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English summaryPeer reviewe

The tumor and plasma cytokine profiles of renal cell carcinoma patients

Renal cell carcinoma (RCC) accounts for 90% of all renal cancers and is considered highly immunogenic. Although many studies have reported the circulating peripheral cytokine profiles, the signatures between the tumor tissue and matching healthy adjacent renal tissue counterparts have not been explored. We aimed to comprehensively investigate the cytokine landscape of RCC tumors and its correlation between the amount and phenotype of the tumor infiltrating lymphocytes (TILs). We analyzed the secretion of 42 cytokines from the tumor (n = 46), adjacent healthy kidney tissues (n = 23) and matching plasma samples (n = 33) with a Luminex-based assay. We further explored the differences between the tissue types, as well as correlated the findings with clinical data and detailed immunophenotyping of the TILs. Using an unsupervised clustering approach, we observed distinct differences in the cytokine profiles between the tumor and adjacent renal tissue samples. The tumor samples clustered into three distinct profiles based on the cytokine expressions: high (52.2% of the tumors), intermediate (26.1%), and low (21.7%). Most of the tumor cytokines positively correlated with each other, except for IL-8 that showed no correlation with any of the measured cytokine expressions. Furthermore, the quantity of lymphocytes in the tumor samples analyzed with flow cytometry positively correlated with the chemokine-family of cytokines, CXCL10 (IP-10) and CXCL9 (MIG). No significant correlations were found between the tumor and matching plasma cytokines, suggesting that circulating cytokines poorly mirror the tumor cytokine environment. Our study highlights distinct cytokine profiles in the RCC tumor microenvironment and provides insights to potential biomarkers for the treatment of RCC.Peer reviewe

Early BCR-ABL1 Transcript Decline after 1 Month of Tyrosine Kinase Inhibitor Therapy as an Indicator for Treatment Response in Chronic Myeloid Leukemia

In chronic myeloid leukemia (CML), early treatment prediction is important to identify patients with inferior overall outcomes. We examined the feasibility of using reductions in BCR-ABL1 transcript levels after 1 month of tyrosine kinase inhibitor (TKI) treatment to predict therapy response. Fifty-two first-line TKI-treated CML patients were included (imatinib n = 26, dasatinib n = 21, nilotinib n = 5), and BCR-ABL1 transcript levels were measured at diagnosis (dg) and 1, 3, 6, 12, 18, 24, and 36 months. The fold change of the BCR-ABL1 transcripts at 1 month compared to initial BCR-ABL1 transcript levels was used to indicate early therapy response. In our cohort, 21% of patients had no decrease in BCR-ABL1 transcript levels after 1 month and were classified as poor responders. Surprisingly, these patients had lower BCR-ABL1 transcript levels at dg compared to responders (31% vs. 48%, p = 0.0083). Poor responders also significantly more often had enlarged spleen (55% vs. 15%; p<0.01) and a higher percentage of Ph+ CD34+CD38- cells in the bone marrow (91% vs. 75%, p<0.05). The major molecular response rates were inferior in the poor responders (at 12m 18% vs. 64%, p<0.01; 18m 27% vs. 75%, p<0.01; 24m 55% vs. 87%, p<0.01). In conclusion, early treatment response analysis defines a biologically distinct patient subgroup with inferior long-term outcomes.Peer reviewe

Age-associated changes in the immune system may influence the response to anti-PD1 therapy in metastatic melanoma patients

Anti-PD1 treatment has improved the survival of metastatic melanoma patients, yet it is unknown which patients benefit from the treatment. In this exploratory study, we aimed to understand the effects of anti-PD1 therapy on the patients' immune system and discover the characteristics that would result in successful treatment. We collected peripheral blood (PB) samples from 17 immuno-oncology-naive metastatic melanoma patients before and after 1 and 3 months of anti-PD1 therapy. In addition, matching tumor biopsies at the time of diagnosis were collected for tissue microarray. The complete blood counts, PB immunophenotype, serum cytokine profiles, and tumor-infiltrating lymphocytes were analyzed and correlated with the clinical data. Patients were categorized based on their disease control into responders (complete response, partial response, stable disease > 6 months, N = 11) and non-responders (progressive disease, stable diseasePeer reviewe

T and NK cell abundance defines two distinct subgroups of renal cell carcinoma

Renal cell carcinoma (RCC) is considered as an immunogenic cancer. Because not all patients respond to current immunotherapies, we aimed to investigate the immunological heterogeneity of RCC tumors. We analyzedthe immunophenotype of the circulating, tumor, and matching adjacent healthy kidney immune cells from 52 nephrectomy patients with multi-parameter flow cytometry. Additionally, we studied the transcriptomic and mutation profiles of 20 clear cell RCC (ccRCC) tumors with bulk RNA sequencing and a customized pan-cancer gene panel. The tumor samples clustered into two distinct subgroups defined by the abundance of intratumoral CD3+ T cells (CD3(high), 25/52) and NK cells (NKhigh, 27/52). CD3(high) tumors had an overall higher frequency of tumor infiltrating lymphocytes and PD-1 expression on the CD8+ T cells compared to NKhigh tumors. The tumor infiltrating T and NK cells had significantly elevated expression levels of LAG-3, PD-1, and HLA-DR compared to the circulating immune cells. Transcriptomic analysis revealed increased immune signaling (IFN-gamma, TNF-alpha via NF-kappa B, and T cell receptor signaling) and kidney metabolism pathways in the CD3(high) subgroup. Genomic analysis confirmed the typical ccRCC mutation profile including VHL, PBRM1, and SETD2 mutations, and revealed PBRM1 as a uniquely mutated gene in the CD3(high) subgroup. Approximately half of the RCC tumors have a high infiltration of NK cells associated with a lower number of tumor infiltrating lymphocytes, lower PD-1 expression, a distinct transcriptomic and mutation profile, providing insights to the immunological heterogeneity of RCC which may impact treatment responses to immunological therapies.Peer reviewe

Spatial immunoprofiling of the intratumoral and peritumoral tissue of renal cell carcinoma patients

While the abundance and phenotype of tumor-infiltrating lymphocytes are linked with clinical survival, their spatial coordination and its clinical significance remain unclear. Here, we investigated the immune profile of intratumoral and peritumoral tissue of clear cell renal cell carcinoma patients (n = 64). We trained a cell classifier to detect lymphocytes from hematoxylin and eosin stained tissue slides. Using unsupervised classification, patients were further classified into immune cold, hot and excluded topographies reflecting lymphocyte abundance and localization. The immune topography distribution was further validated with The Cancer Genome Atlas digital image dataset. We showed association between PBRM1 mutation and immune cold topography, STAG1 mutation and immune hot topography and BAP1 mutation and immune excluded topography. With quantitative multiplex immunohistochemistry we analyzed the expression of 23 lymphocyte markers in intratumoral and peritumoral tissue regions. To study spatial interactions, we developed an algorithm quantifying the proportion of adjacent immune cell pairs and their immunophenotypes. Immune excluded tumors were associated with superior overall survival (HR 0.19, p = 0.02) and less extensive metastasis. Intratumoral T cells were characterized with pronounced expression of immunological activation and exhaustion markers such as granzyme B, PD1, and LAG3. Immune cell interaction occurred most frequently in the intratumoral region and correlated with CD45RO expression. Moreover, high proportion of peritumoral CD45RO+ T cells predicted poor overall survival. In summary, intratumoral and peritumoral tissue regions represent distinct immunospatial profiles and are associated with clinicopathologic characteristics.Peer reviewe

Enlarged Memory T-Cell Pool and Enhanced Th1-Type Responses in Chronic Myeloid Leukemia Patients Who Have Successfully Discontinued IFN-alpha Monotherapy

Peer reviewe

The tumor and plasma cytokine profiles of renal cell carcinoma patients

Renal cell carcinoma (RCC) accounts for 90% of all renal cancers and is considered highly immunogenic. Although many studies have reported the circulating peripheral cytokine profiles, the signatures between the tumor tissue and matching healthy adjacent renal tissue counterparts have not been explored. We aimed to comprehensively investigate the cytokine landscape of RCC tumors and its correlation between the amount and phenotype of the tumor infiltrating lymphocytes (TILs). We analyzed the secretion of 42 cytokines from the tumor (n = 46), adjacent healthy kidney tissues (n = 23) and matching plasma samples (n = 33) with a Luminex-based assay. We further explored the differences between the tissue types, as well as correlated the findings with clinical data and detailed immunophenotyping of the TILs. Using an unsupervised clustering approach, we observed distinct differences in the cytokine profiles between the tumor and adjacent renal tissue samples. The tumor samples clustered into three distinct profiles based on the cytokine expressions: high (52.2% of the tumors), intermediate (26.1%), and low (21.7%). Most of the tumor cytokines positively correlated with each other, except for IL-8 that showed no correlation with any of the measured cytokine expressions. Furthermore, the quantity of lymphocytes in the tumor samples analyzed with flow cytometry positively correlated with the chemokine-family of cytokines, CXCL10 (IP-10) and CXCL9 (MIG). No significant correlations were found between the tumor and matching plasma cytokines, suggesting that circulating cytokines poorly mirror the tumor cytokine environment. Our study highlights distinct cytokine profiles in the RCC tumor microenvironment and provides insights to potential biomarkers for the treatment of RCC

Novel Hemizygous IL2RG p.(Pro58Ser) Mutation Impairs IL-2 Receptor Complex Expression on Lymphocytes Causing X-Linked Combined Immunodeficiency

Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient's clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny.Peer reviewe

Novel oncolytic adenovirus expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitor activates multiple immune effector populations leading to enhanced tumor killing in vitro, in vivo and with patient-derived tumor organoids

Background Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms. Methods The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids. Results Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations. Conclusion Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.Peer reviewe