Increased CD68 and Decreased CD4 Expressing Cells in Tissue Microarray (TMA) in Diagnostic Lymph Node Biopsies Are Associated with Poor Outcome in CLL/SLL.
Authors: Lee Abigail M. , Clear Andrew , Calaminici Maria , Macdougall Finlay , Goff Lindsey , Gribben John G. 
Published: October 2019

Abstract
Increasing evidence supports the importance of the number and location of tumor infiltrating lymphocytes (TILs) in cancer. Although increased circulating T regulatory cells have been found in CLL, no reports to date have examined the nature of the immune microenvironment in the lymph node (LN) in CLL/SLL. We therefore constructed TMAs composed of 1mm cores from initial diagnostic lymph nodes in 35 patients with CLL/SLL. The diagnosis of CLL/SLL was confirmed in all cases. The goals of the study were 1) to examine the nature of TILs in this disease, 2) to compare any changes in protein expression with those we have identified by gene expression profiling in peripheral blood T cells, 3) to examine the immune microenvironment in those cases with and without a leukemic component, ie comparing SLL with CLL, and 4) to assess the ability to discriminate prognostic groups based upon immunohistochemistry of diagnostic lymph nodes. The first group of 17 patients were selected on the basis of poor outcome, with median survival of 38 months from diagnosis, with all patients dying of their disease. The median age of this group at presentation was 62 years, 12 were male and 5 female. The second group of 18 patients were selected on the basis of long survival and had median survival of more than 10 years. Their median age at presentation was 53 years, 11 were male and 7 female. Immunohistochemistry was performed on the TMAs using a panel of monoclonal antibodies including CD4, CD8, CD25, CD6, CD7, FOXP3, CD68, TIA-1 and Granzyme B. The immune infiltrates were scored based on immunophenotype, frequency and location. All cores were analyzed independently by two histopathologists and concordance was achieved in all cases. The majority of cases in both groups had >30 CD8 expressing cells per high power field (hpf) as well as a high frequency of FOXP3 expressing cells, whereas we observed low number of cells expressing TIA-1 and granzyme B. In keeping with our previous gene expression profiling data in peripheral blood T cells, CD6 was expressed in a lower frequency of cells in the poor prognosis group. The poor prognosis group also had fewer CD4 but increased CD25 expressing T cells, as well as increased CD68 expressing monocytes. Ongoing studies are assessing the use of TMA in LN and bone marrow biopsies in CLL/SLL in a larger cohort of patients as well as analyzing additional antibodies, particularly for those antigens previously identified by gene expression profiling. The results are in keeping with the hypothesis that there is a complex interaction between the disease and the immune microenvironment in the LN in CLL/SLL and that the immune microenvironment represents a rational therapeutic target in this disease.
Antigen expression Good Prognosis Pts Poor Prognosis Pts CD4 >5 cells/hpf 14/17 cases (82%) >5 cells/hpf 8/12 cases (67%) <5 cells/hpf 3/17 cases (18%) <5 cells/hpf 4/12 cases (33%) CD25 >5 cells/hpf 11/16 cases (69%) >5 cells/hpf 11/13 cases (85%) <5 cells/hpf 5/16 cases (31%) <5 cells/hpf 2/13 cases(15%) CD68 >5 cells/hpf 9/17 cases (53%) >5 cells/hpf 11/13 cases (85%) <5 cells/hpf 8/17 cases (47%) <5 cells/hpf 2/13 cases(15%) CD6 >5 cells/hpf 14/17 cases (82%) >5 cells/hpf 7/12 cases (58%) <5 cells/hpf 3/17 cases (18%) <5 cells/hpf 5/12 cases (42%)