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PDF file - 193K, Representative flow cytometry analysis of a CIK cell culture generated from PBMC from mMel patients.

Purpose: We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC).Experimental Design: We developed a preclinical autologous model using same patient-generated CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness gene Oct4.Results: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median, 23-fold; range, 11–117). Primary tumor cell cultures established and characterized from the same patients were used as autologous targets. Patient-derived CIK cells efficiently killed autologous metastatic melanoma [up to 71% specific killing (n = 26)]. CIK cells were active in vivo against autologous melanoma, resulting in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic melanoma cultures presented an average of 11.5% ± 2.5% putative mCSCs, which was assessed by Oct4 promoter activity and stemness marker expression (Oct4, ABCG2, ALDH, MITF). Expression was confirmed on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against mCSCs was intense (up to 71%, n = 4) and comparable with results reported against differentiated metastatic melanoma cells (P = 0.8).Conclusions: For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer. Clin Cancer Res; 19(16); 4347–58. ©2013 AACR.

PDF file - 147K, Representative flow cytometry analysis of melanoma primary cell cultures. assessment of CD117 (aka c-kit) and MCSP; c) assessment of MIC A/B and ULBP2; d) assessment of ULBP1 and ULBP3 expression; e) expression assessment of CD34 and VEGFR1 expression; f) assessment of intracellular Oct4 expression in fixed and permeabilized cells; g) assessment of ABCG2 expression; h) assessment of ALDH activity in cells stained with FITC ALDH substrate; i) assessment of MITF negativity in fixed and permeabilized cells stained with a primary specific antibody unconjugated and a PE conjugated secondary antibody; k) assessment of CD117 (also known as c-kit) and MCSP in mMel6, notably highly positive for CD117 expression.

PDF file - 143K, Primary melanoma cell targets.

Supplementary Figure S2: Immunophenotype of patient-derived CIK cells and CIK cell in vitro activity

DNA extraction and Sanger Sequencing In vivo Limiting Dilution Assay Cell growth inhibition assay Analysis of LV.Oct4.eGFP integration in eGFP positive and negative cell fractions

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PDF file - 122K, Representative cell sorting plots of LV-oct4.eGFP transduced cells.

PDF file - 117K, Expression level of ABCG2 (a), NKG2D ligands MIC A/B (b) and ULBP2 (c) in LV-oct4-eGFP transduced cells.

PDF file - 81K, Representative flow cytometry analysis of melanoma primary transduced cells.

Supplementary Table 1: Patients Characteristics Supplementary Table 2: Comparative transduction of melanoma cell cultures

Supplementary Figure S5:Tumor infiltration by CIK cells

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses.Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models.Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01).Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

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Supplementary Figure S6: Sequential chemo-immunotherapy associates with improved survival

Supplementary Figure S3:Expression of NKG2D ligands and PDL1 on putative mCSCs following different treatments.

Cytokine-induced killer lymphocytes (CIK) are a promising alternative to conventional donor lymphocyte infusion (DLI), following allogeneic haematopoietic cell transplantation (HCT), due to their intrinsic anti-tumour activity and reduced risk of graft-versus-host disease (GVHD). We explored the feasibility, anti-leukaemic activity and alloreactive risk of CIK generated from full-donor chimaeric (fc) patients and genetically redirected by a chimeric antigen receptor (CAR) (fcCAR.CIK) against the leukaemic target CD44v6. fcCAR.CIK were successfully ex-vivo expanded from leukaemic patients in complete remission after HCT confirming their intense preclinical anti-leukaemic activity without enhancing the alloreactivity across human leukocyte antigen (HLA) barriers. Our study provides translational bases to support clinical studies with fcCAR.CIK, a sort of biological bridge between the autologous and allogeneic sources, as alternative DLI following HCT.

Malignant melanoma is the third most common type of tumor that causes brain metastases. Patients with cerebral involvement have a dismal prognosis and their treatment is an unmet medical need. Brain involvement is a multistep process involving several signaling pathways such as Janus kinase/signal Transducer and Activator of Transcription (JAK/STAT), Phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT), Vascular Endothelial Growth Factor and Phosphatase and Tensin Homolog (PTEN). Recently therapy that targets the MAPK signaling (BRAF/MEK inhibitors) and immunotherapy (anti-CTLA4 and anti-PD1 agents) have changed the therapeutic approaches to stage IV melanoma. In contrast, there are no solid data about patients with brain metastases, who are usually excluded from clinical trials. Retrospective data showed that BRAF-inhibitors, alone or in combination with MEK-inhibitors have interesting clinical activity in this setting. Prospective data about the combinations of BRAF/MEK inhibitors have been recently published, showing an improved overall response rate. Short intracranial disease control is still a challenge. Several attempts have been made in order to improve it with combinations between local and systemic therapies. Immunotherapy approaches seem to retain promising activity in the treatment of melanoma brain metastasis as showed by the results of clinical trials investigating the combination of anti-CTL4 (Ipilimumab) and anti-PD1(Nivolumab). Studies about the combination or the sequential approach of target therapy and immunotherapy are ongoing, with immature results. Several clinical trials are ongoing trying to explore new approaches in order to overcome tumor resistance. At this moment the correct therapeutic choices for melanoma with intracranial involvement is still a challenge and new strategies are needed.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses. Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models. Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01). Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94(+) cells were CD3(+/–), CD8(+/–), CD4(–), CD21(–), CD5(low), NKp46(+), and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94(+) lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.

Combined direct antineoplastic activity and the long-lasting immunological effects of allogeneic hematopoietic cell transplant (HCT) can cure many hematological malignancies, but broad adoption requires non-relapse mortality (NRM) rates and graft-versus-host disease (GVHD) control. Recently, posttransplant cyclophosphamide (PTCy) given after a bone marrow transplant significantly reduced GVHD-incidence, while PTCy given with tacrolimus/mofetil mycophenolate (T/MMF) showed activity following allogeneic peripheral blood stem cell transplantation (alloPBSCT). Here, we report the experience of a larger cohort (85 consecutive patients) and expanded follow-up period (03/2011–12/2019) with high-risk hematological malignancies who received alloPBSCT from Human-Leukocyte-Antigens HLA-matched unrelated/related donors. GVHD-prophylaxis was PTCy 50 mg/kg (days+3 and +4) combined with T/MMF (day+5 forward). All patients stopped MMF on day+28 with day+110 = median tacrolimus discontinuation. Cumulative incidences were 12% for acute and 7% for chronic GVHD- and no GVHD-attributed deaths. For surviving patients, the 12, 24, and 36-month probabilities of being off immunosuppression were 92, 96, and 96%, respectively. After a 36-month median follow-up, NRM was 4%, median event-free survival (EFS) and overall survival (OS) had yet to occur. One- and two-year chronic GVHD-EFS results were 57% (95% CI, 46–68%) and 53% (95% CI, 45–61%), respectively, with limited late infections and long-term organ toxicities. Disease relapse caused the most treatment failures (38% at 2 years), but low transplant toxicity allowed many patients (14/37, 38%) to receive donor lymphocyte infusions as a post-relapse strategy. We confirmed that PTCy+T/MMF treatment effectively prevented acute and chronic GVHD and limited NRM to unprecedented low rates without loss of disease control efficacy in an expanded patient cohort. This trial is registered at U.S. National Library of Medicine as #NCT02300571.

BRAF signaling is involved in melanoma growth in more than half of metastatic patients. In the last few years, new drugs that block this pathway have significantly improved the outcomes of patients with metastatic melanoma. Ninety percent of BRAF mutations involve exon 15, and the most frequent, V600E, results from the amino acid change from valine (V) to glutamic acid (E). BRAF inhibitor treatments have shown a notable overall response rate and improvements in progression-free and overall survival. Rare BRAF mutations of codon 599 have been also described in a few patients with papillary thyroid cancer and melanoma. Nowadays, no evidence is available in the literature, describing the role of target therapies as treatment in patients with this specific codon mutation. We describe the case of a young woman with metastatic melanoma with a particular BRAF mutation, T599I, who has benefited from treatment with a BRAF inhibitor, vemurafenib.

Allogeneic hematopoietic cell transplant (HCT) remains the only curative therapy for many hematologic malignancies but it is limited by high nonrelapse mortality (NRM), primarily from unpredictable control of graft-versus-host disease (GVHD). Recently, post-transplant cyclophosphamide demonstrated improved GVHD control in allogeneic bone marrow HCT. Here we explore cyclophosphamide in allogeneic peripheral blood stem cell transplantation (alloPBSCT). Patients with high-risk hematologic malignancies received alloPBSCT from HLA-matched unrelated/related donors. GVHD prophylaxis included combination post-HCT cyclophosphamide 50 mg/kg (days +3 and +4) and tacrolimus/mofetil mycophenolate (T/MMF) (day +5 forward). The primary objective was the cumulative incidence of acute and chronic GVHD. Between March 2011 and May 2015, 35 consecutive patients received the proposed regimen. MMF was stopped in all patients at day +28; the median discontinuation of tacrolimus was day +113. Acute and chronic GVHD cumulative incidences were 17% and 7%, respectively, with no grade IV GVHD events, only 2 patients requiring chronic GVHD immunosuppression control, and no deaths from GVHD. Two-year NRM, overall survival, event-free survival, and chronic GVHD event-free survival rates were 3%, 77%, 54%, and 49%, respectively. The graft-versus-tumor effect was maintained as 5 of 15 patients (33%) who received HCT with evidence of disease experienced further disease response. A post-transplant cyclophosphamide + T/MMF combination strategy effectively prevented acute and chronic GVHD after alloPBSCT from HLA-matched donors and achieved an unprecedented low NRM without losing efficacy in disease control or impaired development of the graft-versus-tumor effect. This trial is registered at clinicaltrials.gov as NCT02300571.

Breast cancer is the most common malignancy in women. Despite there being considerable progress in the treatment of this disease, metastatic dissemination is still considered an incurable condition at the present time, causing 500,000 deaths worldwide every year. Although most of the research efforts have been focused on pharmacological approaches, over the last three decades, the use of bone marrow and peripheral blood-derived cell therapy approaches have been attempted and developed. Areas covered: This review will briefly address cell therapy for breast cancer, including autologous stem cell transplantations for overcoming the myelosuppressive effects of high-dose chemotherapy, allogeneic stem cell transplants and adoptive immunotherapy using bone-marrow derived T-cells. Expert opinion: The treatment of breast cancer using bone marrow or peripheral-blood derived cells has evolved from a supportive care approach to allow dose escalation of conventional chemotherapy to a therapeutic strategy aimed at eliciting immune cell mediated anticancer immunity. This latter principle has led to the development of adoptive immunotherapies, either with 'natural' or genetically engineered effectors, which are being intensively investigated for their great potential against several solid tumors, including breast cancer.

Purpose: The goals of this study are i) to analyze the expression of human leukocyte antigen class I (HLA-I) antigen processing machinery (APM) components in surgically removed melanoma tumors, ii) to assess their modulation by Interferons (IFNs) and iii) to determine their value as predictive biomarkers of the clinical response to immunotherapy with checkpoint inhibitors (CI). Methods: The expression levels of HLA class I APM components [HLA-I (ABC) chains, beta(2)microglobulin (β2m), Calnexin, Calreticulin, ERp57, Tapasin, TAP1-2, LMP2, LMP7, LMP10] were evaluated by staining with mAbs and flow cytometry in melanoma cells isolated from surgical biopsies from 16 patients with advanced melanoma. In addition the modulation of these molecules by IFN-γ or IFN-α2 (IFNs) was evaluated. Results: The frequency of cells expressing HLA-I/β2m was >90% in 15/16 Mel tumors. In 1 case extracellular HLA-I/β2m molecules were not detected. The expression levels of extracellular HLA class I APM molecules were quite heterogeneous. Based on Mean Fluorescence Intensity (MFI) of HLA-I/β2m, Mel segregated into 2 groups with high (MFI >200, n=8) and low (MFI Conclusions: We report a wide heterogeneous distribution of HLA-I/β2m expression levels and deficits of APM components in Melanoma, partially restorable by IFNs. Our data support the possibility that defects in HLA expression, not responsive to IFN restoration, may predict low responsiveness to CI. We provide rationale to incorporate the evaluation of HLA-I/β2m and APM in clinical immunotherapy studies with CI. Citation Format: Lidia Giraudo, Loretta Gammaitoni, Bortolot Valentina, Ilenia Iaia, Giulia Cattaneo, Paolo Becco, Susanna Gallo, Alessandro Zaccagna, Alberto Pisacane, Luca Crotto, Soldano Ferrone, Massimo Aglietta, Fabrizio Carnevale Schianca, Dario Sangiolo. Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 569.

Purpose: We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC). Experimental Design: We developed a preclinical autologous model using same patient-generated CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness gene Oct4. Results: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median, 23-fold; range, 11–117). Primary tumor cell cultures established and characterized from the same patients were used as autologous targets. Patient-derived CIK cells efficiently killed autologous metastatic melanoma [up to 71% specific killing (n = 26)]. CIK cells were active in vivo against autologous melanoma, resulting in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic melanoma cultures presented an average of 11.5% ± 2.5% putative mCSCs, which was assessed by Oct4 promoter activity and stemness marker expression (Oct4, ABCG2, ALDH, MITF). Expression was confirmed on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against mCSCs was intense (up to 71%, n = 4) and comparable with results reported against differentiated metastatic melanoma cells (P = 0.8). Conclusions: For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer. Clin Cancer Res; 19(16); 4347–58. ©2013 AACR.

Cytokine-induced killer cells (CIKs) are ex vivo expanded T-NK lymphocytes capable of HLA-unrestricted antitumor activity. CIKs are promising candidates for adoptive cancer immunotherapies; they can be generated and infused in autologous settings of cancer patients, or from donors, after allogeneic hematopoietic cell transplant. Ex vivo expansion rates of CIKs are greatly variable among patients, with consequent potential clinical limitations for "poor expanders." We compared the standard expansion protocol with a new one, which included the timed addition of irradiated allogeneic peripheral blood mononuclear cells. Our hypothesis is that allogeneic stimulation might provide CIK cells with a proliferative boost and simultaneously decrease their alloreactivity versus third parties, if HLA-mismatched from the allogeneic stimulators. Allo-stimulated CIKs (AS-CIK) reached significantly higher expansion rates compared with standard controls, regardless if generated form healthy donors (131- vs. 32-fold) or cancer patients (117- vs. 14-fold). The expansion of the CD3CD56 subset was 2243-fold for AS-CIKs compared with 362 for standard CIKs. AS-CIKs efficiently killed osteosarcoma targets in vitro, results were comparable with that of standard CIKs. Standard and AS-CIKs did not show differences in phenotype and telomere length. The alloreactivity of AS-CIKs against third party HLA-mismatched peripheral blood mononuclear cells was reduced compared with standard CIKs (37% vs. 23%). In conclusion, alloreactivity of CIK cells may be exploited enhancing their final ex vivo expansion. In clinical perspective these findings may facilitate the extension of CIK-based immunotherapy to larger numbers of patients and, translated into hematopoietic cell transplant settings, contribute to reduce the risk of graft versus host disease in the hypothesis of infusions across HLA barriers.

Allogeneic hematopoietic cell transplantation (HCT) is a consolidated treatment for several hematologic malignancies. Donor T lymphocytes can mediate a graft versus tumor (GVT) effect and control opportunistic infections but can also cause severe graft versus host disease (GVHD). Gene-transfer strategies are appealing tools to modulate T cell functions when infused after HCT.The current and potential future applications of T cell gene-transfer approaches to HCT. This review is not limited to GVHD control but covers the issues of GVT and immune reconstitution. Clinical data are used to discuss more general issues, perspectives and concerns common to gene-modification of T cells. An overview of the results and limitations emerging from clinical trials with herpes simplex virus-thymidine kinase (HSV-TK) engineered lymphocytes is provided. The review provides perspectives on additional gene-transfer strategies, currently at preclinical level or that have just entered clinical trials, to increase the efficacy and safety of HCT.Gene-transfer can positively interfere with T cell functions after HCT. TK-lymphocytes have proven effective in controlling GVHD while retaining an acceptable GVT effect. Strategies exploiting new suicide molecules or engineered T cell receptors (TCRs) should be further explored to address current limitations with TK-lymphocytes and augment the efficacy and specificity of GVT and antiviral activity.

Background Metastatic Melanoma (mMel) is considered refractory to conventional chemotherapies. New molecular targeted approaches, inhibiting mutated forms of the serinethreonine kinase B-RAF, significantly increased the response rate but patients almost invariably relapse and prognosis remains severe [1,2]. Open challenge is the characterization and targeting of cancer stem cells (CSCs), considered responsible for chemoresistance and disease relapse. Adoptive immunotherapy holds great promises for the treatment of mMel and research efforts are ongoing to explore its potential activity against melanoma CSCs (mCSCs). Cytokine Induced Killer (CIK) cells are a subset of ex vivo expanded T lymphocytes with mixed CD3CD56 phenotype and endowed with HLA-unrestricted tumor killing activity. We and others recently reported the preclinical activity of CIK cells against several solid tumors including mMel [3]. Aim of our research is to explore the preclinical activity of CIK cells against autologous mCSCs surviving treatments with chemo or molecular targeted therapies.

10061 Background: Cardiovascular events (CVE), i.e. CHF and coronary artery disease (CAD), may occur in up to 10% of patients (pts) affected by GIST and RCC treated with SU. CVE recognition, SU int...

e20514 Background: After a decade of IM, the efficacy and toxicity of this drug are well known. However, there is a surprising paucity of data regarding IM treatment in elderly patients (pts) both ...