The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses.

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

Association of Tumor Cell Metabolic Subtype and Immune Response With the Clinical Course of Hepatocellular Carcinoma.

AIM: Tumor metabolism plays an important role in tumorigenesis and tumor progression. This study evaluated the potential association of tumor cell metabolism and immune cell tumor infiltration with the clinical course of hepatocellular carcinoma (HCC). METHODS: Gene-wise normalization and principal component analysis were performed to evaluate the metabolic system. A tumor microenvironment score system of tumor immune cell infiltration was constructed to evaluate its association with metabolic subtypes. Finally, we analyzed the impact of metabolism and immune cell infiltration on the clinical course of HCC. RESULTS: A total of 673 HCC patients were categorized into cholesterogenic (25.3%), glycolytic (14.6%), mixed (10.4%), and quiescent (49.8%) types based on glycolysis and cholesterol biosynthesis gene expression. The subgroups including the glycolytic genotyping expression (glycolytic and mixed types) showed a higher mortality rate. The glycolytic, cholesterogenic, and mixed types were positively correlated with M0 macrophage, resting mast cell, and naïve B-cell infiltration (P = .013, P = .019, and P = .006, respectively). In TCGA database, high CD8+ T cell and low M0 macrophage infiltration were associated with prolonged overall survival (OS, P = .0017 and P < .0001, respectively). Furthermore, in glycolytic and mixed types, patients with high M0 macrophage infiltration had a shorter OS (P = .03 and P = .013, respectively), and in quiescent type, patients with low naïve B-cell infiltration had a longer OS (P = .007). CONCLUSIONS: Tumor metabolism plays a prognostic role and correlates with immune cell infiltration in HCC. M0 macrophage and CD8+ T cell appear to be promising prognostic biomarker for HCC. Finally, M0 macrophages may represent a useful immunotherapeutic target in patients with HCC.

VT68.2: An Antibody to Chondroitin Sulfate Proteoglycan 4 (CSPG4) Displays Reactivity against a Tumor-Associated Carbohydrate Antigen.

The anti-CSPG4 monoclonal antibodies (mAbs) have shown anti-tumor activity and therapeutic potential for treating breast cancer. In addition, CSPG4 is a dominant tumor-associated antigen that is also involved in normal-tissue development in humans. Therefore, the potential for off-tumor activity remains a serious concern when targeting CSPG4 therapeutically. Previous work suggested that glycans contribute to the binding of specific anti-CSPG4 antibodies to tumor cells, but the specificity and importance of this contribution are unknown. In this study, the reactivity of anti-CSPG4 mAbs was characterized with a peptide mimetic of carbohydrate antigens expressed in breast cancer. ELISA, flow cytometry, and microarray assays were used to screen mAbs for their ability to bind to carbohydrate-mimicking peptides (CMPs), cancer cells, and glycans. The mAb VT68.2 displayed a distinctly strong binding to a CMP (P10s) and bound to triple-negative breast cancer cells. In addition, VT68.2 showed a higher affinity for N-linked glycans that contain terminal fucose and fucosylated lactosamines. The functional assays demonstrated that VT68.2 inhibited cancer cell migration. These results define the glycoform reactivity of an anti-CSPG4 antibody and may lead to the development of less toxic therapeutic approaches that target tumor-specific glyco-peptides.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 2nd - 4th, 2021, Italy).

Advances in immune checkpoint and combination therapy have led to improvement in overall survival for patients with advanced melanoma. Improved understanding of the tumor, tumor microenvironment and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. Combination modalities with other immunotherapy agents, chemotherapy, radiotherapy, electrochemotherapy are also being explored to overcome resistance and to potentiate the immune response. In addition, novel approaches such as adoptive cell therapy, oncogenic viruses, vaccines and different strategies of drug administration including sequential, or combination treatment are being tested. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic theràapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers, but they have yet to be fully characterized and implemented clinically. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. Overall, the future research efforts in melanoma therapeutics and translational research should focus on several aspects including: (a) developing robust biomarkers to predict efficacy of therapeutic modalities to guide clinical decision-making and optimize treatment regimens, (b) identifying mechanisms of therapeutic resistance to immune checkpoint inhibitors that are potentially actionable, (c) identifying biomarkers to predict therapy-induced adverse events, and (d) studying mechanism of actions of therapeutic agents and developing algorithms to optimize combination treatments. During the Melanoma Bridge meeting (December 2nd-4th, 2021, Naples, Italy) discussions focused on the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine as well as the impact of COVID-19 pandemic on management of melanoma patients.

Human Hepatitis B Virus Negatively Impacts the Protective Immune Crosstalk Between Natural Killer and Dendritic Cells.

BACKGROUND AND AIMS: Natural killer (NK) cells play a crucial role in the clearance of human viruses but their activity is significantly impaired in patients infected with chronic hepatitis B (CHB). Cooperation with dendritic cells (DCs) is pivotal for obtaining optimal NK cell antiviral function; thus, we investigated whether HBV might impact the ability of DCs to sustain NK cell functions. APPROACH AND RESULTS: Human DCs were poor stimulators of interferon-gamma (IFN-γ) production by NK cells when exposed to HBV, while maintaining the capability to trigger NK cell cytotoxicity. HBV prevented DC maturation but did not affect their expression of human leukocyte antigen class I, thus allowing DCs to evade NK cell lysis. Tolerogenic features of DCs exposed to HBV were further supported by their increased expression of IL-10 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase, which contributed to the impairment of DC-mediated NK cell IFN-γ production and proliferation, respectively. HBV could also inhibit the expression of inducible immunoproteasome (iP) subunits on DCs. In fact, NK cells could induce iP subunit expression on DCs, but they failed in the presence of HBV. Remarkably, circulating blood DC antigen1 (BDCA1)+ DCs isolated from patients with CHB were functionally compromised, hence altering, in turn, NK cell responses. CONCLUSIONS: The abnormal NK-DC interplay caused by HBV may significantly impair the efficacy of antiviral immune response in patients with CHB.

Integrated Antitumor Activities of Cellular Immunotherapy with CIK Lymphocytes and Interferons against KIT/PDGFRA Wild Type GIST.

Gastrointestinal stromal tumors (GISTs) are rare, mesenchymal tumors of the gastrointestinal tract, characterized by either KIT or PDGFRA mutation in about 85% of cases. KIT/PDGFRA wild type gastrointestinal stromal tumors (wtGIST) account for the remaining 15% of GIST and represent an unmet medical need: their prevalence and potential medical vulnerabilities are not completely defined, and effective therapeutic strategies are still lacking. In this study we set a patient-derived preclinical model of wtGIST to investigate their phenotypic features, along with their susceptibility to cellular immunotherapy with cytokine-induced killer lymphocytes (CIK) and interferons (IFN). We generated 11 wtGIST primary cell lines (wtGISTc). The main CIK ligands (MIC A/B; ULBPs), along with PD-L1/2, were expressed by wtGISTc and the expression of HLA-I molecules was preserved. Patient-derived CIK were capable of intense killing in vitro against wtGISTc resistant to both imatinib and sunitinib. We found that CIK produce a high level of granzyme B, IFNα and IFNγ. CIK-conditioned supernatant was responsible for part of the observed tumoricidal effect, along with positive bystander modulatory activities enhancing the expression of PD-L1/2 and HLA-I molecules. IFNα, but not In, had direct antitumor effects on 50% (4/8) of TKI-resistant wtGISTc, positively correlated with the tumor expression of IFN receptors. wtGIST cells that survived IFNα were still sensitive to CIK immunotherapy. Our data support the exploration of CIK immunotherapy in clinical studies for TKI-resistant wtGIST, proposing reevaluation for IFNα within this challenging setting.

A monocentric phase I study of vemurafenib plus cobimetinib plus PEG-interferon (VEMUPLINT) in advanced melanoma patients harboring the V600BRAF mutation.

BACKGROUND: Studies carried out in vitro and in a mouse model have shown that BRAF inhibitors enhance the effects of IFN-α on BRAFV600E melanoma cells through the inhibition of ERK. Therefore, the combination of vemurafenib and IFN-α in patients with BRAFV600E melanoma may provide therapeutic benefits; MEK inhibition may prevent the reactivation of the MAPK pathway induced by BRAF inhibitor resistance. PATIENTS AND METHODS: In a phase I study, adult patients with advanced BRAFV600-mutated melanoma were treated with vemurafenib + PEG-IFN-α-2b or vemurafenib + cobimetinib + PEG-IFN-α-2b, to assess the safety of the combination and the upregulation of IFN-α/ß receptor-1 (IFNAR1). RESULTS: Eight patients were treated; 59 adverse events with four serious ones (three related to study treatments) were reported. Patients with a pre-treatment IFNAR1 expression on ≤ 35% melanoma cells had a median progression-free survival of 12.0 months (range: 5.6-18.4 months) and a median overall survival of 31.0 months (range: 19.8-42.2 months), while patients with a pre-treatment IFNAR1 expression on > 35% of melanoma cells had a median progression-free survival of 4.0 months (range: 0-8.8; p = 0.03), and a median overall survival of 5 months (p = 0.02). Following treatment, responders had higher levels of growth-suppressor genes, including GAS1 and DUSP1, and genes involved in a metabolically robust immune response, including FAP. CONCLUSION: Our study supports the overall safety of the vemurafenib + PEG-IFN-α-2b + cobimetinib combination. IFNAR1 expression levels correlated with response to treatment, including survival. Vemurafenib + PEG-IFN-α-2b + cobimetinib would have difficulty finding a niche in the current treatment scenario for advanced melanoma, but we speculate that our findings may contribute to identify subjects particularly responsive to treatment. TRIAL REGISTRATION: The study was registered at clinicaltrials.gov (NCT01959633). Registered 10 October 2013, https://clinicaltrials.gov/ct2/show/NCT01959633.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 3rd-5th, 2020, Italy).

Advances in immune checkpoint therapy and targeted therapy have led to improvement in overall survival for patients with advanced melanoma. Single agent checkpoint PD-1 blockade and combination with BRAF/MEK targeted therapy demonstrated benefit in overall survival (OS). Superior response rates have been demonstrated with combined PD-1/CTLA-4 blockade, with a significant OS benefit compared with single-agent PD-1 blockade. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic therapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers but they have yet to be fully characterized and implemented clinically. Overall, the progress in melanoma therapeutics and translational research will help to optimize treatment regimens to overcome resistance and develop robust biomarkers to guide clinical decision-making. During the Melanoma Bridge meeting (December 3rd-5th, 2020, Italy) we reviewed the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine.

Defective HLA Class I Expression and Patterns of Lymphocyte Infiltration in Chordoma Tumors.

BACKGROUND: There are no effective systemic therapies for chordoma. The recent successes of immunotherapeutic strategies in other cancers have resulted in a resurgence of interest in using immunotherapy in chordoma. These approaches rely on a functional interaction between the host's immune system and the expression of tumor peptides via the human leukocyte antigen (HLA) Class I antigen. It is not known whether chordoma cells express the HLA Class I antigen. QUESTIONS/PURPOSES: (1) Do chordoma tumors exhibit defects in HLA Class I antigen expression? (2) What is the pattern of lymphocyte infiltration in chordoma tumors? METHODS: Patients with chordoma treated at Massachusetts General Hospital between 1989 and 2009 were identified with permission from the institutional review board. Of the 75 patients who were identified, 24 human chordoma tumors were selected from 24 distinct patients based on tissue availability. Histology slides from these 24 formalin-fixed paraffin-embedded chordoma tissue samples were deparaffinized using xylene and ethanol and underwent heat-induced antigen retrieval in a citrate buffer. Samples were incubated with monoclonal antibodies directed against HLA Class I antigen processing machinery components. Antibody binding was detected via immunohistochemical staining. Staining intensity (negative, weakly positive, strongly positive) was assessed semiquantitatively and the percentage of chordoma cells stained for HLA Class I antigen subunits was assessed quantitatively. Hematoxylin and eosin-stained histology slides from the same 24 chordoma samples were assessed qualitatively for the presence of tumor-infiltrating lymphocytes and histologic location of these lymphocytes. Immunohistochemical staining with monoclonal antibodies directed against CD4 and CD8 was performed in a quantitative manner to identify the lymphocyte subtype present in chordoma tumors. All results were scored independently by two investigators and were confirmed by a senior bone and soft tissue pathologist. RESULTS: Seven of 24 chordoma samples exhibited no staining by the anti-HLA-A heavy chain monoclonal antibody HC-A2, two had weak staining intensity, and eight had a heterogeneous staining pattern, with fewer than 60% of chordoma cells exhibiting positive staining results. Four of 24 samples tested were not stained by the anti-HLA-B/C heavy chain monoclonal antibody HC-10, five had weak staining intensity, and 11 displayed a heterogeneous staining pattern. For the anti-ß-2-microglobulin monoclonal antibody NAMB-1, staining was detected in all samples, but 11 had weak staining intensity and four displayed a heterogeneous staining pattern. Twenty-one of 24 samples tested had decreased expression in at least one subunit of HLA Class I antigens. No tumors were negative for all three subunits. Lymphocytic infiltration was found in 21 of 24 samples. Lymphocytes were primarily found in the fibrous septae between chordoma lobules but also within the tumor lobules and within the fibrous septae and tumor lobules. Twenty-one of 24 tumors had CD4+ T cells and 11 had CD8+ T cells. CONCLUSION: In chordoma tissue samples, HLA Class I antigen defects commonly were present, suggesting a mechanism for escape from host immunosurveillance. Additionally, nearly half of the tested samples had cytotoxic CD8+ T cells present in chordoma tumors, suggesting that the host may be capable of mounting an immune response against chordoma tumors. The resulting selective pressure imposed on chordoma tumors may lead to the outgrowth of chordoma cell subpopulations that can evade the host's immune system. CLINICAL RELEVANCE: These findings have implications in the design of immunotherapeutic strategies for chordoma treatment. T cell recognition of tumor cells requires HLA Class I antigen expression on the targeted tumor cells. Defects in HLA Class I expression may play a role in the clinical course of chordoma and may account for the limited or lack of efficacy of T cell-based immunity triggered by vaccines and/or checkpoint inhibitors.

CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab.

KRAS mutations hinder therapeutic efficacy of epidermal growth factor receptor (EGFR)-specific monoclonal antibodies cetuximab and panitumumab-based immunotherapy of EGFR+ cancers. Although cetuximab inhibits KRAS-mutated cancer cell growth in vitro by natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), KRAS-mutated colorectal carcinoma (CRC) cells escape NK cell immunosurveillance in vivo. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 colorectal cancer (CRC) cells. We compared four polymorphic Fcγ-CR constructs including CD16158F -CR, CD16158V -CR, CD32131H -CR, and CD32131R -CR transduced into T cells by retroviral vectors. Percentages of transduced T cells expressing CD32131H -CR (83.5 ± 9.5) and CD32131R -CR (77.7 ± 13.2) were significantly higher than those expressing with CD16158F -CR (30.3 ± 10.2) and CD16158V -CR (51.7 ± 13.7) (p < 0.003). CD32131R -CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V -CR T cells released high levels of interferon gamma (IFNγ = 1,145.5 pg/ml ±16.5 pg/ml, p < 0.001) and tumor necrosis factor alpha (TNFα = 614 pg/ml ± 21 pg/ml, p < 0.001) upon incubation with cetuximab-opsonized HCT116 cells. Moreover, only CD16158V -CR T cells combined with cetuximab killed HCT116 cells and A549 KRAS-mutated cells in vitro. CD16158V -CR T cells also effectively controlled subcutaneous growth of HCT116 cells in CB17-SCID mice in vivo. Thus, CD16158V -CR T cells combined with cetuximab represent useful reagents to develop innovative EGFR+KRAS-mutated CRC immunotherapies.

In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab.

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A131R -CR T cells was 74 ± 10%, whereas the percentage of CD16158F -CR T cells was 46 ± 15%. Only CD32A131R -CR T cells bound panitumumab. CD32A131R -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116FcγR+ cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A131R -CR on T cells by cetuximab or panitumumab and CD16158F -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A131R -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR.

A fast, simple, and cost-effective method of expanding patient-derived xenograft mouse models of pancreatic ductal adenocarcinoma.

BACKGROUND: Patient-derived xenograft (PDX) mouse models of cancer have been recognized as better mouse models that recapitulate the characteristics of original malignancies including preserved tumor heterogeneity, lineage hierarchy, and tumor microenvironment. However, common challenges of PDX models are the significant time required for tumor expansion, reduced tumor take rates, and higher costs. Here, we describe a fast, simple, and cost-effective method of expanding PDX of pancreatic ductal adenocarcinoma (PDAC) in mice. METHODS: We used two established frozen PDAC PDX tissues (derived from two different patients) and implanted them subcutaneously into SCID mice. After tissues reached 10-20 mm in diameter, we performed survival surgery on each mouse to harvest 90-95% of subcutaneous PDX (incomplete resection), allowing the remaining 5-10% of PDX to continue growing in the same mouse. RESULTS: We expanded three consecutive passages (P1, P2, and P3) of PDX in the same mouse. Comparing the times required for in vivo expansion, P2 and P3 (expanded through incomplete resection) grew 26-60% faster than P1. Moreover, such expanded PDX tissues were successfully implanted orthotopically into mouse pancreases. Within 20 weeks using only 14 mice, we generated sufficient PDX tissue for future implantation of 200 mice. Our histology study confirmed that the morphologies of cancer cells and stromal structures were similar across all three passages of subcutaneous PDX and the orthotopic PDX and were reflective of the original patient tumors. CONCLUSIONS: Taking advantage of incomplete resection of tumors associated with high local recurrence, we established a fast method of PDAC PDX expansion in mice.

Perspectives in melanoma: meeting report from the "Melanoma Bridge" (December 5th-7th, 2019, Naples, Italy).

The melanoma treatment landscape changed in 2011 with the approval of the first anti-cytotoxic T-lymphocyte-associated protein (CTLA)-4 checkpoint inhibitor and of the first BRAF-targeted monoclonal antibody, both of which significantly improved overall survival (OS). Since then, improved understanding of the tumor microenvironment (TME) and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. The approval of new immune and targeted therapies has further improved outcomes for patients with advanced melanoma and other combination modalities are also being explored such as chemotherapy, radiotherapy, electrochemotherapy and surgery. In addition, different strategies of drugs administration including sequential or combination treatment are being tested. Approaches to overcome resistance and to potentiate the immune response are being developed. Increasing evidence emerges that tissue and blood-based biomarkers can predict the response to a therapy. The latest findings in melanoma research, including insights into the tumor microenvironment and new biomarkers, improved understanding of tumor immune response and resistance, novel approaches for combination strategies and the role of neoadjuvant and adjuvant therapy, were the focus of discussions at the Melanoma Bridge meeting (5-7 December, 2019, Naples, Italy), which are summarized in this report.

Induction of immunogenic cell death in radiation-resistant breast cancer stem cells by repurposing anti-alcoholism drug disulfiram.

BACKGROUND: The current successful clinical use of agents promoting robust anti-tumor immunity in cancer patients warrants noting that radiation therapy (RT) induces immunogenic cell death (ICD) of tumor cells, which can generate anti-tumor immune responses. However, breast cancer stem cells (BCSCs) are resistant to RT and RT alone usually failed to mount an anti-tumor immune response. METHODS: High aldehyde dehydrogenase activity (ALDH)bright and CD44+/CD24-/ESA+ cancer cells, previously shown to have BCSC properties, were isolated from human MDA-MB-231 and UACC-812 breast cancer cell lines by flow cytometer. Flow sorted BCSCs and non-BCSCs were further tested for their characteristic of stemness by mammosphere formation assay. Induction of ICD in BCSCs vs. non-BCSCs in response to different in vitro treatments was determined by assessing cell apoptosis and a panel of damage-associated molecular pattern molecules (DAMPs) by flow and enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that ionizing radiation (IR) triggered a lower level of ICD in BCSCs than non-BCSCs. We then investigated the ability of disulfiram/cooper (DSF/Cu) which is known to preferentially induce cancer stem cells (CSCs) apoptosis to enhance IR-induced ICD of BCSCs. The results indicate that DSF/Cu induced a similar extent of IDC in both BCSCs and non-BCSCs and rendered IR-resistant BCSCs as sensitive as non-BCSCs to IR-induced ICD. IR and DSF/Cu induced ICD of BCSCs could be partly reversed by pre-treatment of BCSCs with a reactive oxygen species (ROS) scavenger and XBP1s inhibitors. CONCLUSION: DSF/Cu rendered IR-resistant BCSCs as sensitive as non-BCSCs to IR-induced ICD. Our data demonstrate the potential of IR and DSF/Cu to induce ICD in BCSCs and non-BCSCs leading to robust immune responses against not only differentiated/differentiating breast cancer cells but also BCSCs, the root cause of cancer formation, progression and metastasis.

Correction to: Induction of immunogenic cell death in radiation-resistant breast cancer stem cells by repurposing anti-alcoholism drug disulfiram.

An amendment to this paper has been published and can be accessed via the original article.

The role of cancer stem cells in the modulation of anti-tumor immune responses.

Tumor lesions comprise multiple subpopulations of cells including those endowed with "stemness" properties. The latter cells are responsible of tumor initiation, metastasis formation, resistance to conventional therapies and disease recurrence. These relatively rare cells denominated cancer stem cells (CSCs) or cancer initiating cells (CICs) are defined based on self-renewing, multipotency and tumorigenicity. These cells through their immunomodulating features can evade from immunesurveillance, persisting in the form of quiescence and dormancy. They can drive the neoplastic growth and recurrence, even after long latency. Moreover, CSCs/CICs due to their ability to modulate and shape immune responses can represent the component of a tumor causing immunotherapy resistance in cancer patients. In this review a general overview of immunological properties of CSCs/CICs is provided, with a special focus on the mechanisms of modulation of T cell mediated responses. The need to further dissect the mechanisms regulating the immunological profile of CSCs/CICs and their interactions with immune cells and tumor microenvironment is discussed. An improved characterization of the immunological properties of CSCs/CICs will contribute to the rationale design of immunotherapeutic interventions which target these cells and may lead to the eradication of malignant diseases.

High IDO1 Expression Is Associated with Poor Outcome in Patients with Anal Cancer Treated with Definitive Chemoradiotherapy.

BACKGROUND: This study characterizes the tumor-immune microenvironment in pretreatment, localized anal squamous cell carcinoma (ASCC), including two markers that have not previously been studied in ASCC: indoleamine 2,3 dioxygenase 1 (IDO1) and human leukocyte antigen (HLA) class I. MATERIALS AND METHODS: Retrospective review identified 63 patients with ASCC receiving definitive chemoradiation between 2005 and 2016 with pretreatment tissue available. Immunohistochemistry was used to quantify cluster of differentiation 8 (CD8), programmed cell death protein 1, programmed death-ligand 1, HLA class I, and IDO1. Cox proportional hazards models evaluated associations between outcomes and immune markers, controlling for clinical characteristics. RESULTS: With a median follow-up of 35 months, 3-year overall survival was 78%. The only marker found to have a robust association with outcome was tumor IDO1. In general, the percentage of tumor cells expressing IDO1 was low (median 1%, interquartile range 0%-20%); however, patients with >50% of tumor cells expressing IDO1 had significantly worse overall survival (hazard ratio [HR] 4.7, p = .007) as well as higher local recurrence (HR 8.6, p = .0005) and distant metastasis (HR 12.7, p = .0002). Tumors with >50% IDO1 were also more likely to have the lowest quartile of CD8 infiltrate (<40 per high-power field, p = .024). CONCLUSION: ASCC has a diverse immune milieu. Although patients generally do well with standard therapy, IDO1 may serve as a prognostic indicator of poor outcome and could help identify a patient population that might benefit from IDO-targeted therapies. IMPLICATIONS FOR PRACTICE: After definitive chemoradiation, patients with locally advanced anal cancer may experience significant treatment morbidity and high risk of recurrence. The goal of the current study is to identify novel prognostic factors in the tumor-immune microenvironment that predict for poor outcomes after definitive chemoradiation. This study characterizes the tumor-immune microenvironment in pre-treatment, localized anal squamous cell carcinoma (ASCC), including two markers which have not previously been studied in ASCC: indoleamine 2,3 dioxygenase 1 (IDO1) and HLA class I. With a median follow-up of 3 years, this study demonstrated that high IDO1 expression is correlated with significantly worse 3-year overall survival (88% vs. 25%). Whereas recent studies of IDO1 inhibitors have shown mixed results, this study suggests that patients with anal cancer with high IDO1 expression have dismal prognosis and may represent a patient population primed for response to targeted IDO1 inhibition.

Addressing current challenges and future directions in immuno-oncology: expert perspectives from the 2017 NIBIT Foundation Think Tank, Siena, Italy.

A collaborative think tank involving panellists from immuno-oncology networks, clinical/translational investigators and the pharmaceutical industry was held in Siena, Italy, in October 2017 to discuss the evolving immune-oncology landscape, identify selected key challenges, and provide a perspective on the next steps required in the translation of current research and knowledge to clinical reality. While there is a trend of combining new agents (e.g., co-stimulator agonists) with a PD-1/PD-L1 treatment backbone, use of alternative combination therapy approaches should also be considered. While the rapid evolution in systems biology provides a deeper understanding of tumor and tumor microenvironment heterogeneity, there remains the need to identify and define genuinely predictive biomarkers to guide treatment and patient selection. Cross-specialty and cross-sector collaboration, along with a broader collective data-sharing approach are key to optimizing immuno-oncology therapy in clinical practice. Continued support of younger research-clinicians is essential for future success in clinical, translational and basic science investigations.

Perspectives in melanoma: meeting report from the Melanoma Bridge (November 29th-1 December 1st, 2018, Naples, Italy).

Diagnosis of melanocytic lesions, correct prognostication of patients, selection of appropriate adjuvant and systemic therapies, and prediction of response to a given therapy remain very real challenges in melanoma. Recent studies have shown that immune checkpoint blockade that represents a forefront in cancer therapy, provide responses but they are not universal. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers they have yet to be fully characterized and implemented clinically. For example, advancements in sequencing and the understanding of the tumor microenvironment in melanoma have led to the use of genome sequencing and gene expression for development of multi-marker assays that show association with inflammatory state of the tumor and potential to predict response to immunotherapy. As such, melanoma serves as a model system for understanding cancer immunity and patient response to immunotherapy, either alone or in combination with other treatment modalities. Overall, the aim for the translational and clinical studies is to achieve incremental improvements through the development and identification of optimal treatment regimens, which increasingly involve doublet as well as triplet combinations, as well as through development of biomarkers to improve immune response. These and other topics in the management of melanoma were the focus of discussions at the fourth Melanoma Bridge meeting (November 29th-December 1st, 2018, Naples, Italy), which is summarised in this report.