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The transcriptional repressor B-cell lymphoma 6 (BCL6) is dysregulated in several neoplasms, but its role in triple negative breast cancer (TNBC), a highly aggressive subtype which lacks effective treatment, is unclear. The presence of intratumoral cancer stem cells (CSCs) is a main cause of tumor relapse. The Notch signaling pathway is crucial for regulating CSC self-renewal and promoting breast cancer (BC) development and resistance to anticancer therapies. Here, we investigated signaling cascades of BCL6 in the CSC compartment of TNBCs, and the mechanisms that govern its activity, mainly through Notch signaling.Gene expression, somatic copy number alterations and clinical data from the Cancer Genome Atlas and METABRIC were accessed through the Xena and cbioportal browsers. Public transcriptome profiles from TNBC datasets were retrieved from the Gene Expression Omnibus. Mammosphere formation efficiency was calculated after BCL6 knockdown via transient siRNA transfection, stable silencing or pharmacological inhibition. The effects exhibited via BCL6 inhibition in putative TNBC stem-like cells were evaluated by immunofluorescence and qRT-PCR analyses. Chromatin immunoprecipitation experiments were performed to validate a putative BCL6 responsive element located in the first intron of the Numb gene and to define the circuit of corepressors engaged by BCL6 following its inhibition. Immunoprecipitation assays were carried out to investigate a novel interaction at the basis of BCL6 control of CSC activity in TNBC.In silico analyses of benchmarked public datasets revealed a significant enrichment of BCL6 in cancer stemness related pathways, particularly of Notch signaling in TNBC. In vitro stable inhibition of BCL6 significantly reduced tumor cell growth and, accordingly, we found that the mammosphere formation efficiency of BCL6 silenced cells was significantly impaired by pharmacological inhibition of Notch signaling. BCL6 was found to be expressed at significantly higher levels in TNBC mammospheres than in their adherent counterparts, and loss of BCL6 function significantly decreased mammosphere formation with preferential targeting of CD44-positive versus ALDH-positive stem-like cells. Functional interplay between BCL6 and the chromatin remodeling factor EZH2 triggered the BCL6/Notch stemness signaling axis via inhibition of Numb transcription.Our results may be instrumental for the prospective design of combination treatment strategies that selectively target novel TNBC-associated biomarker(s) whose activity is implicated in the regulation of cancer stemness (such as BCL6) and molecules in developmentally conserved signaling pathways (such as Notch) to achieve long-lasting tumor control and improve patient outcomes.

Supplementary Figure 1. Represents CD30 serum levels at various time during the therapy in 22 patients

Supplementary Tables. Includes 4 tables: a) Supplementary Table 1 describing clinical characteristics of all HL patients b) Supplementary Table 2 describing disease characteristics of HL responding patients c) Supplementary Table 3 describing adverse events observed during the month of therapy with perifosine alone d) Supplementary Table 4 describing association of serum soluble cytokine levels with therapy response

Legend of Supplementary Figure 1

Purpose: To evaluate safety and activity of perifosine and sorafenib combination therapy in patients with lymphoproliferative diseases.Experimental Design: Patients with relapsed and refractory lymphoproliferative diseases received perifosine (50 mg twice daily) for 1 month. Patients achieving less than partial response (PR) after perifosine alone were administered the combination therapy [perifosine plus sorafenib (400 mg twice daily)] until progressive disease (PD) or unacceptable toxicity occurred. The pERK and pAKT in peripheral blood lymphocytes as well as serum cytokine levels were investigated as predictive biomarkers of response.Results: Forty patients enrolled in this study. After 1 month of perifosine alone, 36 who achieved less than PR went on to combination therapy, whereas four patients with chronic lymphocytic leukemia (CLL) who achieved PR continued with perifosine alone for a median of 10 months (range, 4–21). The most common drug-related toxicities were grade 1–2 anemia (17%), thrombocytopenia (9%), diarrhea (25%), joint pain (22%), and hand–foot skin reaction (25%). Three patients experienced grade 3 pneumonitis. Eight patients (22%) achieved PR, 15 (42%) achieved stable disease, and 13 (36%) experienced PD. A 28% PR rate was recorded for 25 patients with Hodgkin lymphoma. Among all patients, median overall survival and progression-free survival were 16 and 5 months, respectively. Early reductions in pERK and pAKT significantly correlated with the probability of clinical response.Conclusions: Perifosine and sorafenib combination therapy is feasible with manageable toxicity and demonstrates promising activity in patients with Hodgkin lymphoma. The predictive value of pERK and pAKT should be confirmed in a larger patient cohort. Clin Cancer Res; 20(22); 5641–51. ©2014 AACR.

Supplementary Figure 4 from Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Supplementary Figure 1 from Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

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.

Supplementary Figure 5 from Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Supplementary Figure 2C from Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Supplementary Figure 3 from Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Supplementary Methods, Figures 1-4, Tables 1-3 from Mutation-Independent Anaplastic Lymphoma Kinase Overexpression in Poor Prognosis Neuroblastoma Patients

Supplementary Figure 6 from Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Supplementary Table 1, Figure Legends 1-6 from Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Supplementary Figures 1-10 from Tumor-Reactive CD8+ Early Effector T Cells Identified at Tumor Site in Primary and Metastatic Melanoma

Since a non-negligible fraction of patients with metastatic melanoma does not experience long-term disease control, even with immunotherapy and targeted therapy, new biomarkers for patient stratification and treatment tailoring are needed in this setting. We investigated the association of a novel immune-inflammatory blood-based biomarker, the Pan-Immune-Inflammation Value (PIV), with clinical outcomes of patients with metastatic melanoma receiving first-line therapy. We retrospectively included patients treated at the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan and having an available baseline complete blood cell count (CBC). PIV was calculated as: [neutrophil count (103/mm3) × platelet count (103/mm3) × monocyte count (103/mm3)]/lymphocyte count (103/mm3). A total of 228 patients were included: 119 (52%) had been treated with immunotherapy and 109 (48%) with targeted therapy. PIV was significantly higher in patients with ECOG PS ≥ 1, high disease burden, synchronous metastases, and elevated baseline LDH level. High baseline PIV was independently associated with poor overall survival (adjusted hazard ratio [HR]: 2.06; 95% confidence interval [CI]: 1.30–3.29; adjusted P = 0.002) and progression-free survival (adjusted HR 1.56; 95% CI 1.01–2.41; adjusted P = 0.044). High PIV was also associated with primary resistance to both immunotherapy (odds ratio [OR]: 3.98; 95% CI 1.45–12.32; P = 0.005) and targeted therapy (OR: 8.42; 95% CI 2.50–34.5; P < 0.001). PIV showed a promising discrimination ability in terms of AIC and c-index when compared with other CBC-based biomarkers. PIV may guide the treatment decision process and the development of novel first-line treatment strategies in melanoma, but warrants further study and validation.

Breast cancer (BC) is a heterogeneous disease in which the tumor microenvironment (TME) seems to impact the clinical outcome. Here, we investigated whether a combination of gene expression signatures relating to both the structural and immune TME aspects can help predict prognosis in women with high‐grade BC (HGBC). Thus, we focused on a combined molecular biomarker variable that involved extracellular matrix (ECM)‐associated gene expression (ECM3 signature) and interferon (IFN)‐associated metagene (IFN metagene) expression. In 97 chemo‐naive HGBCs from the METABRIC dataset, the dichotomous ECM3/IFN (dECIF) variable identified a group of high‐risk patients (ECM3+/IFN− vs other; hazard ratio = 3.2, 95% confidence interval: 1.5–6.7). Notably, ECM3+/IFN− tumors showed low tumor‐infiltrating lymphocytes, high levels of CD33‐positive cells, absence of PD‐1 expression, or low expression of PD‐L1, as suggested by immune profiles and immune‐histochemical analysis on an independent cohort of 131 HGBCs. To make our results transferable to clinical use, we refined the dECIF biomarker using reduced ECM3 and IFN signatures; notably, the prognostic value of this reduced dECIF was comparable to that of the original dECIF. After validation in a new BC cohort, reduced dECIF was translated into a robust qPCR classifier for real‐world clinical use., This study investigates the combination of two relevant molecular signatures of the tumor microenvironment (TME) in identifying aggressive high‐grade breast cancers (HGBCs). The novel molecular biomarker described here highlighted patients with worst HGBC prognosis and a peculiar TME. Finally, to improve the potential of the identified biomarker in the clinic, we reduced the two original signatures and technically validated the biomarker's expression through a qPCR‐based assay.

© 2019, International Society for Cellular Oncology. 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 CD29High/CD24+/Sca1Low 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.

Simple Summary Conventional anti-cancer treatments for metastatic tumors include chemotherapy and radiation. These approaches can result in harmful side-effects and, in the vast majority of cases, are not curative. Recently, novel treatments have been developed in order to stimulate the host immune system to fight cancer. This type of therapeutic approach, called immunotherapy, has gained a lot of attention in recent years due to discoveries that have deciphered the immunosuppressive role of the tumor microenvironment and underpinning molecular signals. To enhance the delivery of therapeutic drugs to the tumor site, nanoparticle-based delivery systems can be used to reduce off-target effects, and to modulate immune cells present in the tumor microenvironment. This novel therapeutic approach can synergize with other immunotherapies such as immune checkpoint blockade inhibitors and adoptive cell therapy, by enhancing the infiltration of activated immune cells to the tumor site, and by limiting local immunosuppression. Abstract A number of novel cancer therapies have recently emerged that have rapidly moved from the bench to the clinic. Onco-immunotherapies, such as immune checkpoint blockade inhibitors and adoptive cell therapies, have revolutionized the field, since they provide a way to induce strong anti-tumor immune responses, which are able to fight cancer effectively. However, despite showing great efficacy in hematological and some solid tumors, unresponsiveness, development of therapy resistance and the development of serious adverse effects, limit their capacity to impact the vast majority of tumors. Nanoparticle-based delivery systems are versatile vehicles for a wide variety of molecular cargoes and provide an innovative strategy to improve conventional onco-immunotherapies. They can be finely tuned to release their contents in the tumor microenvironment, or to deliver combinations of adjuvants and antigens in the case of nanovaccines. In this review, we summarize the recent advancements in the field of nanobiotechnology, to remodel the tumor microenvironment and to enhance immunotherapies.

During the Tenth Edition of the Annual Congress on "Anticancer Innovative Therapy" [Milan, 23/24 January 2020], experts in the fields of immuno-oncology, epigenetics, tumor cell signaling, and cancer metabolism shared their latest knowledge on the roles of i] epigenetics, and in particular, chromatin modifiers, ii] cancer metabolism, iii] cancer stem cells [CSCs], iv] tumor cell signaling, and iv] the immune system. The novel therapeutic approaches presented included epigenetic drugs, cell cycle inhibitors combined with ICB, antibiotics and other off-label drugs, small-molecules active against CSCs, liposome-delivered miRNAs, tumor-specific CAR-T cells, and T-cell-based immunotherapy. Moreover, important evidence on possible mechanisms of resistance to these innovative therapies were also discussed, in particular with respect to resistance to ICB. Overall, this conference provided scientists and clinicians with a broad overview of future challenges and hopes to improve cancer treatment reasonably in the medium-short term.

In the era of precision medicine, monoclonal antibodies and small molecule inhibitors are the mainstays of the biological therapy in patients with solid tumors. However, resistance to treatment and the “undruggability” of certain key oncogenic proteins emerged as major limitations and jeopardize the clinical benefit of modern therapeutic approaches. Targeted protein degraders are novel molecules entering the early phase of clinical development that exploit the intracellular ubiquitine-proteasome system to promote a specific degradation of target proteins. Since the peculiar mechanism of action, targeted protein degraders have the potential to limit and overcome resistance to treatment and to allow a full actionability of certain cancer drivers that are actually elusive targets. Here, we discuss the state-of-the-art and the open issues in the development of these emerging biological agents from a clinical perspective and with a focus on solid tumors.

BackgroundMyeloid-derived suppressor cells (MDSC), a cornerstone of cancer-related immunosuppression, influence response to therapy and disease outcomes in melanoma patients. Nevertheless, their quantification is far from being integrated into routine clinical practice mostly because of the complex and still evolving phenotypic signatures applied to define the cell subsets. Here, we used a multistep downsizing process to verify whether a core of few markers could be sufficient to capture the prognostic potential of myeloid cells in peripheral blood mononuclear cells (PBMC) of metastatic melanoma patients.MethodsIn baseline frozen PBMC from a total of 143 stage IIIc to IV melanoma patients, we first assessed the relevant or redundant expression of myeloid and MDSC-related markers by flow cytometry (screening set, n=23 patients). Subsequently, we applied the identified panel to the development set samples (n=59 patients undergoing first/second-line therapy) to obtain prognostic variables associated with overall survival (OS) and progression-free survival (PFS) by machine learning adaptive index modeling. Finally, the identified score was confirmed in a validation set (n=61) and compared with standard clinical prognostic factors to assess its additive value in patient prognostication.ResultsThis selection process led to the identification of what we defined myeloid index score (MIS), which is composed by four cell subsets (CD14+, CD14+HLA-DRneg, CD14+PD-L1+ and CD15+ cells), whose frequencies above cut-offs stratified melanoma patients according to progressively worse prognosis. Patients with a MIS=0, showing no over-threshold value of MIS subsets, had the best clinical outcome, with a median survival of >33.6 months, while in patients with MIS 1→3, OS deteriorated from 10.9 to 6.8 and 6.0 months as the MIS increased (pConclusionThe MIS >0 identifies melanoma patients with a more aggressive disease, thus acting as a simple blood biomarker that can help tailoring therapeutic choices in real-life oncology.

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.

Background The role of high-dose chemotherapy with autologous stem cell transplantation (ASCT) in the treatment of soft-tissue sarcoma (STS) remains an unsettled issue. Prospective clinical trials failed to prove a benefit of the procedure but were limited by small and heterogeneous patient cohorts. Thus, it is unknown if ASCT may be a valuable treatment option in specific patient subgroups. Methods The purpose of this study was to investigate the value of ASCT according to histological subtype in STS patients who were registered in the European Society for Blood and Marrow Transplantation database between 1996 and 2016. Results Median progression-free (PFS) and overall survival (OS) in the entire cohort of 338 patients were 8.3 and 19.8 months, respectively, and PFS and OS at 5 years were 13% and 25%, respectively. Analysis of outcomes in different subgroups showed that younger age, better remission status before transplantation and melphalan-based preparative regimen were predictive of benefit from ASCT, whereas histology and grading had no statistically significant impact. Conclusions Outcomes after ASCT compared favorably to those of recent trials on conventional chemotherapies and targeted therapies in STS, including histology-tailored approaches. ASCT, thus, should be reinvestigated in clinical trials focusing on defined patient subgroups.

Triple-negative breast cancers (TNBCs) are characterized by a poor prognosis and lack of targeted treatments, and thus, new therapeutic strategies are urgently needed. Inhibitors against programmed death-1 (PD-1)/PD-1 ligand (PD-L1) have shown significant efficacy in various solid cancers, but their activity against TNBCs remains limited. Here, we report that human TNBCs molecularly stratified for high levels of PD-L1 (PD-L1High) showed significantly enriched expression of immune and cancer stemness pathways compared with those with low PD-L1 expression (PD-L1Low). In addition, the PD-L1High cases were significantly associated with a high stemness score (SSHigh) signature. TNBC cell lines gated for aldehyde dehydrogenase (ALDH) and CD44 stemness markers exhibited increased levels of PD-L1 versus their ALDH-negative and CD44Low counterparts, and PD-L1High cells generated significantly more mammospheres than PD-L1Low cells. Murine mammary SCA-1-positive tumor cells with PD-L1High expression generated tumors in vivo with higher efficacy than PD-L1Low cells. Furthermore, treatment of TNBC cells with selective WNT inhibitors or activators downregulated or upregulated PD-L1 expression, respectively, implying a functional cross-talk between WNT activity and PD-L1 expression. Remarkably, human TNBC samples contained tumor elements co-expressing PD-L1 with ALDH1A1 and/or CD44v6. Additionally, both PD-L1-/SCA1-positive and ALDH1A1-positive tumor elements were found in close contact with CD3-, and PD-1-positive T cells in murine and human tumor samples. Overall, our study suggests that PD-L1-positive tumor elements with a stemness phenotype may participate in the complex dynamics of TNBC-related immune evasion, which might be targeted through WNT signaling inhibition.

Introduction 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. Methods 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). Results 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. Conclusions Bintrafusp alfa had encouraging efficacy and manageable tolerability in patients with NSCLC previously treated with platinum.

Despite different molecular tumor profiles indicate that human epidermal growth factor receptor 2 (HER2) messenger RNA (mRNA) levels mirror HER2 addiction and trastuzumab benefit in HER2-positive breast cancer (BC), the identification of noninvasive clinical predictors of trastuzumab sensitivity remains an unmet clinical need. In the current study, we investigated whether intratumor lactate levels reflect HER2 addiction and, in turn, trastuzumab susceptibility. Accordingly, the gene expression profiles of transgenic murine BC cell lines expressing the human d16HER2 variant (HER2-addicted) or human full-length HER2 (WTHER2; HER2-nonaddicted) revealed a significant enrichment of glycolysis-related gene pathways in HER2-addicted cells. We studied the metabolic content of 22 human HER2-positive BC by quantitative nuclear magnetic resonance spectroscopy and found that those cases with higher lactate levels were characterized by higher HER2 transcript levels. Moreover, gene expression analyses of HER2-positive BC samples from a TCGA data set revealed a significant enrichment in glycolysis-related pathways in high/HER2-addicted tumors. These data were confirmed by metabolic analyses of human HER2-positive BC cell lines with high or low HER2 transcript levels, which revealed significantly more active glycolytic metabolism in high HER2 transcript than in low HER2 transcript cells. Overall, our results provide evidence for noninvasive intratumor lactate detection as a potential metabolic biomarker of HER2 addiction and trastuzumab response suggesting the possibility to use in vivo imaging to assess lactate levels and, in turn, select HER2-positive BC patients who are more likely to benefit from anti-HER2 treatments.

Background Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. Methods The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. Results and conclusions We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. General significance Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis.

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., SCOPUS: re.j, info:eu-repo/semantics/published

Importance Multiple immunostimulatory agonist antibodies have been clinically tested in solid tumors to evaluate the role of targeting glucocorticoid-induced tumor necrosis factor (TNF) receptor–related protein in anticancer treatments. Objective To evaluate the safety and activity of the fully human glucocorticoid-induced TNF receptor–related protein agonist IgG1 monoclonal antibody BMS-986156 with or without nivolumab in patients with advanced solid tumors. Design, Setting, and Participants This global, open-label, phase 1/2a study of BMS-986156 with or without nivolumab enrolled 292 patients 18 years or older with advanced solid tumors and an Eastern Cooperative Oncology Group performance status of 1 or less. Prior checkpoint inhibitor therapy was allowed. Monotherapy and combination dose-escalation cohorts ran concurrently to guide expansion doses beginning October 16, 2015; the study is ongoing. Interventions The protein agonist BMS-986156 was administered intravenously at a dose of 10, 30, 100, 240, or 800 mg every 2 weeks as monotherapy, and in the combination group 30, 100, 240, or 800 mg plus 240 mg of nivolumab every 2 weeks; same-dose cohorts were pooled for analysis. One cohort also received 480 mg of BMS-986156 plus 480 mg of nivolumab every 4 weeks. Main Outcomes and Measures The primary end points were safety, tolerability, and dose-limiting toxic effects. Additional end points included antitumor activity per Response Evaluation Criteria in Solid Tumors, version 1.1, and exploratory biomarker analyses. Results With a follow-up range of 1.4 to 101.7 weeks (follow-up ongoing), 34 patients (16 women and 18 men; median age, 56.6 years [range, 28-75 years]) received monotherapy (4 patients completed initial treatment), and 258 patients (140 women and 118 men; median age, 60 years [range, 21-87 years]) received combination therapy (65 patients completed initial treatment). No grade 3 to 5 treatment-related adverse events occurred with BMS-986156 monotherapy; grade 3 to 4 treatment-related adverse events occurred in 24 patients (9.3%) receiving BMS-986156 plus nivolumab, with no grade 5 treatment-related adverse events. One dose-limiting toxic effect (grade 4 elevated creatine phosphokinase levels) occurred in a patient receiving 800 mg of BMS-986156 plus 240 mg of nivolumab every 2 weeks; BMS-986156 with or without nivolumab exhibited linear pharmacokinetics with dose-related increase after a single dose. Peripheral T-cell and natural killer–cell proliferation increased after administration of BMS-986156 with or without nivolumab. No consistent and significant modulation of intratumoral CD8+T cells and FoxP3+regulatory T cells was observed. No responses were seen with BMS-986156 alone; objective response rates ranged from 0% to 11.1% (1 of 9) across combination therapy cohorts, with a few responses observed in patients previously treated with anti–programmed death receptor (ligand) 1 therapy. Conclusions and Relevance Based on this cohort, BMS-986156 appears to have had a manageable safety profile, and BMS-986156 plus nivolumab demonstrated safety and efficacy comparable to historical data reported for nivolumab monotherapy. Trial Registration ClinicalTrials.gov identifier:NCT02598960

Previous works linked low sodium concentration with mortality risk in cancer. We aimed at weighing the prognostic impact of hyponatremia in all consecutive patients with metastatic solid tumors admitted in a two-years period at our medical oncology department. Patients were included in two cohorts based on serum sodium concentration on admission. A total of 1025 patients were included, of whom 279 (27.2%) were found to be hyponatremic. The highest prevalence of hyponatremia was observed in biliary tract (51%), prostate (45%) and small-cell lung cancer (38.9%). With a median follow-up of 26.9 months, median OS was 2 months and 13.2 months for the hyponatremia versus control cohort, respectively (HR, 2.65; P P

T-cell-based immunotherapy strategies have profoundly improved the clinical management of several solid tumors and hematological malignancies. A recently developed and promising immunotherapy approach is to redirect polyclonal MHC-unrestricted T lymphocytes toward cancer cells by bispecific antibodies (bsAbs) that engage the CD3 complex and a tumor-associated antigen (TAA). The TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) is an attractive immunotherapy target, frequently expressed by neoplastic cells, that we decided to exploit as a TAA. We found that a TRAIL-R2xCD3 bsAb efficiently activates T cells and specifically redirect their cytotoxicity against cancer cells of different origins in vitro, thereby demonstrating its potential as a pan-carcinoma reagent. Moreover, to mimic in vivo conditions, we assessed its ability to retarget T-cell activity in an ex vivo model of ovarian cancer patients' ascitic fluids containing both effector and target cells—albeit with a suboptimal effector-to-target ratio—with remarkable results.

Predictive biomarkers of response to immune-checkpoint inhibitors (ICIs) are an urgent clinical need. The aim of this study is to identify manageable parameters to use in clinical practice to select patients with higher probability of response to ICIs. Two-hundred-and-seventy-one consecutive metastatic solid tumor patients, treated from 2013 until 2017 with anti- Programmed death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) ICIs, were evaluated for baseline lactate dehydrogenase (LDH) serum level, performance status (PS), age, neutrophil-lymphocyte ratio, type of immunotherapy, number of metastatic sites, histology, and sex. A training and validation set were used to build and test models, respectively. The variables&rsquo, effects were assessed through odds ratio estimates (OR) and area under the receive operating characteristic curves (AUC), from univariate and multivariate logistic regression models. A final multivariate model with LDH, age and PS showed significant ORs and an AUC of 0.771. Results were statistically validated and used to devise an Excel algorithm to calculate the patient&rsquo, s response probabilities. We implemented an interactive Excel algorithm based on three variables (baseline LDH serum level, age and PS) which is able to provide a higher performance in response prediction to ICIs compared with LDH alone. This tool could be used in a real-life setting to identify ICIs in responding patients.

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.