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. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Ewing Sarcoma Single-cell Transcriptome Analysis Reveals Functionally Impaired Antigen-presenting Cells.

Novel therapeutic strategies are urgently needed for patients with high-risk Ewing sarcoma and for the reduction of severe side effects for all patients. Immunotherapy may fill this need, but its successful application has been hampered by a lack of knowledge on the composition and function of the Ewing sarcoma immune microenvironment. Here, we explore the immune microenvironment of Ewing sarcoma, by single-cell RNA sequencing of 18 Ewing sarcoma primary tissue samples. Ewing sarcoma is infiltrated by natural killer, T, and B cells, dendritic cells, and immunosuppressive macrophages. Ewing sarcoma-associated T cells show various degrees of dysfunction. The antigen-presenting cells found in Ewing sarcoma lack costimulatory gene expression, implying functional impairment. Interaction analysis reveals a clear role for Ewing sarcoma tumor cells in turning the Ewing sarcoma immune microenvironment into an immunosuppressive niche. These results provide novel insights into the functional state of immune cells in the Ewing sarcoma tumor microenvironment and suggest mechanisms by which Ewing sarcoma tumor cells interact with, and shape, the immune microenvironment. SIGNIFICANCE: This study is the first presenting a detailed analysis of the Ewing sarcoma microenvironment using single-cell RNA sequencing. We provide novel insight into the functional state of immune cells and suggests mechanisms by which Ewing tumor cells interact with, and shape, their immune microenvironment. These insights provide help in understanding the failures and successes of immunotherapy in Ewing sarcoma and may guide novel targeted (immuno) therapeutic approaches.

Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma.

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Comprehensive multiplexed immune profiling of the ductal carcinoma in situ immune microenvironment regarding subsequent ipsilateral invasive breast cancer risk.

BACKGROUND: Ductal carcinoma in situ (DCIS) is treated to prevent subsequent ipsilateral invasive breast cancer (iIBC). However, many DCIS lesions will never become invasive. To prevent overtreatment, we need to distinguish harmless from potentially hazardous DCIS. We investigated whether the immune microenvironment (IME) in DCIS correlates with transition to iIBC. METHODS: Patients were derived from a Dutch population-based cohort of 10,090 women with pure DCIS with a median follow-up time of 12 years. Density, composition and proximity to the closest DCIS cell of CD20+ B-cells, CD3+CD8+ T-cells, CD3+CD8- T-cells, CD3+FOXP3+ regulatory T-cells, CD68+ cells, and CD8+Ki67+ T-cells was assessed with multiplex immunofluorescence (mIF) with digital whole-slide analysis and compared between primary DCIS lesions of 77 women with subsequent iIBC (cases) and 64 without (controls). RESULTS: Higher stromal density of analysed immune cell subsets was significantly associated with higher grade, ER negativity, HER-2 positivity, Ki67 ≥ 14%, periductal fibrosis and comedonecrosis (P < 0.05). Density, composition and proximity to the closest DCIS cell of all analysed immune cell subsets did not differ between cases and controls. CONCLUSION: IME features analysed by mIF in 141 patients from a well-annotated cohort of pure DCIS with long-term follow-up are no predictors of subsequent iIBC, but do correlate with other factors (grade, ER, HER2 status, Ki-67) known to be associated with invasive recurrences.

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.

Breast adipocyte size associates with ipsilateral invasive breast cancer risk after ductal carcinoma in situ.

Although ductal carcinoma in situ (DCIS) is a non-obligate precursor to ipsilateral invasive breast cancer (iIBC), most DCIS lesions remain indolent. Hence, overdiagnosis and overtreatment of DCIS is a major concern. There is an urgent need for prognostic markers that can distinguish harmless from potentially hazardous DCIS. We hypothesised that features of the breast adipose tissue may be associated with risk of subsequent iIBC. We performed a case-control study nested in a population-based DCIS cohort, consisting of 2658 women diagnosed with primary DCIS between 1989 and 2005, uniformly treated with breast conserving surgery (BCS) alone. We assessed breast adipose features with digital pathology (HALO®, Indica Labs) and related these to iIBC risk in 108 women that developed subsequent iIBC (cases) and 168 women who did not (controls) by conditional logistic regression, accounting for clinicopathological and immunohistochemistry variables. Large breast adipocyte size was significantly associated with iIBC risk (odds ratio (OR) 2.75, 95% confidence interval (95% CI) = 1.25-6.05). High cyclooxygenase (COX)-2 protein expression in the DCIS cells was also associated with subsequent iIBC (OR 3.70 (95% CI = 1.59-8.64). DCIS with both high COX-2 expression and large breast adipocytes was associated with a 12-fold higher risk (OR 12.0, 95% CI = 3.10-46.3, P < 0.001) for subsequent iIBC compared with women with smaller adipocyte size and low COX-2 expression. Large breast adipocytes combined with high COX-2 expression in DCIS is associated with a high risk of subsequent iIBC. Besides COX-2, adipocyte size has the potential to improve clinical management in patients diagnosed with primary DCIS.

An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.

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.

Discordant Marker Expression Between Invasive Breast Carcinoma and Corresponding Synchronous and Preceding DCIS.

Ductal carcinoma in situ (DCIS) is considered a potential precursor of invasive breast carcinoma (IBC). Studies aiming to find markers involved in DCIS progression generally have compared characteristics of IBC lesions with those of adjacent synchronous DCIS lesions. The question remains whether synchronous DCIS and IBC comparisons are a good surrogate for primary DCIS and subsequent IBC. In this study, we compared both primary DCIS and synchronous DCIS with the associated IBC lesion, on the basis of immunohistochemical marker expression. Immunohistochemical analysis of ER, PR, HER2, p53, and cyclo-oxygenase 2 (COX-2) was performed for 143 primary DCIS and subsequent IBC lesions, including 81 IBC lesions with synchronous DCIS. Agreement between DCIS and IBC was assessed using kappa, and symmetry tests were performed to assess the pattern in marker conversion. The primary DCIS and subsequent IBC more often showed discordant marker expression than synchronous DCIS and IBC. Strikingly, 18 of 49 (36%) women with HER2-positive primary DCIS developed an HER2-negative IBC. Such a difference in HER2 expression was not observed when comparing synchronous DCIS and IBC. The frequency of discordant marker expression did not increase with longer time between primary DCIS and IBC. In conclusion, comparison of primary DCIS and subsequent IBC yields different results than a comparison of synchronous DCIS and IBC, in particular with regard to HER2 status. To gain more insight into the progression of DCIS to IBC, it is essential to focus on the relationship between primary DCIS and subsequent IBC, rather than comparing IBC with synchronous DCIS.

Predictors of an Invasive Breast Cancer Recurrence after DCIS: A Systematic Review and Meta-analyses.

We performed a systematic review with meta-analyses to summarize current knowledge on prognostic factors for invasive disease after a diagnosis of ductal carcinoma in situ (DCIS). Eligible studies assessed risk of invasive recurrence in women primarily diagnosed and treated for DCIS and included at least 10 ipsilateral-invasive breast cancer events and 1 year of follow-up. Quality in Prognosis Studies tool was used for risk of bias assessment. Meta-analyses were performed to estimate the average effect size of the prognostic factors. Of 1,781 articles reviewed, 40 articles met the inclusion criteria. Highest risk of bias was attributable to insufficient handling of confounders and poorly described study groups. Six prognostic factors were statistically significant in the meta-analyses: African-American race [pooled estimate (ES), 1.43; 95% confidence interval (CI), 1.15-1.79], premenopausal status (ES, 1.59; 95% CI, 1.20-2.11), detection by palpation (ES, 1.84; 95% CI, 1.47-2.29), involved margins (ES, 1.63; 95% CI, 1.14-2.32), high histologic grade (ES, 1.36; 95% CI, 1.04-1.77), and high p16 expression (ES, 1.51; 95% CI, 1.04-2.19). Six prognostic factors associated with invasive recurrence were identified, whereas many other factors need confirmation in well-designed studies on large patient numbers. Furthermore, we identified frequently occurring biases in studies on invasive recurrence after DCIS. Avoiding these common methodological pitfalls can improve future study designs.

Clinicopathological Risk Factors for an Invasive Breast Cancer Recurrence after Ductal Carcinoma In Situ-A Nested Case-Control Study.

Purpose: Ductal carcinoma in situ (DCIS) is treated to prevent progression to invasive breast cancer. Yet, most lesions will never progress, implying that overtreatment exists. Therefore, we aimed to identify factors distinguishing harmless from potentially hazardous DCIS using a nested case-control study.Experimental Design: We conducted a case-control study nested in a population-based cohort of patients with DCIS treated with breast-conserving surgery (BCS) alone (N = 2,658) between 1989 and 2005. We compared clinical, pathologic, and IHC DCIS characteristics of 200 women who subsequently developed ipsilateral invasive breast cancer (iIBC; cases) and 474 women who did not (controls), in a matched setting. Median follow-up time was 12.0 years (interquartile range, 9.0-15.3). Conditional logistic regression models were used to assess associations of various factors with subsequent iIBC risk after primary DCIS.Results: High COX-2 protein expression showed the strongest association with subsequent iIBC [OR = 2.97; 95% confidence interval (95% CI), 1.72-5.10]. In addition, HER2 overexpression (OR = 1.56; 95% CI, 1.05-2.31) and presence of periductal fibrosis (OR = 1.44; 95% CI, 1.01-2.06) were associated with subsequent iIBC risk. Patients with HER2+/COX-2high DCIS had a 4-fold higher risk of subsequent iIBC (vs. HER2-/COX-2low DCIS), and an estimated 22.8% cumulative risk of developing subsequent iIBC at 15 years.Conclusions: With this unbiased study design and representative group of patients with DCIS treated by BCS alone, COX-2, HER2, and periductal fibrosis were revealed as promising markers predicting progression of DCIS into iIBC. Validation will be done in independent datasets. Ultimately, this will aid individual risk stratification of women with primary DCIS. Clin Cancer Res; 24(15); 3593-601. ©2018 AACR.

Finding the balance between over- and under-treatment of ductal carcinoma in situ (DCIS).

With the widespread adoption of population-based breast cancer screening, ductal carcinoma in situ (DCIS) has come to represent 20-25% of all breast neoplastic lesions diagnosed. Current treatment aims at preventing invasive breast cancer, but the majority of DCIS lesions will never progress to invasive disease. Still, DCIS is treated by surgical excision, followed by radiotherapy as part of breast conserving treatment, and/or endocrine therapy. This implies over-treatment of the majority of DCIS, as less than 1% of DCIS patients will go on to develop invasive breast cancer annually. If we are able to identify which DCIS is likely to progress or recur as invasive breast cancer and which DCIS would remain indolent, we can treat the first group intensively, while sparing the second group from such unnecessary treatment (surgery, radiotherapy, endocrine therapy) preserving the quality of life of these women. This review summarizes our current knowledge on DCIS and the risks involved regarding progression into invasive breast cancer. It also shows current knowledge gaps, areas where profound research is highly necessary for women with DCIS to prevent their over-treatment in case of a harmless DCIS, but provide optimal treatment for potentially hazardous DCIS.

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

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.

T cell memory. Skin-resident memory CD8⁺ T cells trigger a state of tissue-wide pathogen alert.

After an infection, pathogen-specific tissue-resident memory T cells (T(RM) cells) persist in nonlymphoid tissues to provide rapid control upon reinfection, and vaccination strategies that create T(RM) cell pools at sites of pathogen entry are therefore attractive. However, it is not well understood how T(RM) cells provide such pathogen protection. Here, we demonstrate that activated T(RM) cells in mouse skin profoundly alter the local tissue environment by inducing a number of broadly active antiviral and antibacterial genes. This "pathogen alert" allows skin T(RM) cells to protect against an antigenically unrelated virus. These data describe a mechanism by which tissue-resident memory CD8(+) T cells protect previously infected sites that is rapid, amplifies the activation of a small number of cells into an organ-wide response, and has the capacity to control escape variants.

Targeting the acute myeloid leukemic stem cell compartment by enhancing tumor cell-based vaccines.

Harvesting the potential of the immune system in order to eradicate (residual) acute myeloid leukemia (AML) cells is the long pursued goal of immunotherapy in AML. Strategies using apoptotic tumor cell vaccines have been explored for many years, without significant clinical improvements. In recent years insight has been gained into the mechanisms activating and interfering with tumor-directed immunity. With the arrival of novel immune-modulating agents allowing for the interference with regulatory molecules and interaction with immune-propelling mechanisms, new doors are opening for increasing vaccination efficacy. Combined with advances in the design of apoptotic tumor-based vaccines, we are on the verge of creating an effective AML vaccine strategy, offering a much needed novel therapeutic option for this devastating disease.