ultrasonography. Core biopsy was carried out, and the histology confirmed melanoma metastases. The molecular analysis of the cutaneous lesion showed gain of function mutation of the BRAF V600 K gene, while we found a wild-type BRAF gene in the metastatic lesion. Based on the recommendation of the oncoteam, hepatic intra-arterial Epirubicin-Platidiam therapy was introduced. He received 11 doses of intra-arterial chemotherapy (IAC), in 21 cycles. IAC was well tolerated without any catheter-related complications or toxicities. Partial regression of the hepatic metastases were documented in February 2016. After completing the eleventh cycle of intrahepatic chemotherapy, the disease remained in complete remission for over a year. The parallel occurrence of cutaneous and ocular melanoma is rare, however, the metastatic progression in such cases make the selection of optimal medical therapy challenging. The distinct genetic background of two melanoma types may help the identification of the source of the metastatic lesions, in order to guide the treatment decisions. Orv Hetil. 2018; 159(16): 642–647.

AIM OF THE STUDY:The arsenal of questions and answers about the minor cancer initiating cancer stem cell (CSC) population put responsible for cancer invasiveness and metastases, has left with an unsolved puzzle. Specific aims of a complex project were partly focused on revealing new biomarkers of cancer. We designed and set up novel techniques to facilitate the detection of cancerous cells.MATERIALS AND METHODS:As a novel approach, we investigated B cells infiltrating breast carcinomas and melanomas (TIL-B) in terms of their tumour antigen binding potential. By developing the TIL-B phage display technology we provide here a new technology for the specific detection of highly tumour-associated antigens. Single chain Fv (scFv) antibody fragment phage ELISA, immunofluorescence (IF) FACS analysis, chamber slide technique with IF confocal laser microscopy and immunohistochemistry (IHC) in paraffin-embedded tissue sections were set up and standardized.RESULTS:We showed strong tumour-associated disialylated glycosphingolipid expression levels on various cancer cells using scFv antibody fragments, generated previously by uniquely invasive breast carcinoma TIL-B phage display library technology.CONCLUSIONS:We report herein a novel strategy to obtain antibody fragments of human origin that recognise tumour-associated ganglioside antigens. Our investigations have the power to detect privileged molecules in cancer progression, invasiveness, and metastases. The technical achievements of this study are being harnessed for early diagnostics and effective cancer therapeutics.

Complete/partial loss of SMARCB1 nuclear‐immunopositivity is characteristic of a certain subset of soft tissue sarcomas (STSs). Our previous work showed that oncomiRs‐206,‐381, and 671‐5p could silence the SMARCB1 mRNA and protein expression and that they display significant overexpression in epithelioid sarcomas (ESs). MiR‐765 was overexpressed too, but functionally was inactive in the silencing. In the current work, using quantitative PCR, we conducted a miRNA study of 51 ESs, 20 rhabdoid tumors (RTs), 20 synovial sarcomas (SSs), 15 malignant peripheral nerve sheath tumors (MPNSTs), 11 myoepithelial carcinomas (MECs), and 10 extraskeletal myxoid chondrosarcomas (EMCSs) with complete/partial loss of SMARCB1 nuclear immunostain, in contrast to controls (SMARCB1‐immunopositive) of 96 STSs, 13 melanomas and 10 sarcomatoid carcinomas. The SMARCB1 genetic status of ESs was determined by MLPA and FISH. A subset of ESs (5/51) showed biallelic deletion of SMARCB1 with no overexpression of any miRNA, suggesting these tumors could be the counterpart of pediatric RT, at least genetically. Another subset (5/51) was genetically either intact or monoallelic deleted with at least threefold overexpression of one of miR‐206,‐381,‐671‐5p, suggesting epigenetic regulation only. 39/51 ESs had a biallelic deletion (>20% by FISH and/or by MLPA) but with overexpressed miR‐206,‐381, and 671‐5p, suggesting intratumoral heterogeneity, i.e., both genetic and epigenetic regulation. At least threefold overexpression of one of miR‐206,‐381, and 671‐5p was detected in all MPNSTs, EMCSs, SSs and 7 MCs. Except for ESs, four SSs and one MPNST, there was no event above threefold overexpression of miR‐765 among all 195 tested tumors. Our results suggest a general role of miR‐206,‐381, and 671‐5p in SMARCB1 gene silencing of ES, MC, EMCS, MPNST and SS. In the future, miR‐765 could possibly be a diagnostic tool for ES because of its 97% specificity and 80% sensitivity. © 2016 Wiley Periodicals, Inc.

The rapidly growing field of gene therapy techniques to modify T cells with chimeric antigen receptors (CARs) for cancer care solutions, reached considerable achievements. However, there is an urgent need of reliable, well tolerable tumor-associated antigen specific antibodies. Tumor-infiltrating B (TIL-B) cell originated single chain Fv (scFv) gene regions could be selected with tumor specificity. DNA sequences of these antibody variable regions were subjects to get engineered into new CAR constructs. Our novel strategy harnesses tumor-infiltrating B cells' unique capacity to reveal highly tumor-associated disialylated glycosphingolipids (GD3 gangliosides). We used these human antibody fragments for generating GD3 ganglioside specific CAR gene constructs for potential usage in solid tumors.

AbsztraktMelanoma malignum solitaer nyelőcsőáttétjének sikeresen operált esetét ismertetjük. 13 évvel a primaer tumor kimetszése után progresszív nyelészavar hátterében a nyelőcső felső harmadában melanoma malignumot igazoltunk. Stagingvizsgálatok alapján az elváltozás solitaernek bizonyult. Transhiatalis oesophagectomiát végeztünk gyomorpótlással, nyaki anastomosissal. A szövettani vizsgálat metastaticus melanomát talált. A beteg szövődménymentesen gyógyult, majd adjuváns dacarbazinkezelésben részesült. 18 hónappal a műtét után a beteg panasz- és daganatmentes.

Meeting abstracts The theory and investigations on cancer stem cells (CSCs) have received growing attention, as these cells are responsible for the failure of cancer therapeutic strategies and the return of cancer. A complex tumorimmunological study on primary and metastatic cancerous tissue

We report a case of metastatic malignant melanoma in the oesophagus. 13 years after the wide excision of primary skin melanoma, we found a polypoid tumor in the upper third of the oesophagus. Biopsy result was melanoma malignum. After negative staging we performed transhiatal oesophagectomy with gastric conduit and cervical anastomosis. Metastatic nature of the oesophageal tumor was proven by histology. After uneventful postoperative course, the patient received adjuvant dacarbazine treatment. The patient was is in good condition, and disease free on the 18 month follow-up.Abstract available from the publisher.

Bar a szentinel nyirokcsomok (SLN) a tumorellenes immunvalasz kialakulasanak fontos szinterei, immunologiai sajatossagaikat kevesen tanulmanyoztak. Vizsgalatainkban melanomas betegek SLN-mintain meghataroztuk az OX40+ aktivalt T-limfocitak, FOXP3+ regulator T-sejtek, DC-LAMP+ erett dendritikus sejtek (DC) es CD123+ plazmacitoid DC-k mennyiseget, es osszevetettuk nem-szentinel csomokban (NSLN) talalt ertekekkel, valamint elemeztuk a beteg- es tumorjellemzők, illetve a betegseg kimenetele fuggvenyeben. A markerek megjelenese az SLN-ekben magasabb szintű volt az NSLN-ekhez viszonyitva, ami az őrszemnyirokcsomok immunologiai kompetenciajara utal. Pozitiv SLN-status eseten a FOXP3+ sejtek magas denzitasa a betegseg progressziojaval es rovidebb tulelessel jart. A tobbi marker eseten nem talaltunk szignifikans osszefuggest a betegseg kimenetelevel. A primer melanomakat infiltralo immunsejtek kozul (a korabbi vizsgalataink szerint prognosztikus ertekű aktivalt T-sejt- es DC-szam mellett) a B-sejtek magas denzitasa onmagaban, illetve az aktivalt T-sejtek nagy mennyisegevel egyuttesen kedvező prognozisra utalt, mig a FOXP3+ limfocitak mennyisege nem mutatott osszefuggest a tulelessel. Eredmenyeink arra utalnak, hogy az immunaktivaciora jellemző markerek megjelenese a primer tumorban erősebben befolyasolja a betegseg kimenetelet, mint az SLN-ekben mert prevalenciajuk. Az őrszemnyirokcsomok magas FOXP3+ T-sejtdenzitasa ugyanakkor az SLN-pozitiv esetekben rossz prognozissal jart egyutt. | Sentinel lymph nodes (SLNs) are critical sites of the development of antitumor immune response, still only few studies have aimed at investigating their immunological status. We determined the prevalence of OX40+ activated T cells, FOXP3+ regulatory T cells, DC-LAMP+ mature dendritic cells (DCs) and CD123+ plasmacytoid DCs in melanoma SLNs. Cell density values were compared to those in non-sentinel nodes (NSLNs), and analyzed with regard to associations with clinicopathological parameters and disease outcome. SLNs contained higher amount of all cell types compared to NSLNs, indicating the functional competence of sentinel nodes. High density of FOXP3+ T lymphocytes showed association with disease progression and shorter survival in SLN-positive patients, while the other cell types studied did not prove of prognostic importance. Of immune cells infiltrating primary melanomas, high amount of B lymphocytes, alone or in combination with high activated T-cell density, correlated with favorable outcome, similarly to DCs and activated T cells found of prognostic importance in our previous studies. Infiltration of FOXP3+ cells in primary melanomas, on the other hand, showed no association with survival. Our results suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of melanoma patients. On the other hand, FOXP3+ cell density in sentinel nodes of SLN-positive cases was found predictive of disease 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.

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.