Search

Objective: To investigate the clinicopathological, immunohistochemical and molecular genetic characteristics of TFE3-rearranged perivascular epithelioid cell tumor (PEComa). Methods: Eight cases of PEComa with TFE3 rearrangement diagnosed in the First Affiliated Hospital of Air Force Medical University from January 2014 to July 2022 were collected. Three were consultation cases and 5 were collected from our hospital; 7 cases were resection specimens and 1 case was a needle biopsy specimen. Routine histolopathological analysis, immunohistochemical staining, fluorescence in situ hybridization (FISH) and the next-generation sequencing were performed. Clinical data were collected and the prognosis was assessed. Results: The 8 patients consisted of 5 females and 3 males with a median age of 45 years (ranged from 25 to 65 years). The tumor location included 1 uterus, 1 liver, 1 urachus, 2 kidneys, 1 abdominal cavity, 1 colon, and 1 retroperitoneum (3 subsequent recurrences in the abdominal cavity, pelvis and ovary, and abdominal cavity, respectively). Morphologically, the tumor cells were uniform and epithelioid with translucent or eosinophilic cytoplasm. They were arranged in nests or sheets, most of which were separated by thin-walled blood vessels. There were no papillary structures, and no overt smooth muscle or fat components. Atypical features were seen in 3 cases, with bizarre nuclei and tumor giant cells. Large areas of necrosis were visible, and mitosis was common (up to 28/50 HPF). Melanin deposition was present in 3 cases. Immunohistochemical staining showed diffuse and strong positivity for TFE3 in 8/8 cases and for HMB45 in 6/8 cases; focal positivity for Cathepsin K and Melan-A in 6/8 cases and for SMA in 2/8 of cases. All cases were negative for CKpan, PAX8 and Desmin. TFE3 gene break-apart was detected by FISH in all 8 cases, 4 of which underwent next-generation sequencing, and it revealed that 2 cases presented with SFPQ::TFE3 fusion, 1 case with ASPSCR1::TFE3 fusion, and 1 case with no chimeric fusion. Seven cases were followed up for 4-94 months. All cases were alive; 4 cases were disease-free, 2 cases showed recurrence, and 1 case had metastasis at initial diagnosis. Conclusions: TFE3-rearranged PEComa has unique histomorphological, immunohistochemical and molecular characteristics. The biological behavior is aggressive, which could lead to recurrence and metastasis, and warrants close clinical follow-up.

HMGA2 overexpression is found in 10-15% of leiomyomas (LM). HMGA2 overexpression is common in variants of hydropic, intravenous and lipo-LM. Cellular or highly cellular LM (CLM) is a LM variant with a less well-defined molecular nature. In this study, we identified and examined 52 hypercellular LM with sclerotic collagen, herein defined as cellular leiomyoma with sclerosis (CLM-S). CLM-S shows large tumour size (average 12.2 cm) and characteristic histology of tumour cells, arranged in cellular fascicles, sheets and trabeculae with abundant dense, pink sclerotic extracellular matrix in bands and nodules and increased vascularity. Tumour cells are uniform with small, round-oval nuclei and scant, pale-eosinophilic to vacuolated cytoplasm reminiscent of pericytes. The differential diagnosis of CLM-S includes conventional CLM, endometrial stromal tumours and perivascular epithelioid cell tumour. Immunohistochemical profile [HMGA2, fumarate hydratase, smooth muscle markers, Melan A and HMB-45] and molecular alterations [by HMGA2 mRNA reverse transcription-polymerase chain reaction (RT-PCR), HMGA2 fluorescence in-situ hybridisation and MED12 sequencing] were analysed in comparison to matched myometrium and CLM controls. Remarkably, 96% (50 of 52) of CLM-S demonstrated diffuse positive immunoreactivity for HMGA2 and up to an 80-fold increase in HMGA2 mRNA, determined by RT-PCR. FISH analysis with break-part probes at intron 3 and the 5' UTR detected HMGA2 rearrangements in 47% (18 of 38) of CLM-S. All CLM-S retained expression of fumarate hydratase. No MED12 mutations were found in any CLM-S. Our findings show that CLM-S has unique and characteristic histomorphology probably driven by HMGA2 overexpression., (© 2022 John Wiley & Sons Ltd.)

Abstract: Dual immunohistochemical (IHC) staining with D2-40 and S100 improves detection of lymphatic invasion (LI) in primary cutaneous melanoma. However, limited data exist evaluating this technique using other melanocytic markers, and thus, the optimal marker for detection of LI is unestablished. To address this knowledge gap, a case-control study was performed comparing melanoma specimens from 22 patients with known lymphatic spread (LS) with a control group of 11 patients without LS. Specimens underwent dual IHC staining with D2-40 and MART-1, SOX-10, and S100 to evaluate for LI. Receiver operating characteristic analysis was used to estimate each stain's accuracy for detection of LI. The LS group was more likely to be ≥65 years (P = 0.04), have a tumor thickness of ≥1 mm (P < 0.01), and have ulcerated tumors (P = 0.02). Detection of LI with D2-40/MART-1 significantly correlated with LS (P = 0.03), and the D2-40/MART-1 stain was most accurate for LI based on receiver operating characteristic curve analysis (area under the curve [AUC] 0.705) in comparison with D2-40/SOX-10 (AUC 0.575) and D2-40/S100 (AUC 0.633). These findings suggest that MART-1 may be the optimal melanocytic marker to combine with D2-40 for detection of LI in melanoma. Further studies are needed to determine the utility of routinely performing these stains for histopathologic analysis of melanoma., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Aims: Blue naevi are uncommon dermal melanocytic neoplasms characterised by GNAQ/GNA11 mutations, which very rarely progress to melanoma. Such melanomas also often have BAP1 mutations, and lack genetic events associated with conventional melanoma. Exceptionally, blue naevi arise in extracutaneous locations; one melanoma arising in this setting has been reported. We report the clinicopathological, immunohistochemical and molecular genetic features of two cases of melanoma arising in extracutaneous blue naevus., Methods and Results: Both arose in males, aged 25 and 63 years, with no history of other melanocytic lesions, and presented as large, painful intra-abdominal masses. The tumours were dark-brown/black, multilobulated, involved small intestinal mesentery and consisted of a predominantly fascicular and spindled, but occasionally nested and epithelioid, proliferation of variably pigmented, relatively monotonous cells with pale cytoplasm and ovoid nuclei with mild to moderate atypia. Mitotic activity was variable but generally low. Both cases showed areas of conventional and cellular blue naevus. Recurrent tumour in one case showed predominantly epithelioid morphology and greater cytological atypia and mitotic activity. One case expressed Melan-A, SOX10 and CD117, with absent expression of S100 protein and DOG1; the other expressed Melan-A, HMB45 and S100 protein. Next-generation sequencing identified GNAQ and BAP1 mutations in one case and GNA11 mutation in the other. Both patients developed widespread metastatic disease., Conclusion: Exceptionally rare, aggressive melanomas arising in extracutaneous blue naevi should be distinguished from metastatic melanoma, gastrointestinal stromal tumour and malignant melanotic nerve sheath tumour, especially given the significant therapeutic and prognostic differences between these different entities., (© 2020 John Wiley & Sons Ltd.)

Objectives: To describe the cytopathological features of clear cell sarcomas (CCSs), including immunohistochemical and molecular results, the latter in selected cases., Methods: Sixteen consecutively diagnosed cases of CCS of soft tissue, over 6-year duration were included. Fine needle aspiration cytology was performed for primary diagnosis in three and for recurrent/metastatic lesions in 12 cases. Cytopathological features in 16 cases (conventional Papanicolaou- and May-Grünwald Giemsa-stained smears) were critically analysed. Corresponding histopathological and immunostained sections were available in 15 cases. Two cases were tested for EWSR1 gene rearrangement by fluorescence in-situ hybridisation., Results: Sixteen tumours occurred in patients with age ranging from 18 to 56 years (median = 33.5); M: F ratio = 1:1; in deep soft tissues, mostly in extremities. Primary cytopathological diagnosis (3 cases) was CCS with a differential diagnosis of melanoma (1 case) and poorly differentiated malignant tumour (2 cases). On review, smears were predominantly hypercellular (n = 14), invariably composed of monomorphic appearing epithelioid/polygonal cells (n = 16), including spindle cells (n = 6); mostly singly scattered (n = 16), in loose clusters (n = 12); with prominent nucleolisation (n = 16); granular to vacuolated, well-defined cytoplasm (n = 12), binucleation/multinucleation (n = 9); mitoses (n = 6); sudden anisonucleosis; racquet-shaped cells (n = 3), against a tigroid background (n = 2), along with focal intracytoplasmic pigment deposition (n = 2). Immunohistochemically, tumour cells were positive for S-100P (15/15), HMB-45 (15/15) and melan-A(6/12). Two cases tested for EWSR1 rearrangement displayed red-green split signals., Conclusions: This constitutes one of the largest series describing the cytomorphological spectrum of CCS of soft tissue. Certain features, such as singly scattered monomorphic, epithelioid cells with prominent nucleolisation are useful diagnostic clues. Immunohistochemical stains are necessary and molecular testing is further helpful in reinforcing a diagnosis in certain cases. A correct diagnosis has crucial treatment implications., (© 2020 John Wiley & Sons Ltd.)

Long-lasting CD8 + T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate-rich elements (AREs) that are located in the 3' untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3' UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Rab27a, Mlph, and MyoVa form a tripartite complex and relate to melanosome distribution. Melanophilin (Mlph) acts as a linker protein between Rab27a and MyoVa. The biological activity and function of 16-kauren on the expression of Mlph has not yet been studied., Objective: We examined the effect of 16-kauren on melanosome transport and skin pigmentation., Methods: Murine Melan-a melanocytes and SP-1 keratinocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, luciferase assay and immunohistochemical staining in 3D pigmented human skin model were performed., Results: We found that 16-kauren inhibits melanosome transport in Melan-a melanocytes without affecting melanin synthesis. Treatment with 16-kauren reduced melanophilin (Mlph), a key protein in melanosome transport, in Melan-a melanocytes, at both the protein and mRNA levels while it did not affect the expression of Rab27a and MyoVa, the other two key proteins for melanosome transport. Notably, the expression of melanogenic proteins, including tyrosinase, trp1, trp2, and MITF, was not affected by 16-kauren. However, 16-kauren attenuated melanosome distribution in co-culture of Melan-a melanocytes and SP-1 keratinocytes as well as in Melan-a monolayer culture. In further confirmation of the depigmenting effects of 16-kauren on Melanoderm™, a 3D pigmented human skin model, treatment with 16-kauren for 12 days increased the brightness of the tissue as determined by lightness value and reduced the distribution of melanosomes as shown in histological examination., Conclusion: These results demonstrated that 16-kauren is a selective modulator of a melangenic target, Mlph expression, and can be employed as a new depigmenting strategy., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

A cutaneous melanocytic tumor with morphologic overlap with clear cell sarcoma, but defined by CRTC1-TRIM11 gene fusion, was recently described in a series of five adult patients. Here, we expand the clinicopathologic features of this entity by four additional cases which include pediatric presentation, exophytic growth, and propensity to occur on the head. Patients (2F; 2M) had a median age of 41 years (range 11-59). Sites of involvement included leg, ear, and face. Tumors were circumscribed, unencapsulated, mostly limited to the dermis, and varied from 5 to 35 mm. One case was exophytic. Lesional cells were arranged in nests and fascicles, and were monomorphic and fusiform with moderate pale to clear cytoplasm, occasional nuclear pseudo-inclusions, and small to prominent nucleoli. Mitotic rate was variable (rare to 12/10 HPF, median 3/10 HPF). The pediatric case showed increased nuclear pleomorphism, tumor necrosis, and mitotic figures. All cases showed strong, diffuse nuclear staining for SOX10, but were negative or focal for S100 protein, HMB45 and Melan-A expression. Cases were positive by FISH technique and/or RNA sequencing for a TRIM11 rearrangement/fusion, and negative for EWSR1 rearrangement. This series is presented to aid in further characterization of this novel melanocytic tumor., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Since mannose receptors (MRs) are expressed on the surfaces of dendritic cells (DCs), the most professional antigen presenting cells in our body, DNA vaccine carriers containing either covalently grafted mannosyl- or mannose-mimicking shikimoyl-ligands are being increasingly used in ex vivo DC-transfection based DNA vaccination. To this end, we have recently demonstrated that ex vivo immunization of mice with liposomes of shikimoylated cationic amphiphiles containing a 6-amino hexanoic acid spacer group in the head-group region in complexation with melanoma antigen (MART1) encoded DNA vaccine (pCMV-MART1) induces long lasting anti-melanoma immune responses (C. Voshavar, et al., J. Med. Chem., 2017, 60, 1605-1610). This finding prompted us to examine, in the present investigation, the efficacies of gold nanoparticles conjugated to the mannose-mimicking shikimoyl ligand (SL) via a 6-amino hexane thiol spacer (AuNPs-SL) for use in ex vivo DC-transfection based genetic immunization. Herein, we report on the design, synthesis, physico-chemical characterization and bioactivities of AuNPs-SL. Dynamic light scattering and transmission electron microscopy studies revealed the hydrodynamic diameters of theAuNPs-SL nanoconjugates to be within the range of 23-44 nm and their surface potentials within the range of 9-28 mV. MTT-assay showed the non-cytotoxic nature of AuNPs-SL and the findings in the electrophoretic gel retardation assays revealed strong DNA binding properties of the AuNPs-SL. Importantly, subcutaneous immunization of C57BL/6J mice with DCs ex vivo transfected with an electrostatic complex of AuNPs-SL & melanoma antigen (MART1) encoded DNA vaccine (p-CMV-MART1) induced a long lasting (100 days) anti-tumor immune response in immunized mice upon subsequent challenge with a lethal dose of melanoma. Notably, mice immunized with either autologous mbmDCs ex vivo pre-transfected with nanoplexes of shikimoylated AuNPs-SL & an irrelevant pCMV-SPORT-β-gal plasmid (without having encoded melanoma antigen) or untransfected DCs showed no lasting protection against subsequent tumor challenge. The presently described shikimoyl-decorated gold nanoparticles (AuNPs-SL) are expected to find future use in ex vivo DC-transfection based genetic immunization against cancer and other infectious diseases.

Robust anti-tumor immunity requires innate as well as adaptive immune responses. We have shown that plasmacytoid dendritic cells develop killer cell-like activity in melanoma cell cocultures after exposure to the infectious but replication-deficient herpes simplex virus 1 (HSV-1) d 106S. To combine this innate effect with an enhanced adaptive immune response, the gene encoding human MelanA/MART-1 was inserted into HSV-1 d 106S via homologous recombination to increase direct expression of this tumor antigen. Infection of Vero cells using this recombinant virus confirmed MelanA expression by Western blotting, flow cytometry, and immunofluorescence. HSV-1 d 106S-MelanA induced expression of the transgene in fibroblast and melanoma cell lines not naturally expressing MelanA. Infection of a melanoma cell line with CRISPR-Cas9-mediated knockout of MelanA confirmed de novo expression of the transgene in the viral context. Dependent on MelanA expression, infected fibroblast and melanoma cell lines induced degranulation of HLA-matched MelanA-specific CD8 + T cells, followed by killing of infected cells. To study infection of immune cells, we exposed peripheral blood mononuclear cells and in vitro -differentiated macrophages to the parental HSV-1 d 106S, resulting in expression of the transgene GFP in CD11c + cells and macrophages. These data provide evidence that the application of MelanA-encoding HSV-1 d 106S could enhance adaptive immune responses and re-direct MelanA-specific CD8 + T cells to tumor lesions, which have escaped adaptive immune responses via downregulation of their tumor antigen. Hence, HSV-1 d 106S-MelanA harbors the potential to induce innate immune responses in conjunction with adaptive anti-tumor responses by CD8 + T cells, which should be evaluated in further studies.

Immunodeficient mice engrafted with human peripheral blood cells are promising tools for in vivo analysis of human patient individual immune responses. However, when human peripheral blood mononuclear cells (PBMCs) are transferred into NOG (NOD/Shi-scid, IL-2rg null ) mice, severe graft versus host disease (GVHD) hinders long term detailed analysis. Administration of human PBMCs into newly developed murine MHC class I- and class II-deficient NOG (NOG-dKO; NOG- Iab, B2m-double-knockout) mice showed sufficient engraftment of human immune cells with little sign of GVHD. Immunization with influenza vaccine resulted in an increase in influenza-specific human IgG Ab, indicating induction of antigen-specific B cells in the NOG-dKO mice. Immunization with human dendritic cells pulsed with HLA-A2 restricted cytomegalovirus peptide induced specific cytotoxic T cells, indicating the induction of antigen-specific T cells in the NOG-dKO mice. Adoptive cell therapies (ACTs) using melanoma antigen recognized by T cells (MART-1)-specific TCR-transduced activated T cells showed strong tumor growth inhibition in NOG-dKO mice without any sign of GVHD accompanied by preferential expansion of the transferred MART-1-specific T cells. ACTs using cultured human melanoma infiltrating T cells also showed anti-tumor effects against autologous melanoma cells in NOG-dKO mice, in which changes in human cancer phenotypes by immune intervention, such as increased CD271 expression, could be evaluated. Therefore, NOG-dKO mice are useful tools for more detailed analysis of both the induction and effector phases of T-cell and B-cell responses for a longer period than regular NOG mice.

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade. A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations. In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes. We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1. This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients. In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy. We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies. Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes. Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire. Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells. In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches.

Most affinity-maturation campaigns for antibodies and T-cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity-determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single-codon libraries of both CDR and non-CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1·HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (Vα and Vβ) and surface-exposed framework residues. Mutational analyses showed that both Vα/Vβ interface and CDR residues were important in maintaining binding to MART-1·HLA-A2, probably due to either structural requirements for proper Vα/Vβ association or direct contact with the ligand. More surprisingly, many Vα/Vβ interface substitutions yielded improved binding to MART-1·HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (F45βY) was most prevalent. Further analysis of F45βY showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the Vα/Vβ interface, at a significant distance from the TCR·peptide·MHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1·HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The detection of primary anorectal melanoma on anal cytology is a rare and challenging diagnosis. We report a case where anorectal cytology showed isolated malignant cells with oval nuclei, prominent nucleoli, and elongated wispy cytoplasmic projections. There was no evidence of squamous dysplasia or melanin pigment identified. To the best of our knowledge, this is the first reported case of a primary anorectal melanoma detected in anorectal cytology. Detection of malignancies other than squamous cell carcinoma can be seen on anorectal cytology and should be considered when there is no evidence of anal intraepithelial neoplasia. Diagn. Cytopathol. 2017;45:452-455. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Melanoma is the second most common non-hematopoietic malignancy after carcinomas to metastasize to the breast and often appears as a well-circumscribed, dense nodule on imaging. Although metastatic lesions presenting as bilateral cysts have been reported, this presentation is not common and may mimic benign breast cysts. We present a challenging case of metastatic melanoma presenting as bilateral breast cysts with spindled cytomorphology in a patient with a history of mammary carcinoma. Discordance between the spindled cytomorphology and the morphology of the core biopsy, which was similar to the patient's primary breast cancer, allowed for entertainment of other tumors and disease processes. Confirmatory immunostaining of the cytology material with HMB-45 was important to establish the diagnosis of metastatic melanoma. Diagn. Cytopathol. 2017;45:446-451. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Conjunctival melanocytic lesions encompass a group of clinically diverse, benign to malignant, neoplasms that may contain overlapping histopathological features, making definitive diagnosis challenging in some cases. In this series, we compared multiple immunohistochemical (IHC) markers in 11 conjunctival nevi, 10 primary acquired melanosis (PAM) lesions, and 11 conjunctival melanomas. Immunostains included the melanocytic markers HMB-45 and Melan-A, as well as the proliferative marker Ki-67. Loss of beta-catenin expression has been associated with more aggressive clinical disease in cutaneous melanoma, but its status in conjunctival melanocytic lesions is not known, therefore we incorporated beta-catenin immunohistochemical staining in our study. In this series, conjunctival melanomas had a higher Ki-67 proliferative index and HMB-45 immunoreactivity than did PAM lesions and conjunctival nevi (P<0.001). Melan-A was highly expressed in all 3 groups. Beta-catenin was more strongly expressed in melanomas and nevi than in PAM (P<0.001). There was high inter-grader reliability (Kappa=0.53). Overall, IHC labeling of HMB-45 and Ki-67 is increased in conjunctival melanomas compared to PAM or conjunctival nevi. Beta-catenin, an IHC marker previously unstudied in conjunctival melanocytic lesions, is not preferentially expressed in benign lesions and may play a different role in conjunctival atypia than it does in cutaneous melanoma., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The development of BRAF V600 and MEK inhibitors constitutes a breakthrough in the treatment of patients with BRAF-mutated metastatic melanoma. However, although there is an increase in overall survival, these patients generally confront recurrence, and several resistance mechanisms have already been described. In the present study we describe a different resistance mechanism. After several weeks of long‑term in vitro treatment of two different V600E BRAF‑mutated melanoma cell lines with MARK inhibitors, PLX4032 and/or GDC-0973, the majority of the cells died whereas some remained viable and quiescent (SUR). Markedly, discontinuing treatment of SUR cells with MAPK inhibitors allowed the population to regrow and these cells retained drug sensitivity equal to that of the parental cells. SUR cells had increased expression levels of CD271 and ABCB5 and presented senescence-associated characteristics. Notably, SUR cells were efficiently lysed by cytotoxic T lymphocytes recognizing MART-1 and gp100 melanoma differentiation antigens. We propose quiescent plasticity as a mechanism of resistance to BRAF and MEK inhibitors while retaining sensitivity to immune effectors.

Objectives: The aim of this study was to describe a method for analysing histological fragments derived from fine- needle aspirate biopsy (FNAB) of salivary gland tumours (SGTs), and to evaluate the use of immunohistochemistry (IHC) on them., Study Design: We reviewed all 509 FNAB pathology reports taken from SGTs at Helsinki University Hospital, Finland, between 1999 and 2009. In 51% of the cases (n = 209) "histo-fragments" had been obtained and 31 had been further analysed by IHC. Of these, 25 (81%) were available for review. We evaluated the benefit of IHC by relating its added value to the preoperative cytological diagnosis and its accuracy compared with the postoperative histological diagnosis., Results: Most of the samples analysed by IHC were assigned a malignant diagnosis, with 12 different types of malignancy represented. IHC was advantageous in 76% of the cases. In the 108 studies using IHC in this series, antibodies to 36 different antigens were used., Conclusion: Analysis of histo-fragments in FNABs using IHC can be valuable in specific differential diagnostics and raises diagnostic accuracy in SGTs., (© 2016 S. Karger AG, Basel.)

Purpose: Development of liver metastasis remains the most common cause of mortality in uveal melanoma (UM). A few cell lines cultured from primary UM tumors have been used widely to investigate the pathobiology of UM. However, the translation of basic knowledge to the clinic for the treatment of the metastatic disease has remained incremental at best. In this study, we examined whether the properties of UM cell lines at various passages were similar to their corresponding primary tumors., Methods: Gene expression profiling by microarray was performed on UM primary tumors and derived cell lines cultured at varying passages. Expression of UM protein markers was monitored by immunohistochemical analyses and Western blotting. The in vivo tumorigenic properties of UM cultures were evaluated using athymic nude mice., Results: Cell passaging severely reduced the expression of genes encoding markers typical of UM, including those of the prognostic gene signature. Marked differences between gene expression profiles of primary tumors and cell lines could be linked to the infiltrating immune and stromal cells in situ. In addition, the tumorigenic properties of UM cell lines also increased with cell passaging in culture as evaluated by their subcutaneous injection into athymic mice., Conclusions: Together, these findings demonstrate that the short-term UM primary cultures exhibit molecular features that resemble the respective surgical material and, thus, represent the best model for in vitro-assessed cancer treatments.

Reliable identification of different melanoma cell lines is important for many aspects of melanoma research. Common markers used to identify melanoma cell lines include: S100; HMB-45; and Melan-A. We explore the expression of these three markers in four different melanoma cell lines: WM35; WM793; SK-MEL-28; and MM127. The expression of these markers is examined at both the mRNA and protein level. Our results show that the metastatic cell line, MM127, cannot be detected using any of the commonly used melanoma-associated markers. This implies that it would be very difficult to identify this particular cell line in a heterogeneous sample, and as a result this cell line should be used with care.

Aim: The detection of circulating tumour cells in the bloodstream before and after surgical manipulation, and the qualitative detection of potential shedding of tumour cells during surgical manipulation of patients with uveal melanoma., Methods: 202 patients treated for a newly diagnosed uveal melanoma were included in the study. Blood samples were acquired 24 h before and 30 min after the basic surgical steps. Detection of potential circulating melanoma cells was extrapolated from the presence of tyrosinase and MelanA/Mart1 transcripts by reverse transcription PCR., Results: Based on the measurement of tyrosinase transcripts, as a result of the first and second surgical manipulation there were three and zero transitions from negative to positive respectively, while there were two and one transitions from positive to negative, respectively. According to MelanA/Mart1 transcripts, there were 19 and 5 transitions from negative to positive respectively, and 15 and 2 transitions from positive to negative, respectively. No statistically significant differences were documented, concerning the presence of circulating tumour cells in the blood samples acquired before and after the first surgical manipulation or the second one., Conclusion: The change in the percentage of patients with detected tumour cells in their bloodstream was not statistically significant. The frequent shifts from negative to positive samples as well as from positive to negative samples comparing preoperative to postoperative samples indicates discontinuous shedding or variation due to measurements close to the threshold of detection. As a conclusion, the surgical manipulation does not seem to have a measurable contribution to the spread of melanoma cells in the bloodstream., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: Molecular staging of sentinel lymph nodes (SLNs) may identify patients who are node-negative by standard microscopic staging but are at increased risk for regional nodal recurrence; such patients may benefit from completion lymph node dissection (CLND)., Study Design: In a multicenter, randomized clinical trial, patients with tumor-negative SLNs by standard pathology (hematoxylin and eosin [H and E] serial sections and immunohistochemistry [IHC]) underwent reverse transcriptase polymerase chain reaction (PCR) analysis of SLNs for melanoma-specific mRNA. Microscopically negative/PCR+ patients were randomized to observation, CLND, or CLND with high-dose interferon (HDI). For this post-hoc analysis, clinicopathologic features and survival outcomes, including overall survival (OS) and disease-free survival (DFS), were compared between PCR+ patients who underwent CLND vs observation. Microscopic and molecular node-negative (PCR-) patients were included for comparison., Results: A total of 556 patients were PCR+: 180 underwent observation, and 376 underwent CLND. An additional 908 PCR- patients were observed. Median follow-up was 72 months. Disease-free survival (DFS) was significantly better for PCR+ patients who underwent CLND compared with observation (p = 0.0218). No statistically significant differences in OS or distant disease-free survival (DDFS) were seen. Regional lymph node recurrence-free survival (LNRFS) was improved in PCR+ patients with CLND compared to observation (p = 0.0065). The PCR+ patients in the observation group had the worst DFS; those with CLND had similar DFS to that in the PCR- group (p = 0.9044)., Conclusions: Patients with microscopically negative/PCR+ SLN have an increased risk of nodal recurrence that was mitigated by CLND. Although CLND did not affect OS, these data suggest that molecular detection of melanoma-specific mRNA in the SLN predicts a greater risk of nodal recurrence and deserves further study., (Copyright © 2016 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Recent high throughput sequencing analysis has revealed that the TCRβ repertoire is largely different between CD8(+) and CD4(+) T cells. Here, we show that the transduction of SIG35α, the public chain-centric HLA-A*02:01(A2)/MART127-35 TCRα hemichain, conferred A2/MART127-35 reactivity to a substantial subset of both CD8(+) and CD4(+) T cells regardless of their HLA-A2 positivity. T cells individually reconstituted with SIG35α and different A2/MART127-35 TCRβ genes isolated from CD4(+) or CD8(+) T cells exhibited a wide range of avidity. Surprisingly, approximately half of the A2/MART127-35 TCRs derived from CD4(+) T cells, but none from CD8(+) T cells, were stained by A2/MART127-35 monomer and possessed broader cross-reactivity. Our results suggest that the differences in the primary structure of peripheral CD4(+) and CD8(+) TCRβ repertoire indeed result in the differences in their ability to form extraordinarily high avidity T cells which would otherwise have been deleted by central tolerance.

To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B' core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8(+) T cells. Second, we generated the two-vector-based "modular promoter" system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B' core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo.

Human pluripotent stem cells (hPSCs) provide a promising platform to produce dendritic cell (DC) vaccine. To streamline the production process, we investigated a unique antigen-loading strategy that suits this novel platform. Specifically, we stably modified hPSCs using tumour antigen genes in the form of a full-length tumour antigen gene or an artificial tumour antigen epitope-coding minigene. Such antigenically modified hPSCs were able to differentiate into tumour antigen-presenting DCs. Without conventional antigen-loading, DCs derived from the minigene-modified hPSCs were ready to prime a tumour antigen-specific T cell response and further expand these specific T cells in restimulation processes. These expanded tumour antigen-specific T cells were potent effectors with central memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs.

Background: Metastasis of sentinel lymph node (SLN) is generally evaluated on histopathological examination and controversy still exists over the usefulness of PCR assay of SLN., Objective: To investigate the prognostic value of triple-marker PCR assay of SLN., Methods: A total of 165 patients with primary cutaneous melanoma who underwent SLN biopsy were included. Clinical and histopathological data were retrieved from each patient's file and triple-marker PCR assay (tyrosinase, GP-100 and MART-1) was performed on the SLN as well as routine histopathological evaluation. PCR positivity was defined as the expression of all three PCR markers. To evaluate melanoma-specific survival (MSS) and disease-free survival (DFS), we used the Kaplan-Meier method and the log-rank test. Multivariate analyses using the Cox proportional hazards regression model were also performed., Results: Sentinel lymph nodes were identified in all 165 patients: 61 patients (37.0%) were male and 104 (63.0%) were female, with a mean age of 60.2 years. Of the 165 melanomas, 81 (49.1%) were acral lentiginous melanomas. Compared with the patients with PCR positivity (1-2 markers) or PCR negativity, patients with PCR positivity (3 markers) had significantly poor MSS (5-year survival rate, 58.7% vs. 84.4%; P < 0.0001) and DFS (5-year survival rate, 25.0% vs. 83.9%; P < 0.0001), with median follow-up of 42 months for MSS and 38 months for DFS. These survival rates of patients with PCR positivity (3 markers) were lower than those of patients with histopathologically positive SLN. In multivariate analysis, PCR positivity (3 markers) was an independent prognostic factor for both MSS (hazard ratio [HR], 2.81; 95% confidence interval [CI], 1.07-7.33; P = 0.035) and DFS (HR, 2.48; 95% CI, 1.08-5.69; P = 0.032)., Conclusions: The expression of three PCR markers was a significant prognostic factor for both MSS and DFS and might be closely correlated to a dismal prognosis., (© 2014 European Academy of Dermatology and Venereology.)

TCRα- and β-chains cooperatively recognize peptide-MHC complexes. It has been shown that a "chain-centric" TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain-centric HLA-A*02:01(A2)/MART127-35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127-35. Although the generated A2/MART127-35-specific T cells used various TRBV genes, TRBV27 predominated with >10(2) highly diverse and unique clonotypic CDR3β sequences. T cells individually reconstituted with various A2/MART127-35 TRBV27 TCRβ genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127-35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Purpose: Melanoma patients with a high risk of recurrence may benefit from immunotherapy with mRNA-electroporated autologous monocyte-derived dendritic cells (DCs). Further benefit may be found in combining DC-therapy with interferon alfa-2b., Patients and Methods: The long-term clinical outcome of AJCC stage III/IV melanoma patients who had no evidence of disease at the time of treatment with autologous mRNA-electroporated DCs in a single-center pilot clinical trial was analyzed. Antigen loading was accomplished by co-electroporation of mRNA encoding a fusion protein between MAGE-A1, -A3, -C2, Tyrosinase, MelanA/MART-1, or gp100, and an HLA class II-targeting sequence. DCs were administered by 4-6 bi-weekly intradermal injections. IFN-α-2b (5 MIU TIW) was initiated either at recurrence (cohort 1), concomitant with DCs (cohorts 2 and 3), or following the fourth DC administration (cohort 4)., Results: Thirty melanoma patients were recruited between April 2006 and June 2009. DC-related adverse events included grade 2 local injection site reactions in all patients, grade 2 fever and flu-like symptoms in one patient, and skin depigmentation in seven patients. After a median follow-up of over 6 years, the median relapse-free survival is 22 months (95% CI 12-32 months). Twelve patients have died. The median overall survival has not been reached; the 2-year and 4-year survival rates are 93 and 70%, respectively., Conclusions: Adjuvant therapy following the resection of melanoma metastases with autologous mRNA-electroporated DCs, combined with interferon alfa-2b, is tolerable and results in encouraging long-term overall survival rates justifying further evaluation in a randomized clinical trial.

MHC anchor residue-modified "heteroclitic" peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26-35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to "pull" the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation., (© 2014 The Authors. European Journal of Immunology Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs) is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs). However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs' immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1), and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

Sentinel lymph node (SLN) biopsies have widely been used for the detection of occult LN metastasis of malignant melanoma (MM). In addition to conventional biomarkers, we assessed the diagnostic and prognostic significance of melanoma-initiating cell (MIC) markers in SLNs of MM. We examined the expressions of gp100, MART-1 and tyrosinase mRNA for routine diagnosis and those of ABCB5, CD133, nestin, KDM5B, NGFR and RANK mRNA as MIC markers. The presence of micrometastasis was confirmed immunohistochemically using antibodies to S-100, HMB-45, MART-1, and tyrosinase. Discordance between immunohistochemical and molecular data was observed in 14 of 70 (20.0%) patients, among whom five (7.1%) were positive for only molecular markers;two of these five patients tested positive for micrometastasis by repeated immunohistochemical stainings. The quantitative expression levels of gp100, MART-1, and tyrosinase mRNA were significantly higher in the metastatic LNs;the cut-off values remain to be elucidated. ABCB5 mRNA expression was detected more frequently in the metastatic SLNs (p＜0.05) and in the group of patients with recurrence. To make a definite diagnosis of metastasis, we still need a combination of immunohistochemical and molecular probes. ABCB5 might be a suitable molecular marker for the detection of melanoma-initiating cells in SLNs.

Binding of a T-cell receptor (TCR) to a peptide/major histocompatibility complex is the key interaction involved in antigen specificity of T cells. The recognition involves up to six complementarity determining regions (CDR) of the TCR. Efforts to examine the structural basis of these interactions and to exploit them in adoptive T-cell therapies has required the isolation of specific T-cell clones and their clonotypic TCRs. Here we describe a strategy using in vitro-directed evolution of a single TCR to change its peptide specificity, thereby avoiding the need to isolate T-cell clones. The human TCR A6, which recognizes the viral peptide Tax/HLA-A2, was converted to TCR variants that recognized the cancer peptide MART1/HLA-A2. Mutational studies and molecular dynamics simulations identified CDR residues that were predicted to be important in the specificity switch. Thus, in vitro engineering strategies alone can be used to discover TCRs with desired specificities.

Although rare, uveal melanoma (UM) is the most common primary intraocular tumor in adults. About half of UM patients develop metastatic disease typically in the liver and die within a short period, due to ineffective systemic therapies. UM has unique and distinct genetic features predictive of metastasis. Animal models are required to improve our understanding of therapeutic options in disseminated UM. Since spontaneous murine UM models are lacking, our aim was to analyze the suitability of the established transgenic melanoma mouse model Tg(Grm1) as a new UM model system. We demonstrated that adult Grm1 transgenic mice develop choroidal thickening and uveal melanocytic neoplasia with expression of the melanocytic markers S100B and MelanA. Further, we showed that GRM1 is expressed in human UM, similar to skin melanoma. This study presents a new mouse model for spontaneous UM and suggests that the glutamate signaling pathway is a possible target for UM therapy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Advances in genetic engineering have made it possible to generate human T-cell products that carry desired functionalities, such as the ability to recognize cancer cells. The currently used strategies for the generation of gene-modified T-cell products lead to highly differentiated cells within the infusion product, and on the basis of data obtained in preclinical models, this is likely to impact the efficacy of these products. We set out to develop a good manufacturing practice (GMP) protocol that yields T-cell receptor (TCR) gene-modified T-cells with more favorable properties for clinical application. Here, we show the robust clinical-scale production of human peripheral blood T-cells with an early memory phenotype that express a MART-1-specific TCR. By combining selection and stimulation using anti-CD3/CD28 beads for retroviral transduction, followed by expansion in the presence of IL-7 and IL-15, production of a well-defined clinical-scale TCR gene-modified T-cell product could be achieved. A major fraction of the T-cells generated in this fashion were shown to coexpress CD62L and CD45RA, and express CD27 and CD28, indicating a central memory or memory stemlike phenotype. Furthermore, these cells produced IFNγ, TNFα, and IL-2 and displayed cytolytic activity against target cells expressing the relevant antigen. The T-cell products manufactured by this robust and validated GMP production process are now undergoing testing in a phase I/IIa clinical trial in HLA-A*02:01 MART-1-positive advanced stage melanoma patients. To our knowledge, this is the first clinical trial protocol in which the combination of IL-7 and IL-15 has been applied for the generation of gene-modified T-cell products.

Adoptive immunotherapy with Ag-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor Ags are nonreactive "self" proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific TCRs can be transduced into normal PBLs, which persist after transfer in ∼30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable antitumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor Ag-MHC (peptide-MHC [pMHC]), Mart-1 (27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related Ags. Our results showed that the mutated TCRs had improved T cell activation potency while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. Although of somewhat limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.

Purpose: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy., Experimental Design: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation., Results: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells., Conclusion: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses., (©2014 AACR.)

T cell receptors (TCRs) are key to antigen-specific immunity and are increasingly being explored as therapeutics, most visibly in cancer immunotherapy. As TCRs typically possess only low-to-moderate affinity for their peptide/MHC (pMHC) ligands, there is a recognized need to develop affinity-enhanced TCR variants. Previous in vitro engineering efforts have yielded remarkable improvements in TCR affinity, yet concerns exist about the maintenance of peptide specificity and the biological impacts of ultra-high affinity. As opposed to in vitro engineering, computational design can directly address these issues, in theory permitting the rational control of peptide specificity together with relatively controlled increments in affinity. Here we explored the efficacy of computational design with the clinically relevant TCR DMF5, which recognizes nonameric and decameric epitopes from the melanoma-associated Melan-A/MART-1 protein presented by the class I MHC HLA-A2. We tested multiple mutations selected by flexible and rigid modeling protocols, assessed impacts on affinity and specificity, and utilized the data to examine and improve algorithmic performance. We identified multiple mutations that improved binding affinity, and characterized the structure, affinity, and binding kinetics of a previously reported double mutant that exhibits an impressive 400-fold affinity improvement for the decameric pMHC ligand without detectable binding to non-cognate ligands. The structure of this high affinity mutant indicated very little conformational consequences and emphasized the high fidelity of our modeling procedure. Overall, our work showcases the capability of computational design to generate TCRs with improved pMHC affinities while explicitly accounting for peptide specificity, as well as its potential for generating TCRs with customized antigen targeting capabilities.

Optimized delivery of antigens combined with sustainable maturation of dendritic cells (DCs) is crucial for generation of effective antitumoral immune responses. Multiple approaches for ex vivo antigen loading and improvement in immunogenicity have been described. We have recently established a single-step protocol consisting of a fusion peptide (a sequence of the melanoma antigen Melan-A and a cationic cell-penetrating HIV TAT domain) bound in complexes with a toll-like receptor agonist. As the exact cellular uptake mechanisms of TAT-coupled antigens have been a matter of considerable debate and significantly depend on cell type, cargo and concentrations, we evaluated internalization routes into human immature DCs in comparison with non-phagocytic cell lines. We found that Melan-A-TAT fusion peptide uptake by DCs is mainly energy dependent, superior compared with polylysine-coupled Melan-A and significantly higher in DCs as compared with Jurkat cells or HUVECs. Furthermore, we could track the uptake of the fusion peptide exclusively through early endosomes to lysosome compartments after 90 min by fluorescence microscopy and immunoelectron microscopy. Specific endocytosis inhibitors revealed major internalization of the fusion peptide by DCs via clathrin-mediated endocytosis, whereas uptake by non-phagocytic HUVECs differed significantly, involving macropinocytosis as well as clathrin-mediated endocytosis. As our understanding of the processes involved in internalization of TAT-coupled cargos by human DCs broadens, and DC activation becomes available by single-step procedures as described, further development of simultaneous DC maturation and intra-cellular peptide targeting is warranted., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%., (Copyright © 2013 Elsevier Inc. All rights reserved.)

RNA transfection is a standard method to load dendritic cells (DC) with antigen for therapeutic cancer vaccination. While electroporation yields high transfection efficiency and satisfying expression levels, lipofection results in only few cells expressing high amounts of antigen. We compared antigen loading of human monocyte-derived DC by MelanA RNA electroporation and lipofection. No differences in phenotype or migrational capacity were detected, but lipofected DC induced stronger cytokine secretion by antigen-specific T cells and were superior in priming and boosting of MelanA-specific CD8(+) T cells. Interestingly, T cells stimulated with the differently transfected DC did not differ in their functional avidity. To determine whether the amount of antigen per cell is indeed responsible for the superiority of the lipofected DC, we increased the amount of MelanA RNA fivefold and mixed those DC with mock-electroporated ones to mimic the antigen distribution of lipofected cells. This significantly improved the stimulatory capacity, indicating that indeed the amount of antigen per cell seems to be the responsible feature for the observed superiority of lipofected DCs. These data suggest that a few DC that express high amounts of antigen are more immunogenic than many DC expressing lower amounts, although this needs to be tested in a two-armed immunogenicity trial.

Background: Metastatic melanoma involving the esophagus is rare; the occurrence of metastatic melanoma in a background of Barrett esophagus is rarer still. We report a case of an 80 year-old male who presented to our institution for workup of Barrett esophagus with high-grade dysplasia and who proved to have metastatic melanoma occurring in the background of Barrett esophagus, the first report of this kind, to our knowledge, in the English literature., Case Presentation: An 80 year-old Caucasian male was diagnosed at an outside institution with Barrett's esophagus with high grade dysplasia and presented to our institution for therapy. The patient underwent endoscopic mucosal resection using a band ligation technique of an area of nodularity within the Barrett esophagus. Microscopic examination demonstrated extensive Barrett esophagus with high-grade dysplasia as well as a second tumor which was morphologically different from the surrounding high-grade dysplasia and which was positive for S-100, HMB 45 and Melan-A on immunohistochemistry, consistent with melanoma. Further workup of the patient demonstrated multiple radiologic lesions consistent with metastases. Molecular studies demonstrated that the melanoma was positive for the 1799T>A (V600E) mutation in the BRAF gene. The overall features of the tumor were most consistent with metastatic melanoma occurring in a background of Barrett esophagus with high-grade dysplasia., Conclusion: This case demonstrates a unique intersection between a premalignant condition (Barrett esophagus with high grade dysplasia) and a separate malignancy (melanoma). This report also shows the utility of molecular testing to support the hypothesis of primary versus metastatic disease in melanoma.

The genetic modification of peripheral blood lymphocytes using retroviral vectors to redirect T cells against tumor cells has been recently used as a means to generate large numbers of antigen-specific T cells for adoptive cell therapy protocols. However, commonly used retroviral vector-based genetic modification requires T cells to be driven into cell division; this potent mitogenic stimulus is associated with the development of an effector phenotype that may adversely impact upon the long-term engraftment potential and subsequent antitumor effects of T cells. To investigate whether the cytokines used during culture impact upon the engraftment potential of gene-modified T cells, a humanized model employing T cells engrafted with a MART-1-specific T cell receptor adoptively transferred into NOD/Shi-scid IL-2rγ(-/-) (NSG) immune-deficient mice bearing established melanoma tumors was used to compare the effects of the common γ chain cytokines IL-2, IL-7, and IL-15 upon gene-modified T cell activity. MART-1-specific T cells cultured in IL-7 and IL-15 demonstrated greater relative in vitro proliferation and viability of T cells compared with the extensively used IL-2. Moreover, the IL-15 culture prolonged the survival of animals bearing melanoma tumors after adoptive transfer. However, the combination of IL-7 and IL-15 produced T cells with improved engraftment potential compared with IL-15 alone; however, a high rate of xenogeneic graft-versus-host disease prevented the identification of a clear improvement in antitumor effect of these T cells. These results clearly demonstrate modulation of gene-modified T cell engraftment in the NSG mouse, which supports the future testing of the combination of IL-7 and IL-15 in adoptive cell therapy protocols; however, this improved engraftment is also associated with the long-term maintenance of xenoreactive T cells, which limits the ultimate usefulness of the NSG mouse model in this situation.

We have previously reported a model for melanoma progression in which oscillation between melanoma cell phenotypes characterized by invasion or proliferation is fundamental to tumor heterogeneity and disease progression. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by promoting a switch from a proliferative to an invasive phenotype. Immunohistochemistry on primary human cutaneous melanoma biopsies showed intratumoral heterogeneity for cells expressing melanocytic markers, and a loss of these markers correlated with hypoxic regions. Furthermore, we show that the downregulation of melanocytic markers is dependent on hypoxia inducible factor 1α (HIF1α), a known regulator of the hypoxic response. In vitro invasion assays showed that a hypoxic environment increases the invasiveness of proliferative melanoma cell cultures in a HIF1α-dependent manner. In contrast, invasive phenotype melanoma cells showed no increase in invasive potential upon exposure to hypoxia. Thus, exposure of proliferative melanoma cells to hypoxic microenvironments is sufficient, in a HIF1α-dependent manner, to downregulate melanocytic marker expression and increase their invasive potential.

Engaging CD4 T cells in antitumor immunity has been quite challenging, especially in an Ag-specific manner, because most human solid tumors usually do not express MHC class II molecules. We have recently shown that human CD4 T cells engineered to express a human melanoma-associated antigenic epitope, MART-127-35, specific MHC class I-restricted transgenic TCR function as polyfunctional effectors that can exhibit a helper as well as cytolytic effector function, in an epitope-specific and MHC class I-restricted manner (Chhabra et al. 2008. J. Immunol. 181: 1063-1070; Ray et al. 2010. Clin. Immunol. 136: 338-347). TCR-engineered (TCReng) CD4 T cells therefore have translational potential, and clinical trials with MHC class I TCReng CD4 T cells are under way. In this study, we show that although TCReng CD4 T cells could be useful in cancer immunotherapy, they are also susceptible to epitope-specific activation-induced cell death (AICD). We also show that the AICD in TCReng CD4 T cells is a death receptor-independent process and that JNK and p53 play critical roles in this process as pharmacological inhibitors targeting JNK activation and p-53-mediated transcription-independent mitochondria-centric death cascade rescued a significant fraction of TCReng CD4 T cells from undergoing AICD without affecting their effector function. Our data offer novel insights toward AICD in TCReng CD4 T cells and identify several potential targets to interfere with this process.

In this short note we report on the shelf-life stability of pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) 2mg lyophilized powder for reconstitution for intradermal administration, used in an in-house, investigator-initiated clinical phase I study. pDERMATT was stored at 25°C/60% relative humidity (6 months), 2-8°C (24 months), and -20°C (66 months) in the dark and analyzed at several timepoints during the conduct of the clinical study for appearance, identity, purity (plasmid topology), content and residual water content. pDERMATT appeared stable at all storage conditions for the periods tested which, although patient inclusion in the study was significantly delayed, ensured the clinical supply needs. This study shows that lyophilization is an useful tool to preserve the quality of the pDNA and can prevent the need for costly and time-consuming additional manufacture of drug product in case of study delays, not uncommon at the early stage of drug development. To our knowledge, this is the first study reporting shelf life stability of a pDNA formulation for more than 5 years., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy.

Malignant melanoma biologically can be divided into non-metastatic and metastatic forms which cannot be predicted precisely using classical clinicopathological parameters, therefore studies on novel genetic or protein markers are abundant in the literature. These studies did not result in clinically useful markers because mostly ignored the results of studies on the genetic basis of metastatic potential of malignant melanoma. Accordingly, the list of promising novel markers is short (BCL2, CDK2, MART-1, OPN). Similar to other solid malignancies, introduction of targeted therapy into clinical practice of melanoma turned the attention toward the genetic basis of resistance to chemo- and targeted therapies. These novel data could lead to the development of molecular diagnostics which can help in designing more effective therapeutic strategies of malignant melanoma.

Elderly cancer patients are often excluded from immune-based clinical trials and therapies based on the belief that they respond poorly to tumor antigens. Using melanoma as a model and melanoma related Mart-127-35 epitope specific T cell receptor (TCR) engineered T cells as a tool we compared the T cell responses from young and elderly to the Mart-127-35 epitope, ex vivo. We also compared the natural Treg (nTreg) activities and the expression of a number of genes associated with immune response by quantitative real-time reverse Transcription Polymerase Chain Reaction (qRTPCR) in formalin fixed primary melanomas, in situ. We detected a significant difference in CD8(+) T cell response to Flu antigen (influenza matrix peptide Flu MP58-66), but the responses of the two cohorts to melanoma antigen were comparable. nTreg activities in the elderly was significantly compromised. The qPCR analyses of tissues from elderly patients revealed lower levels of Fox-P3 expression but comparable levels of expression of IL-2, IFNγ, TNFα, IL-4, IL-10, IDO, and TGFβ. These findings indicate that elderly patients might be capable of responding to tumor antigens, and need not be excluded from immune-based therapies or clinical trials., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)