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Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34 + CD38 - CD26 + and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34 + CD38 - CD26 + and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26 + ( BCR-ABL1 + ) vs. CD26 - ( BCR-ABL1 - ) CD34 + CD38 - fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34 + CD38 - fractions that distinguishes between CD26 + ( BCR-ABL1 + ) and their CD26 - ( BCR-ABL1 - ) counterparts, providing valuable data for future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ruiz, Sánchez, Bonecker, Furtado, Koile, Yankilevich, Cranco, Custidiano, Freitas, Moiraghi, Pérez, Pavlovsky, Varela, Ventriglia, Sánchez Ávalos, Larripa, Zalcberg, Mordoh, Valent and Bianchini.)

Objectives The quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed. Methods The laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories. Results Our field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA. Conclusions In conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.

Triple Negative Breast Cancer (TNBC) treatment is still challenging, and immunotherapy is a potential approach in this tumor subtype. Cetuximab is an IgG1 monoclonal antibody (mAb) directed against Epidermic Growth Factor Receptor (EGFR), a protein overexpressed in a subgroup of TNBC patients and associated with poor prognosis. Previously, we demonstrated in vitro that Cetuximab triggers Ab-dependent cell cytotoxicity against TNBC cells. In this study, using co-cultures including TNBC cells, and NK and Dendritic Cells (DCs) from healthy donors, we studied the effect of Cetuximab-activated NK cells on DC function. Given that we already demonstrated that TNBC has an immunosuppressive effect on NK cells, we also tested Cetuximab combination with IL-15. We determined that Cetuximab opsonization of TNBC cells increased IFN-γ and TNF-α production by NK cells co-cultured with DCs. Moreover, we showed that NK cells activated by TNBC cells opsonized with Cetuximab promoted tumor material uptake and maturation of DCs, as well as their ability to produce IL-12. Furthermore, the stimulation with IL-15 increased the activation of NK cells and the maturation of DCs. These results suggest that IL-15 may enhance the efficacy of Cetuximab in the treatment of TNBC by promoting activation of both NK cells and DCs., Competing Interests: The authors declare no conflict of interest.

The CSF-470 vaccine consists of lethally-irradiated allogeneic cells derived from four cutaneous melanoma cell lines administered plus BCG and GM-CSF as adjuvants. In an adjuvant phase II study vs. IFN-α2b, the vaccine significantly prolonged the distant metastasis-free survival (DMFS) of stages IIB-IIC-III melanoma patients with evidence of the induction of immune responses against vaccine cells. Purpose: The aim of this study was to analyze the antigens against which the immune response was induced, as well as the T-helper profile and lytic ability of immune cells after CSF-470 treatment. Methods: HLA-restricted peptides from tumor-associated antigens (TAAs) were selected from TANTIGEN database for 13 evaluable vaccinated patients. In addition, for patient #006 (pt#006), tumor somatic variants were identified by NGS and candidate neoAgs were selected by predicted HLA binding affinity and similarity between wild type (wt) and mutant peptides. The patient's PBMC reactivity against selected peptides was detected by IFNγ-ELISPOT. T-helper transcriptional profile was determined by quantifying GATA-3, T-bet, and FOXP3 mRNA by RT-PCR, and intracellular cytokines were analyzed by flow cytometry. Autologous tumor cell lysis by PBMC was assessed in an in vitro calcein release assay. Results: Vaccinated patient's PBMC reactivity against selected TAAs derived peptides showed a progressive increase in the number of IFNγ-producing cells throughout the 2-yr vaccination protocol. ELISPOT response correlated with delayed type hypersensitivity (DTH) reaction to CSF-470 vaccine cells. Early upregulation of GATA-3 and Foxp3 mRNA, as well as an increase in CD4+IL4+cells, was associated with a low DMFS. Also, IFNγ response against 9/73 predicted neoAgs was evidenced in the case of pt#006; 7/9 emerged after vaccination. We verified in pt# 006 that post-vaccination PBMC boosted in vitro with the vaccine lysate were able to lyse autologous tumor cells. Conclusions: A progressive increase in the immune response against TAAs expressed in the vaccine and in the patient's tumor was induced by CSF-470 vaccination. In pt#006, we demonstrated immune recognition of patient's specific neoAgs, which emerged after vaccination. These results suggest that an initial response against shared TAAs could further stimulate an immune response against autologous tumor neoAgs., (Copyright © 2020 Podaza, Carri, Aris, von Euw, Bravo, Blanco, Ortiz Wilczyñski, Koile, Yankilevich, Nielsen, Mordoh and Barrio.)

It is estimated that ~50% of patients with melanoma harbour B‑Raf (BRAF)V600 driver mutations, with the most common of these being BRAFV600E, which leads to the activation of mitogen‑activated protein kinase proliferative and survival pathways. BRAF inhibitors are used extensively to treat BRAF‑mutated metastatic melanoma; however, acquired resistance occurs in the majority of patients. The effects of long‑term treatment with PLX4032 (BRAFV600 inhibitor) were studied in vitro on sensitive V600E BRAF‑mutated melanoma cell lines. After several weeks of treatment with PLX4032, the majority of the melanoma cells died; however, a proportion of cells remained viable and quiescent, presenting senescent cancer stem cell‑like characteristics. This surviving population was termed SUR cells, as discontinuing treatment allowed the population to regrow while retaining equal drug sensitivity to that of parental cells. RNA sequencing analysis revealed that SUR cells exhibit changes in the expression of 1,415 genes (P<0.05) compared with parental cells. Changes in the expression levels of a number of epigenetic regulators were also observed. These changes and the reversible nature of the senescence state were consistent with epigenetic regulation; thus, it was investigated as to whether the senescent state could be reversed by epigenetic inhibitors. It was found that both parental and SUR cells were sensitive to different histone deacetylase (HDAC) inhibitors, such as SAHA and MGCD0103, and to the cyclin‑dependent kinase (CDK)9 inhibitor, CDKI‑73, which induced apoptosis and reduced proliferation both in the parental and SUR populations. The results suggested that the combination of PLX4032 with HDAC and CDK9 inhibitors may achieve complete elimination of SUR cells that persist after BRAF inhibitor treatment, and reduce the development of resistance to BRAF inhibitors.

Patients with non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) have shown benefit from anti-PD-1 therapies. However, not all patients experience tumor shrinkage, durable responses or prolonged survival, demonstrating the need to find response markers. In blood samples from NSCLC and RCC patients obtained before and after anti-PD-1 treatment, we studied leukocytes by complete blood cell count, lymphocyte subsets using flow cytometry and plasma concentration of nine soluble mediators, in order to find predictive biomarkers of response and to study changes produced after anti-PD-1 therapy. In baseline samples, discriminant analysis revealed a combination of four variables that helped differentiate stable disease-response (SD-R) from progressive disease (PD) patients: augmented frequency of central memory CD4 + T cells and leukocyte count was associated with response while increased percentage of PD-L1 + natural killer cells and naïve CD4 +  T cells was associated with lack of response. After therapy, differential changes between responders and non-responders were found in leukocytes, T cells and TIM-3 + T cells. Patients with progressive disease showed an increase in the frequency of TIM-3 expressing CD4 + and CD8 + T cells, whereas SD-R patients showed a decrease in these subsets. Our findings indicate that a combination of immune variables from peripheral blood (PB) could be useful to distinguish response groups in NSCLC and RCC patients treated with anti-PD-1 therapy. Frequency of TIM-3 + T cells showed differential changes after treatment in PD vs SD-R patients, suggesting that it may be an interesting marker for monitoring progression during therapy.

The CSF-470 cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-α2b (CASVAC-0401 study). Patient-045 developed a mature vaccination site (VAC-SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start. CDR3-TCRβ repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-γ ELISPOT assay, were performed to analyze the T-cell immune response dynamics throughout the immunization protocol. Histopathological analysis of the VAC-SITE revealed a highly-inflamed granulomatous structure encircled by CD11c + nested-clusters, brisk CD8 + and scarce FOXP3 + , lymphocytes with numerous Langhans multinucleated-giant-cells and macrophages. A large tumor-regression area fulfilled the C-MTS with brisk lymphocyte infiltration, mainly composed of CD8 + PD1 + T-cells, CD20 + B-cells, and scarce FOXP3 + cells. Increasing DTH score and IFN-γ ELISPOT assay signal against the CSF-470 vaccine-lysate was evidenced throughout immunization. TCRβ repertoire analysis revealed for the first time the presence of common clonotypes between a VAC-SITE and a C-MTS; most of them persisted in blood by the end of the immunization protocol. In vitro boost with vaccine-lysate revealed the expansion of persistent clones that infiltrated the VAC-SITE and/or the C-MTS; other persistent clones expanded in the patient's blood as well. We propose that expansion of such persistent clonotypes might derive from two different although complementary mechanisms: the proliferation of specific clones as well as the expansion of redundant clones, which increased the number of nucleotide rearrangements per clonotype, suggesting a functional antigenic selection. In this patient, immunization with the CSF-470 vaccine plus BCG and rhGM-CSF induced a T-cell repertoire at the VAC-SITE that was able to infiltrate an emerging C-MTS, which resulted in the expansion of a T-cell repertoire that persisted in blood by the end of the 2-year treatment., (Copyright © 2019 Aris, Bravo, Garcia Alvarez, Carri, Podaza, Blanco, Rotondaro, Bentivegna, Nielsen, Barrio and Mordoh.)

A major obstacle to obtaining relevant results in cancer vaccination has been the lack of identification of immunogenic antigens. Dendritic cell (DC)-based cancer vaccines used preventively may afford protection against tumor inoculation, but the effect of antigen choice on anti-tumor protection is not clear. When using irradiated syngeneic tumor cells to load DCs, tumor self-antigens are provided, including tumor-associated antigens (TAAs) and neoantigens generated by tumor mutations. On the other hand, allogeneic tumor cells could only supply shared TAAs. To assess the advantages of each source in protective vaccination, we analyzed in C57BL/6 mice the effect of loading DCs with irradiated syngeneic B16-F1 or allogeneic Cloudman melanoma cells; both cell lines were characterized by whole exome sequencing and RNAseq. Tumor cell components from the two irradiated cell lines were efficiently internalized by DCs, and transported to MHC-class II positive tubulovesicular compartments (MIICs). DCs loaded with allogeneic irradiated Cloudman cells (DC-ApoNec ALLO ) induced a partially effective anti-melanoma protection, although Cloudman and B16-F1 cells share the expression of melanocyte differentiation antigens (MDAs), cancer-testis antigens (CTAs) and other TAAs. DCs loaded with syngeneic B16-F1 cells (DC-ApoNec SYN ) established a more potent and long-lasting protection and induced a humoral anti-B16F1 response, thus suggesting that neoepitopes are needed for inducing long-lasting protection., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As cutaneous melanoma (CM) currently remains with a bleak prognosis, thorough investigation of new treatment options are of utmost relevance. In the phase II/III randomized clinical trial (CASVAC-0401), the repeated immunization of stages IIB-III CM patients with the irradiated, allogeneic cellular CSF-470 vaccine plus the adjuvants bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) demonstrated a significant benefit over IFN-alpha2B treatment in distant metastasis-free survival. Here we present on the short and long term immune monitoring results after completing the 2-year protocol; a continuation of the previous report by Mordoh et al. (1). We demonstrate that the repeated CSF-470 vaccinations stimulated a long term cellular and humoral immunity response directed against the vaccine antigens. In the case of 2 patients, we are able to show that a similar immune response was generated against autologous antigens. Evaluation of inhibitory receptor co-expression on patient's T cells indicates that the vaccination protocol did not stimulate T cell exhaustion. In order to better understand the basis for the efficacious vaccine responses observed, we investigated the short term immune events following vaccine injection. A significant increase in C-reactive protein (CRP) and IL-6 was observed 24 h after vaccination, with in vitro studies suggesting IL-6 production occurs in the vaccine site. We demonstrate that CRP enhances the cytotoxicity of peripheral blood mononuclear cells (PBMC) against melanoma cells in an in vitro model. Additionally, CRP stimulates the release of pro and anti-inflammatory cytokines from PBMC. As our results demonstrate that successive vaccinations with CSF-470 plus adjuvants promoted an increase in both anti-tumor innate and adaptive immunity, we propose a subsequent model of action.

The standard treatment for Triple Negative Breast Cancer (TNBC) patients is cytotoxic chemotherapy, but it is restricted since the duration of response is usually short. Blocking the PD-1/PD-L1 pathway through monoclonal antibodies (mAbs) appears to be a promising therapeutic strategy for TNBC patients. Avelumab is a human IgG1 anti-PD-L1 mAb being tested in clinical trials that may also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells as an additional antitumor activity. In the present work, we studied in vitro Avelumab-mediated ADCC against a panel of TNBC cells with different PD-L1 expression using peripheral blood mononuclear cells (PBMC) or purified NK cells from healthy donors. We determined that Avelumab significantly enhanced NK-cell mediated cytotoxicity against TNBC cells and that tumor cells expressing higher levels of PD-L1 were more sensitive to Avelumab-mediated ADCC. IFN-γ treatment upregulated PD-L1 expression in tumor cells but had a variable impact on Avelumab-mediated ADCC, which could be related to the simultaneous effect of IFN-γ on the expression of NK cell ligands. Moreover, IL-2 and IL-15 stimulation of NK cells enhanced Avelumab-triggered cytokine production and degranulation along with increased lytic activity against tumor cells. Improving the treatment of TNBC remains still a considerable challenge. This in vitro study suggests that Avelumab-mediated ADCC, independently of the blockade of the PD-1/PD-L1 pathway, could be a valuable mechanism for tumor cell elimination in TNBC. Avelumab combination with immunomodulators such as IL-15 or IL-2 could be taken into consideration to increase the therapeutic efficacy of Avelumab in TNBC.

Purpose: Breast cancer (BC) is a highly heterogeneous disease presenting a broad range of clinical and molecular characteristics. In the past years, a growing body of evidence demonstrated that immune response plays a significant role in cancer outcome. However, immune prognostic markers are not completely validated in clinical practice in BC patients., Materials and Methods: With the aim to characterize immune features, several parameters were analyzed in peripheral blood at diagnosis of 85 nonmetastatic BC patients between April 2011 and July 2014., Results: With a median follow-up of 38.6 months, peripheral blood analysis of BC patients (stages I, II, and III) showed that total lymphocyte and T lymphocyte counts were augmented in nonrelapsed patients. Also, a higher neutrophil-to-lymphocytes ratio was associated with prolonged disease-free survival. Natural killer cell receptor analysis revealed that early activation receptor CD69 was associated with a better outcome., Conclusion: This preliminary evidence is in accordance with the concept of immune surveillance. We suggest an "immune phenotype" that provides relevant prognostic information in early-stage BC patients and which could be useful in the decision-making process., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB-IIC-III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8 + , CD4 + , and CD20 + lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAF V600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4 + and CD8 + cells by the end of the immunization protocol. By CDR3-T-cell receptor β (TCRβ) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCRβ repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.

Quantification of BCR-ABL1 mRNA levels in peripheral blood of chronic myeloid leukemia patients is a strong indicator of response to tyrosine-kinase inhibitors (TKI) treatment. However, additional prognostic markers are needed in order to better classify patients. The hypothesis of leukemic stem cells (LSCs) heterogeneity and persistence, suggests that their functional evaluation could be of clinical interest. In this work, we assessed the primitive and progenitor fractions in patients at diagnosis and during TKI treatment using functional in vitro assays, defining a "functional leukemic burden" (FLB). We observed that the FLB was reduced in vivo in both fractions upon treatment. However, different FLB levels were observed among patients according to their response to treatment, suggesting that quantification of the FLB could complement early molecular monitoring. Given that FLB assessment is limited by BCR-ABL1 mRNA expression levels, we developed a novel detection method of primitive cells at the DNA level, using patient-specific primers and direct nested PCR in colonies obtained from functional in vitro assays. We believe that this method could be useful in the context of discontinuation trials, given that it is unknown whether the persistent leukemic clone represents LSCs, able to resume the leukemia upon TKI removal., Competing Interests: CONFLICTS OF INTEREST We state that this study was supported financially by a Novartis grant; Dr. Michele Bianchini has received speaker fees from Novartis; all other authors declare no competing financial interests.

In a previous work, we showed that CSF-470 vaccine plus bacillus Calmette-Guerin (BCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) as adjuvants resulted in a significant benefit in the distant metastasis-free survival when comparing vaccinated vs . IFN-α2b-treated high-risk cutaneous melanoma patients in a Phase II study. Immune monitoring demonstrated an increase in anti-tumor innate and adaptive immunities of vaccinated patients, with a striking increase in IFN-γ secreting lymphocytes specific for melanoma antigens (Ags). In an effort to dissect the first steps of the immune response elicited by CSF-470 vaccine plus adjuvants, we evaluated, in an in vitro model, leukocyte migration, cytokine production, and monocyte phagocytosis of vaccine cells. Our results demonstrate that leukocytes recruitment, mostly from the innate immune system, is an early event after CSF-470 vaccine plus BCG plus GM-CSF interaction with immune cells, possibly explained by the high expression of CCL2/MCP-1 and other chemokines by vaccine cells. Early release of TNF-α and IL-1β pro-inflammatory cytokines and efficient tumor Ags phagocytosis by monocytes take place and would probably create a favorable context for Ag processing and presentation. Although the presence of the vaccine cells hampered cytokines production stimulated by BCG in a mechanism partially mediated by TGF-β and IL-10, still significant levels of TNF-α and IL-1β could be detected. Thus, BCG was required to induce local inflammation in the presence of CSF-470 vaccine cells.

In the last few years, there has been a twist in cancer treatment toward immunotherapy thanks to the impressive results seen in advanced patients from several tumor pathologies. Cutaneous melanoma is a highly mutated and immunogenic tumor that has been a test field for the development of immunotherapy. However, there is still a way on the road to achieving complete and long-lasting responses in most patients. It is desirable that immunotherapeutic strategies induce diverse immune reactivity specific to tumor antigens, including the so-called neoantigens, as well as the blockade of immunosuppressive mechanisms. In this review, we will go through the role of promising monoclonal antibodies in cancer immunotherapy with immunomodulatory function, especially blocking of the inhibitory immune checkpoints CTLA-4 and PD-1, in combination with different immunotherapeutic strategies such as vaccines. We will discuss the rational basis for these combinatorial approaches as well as different schemes currently under study for cutaneous melanoma in the clinical trials arena. In this way, the combination of "push and release" immunomodulatory therapies can contribute to achieving a more robust and durable antitumor immune response in patients.

The irradiated, allogeneic, cellular CSF-470 vaccine plus Bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) is being tested against medium-dose IFN-α2b in stages IIB-III cutaneous melanoma (CM) patients (pts) after surgery in an open, randomized, Phase II/III study. We present the results of the Phase II part of the ongoing CASVAC-0401 study (ClinicalTrials.gov: NCT01729663). Thirty-one pts were randomized to the CSF-470 vaccine ( n  = 20) or to the IFN-α2b arm ( n  = 11). During the 2-year treatment, immunized pts should receive 13 vaccinations. On day 1 of each visit, 1.6 × 10 7 irradiated CSF-470 cells plus 10 6 colony-forming units BCG plus 100 µg rhGM-CSF were administered intradermally, followed on days 2-4 by 100 µg rhGM-CSF. IFN-α2b pts should receive 10 million units (MU)/day/5 days a week for 4 weeks; then 5 MU thrice weekly for 23 months. Toxicity and quality of life (QOL) were evaluated at each visit. With a mean and a maximum follow-up of 39.4 and 83 months, respectively, a significant benefit in the distant metastasis-free survival (DMFS) for CSF-470 was observed ( p  = 0.022). Immune monitoring showed an increase in antitumoral cellular and humoral response in vaccinated pts. CSF-470 was well tolerated; 20/20 pts presented grades 1-2 dermic reactions at the vaccination site; 3/20 pts presented grade 3 allergic reactions. Other adverse events (AEs) were grade 1. Pts in the IFN-α2b arm presented grades 2-3 hematological (7/11), hepatic (2/11), and cardiac (1/11) toxicity; AEs in 9/11 pts forced treatment interruptions. QOL was significantly superior in the vaccine arm ( p  < 0.0001). Our results suggest that CSF-470 vaccine plus BCG plus GM-CSF can significantly prolong, with lower toxicity, the DMFS of high-risk CM pts with respect to medium-dose IFN-α2b. The continuation of a Phase III part of the CASVAC-0401 study is encouraged.

Metallothioneins (MTs) are a family of low-molecular-weight, cysteine-rich proteins involved in zinc and redox metabolism, that are epigenetically downregulated during colorectal cancer (CRC) progression, but may be re-induced with a variety of agents. Since loss of MT expression is associated with a worse prognosis, in the present study we investigated the effects of overexpression of the most significantly downregulated isoform in CRC, namely MT1G, on the HT-29 cell line. Overexpression of MT1G resulted in xenograft tumors with an aberrant morphology, characterized by an evident increase in mucin-containing cells that were identified as goblet cells under electron microscopy. Immunohistochemical detection of CDX2 and cytokeratin 20 was also increased, as were goblet‑cell and enterocyte-specific genes by qRT-PCR. Microarray analysis of gene expression confirmed the alteration of several differentiation signaling pathways, including the Notch pathway. Using sodium butyrate and post-confluent growth as inducers of differentiation, we demonstrated that MT1G does indeed play a functional role in promoting goblet over enterocyte differentiation in vitro. Labile zinc is also induced upon differentiation of CRC cells, functionally contributing to enterocyte over goblet differentiation, as revealed using zinc‑specific chelating agents. Overall, our results uncover a new tumor-suppressor activity of MT1G in promoting the differentiation of at least some CRC tumors, and implicate MTs and zinc signaling as new players in colorectal differentiation. This further contributes to the hypothesis that re-induction of MTs may have therapeutic value by diminishing the aggressiveness of CRC tumors.

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.

The clinical outcome of colorectal cancer (CRC) is associated with the immune response; thus, these tumors could be responsive to different immune therapy approaches. Natural killer (NK) cells are key antitumor primary effectors that can eliminate CRC cells without prior immunization. We previously determined that NK cells from the local tumor environment of CRC tumors display a profoundly altered phenotype compared with circulating NK cells from healthy donors (HD). In this study, we evaluated peripheral blood NK cells from untreated patients and their possible role in metastasis progression. We observed profound deregulation in receptor expression even in early stages of disease compared with HD. CRC-NK cells displayed underexpression of CD16, NKG2D, DNAM-1, CD161, NKp46, and NKp30 activating receptors, while inhibitory receptors CD85j and NKG2A were overexpressed. This inhibited phenotype affected cytotoxic functionality against CRC cells and interferon-γ production. We also determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells' antitumor activity. Moreover, NKp46 expression correlated with relapse-free survival of CRC patients with a maximum follow-up of 71 months. CRC-NK cells also exhibited altered antibody-dependent cellular cytotoxicity function responding poorly to cetuximab. IL-2 and IL-15 in combination with cetuximab stimulated NK cell, improving cytotoxicity. These results show potential strategies to enhance CRC-NK cell activity.

Dendritic cells (DCs) are professional APCs used for the development of cancer vaccines because of their ability to activate adaptive immune responses. Previously, we designed the DC/Apo-Nec vaccine using human DCs loaded with dying melanoma cells that primed Ag-specific cytotoxic T cells. Here, we evaluate the effect of a standard pro-inflammatory cytokine cocktail (CC) and adjuvants on DC/Apo-Nec maturation and migration. CC addition to the vaccine coculture allowed efficient Ag uptake while attaining strong vaccine maturation with an immunostimulatory profile. The use of CC not only increased CCR7 expression and the vaccine chemokine responsiveness but also upregulated matrix metalloproteinase-9 secretion, which regulated its invasive migration in vitro. Neither IL-6 nor prostaglandin E2 had a negative effect on vaccine preparation. In fact, all CC components were necessary for complete vaccine maturation. Subcutaneously injected DC/Apo-Nec vaccine migrated rapidly to draining LNs in nude mice, accumulating regionally after 48 h. The migrating cells of the CC-matured vaccine augmented in proportion and range of distribution, an effect that increased further with the topical administration of imiquimod cream. The migrating proportion of human DCs was detected in draining LNs for at least 9 days after injection. The addition of CC during DC/Apo-Nec preparation enhanced vaccine performance by improving maturation and response to LN signals and by conferring a motile and invasive vaccine phenotype both in vitro and in vivo. More importantly, the vaccine could be combined with different adjuvants. Therefore, this DC-based vaccine design shows great potential value for clinical translation.

Clinical studies suggest that triple negative breast cancer (TNBC) patients with epidermal growth factor receptor (EGFR)-expressing tumors could benefit from therapy with Cetuximab, which targets EGFR. NK cells are the primary effectors of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) and thus play a role in Ab-based therapies. We have previously described diminished levels of Cetuximab-mediated ADCC in vitro in patients with advanced breast cancer. Here, we investigated the potential causes of this NK-cell functional deficiency. We characterized NK-cell activating/inhibitory receptors in the peripheral blood of breast cancer patients and found CD85j inhibitory receptor overexpression. The capacity of NK cells to perform Cetuximab-triggered ADCC against TNBC cells correlated inversely with CD85j expression, even in the presence of the stimulatory cytokines IL-2 or IL-15. Hence, patients expressing high levels of CD85j had an impaired ability to lyse TNBC cells in the presence of Cetuximab. We also found that CD85j overexpression was associated with HLA-I and soluble HLA-G expression by tumors. A CD85j functional blockade with a CD85j antagonist Ab restored ADCC levels in breast cancer patients and reverted this negative effect. Our data suggest that strategies that overcome the hurdles of immune activation could improve Cetuximab clinical efficacy., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have developed a therapeutic vaccine consisting of a mixture of lethally-irradiated allogeneic cutaneous melanoma cell lines with BCG and GM-CSF as adjuvants. The CSF-470 vaccine is currently being assayed in a Phase II-III trial against medium-dose IFN-α2b. All vaccinated patients immunized intradermally developed large edematous erythema reactions, which then transformed into subcutaneous nodules active for several months. However, vaccine injection sites were not routinely biopsied. We describe the case of a female patient, previously classified as stage III, but who, due to the simultaneous discovery of bone metastases only received one vaccination was withdrawn from the study, and continued her treatment elsewhere. This patient developed a post-vaccination nodule which was surgically removed 7 weeks later, and allowed to analyze the reactivity and immune profiling of the inoculation site. An inflammatory reaction with zones of fibrosis, high irrigation, and brisk lymphoid infiltration, primarily composed of CD8(+) and CD20(+) lymphocytes, was observed. There were no remaining BCG bacilli, and scarce CD4(+) and Foxp3(+) T cells were determined. MART-1 Ag was found throughout the vaccination site. CD11c(+) Ag presenting cells were either dispersed or forming dense nests. Some CD11c(+) cells proliferated; most of them contained intracellular MART-1 Ag, and some interacted with CD8(+) lymphocytes. These observations suggest a potent, long-lasting local inflammatory response with recruitment of Ag-presenting cells that incorporate melanoma Ags, probably leading to Ag presentation to naïve T cells.

Cancer immunotherapy has emerged as a treatment modality, mainly as the result of discoveries in the immune response regulation, including mechanisms that turn off immune responses. Immunogenic cutaneous melanoma is a canonical model for therapeutic immunotherapy studies. "Passive" immunotherapy with monoclonal antibodies (mAbs) has outpaced "active" immunotherapy with anti-tumor vaccines, and mAbs that antagonize the off responses have been recently introduced in clinical practice. Despite these recent successes, many unresolved practical and theoretical questions remain. Notably unknown are the identity of the lymphocytes that eliminate tumor cells, which white cells enter into tumors, through which endothelium, in what order, and how they perform their task. The parameters of size and location that could be used to determine in which tumors the immune response may be sufficient to eradicate the tumor are yet unknown. Immunotherapy has been so far more efficient to treat solid and hematologic tumors located outside the central nervous system, than primary brain tumors and brain metastases. In contrast to recent advances with mAbs, anti-tumor vaccine development has been lagging behind. The multiplicity of antigens that must be targeted to achieve significant clinical response is partially responsible for this lag, especially in melanoma, one of the most mutated tumors. Further hampering vaccination results is the fact that tumor elimination by the immune system is the result of a race between tumors with different growth rates and the relatively slow development of the adaptive immune response. The enhancement of the native arm of the immune response or the administration of targeted chemotherapy to slow tumor development, are approaches that should be studied. Finally, criteria used to analyze patient response to immunotherapeutic treatments must be perfected, and the patient populations that could benefit the most from this approach must be better defined.

Metallothioneins (MT) are a family of low molecular weight proteins that are silenced during colorectal cancer progression, mainly through epigenetic mechanisms, and this loss is associated with poor survival. In this article, we show that overexpression of the MT1G isoform sensitizes colorectal cell lines to the chemotherapeutic agents oxaliplatin (OXA) and 5-fluorouracil (5-FU), in part through enhancing p53 and repressing NF-κB activity. Despite being silenced, MTs can be reinduced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate. In fact, this induction contributes to the cytotoxicity of these agents, given that silencing of MTs by siRNAs reduces their growth-inhibitory activities. Zinc ions also potently enhance MT expression and are cytotoxic to cancer cells. We show for the first time that OXA and 5-FU induce higher levels of intracellular labile zinc, as measured using the fluorescent probe FLUOZIN-3, and that such zinc contributes to the activation of p53 and repression of NF-κB. Addition of zinc enhanced growth inhibition by OXA and 5-FU, and was also capable of resensitizing 5-FU-resistant cell lines to levels comparable with sensitive cell lines. This effect was MT independent because silencing MTs did not affect zinc cytotoxicity. In conclusion, we show that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents.

CSF470 vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is currently being tested in a Phase II/III Clinical trial in stage II/III melanoma patients. To prepare vaccine doses, irradiated melanoma cell lines are frozen using dimethyl sulfoxide (Me(2)SO) and stored in liquid nitrogen (liqN(2)). Prior to inoculation, doses must be thawed, washed to remove Me(2)SO and suspended for clinical administration. Avoiding the use of Me(2)SO and storage in liqN(2) would allow future freeze-drying of CSF470 vaccine to facilitate pharmaceutical production and distribution. We worked on the development of an alternative cryopreservation methodology while keeping the vaccine's biological and immunogenic properties. We tested different freezing media containing trehalose suitable to remain as excipients in a freeze-dried product, to cryopreserve melanoma cells either before or after gamma irradiation. Melanoma cells incorporated trehalose after 5 h incubation at 37°C by fluid-phase endocytosis, reaching an intracellular concentration that varied between 70-140 mM depending on the cell line. Optimal freezing conditions were 0.2 M trehalose and 30 mg/ml human serum albumin, at -84°C. Vaccine doses could be frozen in trehalose at -84°C for at least four months keeping their cellular integrity, antigen expression and apoptosis/necrosis profile after gamma-irradiation as compared to Me(2)SO control. Non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryopreservation procedures. Trehalose-freezing medium allowed us to cryopreserve melanoma cells, either alive or after gamma irradiation, at -84°C avoiding the use of Me(2)SO and liqN(2) storage. These cryopreservation conditions could be suitable for future freeze-drying of CSF470 vaccine., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Hormonal treatment might affect the immune response to tumor antigens induced in cancer patients who are being vaccinated., Case Presentation: A 33-year-old woman was diagnosed with cutaneous melanoma in May 2009. Her melanoma was located in the intermammary sulcus, had a Breslow thickness of 4 mm, a Clark's level IV, it was ulcerated and highly melanotic. The bilateral sentinel node biopsy was negative. She entered into a randomized Phase II/III clinical study comparing a vaccine composed of irradiated melanoma cells plus BCG plus GM-CSF versus IFN-alpha 2b and she was assigned to the vaccine arm. During the two years treatment she remained disease-free; the final CAT scan being performed in August 2011. Between November and December 2011, her gynecologist treated her with three cycles of 200 mg progesterone/day for ten days, every two weeks, for ovary dysfunction. In November 2011 the patient returned to the Hospital for clinical and imaging evaluation and no evidence of disease was found. At the next visit in March 2012 an ultrasound revealed multiple, large metastases in the liver. A CAT scan confirmed the presence of liver, adrenal glands and spleen metastases. A needle biopsy of a liver lesion revealed metastatic melanoma of similar characteristics to the original tumor. We suggest that progesterone treatment triggered proliferation of so far dormant micrometastases that were controlled during CSF470 vaccine treatment., Conclusion: The use of progesterone in patients with melanoma that are under immunological treatments should be carefully considered, since progesterone could modify the balance of pro-inflammatory and Th1 functions to a regulatory and anti-inflammatory profile of the immune system that could have an impact in tumor progression.

Despite NK cells being originally identified because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltration of malignant tumors, especially in humans. NK cells infiltrating human colorectal carcinomas (CRCs) were analyzed to identify their potential protective role in an antitumor immune response. The expression and function of relevant molecules were analyzed from different sources, comparing tumor-associated NK cells (TANKs) with autologous peripheral blood NK cells (PB-NKs) from CRC patients-the latter in comparison with PB-NKs from normal donors. TANKs displayed a profound alteration of their phenotype with a drastic reduction of NK cell receptor expression. Co-culture experiments showed that CRC cells produce modulation in NK phenotype and functionality. Moreover, PB-NKs from CRC patients also exhibited an altered phenotype and profound defects in the ability to activate degranulation and IFN-γ production. For the first time, TANK and PB-NK cells from CRC patients have been characterized. It is shown that they are not capable of producing relevant cytokines and degranulate. Taken together, our results suggest that NK cells from CRC patients present alterations of phenotype and function therefore supporting the progression of cancer.

We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC-) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC- fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC- fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccine's ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Gamma irradiation is one of the methods used to sterilize melanoma cells prior to coculturing them with monocyte-derived immature dendritic cells in order to develop antitumor vaccines. However, the changes taking place in tumor cells after irradiation and their interaction with dendritic cells have been scarcely analyzed. We demonstrate here for the first time that after irradiation a fraction of tumor cells present large lipid bodies, which mainly contain triglycerides that are several-fold increased as compared to viable cells as determined by staining with Oil Red O and BODIPY 493/503 and by biochemical analysis. Phosphatidyl-choline, phosphatidyl-ethanolamine and sphingomyelin are also increased in the lipid bodies of irradiated cells. Lipid bodies do not contain the melanoma-associated antigen MART-1. After coculturing immature dendritic cells with irradiated melanoma cells, tumor cells tend to form clumps to which dendritic cells adhere. Under such conditions, dendritic cells are unable to act as stimulating cells in a mixed leukocyte reaction. However, when a maturation cocktail composed of TNF-alpha, IL-6, IL-1beta and prostaglandin E2 is added to the coculture, the tumor cells clumps disaggregate, dendritic cells remain free in suspension and their ability to efficiently stimulate allogeneic lymphocytes is restored. These results help to understand the events following melanoma cell irradiation, shed light about interactions between irradiated cells and dendritic cells, and may help to develop optimized dendritic cell vaccines for cancer therapy.

In recent decades, tumor surveillance by the immune system and its impact on disease outcomes in cancer patients in general and in breast cancer (BC) patients in particular has been documented. Natural killer (NK) cells are central components of the innate immunity and existing data indicate that they play a role in preventing and controlling tumor growth and metastasis. Their biological significance was first recognized by their ability to exert direct cellular cytotoxicity without prior sensitization. This is important in tumors, as transforming events are likely to result in downregulation of self-ligands and expression of stress-induced ligands which can be recognized by NK cells. Their activation also leads to secretion of stimulatory cytokines which participate in cancer elimination by several direct mechanisms as well as by stimulating the adaptive immune system. In this regard, it was recently revealed a dendritic cell (DC)-NK-cell crosstalk which provides another novel pathway linking innate and adaptive immunity. In addition, NK cells are feasible targets of stimulation in immunotherapeutic approaches such as antibody-based strategies and adoptive cell transfer. Nevertheless, NK cells display impaired functionality and capability to infiltrate tumors in BC patients. This review compiles information about NK-cell biology in BC and the attempts which aim to manipulate them in novel therapeutic approaches in this pathology.

Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.

Triple negative breast cancers (TNBC) lacking hormone receptors and HER-2 amplification are very aggressive tumors. Since relevant differences between primary tumors and metastases could arise during tumor progression as evidenced by phenotypic discordances reported for hormonal receptors or HER-2 expression, in this analysis we studied changes that occurred in our TNBC model IIB-BR-G throughout the development of IIB-BR-G-MTS6 metastasis to the lymph nodes (LN) in nude mice, using an antibody-based protein array to characterize their expression profile. We also analyzed their growth kinetics, migration, invasiveness and cytoskeleton structure in vitro and in vivo. In vitro IIB-BR-G-MTS6 cells grew slower but showed higher anchorage independent growth. In vivo IIB-BR-G-MTS6 tumors grew significantly faster and showed a 100% incidence of LN metastasis after s.c. inoculation, although no metastasis was observed for IIB-BR-G. CCL3, IL1β, CXCL1, CSF2, CSF3, IGFBP1, IL1α, IL6, IL8, CCL20, PLAUR, PlGF and VEGF were strongly upregulated in IIB-BR-G-MTS6 while CCL4, ICAM3, CXCL12, TNFRSF18, FIGF were the most downregulated proteins in the metastatic cell line. IIB-BR-G-MTS6 protein expression profile could reflect a higher NFκB activation in these cells. In vitro, IIB-BR-G displayed higher migration but IIB-BR-G-MTS6 had more elevated matrigel invasion ability. In agreement with that observation, IIB-BR-G-MTS6 had an upregulated expression of MMP1, MMP9, MMP13, PLAUR and HGF. IIB-BR-G-MTS6 tumors presented also higher local lymphatic invasion than IIB-BR-G but similar lymphatic vessel densities. VEGFC and VEGFA/B expression were higher both in vitro and in vivo for IIB-BR-G-MTS6. IIB-BR-G-MTS6 expressed more vimentin than IB-BR-G cells, which was mainly localized in the cellular extremities and both cell lines are E-cadherin negative. Our results suggest that IIB-BR-G-MTS6 cells have acquired a pronounced epithelial-to-mesenchymal transition phenotype. Protein expression changes observed between primary tumor-derived IIB-BR-G and metastatic IIB-BR-G-MTS6 TNBC cells suggest potential targets involved in the control of metastasis.

To endow the immune system with the capacity to fight cancer has always attracted attention, although the clinical results obtained have been until recently disappointing. Cutaneous melanoma is a highly immunogenic tumor; therefore most of the attempts to produce cancer vaccines have been addressed to this disease. New advances in the comprehension of the mechanisms of antigen presentation by dendritic cells, in the immune responses triggered by adjuvants, as well as the understanding of the role of immunosuppressor molecules such as cytotoxic T-lymphocyte antigen-4 (CTLA-4), which led to the recent approval of the anti-CTLA-4 monoclonal antibody ipilimumab, have opened new hopes about the installment of immunotherapy as a new modality to treat cancer.

MART-1 and gp100 are prototypical melanoma antigen (Ag), but their clinical use as vaccines or as targets of cytotoxic lymphocytes achieved modest success. Possible explanations could be that as MART-1 and gp100 are melanocyte differentiation Ag, clonogenic Ag-non-expressing cells would be spared by immune effectors, or that clonogenic cells would be intrinsically resistant to cytotoxic lymphocytes. We therefore analyzed the proliferative status of MART-1/gp100-expressing and -non-expressing cells in biopsies, and the clonogenicity and sensitiveness to cytotoxic lymphocytes of the human cutaneous melanoma cell lines MEL-XY1 and MEL-XY3. Analysis of MART-1/gp100 and Ki-67 expression in 22 melanoma tumors revealed that MART-1/gp100-expressing and -non-expressing cells proliferated competitively. MART-1, gp100, tyrosinase, and CD271 expression were studied in MEL-XY1 and MEL-XY3 colonies. At 7 days, colonies displayed positive, negative, and mixed expression patterns. By 14 days, colonies of different sizes developed, showing cells with different clonogenic potential, and Ag were downregulated, suggesting Ag plasticity. Subcloning of MEL-XY1 colonies showed that Ag expression varied with time without interfering with clonogenicity. Finally, clonogenic, MART-1/gp100-expressing cells were lysed by specific CD8 lymphocytes. Thus, MART-1 and gp100 expression and plasticity would not interfere with proliferation or clonogenicity, and clonogenic cells may be lysed by cytotoxic lymphocytes.

Metallothioneins are a family of small, cysteine-rich proteins with many functions. Immunohistochemical evaluation of all metallothionein 1 + 2 isoforms in colorectal tumors has demonstrated an important down-regulation compared with normal tissue, although its prognostic significance is unclear. Moreover, the contribution of individual isoforms to overall metallothionein down-regulation is not known. To address these important issues, we analyzed the messenger RNA expression levels of all functional metallothionein 1 + 2 isoforms by quantitative reverse transcription polymerase chain reaction in 22 pairs of normal and tumor-microdissected epithelia and correlated these to the overall immunohistochemical protein expression. Our results showed that 5 isoforms (MT1G, 1E, 1F, 1H, and 1M) were lost during the transition from normal mucosa to tumor, whereas MT1X and MT2A were less down-regulated, and their expression was correlated with overall protein positivity. Second, we showed that MT1G hypermethylation occurred in cell lines and in 29% of tumor samples, whereas histone deacetylase inhibitors are able to induce most isoforms. Furthermore, we analyzed by immunohistochemistry 107 normal mucosae, 25 adenomas, 81 carcinomas, and 19 lymph node metastases to evaluate metallothionein expression during different stages of cancer development and to assess its relationship to patient survival. A lower immunohistochemical expression was associated with poorer survival, although it was not an independent predictor. Overall, this study identifies for the first time the relevant metallothionein isoforms for colorectal cancer progression, supports the concept that their loss is associated with worse prognosis, and suggests 2 mechanisms for epigenetic repression of metallothionein expression in colorectal tumors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We will revisit the dual role of the immune system in controlling and enabling tumor progression, known as cancer immunoediting. We will go through the different phases of this phenomenon, exposing the most relevant evidences obtained from experimental models and human clinical data, with special focus on Cutaneous Melanoma, an immunogenic tumor per excellence. We will describe the different immunotherapeutic strategies employed and consider current models accounting for tumor heterogeneity. And finally, we will propose a rational discussion of the progress made and the future challenges in the therapeutics of Cutaneous Melanoma, taking into consideration that tumor evolution is the resulting from a continuous feedback between tumor cells and their environment, and that different combinatorial therapeutic approaches can be implemented according to the tumor stage.

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8(+) T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8(+) T cell clone. Confocal microscopy with Alexa Fluor®(647)-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8(+) T cell cross-presentation thereafter.

Background: The Lewisx trisaccharide, also referred to as the CD15 antigen, is a diagnostic marker used to distinguish Hodgkin's lymphoma from other lymphocytic cancers. However, the role of such fucosylated structures remains poorly understood, in part because carriers of Lewisx structures on Hodgkin's Reed-Sternberg cells have not been identified., Methods: GalMBP, an engineered carbohydrate-recognition protein that binds selectively to oligosaccharides with paired terminal galactose and fucose residues, has been used in conjunction with proteomic and glycomic analysis to identify glycoprotein carriers of Lewisx and related glycan structures in multiple Hodgkin's Reed-Sternberg cell lines., Results: Multiple glycoproteins that bind to GalMBP and carry CD15/Lewisx have been identified in a panel of six Reed-Sternberg cell lines. The most commonly identified Lewisx-bearing glycoproteins are CD98hc, which was found in all six cell lines tested, and intercellular adhesion molecule-1 and DEC-205, which were detected in five and four of the lines, respectively. Thus, several of the most prominent cell adhesion molecules on the lymphomas carry this characteristic glycan epitope. In addition, the Hodgkin's Reed-Sternberg cell lines can be grouped into subsets based on the presence or absence of less common Lewisx-bearing glycoproteins., Conclusions: CD98 and intercellular adhesion molecule-1 are major carriers of CD15/Lewisx on Reed-Sternberg cells. Binding of DC-SIGN and other glycan-specific receptors to the Lewisx epitopes on CD98 and intercellular adhesion molecule-1 may facilitate interaction of the lymphoma cells with lymphocytes and myeloid cells in lymph nodes.

Natural killer (NK) cells are central components of the innate immunity. In murine models, it has been shown that NK cells can control both local tumor growth and metastasis due to their ability to exert direct cellular cytotoxicity without prior sensitization and to secrete immunostimulatory cytokines like IFN-γ. The latter participates in cancer elimination by inhibiting cellular proliferation and angiogenesis, promoting apoptosis, and stimulating the adaptive immune system, and it is instrumental for enhancing Ag processing and presentation. Nevertheless, NK cells display impaired functionality and capability to infiltrate tumors in cancer patients. Also, NK cells are feasible targets of stimulation to participate in immunotherapeutic approaches like antibody-based strategies and adoptive cell transfer. Thus, multiple attempts currently aim to manipulate NK for utilization in the immunotherapy of cancer.

Antigen presentation by dendritic cells (DC) is of key importance for the initiation of the primary immune response. Mice vaccinated with DC charged with apoptotic/necrotic B16 cells (DC-Apo/Nec) are protected against B16 challenge. The aim of this study was to assess vaccine cell migration in our system and to find out if there is an immunological response taking place at the vaccination site. The formation of a pseudocapsule, peripheral node addresin expression in small venules, and the recruitment of a wide variety of cellular populations, including macrophages, polymorphonuclear lymphocytes, and CD8+ and CD4+ T lymphocytes found in association with DC, evidenced the formation of tertiary lymphoid tissue in the vaccination site in our experimental system., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewis(x) and Lewis(y) structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewis(x) antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Cetuximab, an anti-epidermal growth factor receptor monoclonal antibody, has been shown to increase the median survival of colorectal cancer patients. We previously reported that the expression of HLA-E is significantly increased in primary human colorectal cancer, perhaps contributing to tumour escape from immune surveillance. To establish if HLA-E could be a factor that renders colorectal cancer cells less susceptible to antibody-dependent cellular cytotoxicity (ADCC), in the present study we analysed Cetuximab-mediated cytotoxicity against several colorectal cancer cell lines expressing, or not, HLA-E at the cell surface. We first observed that colorectal cancer cells treated with Cetuximab were killed more efficiently by ADCC. Interestingly, treatment of target cells with recombinant human-beta2-microglobulin inhibits Cetuximab-mediated ADCC through HLA-E membrane stabilization. The specific immunosuppressive role of HLA-E was confirmed using an anti-NKG2A monoclonal antibody, that restored the ability of immune cells to kill their target. This result demonstrates that HLA-E at the cell surface can reliably suppress the ADCC effect. On the other hand, Cetuximab induced a direct growth inhibition but only at high concentrations; furthermore, the CDC effect was quite moderate, and we failed to observe a pro-apoptotic effect. Taking into account that our findings suggest that ADCC activity is the main anti-tumour effect observed at clinically achievable concentrations of Cetuximab at the tumour site, we suggest that determination of HLA-E in colorectal cancer could be relevant to predict success of Cetuximab treatment.

The G1465A polymorphism in the gene of the GABA type B receptor subunit 1 (GABABR1) has been linked to the risk for temporal lobe epilepsy (TLE). However, six replication studies did not show significant association between the G1465A GABABR1 gene variant and TLE. The authors examined this association in a sample of 102 patients with mesial TLE with hippocampal sclerosis (MTLE-HS) and 71 controls. The genotype distribution varied significantly between patients and controls. Heterozygous carriers of the A-allele had a 10-fold increase in risk for MTLE-HS (OR 10.01; 95% CI 3.98-25.18, p=3.788E-08).

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.

HLA-E is a non-classical MHC molecule whose expression by tumour cells has been recently reported in several human cancer types. We studied HLA-E expression in colorectal cancer patients, its clinical significance and prognostic value, as well as characterized its expression in colorectal cancer cell lines. We analysed HLA-E expression at the transcript level by qRT-PCR in micro-dissected samples and at the protein level by semiquantitative immunohistochemistry on paraffin-embedded tissue sections from 42 biopsies of colorectal cancer patients. We observed that HLA-E transcript and protein are spontaneously overexpressed in a significant proportion of colorectal tumour biopsies, as compared to normal mucosae. We also found a negative correlation between HLA-E expression and the CD57+ cells infiltrate. Moreover, we analysed HLA-E expression in several colorectal cancer cell lines and demonstrated that IFN-gamma upregulates the expression of membrane HLA-E in vitro. Interestingly, we demonstrated that colorectal cancer cell lines overexpressing HLA-E at the cell surface inhibited NK-mediated cell lysis. Although IFN-gamma regulatory role needs further investigation, we provide evidence suggesting that this cytokine, within the tumour microenvironment, could promote HLA-E translocation to the surface of tumour epithelial cells. Furthermore, we showed that upregulation of HLA-E could be a marker of shorter disease-free survival in Dukes' C patients and we suggest that this molecule renders tumours less susceptible to immune attack.

Chemokines such as monocyte chemoattractant protein (MCP)-1 are key agonists that attract macrophages to tumors. In melanoma, it has been previously shown that variable levels of MCP-1/CCL2 appear to correlate with infiltrating macrophages and tumor fate, with low to intermediate levels of the chemokine contributing to melanoma development. To work under such conditions, a poorly tumorigenic human melanoma cell line was transfected with an expression vector encoding MCP-1. We found that M2 macrophages are associated to MCP-1+ tumors, triggering a profuse vascular network. To target the protumoral macrophages recruitment and reverting tumor growth promotion, clodronate-laden liposomes (Clod-Lip) or bindarit were administered to melanoma-bearing mice. Macrophage depletion after Clod-Lip treatment induced development of smaller tumors than in untreated mice. Immunohistochemical analysis with an anti-CD31 antibody revealed scarce vascular structures mainly characterized by narrow vascular lights. Pharmacological inhibition of MCP-1 with bindarit also reduced tumor growth and macrophage recruitment, rendering necrotic tumor masses. We suggest that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.

Lewis x (Le(x), CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galbeta(1-4)Fucalpha(1-3)GlcNAc, which is expressed on glycoconjugates in human polymorphonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Le(x) in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Le(x) mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Le(x+)-cells induced by anti-Le(x) mAbs and complement when cells were adhered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF-7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Le(x)-dependent PMN interaction with HUVEC. Although both anti-Le(x) mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blotting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Le(x)-trisaccharide, interacts with specific glycoproteins of M (r ) approximately 28 kD and 10 kD from MCF-7 cells. The interaction between Le(x+)-cancer cells and vascular endothelium is a potential target for cancer treatment.

Purpose: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development., Experimental Design: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done., Results: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log(2) ratio less than -1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis., Conclusions: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.

The causative molecular pathways underlying the pathogenesis of colorectal cancer (CRC) need to be better characterized. The purpose of our study was to better understand the genetic mechanism of oncogenesis for human colorectal cancer and to identify new potential tumor markers of use in clinical practice. We used cDNA microarrays to compare gene expression profiles of colorectal biopsies from 25 CRC patients and 13 normal mucosa from adjacent non-cancerous tissues. Findings were validated by real-time PCR; in addition, western blotting and immunochemistry analysis were carried out as further confirmation of differential expression at a protein level. Comparing cancerous tissues with normal colonic mucosa we identified 584 known genes differentially expressed to a significant degree (p<0.001). Many of the transcripts that were more abundant in tumors than in non-neoplastic tissues appear to reflect important events for colon carcinogenesis. For example, a significant number of these genes serve as apoptotic inhibitors (e.g. BFAR, BIRC1, BIRC6). Furthermore, we observed the simultaneous up-regulation of HLA-E and the down-regulation of beta2-microglobulin; these genes strongly support a potential tumor escape strategy from immune surveillance in colon cancer tissues. Our study provides new gene candidates in the pathogenesis of human CRC disease. From our results we hypothesize that CRC cells escape immune surveillance through a specific gene expression alteration; moreover, over-expression of several survival genes seems to confer a more anti-apoptotic phenotype. These genes are involved in pathways not previously implicated in CRC pathogenesis and they may provide new targets for therapy.