Characterization of a novel fusion gene EML4-NTRK3 in a case of recurrent congenital fibrosarcoma

We describe the clinical course of a recurrent case of congenital fibrosarcoma diagnosed in a 9-mo-old boy with a history of hemimelia. Following complete surgical resection of the primary tumor, the patient subsequently presented with bulky bilateral pulmonary metastases 6 mo following surgery. Molecular characterization of the tumor revealed the absence of the prototypical ETV6-NTRK3 translocation. However, tumor characterization incorporating cytogenetic, array comparative genomic hybridization, and RNA sequencing analyses, revealed a somatic t(2;15)(2p21;15q25) translocation resulting in the novel fusion of EML4 with NTRK3. Cloning and expression of EML4-NTRK3 in murine fibroblast NIH 3T3 cells revealed a potent tumorigenic phenotype as assessed in vitro and in vivo. These results demonstrate that multiple fusion partners targeting NTRK3 can contribute to the development of congenital fibrosarcoma

Interstitial fluid: the overlooked component of the tumor microenvironment?

Background: The interstitium, situated between the blood and lymph vessels and the cells, consists of a solid or matrix phase and a fluid phase, together constituting the tissue microenvironment. Here we focus on the interstitial fluid phase of tumors, i.e., the fluid bathing the tumor and stromal cells. Novel knowledge on this compartment may provide important insight into how tumors develop and how they respond to therapy. Results: We discuss available techniques for interstitial fluid isolation and implications of recent findings with respect to transcapillary fluid balance and uptake of macromolecular therapeutic agents. By the development of new methods it is emerging that local gradients exist in signaling substances from neoplastic tissue to plasma. Such gradients may provide new insight into the biology of tumors and mechanistic aspects linked to therapy. The emergence of sensitive proteomic technologies has made the interstitial fluid compartment in general and that of tumors in particular a highly valuable source for tissue-specific proteins that may serve as biomarker candidates. Potential biomarkers will appear locally at high concentrations in the tissue of interest and will eventually appear in the plasma, where they are diluted. Conclusions: Access to fluid that reliably reflects the local microenvironment enables us to identify substances that can be used in early detection and monitoring of disease

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Supplementary Figure 9 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 9 shows that BMF knockdown rescues ATF5 loss-induced reduction of anchorage-independent cell viability</p

Supplementary Figure 5 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 5 shows that overexpression of ATF5 promotes anoikis resistance of CHLA-255 in vitro</p

Supplementary Table 1 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Table 1. List of antibodies used in this study</p

Supplementary Figure 2 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 2 shows that ATF5 knockdown decreases neuroblastoma cell viability and promotes apoptosis under adherent conditions</p

Supplementary Figure 4 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 4 shows that decreased ATF5 expression does not alter the invasiveness of BE(2)-C cells in vitro</p

FIGURE 5 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

BMF is required for ATF5 depletion–dependent anoikis induction and is upregulated by FOXO3 induction after ATF5 knockdown. A, BMF expression, by immunoblot, in suspension cells 72 hours after transfection with empty vector (EV) or BMF expression vector in BE(2)-C cells and SK-N-DZ cells. β-Actin was used as a loading control. Densitometric analysis was performed using ImageJ. B, Quantification of anoikis 72 hours after transfection with BMF expression vector or EV in BE(2)-C and SK-N-DZ cells. C, Knockdown of BMF in BE(2)-C and SK-N-DZ cells. Adherent cells were transiently transfected with siGFP (negative control), siBMF-1, or siBMF-2. Twenty-four hours later, cells were seeded in nonadherent poly-HEMA–coated plates. A total of 96 hours after transfection, lysates were immunoblotted. Untreated (UT). β-Actin was used as a loading control. Densitometric analysis was performed using ImageJ. D, BMF knockdown suppresses anoikis in detached neuroblastoma cells. Quantification of anoikis in detached BE(2)-C and SK-N-DZ, 96 hours after transfection with siGFP (negative control), siBMF-1, or siBMF-2. BMF knockdown inhibits induction of anoikis following ATF5 depletion. BE(2)-C (E) and SK-N-DZ (F), expressing shATF5-1 (left) or shATF5-2 (right), were transfected with siGFP (negative control), siBMF-1, or siBMF-2, and 24 hours later seeded in poly-HEMA–coated plates. Dox was added to deplete ATF5, and 72 hours later, anoikis was measured. −Dox (open bars), +Dox (closed bars). G, ATF5 knockdown in BE(2)-C and SK-N-DZ suspension cells elevates FOXO3 expression. Immunoblot analyses of ATF5 and FOXO3 in suspension cells expressing shATF5-1 or shATF5-2 at 72 hours ± Dox addition. β-Actin was used as a loading control. Densitometric analysis was performed using ImageJ. H, Immunoblot analyses show that FOXO3 knockdown with siRNA prevents BMF induction in response to ATF5 knockdown. BE(2)-C and SK-N-DZ cells, expressing shATF5-2, were transfected with siFOXO3-1 or siFOXO3-2 and 24 hours later were seeded in poly-HEMA–coated plates. Dox was added, and 72 hours later, cell lysates were immunoblotted. β-Actin was used as a loading control. Densitometric analysis was performed using ImageJ. I, Anoikis induction by ATF5 knockdown requires FOXO3. Percentage of BE(2)-C-shATF5-2 (left) and SK-N-DZ-shATF5-2 (right) cells undergoing anoikis at 72 hours, ± Dox addition and ± FOXO3 knockdown. −Dox (open bars), +Dox (closed bars). Cells were treated as described in H. Mean ± SD. **, P P P < 0.0001.</p