Immunological Landscape Research Articles

Immune dysregulation in children with inherited bone marrow failure syndromes and refractory cytopenia of childhood

Background and AimsInherited bone marrow failure syndromes (IBMFS) are usually identified when patients develop hematologic complications, such as severe BMF, myelodysplastic syndrome or leukemia. In clinical practice, differential diagnosis with refractory cytopenia of childhood (RCC) or autoimmune cytopenia with or without an underlying Inborn Error of Immunity (IEI) may be challenging. To fill this gap, we aim at profiling the immunological landscape associated with pediatric chronic cytopenia(s). MethodsWe enrolled patients presenting with chronic cytopenia (>12 months): isolated autoimmune cytopenia(s) (AIC, N = 11); IBMFS (N = 12: 4 Diamond-Blackfan; 4 Shwackman-Diamond, 2 Fanconi anemia, 1 RTEL1-Dyskeratosis congenita, 1 RUNX1-deficiency) and RCC (N = 6). Extended immunophenotyping from peripheral blood was performed to compare the AIC to IBMFS and RCC group.Table 1(abstract: 86) Clinical features of IBMFS and RCC patient cohorts. A, anemia; L, leukopenia; N, neutropenia; T, thrombocytopenia; PAN, pancytopenia; (*) siblings.DIAGNOSISMUTATED GENECYTOPENIA LINEAGEADDITIONAL CLINICAL FEATURESAGE AT FLOW ANALYSIS (y)IBMFSDiamond-Blackfan anemia (N=4)RPS19APancreas annularis11ongoingA + L/0,5N.A.AGH deficiency, suppurative sialadenitis, interatrial defect16N.A.AAtrial septal defect21Shwachman Diamond syndrome (N=4)SBDSA + NGH deficiency, pancreatic insufficiency, bone abnormalities, nephrocalcinosis12SBDSNGrowth retardation, pancreatic insufficiency, hepatitis (unknown cause), bone abnormalities, osteopenia15SBDSNGrowth retardation, intestinal malrotation, cleft palate16N.A.NGrowth retardation, pancreatic insufficiency, bone abnormalities18Fanconi anemia (N=2*)BRIP1 (FANCJ)A + T/16BRIP1 (FANCJ)/Precocious puberty, congenital melanocytic nevus9RUNX1 deficiencyRUNX1N + T/15Dyskeratosis congenitaRTEL1PANUpper esophageal stricture19RCCHebo deficiencyERCC6L2N + T/13Refractory cytopenia (N=5)/A + T/16/N + T/14N.A.PAN/9NonePANSeronegative autoimmune hepatitis, eczema, hydronephrosis4NonePANSeronegative autoimmune hepatitis11 Results:Lymphocyte subpopulations analysis revealed lower frequencies of CD4 Recent Thymic Emigrants (RTEs) in the IBMFS group (p = 0.020), CD4 naïve T cells in both IBMFS (p = 0.002) and RCC (p = 0.041) patients and CD8 naïve T cells in IBMFS group (p = 0.021), counterbalanced by a higher frequency of CD4 central memory (CM; p = 0.001 for IBMFS; p = 0.022 for RCC) and CD8 CM T cells (p < 0.05 for IBMFS and RCC). A similar profile was detected in both cohorts for naïve Tregs (p < 0.05) towards memory Tregs (p <0.05). ConclusionsWe observed a striking T cell immunedysregulation due to an imbalance towards the memory compartment in pediatric patients with RCC and IBMFS. As this scenario is reminiscent of what observed in patients with AIC associated with signs of IEI, further investigations are required to define the pathogenicity of the altered immune homeostasis. Nevertheless, peripheral immunophenotyping confirms to be a useful screening tool able to facilitate differential diagnosis in pediatric cytopenia. [Display omitted]

Late-stage MC38 tumours recapitulate features of human colorectal cancer - implications for appropriate timepoint selection in preclinical studies.

Anti-tumour T cell responses play a crucial role in controlling the progression of colorectal cancer (CRC), making this disease a promising candidate for immunotherapy. However, responses to immune-targeted therapies are currently limited to subpopulations of patients and specific types of cancer. Clinical studies have therefore focussed on identifying biomarkers that predict immunotherapy responses and elucidating the immunological landscapes of different cancers. Meanwhile, our understanding of how preclinical tumour models resemble human disease has fallen behind, despite their crucial role in immune-targeted drug development. A deeper understanding of these models is therefore needed to improve the development of immunotherapies and the translation of findings made in these systems. MC38 colon adenocarcinoma is a widely used preclinical model, yet how it recapitulates human colorectal cancer remains poorly defined. This study investigated the tumour-T cell immune landscape of MC38 tumours using histology, immunohistochemistry, and flow cytometry. We demonstrate that early-stage tumours exhibit a nascent TME, lacking important immune-resistance mechanisms of clinical interest, while late-stage tumours exhibit a mature TME resembling human tumours, with desmoplasia, T cell exhaustion, and T cell exclusion. Consequently, these findings clarify appropriate timepoint selection in the MC38 model when investigating both immunotherapies and mechanisms that contribute to immunotherapy resistance. Overall, this study provides a valuable resource that will enable appropriate application of the MC38 model and expedite the development and clinical translation of new immunotherapies.

Genomic Profiling of Urothelial Carcinoma in Situ of the Bladder

BACKGROUND AND AIMS Urothelial carcinoma in situ (CIS) of the bladder is an intra-epithelial, high grade, malignant neoplasm characterised by flat (non-papillary) growth, with high probability of disease progression.1,2 CIS has histologic features similar to invasive cancer and is often a precursor to and associated with invasive cancer. Molecularly, bladder cancer can be broadly categorised into luminal and basal subtypes.1-7 However, the molecular features that are unique to CIS, as compared to other high-grade lesions, including papillary Ta and T1 tumours, are underexplored. Bulk RNA expression profiling is a standard for molecularly characterising bladder cancer, and RNA-sequencing protocols utilising formalin-fixed paraffin-embedded (FFPE) tumours are well established. However, CIS presents challenges for RNA sequencing due to the inability to reliably detect, the low incidence rate, and the quality of RNA derived from FFPE samples. While enhanced cystoscopy and narrow band imaging have improved the ability to detect CIS, sampling acquisition techniques still limit the optimisation of RNA sequencing. Furthermore, the authors determined that established dual nucleic acid extraction protocols for FFPE samples were not feasible due to small biopsy sample sizes, and that protocols should be optimised for either RNA or DNA. This study attempts to overcome above stated challenges to understand the molecular landscape of CIS by contrasting against papillary tumours and normal urothelium using RNA-sequencing on FFPE samples, as well as whole exome sequencing, and immunohistochemical and immunofluorescent analyses, with an intent to identify unique molecular signatures associated with CIS. MATERIALS AND METHODS The authors performed whole transcriptome profiling with RNA sequencing of FFPE specimens from 15 CIS, nine high-grade papillary Ta/T1 tumours, and eight normal urothelial samples (Cohort A). Wilcoxon test was used to filter differentially expressed genes and The Cancer Genome Atlas (TCGA) single sample classifier was used to assign molecular subtypes. Whole exome sequencing was performed for 19 patients with matched CIS and papillary tumour samples (Cohort B). Using multiplex immunofluorescence and immunohistochemistry analyses, 24 samples from 15 patients were analysed for the presence of cytotoxic T cells, T helper cells, regulatory T cells, B cells, M1 and M2 macrophages, and programmed cell death protein 1 and programmed death-ligand 1-expressing cells. RESULTS The authors performed molecular subtyping applying the UROMOL classification and as previously shown for CIS, the majority were Class 2a and 2b, with four Class 3 and one Class 1. They applied the TCGA single patient classifier and the majority were luminal with a breakdown of two luminal, five luminal infiltrated, seven luminal papillary, three basal, and one neuronal subtype (Figure 1). A 46-gene signature of differentially expressed genes in CIS samples was identified and included known druggable targets that were selectively upregulated (MTOR, TYK2, AXIN1, CPT1B, GAK, and PIEZO1) or downregulated (BRD2 and NDUFB2; p&lt;0.05). An independent dataset was used to assess the robustness of these markers. High expression of MTOR, GUSBP11, KMT2D, and URB1 was significantly associated with CIS in this independent dataset.Additionally, mutational analysis of 34 matched CIS and 33 papillary tumours revealed a clonal origin of the lesions with mutations shared between both synchronous and metachronous tumours. Inter- and intra-patient mutational heterogeneity was also observed. The most frequently mutated gene was KDM6A, which was observed in 53% of the patient samples. Analysis of the immunological landscape of 24 CIS and papillary tumour lesions showed higher levels of immune cells in stromal compartments compared to carcinoma regions. Furthermore, more programmed cell death protein 1 positive cells were observed in CIS lesions compared with papillary tumours (p=0.03). CONCLUSION Collectively, this study identifies a molecular signature that distinguishes CIS lesions from papillary tumours in terms of gene expression levels, mutational landscape, and proportion of programmed cell death protein 1 positive cells that may contribute to an aggressive feature of progressive disease phenotype of the bladder cancer.

Identification of novel pathways and immune profiles related to sarcopenia.

Sarcopenia is a progressive deterioration of skeletal muscle mass strength and function. To uncover the underlying cellular and biological mechanisms, we studied the association between sarcopenia's three stages and the patient's ethnicity, identified a gene regulatory network based on motif enrichment in the upregulated gene set of sarcopenia, and compared the immunological landscape among sarcopenia stages. We found that sarcopenia (S) was associated with GnRH, neurotrophin, Rap1, Ras, and p53 signaling pathways. Low muscle mass (LMM) patients showed activated pathways of VEGF signaling, B-cell receptor signaling, ErbB signaling, and T-cell receptor signaling. Low muscle mass and physical performance (LMM_LP) patients showed lower enrichment scores in B-cell receptor signaling, apoptosis, HIF-1 signaling, and the adaptive immune response pathways. Five common genes among DEGs and the elastic net regression model, TTC39DP, SLURP1, LCE1C, PTCD2P1, and OR7E109P, were expressed between S patients and healthy controls. SLURP1 and LCE1C showed the highest expression levels among sarcopenic Chinese descent than Caucasians and Afro-Caribbeans. Gene regulatory analysis of top upregulated genes in S patients yielded a top-scoring regulon containing GATA1, GATA2, and GATA3 as master regulators and nine predicted direct target genes. Two genes were associated with locomotion: POSTN and SLURP1. TTC39DP upregulation was associated with a better prognosis and stronger immune profile in S patients. The upregulation of SLURP1 and LCE1C was associated with a worse prognosis and weaker immune profile. This study provides new insight into sarcopenia's cellular and immunological prospects and evaluates the age and sarcopenia-related modifications of skeletal muscle.

Abstract LB203: Targeted inhibition of IRAK-4 with CA-4948 (emavusertib) sensitizes metastatic brain melanoma to anti-PD-1 immune checkpoint blockade

Abstract Introduction: The unique microenvironment within the brain alongside restrictions imparted by the blood brain barrier (BBB) create a sanctuary site for tumors to thrive. Melanoma is often responsive to therapy when present in peripheral tissue, but becomes recalcitrant when sequestered beyond the BBB. An estimated 40-60% of metastatic melanoma patients will develop a brain lesion, even while receiving treatment. Stereotactic radiosurgery (SRS) is the gold standard in intervention for metastatic brain melanoma (MBM), often given with anti-PD1 immune checkpoint blockade (ICB). While this therapy is initially effective, resistance occurs rapidly and ~50% of patients will develop new brain lesions within a year. Treatments at this stage are limited, necessitating the discovery of new therapies. Recent studies have shown that myeloid differentiation primary response protein 88 (MyD88) is activated in melanoma in tumor-associated myeloid cells where chronic myddosome signaling results in defective immunological responses. We have found that myddosome signaling is also prevalent in MBM, likely protecting brain metastases against effector immune detection. We recently identified that CA-4948, a small molecule inhibitor of interleukin (IL)-1 receptor-associated kinase (IRAK-4) and rate-limiting step in the myddosome cascade, can reach therapeutic levels in the brain and demonstrates on-target inhibition in preclinical models of MBM. These data highlight a unique therapeutic opportunity where selective inhibition of pro-tumorigenic cells in the MBM tumor microenvironment (TME) via targeted IRAK-4 inhibition may recondition the immunological landscape of these tumors, sensitizing these difficult-to-treat tumors to immunotherapy. Hypothesis: Selective inhibition of pro-tumorigenic cells via targeted inhibition of IRAK-4 with CA-4948 will recondition the immunological landscape of MBM, improving anti-tumor response to anti-PD-1 ICB. Methods: Patient MBM samples were assessed by IHC to define the distribution of myddosome signaling. Preclinical syngeneic MBM tumor models were treated with CA-4948 alone and in combination with anti-PD-1 ICB. Tumors were examined by flow cytometry and IHC to characterize the composition of the immune TME and evaluate the distribution of T lymphocytes. Overall survival was compared between control, single agent, and combination treated animals. Results: Tumor-associated immune cells and astrocytes comprise the majority of active myddosome signaling in patient MBM. Preclinical models corroborate these findings, with targeted inhibition of IRAK-4 through CA-4948 treatment effective at reducing activation and validating anti-tumor activity in the CNS. Analysis of treated tumors reveal decreased expression of Programmed Death Ligand 1 (PD-L1), and increased numbers of tumor-infiltrating T lymphocytes. Combination CA-4948 with anti-PD-1 ICB resulted in reduced tumor growth, and improved survival over single agent therapy. Conclusions: Therapeutic inhibition of IRAK-4 in MBM mitigates immune suppression mediated by tumor-associated cells, allowing for improved immune surveillance and tumor infiltration by effector T lymphocytes, resulting in enhanced anti-tumor activity. Thus, CA-4948 may be an effective treatment alongside anti-PD-1 ICB for patients with MBM. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Rylynn A. Russell, Tyler V. Elliott, Erica Matich, Vincent Archibald, Lan Hoang-Minh, Reinhard W. von Roemeling, Dora Ferrari, Jamison Hoffman, Duane A. Mitchell. Targeted inhibition of IRAK-4 with CA-4948 (emavusertib) sensitizes metastatic brain melanoma to anti-PD-1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB203.

Abstract 5446: Immune phenotypes classified by deep learning-based H&amp;E tissue analyzer demonstrate distinct immune landscape and transcriptomic features in ovarian cancer

Abstract Introduction: Deep learning-based H&amp;E slide analyzer, Lunit-SCOPE IO can classify the tumor microenvironment as three immune phenotypes (IPs): inflamed, immune-excluded and immune-desert. In our previous study, the IPs demonstrated distinct survival outcome and immunologic landscape in endometrial cancer. To further explore the application of IPs in other type of cancer, we applied Lunit-SCOPE in ovarian cancer (OV) and compared tumor microenvironments based on the IPs. Methods: H&amp;E slide images, RNA-sequencing data, and clinical data were obtained from TCGA OV cohort. Lunit-SCOPE IO is a H&amp;E slide analyzer using deep learning algorithm and was trained with 1,824 H&amp;E slides of advanced NSCLC. We used the analyzer to classify H&amp;E slide images from TCGA OV cohort to three IPs. Cibersort was used to assess immune cell infiltration across the IPs. The cytolytic activity score was obtained from geometric mean of GZMA and PRF1 RNA expression. We further assessed transcriptomic characteristics across IPs via differentially expressed genes analysis and gene set enrichment analysis (GSEA). Additionally, we compared CD274 gene expression across IPs. Finally, survival analysis was performed to evaluate survival outcome based on IPs. We used Mann-Whitney method to compare non-parametric variables between the IPs. Results: The proportion of inflamed, immune-excluded and immune-desert group in TCGA OV were 7 (10.2%), 13 (18.8%), and 49 (71%), respectively. Inflamed group showed significantly higher proportion of M1 macrophage compared with immune-desert group (6.6% vs. 2%, P=.012). We observed higher cytolytic activity score in inflamed group compared to immune-desert group (7.21 vs. 5.27, P=.016). In GSEA, inflamed group is significantly enriched in IL6 JAK STAT3 signaling, interferon gamma response, KRAS signaling up, and inflammatory response pathway (NES 2.19; adjusted P=.021, NES 1.70; adjusted P=.021, NES 1.62; adjusted P=.037, and NES 1.52; adjusted P=.037, respectively). Immune-desert is related to upregulated MYC target V2 pathway (NES -1.95; adjusted P=.037). Inflamed group did not show significant difference in CD274 expression, compared with others (Inflamed vs. immune-excluded, 4.97 vs. 4.19, P=.115 and Inflamed vs. immune-desert, 4.97 vs. 3.95, P=.058). Inflamed group did not show any significant difference in overall survival, compared with non-inflamed group (Inflamed vs. non-inflamed, HR 0.98; 95% CI 0.45 - 2.14; P=.958). Conclusion: Deep learning-based H&amp;E slide analyzer can be used to classify tumor H&amp;E slide images with OV into three IPs. In this study, the classification demonstrated distinct immunologic traits and transcriptomic characteristics in tumor microenvironment. The enrichment of interferon gamma response pathway in inflamed OV group may indicate opportunities for targeted immunotherapy. Citation Format: Horyun Choi, Youjin Oh, Chan-Young Ock, Kyunghyun Paeng, Young Kwang Chae. Immune phenotypes classified by deep learning-based H&amp;E tissue analyzer demonstrate distinct immune landscape and transcriptomic features in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5446.

Comprehensive analysis of glycolysis mediated pattern clusters and immune infiltration characterization of tumor microenvironment in triple-negative breast cancer

BackgroundThe involvement of glycolysis in carcinogenesis and the tumor microenvironment is being increasingly supported by the available data. The aim of this work was to develop a triple-negative breast cancer predictive model based on glycolysis. MethodsGlycolysis mediated pattern clusters were created using the R “ConsensusClusterPlus” package. The variations in the tumor microenvironment between the pattern clusters were examined using the R “GSVA”, “ESTIMATE”, and “CIBERSORT” package. The risk score and nomogram were established to assess clinical outcomes of patients. ResultsSubstantial differences were observed in the immunological landscape between the glycolysis-mediated pattern clusters. When it came to predicting survival and immunotherapy response, the developed risk score showed promising predictive power. Nomogram was designed to be used in therapeutic settings due to its remarkable predictive accuracy. ConclusionsThe immune microenvironment varied among cases of triple-negative breast cancer. The nomogram and the risk score based on glycolysis could both be used to help create more effective treatments.

Targeted multiomics in childhood-onset SLE reveal distinct biological phenotypes associated with disease activity: results from an explorative study

ObjectiveTo combine targeted transcriptomic and proteomic data in an unsupervised hierarchical clustering method to stratify patients with childhood-onset SLE (cSLE) into similar biological phenotypes, and study the immunological cellular landscape...

Sex-biased molecular differences in lung adenocarcinoma are ethnic and smoking specific

BackgroundSex-related differences in cancer epidemiology, tumor biology, immune system activity, and pharmacogenomics have been suggested to be important considerations for precision cancer control. Here we elucidated systematically sex biases in genetic variants, gene expression profiles, and immunological landscapes of lung adenocarcinoma patients (LUADs) with different ancestry and smoking status.MethodsSomatic mutation and mRNA expression data of Asian and Non-Asian LUADs were obtained from public databases. Sex-biased genetic mutations, gene expression, biological pathways, and immune infiltration were identified in the context of smoking status and race.ResultsAmong nonsmokers, male-biased mutations were prevalent in Asian LUADs, while few sex-biased mutations were detected in Non-Asian LUADs. EGFR was the only mutation whose frequency was significantly higher in females than males in both Asian and Non-Asian nonsmokers. More genes exhibited sex-biased expression in Non-Asian LUADs compared to Asian LUADs. Moreover, genes distinctly expressed in females were mainly related to immune-related pathways, whereas those in males were more involved in activation of DNA repair, E2F_targets, and MYC_targets pathways. We also detected sex-specific immune infiltration in the context of genetic variation. In EGFR-mutant LUADs, males had a significantly increased infiltration of CD8 + T cells, whereas resting CD4 + memory T cells were more abundant in females. Additionally, in KRAS-mutant LUADs, CD8 + and CD4 + T cells were more abundant in females than males. In addition, we detected all female patients with high SCGB3A2 expression were exclusively sensitive to immunotherapy, while this phenomenon was not observed in male patients.ConclusionsOur findings provided evidence that sex-related molecular and cellular components are involved in shaping tumor distinct genetic and immune features, which might have important impact on personalized targeted and immune therapy.

NKG2D Fine-Tunes the Local Inflammatory Response in Colorectal Cancer.

Treating colorectal cancer (CRC) is a major challenge due to the heterogeneous immunological, clinical and pathological landscapes. Immunotherapy has so far only proven effective in a very limited subgroup of CRC patients. To better define the immune landscape, we examined the immune gene expression profile in various subsets of CRC patients and used a mouse model of intestinal tumors to dissect immune functions. We found that the NK cell receptor, natural-killer group 2 member D (NKG2D, encoded by KLRK1) and NKG2D ligand gene expression is elevated in the most immunogenic subset of CRC patients. High level of KLRK1 positively correlated with the mRNA expression of IFNG and associated with a poor survival of CRC patients. We further show that NKG2D deficiency in the Apcmin/+ mouse model of intestinal tumorigenesis led to reduced intratumoral IFNγ production, reduced tumorigenesis and enhanced survival, suggesting that the high levels of IFNγ observed in the tumors of CRC patients may be a consequence of NKG2D engagement. The mechanisms governing the contribution of NKG2D to CRC progression highlighted in this study will fuel discussions about (i) the benefit of targeting NKG2D in CRC patients and (ii) the need to define the predictive value of NKG2D and NKG2D ligand expression across tumor types.

The current understanding of the immune landscape relative to radiotherapy across tumor types.

Radiotherapy is part of the standard of care treatment for a great majority of cancer patients. As a result of radiation, both tumor cells and the environment around them are affected directly by radiation, which mainly primes but also might limit the immune response. Multiple immune factors play a role in cancer progression and response to radiotherapy, including the immune tumor microenvironment and systemic immunity referred to as the immune landscape. A heterogeneous tumor microenvironment and the varying patient characteristics complicate the dynamic relationship between radiotherapy and this immune landscape. In this review, we will present the current overview of the immunological landscape in relation to radiotherapy in order to provide insight and encourage research to further improve cancer treatment. An investigation into the impact of radiation therapy on the immune landscape showed in several cancers a common pattern of immunological responses after radiation. Radiation leads to an upsurge in infiltrating T lymphocytes and the expression of programmed death ligand 1 (PD-L1) which can hint at a benefit for the patient when combined with immunotherapy. In spite of this, lymphopenia in the tumor microenvironment of 'cold' tumors or caused by radiation is considered to be an important obstacle to the patient's survival. In several cancers, a rise in the immunosuppressive populations is seen after radiation, mainly pro-tumoral M2 macrophages and myeloid-derived suppressor cells (MDSCs). As a final point, we will highlight how the radiation parameters themselves can influence the immune system and, therefore, be exploited to the advantage of the patient.

Characterization of SHCBP1 to prognosis and immunological landscape in pan-cancer: novel insights to biomarker and therapeutic targets

Previous studies have revealed the significant roles of SHC SH2 domain-binding protein 1 (SHCBP1) in occurrence and progression of cancers, but there is no pan-cancer analysis of SHCBP1. In this study, we explored the potential carcinogenic role of SHCBP1 across 33 tumors from the TCGA and GTEx databases. We investigated SHCBP1 expression, prognosis, genetic alterations, tumor mutational burden (TMB) score, microsatellite instability (MSI) and tumor microenvironment from TIMER2, GEPIA2, UALCAN and cBioPortal databases. Moreover, the cellular functions and potential mechanisms were evaluated by GO and KEGG analysis. Besides, the mRNA expression of SHCBP1 was examined using qRT-PCR assay in gastrointestinal cancers. SHCBP1 was significantly upregulated in various cancers, and apparent relationship existed between SHCBP1 and survival prognosis in patients. The TMB, MSI, and tumor microenvironment analysis indicated that SHCBP1 was closely related to immune checkpoints, immune targets, as well as CD4+ naive T cell, CD8+ T cell, and neutrophil. Moreover, the cellular functions of SHCBP1 were mainly in regulating cell cycle motor protein activity. In addition, we validated that SHCBP1 mRNA expression was over-expressed in gastrointestinal cancers. This study was the first to systematically determine the prognostic value of SHCBP1, providing a forward-looking perspective on immunotherapy and cellular processes in pan-cancer.

Driver Mutations Dictate the Immunologic Landscape and Response to Checkpoint Immunotherapy of Glioblastoma.

The composition of the tumor immune microenvironment (TIME) is considered a key determinant of patients' response to immunotherapy. The mechanisms underlying TIME formation and development over time are poorly understood. Glioblastoma (GBM) is a lethal primary brain cancer for which there are no curative treatments. GBMs are immunologically heterogeneous and impervious to checkpoint blockade immunotherapies. Utilizing clinically relevant genetic mouse models of GBM, we identified distinct immune landscapes associated with expression of EGFR wild-type and mutant EGFRvIII cancer driver mutations. Over time, accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) was more pronounced in EGFRvIII-driven GBMs and was correlated with resistance to PD-1 and CTLA-4 combination checkpoint blockade immunotherapy. We determined that GBM-secreted CXCL1/2/3 and PMN-MDSC-expressed CXCR2 formed an axis regulating output of PMN-MDSCs from the bone marrow leading to systemic increase in these cells in the spleen and GBM tumor-draining lymph nodes. Pharmacologic targeting of this axis induced a systemic decrease in the numbers of PMN-MDSC, facilitated responses to PD-1 and CTLA-4 combination checkpoint blocking immunotherapy, and prolonged survival in mice bearing EGFRvIII-driven GBM. Our results uncover a relationship between cancer driver mutations, TIME composition, and sensitivity to checkpoint blockade in GBM and support the stratification of patients with GBM for checkpoint blockade therapy based on integrated genotypic and immunologic profiles.

Strategies for optimizing CITE-seq for human islets and other tissues.

Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor-matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

Immune Phenotypic Characterization of a TRAIL-Knockout Mouse

The TNF-superfamily member TRAIL is known to mediate selective apoptosis in tumor cells suggesting this protein as a potential antitumor drug target. However, initial successful pr-clinical results could not be translated into the clinic. Reasons for the ineffectiveness of TRAIL-targeting in tumor therapies could include acquired TRAIL resistance. A tumor cell acquires TRAIL resistance, for example, by upregulation of antiapoptotic proteins. In addition, TRAIL can also influence the immune system and thus, tumor growth. We were able to show in our previous work that TRAIL-/- mice show improved survival in a mouse model of pancreatic carcinoma. Therefore, in this study we aimed to immunologically characterize the TRAIL-/- mice. We observed no significant differences in the distribution of CD3+, CD4+, CD8+ T-cells, Tregs, and central memory CD4+ and CD8+ cells. However, we provide evidence for relevant differences in the distribution of effector memory T-cells and CD8+CD122+ cells but also in dendritic cells. Our findings suggest that T-lymphocytes of TRAIL-/- mice proliferate at a lower rate, and that the administration of recombinant TRAIL significantly increases their proliferation, while regulatory T-cells (Tregs) from TRAIL-/- mice are less suppressive. Regarding the dendritic cells, we found more type-2 conventional dendritic cells (DC2s) in the TRAIL-/- mice. For the first time (to the best of our knowledge), we provide a comprehensive characterization of the immunological landscape of TRAIL-deficient mice. This will establish an experimental basis for future investigations of TRAIL-mediated immunology.

The Genetic and Immunologic Landscape Underlying the Risk of Malignant Progression in Laryngeal Dysplasia

(1) Background: The development of laryngeal cancer is a multistep process involving structural alterations of the epithelial mucosa, from dysplasia (LDy) to invasive carcinoma. In this study, we define new biomarkers, prognostic for malignant transformation, in patients affected by LDy. (2) Methods: We used targeted next-generation sequencing and immunohistochemical analysis to define the mutational and immunological landscape of 15 laryngeal dysplasia progressing to invasive cancer (progressing dysplasia), as well as 31 cases of laryngeal dysplasia that did not progress to carcinoma (non-progressing dysplasia). Two pathologists independently analyzed the presence of tumor-infiltrating lymphocytes in LDy pre-embedded paraffin-fixed specimens. The RNA-based next-generation sequencing panel OIRRA was used to evaluate the expression of 395 genes related to immune system activation. (3) Results: High TILs are significantly correlated with a higher risk of malignant transformation. The non-brisk pattern was significantly associated with an 86% reduced risk of malignant progression (OR = 0.16, 95% CI: 0.03-0.5, p = 0.008). TILs showed a highly positive correlation with CCR6, CD83, HLA-DPB1, MX1 and SNAI1, and they were inversely correlated with CD48, CIITA, CXCR4, FCER1G, IL1B, LST1 and TLR8. (4) Conclusions: TILs have a great potential to identify high-risk progression dysplasia and thus to define surveillance protocols and prevention programs.

Genomic and immune landscape of ERBB2/ERBB3 alterations in gastroesophageal adenocarcinoma.

457 Background: HER2/ERBB2 is a known therapeutic target in gastroesophageal adenocarcinoma (GEAC). HER3/ERBB3 is an emerging target in a variety of cancers but less defined in GEAC. Insights into the genomic and immunologic landscape of ERBB2/ERBB3 alterations (ERBB2/3-alt) are required to develop effective treatments. Methods: We analyzed de-identified data from 2,050 GEAC patient tumor samples which underwent next generation sequencing with the Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). Bivariate analyses were performed to compare demographics, immuno-oncology (IO) biomarkers, and co-mutations between ERBB2/3-alt groups. P-values comparing individual co-mutations between groups were adjusted for false discovery. Results: Overall, 17.8% (365/2050) of tumors harbored an ERBB2 or ERBB3-alt, 3 of which had both and were excluded from analyses. Alterations were defined as pathogenic/likely pathogenic mutations, copy number amplifications (CN amp), or copy number loss (CN loss). Tumors were divided into ERBB2 CN amp (n=252, 12.3%), ERBB2 other (CN loss/mutation) (n=71, 3.5%), ERBB3 CN amp (n=17, 0.8%), ERBB3 other (n=22, 1%), and WT (n=1685, 82%) for both. No differences were observed between groups regarding baseline demographics (age of onset, race, ethnicity) aside from the percentage of females (p=0.002). Significant differences in IO markers and the co-occurring genetic alterations were seen. ERBB3 other (N = 22 mutations - No CN loss) had increased TMB, microsatellite instability high (MSI-H), and median neoantigen tumor burden. Conclusions: ERBB2/ERBB3-alt are associated with significant changes in the tumor microenvironment in GEAC. Co-occurring genetic or immunologic alterations can be exploited to develop effective targeted or immune therapies. [Table: see text]

Single-cell spatial landscape of immunotherapy response reveals mechanisms of CXCL13 enhanced antitumor immunity

BackgroundImmunotherapy has revolutionized clinical outcomes for patients suffering from lung cancer, yet relatively few patients sustain long-term durable responses. Recent studies have demonstrated that the tumor immune microenvironment fosters tumorous...

Single-cell spatial immune landscapes of primary and metastatic brain tumours

Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell–cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

Single-cell spatial landscapes of the lung tumour immune microenvironment

Single-cell technologies have revealed the complexity of the tumour immune microenvironment with unparalleled resolution1–9. Most clinical strategies rely on histopathological stratification of tumour subtypes, yet the spatial context of single-cell phenotypes within these stratified subgroups is poorly understood. Here we apply imaging mass cytometry to characterize the tumour and immunological landscape of samples from 416 patients with lung adenocarcinoma across five histological patterns. We resolve more than 1.6 million cells, enabling spatial analysis of immune lineages and activation states with distinct clinical correlates, including survival. Using deep learning, we can predict with high accuracy those patients who will progress after surgery using a single 1-mm2 tumour core, which could be informative for clinical management following surgical resection. Our dataset represents a valuable resource for the non-small cell lung cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. This study also highlights how artificial intelligence can improve our understanding of microenvironmental features that underlie cancer progression and may influence future clinical practice.