Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies

Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5–10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.Pattern Recognition and Bioinformatic

Somatic mutations and copy number variations in breast cancers with heterogeneous HER2 amplification.

Intratumour heterogeneity fuels carcinogenesis and allows circumventing specific targeted therapies. HER2 gene amplification is associated with poor outcome in invasive breast cancer. Heterogeneous HER2 amplification has been described in 5-41% of breast cancers. Here, we investigated the genetic differences between HER2-positive and HER2-negative admixed breast cancer components. We performed an in-depth analysis to explore the potential heterogeneity in the somatic mutational landscape of each individual tumour component. Formalin-fixed, paraffin-embedded breast cancer tissue of ten patients with at least one HER2-negative and at least one HER2-positive component was microdissected. Targeted next-generation sequencing was performed using a customized 53-gene panel. Somatic mutations and copy number variations were analysed. Overall, the tumours showed a heterogeneous distribution of 12 deletions, 9 insertions, 32 missense variants and 7 nonsense variants in 26 different genes, which are (likely) pathogenic. Three splice site alterations were identified. One patient had an EGFR copy number gain restricted to a HER2-negative in situ component, resulting in EGFR protein overexpression. Two patients had FGFR1 copy number gains in at least one tumour component. Two patients had an 8q24 gain in at least one tumour component, resulting in a copy number increase in MYC and PVT1. One patient had a CCND1 copy number gain restricted to a HER2-negative tumour component. No common alternative drivers were identified in the HER2-negative tumour components. This series of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. Many other molecular aberrations are likely to act as alternative or collaborative drivers. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile, of which some genetic aberrations will be crucial for cancer progression, and others will be mere 'passenger' molecular anomalies

A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

<div><p>The CD34⁺ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8⁺ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8⁺ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.</p></div

In vivo stabilization of a less toxic asparaginase variant leads to a durable anti-tumor response in acute leukemia

Asparagine is a non-essential amino acid since it can either be taken up via the diet or synthesized by asparagine synthetase (ASNS). Acute lymphoblastic leukemia (ALL) cells do not or minimally express ASNS which makes them completely dependent on extracellular asparagine for their growth and survival. This dependency makes ALL cells vulnerable to treatment with L-asparaginase, an enzyme that hydrolyzes asparagine. To date, all clinically approved L-asparaginases have a significant L-glutaminase co-activity, associated with non-immune related toxic side effects observed during therapy. Therefore, reduction of L-glutaminase co-activity with concomitant maintenance of its anti-cancer L-asparaginase effect may effectively improve the tolerability of this unique drug. Previously, we designed a new alternative variant of Erwinia chrysanthemi (ErA; Erwinaze) with decreased L-glutaminase co-activity, while maintaining its L-asparaginase activity, by the introduction of three key mutations around the active site (ErA-TM). However, Erwinaze and our ErA-TM variant have a very short half-life in vivo. Here, we show that the fusion of ErA-TM with an albumin binding domain (ABD)-tag significantly increases its in vivo persistence. In addition, we evaluated the in vivo therapeutic efficacy of ABD-ErA-TM in a B-ALL xenograft model of SUP-B15. Our results show a comparable long lasting durable anti-leukemic effect between the standard-of-care PEG-asparaginase and ABD-ErA-TM L-asparaginase, but with fewer co-glutaminase related acute side effects. Since the toxic side effects of current L-asparaginases often result in treatment discontinuation in ALL patients, this novel ErA-TM variant with ultra-low L-glutaminase co-activity and long in vivo persistence may have great clinical potential

Phenotypic and morphologic analysis of IFNα- and IL-4 MUTZ-DC.

<p>(A) Representative example of the flow cytometric analysis of cell surface antigen expression profiles on IFNα MUTZ-DC following 3 days of differentiation and (B) following 24 hours of maturation. Represented are the isotype controls (grey) for each antigen (black). (C) Quantification of the flow cytometric analysis of DC-associated protein expression on IL-4 MUTZ-DC (white bars) and IFNα MUTZ-DC (black bars), directly following differentiation (C; n = 10) or after 24 hours of maturation (D; n = 4). The bars represent the mean values with the standard error of the mean (SEM). (E) Morphologic analysis of IL-4 (upper images) and IFNα MUTZ-DC (lower images), using the bright field channel of the ImageStream (magnification 60x). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.</p

T cell proliferation and activation by IFNα- and IL-4 MUTZ-DC.

<p>(A) Immature or mature IL-4 (white bars) or IFNα MUTZ-DC (black bars) were cultured with CFSE-labeled allogeneic peripheral blood lymphocytes (PBL) for 6 days at a DC:PBL ratio of 1:5, after which the CFSE dilution was determined within the CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell compartment, as a measure for proliferation (n = 6; ** p ≤ 0.01). The MLR supernatant was subsequently analyzed for the produced T cell cytokines (B; n = 3). Shown are the mean proliferation and cytokine production, with the corresponding SEM. (C) Immature IL-4 (circles) or IFNα (triangles) MUTZ-DC were loaded with a concentration titration (ranging from 0 μg/ml to 10 μg/ml) of a 25-mer MART-1 synthetic long peptide (SLP), in the presence of a cytokine maturation cocktail. After an overnight culture period, loaded MUTZ-DC were co-cultured with a MART CTL for 5 hours in the presence of a protein transport inhibitor, and the produced IFNγ was quantified using flow cytometry. (D) Mature IL-4 or IFNα MUTZ-DC were loaded exogenously with MART-1_26–35L peptide for 2 hours, after which they were co-cultured with naïve CD8⁺ T cells. Before every re-stimulation the percentage of MART-1 specific CD8⁺ T cells was determined using Dextramer (Dx) staining. Shown are representative examples of MART-1 specific CD8⁺ T cell outgrowth, following priming and one restimulation with either IL-4 or IFNα MUTZ-DC. (E) MART-1 specific CD8⁺ T cells were sorted and polyclonally expanded, after which they were rechallenged with MART-1_26–35L peptide-loaded JY cells. Intracellular IFNγ production was determined as a measure for activation.</p