Sentinel node based immunotherapy of cancer

The tumor draining lymph node, the sentinel node, is regarded as a valuable station for staging and evaluating prognoses for patients with cancer. The object of this thesis was to investigate the immune response against solid cancers and to explore the sentinel node as a source of naturally occurring tumor reactive lymphocytes, for further activation and expansion in vitro for the purpose of immunotherapy. Since mortality rates in colon cancer and urinary bladder cancer are high, in spite of surgery and chemotheraphy, immunotherapy may offer an appealing complement with possible long-term protection against tumor recurrence through immunological memory.

We have demonstrated that sentinel nodes from patients with urinary bladder cancer and colon cancer contain tumor reactive immune responses. Sentinel node acquired lymphocytes proliferated dose dependently when subjected to autologous tumor extract and produced the Th1 cytokine IFN-ã, whereas tumor infiltrating lymphocytes responded poorly. However, the responses in sentinel node acquired lymphocytes harvested from metastatic lymph nodes were dampened or absent, suggesting tumor induced immunosuppression. Using long term cultures in the presence of IL-2 and autologous tumor extract, the state of anergy was overcome, since proliferation and functional production of IFN-ã were restored. Thus, the sentinel node seems to be the predominant location for activation and expansion of tumor-reactive lymphocytes, cells that can be cultured and expanded in vitro.

Since the sentinel node is the predominant location for metastases, we have developed a flow cytometry based assay, using antibodies directed against the cell surface markers EpCAM and CA19-9 to ensure tumor free expansion. We were able to detect very low levels of tumor cells in single cell suspensions. By using the combination of two cell surface markers, the problem of downregulated protein expression by tumor cells could be avoided.

Sentinel nodes were recovered intraoperatively from 16 patients with colon cancer for adoptive immunotherapy. Sentinel node acquired lymphocytes were collected, activated and expanded against autologous tumour extract. The procedure promoted mainly the clonal expansion of T helper 1 lymphocytes, producing IFN-ã upon stimulation with tumor extract. On average, 71 million activated and clonally expanded autologous T lymphocytes were transfused back to each patient. No toxic side-effects or other adverse advents were observed. Four Dukes D patients displayed complete responses with clearance of tumor burden, they have an overall average survival of 38 months. Interestingly we could demonstrate a dose response since the patients with partial response or stable disease received significantly fewer lymphocytes than patients with complete responses. The cumulative survival of immunotherapy treated patients compared with all Dukes D cases in the Stockholm region during the year of 2003, demonstrates a significant increased survival in the immunotheraphy treated group, with an average survival of 2.6 years compared to 0.8 years for the control group (P=0.048).

In conclusion, sentinel node acquired lymphocytes can be harvested, cultured and clonally expanded in vitro with maintained functionality. Immunotherapy using expanded sentinel node acquired lymphocytes is feasible, safe without apparent side effects and seems to induce tumor regression in patients with colorectal cancer.

List of scientific papers

I. Marits P, Karlsson M, Sherif A, Garske U, Thorn M, Winqvist O (2006). "Detection of immune responses against urinary bladder cancer in sentinel lymph nodes." Eur Urol 49(1): 59-70. Epub 2005 Nov 14
https://pubmed.ncbi.nlm.nih.gov/16321468

II. Marits P, Karlsson M, Dahl K, Larsson P, Wanders A, Thorn M, Winqvist O (2006). "Sentinel node lymphocytes: tumour reactive lymphocytes identified intraoperatively for the use in immunotherapy of colon cancer." Br J Cancer 94(10): 1478-84
https://pubmed.ncbi.nlm.nih.gov/16641897

III. Karlsson M, Dahl K, Marits P, Dagoo T, Enerback S, Thorn M, Winqvist O (2007). "Sentinel node CD4+ Th1-cells induce tumor regression in humans." (Submitted)

IV. Karlsson M, Nilsson O, Thorn M, Winqvist O (2007). "Detection of metastatic colon cancer cells in the sentinel node by flow cytometry." (Submitted)