Alessandro Zaccagna

Mycosis fungoides in patients under 20 years of age : Report of 7 cases, review of the literature and study of the clinical course

Background: Mycosis fungoides (MF) is rare in young patients. Its clinical behavior is still uncertain, as some reports have suggested that it has a more aggressive course than does the adult-onset type. Aim: To ascertain if early-onset MF represents a heterogeneous group of cutaneous T cell lymphomas. Materials and Methods: Clinical, immunohistopathological and follow-up data of early-onset (<20 years of age) MF cases reported in the literature (n = 42) plus 7 described herein were compared with those of a cohort of adult-onset MF patients (n = 252) diagnosed at our institution since 1975. Results: The majority of the 49 early-onset MF patients had patch-plaque stage disease at diagnosis. Ten had hypopigmented lesions. The predominant phenotype was CD3+ CD4+CD7–CD8–. Seven patients had a stage progression, 6 with extracutaneous involvement. Five- and 10-year survival rates were 93 and 74%, respectively. Conclusions: No statistically significant differences were found in the disease course between early- and adult-onset MF.

Effective Activity of Cytokine-Induced Killer Cells against Autologous Metastatic Melanoma Including Cells with Stemness Features

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.

Metastatic melanoma of the heart

Malignant melanoma has an unpredictable biologic behavior and is the neoplasm with the greatest propensity for cardiac involvement. Although relatively frequent at autopsy, cardiac metastases are rarely identified antemortem.

Cutaneous metastasis of neuroendocrine carcinoma of the larynx: report of a case.

Background:  Cutaneous metastasis from neuroendocrine carcinomas of visceral origin is rarely described in indexed literature. The primary sites of origin include: lung (Wick et al., J Am Acad Dermatol 1985; 13: 134), larynx (Zambruno et al., Ann Dermatol Venereol 1989; 116: 855; Schmidt et al., J Laryngol Otol 1994; 108: 272; Guerzider et al., Ann Pathol 1991; 11 (4): 253), mediastinum (Yoshimasu et al., J Dermatol 2001; 28 (3): 168), uterus (Fogaca et al., J Cutan Pathol 1993; 20: 455), and thymus (Wick et al., J Am Acad Dermatol 1985; 13: 134).

Cytokine-Induced Killer Cells Kill Chemo-surviving Melanoma Cancer Stem 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.

Cytokine Induced Killer cells effectively kill chemo-resistant melanoma cancer stem cells

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.

Abstract 2290: Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells

Purpose of the study was to explore the activity of cytokine-induced killer (CIK) cells against autologous chemo-surviving melanoma cancer stem cells (mCSCs) in vivo. Elimination of mCSCs is an ambitious goal as considered responsible for chemo-resistance and relapses. CIK cells are ex vivo expanded T-NK lymphocytes with MHC-independent antitumor activity. We provided proof of concept that CIK cells can kill mCSCs in vitro (Gammaitoni et al. Clin Cancer Res 2013) and demonstration of their activity in vivo, within an autologous model, would be relevant in clinical perspective. Experimental procedures and results. In vivo activity of CIK cells against autologous mCSCs was explored in NOD/SCID mice bearing tumors generated by subcutaneous implantation of primary melanoma cells or alternatively surgically resected tumor samples (patient-derived xenografts, PDX). mCSCs were visualized by a lentiviral CSC-detector encoding eGFP under control of stem-gene promoter OCT4 (Gammaitoni et al. Clin Cancer Res 2013). Melanoma cells were engineered with the CSC-detector before implantation or alternatively, in PDX, after removal of residual tumors at the end of experimental treatments. In vivo chemotherapy (CHT) consisted of intravenous (i.v.) Fotemustine (600μg days 1;15). Immunotherapy consisted of i.v. CIK cells (1×107) every 5 days. Antitumor activity was evaluated assessing tumor proliferative index by Ki67 expression in residual tumors. Activity against mCSCs was evaluated assessing the rate of residual GFP+-mCSCs in residual tumors after treatments. In vivo infusion of CIK cells for 2 weeks determined significant tumor response (n = 6, p Similarly, in the PDX model, the infusion (n = 4) of autologous CIK cells exerted significant antitumor activity that involved eGFP+mCSCs (p = 0.009), confirmed by the absence of their increment in residual tumors (p = 0.3). In vitro CHT treatment of melanoma from 8 different patients significantly enriched eGFP+ mCSCs (2.2 ±0.2 fold, p = 0.0018) compared to untreated controls. Melanoma treated with CHT, enriched in eGFP+mCSCs, were efficiently killed by autologous CIK cells with mean killing values ranging from 94±2% (40:1 E/T) to 21%±4 (1:3 E/T). Conclusions. Our findings provide first demonstration that immunotherapy with CIK cells is active against autologous mCSCs, surviving chemotherapy, in vitro and in vivo. We provide an experimental platform to study mCSCs and rationale to design clinical studies with CIK cells against melanoma. Citation Format: Loretta Gammaitoni, Lidia Giraudo, Marco Macagno, Giulia Cattaneo, Valeria Leuci, Francesco Sassi, Alessandro Zaccagna, Alberto Pisacane, Valentina Coha, Fabrizio Carnevale-Schianca, Massimo Aglietta, Dario Sangiolo. Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2290.