CTLA4 blockade induces frequent tumor infiltration by activated lymphocytes regardless of clinical responses in humans

Abstract

Background: CTLA4 blocking monoclonal antibodies provide durable clinical benefit in a subset of patients with advanced melanoma mediated by intratumoral lymphocytic infiltrates. A key question is defining whether the intratumoral infiltration (ITI) is a differentiating factor between patients with and without tumor responses.

Methods: Paired baseline and postdosing tumor biopsy specimens were prospectively collected from 19 patients with metastatic melanoma, including 3 patients with an objective tumor response, receiving the anti-CTLA4 antibody tremelimumab within a clinical trial with primary endpoint of quantitating CD8(+) cytotoxic T-lymphocyte (CTL) infiltration in tumors. Samples were analyzed for cell density by automated imaging capture and further characterized for functional lymphocyte properties by assessing the cell activation markers HLA-DR and CD45RO, the cell proliferation marker Ki67, and the regulatory T-cell marker FOXP3.

Results: There was a highly significant increase in ITI by CD8(+) cells in biopsy samples taken after tremelimumab treatment. This included increases between 1-fold and 100-fold changes in 14 of 18 evaluable cases regardless of clinical tumor response or progression. There was no difference between the absolute number, location, or cell density of infiltrating cells between clinical responders and patients with nonresponding lesions that showed acquired intratumoral infiltrates. There were similar levels of expression of T-cell activation markers (CD45RO, HLA-DR) in both groups and no difference in markers for cell replication (Ki67) or the suppressor cell marker FOXP3.

Conclusion: CTLA4 blockade induces frequent increases in ITI by T cells despite which only a minority of patients have objective tumor responses.

Trial registration: ClinicalTrials.gov NCT00471887.

Figure 1. Immunohistochemical analysis of CD8⁺ cell infiltration before and after tremelimumab in four representative patients

Specimens of pre- and post-tremelimumab tumor biopsies from two patients who went onto have a durable complete response after tremelimumab (GA18 and GA29) are compared with two representative samples of patients who had disease progression after therapy (GA12 and GA14). Even though there were no intact melanoma cells evident in the post-dosing sample GA18, the histological changes supported that the biopsy derived from a regressing lesion with lymphocytic infiltrates and the values for this case are considered as intratumoral infiltration.

Figure 2. Quantitative immunohistochemical analysis of intra-tumor infiltration (ITI) and peri-tumor infiltration (PTI) by CD4⁺ and CD8⁺ cells

Results are expressed as the absolute number of positively staining cells/mm² averaged over 10 fields. Open circles: Patients with an objective clinical response. Closed triangles: Patients with stable disease or progression.

Figure 3. Immunohistochemical analysis of HLA-DR and CD45RO double staining, Ki67 and FOXP3 single staining in two representative samples with post-dosing intratumoral lymphocytic infiltrates

Specimens of pre- and post-tremelimumab tumor biopsies from a patient who went onto have a durable complete response after tremelimumab (GA18) are compared with samples from a patient who had disease progression after therapy (GA12). Top row: HLA-DR and CD45RO double staining. Middle row: Ki67 staining. Bottom row: FOXP3 staining.