Implementation of paediatric precision oncology into clinical practice: The Individualized Therapies for Children with cancer program 'iTHER'.

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.

LFA-1 activation enriches tumor-specific T cells in a cold tumor model and synergizes with CTLA-4 blockade.

The inability of CD8+ effector T cells (Teffs) to reach tumor cells is an important aspect of tumor resistance to cancer immunotherapy. The recruitment of these cells to the tumor microenvironment (TME) is regulated by integrins, a family of adhesion molecules that are expressed on T cells. Here, we show that 7HP349, a small-molecule activator of lymphocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrin cell-adhesion receptors, facilitated the preferential localization of tumor-specific T cells to the tumor and improved antitumor response. 7HP349 monotherapy had modest effects on anti-programmed death 1-resistant (anti-PD-1-resistant) tumors, whereas combinatorial treatment with anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) increased CD8+ Teff intratumoral sequestration and synergized in cooperation with neutrophils in inducing cancer regression. 7HP349 intratumoral CD8+ Teff enrichment activity depended on CXCL12. We analyzed gene expression profiles using RNA from baseline and on treatment tumor samples of 14 melanoma patients. We identified baseline CXCL12 gene expression as possibly improving the likelihood or response to anti-CTLA-4 therapies. Our results provide a proof-of-principle demonstration that LFA-1 activation could convert a T cell-exclusionary TME to a T cell-enriched TME through mechanisms involving cooperation with innate immune cells.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.

Increased Tumor Glycolysis Characterizes Immune Resistance to Adoptive T Cell Therapy.

Adoptive T cell therapy (ACT) produces durable responses in some cancer patients; however, most tumors are refractory to ACT and the molecular mechanisms underlying resistance are unclear. Using two independent approaches, we identified tumor glycolysis as a pathway associated with immune resistance in melanoma. Glycolysis-related genes were upregulated in melanoma and lung cancer patient samples poorly infiltrated by T cells. Overexpression of glycolysis-related molecules impaired T cell killing of tumor cells, whereas inhibition of glycolysis enhanced T cell-mediated antitumor immunity in vitro and in vivo. Moreover, glycolysis-related gene expression was higher in melanoma tissues from ACT-refractory patients, and tumor cells derived from these patients exhibited higher glycolytic activity. We identified reduced levels of IRF1 and CXCL10 immunostimulatory molecules in highly glycolytic melanoma cells. Our findings demonstrate that tumor glycolysis is associated with the efficacy of ACT and identify the glycolysis pathway as a candidate target for combinatorial therapeutic intervention.

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Prognostic effect of different PD-L1 expression patterns in squamous cell carcinoma and adenocarcinoma of the cervix.

Programmed death-ligand 1 (PD-L1) is expressed in various immune cells and tumor cells, and is able to bind to PD-1 on T lymphocytes, thereby inhibiting their function. At present, the PD-1/PD-L1 axis is a major immunotherapeutic target for checkpoint inhibition in various cancer types, but information on the clinical significance of PD-L1 expression in cervical cancer is largely lacking. Here, we studied PD-L1 expression in paraffin-embedded samples from two cohorts of patients with cervical cancer: primary tumor samples from cohort I (squamous cell carcinoma, n=156 and adenocarcinoma, n=49) and primary and paired metastatic tumor samples from cohort II (squamous cell carcinoma, n=96 and adenocarcinoma, n=31). Squamous cell carcinomas were more frequently positive for PD-L1 and also contained more PD-L1-positive tumor-associated macrophages as compared with adenocarcinomas (both P<0.001). PD-L1-positive tumor-associated macrophages were found to express CD163 and/or CD14 by triple fluorescent immunohistochemistry, demonstrating an M2-like phenotype. Interestingly, disease-free survival (P=0.022) and disease-specific survival (P=0.046) were significantly poorer in squamous cell carcinoma patients with diffuse PD-L1 expression as compared with patients with marginal PD-L1 expression (i.e., on the interface between tumor and stroma) in primary tumors. Disease-specific survival was significantly worse in adenocarcinoma patients with PD-L1-positive tumor-associated macrophages compared with adenocarcinoma patients without PD-L1-positive tumor-associated macrophages (P=0.014). No differences in PD-L1 expression between primary tumors and paired metastatic lymph nodes were detected. However, PD-L1-positive immune cells were found in greater abundance around the metastatic tumors as compared with the paired primary tumors (P=0.001 for squamous cell carcinoma and P=0.041 for adenocarcinoma). These findings point to a key role of PD-L1 in immune escape of cervical cancer, and provide a rationale for therapeutic targeting of the PD-1/PD-L1 pathway.

A beneficial tumor microenvironment in oropharyngeal squamous cell carcinoma is characterized by a high T cell and low IL-17(+) cell frequency.

Patients with HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis than patients with non-HPV-induced OPSCC. The role of the immune response in this phenomenon is yet unclear. We studied the number of T cells, regulatory T cells (Tregs), T helper 17 (Th17) cells and IL-17(+) non-T cells (mainly granulocytes) in matched HPV-positive and HPV-negative OPSCC cases (n = 162). Furthermore, the production of IFN-γ and IL-17 by tumor-infiltrating T cells was analyzed. The number of tumor-infiltrating T cells and Tregs was higher in HPV-positive than HPV-negative OPSCC (p < 0.0001). In contrast, HPV-negative OPSCC contained significantly higher numbers of IL-17(+) non-T cells (p < 0.0001). Although a high number of intra-tumoral T cells showed a trend toward improved survival of all OPSCC patients, their prognostic effect in patients with a low number of intra-tumoral IL-17(+) non-T cells was significant with regard to disease-specific (p = 0.033) and disease-free survival (p = 0.012). This suggests that a high frequency of IL-17(+) non-T cells was related to a poor immune response, which was further supported by the observation that a high number of T cells was correlated with improved disease-free survival in the HPV-positive OPSCC (p = 0.008). In addition, we detected a minor Th17 cell population. However, T cells obtained from HPV-positive OPSCC produced significantly more IL-17 than those from HPV-negative tumors (p = 0.006). The improved prognosis of HPV-positive OPSCC is thus correlated with higher numbers of tumor-infiltrating T cells, more active Th17 cells and lower numbers of IL-17(+) non-T cells.

The tumor area occupied by Tbet+ cells in deeply invading cervical cancer predicts clinical outcome.

BACKGROUND: Deep invasion of the normal surrounding tissue by primary cervical cancers is a prognostic parameter for postoperative radiotherapy and relatively worse survival. However, patients with tumor-specific immunity in the blood at the time of surgery displayed a much better disease free survival. Here we analyzed if this was due to a more tumor-rejecting immune population in the tumor. METHODS: Tumor sections from a group of 58 patients with deep normal tissue-invading cervical tumors were stained for the presence of immune cells (CD45), IFNγ-producing cells (Tbet) and regulatory T cells (Foxp3) by immunohistochemistry. The slides were scanned and both the tumor area and the infiltration of the differently stained immune cells were objectively quantified using computer software. RESULTS: We found that an increased percentage of tumor occupied by CD45+ cells was strongly associated with an enhanced tumor-infiltration by Tbet+ cells and Foxp3+ cells. Furthermore, the area occupied by CD45+ immune cells, Tbet+ cells but not Foxp3+ cells within the tumor were, in addition to the lymph node status of patients, associated with a longer disease free survival and disease specific survival. Moreover, interaction analyses between these immune parameters and lymph node status indicated an independent prognostic effect of tumor infiltrating Tbet+ cells. This was confirmed in a multivariate Cox analysis. CONCLUSIONS: The area occupied by a preferentially type I oriented CD45+ cell infiltrate forms an independent prognostic factor for recurrence-free and disease-specific survival on top of the patient's lymph node status.

Whole-transcriptome analysis of flow-sorted cervical cancer samples reveals that B cell expressed TCL1A is correlated with improved survival.

Cervical cancer is typically well infiltrated by immune cells. Because of the intricate relationship between cancer cells and immune cells, we aimed to identify both cancer cell and immune cell expressed biomarkers. Using a novel approach, we isolated RNA from flow-sorted viable EpCAM+ tumor epithelial cells and CD45+ tumor-infiltrating immune cells obtained from squamous cell cervical cancer samples (n = 24). Total RNA was sequenced and differential gene expression analysis of the CD45+ immune cell fractions identified TCL1A as a novel marker for predicting improved survival (p = 0.007). This finding was validated using qRT-PCR (p = 0.005) and partially validated using immunohistochemistry (p = 0.083). Importantly, TCL1A was found to be expressed in a subpopulation of B cells (CD3-/CD19+/CD10+/CD34-) using multicolor immunofluorescence. A high TCL1A/CD20 (B cell) ratio, determined in total tumor samples from a separate patient cohort using qRT-PCR (n = 52), was also correlated with improved survival (p = 0.027). This is the first study demonstrating the prognostic value of separating tumor epithelial cells from tumor-infiltrating immune cells and determining their RNA expression profile for identifying putative cancer biomarkers. Our results suggest that intratumoral TCL1A+ B cells are important for controlling cervical cancer development.

Galectin-1, -3 and -9 Expression and Clinical Significance in Squamous Cervical Cancer.

Galectins are proteins that bind ß-galactoside sugars and provide a new type of potential biomarkers and therapeutic targets in cancer. Galectin-1, -3 and -9 have become the focus of different research groups, but their expression and function in cervical cancer is still unclear. The aim of this study was to determine the phenotype of galectin-1, -3 and -9 expressing cells and the association with clinico-pathological parameters in cervical cancer. Galectin expression was scored in tumor cells, tumor epithelium infiltrating immune cells and stromal cells in squamous cervical cancer (n = 160). Correlations with clinico-pathological parameters and survival were studied according to the REMARK recommendations. We additionally investigated whether the galectins were expressed by tumor cells, fibroblasts, macrophages and T cells. Galectin-1 and -9 were both expressed by tumor cells in 11% of samples, while 84% expressed galectin-3. Strong galectin-1 expression by tumor cells was an independent predictor for poor survival (hazard ratio: 8.02, p = 0.001) and correlated with increased tumor invasion (p = 0.032) and receiving post-operative radiotherapy (p = 0.020). Weak and positive tumor cell galectin-3 expression were correlated with increased and decreased tumor invasion, respectively (p = 0.012). Tumor cell expression of galectin-9 showed a trend toward improved survival (p = 0.087). The predominant immune cell type expressing galectin-1, -3 and -9 were CD163+ macrophages. Galectin-1 and -3 were expressed by a minor population of T cells. Galectin-1 was mainly expressed by fibroblasts in the tumor stroma. To conclude, while tumor cell expression of galectin-9 seemed to represent a beneficial response, galectin-1 expression might be used as a marker for a more aggressive anti-cancer treatment.

Angels and demons: Th17 cells represent a beneficial response, while neutrophil IL-17 is associated with poor prognosis in squamous cervical cancer.

The role of interleukin (IL)-17 in cancer remains controversial. In view of the growing interest in the targeting of IL-17, knowing its cellular sources and clinical implications is crucial. In the present study, we unraveled the phenotype of IL-17 expressing cells in cervical cancer using immunohistochemical double and immunofluorescent triple stainings. In the tumor stroma, IL-17 was found to be predominantly expressed by neutrophils (66%), mast cells (23%), and innate lymphoid cells (8%). Remarkably, T-helper 17 (Th17) cells were a minor IL-17 expressing population (4%). A similar distribution was observed in the tumor epithelium. The Th17 and granulocyte fractions were confirmed in head and neck, ovarian, endometrial, prostate, breast, lung, and colon carcinoma. An above median number of total IL-17 expressing cells was an independent prognostic factor for poor disease-specific survival in early stage disease (p = 0.016). While a high number of neutrophils showed at trend toward poor survival, the lowest quartile of mast cells correlated with poor survival (p = 0.011). IL-17 expressing cells and neutrophils were also correlated with the absence of vaso-invasion (p < 0.01). IL-17 was found to increase cell growth or tightness of cervical cancer cell lines, which may be a mechanism for tumorigenesis in early stage disease. These data suggest that IL-17, primarily expressed by neutrophils, predominantly promotes tumor growth, correlated with poor prognosis in early stage disease. Strikingly, a high number of Th17 cells was an independent prognostic factor for improved survival (p = 0.026), suggesting Th17 cells are part of a tumor suppressing immune response.

The correlations between IL-17 vs. Th17 cells and cancer patient survival: a systematic review.

Both IL-17 and Th17 cells have been ascribed tumor promoting as well as tumor suppressing functions. We reviewed the literature on correlations between IL-17 versus Th17 cells and survival in human cancer, following the PRISMA guidelines. Serum, formalin-fixed, paraffin-embedded (FFPE) tissue and peripheral blood samples were most frequently studied. High IL-17 quantities were correlated with poor prognosis, whereas high Th17 cell frequencies were correlated with improved prognosis. Since Th17 cells are a subpopulation of IL-17+ cells and had a different correlation with prognosis than total IL-17, we substantiate that a distinction should be made between Th17 and other IL-17+ cells.

Correlations between immune response and vascularization qRT-PCR gene expression clusters in squamous cervical cancer.

BACKGROUND: The tumour microenvironment comprises a network of immune response and vascularization factors. From this network, we identified immunological and vascularization gene expression clusters and the correlations between the clusters. We subsequently determined which factors were correlated with patient survival in cervical carcinoma. METHODS: The expression of 42 genes was investigated in 52 fresh frozen squamous cervical cancer samples by qRT-PCR. Weighted gene co-expression network analysis and mixed-model analyses were performed to identify gene expression clusters. Correlations and survival analyses were further studied at expression cluster and single gene level. RESULTS: We identified four immune response clusters: 'T cells' (CD3E/CD8A/TBX21/IFNG/FOXP3/IDO1), 'Macrophages' (CD4/CD14/CD163), 'Th2' (IL4/IL5/IL13/IL12) and 'Inflammation' (IL6/IL1B/IL8/IL23/IL10/ARG1) and two vascularization clusters: 'Angiogenesis' (VEGFA/FLT1/ANGPT2/ PGF/ICAM1) and 'Vessel maturation' (PECAM1/VCAM1/ANGPT1/SELE/KDR/LGALS9). The 'T cells' module was correlated with all modules except for 'Inflammation', while 'Inflammation' was most significantly correlated with 'Angiogenesis' (p < 0.001). High expression of the 'T cells' cluster was correlated with earlier TNM stage (p = 0.007). High CD3E expression was correlated with improved disease-specific survival (p = 0.022), while high VEGFA expression was correlated with poor disease-specific survival (p = 0.032). Independent predictors of poor disease-specific survival were IL6 (hazard ratio = 2.3, p = 0.011) and a high IL6/IL17 ratio combined with low IL5 expression (hazard ratio = 4.2, p = 0.010). CONCLUSIONS: 'Inflammation' marker IL6, especially in combination with low levels of IL5 and IL17, was correlated with poor survival. This suggests that IL6 promotes tumour growth, which may be suppressed by a Th17 and Th2 response. Measuring IL6, IL5 and IL17 expression may improve the accuracy of predicting prognosis in cervical cancer.

FoxP3(+) and IL-17(+) cells are correlated with improved prognosis in cervical adenocarcinoma.

Cervical adenocarcinoma comprises approximately 15 % of cervical cancer cases. This histological subtype has different characteristics than cervical squamous cell carcinoma, which may influence disease progression. To study whether the infiltration of T cell subpopulations was correlated with cervical adenocarcinoma patient survival, similar to squamous cell carcinoma, the tumor-infiltrating T cells, Tregs, Th17 cells and IL-17(+) cell frequencies were analyzed in a cohort of cervical adenocarcinoma patients (n = 67). Intraepithelial, stromal and total cell frequencies were scored using triple immunofluorescence. The majority of Tregs were present in the tumor stroma, while other T cells and IL-17(+) cells infiltrated the tumor epithelium three times more frequently. A high total number of Tregs were significantly correlated with improved disease-specific and disease-free survival (p = 0.010, p = 0.007). Within the tumor epithelium, a high T cell frequency was significantly correlated with improved disease-free survival (p = 0.034). In particular, a low number of both Tregs and IL-17(+) cells were correlated with poor disease-specific survival (p = 0.007). A low number of Tregs combined with Th17 cells present were also correlated with poor survival (p = 0.018). An increased number of IL-17(+) cells were significantly correlated with the absence of vaso-invasion (p = 0.001), smaller tumor size (p = 0.030) and less infiltration depth (p = 0.021). These results suggest that Tregs and IL-17(+) cells represent a beneficial immune response, whereas Th17 cells might represent a poor response in cervical adenocarcinoma. This contrasts with the correlations described in squamous cell carcinoma, suggesting that the local immune response in cervical adenocarcinoma contributes differently to tumor growth than in squamous cell carcinoma.

The human collagen beta(1-O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein.

BACKGROUND: Glycosyl transferases transfer glycosyl groups onto their substrate. Localization partially defines their function. Glycosyl transferase 25 domain 1 (GLT25D1) was recently shown to have galactosyltransferase activity towards collagens and another well known substrate, mannose binding lectin (MBL). To gain more insight in the role of galactosylation of lysines in the Gly-X-Lys repeats of collagenous proteins, we investigated the subcellular localization of GLT25D1. RESULTS: Immunofluorescence analysis of GLT25D1 expressed in the human hepatoma cell line (Huh7), revealed a perinuclear lattice like staining, resembling localization to the endoplasmic reticulum (ER). Possible targeting signals, an N-terminal signal sequence and a C-terminal ER-retention signal, were identified using prediction programs. These signals were then investigated by constructing a series of epitope-tagged forms of GLT25D1 that were analyzed by immunofluorescence and western blotting. In agreement with the predictions our results show that GLT25D1 is directed to the ER lumen as a soluble protein and retained there. Moreover, using two endoglycosidase enzymes EndoH and EndoF, we demonstrate that the putative bi-functional glycosyl transferase itself is a glycoprotein. Additionally we examined co-localization of GLT25D1 with MBL and lysyl hydroxylase 3 (LH3, PLOD3), which is a protein able to catalyze hydroxylation of lysine residues before they can be glycosylated. We demonstrate overlapping localization patterns of GLT25D1, MBL and LH3. CONCLUSIONS: Taken together our data indicate that galactosylation of collagenous proteins by the soluble GLT25D1 occurs in the early secretory pathway.