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High tumor mutational burden assessed in tissue biopsies (tTMB-H) or blood (bTMB-H) is associated with clinical efficacy in patients treated with immunotherapies. CheckMate 848 (NCT03668119) is a prospective phase 2 study of nivolumab (NIVO) with or without ipilimumab (IPI) in patients with advanced or metastatic solid tumors that are tTMB-H or bTMB-H (≥ 10 mutations/megabase) who were immunotherapy-naive and refractory to standard local therapies. The primary endpoint was objective response rate (ORR) in patients with tTMB-H or bTMB-H, assessed by FoundationOne® CDx-based and Clinical Trial Assays (Foundation Medicine), respectively. The study was not powered to compare NIVO + IPI vs NIVO. We present the interim and final analyses for the tTMB-H and bTMB-H cohorts, respectively (≥ 12 months follow-up, database lock June 2021). Of 1954 screened patients, 212 were randomized 2:1 to NIVO 240 mg Q2W + IPI 1 mg/kg Q6W or NIVO 480 mg Q4W for ≤ 24 months, and 201 (135 tTMB-H; 147 bTMB-H) were refractory to standard therapies. Of > 40 tumor types, colorectal (10.8%), small-cell lung (7.5%), breast (7.1%), and uterine (7.1%) were the most common. ORR and survival outcomes with NIVO + IPI were improved in patients with tTMB-H. The responses were independent of bTMB-H status in the tTMB-H cohort but improved with tTMB-H status in the bTMB-H cohort (Table). The safety profile of NIVO + IPI was manageable, and clinical outcomes with NIVO were comparable with previous studies. The impact of TMB cutoff, PD-L1 expression, and microsatellite instability were explored. In conclusion, NIVO + IPI demonstrated clinical efficacy with a manageable safety profile in patients with advanced or metastatic solid tumors that are tTMB-H or bTMB-H and refractory to standard therapies, with increased efficacy observed in patients with tTMB-H. NIVO + IPI tTMB-H cohort bTMB-H cohorta Patients, n (%)b,c 68 (32.1) 80 (37.7) Number of prior treatments, median (range) 2 (0–7) 2 (1–9) ORR, n (%)c, 95% CI 24 (35.3), 24.1–47.8 18 (22.5), 13.9–33.2 ORR in patients with bTMB-H by tTMBc: < 10 mut/Mb (n = 31), n (%), 95% CI NA 3 (9.7), 2.0–25.8 ≥ 10 mut/Mb (n = 39), n (%), 95% CI NA 13 (33.3), 19.1–50.2 ≥ 10 to < 16 mut/Mb (n = 18), n (%), 95% CI NA 3 (16.7), 3.6–41.4 ≥ 16 mut/Mb (n = 21), n (%), 95% CI NA 10 (47.6), 25.7–70.2 ORR in patients with tTMB-H by bTMBc: < 10 mut/Mb (n = 20), n (%), 95% CI 7 (35.0), 15.4–59.2 NA ≥ 10 mut/Mb (n = 43), n (%), 95% CI 16 (37.2), 23.0–53.3 NA ≥ 10 to < 16 mut/Mb (n = 12), n (%), 95% CI 3 (25.0), 5.5–57.2 NA ≥ 16 mut/Mb (n = 31), n (%), 95% CI 13 (41.9), 24.5–60.9 NA Percentage of responders (≥ 9 months) (95% CI) 91 (68–98) 88 (61–97) Median PFS, months (95% CI)c 4.1 (2.8–11.3) 2.8 (2.3–3.0) Median OS, months (95% CI)c 14.5 (7.7–NE) 8.5 (5.8–10.5) aThe bTMB cohort was randomized prior to December 20, 2019. bOut of 212 randomized patients; data presented in this table are from patients who were refractory to standard therapies. cMinimum follow-up 12 months. bTMB, blood tumor mutational burden; NA, not applicable; NE, not evaluable; PFS, progression-free survival; OS, overall survival; tTMB, tissue tumor mutational burden. Citation Format: Michael Schenker, Mauricio Burotto, Martin Richardet, Tudor Ciuleanu, Anthony Goncalves, Neeltje Steeghs, Patrick Schöffski, Paolo A. Ascierto, Michele Maio, Iwona Lugowska, Lorena Lupinacci, Alexandra Leary, Jean-Pierre Delord, Julieta Grasselli, David S. Tan, Jennifer E. Friedmann, Jacqueline Vuky, Marina Tschaika, Ruta Slepetis, Georgia D. Kollia, Misena Pacius, Ning Huang, Parul Doshi, Jonathan Baden, Massimo Di Nicola. CheckMate 848: A randomized, open-label, phase 2 study of nivolumab in combination with ipilimumab or nivolumab monotherapy in patients with advanced or metastatic solid tumors of high tumor mutational burden [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 CT022.

CD8+ T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8+ T cells expressing FOXP3. CD8+ FOXP3+ CD25+ T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8+ FOXP3+ T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-β1], exhausted (PD-1), or senescent (CD57) CD8+ T lymphocytes. Instead, this subset showed an antigen-experienced “EM1” phenotype (CCR7− CD45RA− CD28+ CD27+) and exhibited a CD127−, KLRG1−, HLA-DR+, CD38+, T-bet+, perforin+ “early effector” profile predicted by current models. CD8+ FOXP3+ T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8+ FOXP3+ T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8+ FOXP3+ “early effector” subset may be an invaluable tool for monitoring immunity at tumor site. Cancer Res; 70(21); 8378–87. ©2010 AACR.

To investigate the therapeutic activity of the fully human anti–HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138+ cells, analyzed the capacity of IFN-γ to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-γ. CD138+HLA-DR+ cells were detected in 31 of 60 patients, with 15 of 60 patients having ≥20% CD138+HLA-DR+ cells (median, 50%; range, 23–100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-γ restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-γ/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P ≤ 0.0001) or mice receiving 1D09C3 alone (147 versus 92 days, P ≤ 0.03). The better therapeutic activity of IFN-γ/1D09C3 treatment over 1D09C3 alone was further shown by a 2-fold increase of mice being disease-free at 150 days after xenograft (47% versus 25%). No mice experienced any apparent treatment-related toxicity. Our data show that (a) one fourth of MM patients express HLA-DR on CD138+ cells and (b) IFN-γ–induced up-regulation of HLA-DR results in a potent enhancement of the in vivo antimyeloma activity of 1D09C3. [Cancer Res 2007;67(7):3269–75]

Background: The standard chemotherapy treatment for Non-Hodgkin lymphoma (NHL) consists in the combination of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). Because of the relevant treatment-related toxicity and the minor therapeutic effectiveness of CHOP therapy variants, the development of novel therapeutic approaches represents an urgent clinical need. Despite treatment with the anti-CD20 monoclonal antibody Rituximab and CHOP has led to favourable results for CD20+ lymphoma, many patients experienced drug resistance. Based on studies indicating that the malignant behaviour of tumors depends on the interactions between tumor and its microenvironment, we tested the effect of the novel heparanase inhibitor Roneparstat (sigma-tau Research Switzerland) in combination with various clinically relevant agents against B-cell lymphoma models, poorly responsive to conventional agents. Methods: We assessed the CD20 cell surface expression by flow cytometry, the heparanase (HPA-1) expression levels by immunoblotting, the VEGF spontaneous release in cell supernatant by proteome array in B-NHL models. The therapeutic activity was evaluated in SCID mice xenografted with CD20+ and VEGF+ B-NHL models. The efficacy of the drug treatment was estimated as tumor volume inhibition percentage (TVI%), log10 cell kill (LCK) and complete regression (CR). To gain insight the mechanisms underlying the anti-tumor activity of Roneparstat plus rituximab histopathological/immunoistochemical analyses were performed on treated tumors versus controls. Results: In a model of aggressive diffuse large B-cell NHL, except for doxorubicin, the combinations cyclophosphamide, dexamethasone and rituximab exhibited significantly superior efficacy over single-agent therapy. The most impressive enhancement of anti-tumor activity was observed with Roneparstat plus rituximab, with a high rate of complete tumor regressions and no evidence of disease at the end of the experiment in 50% of treated animals. Histological analysis revealed inflammatory cell infiltration, stromal destructuration, most pronounced apoptotic changes in Roneparstat plus rituximab-treated tumors versus controls; stromal architecture analysis showed signs of an altered, incomplete reticulin network suggestive of impaired stromal scaffolding that might have promoted the recruitment of complement components (C1q, C5) within tumors increasing the cytotoxic effect of rituximab. This interpretation was also supported by the favourable interaction of Roneparstat with bevacizumab in a Burkitt's lymphoma model characterized by high levels of VEGF.. Conclusion: Our results provide evidence that targeting the tumor microenvironment with Roneparstat may have therapeutic potential in combination with various agents against aggressive lymphoma subtypes. Citation Format: Massimo Di Nicola, Franco Zunino, Anna Rossini, Giusi Ruggiero, Micheleandrea De Cesare, Denis Cominetti, Monica Tortoreto, Cinzia Lanzi, Giuliana Cassinelli, Roberta Zappasodi, Claudio Tripodo, Nadia Zaffaroni. Microenvironment modulation and enhancement of cytotoxic therapy by the heparanase inhibitor Roneparstat against human B-non Hodgkin lymphomas. [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 3289.

BACKGROUND: Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic metabolism, especially in anaerobic condition. High serum levels at baseline seem to be a negative prognostic marker in many tumor types, as well a negative predictive marker for response to treatment with anti CTL4 antibodies(Ipilimumab)in Malignant Melanoma. Recently, the treatment of several advanced tumors with anti PD-1 or PDL-1 monoclonal antibodies (mAbs) showed better outcome in comparison to Ipilimumab. However, biomarkers for a proper selection of responding patients is not yet available. The aim of this study is to correlate LDH serum level at baseline with clinical outcome in patients (pts) with advanced solid tumors treated with anti PDL-1 and anti PD-1 mAbs. MATERIAL AND METHODS: We evaluated baseline LDH serum level in 145 pts affected by advanced solid tumors, treated at our Institute with anti PDL-1 and anti PD-1 mAbs. The rate of clinical responses and progression free survival (PFS) were related to normal or elevated baseline LDH serum level. We stratified the pts in 4 groups: normal value (group A),up to 1.5 x ULN (group B); < 2 > 1.5 x ULN (Group C); > 2 x ULN (Group D). We defined as disease control (DC) any complete (CR) and partial response (PR) or stable disease (SD) lasting > 3 months. Correlation between LDH serum levels and DC was assessed by Fisher's exact test; PFS was estimated using the Kaplan-Meier method. RESULTS: We evaluated a heterogeneous population of 145 patients: 73 pts with lung cancer (50.3%; 67 NSCLC, 6 SCLC); 32 pts with Melanoma (22%) and 40 pts (27.6%) with other solid tumors (8 urothelial, 7 biliary tract, 6 mesothelioma, 4 head and neck, 4 sarcoma, 3 gastric, 3 colon, 2 RCC, 1 thyroid, 1 ovarian, 1 HCC). 79 pts (54.5%) were treated by anti PD-1 and 66 (45.5%) by anti PDL-1 agents. Overall as “best response”, 79 pts (54.5%) achieved DC (1 CR, 26 PR, 52 SD) and 66 pts (45.5%) had progressive disease (PD). DC was achieved in 41/79 (52%) pts treated by anti PD-1 and 38/66 (58%) pts treated by anti PDL-1: only 2/41 (5%) and 5/38 (13%) of them had high LDH levels at baseline, respectively. Among 107 pts in Group A, 72 (67.3%) achieved DC as compared to 7/38 pts (18.4%) with high levels of LDH (Group B-D) (p: 0.0001). In the latter groups, PD occurred in 17/23 (74%), 5/6 (83%) and 9/9 (100%) patients in the B, C and D group respectively. CONCLUSIONS: According to this preliminary analysis high baseline LDH serum levels seems to predict a greater likelihood of obtaining worse clinical outcome in terms of response and PFS also in patients treated by anti PD-1 and anti PDL-1 mAbs. In order to confirm this interesting result, we are evaluating a greater number of patients treated with anti PD-1 and anti PDL-1 mAbs at our Institution. Citation Format: Maria Silvia Cona, Valter Torri, Massimo Di Nicola, Marina Garassino, Michele Del Vecchio, Sara Cresta, Diego Signorelli, Silvia Damian, Matteo Duca, Alice Indini, Monica Niger, Filippo de Braud. Baseline LDH serum level as predictive value of activity in patients treated with anti PD-1 and PDL-1 monoclonal antibodies. [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 3943.

Increasing evidence argues that the success of an anticancer treatment may rely on immunoadjuvant side effects including the induction of immunogenic tumor cell death. Based on the assumption that this death mechanism is a similar prerequisite for the efficacy of an active immunotherapy using killed tumor cells, we examined a vaccination strategy using dendritic cells (DC) loaded with apoptotic and necrotic cell bodies derived from autologous tumors. Using this approach, clinical and immunologic responses were achieved in 6 of 18 patients with relapsed indolent non–Hodgkin's lymphoma (NHL). The present report illustrates an impaired ability of the neoplastic cells used to vaccinate nonresponders to undergo immunogenic death on exposure to a cell death protocol based on heat shock, γ-ray, and UVC ray. Interestingly, when compared with doxorubicin, this treatment increased surface translocation of calreticulin and cellular release of high-mobility group box 1 and ATP in histologically distinct NHL cell lines. In contrast, treated lymphoma cells from responders displayed higher amounts of calreticulin and heat shock protein 90 (HSP90) compared with those from nonresponders and boosted the production of specific antibodies when loaded into DCs for vaccination. Accordingly, the extent of calreticulin and HSP90 surface expression in the DC antigenic cargo was significantly associated with the clinical and immunologic responses achieved. Our results indicate that a positive clinical effect is obtained when immunogenically killed autologous neoplastic cells are used for the generation of a DC-based vaccine. Therapeutic improvements may thus be accomplished by circumventing the tumor-impaired ability to undergo immunogenic death and prime the antitumor immune response. Cancer Res; 70(22); 9062–72. ©2010 AACR.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALKhigh 12.8%, ALKlow 73%, P = 0.0035; cell death: ALKhigh 56.4%, ALKlow 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs. [Cancer Res 2009;69(18):7338–46]

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (Por = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (Por = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (Por = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (Por = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

Deregulated c-Myc expression plays a critical role in the pathogenesis and progression of aggressive B-cell non-Hodgkin lymphomas (B-NHLs) and multiple myeloma. Chromosomal translocations juxtaposing c-Myc to immunoglobulin gene regulatory elements are virtually invariant, early transforming events in Burkitt's lymphoma (BL), thus constituting the prime example of c-Myc-driven oncogenesis. The key role of c-Myc-mediated epigenetic changes for its oncogenic gene expression reprogramming makes histone deacetylases (HDAC) inhibitors novel suitable drug candidates for the treatment of c-Myc-driven lymphomas. Furthermore, an increasing body of evidence is unraveling the ability of these inhibitors to down-regulate c-Myc expression itself in tumor cells. Therefore, we studied the potential antitumor activity of ITF2357, a new-generation pan-HDACs inhibitor, in human BL pre-clinical models. ITF2357 boosted histone and non-histone protein acetylation in Namalwa and Raji cells, and induced G1 cell-cycle arrest followed by apoptosis especially in the Namalwa model. Treated cells displayed a significant decrease of c-Myc protein but not mRNA levels. This effect was only partially explained by the significant up-regulation of let-7a and miR-26a - two microRNA that negatively control c-Myc expression - since it was not as persistent as c-Myc down-regulation. Given tumor cell addiction to cap-dependent translation for the constitutive expression of short-lived oncoproteins, including c-Myc, we investigated whether this mechanism was also affected by ITF2357. Akt and PIM kinases are the best-defined activators of cap-dependent protein translation initiation via 4E-BP1 phosphorylation and derepression of the translation factor eIF4E. We found that 4E-BP1 and eIF4E phosphorylation as well as phosphorylated-Akt and PIM kinases were significantly down-regulated in the treated cells. Since mTOR and PIM kinase inhibitors display only limited clinical efficacy against B-NHLs, ITF2357 treatment may thus exert a therapeutic advantage, given its ability to concurrently block the redundant oncogenic survival signals that control cap-dependent translation initiation, and potentially overcome the selection of chemoresistance mechanisms. In Raji-xenografted immunodeficient mice, ITF2357-mediated biomolecular effects were translated into 45% tumor shrinkage and complete disease eradication in 70% of the animals when the drug was used as maintenance management after its combination with low-dose cyclophosphamide. Taken as a whole, these findings provide the proof-of-concept for the clinical evaluation of ITF2357 in rational combinations with conventional and unconventional anticancer agents for the treatment of c-Myc-overexpressing and/or cap-translation-dependent NHLs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4719. doi:1538-7445.AM2012-4719

We reported that the splice variant of human HER2 lacking exon 16 (delta16HER2) represents a highly penetrating HER2 oncogenic alteration identified in human primary breast tumor specimens and is able to influence the response to Trastuzumab. This HER2 variant forms covalent cysteine bonds that generate constitutively active homodimers, thereby activating multiple oncogenic downstream signaling pathways that we recently found to be mediated through activated Src kinase. To examine the ability of delta16HER2 to transform mammary epithelium in vivo and to monitor delta16HER2-driven tumorigenesis in live mice, we generated a FVB transgenic mouse model for the human delta16HER2 isoform. Transgenic female mice developed multifocal mammary tumors with a rapid onset starting at about 12 weeks of age and progressively thereafter, clearly pointing to the candidacy of the delta16HER2 isoform as the transforming form of the human HER2 oncoprotein. Histological and immunohistochemical analysis (IHC) of primary mammary nodules revealed a population of polygonal cells with classical epithelia-like aspects distinctly expressing HER2 and also a population of smaller spindle-shaped cells arranged in fascicles with lower levels of HER2 expression, suggesting the onset of the epithelial-to-mesenchymal transition (EMT). Consistent with these findings, FACS analysis of delta16HER2-positive tumor cells immunomagnetically purified from disaggregated transgenic primary tumors indicated the increased mean fluorescence intensity of HER2 staining with increasing tumor cell size. IHC analysis of the lung metastases that had formed in the majority of female mice revealed monomorphic and classical epithelial tumor cells homogeneously expressing high levels of delta16HER2. FACS and IHC analyses confirmed the lower binding efficacy of Trastuzumab to delta16HER2-overexpressing primary tumor cells cultured both under bidimensional (2D) and tridimensional (3D) conditions as compared to monoclonal reagents directed to different HER2 extracellular domain epitopes. Experiments in both primary and metastatic in vitro and in vivo delta16HER2-positive models are in progress to determine whether delta16HER2-driven tumor aggressiveness and Trastuzumab susceptibility depend not only on genetic changes intrinsic to the tumor cell, i.e., the EMT process, but also on extrinsic tumor surrounding microenvironment-related factors such as an imbalance between extracellular and intracellular pH, redox state and hypoxia. Preliminary FACS and IHC analyses indicate that delta16HER2-positive primary tumor cells are reactive for known epithelial markers as EpCAM, E-cadherin- and ck-18 and, a small subset of these mammary tumor cells, also stain positive for the mesenchymal differentiation markers vimentin, N-cadherin and ck14 significantly indicating an active EMT program. Supported by AIRC and Ministry of Health Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 916. doi:1538-7445.AM2012-916

Introduction. We have previously demonstrated that adenovirus-transduced CD34+ cells expressing membrane-bound (m)TRAIL (CD34-TRAIL+ cells) exert potent antitumor activity against a variety of hematopoietic tumors by targeting both tumor cells and tumor vasculature (Blood, 115:2231-40, 2010). Recently, we have identified non-Hodgkin lymphoma cell lines which are resistant to the in vivo antitumor activity of mTRAIL. Perifosine has been shown to increase the toxicity of soluble TRAIL against cancer cell lines by inhibiting the PI3K/Akt pathway as well as enhancing pro-apoptotic TRAIL receptors. We therefore investigated the efficacy of Perifosine in modulating the antitumor activity of CD34-TRAIL+ cells using as model systems the TRAIL-resistant SU-DHL-4V and the TRAIL-sensitive KMS-11 cell lines. Methods and Results. In vitro, Perifosine significantly enhanced the cytotoxic activity of CD34-TRAIL+ cells against both SU-DHL-4V and KMS-11 cell lines by increasing the expression of TRAIL receptors and down-modulating phospho-Akt, cFLIP and Mcl-1 expression. In vivo, in NOD/SCID mice bearing subcutaneous nodules, Perifosine significantly increased the antitumor activity of CD34-TRAIL+ cells against both tumor types. In fact, CD34-TRAIL+ cells used as single agent exerted a limited if any activity against TRAIL-resistant SU-DHL-4V nodules, whereas, when combined with Perifosine, transduced cells reduced SU-DHL-4V growth by 43% (p< .001) over controls. TRAIL-sensitive KMS-11 nodules were reduced by 39% (p< .001) following treatment with CD34-TRAIL+ cells, and addition of Perifosine further reduced tumor growth by 65% (p Conclusions. Our results demonstrate that: (i) TRAIL-R2 expression by TECs correlates with the in vivo antivascular activity of CD34-TRAIL+ cells; (ii) Perifosine potentiates the antitumor activity of TRAIL-armed CD34+ cells and is able to overcome in vivo resistance to mTRAIL by inducing TRAIL-R2 expression on tumor endothelial cells. These results may open new perspectives in view of clinical applications of CD34-TRAIL+ cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2011-640