1 1 2 3 4 5 6 7 8 9 10 12 3 13 14 15 17 18 6 9 19 20 The aim of the present study was to investigate lymphatic drainage from metastases and identify first draining lymph nodes. We also analyzed tumor-reactive immunological properties in lymphocytes derived from these lymph nodes. MATERIALS AND METHODS Patients 1 Table 1. a Patient Age (y) Sex Primary tumor Origin metinel node Tracer Number of metinel nodes Metinel node pos/neg for metastases Successful expansion Alive Follow-up time (mo) 1 48 M Colon Intra-abdominal local recurrence PB 3 3 neg Yes Yes 38 2 54 M Rectum Liver metastasis PB 2 2 neg Yes Yes 38 3 50 M Mal melanoma Subcutaneous metastasis PB 2 2 pos No No – 4 77 F Colon Liver metastasis PB 3 3 neg No No – 5 74 M Colon Liver metastasis PB 2 1 pos Yes No – 1 neg 6 66 M Colon Liver metastasis PB 1 MI No Yes 29 7 51 F Ovarian cancer Liver metastasis PB 2 1 pos b No – 1 neg 8 64 F Ovarian cancer Groin lymph node PB/RT 5 5 pos No No – 9 59 F Pancreatic cancer Intra-abdominal local recurrence PB 4 MI No No – 10 39 F Mal melanoma Groin lymph node PB/RT 4 2 neg No Yes 11 11 47 M Mal melanoma Truncal metastasis RT 1 2 pos No No – 12 45 F Breast cancer Cervical lymph node PB/RT 2 2 neg Yes Yes 5 13 33 F Tongue cancer Truncal lymph node RT 3 MI No Yes 6 14 64 F Colon Intra-abdominal local recurrence PB 2 1 pos Yes Yes 4 1 neg 15 41 F Breast cancer Liver metastasis PB 2 2 neg No Yes 4 16 39 M Mal melanoma Cervical lymph node PB/RT 2 2 neg No Yes 2 17 20 M Cholangiocarcinoma Liver metastasis PB 3 3 neg Yes No – 18 65 M Colon Liver metastasis PB 3 1 pos Yes Yes 1 2 neg 19 42 F Leiomyosarcoma Liver metastasis PB 4 4 pos Yes Yes 1 pos, positive; neg, negative; Mal, malignant; PB, Patent blue dye; MI, missing information; RT, radioactive tracer (4 × 10–15 MBq Tc-nanokolloid). a b The lymph nodes that drained metastases were identified by injecting approximately 1 mL Patent blue dye (PB) (Guerbet, Paris) subserosally or subcutaneously in four places around one of the metastasis or in the tissues close to the local recurrences with a 27-gauge needle. After the injection of PB, the first draining lymph nodes turned blue within 3 to 10 minutes and were regarded as metastases-draining lymph nodes. In our previous work, a 10-minute cutoff was sufficient to identify sentinel nodes to primary tumors. We have experienced that during this period of time the first draining lymph nodes turn blue. Waiting longer means that a second echelon of draining lymph nodes may be colored blue, which does not represent the first draining lymph nodes. We arbitrarily applied the same 10-minute limit when detecting lymph nodes that drain metastases. These lymph nodes were either immediately removed or marked with sutures. The sources of the liver metastases in our study were from colorectal cancer in five patients and in one patient each from ovarian cancer, breast cancer, cholangiocarcinoma, and leiomyosarcoma. Four patients had subcutaneous lymph node metastases from disseminated malignant melanoma, and one patient each had metastases from ovarian cancer, breast cancer, and squamous cell carcinoma of the tongue. Two patients had intra-abdominal local recurrences after surgery for colon cancer, and one patient had an intra-abdominal local recurrence after surgery for pancreatic cancer. Because of these liver metastases, five patients underwent partial right-sided liver resections (patients 4, 5, 6, 15, and 17), and three patients underwent partial left-sided liver resection (patients 2, 18, and 19). The patient with metastases resulting from ovarian cancer (patient 7) had several large, bulky, cystic tumors, and surgeons performed an almost complete right-sided liver resection. In four cases, surgery was performed for solitary colorectal liver metastases (patients 2, 4, 5 and 6), and in the remaining five cases, tumor-reducing surgery was performed. In six cases, a preoperative lymphoscintigraphy was performed by a subcutaneous injection of 4 × 10 to 15 MBq Tc-nanokolloid radioactive tracer in quadrants around the metastasis. The lymphoscintigraphies were performed to plan the surgical procedure by localizing the lymph node or nodes draining the metastasis. The position of the metastasis-draining lymph node or nodes was marked on the skin with a felt-tip pen. When the lymphoscintigraphy was not performed on the day of surgery, a new peritumoral injection of radioactive tracer was made at start of surgery. The draining nodes were intraoperatively identified by the use of a handheld gamma detection tube. In four of six cases, a simultaneous intraoperative injection of PB was performed. Preparation of Specimens Lymph nodes draining metastases and nondraining lymph nodes (for analytical purposes) were surgically removed and immediately taken to the laboratory on ice. In addition, one 5-mm slice of the whole metastasis (including the invasive margin) was also dissected and sent for analysis. Frequently, most of the lymph node or nodes were used for the expansion procedure. The remaining specimens underwent routine histopathological examinations with hematoxylin and eosin staining. At the laboratory, slices <1 mm were cut from the central and peripheral part of the lymph nodes for analysis by flow cytometry (FACS) and proliferation analyses. The metastasis was also analyzed by FACS, and a preparation of the whole tumor (homogenate) was used. Immunological Evaluation 19 6 3 6 19 6 The transfusions took place at the surgical ward as an intravenous transfusion over the course of 1 hour. Our criterion for adoptive transfer is a transfusion of lymphocytes to a patient, regardless of the number of transferred cells. In our previous studies (M.K., unpublished data) of adoptive transfer originating from sentinel nodes, on average, 71 million clonally expanded autologous tumor-reactive lymphocytes were transfused to each patient, and in that pilot study, the disease of all 16 patients responded to therapy. We do not know the optimal number of cells to be transfused for the best or maximum immune response, but according to our experience, 39.5 million transfused cells (the average number of transfused cells in the metinel node study) may be sufficient. The explanation may be that the CD4 cells proliferate and promote division of effector T cells and memory T cells when they are stimulated by their antigen (which is derived from the tumor). The cell expansion continues in the patient. RESULTS Metinel Nodes 1 1 1 Fig. 1. A B  In all cases of surgery for liver metastases, lymph nodes draining the area of the metastases were found within the liver hilum or hepatoduodenal ligament. The average number of metinel nodes from liver metastases were 2.4, and 7 (32%) of 22 of the analyzed metinel nodes were positive for metastatic disease. Two patients underwent surgery to treat intra-abdominal local recurrences from colon cancer (patients 1 and 14); resections of the recurrence and reresection of the bowel en bloc were performed. One patient with pancreatic cancer (patient 9) underwent tumor-reducing surgery to treat a local intra-abdominal recurrence after a previous Whipple operation. 1 Immunology 19 20 + + + + 2 + + + + + + Table 2. Immunology of single cell suspensions collected from metinel nodes Patient Time in culture (d) No. of cells at start (millions) No. of cells at end (millions) Ratio of cells start/end Start (% gated), ratio CD4/CD8 and CD19/CD56 End, ratio CD4/CD8 and CD19/CD56 Ratio CD4/CD8 start Ratio CD4/CD8 end 1 31 100 80 .8 24/11 and 55/− 24/16 and −/− 1.8 1.5 2 33 42 40 . 51/15 and 13/− 24 and .12/− 3.4 1.5 3 28 – – – 8/39 and 1/− – .2 – 4 20 65.4 – – 37/9 and −/− – 4.1 – 5 43 130 – . 47/7.1 and 28/1.3 73/22 and .09/.17 6.6 3.3 6 6 5 – – .2/1.6 and .4/.4 – .125 – 7 6 5 .7 . 16.4/6.5 and 24.2/1.6 – 2.5 – 8 52 42.6 – – 15.9/7.6 and 9/3.4 – 2.1 – 9 15 12 – – – – – – 10 74 166.8 5 . 40/7 and .5/.3 – 5.7 – 11 35 209 140 . 12.8/16.3 and 1.9/0 – .8 – 12 31 285 2 . 16.4/31.8 and .1/0 – .5 – 13 24 3 – – – – – – 14 32 30 1 . – – – – 15 48 36 – – 0/.1 and 0/0 – 0 – 16 45 20 – – 28.25/6.95 and 1.15/0 – 4.1 – 17 20 89 4 . 5.8/2.6 and 4.2/.2 34.2/30.4 and 7.6/20.1 2.2 1.1 18 29 67 4 . 9.7/3.8 and 1.3/.1 – 2.6 – 19 43 11 15 . 12.7/15.8 and 3/18.9 85/3 and .9/8.4 .8 28.3 + + + + + + + + + + + + + + + 6 6 6 6 2 2 Fig. 2. +  3 Fig. 3. Amount of interferon gamma secreted into the supernatant measured by enzyme-linked immunosorbent assay. Data presented are for patient 7 after 35 days of in vitro cell culture.  According to more detailed analyses of some cases, we found that some T cells expressed Fox P3, indicating that these cells in fact were T regulatory cells. DISCUSSION We show that it is possible to find the first lymph nodes draining various types of metastases by lymphatic mapping by means of dye or radioactive tracers. We have named these metastasis-draining lymph nodes “metinel nodes” to show both the analogy and difference to the well-known term “sentinel node,” which means the first node to receive lymphatic drainage from a primary tumor. In 19 studied cases, we demonstrated three different ways of identifying these nodes by injection of tracers subserosally around liver or intra-abdominal metastases, close to subcutaneous metastases, or into local lymph node metastases. The fact that it is possible to expand the metinel node–derived lymphocytes via in vitro expansions during several weeks of time, together with our analysis that they contain lymphocytes that show clonal expansion toward tumor antigens and produce high levels of IFN-γ in the expansions, are evidence that they contain tumor-reactive lymphocytes. These cells are mainly T-helper 1 cells that have developed an immunological response toward the metastatic cells. We suggest that these T cells may be used in future trials of adoptive cellular therapy of disseminated solid cancer. 21 22 23 24 25 26 18 Thus, a tumor causes an upregulation of lymphangiogenic factors that leads to an increase in peritumoral lymphatic vessels in both tumors and metastases. It is likely that these newly developed lymph vessels connect to the preexisting lymphatic vessels and lymph nodes. Shed tumor cells may invade these newly formed vessels, and the dissemination of tumor cells is facilitated. The present study shows that this process can be visualized by lymphatic mapping, and the first draining lymph node from the metastasis, the metinel node, may be identified. 16 15 27 28 29 30 14 31 32 33 34 35 36 19 20 37 39 2 1 In this study, we have shown that it is possible to find the draining lymph nodes from different types of metastases. We named these nodes “metinel nodes,” and they contain tumor-reactive lymphocytes, preferentially T-helper cells with Th1 response. We have also demonstrated that it is possible to proliferate these cells in vitro and, if the expansion procedure is successful, to use the cells for adoptive immunotherapy.