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Regulatory T cells (Tregs) have long been considered one-sided suppressors of antitumor immune responses and hence associated to poor patient outcome in cancer. However, evidence is mounting of a paradoxical positive prognostic effect of Tregs on certain malignancies, including urinary bladder cancer (UBC). This discrepancy has partly been attributed to the shear misidentification of Tregs, but also to the inflammatory profile of the tumor. Our aim was to determine whether tumor-infiltrating Forkhead box P3⁺ (FOXP3⁺) cells confer a stable Treg phenotype and to investigate putative beneficial Treg functions, focusing on tumor-promoting inflammatory pathways in UBC. Patients (n = 52) with suspected UBC were prospectively included. We show, by employing a broad range of analytical approaches, that tumor-infiltrating CD4⁺FOXP3⁺ T cells in UBC phenotypically, functionally, and epigenetically represent a true Treg population. At the invasive front of UBC tumors, we found an inverse relationship between Treg frequency and expression of matrix metalloproteinase 2 (MMP2), a key pro-invasive factor induced by tumor-promoting inflammation. Correspondingly, a significant, dose-dependent Treg-mediated downregulation of MMP2 protein and mRNA expression was observed in both macrophages and urinary bladder cancer cells. Also, we found that Treg frequency specifically at the invasive front positively correlated with survival. Thus, we identify Treg-mediated suppression of MMP2 in the tumor microenvironment as a mechanism explaining the paradoxical positive prognostic impact of tumor-infiltrating Tregs in UBC.

Cancer is currently treated by a combination of therapies, including chemotherapy which is believed to suppress the immune system. Combination of immunotherapy and chemotherapy correlates with improved survival but needs careful planning in order to achieve a synergistic effect. In this study, we have demonstrated that doxorubicin treatment of B cells resulted in increased expression of CD86 and concordantly increased CD4(+) T cell activation in the presence of superantigen, an effect that was inhibited by the addition of a CD86 blocking antibody. Furthermore, doxorubicin resulted in decreased expression of the anti-inflammatory cytokines IL-10 and TNF-α. Finally, B cells from urinary bladder cancer patients, treated with a neoadjuvant regiment containing doxorubicin, displayed increased CD86-expression. We conclude that doxorubicin induces CD86 expression on B cells and hence enhances their antigen-presenting ability in vitro, a finding verified in patients. Development of tailored time and dose schedules may increase the effectiveness of combining chemotherapy and immunotherapy.

PURPOSE: To determine whether sentinel node detection (SNd) in muscle-invasive urothelial bladder cancer (MIBC) can be performed in patients undergoing neoadjuvant chemotherapy (NAC) and determine whether SNd is feasible in all pT stages, including pT0.

BACKGROUND: Previous published series of SNd in MIBC have not included patients undergoing NAC, and systematic reports of pT0 patients w/wo NAC were absent. Translational immunological tumor research on MIBC focusing on SNd, in the era of NAC, requires technical feasibility. Additionally, SNd in MIBC requests further evaluations as a method for nodal staging.

MATERIALS AND METHODS: Ninety-nine patients with suspected urothelial MIBC were prospectively selected from six urological centers. After TUR-B and primary staging, 65 MIBC patients qualified for radical cystectomy. Precystectomy staging was cT2a-T4aN0M0, including 47 NAC patients and 18 chemo-naïve patients. All 65 patients underwent intraoperative SNd by peritumoral injection of 80 Mbq Technetium and Geiger probe detection. Postcystectomy staging was pT0-T4aN0-N2M0. SNs were defined by two calculations, SNdef1 and SNdef2.

RESULTS: Totally 1063 lymph nodes were removed (total SNs; 222-227). NAC patients with pT0 (n = 24) displayed a true positive detection in 91.7 % by either SNdef, with a median of 3.0 SNs. NACpT >0 patients had a true positive detection in 87 % (SNdef1) and 91.3 % (SNdef2). In a univariate analysis, patient group neither NAC nor tumor downstaging influenced detection rates, regardless of SN definition. In total eight patients, 4/22 metastatic nodes were SNs while 18/22 were non-SNs.

CONCLUSIONS: Sentinel node detection in MIBC is feasible also in NAC patients, regardless of pT stage. SNd played no role in nodal staging.

Introduction: Overall Survival of patients with muscle invasive urothelial bladder cancer MIBC remains around 50% (5 years), albeit some improvements by combining neoadjuvant chemotherapy with radical surgery. Our previous work has demonstrated that in vitro expansions of sentinel node-acquired autologous tumor specific CD4+ T cells are promising for adoptive immunotherapy [1]. In order for naive T helper cells to become activated, they need effective APCs, presenting tumor antigens. In another study, we observed that B cells in cancer patients were tumor antigen experienced and from their phenotypes we suggested a CD4+ T cell dependent anti-tumoral response [2]. In this study, we report a flow cytometric investigation of tumor draining lymph node (sentinel node) derived B cell activation by autologous tumor extract in patients with MIBC.

Methods: Sentinel nodes (SNs) from 28 patients with MIBC were detected by a Geiger meter at cystectomy after peritumoral injection with radioactive isotope. Lymphocytes were isolated from freshly received SNs where they were stimulated with autologous tumor extract in a sterile environment. After cultivation for 7 days, the cells were analyzed by multi-color flow cytometry using FASCIA (Flow cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood).

Results: Patients displayed an increased B cell activation in SNs after stimulation with autologous tumor extract compared to when SN acquired lymphocytes were stimulated with autologous extract of macroscopically non-malignant bladder. CD4+ T cells from SNs were activated and formed blasts after co-culture with SN acquired B cells in the presence of tumor antigen. However, CD4+ T cells were not activated and did not blast when co-cultured with B cells incubated with HLA-DR-blocking antibodies. This indicates antigen presenting ability of SN acquired B cells.

Conclusions: We demonstrate sentinel node acquired B lymphocytes can be activated in culture upon stimulation with autologous tumor extract but not with extract of non-malignant epithelium of the bladder, after 7 days. Lower number of sentinel node acquired CD4+ T cells cultured with HLA-DR blocked CD19+ cells in presence of tumor antigen, indicate functional antigen presenting ability of B cells in sentinel nodes. The role of B cells as APCs in human T cell anti-tumoral response should be further explored, as well as their usefulness in adoptive immunotherapy.

Study Type - Therapy (case series) Level of Evidence 4 What's known on the subject? and What does the study add? This is the first presented results and second publication on detection of tumour-draining lymph nodes in human renal cell carcinoma. Techniques are displayed and tumour-draining patterns are presented. OBJECTIVE: •  To evaluate the feasibility of performing sentinel node detection in patients with renal cell carcinoma (RCC). MATERIALS AND METHODS: •  An open series of 13 arbitrarily selected patients with T1b-T3b RCC scheduled for radical nephrectomy at a single Tertiary Academic Centre were examined with different modalities of sentinel node detection. •  Preoperative ultrasonography-guided injection of radioactive isotope, lymphoscintigram and single photon emission computed tomography/computed tomography, followed by intraoperative gamma-probe detection and Patent Blue detection, as well as postoperative scintigram of the main specimen were the planned interventions. •  These investigations were performed in conjunction with intended open radical nephrectomy. RESULTS: •  In 10 of the 13 patients sentinel node detection was achieved with 32 sentinel nodes displayed. •  Radio-guided surgery using an intraoperative gamma-probe resulted in the highest realtive detection rate with detection of sentinel nodes in nine patients. •  In total, nine metastatic sentinel nodes were detected in three patients. •  One patient, preoperatively staged as N+, was restaged after sentinel node detection and histopathology as pN0. CONCLUSIONS: •  Sentinel node detection in renal tumours is feasible although evaluation of different modes of detection needs further refinement and standardization. •  All nodes preoperatively detected by routine computed tomography as suspicious metastatic lesions were confirmed as sentinel nodes, including two nodes considered as metastatic by preoperative routine imaging but ultimately staged as non-metastatic sentinel nodes.