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

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.

Fig. S3. In NRASQ61 mutant cells MEKi alone leads to highest levels of PD1+ melanoma cells. Representative flow cytometry plots of a NRASQ61 mutant melanoma cell line (SKMEL2) treated with dabrafenib [1mcM], trametinib [5nM], the combination of dabrafenib + trametinib [1mcM+5nM], and fotemustine [50mcg/ml] for 96 hours. DAPI staining was used to identify viable cells. PD1, PDL1, PDL2 plots were performed considering only viable cells.

PDF file - 143K, Primary melanoma cell targets.

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

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

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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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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.

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In patients with B-RAF-mutated cutaneous melanoma, targeted therapies are the treatment of choice to achieve a rapid response. In this multicentric, prospective, observational study, patients with B-RAF-mutated cutaneous melanoma who were treated with dabrafenib and trametinib were categorized in two cohorts (cohort A: limited disease (n = 104) and cohort B: bulky disease (n = 97)) according to lactate dehydrogenase levels. The primary endpoint was the progression pattern; the secondary endpoints were overall survival (OS), progression-free survival (PFS), and safety data. From baseline to time of progression, there was a progression from nodal to other sites of disease in cohort A and from skin and nodal to other sites in cohort B. In both the cohorts, the number of involved organs and metastases at each location decreased. The median OS was 32.4 months (95% CI: 20.1 months (not estimable)) for cohort A, and 10.5 months (95% CI: 8.3–14.4 months) for cohort B; median PFS was 12.4 months (95% CI: 10.9–17.0 months) for cohort A, and 8.1 months (95% CI: 6.3–9.4 months) for cohort B. No new safety signals were reported. This study describes the patterns of first-line treatment progression with dabrafenib and trametinib in Italian clinical practice. The effectiveness and safety data were consistent with previous trials and extended to a real-world heterogeneous population.

About 50% of melanomas harbour a BRAF mutation. Of these 50%, 10% have a V600K mutation. Although it is the second most common driver mutation after V600E, no specific studies have been conducted to identify a clinical and therapeutic gold standard for this patient subgroup. We analysed articles, including registrative clinical trials, to identify common clinical and biological traits of the V600K melanoma population, including different adopted therapeutic strategies. Melanoma V600K seems to be more frequent in Caucasian, male and elderly populations with a history of chronic sun damage and exposure. Prognosis is poor and no specific prognostic factor has been identified. Recent findings have underlined how melanoma V600K seems to be less dependent on the ERK/MAPK pathway, with a higher expression of PI3KB and a strong inhibition of multiple antiapoptotic pathways. Both target therapy with BRAF inhibitors + MEK inhibitors and immunotherapy with anti-checkpoint blockades are effective in melanoma V600K, although no sufficient evidence can currently support a formal recommendation for first line treatment choice in IIIC unresectable/IV stage patients. Still, melanoma V600K represents an unmet medical need and a marker of poor prognosis for cutaneous melanoma.

Agnostic biomarkers such as gene fusions allow to address cancer patients to targeted therapies, however, the low prevalence of these alterations across common malignancies poses challenges and needs a feasible and sensitive diagnostic process. RNA-based targeted next generation sequencing was performed on 125 samples of patients affected either by colorectal carcinoma, melanoma, or lung adenocarcinoma lacking genetic alterations in canonical driver genes, or by a colorectal carcinoma with microsatellite instability. Gene fusion rates were compared with in silico data from MSKCC datasets. For NTRK gene fusion detection we also employed a multitarget qRT-PCR and pan-TRK immunohistochemistry. Gene fusions were detected in 7/55 microsatellite instable colorectal carcinomas (12.73%), and in 4/70 of the “gene driver free” population (5.71%: 3/28 melanomas, 10.7%, and 1/12 lung adenocarcinomas, 8.3%). Fusion rates were significantly higher compared with the microsatellite stable and “gene driver positive” MSKCC cohorts. Pan-TRK immunohistochemistry showed 100% sensitivity, 91.7% specificity, and the occurrence of heterogeneous and/or subtle staining patterns. The enrichment of gene fusions in this “real-world” cohort highlights the feasibility of a workflow applicable in clinical practice. The heterogeneous expression in NTRK fusion positive tumours unveils challenging patterns to recognize and raises questions on the effective translation of the chimeric protein.

Purpose of this study is to explore the preclinical efficacy of Chondroitin Sulfate Proteoglycan 4 (CSPG4)-specific CAR-engineered Cytokine-Induced Killer lymphocytes (CIK) to eradicate melanoma cells with defective HLA class I (HLA-I). The latter abnormality plays a major role in clinical resistance to Checkpoint Inhibitors (CI). CSPG4 was selected as the target because of its high expression on melanoma and its restricted distribution in normal tissues. Our approach is based on CAR.CIK redirected against CSPG4. CIK, ex vivo expanded T-NK lymphocytes endowed with intrinsic HLA-independent antitumor activity, were used as effectors. Experimental procedure. CAR.CIK were generated by retroviral transduction of patient derived PBMC with a vector encoding a 2nd generation CSPG4-CAR with 4-1BB co-stimulation. Surface HLA-I expression was evaluated by flow cytometry on melanoma cell lines (Mel), derived by surgical biopsies. These cells also served as targets for CSPG4 CAR.CIK. The activity of CSPG4 CAR.CIK was analyzed in vitro and in immunodeficient mice grafted with a patient-derived HLA-defective melanoma. Mice were treated intravenously with 3x106 CAR.CIK (5 cells infusions in total). Results. CAR.CIK were efficiently generated from 4 melanoma patients. Mean expression of CSPG4-CAR was 48±8%, the rate of ex vivo expansion (84 fold) and phenotypic characteristics (CD3+CD8+=71±13%, CD3+ CD56+=22.5±13%, NKG2D+= 68±32.3%) were comparable with unmodified controls. Membrane HLA-I molecules were detected in 23/24 Mel samples, with a variable membrane density per cell (median 21232, range 787-49871). Sample Mel17 did not express HLA-I because of a start lost mutation (p.Met1Ile) in β2microglobulin. CSPG4 was intensely expressed by all Mel (78±5%), with no correlation to HLA-I levels. CSPG4 CAR.CIK efficiently killed 10 Mel samples in vitro, including Mel17 (HLA-I negative) and 2 Mel with the lowest HLA-I density (Mel71, Mel72). Mean Mel-specific killing by CSPG4 CAR.CIK was significantly higher compared with unmodified CIK especially at low effector/target (E/T) ratios (85% vs 40% at E/T=1:1; 46% vs 9% at E/T=1:8, p Conclusions. We reported the activity of CSPG4 CAR.CIK against melanoma, including those with low or defective HLA-I expression. CIK may provide a valid platform for CAR-based strategies against melanoma and solid tumors in general. Our data provide the rationale to implement clinical studies exploring the proposed strategy in melanoma patients not responding or relapsing after immunotherapy with CI. Citation Format: Giulia Cattaneo, Lidia Giraudo, Loretta Gammaitoni, Ilenia Iaia, Fabrizio Carnevale-Schianca, Alberto Pisacane, Enrico Berrino, Caterina Marchiò, Luca Paruzzo, Andrea Michela Biolato, Chiara Donini, Marco Basiricò, Elisa Landoni, Soldano Ferrone, Valeria Leuci, Massimo Aglietta, Gianpietro Dotti, Dario Sangiolo. CSPG4-specific CAR.CIK lymphocyte-based immunotherapy to eliminate HLA class I-defective melanoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2812.

Secondary acute myeloid leukemia (sAML) poorly responds to conventional treatments and allogeneic stem cell transplantation (HSCT). We evaluated toxicity and efficacy of CPX-351 in 71 elderly patients (median age 66 years) with sAML enrolled in the Italian Named (Compassionate) Use Program. Sixty days treatment-related mortality was 7% (5/71). The response rate at the end of treatment was: CR/CRi in 50/71 patients (70.4%), PR in 6/71 (8.5%), and NR in 10/71 (19.7%). After a median follow-up of 11 months relapse was observed in 10/50 patients (20%) and 12 months cumulative incidence of relapse (CIR) was 23.6%. Median duration of response was not reached. In competing risk analysis, CIR was reduced when HSCT was performed in first CR (12 months CIR of 5% and 37.4%, respectively, for patients receiving (=20) or not (=30) HSCT, p = 0.012). Twelve-months OS was 68.6% (median not reached). In landmark analysis, HSCT in CR1 was the only significant predictor of longer survival (12 months OS of 100 and 70.5%, for patients undergoing or not HSCT in CR1, respectively, p = 0.011). In conclusion, we extend to a real-life setting, the notion that CPX is an effective regimen for high risk AML patients and may improve the results of HSCT.

Purpose: BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti–PD-1 antibody. A portion of melanoma cells may express PD-1, and anti–PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1+ melanoma cells, supporting an additional—lymphocyte-independent—basis for their therapeutic combination with anti–PD-1 antibody. Experimental Design: With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE, N = 61; validation cell lines, N = 7) and melanoma tumors (TCGA, N = 214). We explored in vitro how BRAF/MEKi affect rates of PD-1+, PD-L1/2+ melanoma cells, and characterized the proliferative and putative stemness features of PD-1+ melanoma cells. We tested the functional lymphocyte-independent effect of anti–PD-1 antibody alone and in combination with BRAF/MEKi in vitro and in an in vivo immunodeficient murine model. Results: PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1+ cells increase to relevant rates during BRAF/MEKi treatment [7.3% (5.6–14.2) vs. 1.5% (0.7–3.2), P = 0.0156; N = 7], together with PD-L2+ melanoma cells [8.5% (0.0–63.0) vs. 1.5% (0.2–43.3), P = 0.0312; N = 7]. PD-1+ cells proliferate less than PD-1− cells (avg. 65% less; t = 7 days) and are preferentially endowed with stemness features. In vivo, the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse. Conclusions: BRAF/MEKi increase the rates of PD-1+ melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti–PD-1 antibody. Clin Cancer Res; 24(14); 3377–85. ©2018 AACR.

Before the introduction of "new drugs," we designed a trial in which 162 newly diagnosed myeloma patients were biologically randomized to receive either an autologous stem cell transplant (auto-SCT) followed by a nonmyeloablative allogeneic stem cell transplant (allo-SCT) or a double auto-SCT. Fifty-eight patients in the allo-SCT arm and 46 in the double auto-SCT arm completed the assigned treatment. At a median follow-up of 12.3 years from allo-SCT and 12.1 years from second auto-SCT, median overall survival (OS) was 11.4 in the allo-SCT arm and 3.9 years in the auto-SCT -arm (P = .007), whereas event-free survival was 3.6 and 1.5 years (P .001), respectively. A subset of allo-SCT patients showed persistent molecular remission. Two-year cumulative incidence of chronic graft-versus-host disease was 67.2%. At 5 years, 39% of these patients were alive, disease-free, and off immunosuppression; 36.6% had relapsed and 12.2% were still on immunosuppression. Thirty-three of 58 patients (allo-SCT arm) and 39 of 46 (auto-SCT arm) relapsed at least once and were rescued with new drugs. In the allo-SCT arm, 2 patients in biochemical relapse did not reach clinical criteria for treatment. Overall 28 (90%) were treated with new drugs and 14 (45%) received donor lymphocyte infusions (DLIs). In 28 of 31 patients (90%) DLIs were given with new drugs. Median OS from first relapse was 7.5 years in the allo-SCT arm and 2 years in the auto-SCT arm (P = .01). Patients who received DLI showed significantly longer OS (hazard ratio, .38; P = .042) as compared with auto-SCT patients. This difference was slightly lower when only allo-SCT patients who did not receive DLIs were considered (hazard ratio, .56; P = .154). In summary, long-term disease-free survival and survival outcomes after treating relapse with new drugs with or without DLIs were better in allo-SCT patients.

Introduction: The outcome of patients with acute myeloid leukemia (AML) secondary to myelodisplastic syndrome (MDS) or therapy-related (t-AML) receiving conventional treatment and allogeneic stem cell transplantation consolidation (HSCT) is poor. CPX-351 is a new drug composed by liposomal encapsulated cytarabine and daunorubicin, at a fixed molecular ratio of 5:1. It showed superior results, compared to standard 3+7 induction, in a phase III trial (Lancet et al, JCO 2018) in patients affected by t-AML or AML with myelodisplasia-related changes and it is now commercially available for secondary AML (sAML). We recently published results from CPX-351 Italian Named (Compassionate) Use Program (CUP) which enrolled 73 elderly sAML patients (Guolo et al, Blood Cancer J. 2020) showing that CPX-351 is an effective induction regimen for high risk AML patients treated with a curative aim. With a limited follow up, our data suggested the good activity and tolerability of CPX-351. Good quality remissions with acceptable toxicity in the majority of patients was achieved and CPX-351 increased the feasibility of HSCT in a poor risk AML cohort. Scarce data are available on long term outcome of high risk patients receiving CPX-351 in the real life setting. Here we report the results from the extended follow up analysis of the Italian CUP. Results: Seventy three patients were enrolled between December 2018 and June 2019 in a compassionate use program (CUP) in 33 Italian Hematology Centers. Data collection began on July 2019 and included 71/73 patients (97.2%), enrolled in 31 Centers. As previously reported, median age was 65.5 years (52-79). Sixty-two (88%) patients had at least one relevant comorbidity upon enrollment. Six patients (9%) presented with ECOG 3-4 upon enrollment. With a median follow up of 22 months, median overall survival (OS) was 13 months (21.2 - 22.8 95% IC). Two-years OS was 28.6% in the whole cohort. In order to confirm the positive impact of HSCT in first complete remission (CR) and the correlation with the other variables, a landmark model was applied, including only patients alive and in CR at day 90. In landmark analysis, HSCT performed in first CR after CPX-351 was the only significant predictor of longer survival: median OS was not reached for patients transplanted in first CR Vs 12 months for patients who did not undergo HSCT, p < 0.05, Figure1). Two-year OS for patients who received HSCT was 57.6% vs 15.8% for patients who did not undergo HSCT. Conclusions: Results from the extended follow up of Italian CPX-351 CUP confirm the good activity CPX-351 in such a difficult cohort as sAML. Two-year OS for transplanted patients is high despite the high median age, the high frequency of severe comorbidities in this real life cohort of patients and the high frequency of high risk AML. On the contrary, non-transplanted patients show a poor outcome, thus confirming that CPX-351 induction as an optimal bridge to transplant induction therapy. Figure 1 Figure 1. Disclosures Galimberti: Incyte: Speakers Bureau; AbbVie, Janssen: Honoraria, Other: Travel grants. Marco: Insight,: Consultancy; Jazz: Consultancy; Janssen: Consultancy. Fracchiolla: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Speakers Bureau. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Rossi: Novartis: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees. Pagano: Gilead Science, MSD, Pfizer, Basilea, Janssen, Novartis, Jazz Pharmaceutical, Cidara: Membership on an entity's Board of Directors or advisory committees; Menarini: Consultancy; Gilead Sciences, MSD, Pfizer Pharmaceuticals, Astellas Pharma: Speakers Bureau.

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.

Allogeneic hematopoietic stem cell transplantation (HSCT) from an unrelated HLA-mismatched donor (MMUD) is one of the alternatives where an HLA-matched donor is not found. Patients were divided into 4 groups: 43 pediatric with ATG and bone marrow as stem cell source (BM, A), 17 pediatric with ATG and peripheral blood stem cells (PBSC, B), 39 adult patients with ATG and PBSC (C) and 41 adults with PT-Cy-MMF-FK506 and PBSC (D) as GvHD prophylaxis. Overall survival (OS) was 73.9% vs. 31.5% vs. 42.4% vs. 64.1% for A, B, C and D (P

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.

Simple Summary Limited therapeutic results of immune checkpoint inhibitors in definite tumor settings, such as melanoma, call for alternative or complementary approaches. Among these, adoptive cell therapy (ACT) by means of HLA-independent tumor killer lymphocytes is a promising approach. We aimed at developing a pre-clinical 3D model to investigate and visualize the interaction between tumor and immune effectors in melanoma. To this aim, we employed Cytokine-Induced Killer cells (CIK) and NK-92 on patient-derived melanoma samples. By means of imaging-based methods, we measured the effector recruitment on the 3D targets, their infiltration, and cytotoxic activity. Our results and methodologies can be easily generalized to other effectors and other classes of tumors and help elucidate fundamental questions on the basic biology and kinetics of immune effector recruitment in a realistic 3D setting mimicking a solid tumor. Abstract Cancer adoptive cell therapy (ACT) with HLA-independent tumor killer lymphocytes is a promising approach, with intrinsic features potentially addressing crucial tumor-escape mechanisms of checkpoint inhibitors. Cytokine-induced Killer (CIK) and Natural Killer (NK) lymphocytes share similar tumor-killing mechanisms, with preclinical evidence of intense activity against multiple solid tumors and currently testing in clinical studies. To improve the effective clinical translation of such ACT approaches, several fundamental questions still need to be addressed within appropriate preclinical contexts, capable of overcoming limitations imposed by most traditional two-dimensional assays. Here, we developed a novel experimental approach to explore, dissect, and visualize the interactions of CIK and NK lymphocytes with melanoma tumors in vitro in 3D. Primary melanoma cells were assembled into small tumors that were dispersed in a 3D matrix and challenged with patient-derived CIK or the NK-92 cell line. By means of imaging-based methods, we reported, visualized, and quantitatively measured the recruitment of CIK and NK on the 3D targets, their infiltration, and cytotoxic activity. Our results support the effective tumor recruitment and tumor infiltration by CIK and NK. Such features appeared dependent on the specific geometric aspects of the environment but can be explained in terms of directional migration toward the tumor, without invoking major feedback components. Overall, our 3D platform allows us to monitor the processes of tumor recruitment, infiltration, and killing by means of live measurements, revealing important kinetic aspects of ACT with CIK and NK against melanoma.

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.

Allogeneic hematopoietic cell transplantation (allo-HCT) is an adoptive immunotherapy strategy whose effectiveness relies on graft-versus-tumor (GVT) effect. We explored the feasibility of enhancing GVT after allo-HCT by peptide vaccination. Two myeloma patients were transplanted with a fludarabine-total body irradiation conditioning regimen and vaccinated with an HLA-A*0201-restricted modified survivin nonapeptide, plus montanide as adjuvant. At time of first vaccination, one patient had just attained serological remission despite documented relapse after transplant, while the other patient was in stable disease. Both patients had an immune response to vaccination: the frequency of survivin-specific CD8+ T cells increased between second and sixth vaccination and accounted for 0.5–0.8% of CD8+ cells; CD8+ cells were functional in ELISPOT assay. The first patient persists in complete remission with a follow-up of >5 years, while the second patient did not have a clinical response and vaccination was halted. We analyzed the T-cell receptor (TCR) repertoire of the first patient by spectratyping and found that vaccination did not affect the diversity of TCR profile, indicating that survivin clonotypes were probably spread in multiple TCR families. We generated a limited number (n = 4) of survivin-specific T cell clones: three were reactive only against the modified peptide, whereas one clone recognized also the naive peptide. Peptide vaccination is safe and applicable after allo-HCT and elicits an efficient antigen-specific T cell response without causing graft-versus-host disease.

The mesenchymal stem cell (MSC) role after allogeneic hematopoietic stem cell transplantation (HSCT) is still a matter of debate; in particular, MSC engraftment in recipient bone marrow (BM) is unclear. A total of 46 patients were analyzed for MSC and hemopoietic stem cell engraftment after HSCT. The majority of patients had the BM as the stem cell source, and acute leukemia was the main indication for HSCT. Mesenchymal and hematopoietic stem cell chimerism analysis was carried out through specific polymorphic tandemly repeated regions. All patients reached complete donor engraftment; no evidence of donor-derived MSC engraftment was noted. Our data indicate that MSCs after HSCT remain of recipient origin despite the following: (i) myeloablative conditioning; (ii) the stem cell source; (iii) the interval from HSCT to BM analysis; (iv) the underlying disease before HSCT; and (v) the patients' or the donors' age at HSCT.

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.

The prognosis of stage IV metastatic melanoma is poor. An overall 1-year survival of 25.5% and a median survival of 6.2 months were reported without any significant improvement during the last 30 years before the introduction of new drugs (immune checkpoint inhibitors and targeted therapies) which completely modified the therapeutic approach and induced an overwhelming improvement on the survival rates of these patients. This review will analyze the therapeutic tools available for the treatment of patients with metastatic melanoma, including adjuvant interferon and locoregional therapies (surgery, radiotherapy and electrochemotherapy) and will mainly focus on the presentation of results obtained by the new treatments (checkpoint inhibitors and targeted therapies).

Interferons (IFNs) play a key role in modulating anti-microbial and antitumor immune responses. In oncology, past attempts to exploit IFNs therapeutically did not fulfill expectations, and had only modest clinical results, mostly limited to adjuvant melanoma treatment. The recent successes of immunotherapy in oncology have brought new attention to the potential of immune-modulatory agents like the IFNs. Areas covered: The authors review the biological effects of IFN on melanoma and immune cells. Then, the authors summarize the clinical results of adjuvant and therapeutic IFN in melanoma, giving focus to possible prognostic factors and new on-going clinical trials. Expert opinion: IFNs offer intriguing opportunities for synergism between conventional treatments and recently introduced molecular-targeted and immunotherapy approaches. However, the full comprehension of all IFN effects and their multiple biologic links is challenging. A strong commitment toward parallel translational research is needed to facilitate the interpretation of IFN's expected and unexpected effects, guiding the rational design of informative clinical studies.

Conventional treatments reached an unsatisfactory therapeutic plateau in the treatment of advanced unresectable bone and soft tissue sarcomas that remain an unsolved medical need. Several evidences support the concept that adoptive immunotherapy may effectively integrate within the complex and multidisciplinary treatment of sarcomas.In this work we reviewed adoptive immunotherapy strategies that have been explored in sarcoma settings, with specific focus on issues related to their clinic transferability. We schematically divided approaches based on T lymphocytes specific for MHC-restricted tumor-associated antigens or relying on MHC-independent immune effectors such as natural killer (NK), cytokine-induced killer (CIK) or γδ T cells.Preclinical findings and initial clinical reports showed the potentialities and drawbacks of different adoptive immunotherapy strategies. The expansion of tumor infiltrating lymphocytes is difficult to be reproduced outside melanoma. Genetically redirected T cells appear to be a promising option and initial reports are encouraging against patients with sarcomas. Adoptive immunotherapy with MHC-unrestricted effectors such as NK, CIK or γδ T cells has recently shown great preclinical potential in sarcoma setting and biologic features that may favor clinical transferability. Combination of different immunotherapy approaches and integration with conventional treatments appear to be key issues for successful designing of next clinical trials.

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.

Of 93 patients with pretreated, BRAFV600 mutation-positive advanced melanoma who received vemurafenib or dabrafenib before (n = 45) or after (n = 48) treatment with ipilimumab 3 mg/kg, median overall survival (mOS) from first treatment was 9.9 and 14.5 months, respectively. Among patients treated with a BRAF inhibitor first, mOS from the end of BRAF inhibition was 1.2 months for those who did not complete ipilimumab treatment as per protocol, compared with 12.7 months for those who did (p < .001). Prospective, randomized studies are required to determine the optimal sequencing of ipilimumab and BRAF inhibitors in patients with BRAF-mutated metastatic melanoma.

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem–like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease. Cancer Res; 74(1); 119–29. ©2013 AACR.

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.

Background Antibodies blocking the inhibitory checkpoint PD1, expressed by T lymphocytes, and kinase inhibitors of BRAF and MEK (MAPKi) are mainstays of current treatment for patients with metastatic melanoma. Mouse models point towards a synergistic antitumor activity of anti-PD1-antibodies and MAPKi, explaining the findings by an enhanced lymphocytic activity opposing tumor adaptive resistance. Recently PD1 was found expressed by a subpopulation of melanoma cells where it could mediate tumorigenic effects, but no data exist showing how MAPKi could modulate its expression. We hypothesized that MAPKi may upregulate PD1 expression and enhance the direct, immune-cell independent antitumor action of anti-PD1 antibodies. We first explore the exact prevalence and magnitude of PD1 expression in patients and cell lines. Then we test the effect of MAPKi on its expression and explore possible immune-independent synergisms with anti-PD1-antibodies. Methodology and results. Prevalence of PD1 expression by melanoma cells was explored in the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) datasets. CCLE included 61 melanoma cell lines, which all expressed PD1 (Affymetrix mRNA 4.20 [3.81- 4.65]) with values comparable to PDL1 (4.63, range 3.73 -7.84) and PDL2 (3.73, range 3.97-8.50). The TCGA analysis included 114 melanomas, excluding those with evidence of immune infiltrate (n=256) or stromal cells (n=100) potentially interfering with PD1 assessment. PD1 mRNA was expressed by all samples (median FPKM values: 64.7, range 0.4-1461.3). Next we assessed by flow-cytometry the membrane expression of PD1 in 4 melanoma cell lines and 3 patient-derived melanoma cultures, harboring BRAF and NRAS mutations We confirmed that low rates of PD1 were expressed by all melanoma (median 1.2%, range 0.7- 4.3). The median rate of viable PD1+ melanoma cells significantly increased (12.9% range 5.9-20, p=0.0180, n =7) following treatment (96h) with MAPKi (dabrafenib and trametinib) but not with chemotherapy (fotemustine). Longer treatments (8 days) further increased the rate of PD1+ melanoma cells (31.8% range 15.0-50.3, n=3) and the effect was reversed by drug withdrawal. The MAPKi modulatory effect on PD1 expression was therapeutically exploited in vitro. The addition of PD1 blocking antibody enhanced the inhibitory activity of MAPKi on melanoma proliferation (mean inhibition rate: 66% vs 40%, n=2) in the absence of any immune cells. Conclusions We report a new expression pattern of PD1 receptor on melanoma and its potential therapeutic perspective. PD1 is directly expressed by a small but consistent rate of melanoma cells. Treatment with MAPKi significantly enhanced the rate of PD1+ melanoma cells and direct synergism with PD1 blockade was reported without the involvement of immune response. Our findings may be clinical relevant in settings of patients treated with MAPKi. Citation Format: Martina Sanlorenzo, Igor Vujic, Arianna Floris, Mauro Novelli, Chiara Donini, Loretta Gammaitoni, Lidia Giraudo, Marco Macagno, Pietro Quaglino, Maria Teresa Fierro, Silvia Giordano, Fabrizio Carnevale-Schianca, Massimo Aglietta, Dario Sangiolo. Melanoma PD1 expression is upregulated by MAPK inhibitors leading to an immune-independent synergism with anti-PD1 antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5603. doi:10.1158/1538-7445.AM2017-5603

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.