A. Sherif

Lymphatic mapping and detection of sentinel nodes in patients with bladder cancer.

PURPOSE We examined the possibility for detecting sentinel nodes in patients with bladder cancer and whether the histopathological status of identified sentinel nodes reflected that of the lymphatic field. MATERIALS AND METHODS A total of 13 patients with bladder cancer who met the criteria qualifying them for radical cystectomy had intravesical injections of radioactive tracer and blue dye marker around the tumor followed by lymphoscintigraphy to visualize lymphatic drainage and detect sentinel nodes. Sentinel nodes were identified preoperatively by the blue color and increased radioactivity and were compared histopathologically with other routinely excised lymph nodes. RESULTS Sentinel nodes were detected in 85% (11 of 13) of patients. There were 4 patients who had sentinel nodes containing tumor cells, and each metastasis was only seen in the detected sentinel node. There were no false-negative sentinel nodes. Of the metastatic sentinel nodes 3 were located outside the normally excised lymph nodes of the obturator fossa. CONCLUSIONS Sentinel nodes can be detected in patients with bladder cancer. The histopathological status of the identified sentinel nodes was diagnostic for all other excised lymph nodes. Sentinel nodes often seem to be located outside the obturator lymphatic field, which is normally examined during preoperative staging of bladder cancer.

Detection of Immune Responses in Sentinel Nodes Draining Human Urinary Bladder Cancer

Being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour‐reactive lymphocytes. We investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour‐specific proliferation and TCR Vβ repertoires. During tumour surgery, sentinel lymph nodes were identified by peri‐tumoural injection of blue dye. Fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single‐cell suspensions were prepared. Cells were assayed for reactivity against autologous tumour extract in [3H]‐thymidine incorporation assays and characterized by flow cytometry. Parallel analyses of the expression of Vβ gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. Probes were reacted with cDNA prepared from magnetically separated CD4+ cells, and the TCR repertoire was determined by hybridizing the products to oligonucleotide microarrays. Dose‐dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. Common clonal expansions were detected among tumour‐infiltrating lymphocytes and in sentinel lymph nodes. Nonsentinel lymph nodes displayed a divergent TCR Vβ repertoire. These results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. Identification of sentinel lymph nodes makes it possible to obtain tumour‐reactive lymphocytes for use in adoptive immunotherapy.

DETECTION OF IMMUNE RESPONSES AGAINST URINARY BLADDER CANCER IN SENTINEL LYMPH NODES

OBJECTIVE The lymphatic drainage from a tumour is received in the sentinel node where the immune system encounters tumour derived antigens. We investigated anti-tumoural lymphocyte function in sentinel nodes from patients with urinary bladder cancer. METHODS In 14 patients undergoing cystectomy due to bladder cancer, radioactive tracer and blue dye were used to identify the sentinel node. Cell suspensions from the tumour, sentinel- and non-sentinel nodes and peripheral blood were analyzed by flow cytometry with antibodies against lymphocyte surface antigens and against the tumour cell marker cytokeratin-20. Reactivity against autologous tumour extract and the mitogen Concanavalin A was tested in proliferation assays with 3H-Thymidine incorporation. Lymphocytes were put in long-term culture with IL-2 and autologous tumour extract. RESULTS Sentinel nodes were detected in 12 of the 14 patients. Antigen dependent proliferation in response to autologous tumour extract was detected in 6 patients, in 5 cases in sentinel nodes, in the remaining case in a non-sentinel node. Proliferation against Concanavalin A was vigorous in lymph nodes from all patients, whereas tumour infiltrating lymphocytes were unresponsive. Lymphocytes from sentinel nodes could be expanded in vitro. CONCLUSION Tumour reactive lymphocytes are present in sentinel nodes draining human bladder cancers. These cells display immunologic function upon restimulation in vitro, and provide a promising source for expansion and subsequent adoptive T cell immunotherapy.