Purpose
Isothiocyanates elicit anticancer effects by targeting cancer stem cells (CSCs). Here, we tested the antitumor activity of phenethyl-isothiocyanate (PEITC), either alone or in combination ...with trastuzumab, in HER2-positive tumor models.
Methods
We assessed the
in vitro
anticancer activity of PEITC, alone or combined with trastuzumab, in HER2-positive BT474, SKBR3, HCC1954 and SKOV3 cancer cells by measuring their sphere forming efficiency (SFE). The expression of the human/rodent CSC biomarkers aldehyde-dehydrogenase (ALDH) and CD29
High
/CD24
+
/Sca1
Low
was evaluated by cytofluorimetric analysis. The expression of wild type HER2 (WTHER2), its splice variant d16HER2 and NOTCH was analysed by quantitative RT-PCR and Western blotting. The
in vivo
activity of PEITC and trastuzumab was evaluated in mice orthotopically implanted with MI6 tumor cells transgenic for the human d16HER2 splice isoform. Magnetic resonance imaging/spectroscopy and immunohistochemistry were used to assess morpho-functional and metabolic profiles of treated
versus
untreated mice.
Results
We found that PEITC significantly impaired the SFE of HER2-positive human cancer cells by decreasing their ALDH-positive compartments. The anti-CSC activity of PEITC was demonstrated by a reduced expression/activation of established cancer-stemness biomarkers. Similar results were obtained with MI6 cells, where PEITC, alone or in combination with trastuzumab, significantly inhibited their SFE. We also found that PEITC hampered the
in vivo
growth of MI6 nodules by inducing hemorrhagic and necrotic intra-tumor areas and, in combination with trastuzumab, by significantly reducing spontaneous tumor development in d16HER2 transgenic mice.
Conclusions
Our results indicate that PEITC targets HER2-positive CSCs and that its combination with trastuzumab may pave the way for a novel therapeutic strategy for HER2-positive tumors.
To develop high-dose myeloablative therapy for CD20(+) non-Hodgkin's lymphoma (NHL) as a safe and widely applicable regimen.
Patients with relapsed/refractory (n = 25) or de novo high-risk (n = 5) ...NHL received one myeloablative dose of yttrium-90 ((90)Y)-ibritumomab tiuxetan after five chemotherapy courses, including three cycles of anthracycline- or platinum-containing regimens, one cycle of cyclophosphamide (4 to 7 g/m(2)), and one cycle of cytarabine (12 to 24 g/m(2)). The only exclusion criteria were CNS lymphoma and Eastern Cooperative Oncology Group performance status of more than 3. Primary end points were overall survival (OS) and event-free survival (EFS). Secondary end points included safety and applicability of high-dose (90)Y-ibritumomab tiuxetan. To minimize hematologic toxicity, stem cells were reinfused at days 7 and 14 after (90)Y-ibritumomab tiuxetan.
Thirteen patients received (90)Y-ibritumomab tiuxetan 0.8 mCi/kg, and 17 patients received 1.2 mCi/kg. At 1.2 mCi/kg, the radiation absorbed by critical nonhematologic organs approached the protocol-defined upper safety limit, defining this as the recommended dose for subsequent studies. Hematologic toxicity was mild to moderate and of short duration. Infections occurred in 27% of patients (none had a severity grade greater than 3). After a median observation time of 30 months (range, 22 to 48 months), no myeloid secondary malignancy or chromosomal abnormality was observed, the OS rate was 87%, and the EFS rate was 69%.
High-dose (90)Y-ibritumomab tiuxetan seems to be an innovative myeloablative regimen with unprecedented short-term toxicity and wide applicability. Further studies are required to assess its long-term safety and role in the management of CD20(+) NHL.
Adoptive cell therapy with ex vivo expanded autologous antitumor cytotoxic T lymphocytes represents an important therapeutic option as an anticancer strategy. In order to identify a reliable method ...for producing adequate amounts of functional antitumor cytotoxic T lymphocytes with a potentially long in vivo lifespan, we tested the T-cell expansion efficiency of a new artificial antigen-presenting cell-based system.
Our artificial antigen-presenting cells were generated with activating (anti-CD3), co-stimulating (anti-CD28) and adhesion (anti-LFA-1) biotinylated monoclonal antibodies preclustered in microdomains held on a liposome scaffold by neutravidin rafts. The co-localization of T-cell ligands in microdomains and the targeting of an adhesion protein, increasing the efficiency of immunological synapse formation, represent the novelties of our system. The activity of our artificial antigen-presenting cells was compared with that of anti-CD3/-CD28 coated immunomagnetic microbeads and immobilized anti-CD3 monoclonal antibody (OKT3 clone), the only two commercially available artificial systems.
Our artificial antigen-presenting cells expanded both polyclonal T cells and MART-1-specific CD8(+) T cells in a more efficient manner than the other systems. Stimulation with artificial antigen-presenting cells allows for the generation of viable T cells displaying an immunophenotype consistent with in vivo potential for persistence, without increasing the frequency of regulatory T cells. The starting specificity of anti MART-1 CD8(+) T cells was preserved after stimulation with artificial antigen-presenting cells and it was statistically greater when compared to the activity of the same cells expanded with the other systems. Finally, our artificial antigen-presenting cells proved to be suitable for large-scale application, minimizing the volume and the costs of T-cell expansion.
Our artificial antigen-presenting cells might represent an efficient tool to rapidly obtain a sufficient number of functional T cells for adoptive immunotherapy in patients with cancer.
During the last years, there has been a dramatic increase in the administrative and bureaucratic burden associated with clinical research, which has clearly had an impact on its overall efficiency ...and on the activity of clinical investigators and research teams. Indeed, the supervision of the adherence of clinical research to Good Clinical Practice (GCP) guidelines and legal regulations is of the utmost importance. Yet, while such regulations have remained largely unchanged during recent years, the number of administrative tasks and their complexity have grown markedly, as supported by the results of a survey performed among 940 clinical investigators that we report in this manuscript. Therefore, many investigators believe that it has become necessary to undertake a rigorous analysis of the causes and consequences of this issue, and to create a conduit to channel the advice from experienced investigators regarding clinical research procedures, in order to improve them. Based on these premises, ESMO has launched the ESMO Clinical Research Observatory (ECRO), a task force that will analyse different aspects of clinical research. ECRO will aim to provide the views of ESMO on clinical research procedures based on the feedback from clinical investigators, under complete adherence to the Declaration of Helsinki, the GCP guidelines and any other applicable legal regulations, while at the same time showing profound respect for all the stakeholders involved in clinical research. This manuscript provides the background and rationale for the creation of ECRO, its planned activity and an analysis of the current administrative burden in clinical research with recommendations to rationalise it. Indeed, we expect that this effort shall lead to a relevant improvement in the care of patients and in the development of clinical research.
We reported that the clinical efficacy of dendritic cell–based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now ...investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B+ killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.
We previously published the results of a pilot study showing that vaccination with tumor-loaded dendritic cells (DCs) induced both T and B cell response and produced clinical benefit in the absence ...of toxicity in patients with relapsed, indolent non-Hodgkin lymphoma (iNHL). The purpose of the present short report is to provide a 15-year follow-up of our study and to expand the biomarker analysis previously performed. The long-term follow-up highlighted the absence of particular or delayed toxicity and the benefit of active immunization with DCs loaded with autologous, heat-shocked and UV-C treated tumor cells in relapsed iNHL (5-year and 10-year progression-free survival (PFS) rates: 55.6% and 33.3%, respectively; 10-year overall survival (OS) rate: 83.3%). Female patients experienced a better PFS (p=0.016) and a trend towards a better OS (p=0.185) compared with male patients. Of note, we observed a non-negligible fraction of patients (22%) who experienced a long-lasting complete response. In a targeted gene expression profiling of pre-treatment tumor biopsies in 11 patients with available formalin-fixed, paraffin-embedded tissue, we observed that KIT, ATG12, TNFRSF10C, PBK, ITGA2, GATA3, CLU, NCAM1, SYT17 and LTK were differentially expressed in patients with responder versus non-responder tumors. The characterization of peripheral monocytic cells in a subgroup of 14 patients with available baseline blood samples showed a higher frequency of the subset of CD14++CD16+ cells (intermediate monocytes) in patients with responding tumors. Since in patients with relapsed iNHL the available therapeutic options are often incapable of inducing a long-lasting complete remission and can be sometimes characterized by intolerable toxicity, we think that the encouraging results of our long-term follow-up analysis represent a stimulus to further investigate the role of active vaccination in this specific setting and in earlier lines of therapy and to explore novel combinatorial strategies encompassing other innovative immunotherapy agents, such as immune-checkpoint inhibitors.
The safety and efficacy of bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor β (TGF-β) receptor II (a TGF-β “trap“) ...fused to a human immunoglobulin G1 antibody blocking programmed death-ligand 1 (PD-L1), was evaluated in patients with advanced NSCLC.
This expansion cohort of NCT02517398, an ongoing, phase 1, open-label trial, includes 80 patients with advanced NSCLC that progressed after platinum doublet therapy or after platinum-based adjuvant or neoadjuvant treatment and those who also have not received previous immunotherapy. Patients were randomized at a one-to-one ratio to receive either bintrafusp alfa 500 mg or the recommended phase 2 dosage of 1200 mg every 2 weeks. The primary end point was the best overall response (by Response Evaluation Criteria in Solid Tumors 1.1 as adjudicated by independent review committee) and was assessed by the objective response rate (ORR).
A total of 80 patients were randomized to receive bintrafusp alfa 500 or 1200 mg (n = 40 each). Median follow-up was 51.9 weeks (IQR, 19.6–74.0). The ORR in all patients was 21.3% (17 of 80). The ORR was 17.5% (seven of 40) and 25.0% (10 of 40) for the 500 mg dose and the 1200 mg dose (recommended phase 2 dose), respectively. At the 1200 mg dose, patients with PD-L1–positive and PD-L1–high (≥80% expression on tumor cells) had ORRs of 36.0% (10 of 27) and 85.7% (six of seven), respectively.
Treatment-related adverse events occurred in 55 of the 80 patients (69%) and were graded as greater than or equal to 3 in 23 of the 80 patients (29%). Of the 80 patients, eight (10%) had a treatment-related adverse event that led to treatment discontinuation; no treatment-related deaths occurred.
Bintrafusp alfa had encouraging efficacy and manageable tolerability in patients with NSCLC previously treated with platinum.
BackgroundTransforming growth factor beta (TGFβ) is a bifunctional regulator of tumor growth playing a role in tumor immune evasion and resistance to checkpoint blockade. Increased activation of TGFβ ...pathway correlated with reduced overall survival in patients with PD-1 resistance/refractory tumors. Therefore, the combination of a TGFβ inhibitor with an anti-PD-1 agent may benefit patients who are resistant to checkpoint blockade. SAR439459 is a ”second generation” human anti-TGFβ IgG4 monoclonal antibody. Here we report the preliminary PD results and patient selection strategy (mesenchymal CRC) of SAR439459 ± anti-PD-1 cemiplimab in patients with advanced tumors from an on-going phase 1 study (NCT03192345).MethodsPeripheral blood, serum and tumor biopsies from patients were collected for the assessment of both predictive and PD biomarkers. A consensus molecular subtyping 4 (CMS4) gene classifier was developed and used to identify mesenchymal CRC tumors based on an in-silico experiment followed by a validation using ~200 procured CRC tumor biopsy samples with customized NanoString assay. TGFβ level in plasma and tumor was measured by ELISA to assess target engagement of SAR439459. Well-known immune modulation events as the PD readout were measured: 1) immunophenotyping of circulating immune cells ; 2) cytokine/chemokine production by MSD assay; 3) PD-L1, CD8+ T cells and FoxP3+ Tregs in tumor micro-environment (TME) by immunohistochemistry; 4) TGFβ pathway activation gene signature in TME by RNAseq.ResultsSAR439459 ± cemiplimab, induced inhibition of plasma TGFβ level ≥ 90% at doses ≥ 0.25mg/kg Q2W, together with a clear trend of decrease in intra-tumoral TGFβ. RNAseq data from paired biopsies revealed concomitant down-regulation of TGFβ pathway. In periphery, SAR439459 ± cemiplimab increased proliferating T and NK cells. Concomitantly, enhanced production of pro-inflammatory cytokines/chemokines confirmed peripheral immune activation. In TME, a trend of increased CD8+ T cell infiltration and conversion from ”immune-excluded” to ”immune-inflamed” phenotype was observed following the combination treatment in several cases. No significant modulation of PD-L1 or FoxP3 was observed from the available paired biopsies. Out of 137 pre-screened CRC patients, 58 (42%) were identified as carrying the CMS4 phenotype based on the gene classifier.ConclusionsClinical modulation of TGFβ level and the related pathway demonstrated SAR439459’s target engagement. Further analysis confirmed the peripheral immune activation in patients treated with SAR439459 ± cemiplimab. Coupled with CD8+ T cell modulation in TME, these findings suggest the identification of early PD biomarkers impacted by SAR439459 which is consistent with the mechanism of action and biological activity of TGFβ blockade therapy.Trial RegistrationNCT03192345Ethics ApprovalThe study protocols were approved by the institutional review board or independent ethics committee of each participating institution. All patients provided written informed consent priorto enrollment.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of transformed cells while sparing normal cells. To enhance the therapeutic index of soluble ...(s)TRAIL, we used CD34+ cells transduced with a replication-deficient adenovirus encoding the human TRAIL gene (CD34-TRAIL+) for the systemic delivery of membrane-bound (m)TRAIL to lymphoid tumors. CD34-TRAIL+ cells were evaluated for their activity in vitro and in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with sTRAIL-sensitive and -resistant tumors. In vitro, coculturing CD34-TRAIL+ cells with sTRAIL-sensitive or -resistant lymphoma cell lines induced significant levels of caspase-dependent tumor cell death. In vivo, CD34-TRAIL+ cells significantly increased the survival of NOD/SCID mice bearing sTRAIL-sensitive or -resistant lymphoid tumors at an early or advanced stage of disease. No obvious toxicity was observed on administration of CD34-TRAIL+ cells. Histological analysis revealed high-level expression of the agonistic receptor TRAIL-R2 by tumor endothelial cells, and efficient tumor homing of transduced cells. Injection of CD34-TRAIL+ cells resulted in extensive damage of tumor vasculature followed by hemorrhagic necrosis exhibiting a perivascular distribution. These results show that CD34-TRAIL+ cells might be an efficient vehicle for mTRAIL delivery to tumors, where they exert a potent antitumor effect possibly mediated by both direct tumor cell killing and indirect vascular-disrupting mechanisms.
Summary
A very short, intensive paediatric chemotherapy programme was tested in a consecutive monoinstitutional group of 22 adult Burkitt's lymphoma (BL) patients. After a 5‐week induction phase of ...weekly infusions consisting of vincristine, cyclophosphamide, doxorubicin, high‐dose (HD) methotrexate (MTX) plus leukovorin rescue, and intrathecal MTX or cytarabine (ARA‐C), a consolidation phase including HD ARA‐C plus cisplatin was given. Responding patients achieving less than complete response (CR) after completion of the initial induction phase, were promptly shifted to a high‐dose, stem cell supported sequential chemotherapy schema (R‐HDS). Patient characteristics: median age, 35·5 (range 18–76) years; Ann Arbor stage I–II/III–IV, 11/11; bulky disease, 15 patients; LDH ≥ 460 U/l, 11 patients. The median duration of the chemotherapy programme was 62 d (range, 43–94 d). Seventeen patients achieved a CR (77%), one patient died of progressive disease and four partial responders following induction were converted to CR following R‐HDS. Of 17 patients in CR, one died of infectious toxicity while in CR, and one relapsed at 30 months and died of progressive disease. After a median follow‐up of 28·7 months (range, 6–158 months), 16 patients (73%) were in continued CR. Overall survival and progression‐free survival were 77% 95% confidence interval (CI), 52–99% and 68% (95% CI, 43–99%) respectively. Confirmation of these excellent efficacy and feasibility results by larger, multicentre and prospective studies is warranted.
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